Jennifer S Carew

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• Bao, C., Liang, S., Han, Y., Yang, Z., Liu, S., Sun, Y., Zheng, S., Li, Y., Wang, T., Gu, Y., Wu, K., Black, S. M., Wang, J., Nawrocki, S. T., Carew, J. S., Yuan, J. X., & Tang, H. (2023). The Novel Lysosomal Autophagy Inhibitor (ROC-325) Ameliorates Experimental Pulmonary Hypertension. Hypertension (Dallas, Tex. : 1979), 80(1), 70-83.
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  Autophagy plays an important role in the pathogenesis of pulmonary hypertension (PH). ROC-325 is a novel small molecule lysosomal autophagy inhibitor that has more potent anticancer activity than the antimalarial drug hydroxychloroquine, the latter has been prevalently used to inhibit autophagy. Here, we sought to determine the therapeutic benefit and mechanism of action of ROC-325 in experimental PH models.
• Jones, T. M., Espitia, C. M., Chipollini, J., Lee, B. R., Wertheim, J. A., Carew, J. S., & Nawrocki, S. T. (2023). Targeting NEDDylation is a Novel Strategy to Attenuate Cisplatin-induced Nephrotoxicity. Cancer research communications, 3(2), 245-257.
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  Although cisplatin remains a backbone of standard-of-care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30%-40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models. We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin-interacting protein (TXNIP)-mediated mechanism. Cotreatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism.
• Nawrocki, S. T., Olea, J., Villa Celi, C., Dadrastoussi, H., Wu, K., Tsao-Wei, D., Colombo, A., Coffey, M., Fernandez Hernandez, E., Chen, X., Nuovo, G. J., Carew, J. S., Mohrbacher, A. F., Fields, P., Kuhn, P., Siddiqi, I., Merchant, A., & Kelly, K. R. (2023). Comprehensive Single-Cell Immune Profiling Defines the Patient Multiple Myeloma Microenvironment Following Oncolytic Virus Therapy in a Phase Ib Trial. Clinical cancer research : an official journal of the American Association for Cancer Research, 29(24), 5087-5103.
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  Our preclinical studies showed that the oncolytic reovirus formulation pelareorep (PELA) has significant immunomodulatory anti-myeloma activity. We conducted an investigator-initiated clinical trial to evaluate PELA in combination with dexamethasone (Dex) and bortezomib (BZ) and define the tumor immune microenvironment (TiME) in patients with multiple myeloma treated with this regimen.
• Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., & Lee, B. R. (2023). Targeting NEDDylation is a novel strategy to attenuate cisplatin-induced nephrotoxicity. . American Association for Cancer Research.
• Ogbuji, V., Paster, I. C., Recio-Boiles, A., Carew, J. S., Nawrocki, S. T., & Chipollini, J. (2023). Current Landscape of Immune Checkpoint Inhibitors for Metastatic Urothelial Carcinoma: Is There a Role for Additional T-Cell Blockade?. Cancers, 16(1).
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  Urothelial carcinoma (UC) is the most common form of bladder cancer (BC) and is the variant with the most immunogenic response. This makes urothelial carcinoma an ideal candidate for immunotherapy with immune checkpoint inhibitors. Key immune checkpoint proteins PD-1 and CTLA-4 are frequently expressed on T-cells in urothelial carcinoma. The blockade of this immune checkpoint can lead to the reactivation of lymphocytes and augment the anti-tumor immune response. The only immune checkpoint inhibitors that are FDA-approved for metastatic urothelial carcinoma target the programmed death-1 receptor and its ligand (PD-1/PD-L1) axis. However, the overall response rate and progression-free survival rates of these agents are limited in this patient population. Therefore, there is a need to find further immune-bolstering treatment combinations that may positively impact survival for patients with advanced UC. In this review, the current immune checkpoint inhibition treatment landscape is explored with an emphasis on combination therapy in the form of PD-1/PD-L1 with CTLA-4 blockade. The investigation of the current literature on immune checkpoint inhibition found that preclinical data show a decrease in tumor volumes and size when PD-1/PD-L1 is blocked, and similar results were observed with CTLA-4 blockade. However, there are limited investigations evaluating the combination of CTLA-4 and PD-1/PD-L1 blockade. We anticipate this review to provide a foundation for a deeper experimental investigation into combination immune checkpoint inhibition therapy in metastatic urothelial carcinoma.
• Adema, V., Ma, F., Kanagal-Shamanna, R., Thongon, N., Montalban-Bravo, G., Yang, H., Peslak, S. A., Wang, F., Acha, P., Sole, F., Lockyer, P., Cassari, M., Maciejewski, J. P., Visconte, V., Gañán-Gómez, I., Song, Y., Bueso-Ramos, C., Pellegrini, M., Tan, T. M., , Bejar, R., et al. (2022). Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1-Mutant Myelodysplastic Syndromes With Ringed Sideroblasts. Blood cancer discovery, 3(6), 554-567.
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  SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production.
• Jones, T. M., Espitia, C. M., Ooi, A., Bauman, J. E., Carew, J. S., & Nawrocki, S. T. (2022). Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cell death & disease, 13(4), 350.
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  Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.
• Adema, V., Khouri, J., Ni, Y., Rogers, H. J., Kerr, C. M., Awada, H., Nagata, Y., Kuzmanovic, T., Advani, A. S., Gerds, A. T., Mukherjee, S., Nazha, A., Saunthararajah, Y., Madanat, Y., Patel, B. J., Solé, F., Nawrocki, S. T., Carew, J. S., Sekeres, M. A., , Maciejewski, J. P., et al. (2020). Analysis of distinct hotspot mutations in relation to clinical phenotypes and response to therapy in myeloid neoplasia. Leukemia & lymphoma, 1-4.
• Advani, A. S., Tse, W., Li, H., Jia, X., Elson, P., Cooper, B., Ali-Osman, F., Park, J., Rao, A. V., Rizzieri, D. A., Wang, E. S., Cotta, C. V., Kalaycio, M., Sobecks, R. M., Rouphail, B., Maciejewski, J. P., Fensterl, J., Carew, J. S., Foster, B., , Rush, M. L., et al. (2020). A Phase II Trial of Imatinib Mesylate as Maintenance Therapy for Patients With Newly Diagnosed C-kit-positive Acute Myeloid Leukemia. Clinical lymphoma, myeloma & leukemia.
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  Adults with acute myeloid leukemia (AML) have a high rate of remission; however, more than 50% relapse. C-kit is expressed in approximately 60% of patients with de novo AML and represents a potential therapeutic target.
• Islam, S., Espitia, C. M., Persky, D. O., Carew, J. S., & Nawrocki, S. T. (2020). Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy. Blood advances, 4(20), 5297-5310.
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  Despite the promising antilymphoma activity of histone deacetylase (HDAC) inhibitors as a drug class, resistance is a significant clinical issue. Elucidating the molecular mechanisms driving HDAC inhibitor resistance and/or the specific targets that are altered in drug-resistant cells may facilitate the development of strategies that overcome drug resistance and are more effective for refractory patients. We generated novel T-cell lymphoma (TCL) cell line models of acquired resistance to the HDAC inhibitor belinostat to identify potential effective therapies. Belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including romidepsin, panobinostat, and vorinostat. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones. Drug-resistant cells featured significantly decreased expression of the key antiviral mediators IRF1 and STAT1. On the basis of these findings, we investigated the efficacy of the clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that HDAC inhibitor-resistant cells displayed enhanced vulnerability to reovirus replication and cell death in both in vitro and in vivo models compared with their parental counterparts. Importantly, Reolysin also significantly increased the antilymphoma activity of belinostat in HDAC inhibitor-resistant cells. Our data demonstrate that Reolysin alone or in combination with belinostat is a novel therapeutic strategy to treat TCL patients who develop resistance to HDAC inhibitors.
• Jones, T. M., Carew, J. S., & Nawrocki, S. T. (2020). Therapeutic Targeting of Autophagy for Renal Cell Carcinoma Therapy. Cancers, 12(5).
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  Kidney cancer is the 7th most prevalent form of cancer in the United States with the vast majority of cases being classified as renal cell carcinoma (RCC). Multiple targeted therapies have been developed to treat RCC, but efficacy and resistance remain a challenge. In recent years, the modulation of autophagy has been shown to augment the cytotoxicity of approved RCC therapeutics and overcome drug resistance. Inhibition of autophagy blocks a key nutrient recycling process that cancer cells utilize for cell survival following periods of stress including chemotherapeutic treatment. Classic autophagy inhibitors such as chloroquine and hydroxychloroquine have been introduced into phase I/II clinical trials, while more experimental compounds are moving forward in preclinical development. Here we examine the current state and future directions of targeting autophagy to improve the efficacy of RCC therapeutics.
• Kathawala, R. J., Espitia, C. M., Jones, T. M., Islam, S., Gupta, P., Zhang, Y. K., Chen, Z. S., Carew, J. S., & Nawrocki, S. T. (2020). ABCG2 Overexpression Contributes to Pevonedistat Resistance. Cancers, 12(2).
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  MLN4924 (pevonedistat) is a first-in-class NEDD8-activating enzyme (NAE) inhibitor in clinical trials for the treatment of solid tumors and hematologic malignancies. Despite the promising activity of MLN4924 observed in early trials, drug resistance has been noted in some patients. Identifying the underlying cause of treatment failure may help to better stratify patients that are most likely to benefit from this novel agent. Early preclinical studies revealed that the development of NAE mutations promotes resistance to MLN4924. However, these mutations have not been detected in patients that are relapsed/refractory to MLN4924, suggesting that other mechanisms are driving clinical resistance. To better understand the potential mechanisms of MLN4924 resistance, we generated MLN4924-resistant ovarian cancer cells. Interestingly, these cells did not develop mutations in NAE. Transcriptome analyses revealed that one of the most upregulated genes in resistant cells was . This result was validated by quantitative real-time PCR and immunoblotting. Importantly, the sensitivity of MLN4924-resistant cells was restored by lentiviral short hairpin RNA (shRNA) targeting . Further investigation using ABCG2-overexpressing NCI-H460/MX20 cells determined that these cells are resistant to the anticancer effects of MLN4924 and can be sensitized by co-treatment with the ABCG2 inhibitors YHO-13351 and fumitremorgin C. Finally, HEK293 models with overexpression of wild-type ABCG2 (R482) and variants (R482G and R482T) all demonstrated significant resistance to MLN4924 compared to wild-type cells. Overall, these findings define an important molecular resistance mechanism to MLN4924 and demonstrate that ABCG2 may be a useful clinical biomarker that predicts resistance to MLN4924 treatment.
• Nawrocki, S. T., Wang, W., & Carew, J. S. (2020). Autophagy: New Insights into Its Roles in Cancer Progression and Drug Resistance. Cancers, 12(10).
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  Autophagy is a mechanism of lysosomal proteolysis that is utilized to degrade damaged organelles, proteins, and other cellular components. Although key studies demonstrate that autophagy functions as a mechanism of tumor suppression via the degradation of defective pre-malignant cells, autophagy can also be used as a mechanism to break down cellular components under stress conditions to generate the required metabolic materials for cell survival. Autophagy has emerged as an important mediator of resistance to radiation, chemotherapy, and targeted agents. This series of articles highlight the role of autophagy in cancer progression and drug resistance and underscores the need for new and more effective agents that target this process.
• Usman, R. M., Razzaq, F., Akbar, A., Farooqui, A. A., Iftikhar, A., Latif, A., Hassan, H., Zhao, J., Carew, J. S., Nawrocki, S. T., & Anwer, F. (2020). Role and mechanism of autophagy-regulating factors in tumorigenesis and drug resistance. Asia-Pacific journal of clinical oncology.
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  A hallmark feature of tumorigenesis is uncontrolled cell division. Autophagy is regulated by more than 30 genes and it is one of several mechanisms by which cells maintain homeostasis. Autophagy promotes cancer progression and drug resistance. Several genes play important roles in autophagy-induced tumorigenesis and drug resistance including Beclin-1, MIF, HMGB1, p53, PTEN, p62, RAC3, SRC3, NF-2, MEG3, LAPTM4B, mTOR, BRAF and c-MYC. These genes alter cell growth, cellular microenvironment and cell division. Mechanisms involved in tumorigenesis and drug resistance include microdeletions, genetic mutations, loss of heterozygosity, hypermethylation, microsatellite instability and translational modifications at a molecular level. Disrupted or altered autophagy has been reported in hematological malignancies like lymphoma, leukemia and myeloma as well as multiple solid organ tumors like colorectal, hepatocellular, gall bladder, pancreatic, gastric and cholangiocarcinoma among many other malignancies. In addition, defects in autophagy also play a role in drug resistance in cancers like osteosarcoma, ovarian and lung carcinomas following treatment with drugs such as doxorubicin, paclitaxel, cisplatin, gemcitabine and etoposide. Therapeutic approaches that modulate autophagy are a novel future direction for cancer drug development that may help to prevent issues with disease progression and overcome drug resistance.
• Advani, A. S., Cooper, B., Visconte, V., Elson, P., Chan, R., Carew, J., Wei, W., Mukherjee, S., Gerds, A., Carraway, H., Nazha, A., Hamilton, B., Sobecks, R., Caimi, P., Tomlinson, B., Malek, E., Little, J., Miron, A., Pink, J., , Maciejewski, J., et al. (2019). A Phase I/II Trial of MEC (Mitoxantrone, Etoposide, Cytarabine) in Combination with Ixazomib for Relapsed Refractory Acute Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(14), 4231-4237.
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  The prognosis of patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor, and novel therapies are needed. The proteasome pathway represents a potential therapeutic target. A phase I trial of the second-generation proteasome inhibitor ixazomib in combination with MEC (mitoxantrone, etoposide, and cytarabine) was conducted in patients with R/R AML.
• Anwer, F., Gee, K. M., Iftikhar, A., Baig, M., Russ, A. D., Saeed, S., Zar, M. A., Razzaq, F., Carew, J., Nawrocki, S., Al-Kateb, H., Cavalcante Parr, N. N., McBride, A., Valent, J., & Samaras, C. (2019). Future of Personalized Therapy Targeting Aberrant Signaling Pathways in Multiple Myeloma. Clinical lymphoma, myeloma & leukemia, 19(7), 397-405.
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  Multiple myeloma (MM) is a genetically complex disease. Identification of mutations and aberrant signaling pathways that contribute to the progression of MM and drug resistance has potential to lead to specific targets and personalized treatment. Aberrant signal pathways include RAS pathway activation due to RAS or BRAF mutations (targeted by vemurafenib alone or combined with cobimetinib), BCL-2 overexpression in t(11:14) (targeted by venetoclax), JAK2 pathway activation (targeted by ruxolitinib), NF-κB pathway activation (treated with DANFIN combined with bortezomib), MDM2 overexpression, and PI3K/mTOR pathway activation (targeted by BEZ235). Cyclin D1 (CCND1) and MYC are also emerging as key potential targets. In addition, histone deacetylase inhibitors are already in use for the treatment of MM in combination therapy, and targeted inhibition of FGFR3 (AZD4547) is effective in myeloma cells with t(4;14) translocation. Bromodomain and extra terminal (BET) protein antagonists decrease the expression of MYC and have displayed promising antimyeloma activity. A better understanding of the alterations in signaling pathways that promote MM progression will further inform the development of precision therapy for patients.
• Carew, J. S., Espitia, C. M., Zhao, W., Visconte, V., Anwer, F., Kelly, K. R., & Nawrocki, S. T. (2019). Rational cotargeting of HDAC6 and BET proteins yields synergistic antimyeloma activity. Blood advances, 3(8), 1318-1329.
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  Inhibition of bromodomain and extra terminal (BET) protein family members, including BRD4, decreases the expression of c-MYC and other key oncogenic factors and also significantly induces histone deacetylase 6 (HDAC6) expression. On the basis of the role of HDAC6 in malignant pathogenesis, we hypothesized that rational cotargeting of HDAC6 and BET family proteins may represent a novel approach that yields synergistic antimyeloma activity. We used genetic and pharmacologic approaches to selectively impair HDAC6 and BET function and evaluated the consequential impact on myeloma pathogenesis. These studies identified HDAC6 upregulation as an efficacy reducing mechanism for BET inhibitors because antagonizing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of multiple myeloma (MM) cell lines and primary CD138 cells obtained from patients with MM. The synergy of this therapeutic combination was linked to significant reductions in c-MYC expression and increases in apoptosis induction. Administration of the clinical HDAC6 inhibitor ricolinostat was very well tolerated and significantly augmented the in vivo antimyeloma activity of JQ1. Ex vivo pharmacodynamic analyses demonstrated that the combination of JQ1 and ricolinostat led to significantly lower MM cell proliferation and increased apoptosis and diminished expression of c-MYC and BCL-2. These data demonstrate that cotargeting of HDAC6 and BET family members is a novel and clinically actionable approach to augment the efficacy of both classes of agents that warrants further investigation.
• Cimino, P. J., Huang, L., Du, L., Wu, Y., Bishop, J., Dalsing-Hernandez, J., Kotlarczyk, K., Gonzales, P., Carew, J., Nawrocki, S., Jordan, M. A., Wilson, L., Lloyd, G. K., & Wirsching, H. G. (2019). Plinabulin, an inhibitor of tubulin polymerization, targets KRAS signaling through disruption of endosomal recycling. Biomedical reports, 10(4), 218-224.
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  Constitutive activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogenic event in certain types of human cancer and is associated with poor patient survival. Small molecule signaling inhibitors have improved the clinical outcomes of patients with various cancer types but attempts to target KRAS have been unsuccessful. Plinabulin represents a novel class of agents that inhibit tubulin polymerization with a favorable safety profile in clinical trials. In the present study, the potency of plinabulin to inhibit tubulin polymerization and growth of KRAS-driven cancer cells was characterized. efficacy of plinabulin was tested in two different mouse models; one being the RCAS/t-va gene transfer system and the other being a xenograft model. cell culture tubulin polymerization assays were used to complement the mouse models. There was improved survival in a KRAS-driven mouse gene transfer glioma model, but lack of benefit in a similar model, without constitutively active KRAS, which supports the notion of a KRAS-specific effect. This survival benefit was mediated, at least in part, by the ability of plinabulin to inhibit tubulin polymerization and disrupt endosomal recycling. It was proposed a mechanism of compromised endosomal recycling of displaced KRAS through targeting microtubules that yields inhibition of protein kinase B, but not extracellular signal regulated kinase (ERK) signaling, therefore lending rationale to combination treatments of tubulin- and ERK-targeting agents in KRAS-driven cancer.
• Jones, T. M., Espitia, C., Wang, W., Nawrocki, S. T., & Carew, J. S. (2019). Moving beyond hydroxychloroquine: the novel lysosomal autophagy inhibitor ROC-325 shows significant potential in preclinical studies. Cancer communications (London, England), 39(1), 72.
• Mahalingam, D., Carew, J. S., Espitia, C. M., Cool, R. H., Giles, F. J., de Jong, S., & Nawrocki, S. T. (2019). Heightened JNK Activation and Reduced XIAP Levels Promote TRAIL and Sunitinib-Mediated Apoptosis in Colon Cancer Models. Cancers, 11(7).
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  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib and would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.
• Nawrocki, S. T., Han, Y., Visconte, V., Przychodzen, B., Espitia, C. M., Phillips, J., Anwer, F., Advani, A., Carraway, H. E., Kelly, K. R., Sekeres, M. A., Maciejewski, J. P., & Carew, J. S. (2019). The novel autophagy inhibitor ROC-325 augments the antileukemic activity of azacitidine. Leukemia, 33(12), 2971-2974.
• Calton, C. M., Kelly, K. R., Anwer, F., Carew, J. S., & Nawrocki, S. T. (2018). Oncolytic Viruses for Multiple Myeloma Therapy. Cancers, 10(6).
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  Although recent treatment advances have improved outcomes for patients with multiple myeloma (MM), the disease frequently becomes refractory to current therapies. MM thus remains incurable for most patients and new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics that provide tumor-targeted therapy by specifically infecting and replicating within cancerous cells. Oncolytic therapy yields results from both direct killing of malignant cells and induction of an anti-tumor immune response. In this review, we will describe oncolytic viruses that are being tested for MM therapy with a focus on those agents that have advanced into clinical trials.
• Russ, A., Hua, A. B., Montfort, W. R., Rahman, B., Riaz, I. B., Khalid, M. U., Carew, J. S., Nawrocki, S. T., Persky, D., & Anwer, F. (2018). Blocking "don't eat me" signal of CD47-SIRPα in hematological malignancies, an in-depth review. Blood reviews, 32(6), 480-489.
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  Hematological malignancies express high levels of CD47 as a mechanism of immune evasion. CD47-SIRPα triggers a cascade of events that inhibit phagocytosis. Preclinical research supports several models of antibody-mediated blockade of CD47-SIRPα resulting in cell death signaling, phagocytosis of cells bearing stress signals, and priming of tumor-specific T cell responses. Four different antibody molecules designed to target the CD47-SIRPα interaction in malignancy are currently being studied in clinical trials: Hu5F9-G4, CC-90002, TTI-621, and ALX-148. Hu5F9-G4, a humanized anti-CD47 blocking antibody is currently being studied in four different Phase I trials. These studies may lay the groundwork for therapeutic bispecific antibodies. Bispecific antibody (CD20-CD47SL) fusion of anti-CD20 (Rituximab) and anti-CD47 also demonstrated a synergistic effect against lymphoma in preclinical models. This review summarizes the large body of preclinical evidence and emerging clinical data supporting the use of antibodies designed to target the CD47-SIRPα interaction in leukemia, lymphoma and multiple myeloma.
• Carew, J. S., & Nawrocki, S. T. (2017). Drain the lysosome: development of the novel orally available autophagy inhibitor ROC-325. Autophagy, 0.
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  Although macroautophagy/autophagy is a key contributor to malignant pathogenesis and therapeutic resistance, there are few FDA-approved agents that significantly affect this pathway. We used medicinal chemistry strategies to develop ROC-325, an orally available novel inhibitor of lysosomal-mediated autophagy. Detailed in vitro and in vivo studies in preclinical models of renal cell carcinoma demonstrated that ROC-325 triggered the hallmark features of lysosomal autophagy inhibition, was very well tolerated, and exhibited significant superiority with respect to autophagy inhibition and anticancer activity over hydroxychloroquine. Our findings support the clinical investigation of the safety and preliminary efficacy of ROC-325 in patients with autophagy-dependent malignancies and other disorders where aberrant autophagy contributes to disease pathogenesis.
• Carew, J. S., Espitia, C. M., Zhao, W., Mita, M. M., Mita, A. C., & Nawrocki, S. T. (2017). Oncolytic reovirus inhibits angiogenesis through induction of CXCL10/IP-10 and abrogation of HIF activity in soft tissue sarcomas. Oncotarget, 8(49), 86769-86783.
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  The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-γ-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1α, HIF-2α and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.
• Carew, J. S., Espitia, C. M., Zhao, W., Han, Y., Visconte, V., Phillips, J. G., & Nawrocki, S. T. (2016). Disruption of autophagic degradation with ROC-325 antagonizes renal cell carcinoma pathogenesis. Clinical cancer research : an official journal of the American Association for Cancer Research.
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  Although autophagy plays important roles in malignant pathogenesis and drug resistance, there are few clinical agents that disrupt this pathway and the potential therapeutic benefit of autophagy inhibition remains undetermined. We used medicinal chemistry approaches to generate a series of novel agents that inhibit autophagic degradation.
• Carew, J. S., Espitia, C. M., Zhao, W., Han, Y., Visconte, V., Phillips, J., & Nawrocki, S. (2016). Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms.. Leukemia.
• Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., Adachi, H., Adams, C. M., Adams, P. D., Adeli, K., Adhihetty, P. J., Adler, S. G., Agam, G., Agarwal, R., Aghi, M. K., Agnello, M., Agostinis, P., Aguilar, P. V., Aguirre-Ghiso, J., , Airoldi, E. M., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy, 12(1), 1-222.
• Liu, P. P., Liu, J., Jiang, W. Q., Carew, J. S., Ogasawara, M. A., Pelicano, H., Croce, C. M., Estrov, Z., Xu, R. H., Keating, M. J., & Huang, P. (2016). Elimination of chronic lymphocytic leukemia cells in stromal microenvironment by targeting CPT with an antiangina drug perhexiline. Oncogene, 35(43), 5663-5673.
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  Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due, in part, to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) have a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that perhexiline, an antiangina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity, leading to rapid depolarization and massive CLL cell death. The therapeutic activity of perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only four drug injections. Our study suggests that targeting CPT using an antiangina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.
• Liu, P. P., Liu, J., Jiang, W. Q., Carew, J. S., Ogasawara, M. A., Pelicano, H., Croce, C. M., Estrov, Z., Xu, R. H., Keating, M. J., & Huang, P. (2016). Elimination of chronic lymphocytic leukemia cells in stromal microenvironment by targeting CPT with an antiangina drug perhexiline. Oncogene, 35:5663-5673.
• Visconte, V., Nawrocki, S. T., Espitia, C. M., Kelly, K. R., Possemato, A., Beausoleil, S. A., Han, Y., Carraway, H. E., Nazha, A., Advani, A. S., Maciejewski, J. P., Sekeres, M. A., & Carew, J. S. (2016). Comprehensive quantitative proteomic profiling of the pharmacodynamic changes induced by MLN4924 in acute myeloid leukemia cells establishes rationale for its combination with azacitidine. Leukemia, 30(5), 1190-4.
• Visconte, V., Przychodzen, B., Han, Y., Nawrocki, S. T., Thota, S., Kelly, K. R., Patel, B. J., Hirsch, C., Advani, A. S., Carraway, H. E., Sekeres, M. A., Maciejewski, J. P., & Carew, J. S. (2016). Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms. Leukemia.
• Carew, J. S., Espitia, C. M., Zhao, W., Mita, M. M., Mita, A. C., & Nawrocki, S. T. (2015). Targeting Survivin Inhibits Renal Cell Carcinoma Progression and Enhances the Activity of Temsirolimus. Molecular cancer therapeutics, 14(6), 1404-13.
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  Elevated expression of the antiapoptotic factor survivin has been implicated in cancer cell survival and disease progression. However, its specific contribution to renal cell carcinoma (RCC) pathogenesis is not well defined. We investigated the roles of survivin in RCC tumor progression, resistance to mTOR inhibitors, and evaluated the therapeutic activity of the survivin suppressant YM155 in RCC models. Here, we report that survivin expression levels were significantly higher in RCC cell lines compared with normal renal cells. Stable targeted knockdown of survivin completely abrogated the ability of 786-O RCC tumors to grow in mice, thus demonstrating its importance as a regulator of RCC tumorigenesis. We next explored multiple strategies to therapeutically inhibit survivin function in RCC. Treatment with the mTOR inhibitor temsirolimus partially diminished survivin levels and this effect was augmented by the addition of YM155. Further analyses revealed that, in accordance with their combined anti-survivin effects, YM155 significantly improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in xenograft models in vivo. Similar to pharmacologic inhibition of survivin, shRNA-mediated silencing of survivin expression not only inhibited RCC tumor growth, but also significantly sensitized RCC cells to temsirolimus therapy. Subsequent experiments demonstrated that the effectiveness of this dual survivin/mTOR inhibition strategy was mediated by a potent decrease in survivin levels and corresponding induction of apoptosis. Our findings establish survivin inhibition as a novel approach to improve RCC therapy that warrants further investigation.
• Carew, J. S., Nawrocki, S. T., & Cleveland, J. L. (2015). Modulating autophagy for therapeutic benefit. Autophagy, 3(5), 464-7.
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  Autophagy is an ancient cell survival pathway that allows cells to recoup ATP and essential building blocks for biosynthesis when they are starved of nutrients or when they are exposed to hypoxia, which are hallmarks of the tumor microenvironment. This pathway involves the formation of double-membraned vesicles, coined autophagosomes, which envelop bulk cellular material and/or organelles and that subsequently fuse with lysosomes that degrade their cargo. Autophagy has been suggested to play important roles in chemoresistance of cancer to some therapeutic agents, which typically induce an apoptotic response. For example, the histone deacetylase inhibitor SAHA induces both apoptosis and autophagy, suggesting that agents that disrupt the autophagy pathway might augment its efficacy as a therapeutic agent. We tested this notion in a model of Imatinib-refractory chronic myelogenous leukemia (CML) and in imatinib-resistant primary CML cells from patients bearing mutations in Bcr-Abl, including the T315I mutation that causes resistance to currently utilized tyrosine kinase inhibitors and translates into a very poor clinical prognosis. Agents that disrupt autophagy were shown to synergize with SAHA in provoking apoptotic death of these refractory tumors. These findings support the use of agents that disrupt the autophagy pathway in settings of chemorefractory malignancies.
• Kelly, K. R., Espitia, C. M., Zhao, W., Wendlandt, E., Tricot, G., Zhan, F., Carew, J. S., & Nawrocki, S. T. (2015). Junctional adhesion molecule-A is overexpressed in advanced multiple myeloma and determines response to oncolytic reovirus. Oncotarget, 6(38), 41275-89.
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  Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.
• Kelly, K. R., Espitia, C. M., Zhao, W., Wu, K., Visconte, V., Anwer, F., Calton, C. M., Carew, J. S., & Nawrocki, S. T. (2018). Oncolytic reovirus sensitizes multiple myeloma cells to anti-PD-L1 therapy. Leukemia, 32(1), 230-233.
• Nawrocki, S. T., Kelly, K. R., Smith, P. G., Keaton, M., Carraway, H., Sekeres, M. A., Maciejewski, J. P., & Carew, J. S. (2015). The NEDD8-activating enzyme inhibitor MLN4924 disrupts nucleotide metabolism and augments the efficacy of cytarabine. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(2), 439-47.
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  New therapies are urgently needed for patients with acute myelogenous leukemia (AML). The novel NEDDylation inhibitor MLN4924 (pevonedistat) has demonstrated significant preclinical antileukemic activity and preliminary efficacy in patients with AML in a phase I trial. On the basis of its antimyeloid and DNA-damaging properties, we investigated the ability of MLN4924 to augment conventional cytarabine (ara-C) therapy.
• Nazha, A., Seastone, D., Radivoyevitch, T., Przychodzen, B., Carraway, H. E., Patel, B. J., Carew, J., Makishima, H., Sekeres, M. A., & Maciejewski, J. P. (2015). Genomic patterns associated with hypoplastic compared to hyperplastic myelodysplastic syndromes. Haematologica, 100(11), e434-7.
• Bansal, H., Yihua, Q., Iyer, S. P., Ganapathy, S., Proia, D. A., Proia, D., Penalva, L. O., Uren, P. J., Suresh, U., Carew, J. S., Karnad, A. B., Weitman, S., Tomlinson, G. E., Rao, M. K., Kornblau, S. M., & Bansal, S. (2014). WTAP is a novel oncogenic protein in acute myeloid leukemia. Leukemia, 28(5), 1171-4.
• Mahalingam, D., Mita, M., Sarantopoulos, J., Wood, L., Amaravadi, R. K., Davis, L. E., Mita, A. C., Curiel, T. J., Espitia, C. M., Nawrocki, S. T., Giles, F. J., & Carew, J. S. (2014). Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors. Autophagy, 10(8), 1403-14.
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  We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.
• Carew, J. S., Espitia, C. M., Zhao, W., Kelly, K. R., Coffey, M., Freeman, J. W., & Nawrocki, S. T. (2013). Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer. Cell death & disease, 4, e728.
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  Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.
• Nawrocki, S. T., Kelly, K. R., Smith, P. G., Espitia, C. M., Possemato, A., Beausoleil, S. A., Milhollen, M., Blakemore, S., Thomas, M., Berger, A., & Carew, J. S. (2013). Disrupting protein NEDDylation with MLN4924 is a novel strategy to target cisplatin resistance in ovarian cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 19(13), 3577-90.
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  Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure and novel therapeutic strategies are urgently needed. MLN4924 is a NEDDylation inhibitor currently under investigation in multiple phase I studies. We investigated its anticancer activity in cisplatin-sensitive and -resistant ovarian cancer models.
• Abraham, J., Chua, Y. X., Glover, J. M., Tyner, J. W., Loriaux, M. M., Kilcoyne, A., Giles, F. J., Nelon, L. D., Carew, J. S., Ouyang, Y., Michalek, J. E., Pal, R., Druker, B. J., Rubin, B. P., & Keller, C. (2012). An adaptive Src-PDGFRA-Raf axis in rhabdomyosarcoma. Biochemical and biophysical research communications, 426(3), 363-8.
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  Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.
• Carew, J. S., Esquivel, J. A., Espitia, C. M., Schultes, C. M., Mülbaier, M., Lewis, J. D., Janssen, B., Giles, F. J., & Nawrocki, S. T. (2012). ELR510444 inhibits tumor growth and angiogenesis by abrogating HIF activity and disrupting microtubules in renal cell carcinoma. PloS one, 7(1), e31120.
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  Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC.
• Carew, J. S., Kelly, K. R., & Nawrocki, S. T. (2012). Autophagy as a target for cancer therapy: new developments. Cancer management and research, 4, 357-65.
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  Autophagy is an evolutionarily conserved lysosomal degradation pathway that eliminates cytosolic proteins, macromolecules, organelles, and protein aggregates. Activation of autophagy may function as a tumor suppressor by degrading defective organelles and other cellular components. However, this pathway may also be exploited by cancer cells to generate nutrients and energy during periods of starvation, hypoxia, and stress induced by chemotherapy. Therefore, induction of autophagy has emerged as a drug resistance mechanism that promotes cancer cell survival via self-digestion. Numerous preclinical studies have demonstrated that inhibition of autophagy enhances the activity of a broad array of anticancer agents. Thus, targeting autophagy may be a global anticancer strategy that may improve the efficacy of many standard of care agents. These results have led to multiple clinical trials to evaluate autophagy inhibition in combination with conventional chemotherapy. In this review, we summarize the anticancer agents that have been reported to modulate autophagy and discuss new developments in autophagy inhibition as an anticancer strategy.
• Kelly, K. R., Espitia, C. M., Mahalingam, D., Oyajobi, B. O., Coffey, M., Giles, F. J., Carew, J. S., & Nawrocki, S. T. (2012). Reovirus therapy stimulates endoplasmic reticular stress, NOXA induction, and augments bortezomib-mediated apoptosis in multiple myeloma. Oncogene, 31(25), 3023-38.
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  Oncolytic virotherapy with reovirus has demonstrated anti-cancer activity and minimal toxicity in clinical trials, but the mechanisms underlying these effects have not been fully elucidated. Reolysin, a proprietary formulation of reovirus for cancer therapy, stimulated selective viral replication and apoptosis in multiple myeloma (MM) cells. Reolysin-mediated apoptosis was associated with an induction of endoplasmic reticular (ER) stress-related gene expression, swelling of the endoplasmic reticulum, increases in intracellular calcium levels and a strong induction of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knockdown of NOXA expression by short hairpin RNA significantly reduced the pro-apoptotic effects of Reolysin. We next showed that co-administration of Reolysin and bortezomib resulted in the dual accumulation of viral and ubiquitinated proteins, which led to enhanced ER stress, NOXA induction and apoptosis. Importantly, the combination of reovirus infection and proteasomal inhibition significantly decreased tumor burden in a xenograft and syngeneic bone disease model of MM without exhibiting adverse side effects. Our study establishes ER stress stimulation and NOXA induction as novel mediators of reovirus-induced apoptosis. Furthermore, reovirus infection can be used as a promising approach to augment the anti-myeloma activity of bortezomib by promoting additional stress to the endoplasmic reticulum of MM cells.
• Kelly, K. R., Espitia, C. M., Taverna, P., Choy, G., Padmanabhan, S., Nawrocki, S. T., Giles, F. J., & Carew, J. S. (2012). Targeting PIM kinase activity significantly augments the efficacy of cytarabine. British journal of haematology, 156(1), 129-32.
• Kelly, K. R., Nawrocki, S. T., Espitia, C. M., Zhang, M., Yang, J. J., Padmanabhan, S., Ecsedy, J., Giles, F. J., & Carew, J. S. (2012). Targeting Aurora A kinase activity with the investigational agent alisertib increases the efficacy of cytarabine through a FOXO-dependent mechanism. International journal of cancer, 131(11), 2693-703.
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  Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara-C. Accordingly, alisertib significantly potentiated the antileukemic activity of ara-C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro-apoptotic effects of the alisertib/ara-C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara-C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara-C that warrants further investigation.
• Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., Ahn, H. J., Ait-Mohamed, O., Ait-Si-Ali, S., Akematsu, T., Akira, S., Al-Younes, H. M., Al-Zeer, M. A., Albert, M. L., Albin, R. L., , Alegre-Abarrategui, J., et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8(4), 445-544.
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  In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
• Nawrocki, S. T., Griffin, P., Kelly, K. R., & Carew, J. S. (2012). MLN4924: a novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy. Expert opinion on investigational drugs, 21(10), 1563-73.
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  The small ubiquitin-like molecule NEDD8 has been identified as an essential regulator of the activity of the cullin-RING E3 ubiquitin ligases (CRLs), which control the turnover of multiple proteins with fundamental roles in cancer biology. The aberrant function of the NEDD8 cascade within the context of malignancy makes it an attractive target for the development of novel anticancer agents. MLN4924 is a first-in-class inhibitor of the proximal regulator of the NEDD8 system (NEDD8-activating enzyme, NAE) that has entered Phase-I trials for cancer therapy and has established that significant therapeutic benefit can be achieved by antagonizing NEDD8-mediated protein degradation.
• Rickles, R. J., Tam, W. F., Giordano, T. P., Pierce, L. T., Farwell, M., McMillin, D. W., Necheva, A., Crowe, D., Chen, M., Avery, W., Kansra, V., Nawrocki, S. T., Carew, J. S., Giles, F. J., Mitsiades, C. S., Borisy, A. A., Anderson, K. C., & Lee, M. S. (2012). Adenosine A2A and beta-2 adrenergic receptor agonists: novel selective and synergistic multiple myeloma targets discovered through systematic combination screening. Molecular cancer therapeutics, 11(7), 1432-42.
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  The use of combination drug regimens has dramatically improved the clinical outcome for patients with multiple myeloma. However, to date, combination treatments have been limited to approved drugs and a small number of emerging agents. Using a systematic approach to identify synergistic drug combinations, combination high-throughput screening (cHTS) technology, adenosine A2A and β-2 adrenergic receptor (β2AR) agonists were shown to be highly synergistic, selective, and novel agents that enhance glucocorticoid activity in B-cell malignancies. Unexpectedly, A2A and β2AR agonists also synergize with melphalan, lenalidomide, bortezomib, and doxorubicin. An analysis of agonists, in combination with dexamethasone or melphalan in 83 cell lines, reveals substantial activity in multiple myeloma and diffuse large B-cell lymphoma cell lines. Combination effects are also observed with dexamethasone as well as bortezomib, using multiple myeloma patient samples and mouse multiple myeloma xenograft assays. Our results provide compelling evidence in support of development of A2A and β2AR agonists for use in multi-drug combination therapy for multiple myeloma. Furthermore, use of cHTS for the discovery and evaluation of new targets and combination therapies has the potential to improve cancer treatment paradigms and patient outcomes.
• Carew, J. S., Espitia, C. M., Esquivel, J. A., Mahalingam, D., Kelly, K. R., Reddy, G., Giles, F. J., & Nawrocki, S. T. (2011). Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. The Journal of biological chemistry, 286(8), 6602-13.
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  Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.
• Carew, J. S., Kelly, K. R., & Nawrocki, S. T. (2011). Mechanisms of mTOR inhibitor resistance in cancer therapy. Targeted oncology, 6(1), 17-27.
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  Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that regulates cell cycle progression, protein translation, metabolism, and cellular proliferation. The mTOR pathway promotes cell proliferation under energy or nutrient-rich conditions by increasing ribosomal biogenesis and protein synthesis. Since enhanced activity of the mTOR pathway is frequently observed in malignant cells, inhibition of this kinase has become an attractive strategy to treat cancer. Rapamycin and its analogs temsirolimus, everolimus, and ridaforolimus referred to as "rapalogs" have demonstrated promising efficacy against renal cell carcinoma and are under investigation for the treatment of other malignancies. However, the emergence of drug resistance may ultimately limit the utility of rapalog therapy. Here we summarize the known mechanisms of resistance to mTOR-inhibitor therapy and describe potential strategies to overcome these for the current agents that target this pathway.
• Kelly, K. R., Ecsedy, J., Mahalingam, D., Nawrocki, S. T., Padmanabhan, S., Giles, F. J., & Carew, J. S. (2011). Targeting aurora kinases in cancer treatment. Current drug targets, 12(14), 2067-78.
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  The Aurora family of serine/threonine kinases is essential for chromosome alignment, segregation, centrosomal maturation, mitotic spindle formation, and cytokinesis during mitosis. Their fundamental role in cell cycle regulation and aberrant expression in a broad range of malignancies prompted the development of small molecules that selectively inhibit their activity. Recent studies have revealed new insights into the cellular effects of Aurora kinase inhibition. Moreover, early phase clinical studies have shown that these agents have therapeutic efficacy. In this review, we will outline the functions of Aurora kinases in normal cell division and in malignancy. We will focus on recent preclinical and clinical studies that have explored the mechanism of action and clinical effect of Aurora inhibitors in cancer treatment.
• Kelly, K. R., Ecsedy, J., Medina, E., Mahalingam, D., Padmanabhan, S., Nawrocki, S. T., Giles, F. J., & Carew, J. S. (2011). The novel Aurora A kinase inhibitor MLN8237 is active in resistant chronic myeloid leukaemia and significantly increases the efficacy of nilotinib. Journal of cellular and molecular medicine, 15(10), 2057-70.
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  Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region-Abelson kinase (BCR-ABL). Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose-dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR-ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib-induced apoptosis, indicating that this is an important factor underlying MLN8237's ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.
• Kelly, K. R., Rowe, J. H., Padmanabhan, S., Nawrocki, S. T., & Carew, J. S. (2011). Mammalian target of rapamycin as a target in hematological malignancies. Targeted oncology, 6(1), 53-61.
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  The mammalian target of rapamycin (mTOR) regulates protein synthesis in addition to cell growth and cell proliferation. Elucidation of the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway in the regulation of the pathogenesis of hematological neoplasms has led to the development and clinical evaluation of agents targeting this pathway for the treatment of leukemia and lymphomas. Clinical trials conducted to date have shown modest responses to mTOR inhibition in patients with various hematological malignancies. Novel agents that simultaneously target mTOR complex 2 (mTORC2) or AKT in addition to mTOR complex 1 (mTORC1) may offer an opportunity to improve therapeutic efficacy.
• Mahalingam, D., Espitia, C. M., Medina, E. C., Esquivel, J. A., Kelly, K. R., Bearss, D., Choy, G., Taverna, P., Carew, J. S., Giles, F. J., & Nawrocki, S. T. (2011). Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma. British journal of cancer, 105(10), 1563-73.
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  Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib.
• Swords, R., Kelly, K., Carew, J., Nawrocki, S., Mahalingam, D., Sarantopoulos, J., Bearss, D., & Giles, F. (2011). The Pim kinases: new targets for drug development. Current drug targets, 12(14), 2059-66.
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  The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.
• Carew, J. S., Medina, E. C., Esquivel, J. A., Mahalingam, D., Swords, R., Kelly, K., Zhang, H., Huang, P., Mita, A. C., Mita, M. M., Giles, F. J., & Nawrocki, S. T. (2010). Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. Journal of cellular and molecular medicine, 14(10), 2448-59.
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  Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin-conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ-induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ-induced aggresome formation, but promoted CQ-mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR-mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR-induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ-induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ-mediated aggregate formation, superoxide generation and apoptosis.
• Mahalingam, D., Medina, E. C., Esquivel, J. A., Espitia, C. M., Smith, S., Oberheu, K., Swords, R., Kelly, K. R., Mita, M. M., Mita, A. C., Carew, J. S., Giles, F. J., & Nawrocki, S. T. (2010). Vorinostat enhances the activity of temsirolimus in renal cell carcinoma through suppression of survivin levels. Clinical cancer research : an official journal of the American Association for Cancer Research, 16(1), 141-53.
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  The mammalian target of rapamycin (mTOR) inhibitor temsirolimus has exhibited promising anticancer activity for the treatment of renal cell cancers (RCC). Survivin expression has been implicated in drug resistance and reducing its levels with the histone deacetylase (HDAC) inhibitor vorinostat may enhance the anticancer activity of temsirolimus.
• Swords, R. T., Kelly, K. R., Smith, P. G., Garnsey, J. J., Mahalingam, D., Medina, E., Oberheu, K., Padmanabhan, S., O'Dwyer, M., Nawrocki, S. T., Giles, F. J., & Carew, J. S. (2010). Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia. Blood, 115(18), 3796-800.
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  NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.
• Swords, R., Mahalingam, D., O'Dwyer, M., Santocanale, C., Kelly, K., Carew, J., & Giles, F. (2010). Cdc7 kinase - a new target for drug development. European journal of cancer (Oxford, England : 1990), 46(1), 33-40.
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  The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.
• Mahalingam, D., Kelly, K. R., Swords, R. T., Carew, J., Nawrocki, S. T., & Giles, F. J. (2009). Emerging drugs in the treatment of pancreatic cancer. Expert opinion on emerging drugs, 14(2), 311-28.
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  Pancreatic cancer is the fourth leading cause of cancer-related death in the US. However, there is a growing belief that novel biological agents could improve survival of patients with this cancer. Gemcitabine-based chemotherapy remains the cornerstone treatment for advanced pancreatic cancers. So far, the current targeted agents that have been used in combination with gemcitabine have failed to improve clinical outcomes. This failure may stem from the heterogeneous molecular pathogenesis of pancreatic cancers, which involves several oncogenic pathways and defined genetic mutations.
• Mahalingam, D., Swords, R., Carew, J. S., Nawrocki, S. T., Bhalla, K., & Giles, F. J. (2009). Targeting HSP90 for cancer therapy. British journal of cancer, 100(10), 1523-9.
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  Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.
• Swords, R., Mahalingam, D., Padmanabhan, S., Carew, J., & Giles, F. (2009). Nilotinib: optimal therapy for patients with chronic myeloid leukemia and resistance or intolerance to imatinib. Drug design, development and therapy, 3, 89-101.
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  Chronic myeloid leukemia (CML) is the consequence of a single balanced translocation that produces the BCR-ABL fusion oncogene which is detectable in over 90% of patients at presentation. The BCR-ABL inhibitor imatinib mesylate (IM) has improved survival in all phases of CML and is the standard of care for newly diagnosed patients in chronic phase. Despite the very significant therapeutic benefits of IM, a small minority of patients with early stage disease do not benefit optimally while IM therapy in patients with advanced disease is of modest benefit in many. Diverse mechanisms may be responsible for IM failures, with point mutations within the Bcr-Abl kinase domain being amongst the most common resistance mechanisms described in patients with advanced CML. The development of novel agents designed to overcome IM resistance, while still primarily targeted on BCR-ABL, led to the creation of the high affinity aminopyrimidine inhibitor, nilotinib. Nilotinib is much more potent as a BCR-ABL inhibitor than IM and inhibits both wild type and IM-resistant BCR-ABL with significant clinical activity across the entire spectrum of BCR-ABL mutants with the exception of T315I. The selection of a second generation tyrosine kinase inhibitor to rescue patients with imatinib failure will be based on several factors including age, co-morbid medical problems and ABL kinase mutational profile. It should be noted that while the use of targeted BCR-ABL kinase inhibitors in CML represents a paradigm shift in CML management these agents are not likely to have activity against the quiescent CML stem cell pool. The purpose of this review is to summarize the pre-clinical and clinical data on nilotinib in patients with CML who have failed prior therapy with IM or dasatinib.
• Carew, J. S., Giles, F. J., & Nawrocki, S. T. (2008). Histone deacetylase inhibitors: mechanisms of cell death and promise in combination cancer therapy. Cancer letters, 269(1), 7-17.
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  Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression by catalyzing the removal of acetyl groups, stimulating chromatin condensation and promoting transcriptional repression. Since aberrant epigenetic changes are a hallmark of cancer, HDACs are a promising target for pharmacological inhibition. HDAC inhibitors can induce cell-cycle arrest, promote differentiation, and stimulate tumor cell death. These properties have prompted numerous preclinical and clinical investigations evaluating the potential efficacy of HDAC inhibitors for a variety of malignancies. The preferential toxicity of HDAC inhibitors in transformed cells and their ability to synergistically enhance the anticancer activity of many chemotherapeutic agents has further generated interest in this novel class of drugs. Here we summarize the different mechanisms of HDAC inhibitor-induced apoptosis and discuss their use in combination with other anticancer agents.
• Carew, J. S., Nawrocki, S. T., Giles, F. J., & Cleveland, J. L. (2008). Targeting autophagy: a novel anticancer strategy with therapeutic implications for imatinib resistance. Biologics : targets & therapy, 2(2), 201-4.
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  Autophagy is an ancient, intracellular degradative system which plays important roles in regulating protein homeostasis and which is essential for survival when cells are faced with metabolic stress. Increasing evidence suggests that autophagy also functions as a tumor suppressor mechanism that harnesses the growth and/or survival of cells as they transition towards a rapidly dividing malignant state. However, the impact of autophagy on cancer progression and on the efficacy of cancer therapeutics is controversial. In particular, although the induction of autophagy has been reported after treatment with a number of therapeutic agents, including imatinib, this response has variously been suggested to either impair or contribute to the effects of anticancer agents. More recent studies support the notion that autophagy compromises the efficacy of anticancer agents, where agents such as chloroquine (CQ) that impair autophagy augment the anticancer activity of histone deacetylase (HDAC) inhibitors and alkylating agents. Inhibition of autophagy is a particularly attractive strategy for the treatment of imatinib-refractory chronic myelogenous leukemia (CML) since a combination of CQ with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) compromises the survival of even BCR-ABL-T315I+ imatinib-resistant CML. Additional studies are clearly needed to establish the clinical utility of autophagy inhibitors and to identify patients most likely to benefit from this novel therapeutic approach.
• Carew, J. S., Nawrocki, S. T., Reddy, V. K., Bush, D., Rehg, J. E., Goodwin, A., Houghton, J. A., Casero, R. A., Marton, L. J., & Cleveland, J. L. (2008). The novel polyamine analogue CGC-11093 enhances the antimyeloma activity of bortezomib. Cancer research, 68(12), 4783-90.
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  Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the in vitro and in vivo antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH(2)-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy.
• Nawrocki, S. T., Carew, J. S., Maclean, K. H., Courage, J. F., Huang, P., Houghton, J. A., Cleveland, J. L., Giles, F. J., & McConkey, D. J. (2008). Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA. Blood, 112(7), 2917-26.
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  The histone deacetylase inhibitor SAHA enhances cell death stimulated by the proteasome inhibitor bortezomib (BZ) by disrupting BZ-induced aggresome formation. Here we report that Myc regulates the sensitivity of multiple myeloma (MM) cells to BZ + SAHA-induced cell death. In MM cells, Myc expression directly correlated with intracellular ER content, protein synthesis rates, the percentage of aggresome-positive cells, and the sensitivity to BZ + SAHA-induced cell death. Accordingly, Myc knockdown markedly reduced the sensitivity of MM cells to BZ + SAHA-mediated apoptosis. Furthermore, activation of Myc was sufficient to provoke aggresome formation and thus sensitivity to BZ + SAHA, and these responses required de novo protein synthesis. BZ + SAHA-mediated stimulation of apoptosis includes the induction of the proapoptotic BH3-only protein Noxa as well as endoplasmic reticular stress, a disruption of calcium homeostasis, and activation of capase-4. Finally, knockdown studies demonstrated that both caspase-4 and Noxa play significant roles in Myc-driven sensitivity to BZ + SAHA-induced apoptosis. Collectively, our results establish a mechanistic link between Myc activity, regulation of protein synthesis, increases in HDAC6 expression and aggresome formation, induction of Noxa, and sensitivity to BZ + SAHA-induced apoptosis. These data suggest that MM patients with elevated Myc activity may be particularly sensitive to the BZ + SAHA combination.
• Zhang, H., Trachootham, D., Lu, W., Carew, J., Giles, F. J., Keating, M. J., Arlinghaus, R. B., & Huang, P. (2008). Effective killing of Gleevec-resistant CML cells with T315I mutation by a natural compound PEITC through redox-mediated mechanism. Leukemia, 22(6), 1191-9.
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  Mutation of Bcr-Abl is an important mechanism by which chronic myelogenous leukemia (CML) cells become resistant to Gleevec. The T315I mutation is clinically significant since CML cells harboring this mutation are insensitive to Gleevec and other Bcr-Abl-targeted drugs. Identification of new agents capable of effectively killing CML cells with T315I mutation would have important therapeutic implications in Gleevec-resistant CML. Here, we showed that beta-phenylethyl isothiocyanate (PEITC), a natural compound found in vegetables, is effective in killing CML cells expressing T315I BCR-ABL. Treatment of leukemia cell lines harboring wild-type or mutant Bcr-Abl with 10 microM PEITC resulted in an elevated ROS stress and a redox-mediated degradation of the BCR-ABL protein, leading to massive death of the leukemia cells. Antioxidant NAC attenuated the PEITC-induced oxidative stress in CML cells and prevented the degradation of BCR-ABL, caspase-3 activation and cell death. We further showed that the ROS-induced degradation of BCR-ABL was mediated partially by caspase-3 and the proteasome pathway. The ability of PEITC to effectively kill T315I-positive CML cells was further confirmed using primary leukemia cells isolated from CML patients. Our results suggest that PEITC is a promising compound capable of killing Gleevec-resistant CML cells through a ROS-mediated mechanism and warrants further investigations.
• Carew, J. S., Nawrocki, S. T., Kahue, C. N., Zhang, H., Yang, C., Chung, L., Houghton, J. A., Huang, P., Giles, F. J., & Cleveland, J. L. (2007). Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance. Blood, 110(1), 313-22.
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  Novel therapeutic strategies are needed to address the emerging problem of imatinib resistance. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for imatinib-resistant chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of autophagy and apoptosis. Considering that autophagy may promote cancer cell survival, we hypothesized that disrupting autophagy would augment the anticancer activity of SAHA. Here we report that drugs that disrupt the autophagy pathway dramatically augment the antineoplastic effects of SAHA in CML cell lines and primary CML cells expressing wild-type and imatinib-resistant mutant forms of Bcr-Abl, including T315I. This regimen has selectivity for malignant cells and its efficacy was not diminished by impairing p53 function, another contributing factor in imatinib resistance. Disrupting autophagy by chloroquine treatment enhances SAHA-induced superoxide generation, triggers relocalization and marked increases in the lysosomal protease cathepsin D, and reduces the expression of the cathepsin-D substrate thioredoxin. Finally, knockdown of cathepsin D diminishes the potency of this combination, demonstrating its role as a mediator of this therapeutic response. Our data suggest that, when combined with HDAC inhibitors, agents that disrupt autophagy are a promising new strategy to treat imatinib-refractory patients who fail conventional therapy.
• Nawrocki, S. T., Carew, J. S., Douglas, L., Cleveland, J. L., Humphreys, R., & Houghton, J. A. (2007). Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels. Cancer research, 67(14), 6987-94.
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  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21(+/+) cells but not in the HCT116 p21(-/-) cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA-mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab.
• Carew, J. S., Nawrocki, S. T., Krupnik, Y. V., Dunner, K., McConkey, D. J., Keating, M. J., & Huang, P. (2006). Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL. Blood, 107(1), 222-31.
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  Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.
• Pelicano, H., Carew, J. S., McQueen, T. J., Andreeff, M., Plunkett, W., Keating, M. J., & Huang, P. (2006). Targeting Hsp90 by 17-AAG in leukemia cells: mechanisms for synergistic and antagonistic drug combinations with arsenic trioxide and Ara-C. Leukemia, 20(4), 610-9.
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  17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.
• Pelicano, H., Xu, R. H., Du, M., Feng, L., Sasaki, R., Carew, J. S., Hu, Y., Ramdas, L., Hu, L., Keating, M. J., Zhang, W., Plunkett, W., & Huang, P. (2006). Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. The Journal of cell biology, 175(6), 913-23.
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  Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.
• Achanta, G., Sasaki, R., Feng, L., Carew, J. S., Lu, W., Pelicano, H., Keating, M. J., & Huang, P. (2005). Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma. The EMBO journal, 24(19), 3482-92.
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  Mitochondrial DNA (mtDNA) mutations and deletions are frequently observed in cancer, and contribute to altered energy metabolism, increased reactive oxygen species (ROS), and attenuated apoptotic response to anticancer agents. The mechanisms by which cells maintain mitochondrial genomic integrity and the reason why cancer cells exhibit more frequent mtDNA mutations remain unclear. Here, we report that the tumor suppressor molecule p53 has a novel role in maintaining mitochondrial genetic stability through its ability to translocate to mitochondria and interact with mtDNA polymerase gamma (pol gamma) in response to mtDNA damage induced by exogenous and endogenous insults including ROS. The p53 protein physically interacts with mtDNA and pol gamma, and enhances the DNA replication function of pol gamma. Loss of p53 results in a significant increase in mtDNA vulnerability to damage, leading to increased frequency of in vivo mtDNA mutations, which are reversed by stable transfection of wild-type p53. This study provides a mechanistic explanation for the accelerating genetic instability and increased ROS stress in cancer cells associated with loss of p53.
• Nawrocki, S. T., Carew, J. S., Dunner, K., Boise, L. H., Chiao, P. J., Huang, P., Abbruzzese, J. L., & McConkey, D. J. (2005). Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer research, 65(24), 11510-9.
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  Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2alpha (eif2alpha), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2alpha phosphorylation, ubiquitylated protein accumulation, and proteotoxicity.
• Nawrocki, S. T., Carew, J. S., Pino, M. S., Highshaw, R. A., Dunner, K., Huang, P., Abbruzzese, J. L., & McConkey, D. J. (2005). Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis. Cancer research, 65(24), 11658-66.
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  Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.
• Xu, R. H., Pelicano, H., Zhou, Y., Carew, J. S., Feng, L., Bhalla, K. N., Keating, M. J., & Huang, P. (2005). Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer research, 65(2), 613-21.
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  Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser(112), relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently induced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications.
• Carew, J. S., Nawrocki, S. T., Xu, R. H., Dunner, K., McConkey, D. J., Wierda, W. G., Keating, M. J., & Huang, P. (2004). Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabine. Leukemia, 18(12), 1934-40.
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  B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.
• Carew, J. S., Zhou, Y., Albitar, M., Carew, J. D., Keating, M. J., & Huang, P. (2003). Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and therapeutic implications. Leukemia, 17(8), 1437-47.
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  Mitochondrial DNA (mtDNA) codes for 13 respiratory chain subunits and is more vulnerable to damage than nuclear DNA due, in part, to a lack of histone protection and a weak repair capacity. While mtDNA alterations have been observed in human cancer, their roles in oncogenesis and chemosensitivity remain unclear. We investigated the relationship between mtDNA mutations, reactive oxygen species (ROS) generation, and clinical outcomes in chronic lymphocytic leukemia (CLL) patients. An analysis of mtDNA from 20 CLL patients revealed that primary CLL cells from patients with prior chemotherapy had a significantly higher frequency of heteroplasmic mutations than did those from untreated patients. Overall, mtDNA mutations appeared to be associated with increased ROS generation. Patients refractory to conventional therapeutic agents tended to have higher mutation rates than patients who responded to treatment. Analysis of paired blood samples from the same patient led to the identification of a heteroplasmic mutation in the cytochrome c oxidase II gene several months after chemotherapy. The mutation was associated with increased ROS generation. Our results suggest for the first time that chemotherapy with DNA-damaging agents may cause mtDNA mutations in primary leukemia cells, which often exist in heteroplasmy, and are associated with increased ROS generation.
• MacEwen, E. G., Kutzke, J., Carew, J., Pastor, J., Schmidt, J. A., Tsan, R., Thamm, D. H., & Radinsky, R. (2003). c-Met tyrosine kinase receptor expression and function in human and canine osteosarcoma cells. Clinical & experimental metastasis, 20(5), 421-30.
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  To further characterize the role of hepatocyte growth factor-scatter factor (HGF-SF) and its receptor (c-Met) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential, and cell lines derived from spontaneous canine OS were studied. All cell lines were evaluated for c-Met and HGF-SF expression and receptor activation using Northern, RT-PCR, and Western blot analyses, respectively. Functional activity of receptor-ligand interaction was measured using c-Met phosphorylation status, proliferation assays (anchorage-dependent and -independent), Matrigel invasion, modulation of urokinase plasminogen activator (uPA) expression, and cell dispersion (scattering). All cell lines exhibited steady-state mRNA expression of c-Met. The canine OS cell lines also expressed HGF-SF mRNA as determined by RT-PCR analysis. Western analysis showed c-Met protein expression and HGF-stimulated (human) or constitutive (canine) receptor autophosphorylation. Treatment with recombinant human HGF resulted in enhanced proliferation in 3 of 5 OS cell lines and enhanced colony formation in 2 of 5 OS cell lines. Matrigel invasion was significantly enhanced in 3 of the cell lines and uPA levels were significantly increased in the SAOS-2 cells following HGF treatment. Scattering was enhanced in both the SAOS-2 and SAOS-LM2 cells. These data support the involvement of c-Met and HGF-SF in the growth and progression of human and canine OS, and may offer new targets for the development of therapeutic strategies for OS.
• Pelicano, H., Feng, L., Zhou, Y., Carew, J. S., Hileman, E. O., Plunkett, W., Keating, M. J., & Huang, P. (2003). Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. The Journal of biological chemistry, 278(39), 37832-9.
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  Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide O2. radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I. Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to an increase in O2. generation and sensitization of the leukemia cells to anticancer agents whose action involve free radical generation. Using leukemia cells with genetic alterations in mitochondrial DNA and biochemical approaches, we further demonstrated that As2O3, a clinically active anti-leukemia agent, inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another O2.-generating agent against cultured leukemia cells and primary leukemia cells isolated from patients. Our study shows that interfering mitochondrial respiration is a novel mechanism by which As2O3 increases generation of free radicals. This novel mechanism of action provides a biochemical basis for developing new drug combination strategies using As2O3 to enhance the activity of anticancer agents by promoting generation of free radicals.
• Carew, J. S., & Huang, P. (2002). Mitochondrial defects in cancer. Molecular cancer, 1, 9.
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  Mitochondria play important roles in cellular energy metabolism, free radical generation, and apoptosis. Defects in mitochondrial function have long been suspected to contribute to the development and progression of cancer. In this review article, we aim to provide a brief summary of our current understanding of mitochondrial genetics and biology, review the mtDNA alterations reported in various types of cancer, and offer some perspective as to the emergence of mtDNA mutations, their functional consequences in cancer development, and therapeutic implications.

Presentations

• Carew, J. S. (2018, 12). Oral Session Co-Chair, Molecular Pharmacology and Drug Resistance in Myeloid Diseases. American Society of Hematology.