Andrew Kraft

Interests

Research

I have both clinical, translational, and basic research interests. In the clinical sphere I have been working to develop new approaches to Sarcoma therapy including immunotherapies and DNA damaging agents. I am currently working on a translational effort focused on breast cancer combining an inhibitor of a protein kinase with standard endocrine therapy for hormone positive breast cancer. In the laboratory we have two major projects. One is to understand how the control of RNA degradation regulates tumor growth and invasion, and a second that investigates how adenocarcinoma of the prostate transdifferentiates to neuroendocrine prostate cancer.

Teaching

I have taught fellows both in the classroom and in the clinic. I have interacted with both Radiation Therapy and Medical Oncology fellows. In the classroom I have focused on explaining basic concepts of cancer biology including what causes cancer and how it grows and metastasizes. Additionally, in the classroom I have lectured on basic principles of Sarcoma diagnosis and treatment. In the clinical setting I have focused efforts on teaching approach to patients with cancer and specifically with those who have sarcoma.

Courses

Scholarly Contributions

Journals/Publications

• Lim, J. T., Singh, N., Leuvano, L. A., Calvert, V. S., Petricoin, E. F., Teachey, D. T., Lock, R. B., Padi, M., Kraft, A. S., & Padi, S. K. (2020). PIM Kinase Inhibitors Block the Growth of Primary T-cell Acute Lymphoblastic Leukemia: Resistance Pathways Identified by Network Modeling Analysis. Molecular cancer therapeutics, 19(9), 1809-1821.
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  Despite significant progress in understanding the genetic landscape of T-cell acute lymphoblastic leukemia (T-ALL), the discovery of novel therapeutic targets has been difficult. Our results demonstrate that the levels of PIM1 protein kinase is elevated in early T-cell precursor ALL (ETP-ALL) but not in mature T-ALL primary samples. Small-molecule PIM inhibitor (PIMi) treatment decreases leukemia burden in ETP-ALL. However, treatment of animals carrying ETP-ALL with PIMi was not curative. To model other pathways that could be targeted to complement PIMi activity, HSB-2 cells, previously characterized as a PIMi-sensitive T-ALL cell line, were grown in increasing doses of PIMi. Gene set enrichment analysis of RNA sequencing data and functional enrichment of network modules demonstrated that the HOXA9, mTOR, MYC, NFκB, and PI3K-AKT pathways were activated in HSB-2 cells after long-term PIM inhibition. Reverse phase protein array-based pathway activation mapping demonstrated alterations in the mTOR, PI3K-AKT, and NFκB pathways, as well. PIMi-tolerant HSB-2 cells contained phosphorylated RelA-S536 consistent with activation of the NFκB pathway. The combination of NFκB and PIMis markedly reduced the proliferation in PIMi-resistant leukemic cells showing that this pathway plays an important role in driving the growth of T-ALL. Together these results demonstrate key pathways that are activated when HSB-2 cell line develop resistance to PIMi and suggest pathways that can be rationally targeted in combination with PIM kinases to inhibit T-ALL growth.
• Singh, N., Padi, S. K., Bearss, J. J., Pandey, R., Okumura, K., Beltran, H., Song, J. H., Kraft, A. S., & Olive, V. (2020). PIM protein kinases regulate the level of the long noncoding RNA H19 to control stem cell gene transcription and modulate tumor growth. Molecular oncology, 14(5), 974-990.
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  The proviral integration site for Moloney murine leukemia virus (PIM) serine/threonine kinases have an oncogenic and prosurvival role in hematological and solid cancers. However, the mechanism by which these kinases drive tumor growth has not been completely elucidated. To determine the genes controlled by these protein kinases, we carried out a microarray analysis in T-cell acute lymphoblastic leukemia (T-ALL) comparing early progenitor (ETP-ALL) cell lines whose growth is driven by PIM kinases to more mature T-ALL cells that have low PIM levels. This analysis demonstrated that the long noncoding RNA (lncRNA) H19 was associated with increased PIM levels in ETP-ALL. Overexpression or knockdown of PIM in these T-ALL cell lines controlled the level of H19 and regulated the methylation of the H19 promoter, suggesting a mechanism by which PIM controls H19 transcription. In these T-ALL cells, the expression of PIM1 induced stem cell gene expression (SOX2, OCT-4, and NANOG) through H19. Identical results were found in prostate cancer (PCa) cell lines where PIM kinases drive cancer growth, and both H19 and stem cell gene levels. Small molecule pan-PIM inhibitors (PIM-i) currently in clinical trials reduced H19 expression in both of these tumor types. Importantly, the knockdown of H19 blocked the ability of PIM to induce stem cell genes in T-ALL cells, suggesting a novel signal transduction cascade. In PCa, increases in SOX2 levels have been shown to cause both resistance to the androgen deprivation therapy (ADT) and the induction of neuroendocrine PCa, a highly metastatic form of this disease. Treatment of PCa cells with a small molecule pan-PIM-i reduced stem cell gene transcription and enhanced ADT, while overexpression of H19 suppressed the ability of pan-PIM-i to regulate hormone blockade. Together, these results demonstrate that the PIM kinases control the level of lncRNA H19, which in turn modifies stem cell gene transcription regulating tumor growth.
• Somaiah, N., Van Tine, B. A., Wahlquist, A. E., Milhem, M. M., Hill, E. G., Garrett-Mayer, E., Armeson, K. E., Schuetze, S. M., Meyer, C. F., Reuben, D. Y., Elias, A. D., Read, W. L., Chawla, S. P., & Kraft, A. S. (2020). A randomized, open-label, phase 2, multicenter trial of gemcitabine with pazopanib or gemcitabine with docetaxel in patients with advanced soft-tissue sarcoma. Cancer.
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  Therapeutic options for patients with advanced soft-tissue sarcoma (STS) are limited. The goal of the current phase 2 study was to examine the clinical activity and safety of the combination of gemcitabine plus pazopanib, a multityrosine kinase inhibitor with activity in STS.
• Chatterjee, S., Chakraborty, P., Daenthanasanmak, A., Iamsawat, S., Andrejeva, G., Luevano, L. A., Wolf, M., Baliga, U., Krieg, C., Beeson, C. C., Mehrotra, M., Hill, E. G., Rathmell, J. C., Yu, X. Z., Kraft, A. S., & Mehrotra, S. (2019). Targeting PIM Kinase with PD1 Inhibition Improves Immunotherapeutic Antitumor T-cell Response. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(3), 1036-1049.
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  Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of -engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor.
• Padi, S. K., Singh, N., Bearss, J. J., Olive, V., Song, J. H., Cardó-Vila, M., Kraft, A. S., & Okumura, K. (2019). Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth. Proceedings of the National Academy of Sciences of the United States of America, 116(41), 20505-20510.
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  The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.
• Song, J. H., Kang, H. J., Luevano, L. A., Gokhale, V., Wu, K., Pandey, R., Sherry Chow, H. H., Hurley, L. H., & Kraft, A. S. (2019). Small-Molecule-Targeting Hairpin Loop of hTERT Promoter G-Quadruplex Induces Cancer Cell Death. Cell chemical biology, 26(8), 1110-1121.e4.
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  Increased telomerase activity is associated with malignancy and poor prognosis in human cancer, but the development of targeted agents has not yet provided clinical benefit. Here we report that, instead of targeting the telomerase enzyme directly, small molecules that bind to the G-hairpin of the hTERT G-quadruplex-forming sequence kill selectively malignant cells without altering the function of normal cells. RG260 targets the hTERT G-quadruplex stem-loop folding but not tetrad DNAs, leading to downregulation of hTERT expression. To improve physicochemical and pharmacokinetic properties, we derived a small-molecule analog, RG1603, from the parent compound. RG1603 induces mitochondrial defects including PGC1α and NRF2 inhibition and increases oxidative stress, followed by DNA damage and apoptosis. RG1603 injected as a single agent has tolerable toxicity while achieving strong anticancer efficacy in a tumor xenograft mouse model. These results demonstrate a unique approach to inhibiting the hTERT that functions by impairing mitochondrial activity, inducing cell death.
• Casillas, A. L., Toth, R. K., Sainz, A. G., Singh, N., Desai, A. A., Kraft, A. S., & Warfel, N. A. (2018). Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 169-180.
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  Patients develop resistance to antiangiogenic drugs, secondary to changes in the tumor microenvironment, including hypoxia. PIM kinases are prosurvival kinases and their expression increases in hypoxia. The goal of this study was to determine whether targeting hypoxia-induced PIM kinase expression is effective in combination with VEGF-targeting agents. The rationale for this therapeutic approach is based on the fact that antiangiogenic drugs can make tumors hypoxic, and thus more sensitive to PIM inhibitors. Xenograft and orthotopic models of prostate and colon cancer were used to assess the effect of PIM activation on the efficacy of VEGF-targeting agents. IHC and imaging were used to analyze angiogenesis, apoptosis, proliferation, and metastasis. Biochemical studies were performed to characterize the novel signaling pathway linking PIM and HIF1. PIM was upregulated following treatment with anti-VEGF therapies, and PIM1 overexpression reduced the ability of these drugs to disrupt vasculature and block tumor growth. PIM inhibitors reduced HIF1 activity, opposing the shift to a pro-angiogenic gene signature associated with hypoxia. Combined inhibition of PIM and VEGF produced a synergistic antitumor response characterized by decreased proliferation, reduced tumor vasculature, and decreased metastasis. This study describes PIM kinase expression as a novel mechanism of resistance to antiangiogenic agents. Our data provide justification for combining PIM and VEGF inhibitors to treat solid tumors. The unique ability of PIM inhibitors to concomitantly target HIF1 and selectively kill hypoxic tumor cells addresses two major components of tumor progression and therapeutic resistance. .
• Daenthanasanmak, A., Wu, Y., Iamsawat, S., Nguyen, H. D., Bastian, D., Zhang, M., Sofi, M. H., Chatterjee, S., Hill, E. G., Mehrotra, S., Kraft, A. S., & Yu, X. Z. (2018). PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity. The Journal of clinical investigation, 128(7), 2787-2801.
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  PIM kinase family members play a crucial role in promoting cell survival and proliferation via phosphorylation of their target substrates. In this study, we investigated the role of the PIM kinases with respect to T cell responses in transplantation and tumor immunity. We found that the PIM-2 isoform negatively regulated T cell responses to alloantigen, in contrast to the PIM-1 and PIM-3 isoforms, which acted as positive regulators. T cells deficient in PIM-2 demonstrated increased T cell differentiation toward Th1 subset, proliferation, and migration to target organs after allogeneic bone marrow transplantation, resulting in dramatically accelerated graft-versus-host disease (GVHD) severity. Restoration of PIM-2 expression markedly attenuated the pathogenicity of PIM-2-deficient T cells to induce GVHD. On the other hand, mice deficient in PIM-2 readily rejected syngeneic tumor, which was primarily dependent on CD8+ T cells. Furthermore, silencing PIM-2 in polyclonal or antigen-specific CD8+ T cells substantially enhanced their antitumor response in adoptive T cell immunotherapy. We conclude that PIM-2 kinase plays a prominent role in suppressing T cell responses, and provide a strong rationale to target PIM-2 for cancer immunotherapy.
• Song, J. H., Singh, N., Luevano, L. A., Padi, S. K., Okumura, K., Olive, V., Black, S. M., Warfel, N. A., Goodrich, D. W., & Kraft, A. S. (2018). Mechanisms Behind Resistance to PI3K Inhibitor Treatment Induced by the PIM Kinase. Molecular cancer therapeutics, 17(12), 2710-2721.
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  Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT-inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.
• Padi, S. K., Luevano, L. A., An, N., Pandey, R., Singh, N., Song, J. H., Aster, J. C., Yu, X. Z., Mehrotra, S., & Kraft, A. S. (2017). Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset. Oncotarget, 8(18), 30199-30216.
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  New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.
• Sindhu, S., Gimber, L. H., Cranmer, L., McBride, A., & Kraft, A. S. (2017). Angiosarcoma treated successfully with anti-PD-1 therapy - a case report. Journal for immunotherapy of cancer, 5(1), 58.
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  Angiosarcomas are tumors of malignant endothelial origin that have a poor prognosis with a five-year survival of less than 40%. These tumors can be found in all age groups, but are more common in older patients; with the cutaneous form most common in older white men. Combined modality therapy including surgery and radiation appears to have a better outcome than each modality alone. When metastatic, agents such as liposomal doxorubicin, paclitaxel and ifosfamide have activity but it is short-lived and not curative. Immunotherapy targeting either the PD-1 receptor or PD-L1 ligand has recently been shown to have activity in multiple cancers including melanoma, renal, and non-small lung cancer. Although these agents have been used in sarcoma therapy, their ability to treat angiosarcoma has not been reported.
• Song, J. H., Padi, S. K., Luevano, L. A., Minden, M. D., DeAngelo, D. J., Hardiman, G., Ball, L. E., Warfel, N. A., & Kraft, A. S. (2016). Insulin receptor substrate 1 is a substrate of the Pim protein kinases. Oncotarget, 7(15), 20152-65.
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  The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.
• Warfel, N. A., Sainz, A. G., Song, J. H., & Kraft, A. S. (2016). PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species. Molecular cancer therapeutics, 15(7), 1637-47.
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  Intratumoral hypoxia is a significant obstacle to the successful treatment of solid tumors, and it is highly correlated with metastasis, therapeutic resistance, and disease recurrence in cancer patients. As a result, there is an urgent need to develop effective therapies that target hypoxic cells within the tumor microenvironment. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a prosurvival pathway that is upregulated in response to hypoxia, in a HIF-1-independent manner. We demonstrate that pharmacologic or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia as compared with normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors prevent Nrf2 from accumulating in the nucleus, reducing the transcription of cytoprotective genes and leading to the build-up of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be successfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediated therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Mol Cancer Ther; 15(7); 1637-47. ©2016 AACR.
• An, N., Xiong, Y., LaRue, A. C., Kraft, A. S., & Cen, B. (2015). Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small-Molecule Inhibitors. Cancer research, 75(24), 5318-28.
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  Mesenchymal-epithelial transition (MET) blockade offers a new targeted therapy particularly in those cancers with MET amplification. However, the efficacy and the duration of the response to MET inhibitors are limited by the emergence of drug resistance. Here, we report that resistance to small-molecule inhibitors of MET can arise from increased expression of the prosurvival Pim protein kinases. This resistance mechanism was documented in non-small cell lung cancer and gastric cancer cells with MET amplification. Inhibition of Pim kinases enhanced cell death triggered by short-term treatment with MET inhibitors. Pim kinases control the translation of antiapoptotic protein Bcl-2 at an internal ribosome entry site and this mechanism was identified as the basis for Pim-mediated resistance to MET inhibitors. Protein synthesis was increased in drug-resistant cells, secondary to a Pim-mediated increase in cap-independent translation. In cells rendered drug resistant by chronic treatment with MET inhibitors, genetic or pharmacologic inhibition of Pim kinases was sufficient to restore sensitivity in vitro and in vivo. Taken together, our results rationalize Pim inhibition as a strategy to augment responses and blunt acquired resistance to MET inhibitors in cancer.
• Song, J. H., An, N., Chatterjee, S., Kistner-Griffin, E., Mahajan, S., Mehrotra, S., & Kraft, A. S. (2015). Deletion of Pim kinases elevates the cellular levels of reactive oxygen species and sensitizes to K-Ras-induced cell killing. Oncogene, 34(28), 3728-36.
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  The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-Ras(G12V)) and undergo cell death. Transduction of K-Ras(G12V) into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-Ras(G12V) in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-Ras(G12V)-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-Ras(G12V)-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth.
• Warfel, N. A., & Kraft, A. S. (2015). PIM kinase (and Akt) biology and signaling in tumors. Pharmacology & therapeutics, 151, 41-9.
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  The initiation and progression of human cancer is frequently linked to the uncontrolled activation of survival kinases. Two such pro-survival kinases that are commonly amplified in cancer are PIM and Akt. These oncogenic proteins are serine/threonine kinases that regulate tumorigenesis by phosphorylating substrates that control the cell cycle, cellular metabolism, proliferation, and survival. Growing evidence suggests that cross-talk exists between the PIM and Akt kinases, indicating that they control partially overlapping survival signaling pathways that are critical to the initiation, progression, and metastatic spread of many types of cancer. The PI3K/Akt signaling pathway is activated in many human tumors, and it is well established as a promising anticancer target. Likewise, based on the role of PIM kinases in normal and tumor tissues, it is clear that this family of kinases represents an interesting target for anticancer therapy. Pharmacological inhibition of PIM has the potential to significantly influence the efficacy of standard and targeted therapies. This review focuses on the regulation of PIM kinases, their role in tumorigenesis, and the biological impact of their interaction with the Akt signaling pathway on the efficacy of cancer therapy.
• Zemskova, M. Y., Song, J. H., Cen, B., Cerda-Infante, J., Montecinos, V. P., & Kraft, A. S. (2015). Regulation of prostate stromal fibroblasts by the PIM1 protein kinase. Cellular signalling, 27(1), 135-46.
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  The PIM1 oncogene is over-expressed in human prostate cancer epithelial cells. Importantly, we observe that in human hyperplastic and cancerous prostate glands PIM1 is also markedly elevated in prostate fibroblasts, suggesting an important role for this kinase in epithelial/stromal crosstalk. The ability of PIM1 to regulate the biologic activity of stromal cells is demonstrated by the observation that expression of PIM1 kinase in human prostate fibroblasts increases the level and secretion of the extracellular matrix molecule, collagen 1A1 (COL1A1), the pro-inflammatory chemokine CCL5, and the platelet-derived growth factor receptors (PDGFR). PIM1 is found to regulate the transcription of CCL5. In co-cultivation assays where PIM1 over-expressing fibroblasts are grown with BPH1 prostate epithelial cells, PIM1 activity markedly enhances the ability of these fibroblasts to differentiate into myofibroblasts and express known markers of cancer-associated fibroblasts (CAFs). This differentiation can be reversed by the addition of small molecule PIM kinase inhibitors. Western blots demonstrate that PIM1 expression in prostate fibroblasts stimulates the phosphorylation of molecules that regulate 5'Cap driven protein translation, including 4EBP1 and eIF4B. Consistent with the hypothesis that the kinase controls translation of specific mRNAs in prostate fibroblasts, we demonstrate that PIM1 expression markedly increases the level of COL1A1 and PDGFRβ mRNA bound to polysomes. Together these results point on PIM1 as a novel factor in regulation of the phenotype and differentiation of fibroblasts in prostate cancer by controlling both the transcription and translation of specific mRNAs.
• Cen, B., Xiong, Y., Song, J. H., Mahajan, S., DuPont, R., McEachern, K., DeAngelo, D. J., Cortes, J. E., Minden, M. D., Ebens, A., Mims, A., LaRue, A. C., & Kraft, A. S. (2014). The Pim-1 protein kinase is an important regulator of MET receptor tyrosine kinase levels and signaling. Molecular and cellular biology, 34(13), 2517-32.
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  MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.
• Chatterjee, S., Thyagarajan, K., Kesarwani, P., Song, J. H., Soloshchenko, M., Fu, J., Bailey, S. R., Vasu, C., Kraft, A. S., Paulos, C. M., Yu, X. Z., & Mehrotra, S. (2014). Reducing CD73 expression by IL1β-Programmed Th17 cells improves immunotherapeutic control of tumors. Cancer research, 74(21), 6048-59.
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  T cells of the T helper (Th)17 subset offer promise in adoptive T-cell therapy for cancer. However, current protocols for ex vivo programming of Th17 cells, which include TGFβ exposure, increase the expression of CD39 and CD73, two cell surface ATP ectonucleotidases that reduce T-cell effector functions and promote immunosuppression. Here, we report that ATP-mediated suppression of IFNγ production by Th17 cells can be overcome by genetic ablation of CD73 or by using IL1β instead of TGFβ to program Th17 cells ex vivo. Th17 cells cultured in IL1β were also highly polyfunctional, expressing high levels of effector molecules and exhibiting superior short-term control of melanoma in mice, despite reduced stem cell-like properties. TGFβ addition at low doses that did not upregulate CD73 expression but induced stemness properties drastically improved the antitumor effects of IL1β-cultured Th17 cells. Effector properties of IL1β-dependent Th17 cells were likely related to their high glycolytic capacity, since ex vivo programming in pyruvate impaired glycolysis and antitumor effects. Overall, we show that including TGFβ in ex vivo cultures used to program Th17 cells blunts their immunotherapeutic potential and demonstrate how this potential can be more fully realized for adoptive T-cell therapy.
• Din, S., Konstandin, M. H., Johnson, B., Emathinger, J., Völkers, M., Toko, H., Collins, B., Ormachea, L., Samse, K., Kubli, D. A., De La Torre, A., Kraft, A. S., Gustafsson, A. B., Kelly, D. P., & Sussman, M. A. (2014). Metabolic dysfunction consistent with premature aging results from deletion of Pim kinases. Circulation research, 115(3), 376-87.
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  The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention because Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight about cardiac mitochondrial biology and the aging phenotype.
• Marshall, D. T., Ramey, S., Golshayan, A. R., Keane, T. E., Kraft, A. S., & Chaudhary, U. (2014). Phase I trial of weekly docetaxel, total androgen blockade, and image-guided intensity-modulated radiotherapy for localized high-risk prostate adenocarcinoma. Clinical genitourinary cancer, 12(2), 80-6.
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  This was a phase I study to find the maximum tolerable dose (MTD) of weekly docetaxel combined with high-dose intensity-modulated radiotherapy (IMRT) and androgen deprivation therapy (ADT).
• Saurabh, K., Scherzer, M. T., Shah, P. P., Mims, A. S., Lockwood, W. W., Kraft, A. S., & Beverly, L. J. (2014). The PIM family of oncoproteins: small kinases with huge implications in myeloid leukemogenesis and as therapeutic targets. Oncotarget, 5(18), 8503-14.
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  PIM kinases are a family of serine/threonine kinases involved in cell survival and proliferation. There is significant structural similarity between the three PIM kinases (PIM1, PIM2 and PIM3) and only few amino acid differences. Although, several studies have specifically monitored the role of PIM1 in tumorigenesis, much less is known about PIM2 and PIM3. Therefore, in this study we have used in vitro cell culture models and in vivo bone marrow infection/transplantation to assess the comparative signaling and oncogenic potential of each of the three PIM kinases. All three PIM kinases were able to protect FL5.12 cells from IL3 withdrawal induced death. Interestingly, the downstream signaling cascades were indistinguishable between the three kinases. Transplantation of murine bone marrow co-expressing MYC and PIM1, PIM2 or PIM3 caused rapid and uniformly lethal myeloid leukemia. De-induction of MYC 18 days following transplantation significantly increased the survival of mice, even with continual expression of PIM kinases. Alternatively, mice treated at the pre-leukemic stage with a PIM kinase inhibitor increased the lifespan of the mice, even with continual expression of the MYC transgene. These data demonstrate the role of PIM kinases in driving myeloid leukemia, and as candidate molecules for therapy against human malignancies.
• Warren, G. W., Alberg, A. J., Kraft, A. S., & Cummings, K. M. (2014). The 2014 Surgeon General's report: "The health consequences of smoking--50 years of progress": a paradigm shift in cancer care. Cancer, 120(13), 1914-6.
• An, N., Kraft, A. S., & Kang, Y. (2013). Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice. Journal of hematology & oncology, 6, 12.
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  Pim (proviral insertion in murine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown.
• An, N., Lin, Y. W., Mahajan, S., Kellner, J. N., Wang, Y., Li, Z., Kraft, A. S., & Kang, Y. (2013). Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells. Stem cells (Dayton, Ohio), 31(6), 1202-12.
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  The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.
• Cen, B., Mahajan, S., Wang, W., & Kraft, A. S. (2013). Elevation of receptor tyrosine kinases by small molecule AKT inhibitors in prostate cancer is mediated by Pim-1. Cancer research, 73(11), 3402-11.
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  The PI3K/AKT pathway is hyperactivated in prostate cancer but its effective therapeutic targeting has proven difficult. In particular, the antitumor activity of AKT inhibitors is attenuated by upregulation of receptor tyrosine kinases (RTK) through an uncharacterized feedback mechanism. In this report, we show that RNA interference-mediated silencing or pharmacologic inhibition of Pim-1 activity curtails AKT inhibitor-induced upregulation of RTKs in prostate cancer cells. Although Pim kinases have been implicated in cap-dependent translational control, we find that in the context of AKT inhibition, the expression of RTKs is controlled by Pim-1 in a cap-independent manner by controlling internal ribosome entry. Combination of Pim and AKT inhibitors resulted in synergistic inhibition of prostate tumor growth in vitro and in vivo. Together, our results show that Pim-1 mediates resistance to AKT inhibition and suggest its targeting to improve the efficacy of AKT inhibitors in anticancer therapy.
• Roper, S. M., Zemskova, M., Neely, B. A., Martin, A., Gao, P., Jones, E. E., Kraft, A. S., & Drake, R. R. (2013). Targeted glycoprotein enrichment and identification in stromal cell secretomes using azido sugar metabolic labeling. Proteomics. Clinical applications, 7(5-6), 367-71.
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  Effectively identifying the proteins present in the cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum.
• Schwartz, G. K., Tap, W. D., Qin, L. X., Livingston, M. B., Undevia, S. D., Chmielowski, B., Agulnik, M., Schuetze, S. M., Reed, D. R., Okuno, S. H., Ludwig, J. A., Keedy, V., Rietschel, P., Kraft, A. S., Adkins, D., Van Tine, B. A., Brockstein, B., Yim, V., Bitas, C., , Abdullah, A., et al. (2013). Cixutumumab and temsirolimus for patients with bone and soft-tissue sarcoma: a multicentre, open-label, phase 2 trial. The Lancet. Oncology, 14(4), 371-82.
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  Preclinical studies have shown synergistic antitumour activity by inhibition of insulin-like growth factor-1 receptor (IGF-1R) and mTOR. The expression of IGF-1R seems to be crucial for this effect. We investigated the safety and efficacy of the combination of the IGF-1R antibody cixutumumab and the mTOR inhibitor temsirolimus in patients with chemotherapy-refractory bone and soft-tissue sarcomas according to IGF-1R expression by immunohistochemistry.
• Twardowski, P. W., Beumer, J. H., Chen, C. S., Kraft, A. S., Chatta, G. S., Mitsuhashi, M., Ye, W., Christner, S. M., & Lilly, M. B. (2013). A phase II trial of dasatinib in patients with metastatic castration-resistant prostate cancer treated previously with chemotherapy. Anti-cancer drugs, 24(7), 743-53.
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  There is a need for efficacious therapies for metastatic castration-resistant prostate cancer (mCRPC) after disease progression on docetaxel. The SRC tyrosine kinase and its related family members may be important drivers of prostate cancer and can be inhibited by dasatinib. mCRPC patients, after one previous chemotherapy, started dasatinib at 70 mg twice daily, amended to 100 mg daily. The primary endpoint was the disease control (DC) rate, defined as complete response (CR), partial response (PR), or stable disease (SD) in prostate specific antigen (PSA), RECIST, bone scan, and FACT-P score. Up to 41 patients were to be accrued (two-stage design, 21+20) to rule out a null-hypothesized effect of 5 versus 20% (α=0.05, β=0.1). Secondary endpoints included progression-free survival, toxicity, and pharmacokinetic and pharmacodynamic correlatives. Of 38 patients, 27 were evaluable for response or toxicity. The median duration of therapy was 55 days (6-284). Five patients showed DC after 8 weeks of therapy (18.5% DC, 95% CI: 6.3-38.1%). One PR (3.7% response rate, 95% CI: 0.1-19.0%) was observed in a patient treated for 284 days. Twelve patients (43%) discontinued treatment for toxicity. Dasatinib induced a decrease in phytohemagglutinin-stimulated CSF2, CD40L, GZMB, and IL-2 mRNAs in blood cells, indicating target engagement. Decreases in plasma IL-6 and bone alkaline phosphatase, and in urinary N-telopeptide, were associated with DC. Dasatinib has definite but limited activity in advanced mCRPC, and was poorly tolerated. The observation of a patient with prolonged, objective, clinically significant benefit warrants molecular profiling to select the appropriate patient population.

Proceedings Publications

• Gokhale, V., Kang, H., Luevano, L., Kraft, A., Hurley, L., & Song, J. (2018, April, 2018). Prostate cancer cell death triggered by a small molecule interacting with the hTERT promoter G-quadruplex element. In AACR Annual Meeting 2018, 78(13 Supplement), 4841-4841.
• Song, J., Leuevano, L., Singh, N., Kraft, A., Song, J., Leuevano, L., Singh, N., & Kraft, A. (2017, Dec 2- 5). NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy. In AACR; Prostate Cancer: Advances in Basic, Translational, and Clinical Research, 132.
• Padi, S., Luevano, L. A., An, N., Pandey, R., Singh, N., Song, J. H., Aster, J., Yu, X., Mehrotra, S., Kraft, A. S., Padi, S., Luevano, L. A., An, N., Pandey, R., Singh, N., Song, J. H., Aster, J., Yu, X., Mehrotra, S., & Kraft, A. S. (2016, 12). Therapeutic Targeting of PIM Protein Kinases in a Subset of T-Cell Acute Lymphoblastic Leukemia. In American Society of Hematology, Blood, 128, 2742.
• Sainz, A. G., Song, J., Kraft, A., & Warfel, N. A. (2016, 07-15-2016). PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species. In AACR Annual Meeting, Cancer Research, Volume 76, Issue 14.
• Song, J., & Kraft, A. (2016, 07-15-2016). Insulin receptor substrate 1 is a key substrate for Pim protein kinases. In AACR Annual Meeting, Cancer Research, Volume 76, Issue 14.