Zanna Beharry

Synthesis and Evaluation of Novel Inhibitors of Pim-1 and Pim-2 Protein Kinases

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a) was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although 4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC(50)s of 13 nM for Pim-1 and 2.3 microM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma

The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL.

PIM1 Protein Kinase regulates PRAS40 phosphorylation and mTOR activity in FDCP1 cells

PIM1 is a serine /threonine kinase that has diverse biological roles in cell survival, proliferation and differentiation. PIM1 has been implicated in early transformation and tumor progression in haematopoietic malignancies and prostate carcinomas. The ability of PIM1 to regulate these processes is thought to be in part secondary to its activity in stimulating 4EBP1 phosphorylation and enhancement of protein synthesis. Because 4EBP1 is an mTOR substrate, we have investigated how PIM1 might regulate this latter enzyme. We have examined the ability of PIM1 to modulate PRAS40, a protein known to negatively regulate mTOR activity in FDP1 cells. Upon phosphorylation, PRAS40 dissociates from the mTOR complex and increases mTOR kinase activity. We find that enforced overexpression of PIM1 increases PRAS40 phosphorylation at Thr246, an AKT phosphorylation site, whether grown in complete media or deprived of IL-3 and serum. The increase in PRAS40 phosphorylation was independent of AKT activation and not inhibited by wortmannin. In vitro kinase assays indicate that the PIM1 protein kinase is capable of directly phosphorylating Thr246 in PRAS40. PIM1 protein kinase overexpression reduced the association of PRAS40 with mTOR, and increased the mTOR directed phosphorylation of 4EBP1 and p70S6Kinase. Treatment of FDCP1 cells transfected with PIM1 (FD/mpim44) with small molecule inhibitors of PIM1 kinase activity reduced both PRAS40 and 4EBP1 phosphorylation. These results suggest that PIM1 regulates mTOR activity through phosphorylation of PRAS40. Thus, increases in mTOR activity mediated by the PIM protein kinase may have the potential to control cell growth.

The Pim protein kinases regulate energy metabolism and cell growth

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMP∶ATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap–dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

Novel benzylidene-thiazolidine-2,4-diones inhibit Pim protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells

The Pim protein kinases play important roles in cancer development and progression, including prostate tumors and hematologic malignancies. To investigate the potential role of these enzymes as anticancer drug targets, we have synthesized novel benzylidene-thiazolidine-2,4-diones that function as potent Pim protein kinase inhibitors. With IC50 values in the nanomolar range, these compounds block the ability of Pim to phosphorylate peptides and proteins in vitro and, when added to DU145 prostate cancer cells overexpressing Pim, inhibit the ability of this enzyme to phosphorylate a known substrate, the BH3 protein BAD. When added to prostate cancer cell lines, including PC3, DU145, and CWR22Rv1, and human leukemic cells, MV4;11, K562, and U937 cells, these compounds induce G1-S cell cycle arrest and block the antiapoptotic effect of the Pim protein kinase. The cell cycle arrest induced by these compounds is associated with an inhibition of cyclin-dependent kinase 2 and activity and translocation of the Pim-1 substrate p27Kip1, a cyclin-dependent kinase 2 inhibitory protein, to the nucleus. Furthermore, when added to leukemic cells, these compounds synergize with the mammalian target of rapamycin inhibitor rapamycin to decrease the phosphorylation level of the translational repressor 4E-BP1 at sites phosphorylated by mammalian target of rapamycin. Combinations of rapamycin and the benzylidene-thiazolidine-2,4-diones synergistically block the growth of leukemic cells. Thus, these agents represent novel Pim inhibitors and point to an important role for the Pim protein kinases in cell cycle control in multiple types of cancer cells. [Mol Cancer Ther 2009;8(6):1473–83]

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCFSkp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser64 and Ser72, we have identified Thr417 as a unique Pim-1 phosphorylation target. Phosphorylation of Thr417 controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.

Collaboration of Kras and Androgen Receptor Signaling Stimulates EZH2 Expression and Tumor-Propagating Cells in Prostate Cancer

Elevation of the chromatin repression factor enhancer of zeste homolog (EZH2) is associated with progression and poor prognosis in several human cancers including prostate cancer. However, the mechanisms driving EZH2 expression are not fully understood. In this study, we investigated the functional synergy in prostate cancers in mice resulting from activation of the androgen receptor, Kras, and Akt, which drives three of the most frequently activated oncogenic signaling pathways in prostate cancer. Although, any two of these three events were sufficient to promote the formation and progression of prostate cancer, only the synergy of androgen receptor and Kras signaling could elevate EZH2 expression and expand prostate cancer progenitor cells in vivo. Our findings have revealed a genetic mechanism resulting in enhanced EZH2 expression during the progression of aggressive prostate cancer, with important implications for understanding how to target advanced disease where cancer progenitor cells may be critical.

Abstract 1258: Regulation of energy metabolism and protein synthesis by the Pim protein kinases

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked mTORC1 activity by stimulating the phosphorylation, and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases (TKO MEFs) demonstrated activated AMPK driven by elevated ratios of AMP:ATP relative to wild type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and PGC-1α, enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. These results demonstrate the Pim kinase -mediated control of energy metabolism through regulation of AMPK activity, and identify a new and important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth. In prostate cancer cell lines we find that the Pim-3 protein kinase is elevated in those cells that are highly metastatic including PC3-LN4, ARCaPM and M12 when compared to PC-3, ARCaPE and p69 and the amount of Pim-3 correlates with c-Myc levels. Further experiments will be needed to determine whether the Pim-3 protein kinase expression regulates the metastatic potential of prostate cancer cells through control of energy metabolism and protein synthesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1258. doi:10.1158/1538-7445.AM2011-1258

Abstract C64: Treatment with the Pim protein kinase inhibitor SMI‐4a enhances AMPK phosphorylation, decreases Raptor levels, and blocks mTORC1 activity

We have identified a class of small molecule inhibitors of the Pim protein kinases, benzylidene thiazolidine‐2‐4 diones (J. Med. Chem. (2009) 52:74) with the most potent members having IC50s of 13 nM for Pim‐1 and 2.3 µM for Pim‐2. Compounds in this chemotype demonstrated selectivity of more than 2500‐fold and 400‐fold for Pim‐1 or Pim‐2 respectively while other congeners had equivalent potency towards both isozymes. In vivo, these molecules inhibited Pim kinase autophosphorylation and in a murine model inhibited the growth of subcutaneously implanted murine adenocarcinoma JC cells. One of the members of this chemotype, SMI‐4a, has been shown to block the phosphorylation of the mTOR regulatory protein PRAS40 and subsequently the activity of the mTOR pathway (Mol. Cancer. Ther. (2009) 8: 1473; Cancer Biol. Ther. (2009) 8: 846). Now we show that the addition of SMI‐4a to malignant cells increases the phosphorylation of AMPKα on Thr 172 in a LKB1‐dependent manner, induces the phosphorylation of Raptor on Ser792, decreases the levels of Raptor protein, and inhibits mTORC1 activity. Immunoprecipitation of mTOR from SMI‐4a treated cells consistently showed lower levels of bound Raptor and in vitro mTOR kinase assays from treated cells demonstrated a decreased ability to phosphorylate 4E‐BP1. Knockdown of PIM‐1 via siRNA in K562 leukemic cells showed increased AMPK phosphorylation and decreased Raptor protein levels, further demonstrating an important role for Pim kinase in regulating AMPK phosphorylation and Raptor levels. Additionally, mouse embryo fibroblasts (MEFs) deficient for Pim‐1, Pim‐2 and Pim‐3 kinase (TKO MEFs) showed a significantly increased level of AMPK phosphorylation compared to wild type MEFs, which correlated with decreased mTORC1 activity and increased binding of 4E‐BP1 with eIF‐4E. The TKO MEFs grew significantly more slowly than wild type. The decreased mTORC1 activity correlated with an increase in the cellular level of AMP in TKO MEFs. Furthermore, the correlation between increased AMPK phosphorylation and a lower level of Raptor protein observed with SMI‐4a treatment was also observed in TKO MEFs. Infection of TKO MEFs with lentiviruses expressing Pim 1 or Pim2 was able to reverse these effects, decreasing AMPK phosphorylation, and increasing Raptor protein levels. The cellular activity of mTORC1 was difficult to assess in TKO MEFs as we found substantially lower protein levels of the mTORC1 substrates 4E‐BP1 and p70S6K. Akt was readily phosphorylated upon serum stimulation of TKO MEFs and mTORC2 activity was unchanged. Given the role of Pim kinase in regulating mTORC1 activity, we have combined SMI‐4a and the mTOR inhibitor rapamycin inducing synergistic blockade of this pathway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C64.

Abstract B249: Small molecule inhibitors of the Pim protein kinases block the growth of T‐acute lymphoblastic leukemias

The Pim protein kinases, first identified as a proviral integration site in c‐Myc overexpressor mice, are increased in multiple human hematopoietic neoplasms including myeloid leukemia, diffuse large B‐cell lymphoma, and T‐cell lymphoma. We have developed novel benzylidene‐thiazolidine‐2, 4‐diones (J. Med. Chem. (2009) 52:74) inhibitors of Pim kinases that kill a wide range of both myeloid and lymphoid cell lines with precursor T‐cell lymphoblastic leukemia/lymphoma (pre T‐LBL/T‐ALL) being the most sensitive. The most potent members of this chemotype have IC50s of 13 nM for Pim‐1 and 2.3 M for Pim‐2 while some compounds in this chemotype demonstrated selectivity9s of more than 2500‐fold and 400‐fold for Pim‐1 or Pim‐2 respectively while other congeners had equivalent potency towards both isozymes. Of the 47 additional protein kinases tested, only DYRK1a was sensitive to these agents. Incubation of pre T‐LBL cells with one of these Pim inhibitors, SMI‐4a, induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27Kip1 and translocation of this protein to the nucleus. Additionally, SMI‐4a induced apoptosis in these leukemic cells through the mitochondrial pathway, and inhibited mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E‐BP1, two substrates of this enzyme. Using immuno‐deficient animals, we demonstrate that treatment 5/7 days with 60 mg/kg twice daily by oral gavage of SMI‐4a inhibits subcutaneous growth of pre T‐LBL tumors by an average of 47.9% (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B249.