Alexis Mollard

A Small-Molecule Inhibitor of PIM Kinases as a Potential Treatment for Urothelial Carcinomas

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

Background. Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. Materials and Methods. To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. Results. Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. Conclusion. Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma.

Tumor targeting, trifunctional dendritic wedge.

We report in vitro and in vivo evaluation of a newly designed trifunctional theranostic agent for targeting solid tumors. This agent combines a dendritic wedge with high boron content for boron neutron capture therapy or boron MRI, a monomethine cyanine dye for visible-light fluorescent imaging, and an integrin ligand for efficient tumor targeting. We report photophysical properties of the new agent, its cellular uptake and in vitro targeting properties. Using live animal imaging and intravital microscopy (IVM) techniques, we observed a rapid accumulation of the agent and its retention for a prolonged period of time (up to 7 days) in fully established animal models of human melanoma and murine mammary adenocarcinoma. This macromolecular theranostic agent can be used for targeted delivery of high boron load into solid tumors for future applications in boron neutron capture therapy.

Abstract 5543: Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells.

Pancreatic cancer is virtually a uniformly lethal disease and a better understanding of the molecular basis of this malignancy is needed to discover new ways to prevent or treat this deadly disease. A central feature of malignant cells is their ability to disseminate from the primary tumor and establish local and distant metastases. In most cases, cancer patients with localized disease have significantly better prognosis than those with metastatic tumors and the majority of cancer mortality is associated with metastatic disease rather than the primary tumor. The receptor tyrosine kinase Axl is overexpressed in over 50% of pancreatic cancers and expression of Axl in these cancers is highly associated with a poor prognostic outcome for patients. Axl is a TAM family receptor tyrosine kinase involved in multiple aspects of tumorigenesis. Increased expression of Axl is associated with increased oncogenic transformation, cell survival, proliferation, migration, angiogenesis, and cellular adhesion. The known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and it9s binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. We discovered and developed a small molecule Axl kinase inhibitor, HCI-2084, and explored it for the effectiveness of targeting the Axl kinase in cell-based models of pancreatic cancer. HCI-2084 is a 2-((2,5-substitutedpyrimidin-4-yl)amino)-N,N-dimethyl benzene sulfonamide that has low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assays. In further biochemical evaluation, HCI-2084 was shown to inhibit the entire TAM family of kinases (IC50 Axl = 12 nM; IC50 Mer = 60 nM; Tyro3 = 71% inhibition at 200 nM). HCI-2084 inhibits a small number of additional kinases when screened in a kinase panel of over 500 kinases and has demonstrated an ADMET profile suggesting it may be a potential clinical candidate. In cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro and potently induces apoptosis. Mechanistically, HCI-2084 decreases the expression of genes involved in Epithelial-Mesenchymal Transition (EMT) and induces cells to take on more epithelial phenotypes. HCI-2084 also significantly inhibited the growth of pancreatic cancer cell lines grown in xenograft tumor mouse model and taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is a potential agent treat this disease. Citation Format: Malia Anderson, Alex Ober, Alexis Mollard, Lee Call, Jared J. Bearss, Hariprasad Vankayalapati, Sunil Sharma, Steven Warner, David J. Bearss. Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2013-5543

Abstract 1875: A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism.

Rather than relying on oxidative phosphorylation for the generation of ATP, human tumor cells primarily utilize aerobic glycolysis to metabolize glucose (the Warburg effect). Pyruvate kinase is a metabolic enzyme that converts phosphoenolpyruvate (PEP) to pyruvate, catalyzing the rate-limiting step of glycolysis. The M1 isoform of pyruvate kinase (PKM1) that is the principal isoform in most adult differentiated tissues, while the PKM2 splice variant is the main isoform in embryonic tissues and in all cancer cells. Unlike the M1 splice form (PKM1), which is found in its tetrameric active form in cells, PKM2 is found in cells as an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Regulation of PKM2 activity in cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. PKM2 expression enhances tumorigenicity of cells while PKM1 expression represses it. This suggests that activators of PKM2 may have anti-tumor properties by forcing PKM2 to act more like PKM1. We have a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays. These compounds increase pyruvate kinase activity in cancer cells and lead to an increase in pyruvate and ATP production. Our studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro and in vivo and can reverse the metabolic changes induced by oncogenes such as k-Ras and c-Myc in lung cancer cells. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant 54% decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies. Citation Format: Brigham L. Bahr, Jenny Stevens, Spencer Squire, Christopher Moreno, Lee T. Call, Bret J. Stephens, Alexis Mollard, Steven L. Warner, David J. Bearss. A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1875. doi:10.1158/1538-7445.AM2013-1875

Abstract LB-303: Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite the recent advances made in the treatment and management of B cell malignancies, these diseases are not curable and overall survival is limited. Brutons Tyrosine Kinase (BTK) is a member of the Tec family of intracellular kinases first identified for its signaling via the B-cell Receptor (BCR) and its role in the immune system. More recently, BTK was found to play an important role in B cell malignancies and select solid tumors. Preclinical and clinical results with selective irreversible BTK inhibitors provide validation for BTK as a therapeutic target in B cell malignancies. Aiming to leverage the contribution of the BTK signaling pathway to tumor growth, and its role in progression and drug resistance, we have developed a series of relatively selective, reversible, small molecule BTK inhibitors and evaluated their activity in enzyme, cell-based, and in vivo studies. Data obtained with the orally available MKC4659 compound illustrates our findings. In biochemical assays, compound MKC4659 demonstrated a relatively select targeting profile focused on a narrow group of PTKs including significant activity against BTK with IC50 less than 25 nM. In cellular assays the compound demonstrated significant in vitro potency against B cell lymphoma cell lines, inhibiting the growth of several B cell tumor cell lines including ones unresponsive to currently known BTK inhibitors. Importantly, MKC4659 showed a differential effect on B cell lymphoma, with no significant activity detected in control cells lacking detectable BTK expression. In vitro mode of action studies demonstrated that MKC4659 induces apoptosis and PARP cleavage in B cell lymphoma but not in control cells. Assays evaluating the in vitro on-target effect of compounds showed significant inhibition of the B cell receptor-mediated activation of the BTK pathway. In addition to inhibiting the phosphorylation of BTK, MKC4659 inhibited the phosphorylation of PLCγ in several B cell lymphoma cell lines. With in vitro potency demonstrated, and PK and ADMET profiles amenable to in vivo dosing, MKC4659 was evaluated for in vivo efficacy in a xenograft model of B cell lymphoma. In vivo dosing of MKC4659 inhibited growth of DOHH2 xenograft tumors in a dose dependent manner. In summary, our team has identified BTK pathway inhibitors with demonstrated on-target and anti-tumor activity in cellular assays, and efficacy in a preclinical model of B cell malignancy. This effort provides a platform for compound development and evaluation for the treatment of hematologic malignancies. Optimization efforts on the MKC4659 series are ongoing and have yielded potent and drug-like preclinical candidates that are now moving into advanced animal studies. Citation Format: Mary Faris, Uriel M. Malyankar, Victor Tam, Colleen Schweitzer, Diljeet Joea, Alexis Mollard, Bret Stephens, Steven L. Warner, David J. Bearss, Qingping Zeng. Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2013-LB-303

Abstract 904: TP-0413 is a dual-specific inhibitor against Jak2 and Alk2 with therapeutic potential for treating anemia of chronic disease.

Anemia of chronic disease is a debilitating condition commonly found in cancer patients. Studies suggest that this syndrome is largely the result of the body9s production of hepcidin, a master regulator of human iron metabolism. TP-0413 is a small molecule that targets two important components of the hepcidin pathway, Janus Kinase 2 (Jak2) and Activin A receptor, type I (Alk-2). Jak2 facilitates hepcidin function by phosphorylating ferroportin upon hepcidin binding, inducing its degradation and thus preventing iron entry into plasma. Alk2 on the other hand, is a component of the signal transduction pathway that leads to hepcidin expression. Here we report on recent efforts aimed at further characterization of TP-0413. In biochemical assays TP-0413 inhibits Jak2 and Alk2 at 3.11 nM and 70 nM, respectively (IC50). In cell culture, HepG2 cells treated with TP-0413 exhibit reduced hepcidin levels, and TP-0413 is able to inhibit BMP-2 induction of hepcidin. Furthermore, TP-0413 ameliorates hypoferremia induced by LPS treatment in mice, and modulates cytokine levels in these animals. TP-0413 shows favorable drug-like properties in pharmacokinetic models, and further studies are being undertaken to advance TP-0413 towards clinical trials. Citation Format: Bret J. Stephens, Alexis Mollard, Lingyao Meng, David J. Bearss, Steven L. Warner. TP-0413 is a dual-specific inhibitor against Jak2 and Alk2 with therapeutic potential for treating anemia of chronic disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 904. doi:10.1158/1538-7445.AM2013-904