Stephen C. Cosenza

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing axa0common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

A Non–ATP-Competitive Dual Inhibitor of JAK2V617F and BCR-ABLT315I Kinases Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition

Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

Hydrothiolation of benzyl mercaptan to arylacetylene: application to the synthesis of (E) and (Z)-isomers of ON 01910·Na (Rigosertib®), a phase III clinical stage anti-cancer agent

A stereoselective and efficient method for free radical addition of benzyl thiol to aryl acetylene in the presence of Et3B-hexane has been developed for the synthesis of (Z) and (E)-styryl benzyl sulfides where base catalyzed hydrothiolations have failed. The scope of this reaction was successfully extended for the synthesis of (E)-ON 01910·Na, a phase III clinical stage anti-cancer agent and its inactive geometrical isomer (Z)-ON 01910·Na. It is interesting to note that all the E-isomers synthesized have shown better cytotoxicity profile on cancer cells compared to the Z-isomers.

Preclinical Pharmacokinetic and Pharmacodynamic Evaluation of Novel Anticancer Agents, ON01910.Na (Rigosertib, Estybon™) and ON013105, for Brain Tumor Chemotherapy

ABSTRACTPurposeTo evaluate a mitotic inhibitor, ON01910.Na, as a potential chemotherapeutic agent for brain tumors using a series of PK/PD studies, which led to the evaluation of its structural analog, ON013105, a prodrug of the more lipophilic product, ON013100.MethodsSystemic PK characterization of ON01910 and ON013105 was completed in healthy mice. Using an orthotopic U87 glioma mouse model, brain and brain tumor distribution under steady-state conditions were evaluated for ON01910.Na and ON013105/ON013100; anticancer potential following a multiple-dose schedule of 250xa0mg/kg/day IP for 7xa0days was evaluated for ON01910.Na.ResultsON01910 exhibited low brain and brain tumor distribution with quasi-steady-state brain/plasma (Cssbrain/Cssplasma) and brain tumor/plasma (Cssbrain tumor/Cssplasma) concentration ratios of 0.03u2009±u20090.02 and 0.14u2009±u20090.08, respectively. Significant antiangiogenic potential and antiproliferative capacity of ON01910 in the intracerebral model was absent. ON013100 showed high brain and brain tumor penetration with Cssbrain/Cssplasma and Cssbrain tumor/Cssplasma ratios of 0.92u2009±u20090.26 and 1.35u2009±u20090.40, respectively; its prodrug ON013105 showed negligible brain and brain tumor penetration.ConclusionsON013105, not ON01910.Na, was identified as a potential anticancer drug candidate for further investigation in brain tumor chemotherapy based on the properties of ON013100.

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.

Abstract 4519: Targeting of cyclin D/Rb/E2F and PI3K/AKT/MTOR pathways with ON 123300 as a therapeutic strategy for mantle cell lymphoma

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against Mantle Cell Lymphomas (MCLs) both in vitro and in vivo. Mantle cell lymphoma is genetically characterized by the t(11;14)(q13;q32) chromosomal translocation which results in constitutive overexpression of cyclin D1. In addition, MCLs also activate other pathways, including aberrant B-Cell Receptor and PI3K/AKT/mTOR signaling. As a result, MCL has a poor clinical outcome with a median survival of 4-5 years. In this study, we show that ON123300, which inhibits both CDK4/6 and PI3K-α (the predominant PI3K catalytic subunit expressed in MCL cells), is a superior inducer of apoptosis of MCL cells when compared to PD0332991, a selective inhibitor of CDK4/6 kinases. Experimental Procedures: We examined the effects of PD 0332991 and ON123300 on cell cycle progression, modulation of the Rb and PI3K/AKT pathways, and the induction of apoptosis in the Granta 519 and Z138C mantle cell lymphoma cell lines. When Granta 519 and Z138C cells were incubated with increasing concentrations of PD 0332991 and ON 123300, both compounds efficienty inhibited the phosphorylation of the Rb family of proteins. However, ON123300 showed concentration-dependent inhibition of MTOR, AKT, 4EBP1 and S6RB phosphorylation while PD 0332991 had no effect on the phosphorylation status of these proteins. While cells treated with PD 0332991 rapidly accumulated in the G0/G1 stage of cell cycle, cells treated with ON123300 showed an accumulation of cells with a sub-G1 DNA content. These ON123300 treated cells showed cleavage of PARP as well as Caspases 3, 7 and 9 and inhibition of FOXO1 phosphorylation, which was not observed in cells treated with PD 0332991. We tested the effects of ON 123300 in nude mouse xenograft assays using Z138 MCL cells. These studies revealed a strong inhibition of tumor growth when tumor-bearing mice were treated daily with 100 mg/kg of ON123300. In addition, there was little evidence of toxicity as measured by change in the body weight in ON123300-treated mice. Conclusions: ON123300 targets the CyclinD/CDK/Rb pathway as well as the PI3K/AKT/MTOR pathway to induce apoptosis of MCL cells via intrinsic apoptotic pathways. Mouse xenograft assays show that ON 123300 is a strong inhibitor of MCL tumor growth in vivo. This dual activity against Rb and AKT pathways appears to be an effective therapeutic strategy for the treatment of MCL. Citation Format: E. Premkumar Reddy, Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Balaiah Akula, Samir Parekh. Targeting of cyclin D/Rb/E2F and PI3K/AKT/MTOR pathways with ON 123300 as a therapeutic strategy for mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2014-4519

Abstract 4453: The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells

Triple negative breast cancer (TNBC) is associated with a poor prognosis and high frequency of recurrence. Because the molecular mechanisms that are deregulated in this tumor type are not well understood, there is a lack of targeted therapies that can effectively treat this disease. An unfortunate limitation of existing TNBC therapies is the frequency of relapse, which is highly resistant and metastatic and has been attributed to tumor-initiating stem cells (T-ICs). In patients with relapsed TNBC, T-ICs with a CD44high/CD24-/low antigenic phenotype are enriched in the tumor cell population. Our previous data illustrated that a small molecule kinase inhibitor, ON108600, potently inhibited the survival and growth of TNBC cell lines and mouse xenografts. To investigate whether ON108600 has a similar inhibitory effect on T-ICs, we performed clonogenic survival assays using sorted CD44high CD24-/low cells isolated from TNBC cell lines. ON108600 potently inhibited the stem cell activity and self-renewal ability of these T-ICs. Although paclitaxel (PTX) treatment improves survival of TNBC patients, acquired resistance to this drug is a common occurrence. Furthermore, strategies that target PTX resistant cells remain elusive. To investigate the molecular mechanisms underlying acquired PTX-resistance in TNBC and evaluate the efficacy of ON108600, we generated PTX-resistant cell lines using the MDA-MB-231 and BT-20 TNBC cell lines. Drug-resistant cells were established by exposure to increasing concentrations of Paclitaxel, and resistance was validated by cell viability and colony formation in the presence of high concentrations of PTX. PTX-resistant MDA-MB-231 and BT-20 cells exhibited an approximate 1000-fold increase in resistance to PTX as compared to the parental cells. Importantly, PTX-resistant TNBC cells displayed a stem-like phenotype characterized by loss of epithelial differentiation markers (e-Cadherin, CD24) and a gain of epithelial-mesenchymal transition markers (N-Cadherin, CD44, Oct4, Snail). Kinase profiling studies have indicated that ON108600 targets kinases CK2, and Traf2- and Nck-interacting kinase, TNIK. Although CK2 subunit levels in resistant cells were unchanged, PTX-resistant cells showed a marked upregulation of TNIK, an activating kinase for T-cell factor-4 (TCF-4) and consequently, a marked increase in β-catenin and Wnt target genes Axin-2 and Cyclin D1. We therefore examined whether dual inhibition of CK2 and TNIK was effective in killing PTX-resistant cells. Treatment of PTX-resistant TNBC cells with ON108600 resulted in marked increase in apoptosis and inhibition of clonogenic survival and mamosphere forming ability of these cells, suggesting that dual CK2/TNIK inhibition may be an effective way to overcome PTX-resistance in TNBC. We are currently testing the efficacy of ON108600 in paclitaxel resistant xenograft models. Citation Format: Amol Padgaonkar, Stephen Cosenza, Venkat Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, E Premkumar Reddy. The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4453. doi:10.1158/1538-7445.AM2015-4453

Determination of the glucuronide metabolite of ON 013100, a benzylstyrylsulfone antineoplastic drug, in colon cancer cells using LC/MS/MS.

ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6 Da) was found in the drug-resistant cells upon a 1h incubation with ON 013100 (20 μg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84 μg/ml at 0 h through 0.21 μg/ml at 6h to below detection limit of 8.0 ng/ml at 9 h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41 μg/ml (0 h) to 0.06 μg/ml (9 h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88 μg/ml to 0.94 μg/ml.

Abstract 3029: Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against colorectal cancers both in vitro and in vivo. While overexpression of Cyclin D1 is closely correlated with the proliferative rate of these tumor cells, metastatic colorectal cancers over-express ARK5, a member of the AMPK family and mediator of AKT activation. In this study, we show that ON 123300, which inhibits both CDK4/6 and ARK5, is a potent inducer of apoptosis of colorectal cancer cells when compared to palbociclib, a highly selective inhibitor of CDK4/6 kinases that does not target ARK5. Results & Conclusions: We examined the effects of palbociclib and ON 123300 on cell cycle progression, modulation of Rb and PI3K/AKT pathways, and induction of apoptosis in multiple colorectal cancer cell lines. Comparative kinase inhibition assays showed that while palbociclib and ON 123300 exhibited equivalent inhibition against CDK4/CDK6, ARK5 activity was inhibited only by ON 123300. When DLD1 and SW480 cells were incubated with increasing concentrations of palbociclib or ON 123300, both compounds were equally efficient in their ability to inhibit phosphorylation of all three members of the Rb family of proteins. However, when the phosphorylation status of proteins associated with the PI3K/AKT pathway was measured by western blot, ON 123300 showed concentration-dependent inhibition of mTOR, AKT, 4EBP1 and S6RB phosphorylation while palbociclib had little or no effect on the phosphorylation of these proteins. Cells treated with palbociclib rapidly accumulated in the G0/G1 stage of the cell cycle with increasing drug concentrations. Although cells treated with ON 123300 also arrested in the G0/G1 phase at lower concentrations (01-0.5 uM), with increasing concentrations of drug there was an accumulation of cells with sub-G1 DNA content, suggesting induction of apoptosis. ON 123300-treated cells showed cleavage of PARP and Caspases (3, 7 and 9) as well as inhibition of FOXO1 phosphorylation, which was not observed in cells treated with palbociclib. Since ARK5 belongs to the AMPK family of kinases, we next examined the effects of ON 123300-mediated ARK5 inhibition on metabolic changes of tumor cells that over-express this gene. Treatment of SW-480 colorectal cancer cells with ON 123300 resulted in an increase in glucose uptake, profound inhibition of glutamine uptake and reduced ATP production. A detailed metabolomic study revealed significant alterations in the levels of metabolites associated with glutamine metabolism. Nude mouse xenograft assays using Colo-205 cells revealed strong inhibition of tumor growth following 100 mg/kg of ON 123300 given QD or QOD, with little evidence of toxicity as measured by change in body weight. Thus, dual inhibition of ARK5 and CDK4 pathways could be an effective therapeutic strategy for the treatment of colorectal cancers. Citation Format: Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Vinee Purohit, Venugopal Gunda, Pankaj K. Singh, E. Premkumar Reddy. Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3029.

Abstract 4537: Pharmacokinetics of PLK2 inhibitor GBO-006-1, developed as a novel first-in-class molecule to treat triple negative breast cancer

Introduction: The goal of this study was to develop a targeted therapy for triple negative breast cancer (TNBC) since these tumors do not respond to hormonal-related therapies or medications that target HER2. TNBC is typically treated with a combination of surgery, radiation, and chemotherapy albeit with worse outcome than patients with breast cancers of other subtypes. We have previously shown that GBO-006-1 (ON 1231320) is a highly selective, ATP competitive PLK2 inhibitor, which induces irreversible mitotic arrest of triple negative breast cancer (TNBC) cell lines and results in their apoptotic death. The goal of our current study was to characterize the drug-like and ADME properties of this molecule, and to optimize the in vivo exposure for preclinical evaluation. Experimental procedures: Drug-like properties were characterized by determining the pH solubility profile, log D, oral absorption potential using PAMPA, metabolic stability using rat and liver microsomes, plasma protein binding, and the inhibitory effect on various CYP isozymes. GBO-006-1 has weakly ionizable groups and did not show any improvement of solubility at various pH values. To overcome this limitation, we developed a co-solvent-based formulation that increased the solubility of the compound. A preclinical formulation was subsequently developed that allowed us to perform PK studies in mice, rats (IV, PO & IP) and dogs (IV) and to conduct efficacy studies with GBO-006-1 as a single agent (10, 30 and 75 mg/kg) and in combination using nude mouse xenograft models. In vitro safety pharmacology was assessed by performing hERG (patch clamp), and genotoxic potential was evaluated using the Ames test. Summary: The solubility of GBO-006-1 was enhanced using a co-solvent approach, which permitted the determination of ADME properties. The solution formulation provided good exposure and bioavailability upon IP administration (41% bioavailability at 3mg/Kg) and dose dependency up to 100 mg/kg. The compound showed moderate to high in vivo clearance in rat and mouse, respectively, and has high plasma protein binding and volume of distribution, with no CYP or hERG inhibition. Exploratory non-GLP toxicity studies are currently ongoing in three species, along with other safety pharmacological studies and ex vivo surrogate biomarker analysis. Conclusion: We were successful in developing a formulation forGBO-006-1, which allowed for parenteral administration. GBO-006-1 showed linear dose escalation in mice that translated into marked efficacy in a triple negative breast cancer xenograft model. Initial safety profiling suggested no hERG inhibition. IND directed studies are currently being conducted to facilitate Phase 1 clinical trials in the near future. Citation Format: ARNAB ROYCHOWDHURY, ATHISAYAMANI JEYARAJ DURAISWAMY, SRINIVASARAO MADDI, CHANDRA DEB, SAYAN MITRA, RAMANA REDDY, MANOJ MANIAR, SHASHIDHAR JATIANI, STEPHEN C. COSENZA, AMOL PADGAONKAR, PREMKUMAR REDDY. Pharmacokinetics of PLK2 inhibitor GBO-006-1, developed as a novel first-in-class molecule to treat triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4537. doi:10.1158/1538-7445.AM2014-4537