Vijay P. Kale

The Apoptotic Mechanism of Action of the Sphingosine Kinase 1 Selective Inhibitor SKI-178 in Human Acute Myeloid Leukemia Cell Lines

We previously developed SKI-178 (N′-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178–induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178–induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178–induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

The regulatory roles of ROCK and MRCK kinases in the plasticity of cancer cell migration

Metastatic cancer cells show great plasticity in their migratory mechanisms. In this review we briefly describe the signal transduction pathways associated with the ROCK and MRCK kinases and their roles in cancer cell migration and in its plasticity. With respect to therapeutic strategies targeting metastatic cancers, selectively blocking a single target, such as ROCK or MRCK, can induce alternate modes of cancer cell migration (i.e. plasticity) making the treatment ineffective. To address the problem of plasticity, we will discuss the strategy of simultaneous targeting of both ROCK and MRCK as an effective anti-metastatic therapeutics.

A novel selective multikinase inhibitor of ROCK and MRCK effectively blocks cancer cell migration and invasion

Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases. DJ4 potently inhibited activities of ROCK and MRCK in an ATP competitive manner. In cellular functional assays, DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. Our results strongly indicate that DJ4 may be further developed as a novel anti-metastatic chemotherapeutic agent for multiple cancers.

Targeting CXCL12/CXCR4 Axis in Multiple Myeloma

Multiple Myeloma (MM) is malignant haematological disease characterized by uncontrolled proliferation of monoclonal plasma cells (PC) in bone marrow (BM). The receptor, CXCR4 is widely expressed on hematopoietic cells including MM cells and respond to CXCL12 gradients for mobilization through blood stream and bone marrow. CXCL12 plays an important role in recruitment of MM cells to bone marrow microenvironment and formation of neoangiogenic niches supporting tumor growth, survival and metastasis. The integral role of this chemokine-receptor axis in development of MM makes it a desirable target for therapeutics. In this review, we outline the background on MM and role of specific chemokine CXCL12 in the disease with an attempt to highlight the targeted therapeutics for its signaling receptor CXCR4 in MM.

Abstract 4805: In vitro characterization of novel inhibitors of ROCK and MRCK kinases as anticancer agents

Metastatic cancers are the second leading cause of deaths in the USA. RhoA and Cdc42 play critical roles in the regulation of plasticity of cancer cell migration/invasion and cell proliferation. ROCK1/2 and MRCKa/â are downstream kinases in the signaling pathways associated with cancer cell migration and invasion. Hence, we hypothesized that simultaneous targeting of these two kinase families would be an effective therapeutic strategy to block migration, invasion, and growth of metastatic cancers. We have identified DJ4 as a novel inhibitor of ROCK and MRCK kinases. In the cellular functional assays, DJ4 treatment significantly blocked stress fiber formation, and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. To study the critical functional groups required for its activity, we have modified the chemical structure of DJ4 at various functional groups and synthesized several analogs of DJ4 to perform the structural activity relationship (SAR) study for their ROCK1 inhibition. The effectiveness of these compounds were further investigated using National Cancer Institute9s drug screening program in 60 human cancer cell lines representing nine different cancer types. These compounds effectively inhibit migration and invasion of multiple cancer cell types. Selected analogs were tested for their anti migration, pro-apoptotic, and anti-proliferative effects in breast cancer cells. Our studies strongly indicate that DJ4 and its analog, DJ110, are potent inhibitors of ROCK1, ROCK2, MERKa and MRCKâ. The results of our finding will be discussed. Citation Format: Dhimant H. Desai, Vijay P. Kale, Jeremy A. Hengst, Taryn E. Dick, Ashley L. Colledge, Shantu G. Amin, Jong K. Yun. In vitro characterization of novel inhibitors of ROCK and MRCK kinases as anticancer agents. [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 4805.

Abstract 2933: The apoptotic mechanism of action of SKI-178, a novel Sphingosine kinase 1 selective inhibitor

We previously developed SKI-178 as a novel Sphingosine kinase 1 (SphK1) selective inhibitor that is cytotoxic toward a broad panel of cancer types. Herein, we sought to determine the mechanism-of-action of SKI-178-induced cell death. We present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism-of-action (MOA) of SKI-178 implicated c-Jun NH 2 -terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the pro-survival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multi-drug resistance mediated by MDR-1 and/or pro-survival Bcl-2 family member over-expression did not affect the sensitivity of AML cells to SKI-178. In addition to AML, we extend the MOA of SKI-178 to include various solid tumor cell lines, including pancreatic cancer and glioblastoma. Taken together, we provide evidence that SKI-178 induces apoptosis in a CDK1-dependent manner and is not a substrate for MDR1, making it a promising chemotherapeutic candidate for the treatment of various cancer types, including those known to be drug resistant. Citation Format: Taryn E. Dick, Jeremy Hengst, Vijay Kale, Ashley Colledge, Jong K. Yun. The apoptotic mechanism of action of SKI-178, a novel Sphingosine kinase 1 selective inhibitor. [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 2933. doi:10.1158/1538-7445.AM2015-2933

Abstract 4467: Targeting of chemokine receptor CXCR4 inhibits invasion and sensitizes melanoma cells to chemotherapeutic agents

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Malignant melanoma is highly metastatic and resistant to conventional therapies. Although numerous clinical approaches for treating melanoma have been adopted, none led to a remarkable therapeutic effect. Recently new drugs have been developed for treatment of metastatic melanoma, and hold great promise; however complete effects are still distant dream because of resistance. We have learnt that the most aggressive melanomas are generally resistant to strategies which target one signaling pathway. Thus an agent targeting multiple signaling pathways would maximize therapeutic outcome. We have identified one such agent named as gambogic acid (GA) that is natural and derived from medicinal plant Garcinia hanburyi (also called mangosteen or kokum). Moreover this agent has a potential to target several pathways responsible for survival and chemoresistance of several cancers including melanoma. Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been associated with inferior survival, poor prognosis, chemoresistance and metastasis of malignant melanoma. We, therefore, investigated whether GA could inhibit CXCR4 signaling and suppress invasion of malignant melanoma cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on melanoma cells in a dose- and time-dependent manner. Suppression of CXCR4 expression by GA correlated with both inhibition of SDF-1α-induced invasion of melanoma cells. GA also inhibited expression of MMP-9, VEGF, c-Myc and phosphorylation of Erk1/2 in melanoma cells. Importantly, GA markedly enhances the efficacy of PLX-4032 and dacarbazine against melanoma cells. Citation Format: Manoj K. Pandey, Deepkamal Karelia, Vijay P. Kale, Krishne Gowda, Arun K. Sharma, Rogerio I. Neves, Shantu G. Amin. Targeting of chemokine receptor CXCR4 inhibits invasion and sensitizes melanoma cells to chemotherapeutic agents. [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 4467. doi:10.1158/1538-7445.AM2015-4467

Abstract 3786: Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation, as indicated by down-regulation of CXCR4 mRNA expression, inhibition of nuclear factor-kappa B (NF-κB) activity, and suppression of p65 binding at CXCR4 promoter analyzed by quantitative chromatin immunoprecipitation (qChIP) assay. Suppression of CXCR4 expression by GA correlated with both inhibition of SDF-1α-induced invasion of MM cells. GA also inhibited phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA suppressed the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. Finally, we found that MM cells, induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and thus has a strong potential to suppress osteoclastogenesis mediated by MM cells. Citation Format: Manoj K. Pandey, Vijay P. Kale, Chunhua Song, Shen-shu Sung, Arun K. Sharma, Sinisa Dovat, Shantu G. Amin. Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma. [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 3786. doi:10.1158/1538-7445.AM2014-3786