Expression of the GTPase-de®cient, activated mutant asubunit of the heterotrimeric G protein G12 (Ga 12 QL) leads to the neoplastic transformation of ®broblast cell lines. The mitogenic pathway regulated by Ga 12 QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Ga 12 QL has been shown to potentiate the serum-induced phospholipase-A 2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Ga 12 QL. Expression of Ga 12 QL and not Ga 13 QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Ga 12 QL or the stimulation of wild-type Ga 12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Ga 12 QL in NIH3T3 cells. These studies, for the ®rst time, identify the crucial role of COX-2 in Ga 12 -mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Ga 12 -mediated signaling pathways.

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

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Rigosertib (ON 01910.Na) is a member of a broader class of unsaturated sulfone kinase inhibitors capable of inducing profound mitotic spindle abnormalities, abnormal centrosome localization, G2-M cell cycle phase arrest and mitotic catastrophe, culminating in apoptosis. Rigosertib is a Ras mimetic that interferes with phosphoinositide 3-kinase (PI-3K)/Akt, reactive oxygen species and Ras/Raf/polo-like kinase (PLK) signaling pathways. Although broadly cytotoxic against malignant cells, it is remarkably non-toxic for non-neoplastic cells. For this reason, this is a particularly attractive compound to test against neoplastic diseases of the bone marrow such as MDS and acute leukemia. This is a report of an unexpected reduction in monoclonal IgG, during a subject participation in a Phase III, randomized study of rigosertib, in patients with MDS who have either failed to respond, or progressed after receiving hypomethylating agents (ONTIME Trial). A 75-year-old man with CMML-2 had a CBC on day 1 of the trial that demonstrated leukocytosis, with absolute monocytosis, 7% blasts in the peripheral blood, Hgb of 9.4 gm/dl, and platelets of 7 K. He was transfusion dependent for both pRBCs and platelets. His chemistry panel demonstrated a high total protein of 9.9 (NL: 6.0 - 8.2 G/DL) with low albumin at 2.4 (NL: 3.5 - 5.0 G/DL); therefore, an SPEP/IPEP was performed, reporting the presence of monoclonal IgG kappa. Quantitative immunoglobulins showed an elevated IgG of 3594 mg/dl (NL: 596 - 1584 MG/DL). Serum free light chains were remarkable for an elevated Kappa fraction at 38.94 (NL: 0.33 - 1.94 MG/DL). On day 1 of cycle 5 of rigosertib, he was started on pulse decadron for 2 months, after which his disease progressed to AML, and he died shortly thereafter. Neither his bone marrow biopsies, nor his hematological parameters demonstrated a response to treatment with rigosertib. In contrast and interestingly, his total protein, serum kappa light chain load, and total IgG, all were drastically reduced shortly after initiation of rigosertib, as can be seen in the graph below depicting a substantial drop in his kappa light chain as well as the kappa/light chain ratio. Importantly, reduction in the monoclonal protein was noted prior to initiation of pulse decadron. Even though his initial bone marrow biopsy did not note a monoclonal plasma cell population, a subsequent bone marrow reported a low-level involvement with a plasma cell dyscrasia, with kappa light chain restriction. His final bone marrow biopsy confirmed progression to AML, but the previously seen plasma cell dyscrasia was no longer present. Conclusion: We are not aware of prior reports describing a similar effect of rigosertib on M-proteins. However, in vitro studies with rigosertib have demonstrated antitumor effects and induction of apoptosis in myeloma cell lines1. This observation merits further exploration of this agent in multiple myeloma. References: 1. Reddy MV, et al. Discovery of a Clinical Stage Multi-Kinase Inhibitor Sodium (E)-2-{2-Methoxy-5-[(2',4',6'-trimethoxystyrylsulfonyl)methyl]phenylamino}acetate (ON01910.Na): Synthesis, Structure-Activity Relationship, and Biological Activity. J Med Chem, 2011, 54(18):6254-76. Figure 1. Decrease in serum free kappa light chains following initiation of rigosertib. Figure 1. Decrease in serum free kappa light chains following initiation of rigosertib. Figure 2. Decrease in kappa/lambda ratio following initiation of rigosertib. Figure 2. Decrease in kappa/lambda ratio following initiation of rigosertib. Disclosures Shammo: Onconova: Research Funding.

Protein kinase CK2 (Casein kinase 2) is a highly conserved, constitutively active Ser/Thr protein kinase implicated in cellular transformation and tumorigenesis. CK2 regulates multiple oncogenic pathways involved in cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA repair. Unlike other signaling molecules such as PI3K, RAF, RAS, where genetic alterations lead to deregulated signaling pathways, in the case of CK2, only its high expression levels have been associated with a disease state and no mutations have been found to date. Here we describe the mechanism of action of ON108600 a potent small molecule inhibitor of both catalytic subunits of protein kinase CK2. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple cancer cell lines while having little or no effect on normal cells. CK2 regulates Akt signaling through phosphorylation and inactivation of PTEN and via direct and specific phosphorylation of Akt at S129. Treatment with ON108600 resulted in dramatic reductions of phosphorylation of PTEN and Akt S129 and downstream targets of Akt in multiple cancer cell lines. Further, phosphorylation of p21Cip1/Waf1 at Thr145 and its cytoplasmic localization has been shown to be a poor prognostic marker in breast cancer. ON108600 showed a dose and time dependant dephosphorylation of p21 at Thr145 and induced a potent mitotic cell cycle arrest in most cancer cell lines. CK2 has also been implicated in regulating Caspase signaling via phosphorylation of caspase cleavage sites and thus exerting its pro-survival effects. ON108600 treatment induced potent apoptosis by activating the Caspase 3/7 signaling cascade. To understand the structural basis of CK2 inhibition by ON108600 we performed x-ray crystallographic studies of ON108600-CK2. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of GTP in the CK2 active site. Structural studies also revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit which is known to interact with the β-regulatory subunit of CK2 and critical for substrate recognition and activation. CK2α has been reported to be a structural microtubule associated protein and the CK2 holoenzyme has been shown to regulate microtubule dynamics. Interestingly, ON108600 was found to inhibit CK2 holoenzyme mediated polymerization of β-tubulins in in-vitro assays. CK2 has emerged as novel druggable target and selective inhibitors of CK2 such as ON108600 may prove as a potential anti-cancer therapy. Our ongoing studies are focused towards identifying novel combinations of ON108600 with existing chemotherapeutic agents in multiple myeloma, prostate and breast cancer. Citation Format: Amol Padgaonkar, Olga Rechkoblit, Stephen Cosenza, Venkat R. Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, Aneel Aggarwal, E Premkumar Reddy. Discovery and biological characterization of ON108600, a small molecule inhibitor of protein kinase CK2. [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 3239. doi:10.1158/1538-7445.AM2013-3239 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.