Ramakrishnan, Vijay

Abstract Multiple myeloma (MM) is an incurable malignancy of plasma cells. It is the second most common hematologic cancer, affecting nearly 30,000 people in the United States annually. Substantial progress has been made in the past fifteen years in the treatment of MM due to the approval of several new classes of drugs. However, patients inevitably relapse and become refractory to existing therapies. Hence, there is an immediate unmet need to develop novel therapies for MM based on a better understanding of the disease biology. Mutations in RAS have been found to occur in about 40% of newly diagnosed MM patients, with the frequency increasing to around 70% in relapsed/refractory patients. Such mutations are absent in patients with the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Clearly, RAS mutations contribute to both disease progression and relapse. However, targeting the MEK/ERK pathway has been unsuccessful in MM patients to date. Given the high frequency of RAS mutations in MM, we hypothesized that targeting this pathway could still be a promising strategy when combined with existing agents that have multifaceted mechanisms to promote tumor cell death, such as the recently approved histone deacetylase (HDAC) inhibitor LBH589 (panobinostat). Our results clearly demonstrate that low doses of LBH589 in combination with the MEK inhibitor AZD6244 induce BIM-dependent synergistic cell death in several MM cell lines and patient cells. Our studies also suggest that mutations in RAS/RAF could serve as a predictive biomarker for sensitivity to AZD6244/LBH589. RAS/RAF mutations appear to confer Mcl-1 dependence in MM cells, in part by driving up the phosphorylation of Mcl-1. The AZD6244/LBH589 combination is able to decrease the phosphorylation of Mcl-1 at several sites, which dissociates BIM-Mcl-1 complexes, ultimately leading to activation of the intrinsic apoptosis pathway. Additionally, we identified that wild-type RAS/RAF cells have relatively lower levels of phospho-Mcl-1, as well as higher levels of Bcl-2 and phospho-Bcl-2 when compared to mutated RAS/RAF cells. This seems to confer functional Bcl-2 dependence. Consequently, we found that wild-type RAS/RAF cells are sensitive to the BH3-mimetic ABT199 (venetoclax) when combined with LBH589. Through ongoing experiments, we hope to further confirm the mechanism of action of both these combinations, identify the particular HDAC that is required to be inhibited for the observed synergy, and validate RAS/RAF mutational status as a biomarker for predicting sensitivity to either combination. Our findings have broad therapeutic potential given the prevalence of RAS mutations in MM. Moreover, the ABT199/LBH589 combination could emerge as a targeted therapy for wild-type RAS patients, perhaps broadening the scope and capacity of Bcl-2 inhibition in MM. Citation Format: Kevin C. Miller, Jessica Haug, Teresa Kimlinger, Sanjay Kumar, Wilson Gonsalves, S. Vincent Rajkumar, Shaji K. Kumar, Vijay Ramakrishnan. HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3909.

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13 C stable isotope resolved metabolomics (SIRM) using U[ 13 C 6 ]Glucose and U[ 13 C 5 ]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13 C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Abstract Introduction: Relapsed and refractory multiple myeloma (MM) remains a significant clinical challenge and drugs with new mechanisms of action to overcome resistance are needed. Constitutive activation of the PI3K/Akt/mTOR pathway in MM promotes tumorigenesis through propagation of the cell cycle, protein synthesis, and inhibition of apoptosis. Anti-MM effects of rapalogs are limited due to feedback activation of the PI3K/Akt signaling pathway. Agents capable of targeting PI3K and mTORC1 or both mTORC1/C2 are able to partially overcome such resistance mechanisms. However, the mTORC2 mediated increase in pAkt (Ser473) after PI3K/mTORC1 inhibition and the activation of PI3K after mTORC1/C2 inhibition could still contribute to resistance to such agents. We sought to investigate the effects of SPR965 (synthesized and provided by Sphaera Pharma, Singapore under an MTA), an orally bioavailable novel small molecule with inhibition of class 1 PI3 kinase and mTORC1/C2 kinases on MM cell lines and patient cells. Results: SPR965 induced cytotoxicity and inhibited proliferation in all MM cell lines examined with IC50 values between 25-500nM. To understand if SPR965 induced apoptotic cell death, annexin/PI staining followed by flow cytometric assays were performed, which showed a clear increase in cells undergoing apoptosis. Western blots showed increase in caspase-3, -9, and PARP cleavage confirming apoptotic induction by SPR965. Importantly, SPR965 caused potent increase in apoptosis in cytogenetically distinct MM patient cells. Next, we examined the mechanism of action of SPR965. SPR965 caused potent mTORC1 inhibition evidenced by decreased p4E-BP1, p-p70S6K, and pS6 at doses as low as 25nM. SPR965 was able to inhibit PI3K activity as shown by decreased pPDK1 and pBTK at slightly higher concentrations of 75nM. Phosphorylation of Akt S473, a TORC2 substrate, increased at initial low concentrations of SPR965, but was attenuated at concentrations of 100 nM and above, demonstrating dose-dependent inhibition of mTORC2. Such effects were also observed when we performed western blots on MM patient derived primary plasma cells. Increased levels of p27 (KIP1) were observed suggestive of G1 growth arrest, which was confirmed on cell cycle analysis. Since SPR965 caused an increase in pAkt (both T308 and S473) at doses up to 75nM, we examined if SPR965 was able to synergize with an Akt inhibitor MK2206 at doses lower than 75nM. Our results showed potent synergy suggesting that the pAkt up regulation contributes to partial resistance, which is inhibited by SPR965 at doses of 100nM and higher, doses that are still clinically achievable. Conclusion: Our findings demonstrate SPR965 induces apoptosis of MM cells through the inhibition of PI3K and mTORC1/C2 activity. Further studies are underway to better characterize the mechanism of action of SPR965, all of which will be informative for the drug to be used as a single agent or in combination with other agents in relapsed MM. Citation Format: Jeremy T. Larsen, Vijay Ramakrishnan, Jessica Haug, Teresa Kimlinger, Somdutta Sen, Dinesh Mahajan, Sundeep Dugar, S. Vincent Rajkumar, Shaji K. Kumar. Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma. [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 2653. doi:10.1158/1538-7445.AM2015-2653

Abstract Background: Therapy with proteasome inhibitors (PIs) is an effective strategy against multiple myeloma (MM), but a significant proportion of patients is primarily resistant or eventually becomes refractory. Paradoxical activation of phospho-Akt, induction of HSP27 and excessive proteasome activity in relation to polyubiquitinated protein burden are all mechanisms for bortezomib resistance in MM cells in vitro. Preclinical data showed investigational drug MLN9708, a specific, reversible, orally bioavailable, small molecule PI, to be active in MM cell lines, patient cells and mouse models and is currently under clinical investigation. We compared the signaling pathways induced by MLN2238, the biologically active form of MLN9708, and bortezomib to outline differences that could be employed to design rational combinations to overcome resistance. Results: We performed time course experiments in MM.1S cells with EC50 doses of MLN2238 and bortezomib and analyzed signaling pathways via western blotting. Compared to bortezomib, MLN2238 induced earlier cleavage of caspase 8, 9, 3 and PARP and a significant downregulation of XIAP starting at 12 hours. Early and persistent phosphorylation of Bad, known to protect from mitochondrial apoptosis, was observed in bortezomib-, but not MLN2238-treated cells, in which phospho-Bad was reduced at 24 hours. Similarly, MLN2238 decreased phosphorylated Akt and mTOR at 24 hours, while bortezomib-treated cells only showed subtle changes. Interestingly, both drugs caused downregulation of the mTOR downstream targets phospho-p70 S6 kinase and phospho-4E-BP1, albeit of different intensity. Induction of heat shock proteins was comparable with both PI, opening the possibilities for combination therapy of MLN2238 with HSP90 inhibitors. Importantly, the inhibitory effect on phosphorylation of Akt, mTOR and Bad persisted when MM.1S cells were treated with MLN2238 in the presence of bone marrow stromal cells (BMSC). Conclusion: We observed differences in several crucial signaling pathways upon treatment of MM.1S cells with bortezomib or MLN2238. In particular, treatment with the latter inhibited phosphorylation of Bad, Akt and mTOR, suggesting a broader pro-apoptotic and anti-proliferative profile, compared to bortezomib. MLN2238 retained the capability of targeting these substrates also in the presence of BMSC. Confirmatory data on patient-derived cells are under current investigation in our lab. We believe that these data help understanding the differences in PI pharmacodynamics and bortezomib escape. This will allow us to design new drug combinations in the future that overcome clinical resistance to PIs and improve MM patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5669. doi:1538-7445.AM2012-5669

Abstract Background: Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. The PI3K/Akt/mTOR pathway is up-regulated in MM and appears to be critical for cell proliferation and blocking this pathway might have profound impact on disease progression. In addition, this pathway also has been found to cross talk with other signal transduction pathways preventing drug-induced apoptosis. Methods: MK2206 was obtained from Merck Inc. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Results: MK2206 treatment led to dose and time dependent decrease in cell viability as observed by MTT assay with an IC50 range of 1-5µM on most MM cell lines tested. Most of the cytotoxicity was observed by 48hours with minimal increase in cytotoxicity beyond that point. The drug was able to induce cytotoxicity in myeloma cell lines when cells were cultured with IGF, IL6, or VEGF with minimal increase in IC50 values. MK2206 also inhibited MM cell proliferation as observed by reduced thymidine uptake. This reduction in proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells. When myeloma cell lines were treated with the drug for various time points, percentage of apoptotic cells increased with time of incubation with the drug as observed by flow cytometry and annexin V-FITC/PI staining. There was also a time dependent increase in caspase cleavage observed indicating that MK2206 might elicit its functions by inducing caspase dependent pathways. Further studies are being carried out to understand the importance of caspase dependent pathways for the success of anti-MM effects of MK2206. Also, MK2206 was able to induce apoptosis in patient derived primary cells, though with varying degrees of response observed between patients. Examining signaling pathways by immunoblotting demonstrated clear down regulation of pAkt and Bcl-xl levels post treatment with the drug with minimal decrease in proteins associated with other signaling pathways, like pSTAT3 and pERK. Conclusions: Taken together, our studies demonstrate significant anti-MM activity of MK2206 in-vitro. We are currently testing MK2206 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of MK2206 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1796.