Statins have shown promise as anticancer agents in experimental and epidemiologic research. However, any benefit that they provide is likely context-dependent, for example, applicable only to certain cancers or in combination with specific anticancer drugs. We report that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) using statins enhances the proapoptotic activity of the B cell lymphoma-2 (BCL2) inhibitor venetoclax (ABT-199) in primary leukemia and lymphoma cells but not in normal human peripheral blood mononuclear cells. By blocking mevalonate production, HMGCR inhibition suppressed protein geranylgeranylation, resulting in up-regulation of proapoptotic protein p53 up-regulated modulator of apoptosis (PUMA). In support of these findings, dynamic BH3 profiling confirmed that statins primed cells for apoptosis. Furthermore, in retrospective analyses of three clinical studies of chronic lymphocytic leukemia, background statin use was associated with enhanced response to venetoclax, as demonstrated by more frequent complete responses. Together, this work provides mechanistic justification and clinical evidence to warrant prospective clinical investigation of this combination in hematologic malignancies.

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Metastasis of breast cancer to the brain is increasing and is a significant cause of morbidity and mortality for breast cancer patients. In order to understand the molecular mechanisms controlling metastasis to the brain, we have generated two mouse models of breast cancer brain metastasis (4T1.luc2.Br5, Eo771.Br5) via serial selection of these cells through the brains of syngeneic mice following carotid artery injection. In vitro, the 4T1.luc2.Br5 cells exhibit increased proliferation and invasion through basement membrane proteins, while both models demonstrate increased ability to adhere to brain microvascular endothelial cells. Transcriptional profiling followed by Western analysis determined that the p75NTR neurotrophin receptor and its ligand BDNF are highly upregulated in brain-metastatic 4T1.luc2.Br5 cells. The TrkB neurotrophin receptor for BDNF is expressed by these cells as well. Knockdown of p75NTR abolishes the increased invasive capacity of brain-selected 4T1.luc2.Br5 in vitro, confirming a role for p75NTR signaling in invasion. While p75NTR expression in primary breast epithelial cells is reportedly low, we observed that 8 of 12 (67%) human breast cancer brain metastasis specimens express high levels of p75NTR, further suggesting an important role in metastasis to brain. Interestingly, while the brain metastatic Eo771.Br5 cells do not express p75NTR in vitro, both the TrkB receptor and its BDNF ligand are upregulated compared to parentals. Together these data suggest a role for activation of p75NTR/TrkB-BDNF signaling in breast cancer brain metastatic cells, and that targeting this pathway may be a means to prevent or treat breast cancer brain metastasis. Citation Format: Wenhong Chen, Christine N. McMahan, Keith D. Barlow, Dennis Juarez, Linda J. Metheny-Barlow. Identification of p75NTR/TrkB-BDNF axis in breast cancer brain metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A64.

The BCL-2 inhibitor venetoclax has begun to change the landscape of therapy for diverse blood cancers. Combining venetoclax with standard of care agents has led to impressive response rates, and increased depth and durability of response compared to monotherapy. However, there is a continuing need to develop safe and effective combinations in multiple myeloma (MM), where the BELLINI triple combination trial (venetoclax-dexamethasone-bortezomib) was closed to accrual due to adverse events. We reported that HMG-CoA-reductase inhibitors (statins), a safe and widely prescribed class of drugs, enhance the efficacy of venetoclax in human leukemia and lymphoma cell lines and in a retrospective study of patients with chronic lymphocytic leukemia (JS Lee et al., Sci. Transl. Med.2018, 10(445)). In MM, the subset with t11;14 translocation is BCL-2-dependent and sensitive to venetoclax, whereas other MM subtypes are more dependent on MCL-1. Here we show that statins sensitize MM cell lines and primary cells to killing by either venetoclax or the MCL-1 inhibitor S63845. The compound pitavastatin is particularly potent, active in vitro at a concentration (100 - 300 nM) that is clinically achievable. In OPM2 cells, pitavastatin synergizes with venetoclax and increases apoptosis in triple combination with dexamethasone. Pitavastatin also potentiates venetoclax killing in dexamethasone-resistant MM1R cells. In MCL-1-dependent MM cells, statins increase sensitivity to S63845. Notably, statins do not increase killing of normal human lymphocytes by either venetoclax or S63845. Statin responsiveness in MM cell lines correlates with p53-independent upregulation of PUMA, BAX and BAK, and gene editing experiments suggest that all three contribute to the apoptotic mechanism. Statin-induced apoptosis and PUMA expression are rescued by addition of geranylgeranyl-pyrophosphate, and recapitulated by an inhibitor of geranylgeranyl-transferase I. Together these findings establish a mechanistic framework to explain how statins promote apoptosis by BH3 mimetic drugs like venetoclax and S63845. Further, these results highlight the potential of statins to enhance therapeutic efficacy of BCL-2 and MCL-1 inhibitor regimens in MM, with possibly lesser toxicity than other combination strategies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is approved for some indications. I will discuss the use of venetoclax for multiple myeloma, a disease for which venetoclax is not approved.

Multiple Myeloma (MM) is a disease of malignant plasma cells for which if left untreated, patients face a 6 month median survival (Osgood, 1960). New agents and combinations continue to improve MM outcomes, extending the median survival to 5 years; however, patients will ultimately succumb to the disease after exhausting treatment options (Fonseca et al., 2017). A novel class of drugs, BH3 mimetics, specifically inhibit the gatekeepers of apoptosis known as pro-survival BCL2 family members BCL2, MCL1 and BCL-xL (Figure 1A). BCL-2 selective BH3 mimetic, venetoclax (ABT-199), has shown promise in a subset of MM patients; however, combinations are required to improve the depth of response. (Moreau et al., 2017) Our lab is pioneering the novel combination of apoptosis-promoting BH3 mimetics with the cancer-killing effect of statins in blood cancers (Lee et al., 2018). Statins are FDA-approved for their ability to lower cholesterol by blocking the synthesis of a key precursor: mevalonate, a major biosynthetic substrate (Figure 1B). Inhibition of the mevalonate pathway by statins stops the synthesis of intermediates necessary for small GTPase function, providing an attractive avenue for targeting these "undruggable" oncogenes (Berndt, Hamilton, & Sebti, 2011; ten Klooster & Hordijk, 2007). Though this potential has been long recognized, previous work on repurposing statins reported effective concentrations that are well above the plasma levels typically achieved in patients receiving standard doses for hypercholesterolemia (Ahmed, Hayslip, & Leggas, 2013; Björkhem-Bergman, Lindh, & Bergman, 2011; van der Spek et al., 2006). Furthermore, previous studies provided insufficient rationale for mechanism-based combination strategies. We have achieved a breakthrough towards this goal by combining statins with the BCL2-specific BH3 mimetic, venetoclax. Our retrospective analysis of chronic lymphocytic leukemia (CLL) clinical trial data revealed that subjects taking statins at the FDA-approved dose had significantly higher frequency of complete remissions following venetoclax treatment (Lee et al., 2018). Through mechanistic studies we showed that statins increase expression of the pro-apoptotic, BH3-only sensitizer PUMA, thereby synergizing with venetoclax to induce apoptosis in various leukemia and lymphoma cell types. Our current work leverages these findings to treat MM. Retrospective analysis of clinical trials of venetoclax in combination with bortezomib and dexamethasone in the venetoclax-sensitive subset of MM demonstrates a similar trend as in the CLL clinical trials in support of background doses of statins (Roberts et al., 2017). Pre-clinical results of MCL-1 BH3 mimetics in development support their future use in MM (Kotschy et al., 2016). Based on these data, we tested two BH3 mimetics, venetoclax and a pre-clinical MCL-1 inhibitor, S63845. Using a panel of 9 human MM cell lines, the data demonstrate that statins potentiate BH3 mimetic-induced apoptosis as measured by reductions in BH3 mimetic IC50. Sensitivity to the combination was maintained in both MM1R and MM1S, MM lines from the same origin that model glucocorticoid resistance and sensitivity respectively, while venetoclax combination with dexamethasone was lost in MM1R cells (Greenstein et al., 2003; Matulis et al., 2016). Additionally, combination-sensitive cell lines corroborate a p53-independent induction of PUMA expression. These data suggest that BH3 mimetic and statin combination may be useful in the clinical settings of dexamethasone resistance and high risk p53-null (17p deleted) patients. To further guide the appropriate application of BH3 mimetics and statins, we are validating that dynamic BH3 profiling can predict efficacy of statins in combination-sensitive versus non-sensitive MM lines. Moreover, preliminary ex vivo studies on freshly isolated CD138+ MM cells from patient bone-marrow aspirates demonstrate combined killing with BH3 mimetics and statins. Ongoing studies will investigate the dependence on upstream GTPase signaling on survival and PUMA upregulation. Disclosures Brem: Bayer: Membership on an entity's Board of Directors or advisory committees; Genetech: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharamcyclics: Membership on an entity's Board of Directors or advisory committees.