ALBERT

Description: Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non–caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Description: Skeletal related events impact both quality of life and overall survival of multiple myeloma (MM) patients. The underlying cause of osteolytic bone disease is an imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts. Bone cells are regulated by a complex milieu of bone marrow microenvironmental components including the immune system via the effect of cytokines, signaling molecules and regulatory proteins. For example, osteoclastogenesis is tightly regulated by T cells through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, the effect of osteoclasts (OCs) on the immune system is less well defined. Here we investigated the effect of osteoclasts on immune cells including T cells, Th17 cells, NK cells and myeloid-derived suppressor cells (MDSCs) in MM. To gain insight into the functional impact of OCs on the immune cells, each type of immune cell was isolated from peripheral blood mononuclear cells (PBMNCs) and examined further in co-cultures with OCs. Our preliminary data showed that the frequency of NK cells and MDSCs in PBMNCs derived from myeloma patients increased when co-cultured with autologous OCs. Activation of the inhibitory immune checkpoints suppresses T cell-mediated antitumor immunity. Up-regulation of several co-inhibitory molecules has also been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, LAG-3 and B7-H3, on T cells in co-culture with or without OCs by multi-color flow cytometry. OC co-culture significantly increased PD-1 expression in both CD4+and CD8+ T cell populations. The presence of OCs also enhanced PD-L1 protein expression on MM cells. Th17 cells, a newsubset of helper T cells, have recently been identified as immunosuppressive cells. Th17 cells are increased in myeloma; morevover they secrete IL-17 which promotes myeloma cell growth (Prabhala RH et al., Blood 2010). Interestingly, we found that naïve CD4 T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Furthermore, we also observed an increased expansion of Th17 cells in co-culture with OCs. NK cell cytotoxic function is severely impaired in myeloma (Fauriat C et al., Leukemia. 2006). In our experiments, NK cells co-cultured in the presence of OCs demonstrated significant decrease in NK cell cytotoxicity, despite increase in NK cell numbers. Our study demonstrated that OCs suppress the cytotoxic function of T cells and NK cells and induce the expansion of immunosuppressive Th17 cells and MDSCs through direct contact. These findings indicate that OCs play an important role in tumor progression in part by enhancing immune suppression. Targeting OCs represent an attractive therapeutic option not only for controlling osteolytic bone disease but also for restoring the impaired immune surveillance in MM. Ongoing studies will focus on understanding direct-contact-mediated interactions between OCs and immune cells. Furthermore, the effect of OC inhibition using anti-resorptive drugs on the immune system will be interrrogated. Disclosures Raje: Medscape: Honoraria; Research to Practice: Honoraria; AstraZeneca: Research Funding; Takeda: Consultancy; Merck: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Amgen Inc.: Consultancy.

Description: Disruption of the intrinsic apoptotic pathway by the aberrant expression of the BCL2 family members are frequent events in multiple myeloma (MM). The anti-apoptotic protein myeloid cell leukemia-1 (MCL1) is highly expressed in MM and plays a crucial role in disease progression. Inhibition of MCL1, thus, represents a unique therapeutic opportunity for the control of the disease. Currently, there is no FDA-approved drug with the ability to selectively target MCL1. Because of its pivotal role in MM, MCL1 is considered a high-value therapeutic target in the clinic. In this report, we use a selective small-molecule inhibitor of MCL1, AZD5991, to examine the therapeutic consequences of MCL1 inhibition in MM. AZD5991 treatment resulted in dose-dependent cytotoxicity with EC50 values ranging from 64 and 417 nM at 24 hours for MCL1-sensitive cell lines (H929, MM.1S, RPMI-8226, U266, LP-1 and ANBL6VR). Two cell lines DOX40 and KMS-12-PE remained resistant to MCL1 inhibition. For the MCL1-sensitive MM.1S and H929 cells, the decrease in cell viability upon MCL1 inhibition was due to an increase in apoptosis. AZD5991 treatment led to 40-82% decrease in viability of CD138+ cells isolated from relapsed and refractory MM patients at a dose of 300 nM at 24 hours. These results indicate that AZD5991 has promising single-agent activity, but it would be prudent to study it in combination with other anti-MM therapies. The BM microenvironment enhances tumor cell growth and survival in MM. We found that soluble factors produced during the MM-BMSC interaction reduced the sensitivity of MM cells to AZD5991, and direct MM-BMSC contact blunted the cytotoxic effect of AZD5991. A comprehensive cytokine array analysis revealed an enrichment of a panel of pro-survival cytokines and growth factors, with the cytokines IL-6, IL-8 and GROα/β/γ being among the most highly up-regulated proteins, upon cell-cell contact between MM.1S cells and BMSCs. Enrichment of these cytokines in the BM milieu, at least in part, confer a protective effect on MM cells and endow them with the ability to resist MCL1 inhibition. A shift in the balance of BCL2 family members is often the primary reason for drug resistance. We found that the baseline BCL2 mRNA expression and the BCL2:MCL1 ratios in the MM cell lines examined are negatively correlated to their corresponding sensitivity to AZD5991. In other words, MM cells with a high BCL2 reservoir are more likely to circumvent cell death elicited by MCL1 inhibition. Treatment with AZD5991 alone leads to release of Bim from MCL1 and an increased Bim bound to BCL2. Venetoclax, a BH3 mimetic that selectively binds BCL2, treatment alone releases Bim from BCL2 and results in an increased Bim bound to MCL1. Bim binding to MCL1 and BCL2 was significantly diminished upon co-treatment. The free-floating Bim proteins subsequently activate the intrinsic apoptotic pathway by facilitating cytochrome c release. These results suggested that MM cells switch their survival dependency to BCL2 upon MCL1 inhibition, and that BCL2 blockade could be an effective way to overcome MCL1 resistance in MM. Based on these observations, we combined AZD5991 with Venetoclax in MM cells. A significant decrease in cell viability was observed with the combined therapy compared with both drugs used alone. Isobologram analysis confirmed greater than additive or synergistic effect upon co-treatment. The enhanced cytotoxic effect of the combined therapy retains even when the MM cells are in co-culture with BMSCs. Remarkably, the pro-survival cytokines IL-6, IL-8 and GROα/β/γ, which were expressed at high levels in the co-culture settings, were among the ones being most reduced after the combined therapy. Our results demonstrated that the combined AZD5991/Venetoclax therapy overcomes the inherent MCL1-resistance in MM via two independent mechanisms. First, the concomitant suppression of the anti-apoptotic proteins MCL1 and BCL2 prevent MM cells from escaping apoptosis by releasing Bim to trigger mitochondrial outer membrane potential and the subsequent release of cytochrome c to activate the intrinsic apoptotic pathway. Second, this combined therapy prevents MM cell growth by causing down-regulation of pro-survival cytokines and growth factors in the BM microenvironment. As a proof of concept, our data indicate combining therapeutics that selectively target the anti-apoptotic proteins MCL1 and BCL2 could be an effective therapy for MM patients. Disclosures Cidado: AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment.

Description: Mounting evidence suggests positive correlation of obesity and bone marrow (BM) adipogenesis with multiple myeloma (MM) initiation and progression. MM is more prevalent in patients with metabolic disorders including obesity and Gaucher's disease. Adipocytes constitute 70% of BM cellular volume in human adults making them one of the prominent players in MM progression. However, the underlying mechanisms involved remain unknown. Fat depots are increasingly recognized as the key endocrine organs for their ability to secrete various hormones (e.g. leptin, adiponectin, resistin), lipid substrates (polyunsaturated fatty acids), and cytokines (e.g. IL-6, IL-1, and TNFα). For example, leptin levels are increased in newly diagnosed MM patients that allow MM initiation by inhibiting NK T-cells (Favreaue 2017, Leukemia). Lipid substrates are also deregulated in the serum of cancer patients, including MM patients. These fatty acids are important initiators of many key downstream drug-targetable signaling pathways such as cyclooxygenase, cytochrome P450, and lipoxygenase pathways. In this study, we hypothesized that the changes in lipid profiles in MM patients contribute to disease progression. PPARγ is the key transcription factor that potentiates adipocyte differentiation and its key functions including lipid synthesis and transportation. We found that the BM adipose tissue (BMAT) fraction from MGUS, and SMM patients showed significantly increased PPARγ expression compared to the healthy donors. In vitro, MGUS and SMM BM stromal cells (BMSCs) showed increased adipogenic differentiation potential compared to the healthy donors. In-vitro, the proliferation of MM.1S human MM cells was significantly increased when co-cultured with BMSCs from MGUS/SMM patients compared to healthy donors. Taken together these data demonstrate a vicious cycle in which early stage MM cells and BM adipocytes support each other's growth. Multicomponent gene expression analysis of the BMAT from MGUS and MM patients showed altered genes involved mostly in fatty acid synthesis and metabolism. Lipidomics analysis revealed significantly decreased Arachidonic Acid (AA) levels in the BMSCs of SMM, the BM supernatant (BMS) of NDMM patients, and the blood serum of MGUS and SMM patients. This led us to hypothesize that increasing AA levels around MM cells may decrease MM progression. Physiological-range AA treatment of human MM cell lines, MM1S, H929, and U266, dose-dependently decreased proliferation and viability after 72-hours in all three cell lines. For in vivo studies, we generated humanized MM tumor growth in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with either 100µg/g or 500µg/g of AA daily along with vehicle control via subcutaneous injection for 3-weeks near the tumor mass. Tumor volume continued to increase in vehicle control and 100 µg/g AA treatment groups but was significantly decreased in 500µg/g AA treatment group beginning 10-days after starting treatment. Gross examination of the tumor mass showed dramatically increased tumor necrosis. Flow cytometry analysis of CD138+ myeloma cells from non-necrotic part of the tumors showed significantly increased number of dead cells and the cells undergoing apoptosis in 500µg/g AA treatment group. AA is metabolized to primarily activate cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) signaling pathways. Recent studies also show that AA induces ferroptosis, a programmed cell death in response to accumulation of lipid peroxides and mediated by iron (Kagan et al., 2017). To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used various inhibitors of each of these signaling pathways including ibuprofen, baicalein, BW B70C, 1-aminobenzotriazole, and ferrostatin. Among these compounds, ferrostatin completely rescued AA induced apoptosis in the human MM cells. Taken together, here we show that the adipogenesis is involved in myeloma pathogenesisis. Furthermore, AA signaling induces ferroptosis-mediated cell death in MM cells. Therefore, therapeutically targeting members of this signaling pathway is a potential novel treatment strategy for MM, especially in the MGUS and SMM stages. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 571 Despite advances in the treatment of multiple myeloma (MM), this disease remains incurable and novel therapeutic strategies are urgently needed. Ideal strategies would overcome resistance factors from the bone-marrow microenvironment (niche) since a variety of inhibitors are rendered less effective by bone-marrow stromal cells (BMSCs) of the MM niche (McMillin et al., Nat Med. 2010 Apr;16(4):483–9). Drug discovery often entails a target-based approach but identifying targets in MM is challenging because of its complex genome and multiple niche interactions. We used a chemical biology approach in which small-molecule inhibitors of MM cells, grown within their niche, are first identified and then used to discover targets within MM or its niche. These compounds also serve as leads for future drug discovery. To model myeloma/niche interactions, we chose an MM cell line MOLP5 that has an obligate dependence on BMSCs to maintain viability. Small-molecule inhibitors were identified by screening ∼25,000 structurally diverse small molecules on GFP-labeled MOLP5 cells co-cultured with primary BMSCs derived from hip replacement samples. MOLP5 growth inhibition was measured by quantifying GFP(+) cells with automated high-throughput microscopy. About 800 hits were counter-screened on BMSCs alone to exclude non-specifically toxic compounds. The remaining 182 MOLP5-selective inhibitors were then tested on 2 other GFP-labeled MM cell-lines, MM1S and INA6, in the presence or absence of BMSCs to exclude compounds that are less effective in the presence of BMSCs. The 64 compounds that overcome BMSC resistance were tested on CD34+ human hematopoietic progenitors to prioritize compounds with selectivity between MM and normal blood cells. The 8 compounds that met these criteria fell into 3 categories: 1) compounds with equal activity in the presence or absence of BMSCs (overcome stromal resistance); 2) compounds with selectivity for BMSC-dependent MOLP5 cells (block stromal viability factors); and 3) compounds with increased activity in the presence of BMSCs (enhance stromal inhibitory factors). Because most efficacious clinical compounds like bortezomib act like compounds in category 1, compound BRD9876 was chosen from this category for mechanistic studies. Gene-expression profiling of BRD9876-treated MM1S cells suggested possible links to mitotic arrest and cell cycle analyses revealed a rapid accumulation of cells in the G2/M phase. Treated cells were stained for the mitotic spindle protein α-tubulin and found to exhibit an aberrant mono-astral mitotic phenotype, reminiscent of the kinesin-5 (Eg5; KIF11) inhibitor monastrol. This was encouraging because a kinesin-5 inhibitor ARRY-520 has shown promising durable responses in multiple myeloma (Shah et al, ASH Annual Meeting 2011; Abstract 1860). To determine if BRD9876 was a kinesin-5 inhibitor, a BRD9876-resistant sub-line of MM1S was developed and the kinesin-5 gene sequenced. BRD9876-resistant cells have a novel kinesin-5 mutation (Y104C) at a site that is distant from the monastrol-binding pocket. Most kinesin-5 inhibitors in clinical development bind the monastrol pocket, and the BRD9876-resistant cells were not cross-resistant to one such inhibitor, ispinesib, suggesting a distinct mode of kinesin-5 inhibition by BRD9876. To identify biomarkers of sensitivity to BRD9876, quantitative dose/response measurements in 98 genetically characterized cell lines (Schreiber & co-workers, submitted) comprising a subset of the Cancer Cell Line Encyclopedia (CCLE) were analyzed. Unbiased analyses correlating genetic features with sensitivity revealed that mutations in the mitotic regulator WEE1 were associated with sensitivity to BRD9876. Validation studies comparing WEE1 mutant to wild-type cell lines confirmed enhanced sensitivity of mutant cells to both BRD9876 and ispinesib suggesting that WEE1 mutations could be a useful biomarker for different kinesin-5 inhibitors. In contrast, co-treatment of WEE1 WT cells with sub-toxic concentrations of the WEE1 inhibitor MK1775 led to marked enhancement of BRD9876 activity but had little effect on ispinesib activity, suggesting a unique synergistic relationship between WEE1 inhibitors and BRD9876. In summary, niche-based screening in multiple myeloma has revealed a novel therapeutic candidate and can complement other drug-discovery approaches against this disease. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Description: Altered cellular metabolic pathways are the hallmark of tumor cells. Multiple myeloma (MM) is positively correlated with metabolic disorders such as obesity and Gaucher's disease. The local bone marrow (BM) microenvironment (TME) majorly influences the initiation and progression of MM. In a typical MM patient, BM adipocytes make up 70% of the cellular volume. The abundance of adipocyte-secreted free fatty acids (FFA) may shift myeloma cellular metabolism from aerobic glycolysis to more energy-producing fatty acid oxidation. The FFAs are important catalysts of key downstream drug-targetable signaling pathways such as cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) pathways. In this study, we hypothesized that altered lipid profile in the local BM TME contributes to MM progression. BM-Fat enriched tissue isolated from BM aspirates of Monoclonal Gammopathy of Undetermined Significance (MGUS) and smoldering MM (SMM) patients showed a significant increase in adipogenic PPARγ gene expression compared to aged-matched healthy donors (N≥3). The BM mesenchymal stem/progenitor cells (BMSCs) from MGUS/SMM patients expressed normal levels of BMSC markers CD271, CD105, CD44, CD106, CD29, CD90, CD49e, and Notch4 but showed significantly increased expression of adipogenic markers including Preadipocyte factor 1, Leptin Receptor, and Perilipin A (N=6). This also translated into significantly increased adipogenic differentiation of patient BMSCs when cultured alone or with the human MM cell-line MM.1S (N≥3). Furthermore, MM.1S showed significantly increased proliferation when co-cultured with BMSCs from MGUS/SMM patients (N=5). These data demonstrate a vicious cycle where adipogenesis is increased in early precursor MM stages that further support the growth of myeloma cells. We performed gas chromatometry based lipidomics analysis on the supernatant of BM aspirates from MGUS, SMM, and newly diagnosed MM (NDMM) patients. The analysis identified significant decreases in key polyunsaturated fatty acids (PUFA) including Arachidonic Acid (AA) and Docosatetraenoic acid (N≥5). Lipid metabolism specific gene array on RNA from adipose tissue fraction of BM aspirates from MGUS, SMM and NDMM patients showed altered changes in genes responsible for fatty acid synthesis and metabolism. PUFA are involved in anti-inflammatory mechanisms in cancer. We hypothesized that increased levels of certain PUFA, such as AA, in the BM TME may decrease MM progression. To test this hypothesis, we treated MM cells with physiological doses of AA. AA dose-dependently decreased proliferation and viability of human MM cell lines, MM1S, H929, and U266, and CD138+ patient myeloma cells. For in vivo studies, humanized MM tumor model was generated in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with daily localized injections of vehicle, 100µg/g AA, 500µg/g of AA, or IV with 2mg/kg/biweekly Carfilzomib (CFZ), or CFZ with 500µg/g of AA (COMBO). Tumor volume significantly decreased in 500µg/g AA treatment group beginning 10-days and was comparable to the CFZ treatment. Gross examination and flow cytometry analysis of CD138+ myeloma cells showed dramatically increased tumor-cell apoptosis in 500µg/g AA and COMBO treatment groups. To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used specific inhibitors of each of these signaling pathways including ibuprofen (Cox inhibitor), baicalein (12-LOX inhibitor), BW B70C (5,15-LOX inhibitor), 1-aminobenzotriazole (CYP450 inhibitor), and ferrostatin (Ferroptosis/lipid peroxidation inhibitor). Among these compounds, ferrostatin treatment completely rescued AA induced apoptosis in the human MM.1S cells. Ferroptotic cell death is the result of an accumulation of lipid peroxides which is generally prevented by the enzyme Glutathione peroxidase 4 (GPX4). We, therefore investigated the role of AA on GPX4 and found that all MM cell lines partially or completely lost the expression of GPX4 when exposed to AA and that this effect was completely prevented when cotreated with Ferrostatin. Taken together, we show that BM adipocytes promote myeloma cell proliferation at least in part through secreted FFAs. Therapeutically targeting members of this signaling pathway, such as ferroptosis, is a potential novel treatment strategy for MM especially in the MGUS and SMM stages. Disclosures Raje: Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy.