Chimeric antigen receptor (CAR) T cells have shown promising results in patients (pts) with B cell malignancies, yet approximately 60% of pts with diffuse large B cell lymphoma (DLBCL) will relapse. Therefore, future efforts are needed to improve the outcomes of these pts. A total of 18 pts with relapsed/refractory B cell malignancies, DLBCL (n=17) and ALL (n=1), were enrolled on a phase 1b/2 study (NCT02772198) of locally produced CD19 CAR T cells. The median age was 40.5 years (range, 23-70). All pts received a lymphodepleting preparative regimen with cyclophosphamide and fludarabine, followed by intravenous infusion of autologous CD19 CAR T cells with a CD28 costimulatory domain. Dosing of CAR T cells was 1-1.5 million CAR+ cells per kg. Clinical response was determined at 28 days following cell administration. Blood samples obtained prior to the lymphodepleting conditioning and at days 7, 14, 21, 30 and 60 after CAR T administration were collected. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. The manufactured CAR T products (n=9) were also subjected to immunophenotypic analysis. Clinically, 11 of 18 pts (61%) responded to CAR T therapy, 6 (33%) with complete response (CR), and 5 (28%) with partial response (PR). Analysis of manufactured CAR T products (n=9) revealed high CD3+ purity (99%), composed with CD4+ (28%) and CD8+ (72%) T cells. Phenotypic characterization demonstrated marked heterogeneity in the percentages of naïve, central memory, effector memory and effector cells within the CD4+ and CD8+ subsets in the CAR T product. In order to assess the homing potential of CAR T cells, expression of chemokine receptors was evaluated in the product cells. High CXCR3 expression was detected (77% and 96% positive within CD4+ and CD8+ subsets, respectively), indicating high migratory capacity of CAR T cells toward inflamed tissues with high levels of CXCL9 and CXCL10 ligands. Furthermore, co-expression of CXCR4 (56% and 54% positive within CD4+ and CD8+ cells) suggests increased homing ability of the manufactured CAR T toward CXCL12-rich bone marrow microenvironment and lymph nodes. Interestingly, higher CCR7 expression (32% vs 8.5%) and lower CCR6 levels (15% vs 28%) were detected on CD8+ CAR T cells from responding pts who achieved CR and PR (n=6) in comparison to non-responders (n=3), suggesting that less differentiated phenotype together with increased trafficking of CAR T to lymphoid tissue corresponds with improved clinical outcome. Additionally, we assessed the immunoregulatory and senescent/exhausted phenotype in CAR T products. Low percentage of CD4+CD25+CD127- Treg cells (13.5%) was detected, with no correlation to clinical response. However, significantly higher frequency of exhausted CD57+CD39+CD28- cytotoxic CD8+ cells stand out as signature population in CAR T products of non-responders in comparison to CR pts (37% vs 9.5%, p<0.02). It is known that immunosuppressive environment affects CAR T cell activation, dampening anti-tumor responses. Therefore, we next evaluated the frequency and kinetics of regulatory T cells and myeloid suppressor cells in the peripheral blood of the CAR T treated pts. Notably, responding and non-responding pts presented distinct Treg patterns. Pts achieving CR demonstrated modest and delayed increase in Treg cells, reaching maximal frequency of 23% Treg out of CD4+ cells at day 21 post CAR T infusion, declining to basal low levels (12.5%) at day 30. In contrast, non-responders possessed rapidly increasing percentage of Treg cells (35%) at day 14 post-infusion. In line with this finding, notable increase in proportion of immunosuppressive CD11b+CD14+ myeloid cells expressing CD163, CD206 and MERTK M2 markers was detected in blood of non-responders, while pts achieving CR experienced transient increase in myeloid suppressor cells at day 7 that went back to normal levels at day 14. Overall, these results elucidate in part the mechanisms of CAR T traffic, immunosuppressive responses as well as induction of T cell senescence/exhaustion that most probably downregulate CAR T effectiveness as observed in non-responding pts. It is conceivable that deeper understanding of these processes will help not just establishing surrogate markers predicting clinical responses but may lead to new strategies for restoration of CAR T activation, thereby improving their efficacy and patient's prognosis. Disclosures No relevant conflicts of interest to declare.

ABSTRACT

Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Acquired or de novo resistance to current anti-MM therapy remains a major treatment obstacle. Novel new therapies are thus in need. Recent data have highlighted the contribution of Ca2+channels in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. As no data is available evaluating TRPV1 in MM, the aim of the current study was to evaluate its possible role in MM. Results: Elevated levels of TRPV1 transcript was detected in MM cell lines (n=8) and BM aspirates from MM patients (n=24) in comparison to normal BM (n=5). AMG9810 a specific antagonist of TRPV1, significantly reduced the viability of MM cell lines (n=8) and primary CD138+ cells (n=6),in a time- and dose-dependent manner (p&lt;0.01) and induced apoptosis manifested by phosphatidylserine externalization, loss of mitochondrial membrane potential (ψm), caspase 3 cleavage and DNA fragmentation. AMG9810-triggered apoptosis could be partially blocked by inhibition of calpains and cathepsins, indicating the role of lysosomal rapture in AMG9810-mediated cell death. Indeed, treatment with TRPV1 antagonist induced rapid lysosomal acidification and increased the number of acidic vesicles (detected by acridine orange stain). The acidic vesicles appeared as early as 1 hour post exposure to AMG9810 preceding the mitochondrial destabilization and apoptosis, thus suggesting that TRPV1 blockade induces lysosomal-induced cell death in MM. Furthermore, TRPV1 inhibition with AMG9810 completely suppressed the pro-survival AKT/mTOR pathway and significantly reduced the levels of anti-apoptotic factors BCL-2 and BCL-XL. Combining AMG9810 with the proteasome inhibitor bortezomib (Bort) induced synergistic cell death in both native and Bort-resistant cells (CI&lt;0.4). Moreover, TRPV1 inhibition successfully overcame the CXCR4-mediated protection from Bort provided by BM stromal cells. This finding suggests that the TRPV1 channel may regulate the activity of CXCR4 chemokine receptor in MM cells affecting the MM-microenvironment interactions. In accordance, the TRPV1 antagonist AMG8910 prevented the responsiveness of CXCR4-expressing MM cells to CXCL12 stimulation, decreased the phosphorylation of signaling mediators like Erk1/2 and AKT and suppressed cell migration, while TRPV1 activator capsaicin promoted the CXCR4-mediated signaling and migration. Gene and protein expression analysis were next performed to delineate the molecular mechanisms underlying the observed synergism between Bort and AMG9810. Bort treatment resulted in robust induction of endoplasmic reticulum (ER) stress genes including the increase in pro-apoptotic factors ATF4, CHOP and GADD34. Compensatory unfolded protein response (UPR) was activated as well, with increase in chaperons HSP27, HSP70, HSP90, and lysosomal chaperon LAMP3 known to stabilize lysosome, protecting cells against lysosomal membrane permeabilization (LMP) and subsequent cell death. AMG9810 further increased ER stress, elevating CHOP and GADD34 expression, while significantly reducing both basal and Bort-increased levels of HSP70 and LAMP3, thus overcoming the protective response to Bort treatment and prompting lethal LMP. Finally, combining Bort with AMG9810 resulted in significantly reduced ROS that was correlated with impaired mitochondria and increased MM apoptosis, suggesting that dissipation of intracellular ROS may be involved in AMG9810-promoted cytotoxicity. Conclusions: Altogether, our data indicate that TRPV1 is implicated in MM cell survival, proliferation, migration, microenvironment interactions and stress response. TRPV1 inhibition by AMG9810 inhibits CXCR4-mediated migration and stromal protection, synergizes with Bort, amplifies ER stress, targets cytoprotective HSP70 and LAMP3, destabilizes lysosome, impairs mitochondria and promotes MM cell death. These results unravel the mechanism mediating the strong synergistic anti-MM activity of Bort in combination with TRPV1 inhibition which may be translated into the clinic. Disclosures Peled: Biokine: Consultancy; Biosight: Consultancy.

Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI&lt;0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation. Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.