ALBERT

Description: The functional characterization of kinases in multiple myeloma (MM) cells has mainly involved the use of RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. Furthermore, in the bone marrow (BM) microenvironment, where MM cells primarily reside, BM stromal cells (BMSCs) and other nonmalignant cell populations can function as "accessory" cells and alter the vulnerability of MM cells to diverse targeted therapies, including kinase inhibitors. In order to characterize how the kinase dependencies of MM cells differ in the presence vs. absence of BMSCs, we evaluated 16 MM cell lines and their response, in monoculture vs. co-culture conditions, to a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. We observed that virtually all tested cell lines showed no response, in either monocultures or co-cultures, to inhibitors of c-met, ALK, Abl, EGFR superfamily members (EGFR, HER2), c-kit, PDGFR, Flt3, FAK, VEGFR and Syk, including cell lines with detectable transcript levels for the respective kinases at baseline or stroma-induced increase in co-cultures. These results suggest that the respective kinases do not represent major dependencies for MM cell lines in either the cell-autonomous state or in the context of interaction with stromal cells. Dual PI3K/mTOR inhibitors exhibited similarly potent anti-tumor activity in both monocultures and stromal co-cultures of essentially all MM cell lines tested; while mTOR inhibitors devoid of PI3K activity had examples (e.g. in U266 and KMM2 cells) of stroma-induced resistance, likely reflecting a role of PI3K signaling in mediating, at least in some MM cells, stroma-induced decrease in dependence to mTOR function. Inhibitors of IKK or JAK (e.g. ruxolitinib, baricitinib) exhibited higher activity against several MM cell lines in the presence compared to absence of BMSCs. This stroma-induced sensitization to these kinase inhibitors is consistent with the role of BMSC-derived cytokines or cell adhesion molecules in inducing in MM cells NF-kappaB transcriptional activation or JAK/STAT3 signaling. Interestingly, several other classes of kinase inhibitors, e.g. against Aurora, PLK, MEK, Akt, exhibited more heterogeneous stroma-induced effects on their activity (decrease against some cell lines vs. sensitization against others; without an obvious common pattern of these effects for each of these kinase inhibitors across different cell lines). These results suggest that the dependence of MM cells on the corresponding kinases and the signaling networks they regulate are not only subject to stroma-induced changes, but also exhibit a higher degree of cell type-dependent plasticity than previously appreciated. In further support of the complexity of these stoma-induced events, we also observed that BRAF inhibitors (e.g. vemurafenib, dabrafenib, PLX-4720, AZ-628, et.c.) induced at least modest increase in proliferation of several BRAF-wild-type MM cell lines cultured in the absence of stromal cells, but co-culture with BMSC blunted this effect in some cell lines (JJN3, AMO1, OPM2), and modestly enhanced it in others (e.g. KMS34). BRAF inhibitor-induced proliferation of BRAF-wild-type cell lines has been previously reported in different tumor types and has been attributed to activation and signaling through C-RAF: our current observations suggest that, in MM patients harboring both V600E-BRAF mutant and wild-type clones, the impact of treatment with BRAF inhibitor on decreasing the burden of the former clone(s) vs. selecting for outgrowth of the latter may actually be subject to complex, genotype-dependent, influence of the BMSCs of the local microenvironment. In summary, our studies demonstrate the feasibility of functionally annotating the kinase dependencies in MM and potentially other neoplasias by using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines. Furthermore, our studies suggest that future efforts to individualize the administration of kinase inhibitors in MM should take into account not only the genotype of MM cells in the respective patient, but also the heterogeneous impact that nonmalignant "accessory" cells such as BMSCs can have on the MM cell dependence on each respective kinase and its downstream targets. Disclosures Aftab: Cleave Biosciences, Inc.: Research Funding; Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding.

Description: In multiple myeloma (MM) and other neoplasias, several kinases have been extensively evaluated as potential therapeutic targets using RNAi-based approaches or pharmacological inhibitors. Attempts to map the functional dependence of MM cells on individual kinases have primarily utilized RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. For many of these oncogenic kinases, large numbers of such inhibitors have been designed: these inhibitors often exhibit very similar effect on their primary designated target(s), but also perturb other secondary kinases, which may vary for different inhibitors within the same class. Using large sets of such inhibitors can enable comparative analyses to reveal the functional roles of both the respective primary target(s), as well as non-overlapping secondary targets. We therefore pursued the functional mapping of the kinome dependencies of 16 MM cell lines, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. In this study, we observed universally potent activity of Aurora (n=18 compounds), PLK (n=5), and mTORC1/2 (n=20) inhibitors; this observation is consistent with the high proliferative rate of MM cell lines in vitro. In contrast, we observed modest to minimal cell-autonomous susceptibility of MM cells to selective inhibitors of PDK1, PI3K (excluding those that also inhibit mTOR), and Akt: this suggests that PDK1- and Akt-independent mechanisms mediate the effect of PI3K signaling on the survival of most of these cell lines. In addition, we observed lack of response in virtually all tested cell lines to inhibitors of c-met (n=17 inhibitors), ALK (n=2), EGFR superfamily members (EGFR, HER2; n=25 inhibitors), c-kit (n=3), PDGFR(n=5), VEGFR (n=21), Flt3 (n=7), FAK (n=2), Syk (n=5), Src (n=5) and BTK: this result was observed even in those cell lines with detectable transcript against the respective kinases. Notable exceptions to this pattern were inhibitors that, in addition to their primary target, also possess activity to other kinases with known roles in MM (e.g. potent activity of FAK or ALK inhibitors that also target IGF1R, such as TAE226 and GSK1838705A, respectively). Consistent with prior experience, several FGFR3 inhibitors showed modest activity against FGFR3- expressing cell lines (e.g. KMS11, KMS18, OPM2, KMS34). Our screen also revealed several previously underappreciated classes of inhibitors with "non-consensus", heterogeneous, activity across the tested MM cell lines. For instance, we identified 3 clusters of cell lines with high (e.g. AMO1, Karpas-620); intermediate (e.g. KMS20, MM1S), and low responsiveness, to 8 different MEK1/2 inhibitors. Notably, both Karpas-620 and AMO1 cells are KRAS-mutant, BRAF-wild-type and have inherently high levels of p-ERK; while AMO1 cells also harbor a MEK2-Q60P mutation, previously reported to positively regulate the kinase domain activity of MEK2 and induce resistance of BRAF-V600E mutant melanoma cells to MEK1/2 inhibitors. These results raise the possibility that the response to MEK1/2 inhibitors and the role of specific mutations, such as MEK2-Q60P, are tumor-type dependent and/or influenced by concurrent BRAF mutation status. Notably, BRAF inhibitors (n=7) were inactive as cytoreductive agents against our cell line panel of BRAF wild-type cells; while several MM cell lines exhibited significantly increased proliferation upon treatment with these inhibitors. This stimulation has been previously noted in melanoma and has been attributed to activation and signaling through C-RAF; it also suggests that treatment of MM patients harboring both V600E-BRAF mutant and wild-type clones with BRAF inhibitor may decrease the burden of the former clone(s), but select for outgrowth of the latter. In summary, our studies establish the value of using large libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines, as an approach to functionally annotate the kinome dependencies across a given neoplasia, such as MM. Furthermore, our studies provide insight into the possible clinical implications that specific molecular lesions (e.g. mutation status of MEK2 or BRAF) can have on the individualized administration of kinase inhibitors targeting the respective pathway. Disclosures Mitsiades: Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Research Funding; Johnson & Johnson: Research Funding.

Description: Introduction: Chimeric Antigen Receptor (CAR) based cellular-immunotherapies have demonstrated significant clinical efficacy in haematological malignancies. However, the progress of cellular-immunotherapy for the treatment of Acute Myeloid Leukaemia (AML) has failed to gain momentum due to the lack of targetable tumour specific antigens. CD38 is a transmembrane glycoprotein expressed in lymphoid and myeloid cells with high expression in plasma B-cells, and is a well validated target for anti-CD38 therapy in Myeloma. A recent study has furthermore shown that a proportion of AML patients express CD38 on their leukemic blasts. TNF-related apoptosis-inducing ligand (TRAIL) receptor DR4 is another targetable antigen which has been shown to be expressed in 70% of AML patients. In this study, we investigate the therapeutic efficacy of "affinity-optimized" variant(s) of CD38 CAR and membrane bound TRAIL on NK-cell based platforms which can target AML blasts with high expression of CD38 (CD38high AML). The CAR variant is a CAR which binds with lower affinity to CD38 expressed on healthy immune cells such as CD38positive NK cells, while targeting CD38high AML. The membrane bound TRAIL variant (TRAIL4c9) is a mutant which binds with higher affinity to TRAIL-DR4 on AML cells, whilst avoiding binding to decoy receptors. We hypothesize that genetically modifying NK cells to express "affinity optimized" CD38 CARand/or TRAIL4c9 can effectively eliminate CD38high AML cells. Methods: AML cell lines THP-1, U937, and KG1a were immunophenotyped for CD38 and TRAIL-DR4 expression. Retrovirally transduced CD38 CAR-KHYG1 NK cells were used as immune effector cells and were co-cultured with AML cell lines in cytotoxicity assays. CD38low AML cell line KG1a was pre-treated with 10nM all-trans-retinoic acid (ATRA) to upregulate CD38 expression and were subsequently co-cultured with CD38 CAR-KHYG1 in cytotoxicity assays. CD38 CAR-KHYG1 was also co-cultured with n=4 patient derived AML cells in cytotoxicity assays. Using Maxcyte GT electroporation system primary donor derived IL-2 activated NK cells were either mock electroporated, or electroporated with TRAIL4c9 m-RNA orCD38 CAR m-RNA and subsequently co-cultured with THP-1 or ATRA pre-treated KG1a in a cytotoxicity assay. Expression of pro-apoptotic, anti-apoptotic and ligands for checkpoint inhibitory receptors was analysed by immunoblotting or flowcytometry. Results: Based on immunophenotyping, we classified AML cell lines as CD38high (THP-1), CD38moderate (U937) and CD38low (KG1a). CD38 CAR-KHYG1 was significantly more cytotoxic than MOCK KHYG1 against CD38high THP-1, at E:T ratios of 2.5:1, 5:1 and 10:1. CD38 CAR-KHYG1 were also more cytotoxic than MOCK KHYG1 against CD38moderate U937 at multiple E:T ratios; albeit the increase in cytotoxicity was at a much lower level in comparison to THP-1 (Fig 1a). Pre-treatment of CD38low KG1a cells with 10nM ATRA upregulated the cell surface expression of CD38, which were subsequently eliminated by CD38 CAR KHYG1 at E:T ratios of 2.5:1, 5:1 and 10:1. KG1a was intrinsically resistant to NK cells as compared to THP-1 and U937 (Fig 1b). This could partly be explained by the high intracellular expression of Bcl-xL, and higher cell surface expression of Nectin-1 and Sialic acid which are the ligands for checkpoint inhibitory receptors CD96 and Siglec-7/9 respectively on NK cell (Fig 1c). CD38 CAR-KHYG1 mounted a potent cytotoxic response against primary CD45intermediate AML blasts (n=4 patients) at multiple E:T ratios, and the extent of CAR induced cytotoxicity correlated with the cell surface CD38 expression on the primary AML blasts (R2=0.87) (Fig 1d,e). TRAIL4c9 or CD38 CAR m-RNA electroporated primary donor-derived NK cells were also potent in eliminating THP-1 and ATRA pre-treated KG1a at multiple E:T ratios (Fig 1f). This demonstrates the potential of therapeutically treating AML patients, with high CD38 expression, with a combination of NK cells expressing "affinity-optimized" CD38 CAR and membrane bound TRAIL variant. Conclusion: The study demonstrates the therapeutic potential of an "affinity-optimized" CD38 CAR NK cell-based therapy, which can potentially be combined with membrane bound TRAIL expressing NK cells to target CD38high AML. In patients with CD38low expressing AML blasts, patients could be pre-treated with ATRA followed by the combination therapy of CD38 CAR and TRAIL expressing NK cells. Disclosures Stikvoort: Onkimmune Ltd., Ireland: Research Funding. Kirkham-McCarthy:Onkimmune Ltd., Ireland: Research Funding. Van De Donk:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Mutis:Celgene: Research Funding; Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; BMS: Research Funding; Novartis: Research Funding; Aduro: Research Funding; Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy; BMS: Research Funding.

Description: Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody-derived antigen recognition domain linked to intracellular cell signaling domains. CAR engineered autologous T cells have been successful in the treatment of a variety of hematologic malignancies. However, several major caveats, including lack of universal donors, long manufacturing times, and absence of a donor in immunologically frail patients, have limited the successful translation of CAR-T cell based therapy to a larger pool of patients. A universal, easy to manufacture, "off the shelf" CAR-based product could potentially address these limitations and result in a lower cost of goods. Towards developing an "off the shelf" CAR-based therapy for Multiple Myeloma (MM), we explored the feasibility and preclinical efficacy of expressing CD38 CARs in KHYG-1 cells, a natural killer (NK) cell line, first established by Yagita et al from a patient with aggressive NK leukemia (Leukemia, 2000). To this end, we effectively transduced KHYG-1 cells with high-affinity CD38 CARs as well as our recently reported affinity-optimized CD38 CARs, which can readily target MM cells with high CD38 expression, while ignoring non-malignant cells with intermediate, low or no CD38 expression when brought to expression on T cells (Drent et al, Molecular Therapy 2017). Moreover, we assessed performance of first and second generation CARs, with co-stimulatory domains CD28 and 4-1BB, and found the combination of CD28/CD3ζ to lead to the best results. After expanding the CAR transduced KHYG-1 cells, we analyzed their phenotype and efficacy in MM by analyzing their cytotoxic activity against CD38+ and CD38- MM and AML cell lines (UM9/THP-1 and U266/HL60, respectively), and against primary MM cells. The CD38-CAR transduced KHYG-1 cells showed no phenotypic alterations, and at effector to target ratios as low as 1:1, induced a high cytotoxicity towards CD38+ cell lines as compared to mock or non-transduced KHYG-1, demonstrating the important contribution of the CD38 CAR on the KHYG-1 NK cell surface. CD38- cell lines were unaffected by both CD38-CAR transduced KHYG-1 cells and mock or non-transduced KHYG-1 cells, indicating the specificity towards CD38 of the CAR and thus the potential safety of the CD38-CAR KHYG-1 cell. Similarly, ex vivo assays using primary MM cells revealed superior cytotoxic activity of CD38-CAR KHYG-1 cells as compared to mock or non-transduced KHYG-1 cells (median 86,5% vs 14% at 1:1 E:T ratio, n=2, Figure 1A). Confirming our previous results we identified an affinity-optimized CD38-CAR which mediated strong primary MM cell cytotoxicity with little or no "off tumor" effect. Normal immune cells (B, T, monocytes), which were either CD38 negative or only intermediate positive, were unaffected (Figure 1B-D), suggesting the potential safety of the CAR-NK cell therapy for clinical applications. As clinical administration would require irradiation of CD38-CAR KHYG-1 cells, we tested the effect of irradiation on their proliferative and cytotoxicity potential. Irradiation with 10Gy, while drastically inhibiting proliferative activity and viability (50% survival after 3 days), did not affect cytotoxicity, suggesting that repeated administrations of irradiated, CD38-CAR transduced KYHG-1 cells may exert effective in vivo anti-tumor activity, which is currently being evaluated in appropriate in vivo models, specifically the humanized bone scaffold in vivo model published by Groen et al (Blood, 2012). In conclusion, we demonstrate that the incorporation of CAR technology into the immortal NK cell line KHYG-1 has enormous potential to become a safe and effective "off the shelf" therapy for MM. Disclosures Stikvoort: Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. van de Donk:Amgen: Research Funding; Janssen Pharmceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. O'Dwyer:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Mutis:Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (〉85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p

Description: Acquired or de novo resistance to established and investigational therapies represents a major clinical challenge for multiple myeloma (MM) and other neoplasias. Despite extensive efforts, clinically-validated molecular markers that predict for proteasome inhibitor (PSI) resistance in most MM patients remain elusive. This challenge is partly due to limited availability so far of molecular data on MM patients before the start of PSI treatment vs. immediately after resistance to it develops; this challenge may also reflect the heterogeneity of the complex molecular mechanisms regulating MM cell response to PSIs. We hypothesized that resistance to PSIs can be mediated by disruption of several functionally overlapping genes, and that the prevalence of any of these lesions may be too low to detect in datasets available thus far. To examine this latter hypothesis, we performed a genome-wide screen for genes whose loss confers to MM cells resistance against bortezomib, through the use of the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 system. Specifically RPMI-8226 MM cells were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of a genome-scale pooled library of 123,411 single-guide RNAs (sgRNAs), which selectively align to target sequences at the 5′ constitutive exons of 18,080 genes and direct the Cas9 nuclease to cause double-stranded cleavage and loss of function of the respective gene. From the pool of MM cells transduced with the sgRNA library and treated with bortezomib, treatment-resistant cells were processed for deep sequencing, to identify enriched sgRNAs and their corresponding genes. We identified that loss-of-function of 33 candidate genes is associated with bortezomib resistance. We observed a high level of consistency between independent sgRNAs targeting the same gene, as well as a high rate of hit confirmation across different biological replicates. Notably, this set of candidate bortezomib-resistance genes was distinct from the "hits" we identified through a parallel CRISPR screen on the same cell line for resistance to a different targeted therapy (namely the bromodomain inhibitor JQ1), supporting the ability of this approach to identify treatment-specific resistance genes. These candidate bortezomib-resistance genes have documented or presumed roles in the regulation of extrinsic and intrinsic apoptotic cascades, autophagy, Toll-like receptor and NF-kappaB signaling, aggresome function, heat shock protein expression, chromatin remodeling, nutrient sensing, and tumor suppressor gene networks. Importantly, information from several publically available molecular profiling datasets converge to support the putative clinical relevance of these genes. For instance, gene expression data from tumor cells of bortezomib-naive patients with advanced MM revealed several transcriptional signatures of these candidate genes (defined by low transcript levels for any of the genes in the signature) which correlated with shorter time to disease progression after treatment with bortezomib (p0.426). Congruent with these findings, the highly bortezomib-responsive clinical setting of newly-diagnosed MM is associated with low cumulative frequency of mutations of these bortezomib-resistance genes (e.g. cumulative mutation rate of 3.9%, 95% confidence interval [CI] 1.25-6.55%). Notably, in other malignancies that are typically PSI-resistant, a higher cumulative frequency of such lesions is observed (average of ~28%, range 0-76%, 95% CI 22.46-32.70%; 57 datasets from 20+ neoplasias examined). In summary, this first application of the CRISPR/Cas9-based technology in MM illustrates its power to interrogate gene function on a genome-wide scale. This approach identifies bortezomib-resistance genes that are associated with pathways linked with the regulation of proteasome inhibitor response. Results from molecularly-annotated clinical samples converge to support a possible role for these genes in bortezomib resistance. This experience supports the value of CRISPR/Cas9-based studies to dissect the molecular mechanisms of treatment resistance in MM and other hematologic neoplasias (* equal contribution of M.S. and Y.H.). Disclosures Shalem: Broad Institute: Patent application for CRISPR technology Patents & Royalties. Sanjana:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Zhang:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria.

Description: Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant "accessory" cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure. Disclosures McMillin: Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.