Abstract: Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. Disclosures Richardson: Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Abstract: Abstract 578 Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating residual disease. A fundamental challenge to developing an effective anti-tumor immune response is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to tumor vaccines. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (CT-011) alone (Cohort 1) and in combination with a dendritic cell/myeloma fusion cell vaccine (Cohort 2) following ASCT. To date, 27 patients have been enrolled into Cohort 1, in which patients receive three infusions of CT-011 at doses of 3mg/kg given at 6 week intervals beginning 1–3 months following ASCT. Mean age of the patients is 57 years; 61% are male. 11 patients have received at least two infusions of CT-011. The remaining patients are undergoing pre-transplant therapy/transplant. CT-011 has been well tolerated, with possibly related adverse events consisting of transient grade 1–2 leukopenia, diarrhea, fatigue, arthralgia, rash, and peri-orbital edema. One patient developed grade 3 neutropenia, which resolved after two days without growth factor. Immunologic response was determined by quantifying circulating tumor reactive T cells prior to each dose of CT-011 and at 1, 3, 6 months following the last infusion, as defined by the percentage of T cells expressing IFNg in response to ex vivo exposure to autologous tumor lysate. 4 patients have completed 6 months of follow up after the third dose of CT-011, and are evaluable for immune response. CT-011 therapy was associated with the dramatic expansion of myeloma specific T cells. Mean percentage of circulating tumor reactive CD4+ and CD8+ T cells increased from 1.5 and 1.96 respectively prior to the first infusion of CT-011, to 4.26 and 8.28 respectively 1 month following the third infusion. As determined by tetramer staining in the subset of patients who are HLA A2.1, infusion of CT-011 resulted in a mean 9 fold expansion of T cells specific to the MUC1 antigen, which is aberrantly expressed by myeloma cells. Notably, immunologic response to CT-011 persists at 6 months following completion of therapy. Clinical response, as determined by time to disease progression, will be determined with longer follow up, as the median time from transplant is presently 8 months. We are initiating enrollment to Cohort 2, in which patients will be vaccinated with an autologous DC/myeloma fusion vaccine 1 week prior to each dose of CT-011. These data demonstrate that CT-011 results in the expansion of tumor reactive lymphocytes in the early post-transplant period, providing an ideal platform for combination with a tumor vaccine. Disclosures: Rosenblatt: CureTech Ltd.: Research Funding. Schickler:CurTech Ltd.: Employment, Research Funding. Rotem-Yehudar:CureTech Ltd: Employment, Research Funding. Avigan:CureTech Ltd: Research Funding.

Abstract: We report the long-term findings and final analysis of a pivotal multicenter trial of ibrutinib monotherapy in previously treated patients with Waldenström macroglobulinemia (WM). PATIENTS AND METHODS Sixty-three symptomatic patients with median prior therapies of two (range, one to nine therapies), of whom 40% were refractory to their previous therapy, received ibrutinib at 420 mg/d. Dose reduction was permitted for toxicity. RESULTS The median follow-up was 59 months, and overall and major response rates were 90.5% and 79.4%, respectively. At best response, median serum immunoglobulin M declined from 3,520 to 821 mg/dL, bone marrow disease involvement declined from 60% to 20%, and hemoglobin rose from 10.3 to 14.2 g/dL ( P 〈 .001 for all comparisons). Responses were impacted by mutated (Mut) MYD88 and CXCR4 status. Patients with MYD88 Mut , wild-type (WT) CXCR4 showed higher major (97.2% v 68.2%; P 〈 .0001) and very good partial (47.2% v 9.1%; P 〈 .01) response rates and a shorter time to major response (1.8 v 4.7 months; P = .02) versus patients with MYD88 Mut CXCR4 Mut . Conversely, four patients who had MYD88 WT disease showed no major responses. The median 5-year progression-free survival (PFS) rate for all patients was not reached, and was 70% and 38% for those with MYD88 Mut CXCR4 WT and MYD88 Mut CXCR4 Mut WM, respectively ( P = .02). In patients with MYD88 WT , the median PFS was 0.4 years ( P 〈 .01 for three-way comparisons). The 5-year overall survival rate for all patients was 87%. Grade ≥ 3 adverse events in more than one patient at least possibly related included neutropenia (15.9%), thrombocytopenia (11.1%), and pneumonia (3.2%). Eight patients (12.7%) experienced atrial arrhythmia, and seven of the eight continued therapy with medical management. CONCLUSION Ibrutinib is highly active and produces long-term disease control in previously treated patients with WM. Treatment is tolerable. Response depth, time to major response, and PFS are impacted by MYD88 and CXCR4 mutation status.

Abstract: Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software. Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented. Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy, accounting for 10% of all hematological malignancies. Genetic analyses of large populations revealed that blood-specific somatic mutations in hematopoietic stem cells (HSCs) are commonly acquired during aging, a new entity labeled: clonal hematopoiesis of indeterminate potential (CHIP). We sought to determine the role of CHIP on survival of MM patients, specifically those receiving immunomodulators (IMiDs) maintenance (Lenalidomide or Thalidomide) post autologous stem cell transplant (ASCT). Methods: We tested cryopreserved HSCs of 629 MM patients who underwent ASCT between 2003 and 2011 at the Dana-Farber Cancer Institute. We used a target bait panel of 224 genes and performed deep-targeted sequencing at 978x coverage and ultra-low pass whole-genome sequencing at 0.1x to account for tumor contamination. Sequencing data was analyzed using ichorCNA, MuTect, and Strelka and mutation annotations were based on reported mutations in the literature and databases (ClinVar, COSMIC, cBioPortal, TCGA, and ExAC). Results: Our cohort had a median age of 58 years [24-83] at time of ASCT and median follow up post ASCT of 8 years [0.1-14.5] . 24% of patients had CHIP at time of ASCT, which is statistically similar to the 30% reported in non-Hodgkin's lymphoma (NHL), (Gibson et. al, JCO, 2017). The most commonly detected mutated genes were DNMT3A, TET2, TP53 and ASXL1. Acquiring mutations positively correlated with age (p=0.004). In contrast to NHL, PPM1D was not significantly mutated in MM (40% vs. 3.3%). 27 patients (4.3%) developed a second hematological malignancy at median of 4 years [1-10] post ASCT, of which 10 had CHIP. 22% received at least 3 years [0.06-12.8] of IMiD maintenance. Among those who did not receive IMiD maintenance, CHIP was associated with worse progression free survival (PFS) (p=0.047) where PFS at 3 years post ASCT was 31% (95%CI: 25-38) for those without CHIP vs. 15% with CHIP (95%CI: 7-25). In patients with IMiD maintenance, CHIP had no effect on PFS or overall survival (OS) (p=0.9). In patients with CHIP, receiving IMiD was associated with a better OS and PFS below the age of 58 and better PFS only in those above 58. In the overall cohort, CHIP was not associated with more adverse outcomes, which could be attributed to low OS and PFS in MM or the use of IMiD in 56% of this cohort. IMiD maintenance was associated with better OS (p & lt;0.001) and PFS (p & lt;0.001), consistent with prior studies, even in the presence of CHIP mutations. Conclusion: CHIP is a common entity among MM patients that predicts a worse PFS in those who do not receive IMiD maintenance therapy post ASCT. The use of IMiDs abrogated the deleterious effect imposed by CHIP in this cohort. Larger cohorts with longer follow up are needed, especially in the era of novel agents and long-term use of Lenalidomide maintenance. Citation Format: Tarek H. Mouhieddine, Jihye Park, Robert Redd, Christopher J. Gibson, Salomon Manier, Amin Nassar, Kalvis Hornburg, Marzia Capelletti, Daisy Huynh, Romanos Sklavenitits Pistofidis, Mark W. Bustoros, Saud H. AlDubayan, Brendan Reardon, Cody J. Boehner, Henry Dumke, Chia-Jen Lui, Darlys Schott, Eliezer M. Van Allen, Robert L. Schlossman, Nikhil C. Munshi, Kenneth C. Anderson, David P. Steensma, Jacob P. Laubach, Paul G. Richardson, Jerome Ritz, Benjamin L. Ebert, Robert J. Soiffer, Donna Neuberg, Irene M. Ghobrial. Immunomodulator maintenance post autologous stem cell transplant predicts better outcome in multiple myeloma patients with clonal hematopoiesis of indeterminate potential [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 2954.

Abstract: The discovery that thalidomide derivatives recruit the E3 ligase CRBN to induce neomorphic degradation of proteins critical for multiple myeloma (MM) cells stimulated the research into proteolysis-targeting chimeric compounds (PROTACs), led to development of several CRBN- or VHL-based PROTACs against various oncoproteins and put a new spotlight on the biology and therapeutic targeting of E3 ligases in human neoplasias. However, so far only a few of the ~600 known/presumed E3 ligases have been leveraged for generation of PROTACs. The mechanisms regulating the function of most E3 ligases have not been systematically examined. Because the function of an E3 ligase is considered essential for anti-tumor activity of its respective PROTACs, we applied CRISPR knock-out (KO) systems to identify candidate regulators of E3 ligase function, via characterizing the the network of genes which modulate MM cell responses to PROTACs. We thus performed genome-scale CRISPR-based gene editing (for loss-of-function, LOF) studies in MM.1S cells treated with PROTACs targeting BET bromodomain proteins through MDM2 (A1874), CRBN (dBET6) or VHL (ARV-771 or MZ-1) or targeting CDK9 through CRBN (Thal-SNS-032); and validated key hits with individual sgRNAs in different MM cell lines. The top individual LOF events conferring resistance to PROTACs did not involve a compensatory mechanism or "work-around" the loss of the respective oncoprotein, but were predominantly associated with LOF of the respective E3 ligase; or with LOF for genes with known or plausible role in regulating the respective E3 ligases. For instance, sgRNAs against members of the COP9 signalosome complex decreased MM cell responses to CRBN- and (to a lesser extent) VHL-, but not MDM2-based PROTACs. PROTACs leveraging different E3 ligases were regulated by different cullin ring ligase (CRL) complex members (e.g. CUL2, RBX1, TCEB1, TCEB2 for VHL- vs. DDB1 for CRBN- vs. no CRL member for MDM2-based PROTACs) or E2 conjugating enzymes (UBE2R2 vs. UBE2G1 for VHL- vs. CRBN-based PROTACs). Collectively, these results suggest that MDM2 regulation is largely CRL- and COP9-signalosome independent; while VHL regulation is less COP9 signalosome-dependent compared to CRBN. These mechanistic differences suggest that PROTACs targeting the same oncoprotein through different E3 ligases should not be associated with cross-resistance, a result which we validated in experiments involving sequential administration of different PROTACs against BRD4/3/2. In turn, this observation implied that developing PROTACs that leverage a more extended spectrum of E3 ligases may facilitate sequential uses of existing and these new PROTACs to delay or prevent treatment resistance. Building on results of our genome-scale CRISPR essentiality screens, we examined the dependency landscape of known E3 ligases of MM (n=20 cell lines) and 500+ non-MM cell lines. CRBN is redundant for nearly all MM or non-MM cell lines tested, while most other E3 ligases leveraged for PROTACs (e.g. MDM2, BIRC2, DCAF15, DCAF16, RNF114) are essential for only modest or small subsets of human cancer cell lines, suggesting that resistance to respective PROTACs may readily emerge through LOF of these E3 ligases without major fitness cost to tumor cells. We thus sought to identify E3 ligases which are highly expressed in subsets of human tumor cell lines (at levels well above the large majority of normal tissues) and are major dependencies for these "high expressor" cell lines: we identified MDM2 as a major dependency for p53-wild-type cell lines (consistent with MDM2 role as E3 ligase for p53) and we validated this result by documenting the preferential activity of a MDM2-based PROTAC for BRD4/3/2 against p53 wild-type cells. We also identified other E3 ligases genes with well-known roles in tumor cell biology (e.g. members of anaphase promoting complex/cyclosome); as well as E3 ligases (e.g. KCMF1, RNF4) which, to our knowledge, have not been leveraged for design of PROTACs, but warrant consideration given their patterns of essentiality in "high expressor" tumor cells. Our study provides insights on differential regulation and distinct patterns of essentiality for different E3 ligases and informs the design of new PROTACs which leverage different E3 ligases to help delay/overcome treatment resistance in MM and beyond. Disclosures Schlossman: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.