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Supplementary Figure 1. In-vivo mouse model's schema; Supplementary Figure 2. Total BLI Flux of SCID.beige myeloma xenograft model and Kaplan-Meier survival analysis of the NSG xenograft model; Supplementary Figure 3. Elotuzumab does not affect myeloma cell proliferation and homing to the bone marrow.

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma, has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity by natural killer (NK) cells toward myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody-dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to antitumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor-associated macrophages using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant antitumor efficacy of single-agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc–FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in vivo and mediates ADCP of myeloma cells though a FcγR-dependent manner in vitro. Together, these findings propose a novel immune-mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Mol Cancer Ther; 17(7); 1454–63. ©2018 AACR.

5TGM1 cells co-localized with platelets in femur of syngeneic murine model.

Purpose: Tumor cell–platelet interactions contribute to tumor progression and metastasis in solid tumors. However, the role of platelets in hematological malignancies is not clear. We investigated the association of platelet activation status with clinical stages in multiple myeloma (MM) patients and explored the role of platelets in MM progression.Experimental Design: Platelets were obtained from healthy donors and MM patients. We examined platelet activation status in MM patients by flow cytometry and transmission electron microscopy. We also observed the enriched pathways that are involved with platelet activation in RNA sequencing of platelets. MM cell lines were used to assess the effect of platelets on MM cell proliferation in vitro and their engraftment in vivo. RNA sequencing of MM cell lines was performed to explore molecular mechanisms underlying MM cell–platelet interaction and a CRISPR/Cas9 knockout approach was used for validation.Results: Platelets from MM patients were highly activated with disease progression. RNA sequencing of platelets revealed that genes involved in platelets were enriched in patients with smoldering MM (SMM) or MM. Platelets promoted MM cell proliferation in vitro and contributed to tumor engraftment in bone marrow in vivo. RNA sequencing revealed that IL-1β was upregulated in MM cell lines co-cultured with platelets, whereas IL-1β knockout in MM cell lines abrogated the effects of platelets on MM cell proliferation and engraftment in vivo.Conclusions: Platelets from MM patients were highly activated with disease progression. IL-1β is critical to platelet-mediated MM progression and might be a potential target for MM treatment. Clin Cancer Res; 24(10); 2430–9. ©2018 AACR.

Treatment of CD41 Ab reduced the platelet count in SCID-beige mice.

IL-1b knockout MM cells were generated and confirmed.

Fifteen cytokines were secreted by the MM cell-mediated platelet activation.

Tumor-interacting platelets could be removed by wash process completely.

Clinical information of patients with MGUS, SMM, or MM.

Murine platelets enhance proliferation rate of murine multiple myeloma cells in vitro.

Co-culture with platelets increases the IL-1β mRNA levels in MM cells.

Supplemental Figure 1. Hypoxia-induced drug resistance on MM cells. Supplemental Figure 2. HK2, PFKFB3, PFKFB4 and LDHA expression in MM cell lines. Supplemental Figure 3. (A-B) HIF1A expression in MM1S-shHIF1A. (C) HIF2A expression in MM1S-shHIF2A. (D) Stable knockdown of HIF1A restores the effect of melphalan under hypoxic conditions. (E) HIF1A expression in MM1S-shHIF1A. Supplemental Figure 4. (A-B) HK2 and LDHA expression in MM1S-shHK2, MM1S-shLDHA. (C) Relative mRNA levels of HK2 and LDHA in CD138 + cells 3 from normal donors bone marrow, MGUS, Smoldering myeloma and newly diagnosed myeloma (GSE6477). (D) Stable knockdown of LDHA partially restore the effect of melphalan under hypoxic conditions. (E) PDK1 expression in MM1S-PDK1 expressing cells. (F) Stable overexpression of PDK1 induces resistance to bortezomib in MM1S cells. Supplemental Figure 5. LDHA and HIF1A expression in ANBL6-BRshLDHA, MM1S-HIF1A OE and in MM1S-LDHA OE cells.

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma, has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity by natural killer (NK) cells toward myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody-dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to antitumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor-associated macrophages using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant antitumor efficacy of single-agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc–FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in vivo and mediates ADCP of myeloma cells though a FcγR-dependent manner in vitro. Together, these findings propose a novel immune-mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Mol Cancer Ther; 17(7); 1454–63. ©2018 AACR.

Purpose: Tumor cell–platelet interactions contribute to tumor progression and metastasis in solid tumors. However, the role of platelets in hematological malignancies is not clear. We investigated the association of platelet activation status with clinical stages in multiple myeloma (MM) patients and explored the role of platelets in MM progression. Experimental Design: Platelets were obtained from healthy donors and MM patients. We examined platelet activation status in MM patients by flow cytometry and transmission electron microscopy. We also observed the enriched pathways that are involved with platelet activation in RNA sequencing of platelets. MM cell lines were used to assess the effect of platelets on MM cell proliferation in vitro and their engraftment in vivo. RNA sequencing of MM cell lines was performed to explore molecular mechanisms underlying MM cell–platelet interaction and a CRISPR/Cas9 knockout approach was used for validation. Results: Platelets from MM patients were highly activated with disease progression. RNA sequencing of platelets revealed that genes involved in platelets were enriched in patients with smoldering MM (SMM) or MM. Platelets promoted MM cell proliferation in vitro and contributed to tumor engraftment in bone marrow in vivo. RNA sequencing revealed that IL-1β was upregulated in MM cell lines co-cultured with platelets, whereas IL-1β knockout in MM cell lines abrogated the effects of platelets on MM cell proliferation and engraftment in vivo. Conclusions: Platelets from MM patients were highly activated with disease progression. IL-1β is critical to platelet-mediated MM progression and might be a potential target for MM treatment. Clin Cancer Res; 24(10); 2430–9. ©2018 AACR.

Drug resistance remains a major clinical challenge for cancer treatment. Multiple myeloma is an incurable plasma cell cancer selectively localized in the bone marrow. The main cause of resistance in myeloma is the minimal residual disease cells that are resistant to the original therapy, including bortezomib treatment and high-dose melphalan in stem cell transplant. In this study, we demonstrate that altered tumor cell metabolism is essential for the regulation of drug resistance in multiple myeloma cells. We show the unprecedented role of the metabolic phenotype in inducing drug resistance through LDHA and HIF1A in multiple myeloma, and that specific inhibition of LDHA and HIF1A can restore sensitivity to therapeutic agents such as bortezomib and can also inhibit tumor growth induced by altered metabolism. Knockdown of LDHA can restore sensitivity of bortezomib resistance cell lines while gain-of-function studies using LDHA or HIF1A induced resistance in bortezomib-sensitive cell lines. Taken together, these data suggest that HIF1A and LDHA are important targets for hypoxia-driven drug resistance. Novel drugs that regulate metabolic pathways in multiple myeloma, specifically targeting LDHA, can be beneficial to inhibit tumor growth and overcome drug resistance. Cancer Res; 75(10); 2071–82. ©2015 AACR.

Multiple myeloma is a malignancy of terminally differentiated plasma cells with a strikingly heterogeneous genomic landscape. Genetic alterations including t(4;14), and t(14;16) translocations alongside amplification of chromosome 1q21 are particularly associated with a poor outcome. Amplification of the long arm of chromosome 1 (1q) is among the most frequent copy number alterations encountered, with a confirmed adverse effect on survival. Gene expression profiling has identified a minimal common amplified region between 1q21 and 1q23 as a probable target of the amplification event, however the actionable gene dependencies in that region have not been explored. In this study, we employ a large number of inhouse and publicly available CRISPR, shRNA and drug screens in an effort to characterize the genetic dependencies of 1q-amplified myeloma and discover drugs that target them. Ultimately, we hope to propose a tailored therapeutic strategy for patients with 1q-amplified multiple myeloma. We employed a combination of publicly available (Project Achilles, Dependency Map) and in-house CRISPR and RNAi screens to identify differential dependencies of 1q+ MM. All data were pre-processed and analyzed consistently. Genes were filtered for evidence of both differential dependency and differential expression in 1q+ cell lines. A frequentist approach, prioritizing genes by the number of datasets they hit in, was combined with a gene-wise weighted linear model approach. Genes that hit in at least two datasets and were confirmed by the linear model were taken into consideration for Hallmark pathway analysis with GSEA. Cell cycle, c-Myc, mTOR/PI3K, DNA repair, p53, protein secretion and degradation all constitute major addictions in 1q+ MM and suggest differential sensitivity to corresponding compounds. We searched for differential drug sensitivities utilizing the Drug Repurposing Library as well as a CMap-guided screen. We identified as hits several compounds targeting both the ubiquitin and the cell cycle pathway, including PI3 kinase, PARP, NAMPT and GSK-3 inhibitors which act primarily through a G2/M block thus validating the dependencies discovered in our datasets. In conclusion, here we employed a combination of multiple in-house and publicly available CRISPR, shRNA and drug screens, in the largest to date effort to characterize and target the genetic dependencies of 1q-amplified multiple myeloma. Cell cycle and the ubiquitin pathway came up as strong dependencies, while the drugs that target them were indeed shown to preferentially kill 1q-amplified myeloma cell lines. Thus, for the first time, our results suggest that patients with 1q-amplified myeloma might benefit from genetically tailored treatment involving cell cycle and ubiquitin inhibitors or a combination. As 1q amplification is one of myeloma’s few frequent alterations, this discovery has the exciting potential to affect change in a large number of patients. Citation Format: Mairead Reidy, Romanos Sklavenitis-Pistofidis, Daisy Huynh, Karma Ziad Salem, Jihye Park, Siobhan Glavey, Salomon Manier, Irene Ghobrial. Study of genetic dependencies associated with 1q-amplified multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2986.

In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict disease progression. Most genomic analyses have studied the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. To characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed single-cell RNA sequencing on 48K plasma and 40.8K immune microenvironmental cells from a cohort of 22 patients with varying stages of disease progression and 9 healthy donors. Expression profiles of plasma cells revealed clear tumor-specific differences in known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as other clonally expressed genes (LAMP5, HIST1H1C, and AREG), distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells in malignant samples, observing a range of proliferative capacity across disease stages. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells from the same individual, revealing early alterations in genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1). Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort. Analysis of the BM microenvironment demonstrated significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, even in the earliest stages of the disease. Further investigation revealed upregulation of MHC II expression at the mRNA level in CD14+ monocytes/macrophages and yet, intriguingly, analysis by CyTOF and immunohistochemistry revealed a shift towards intracellular localization of MHC II in these cells. Co-culture with MM cell lines was sufficient to induce the decrease of extracellular MHC II, providing strong evidence for MM-induced compromised antigen presentation by macrophages, and hinting at a mechanism of immune evasion. Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy. Citation Format: Nicholas J. Haradhvala, Oksana Zavidij, Tarek H. Mouhieddine, Romanos Sklavenitis-Pistofidis, Jihye Park, Mairead Reidy, Abdallah Flaifel, Benjamin Ferland, Salomon Manier, Mark Bustoros, Daisy Huynh, Marzia Capelletti, Brianna Berrios, Mahshid Rahmat, Chia-Jen Liu, Meng Xiao He, Esteban Braggio, Rafael Fonseca, Yosef Maruvka, Jennifer Guerriero, Melissa Goldman, Eliezer Van Allen, Steven McCarroll, Jamil Azzi, Gad Getz, Irene M. Ghobrial. Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 139.

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 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.

Introduction: Genomic sequencing of tumor cells obtained from the bone marrow (BM) of patients with multiple myeloma (MM) has demonstrated significant clonal heterogeneity. However, it could be envisioned that such clonal diversity may be even higher since the pattern of BM infiltration in MM is typically patchy. In addition, BM biopsies cannot be repeated multiple times during the course of therapy, indicating a need for less invasive methods to genomically characterize MM patients. In this study, we aimed to determine the overall applicability of performing genomic characterization of MM patients non-invasively using circulating tumor cells (CTC). Methods: We performed CTC enumeration using multi-parameter flow cytometry (MFC) in 50 newly-diagnosed patients with symptomatic MM who were prospectively enrolled on the Spanish clinical trial PETHEMA/GEM2010MAS65 as well as 64 patients with MM with relapsed disease or in remission/on maintenance therapy seen at the Dana-Farber Cancer Institute. For sequencing studies, we obtained 8 samples of newly-diagnosed untreated patients. We sequenced BM clonal PCs and CTCs up to 200x, and germline cells up to 50x. Whole genome amplification (WGA) was performed for CTCs, and two independent libraries were sequenced up to 100x for each duplicate. Only single nucleotide variants (SNVs) shared in both parallel WGA libraries were used. Results: Using sensitive MFC, we showed that CTCs were detectable in 40/50 (80%) newly-diagnosed MM patients, and in 71/130 (55%) of multiple sequential samples from patients with relapsed disease or in remission/on maintenance. Nineteen of the 40 newly-diagnosed cases displaying PB CTCs had relapsed (median TTP of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed (median TTP not reached; P = .08). Afterward, increasing CTC counts in sequential PB samples from patients with relapsed disease or in remission/on maintenance therapy were associated with poor overall survival (P = .01), indicating that both the absolute numbers of CTCs and trend of CTC are predictive of outcome in MM. After demonstrating that CTCs can be readily detected in the majority of MM patients, we then determined the mutational profile of CTCs and compared it to that of patient-paired BM clonal PCs. We identified a median of 223 and 118 SNVs in BM clonal PCs and CTCs, respectively. The concordance of somatic variants found in matched BM clonal PCs and CTCs was of 79%. Noteworthy, upon investigating specific mutations implicated in MM (eg. KRAS, NRAS, BRAF) a total of 18 non-synonymous SNVs (NS-SNVs) in 13 genes were identified in our cohort, and most of these NS-SNVs were simultaneously detected in matched BM clonal PCs and CTCs. That notwithstanding, we also identified several unique mutations present in CTC or BM clonal PCs; of those, up to 39 NS-SNV were identified as CTC specific, and 6 NS-SNVs in 4 genes (CR1, DPY19L2, TMPRSS13, HBG1) were detected in multiple patient samples. A significant concordance for the pattern of copy number variations (CNVs) between matched BM and PB tumor cells was also observed. Conclusion: This study defines a new role for CTCs in the prognostic and molecular profiling of MM patients, and provides the rational for an integrated flow-molecular algorithm to detect CTCs in PB and identify candidate patients for noninvasive genomic characterization to predict outcomes. Citation Format: Yuji Mishima, Bruno Paiva, Jiantao Shi, Mira Massoud, Salomon Manier, Adriana Perilla-Glen, Yosra Aljawai, Satoshi Takagi, Daisy Huynh, Daisy Huynh, Aldo Roccaro, Antonio Sacco, Diego Alignani, Maria-Victoria Mateos, Joan Blade, Juan-Jose Lahuerta, Paul Richardson, Jacob Laubach, Robert Schlossman, Kenneth Anderson, Nikhil Munshi, Felipe Prosper, Jesus San Miguel, Franziska Michor, Irene M. Ghobrial. Prognostic relevance and genomic profile of circulating tumor cells in multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A82.