Bone Marrow Of Myeloma Patients Research Articles

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1237.

Abstract 2958: Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2958.

The Ratio of Urea Nitrogen to Albumin Is a Better Predictor of OS in MM Patients Than That of Urea Nitrogen Alone

Background Multiple myeloma (MM) is a malignant proliferative disease of plasma cells. Abnormally cloned plasma cells secrete large amounts of monoclonal immunoglobulins in the bone marrow of MM patients. Serum urea nitrogen (sUN) is a byproduct of protein metabolism, and its effect on MM patients' prognoses remains unknown. Therefore, we analysed MM patients' clinical data to explore the role of sUN and serum urea nitrogen/serum albumin (sUAR) in the baseline tumor load and MM prognosis of MM patients. Methods We downloaded the clinical data of 762 MM patients from the MMRF database. After excluding those without baseline sUN, 452 patients were finally included in the study. Smoothed curve fitting, threshold analysis, Tamhane's T2 test, multivariate adjusted Cox regression analysis, Kaplan‒Meier (K-M) curves, and receiver operating characteristic (ROC) analysis were applied in the study. Results There were 452 newly diagnosed MM patients included in this study. In most patient groups, sUN and sUAR were positively linked with β2-microglobulin (β2-MG) and lactic dehydrogenase (LDH) according to smoothing curve fitting and threshold analysis. The higher the ISS stage, the greater the values of sUN and sUAR. Furthermore, smoothed curve fitting and threshold analysis showed that sUN was correlated with OS, although sUAR had a stronger correlation with OS and could be applied to a broader group. The results of a multivariate adjusted Cox regression analysis demonstrated that sUN and sUAR were independent prognostic factors for OS. The K-M curve confirmed the correlation between higher sUN and sUAR levels and worse OS. β2-MG and LDH are generally recognized prognostic factors of OS. ROC analysis revealed that sUN might boost β2-MG and LDH's predictive value and sUAR had a higher predictive value. Conclusion This retrospective study based on the MMRF database showed that high sUN and sUAR levels were positively associated with β2-MG, LDH, and ISS staging, and sUAR exhibited a stronger correlation with OS than sUN alone. Key words Serum urea nitrogen, ratio of serum urea nitrogen to serum albumin, multiple myeloma, overall survival, β2-microglobulin, lactic dehydrogenase

Abstract 2959: Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms

Abstract Multiple myeloma (MM), a cancer of plasma cells, occurs in ~34,000 new patients every year in the USA. Although therapeutic regimens, including anti-CD38 monospecific IgG antibodies such as daratumumab and isatuximab in combination with chemotherapies, demonstrate clinical efficacy, most patients eventually develop resistance. Several bispecific (CD38xCD3) or trispecific (CD38xCD3xCD28) T cell engager (TCE) antibody therapies are currently in development to improve upon the efficacy of CD38 targeted therapies by leveraging T cell dependent cellular cytotoxicity (TDCC) of MM cells. However, CD38 is also expressed on some normal hematopoietic cells which could potentially lead to undesired pharmacological activity such as depleting CD38+ immune cells, including activated cytotoxic T cells (i.e., fratricide). IGM-2644 is a novel, pentameric IgM antibody engineered to have ten CD38 binding sites, and an anti-CD3ε single chain Fv domain fused to a joining chain to engage CD3 on T cells, and retains the potential for complement-dependent cytotoxicity (CDC). Here we report the functional characterization of IGM-2644 using in vitro, ex vivo and in vivo anti-tumor efficacy studies with a safety evaluation of this novel IgM TCE. In vitro, IGM-2644 demonstrated significantly improved CDC activity in comparison with daratumumab and isatuximab, with >30-fold increased potency on CD38+ MM and lymphoma cell lines. IGM-2644 induced TDCC similar to a bispecific CD38xCD3 IgG on low CD38 expressing cell lines resistant to daratumumab, while demonstrating significantly lower levels of cytokine release than the bispecific IgG. In ex vivo colony forming unit (CFU) assays, IGM-2644 was able to reduce MM CFUs using primary MM patient bone marrow samples containing autologous T cells and myeloma cells, while no effect was observed on erythroid, granulocyte and macrophage CFUs in normal bone marrow samples.​ IGM-2644 dose dependently inhibited tumor growth in humanized xenograft models of CD38+ NCI-H929 (myeloma) and Raji (lymphoma). Importantly, IGM-2644 also demonstrated significantly reduced T cell fratricide compared to bispecific IgGs both ex vivo and in vivo. Significantly reduced activity of IGM-2644 on normal CD38+ innate immune cells was observed in ex vivo studies. CD38 expression has also been reported on human RBCs and platelets. However, minimal IGM-2644 binding was observed and at levels lower than daratumumab. In summary, IGM-2644 is a novel, potent, bispecific IgM TCE that has both CDC and TDCC mechanisms of cytotoxicity with the potential to be active in daratumumab resistant tumors. The balance of potent TDCC and CDC cytotoxic activity, along with an improved preclinical safety profile compared to other CD38xCD3 bispecific IgG TCEs, supports the clinical development of IGM-2644 in MM. Citation Format: Keyu Li, Rui Yun, Min Chai, Poonam Yakkundi, Rodnie Rosete, Gene Li, Liqin Liu, Mandy Li, Daniel Santos, Kevin C. Hart, Dean Ng, Paul R. Hinton, Umesh Muchhal, Thomas Manley, Maya F. Kotturi, Stephen F. Carroll, Angus M. Sinclair, Bruce A. Keyt. Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2959.

Novel Antigens LMA and Kma Are Expressed on Malignant Bone Marrow Plasma Cells from Patients with Relapsed Refractory Multiple Myeloma and Plasma Cell Dyscrasias

Introduction Previous studies have identified the lambda myeloma antigen (LMA) and kappa myeloma antigen (KMA) on the surface of malignant plasma cells in myeloma patient bone marrow samples, human myeloma cell lines and other plasma cell dyscrasias1-3. LMA and KMA are not present on normal B cells and are found on occasional mononuclear cells in tonsillar tissue and secondary mucosal lymphoid tissue. Targeting KMA and LMA through cellular and other immunological therapies has the potential advantage of leaving a population of normal plasma cells untouched and minimising the risk of serious post-treatment infection. Characteristics of the antibodies called KappaMab and LambdaMabs (10B3 and 7F11) that bind to specific conformational epitopes in the constant regions of these surface-bound light chains have been described in clinical trials and in vitro experiments1, 2, 4-6. Here, we present data on a larger series of patients with plasma cell dyscrasias, demonstrating the expression of LMA on plasma cells from patients in whom myeloma evolves from monoclonal gammopathy of unknown significance (MGUS). We also describe the difference in phenotype between LMA and KMA expressing malignant plasma cells and the frequent expression of KMA in relapsed refractory myeloma patient (RRMM) samples. Methods Patient BM samples (κ=49 and λ=31) were analysed using multiparametric FCM immunophenotyping with APC labelled LambdaMab (10B3) and KappaMab Fab'2 fragments, CD38, CD138, CD269 (BCMA), CD319 (SLAM F7), CD56 and CD45 monoclonal antibodies. Plasma cells were identified as previously described by initial gating using CD38 and CD138. LMA and KMA expression was determined and compared with the other cell markers. Results Nine of 12 (75%) newly diagnosed λ-type patients with MGUS or SMM expressed LMA on the surface of BM plasma cells. Longitudinal data from two patients was available. One patient showed LMA expression and antigen density increasing on the surface of bone marrow plasma cells during progression from MGUS (2020) to smoldering myeloma (SMM) (2022) (% of plasma cells positive for LMA rose from 61% to 92%, antigen density increased from 532 to 5164 molecules of PE/cell). In a second individual, LMA expression and antigen density increased on bone marrow plasma cells during progression from SMM (2020) to myeloma (2022) (% of plasma cells positive for LMA rose from 94% to 100%, antigen density increased from 3981 to 8941 molecules of PE/cell). KMA expression continued to be observed on plasma cells in 4 of 6 patients after autologous stem cell transplant. KMA was expressed in 86% of patients with ĸRRMM (n=14). LMA expression was observed in 2 of 4 patients with λRRMM. Both KMA and LMA antigen densities were higher than BCMA in the RRMM population (KMA mean 1802, range 468-7943 vs BCMA mean 1306, range 350-2630 molecules of PE/cell), (LMA mean 2439, range 263-6664 vs BCMA mean 817, range 537-1065 molecules of PE/cell). Conclusions In an enlarged cohort of patients with myeloma, we have confirmed that KMA and LMA are expressed at all stages of disease. Our data examining longitudinal expression in individual patients suggests that expression of LMA on BM plasma cells increases from MGUS to MM. The majority of patients with relapsed refractory kappa myeloma expressed KMA. Further data is required to confirm LMA expression in this population. The antigen density of both LMA and KMA is higher than BCMA on RRMM BM plasma cells. KMA and LMA expression is independent of serum Ig and FLC levels and % marrow plasma cells. These data show that KappaMab and LambdaMab have therapeutic potential in the treatment of myeloma patients, especially in the setting of relapsed refractory disease where the combination of high antigen density and restriction of the target antigen to the malignant subpopulation of plasma cells will confer valuable clinical benefit.

In This Issue

B cell–depleting therapies impair antibody responses in 2-dose series of mRNA vaccination against SARS-CoV-2, urging research on additional protection against COVID-19 in patients with B-cell malignancies. Greenberger et al. characterize T-cell responses after 2 doses of mRNA vaccines, and antibody responses to a 3rd vaccine dose, in a real-world cohort of over 1,000 patients with hematologic cancers. The third dose boosted antibody titers of patients seropositive after 2 doses, with 97% reaching physiologically meaningful titers. Among the 40% of patients who were seronegative after two doses, the third dose elicited antibodies in quantities sufficient for virus neutralization in approximately one in five cases. T-cell responses to the 2nd vaccine dose in 514 patients correlated with humoral immunity but were less impacted by anticancer treatments.See article, p. 481.Although BCMA-directed CAR T cell therapy (BCMA-CART) leads to high rates of response in myeloma, many of these patients experience disease relapse. There is an urgent unmet need to identify determinants of durable responses. Dhodapkar, Cohen et al. combine several high-dimensional tools to analyze tumor and immune cells in the bone marrow of myeloma patients receiving BCMA-CART. They identify distinct features of tumor and immune cells both at baseline and following therapy that correlate with durability of clinical responses. These data illustrate dynamic cross-talk between CAR T cells, endogenous immunity, and residual tumors, and suggest novel strategies to improve durability of CAR T-cell therapy in myeloma.See article, p. 490.The functional precision medicine approach screens patient cell sensitivity to hundreds of drugs in a dish in order to identify a drug that kills cancer cells but not normal cells. The results are scored based on cancer markers by clinicians, limiting scalability. In a computational screen, Heinemann, Kornauth et al. identified a neural network distinguishing cancer cells from stroma by morphology features. When compared to marker-based scoring in a clinical trial, concordant recommendations by both approaches had best outcomes. In cases with discordant recommendations, progression-free survival was 144 days in deep morphology learning–concordant cases as compared to 72 days in marker-concordant therapy cases.See article, p. 502.In the past decade, numerous revolutionary therapeutic options were made available for adults with acute myeloid leukemia (AML) but direct extrapolation to children is particularly challenging due to the marked differences in leukemia genetics between young and old. Wang, Chan, Cheng, Ng et al. performed integrated drug and genomic profiling of patient biopsies and linked to clinical parameters to build the functional genomic landscape of childhood AML, representing a high-dimensional data set to characterize the biological uniqueness of the disease. Age-specific differences in drug response, new gene-drug interactions, and predictive biomarkers were identified. Feasibility of functional precision medicine-guided treatment of children with high-risk AML was successfully demonstrated in the clinic, marking a new approach to manage difficult cases.See article, p. 516.Myeloblast expansion is a clinical hallmark of myeloid neoplasms. Ferrall-Fairbanks, Dhawan et al. resolve myeloblasts and other hematopoietic stem and progenitor cell (HSPC) populations in chronic myelomonocytic leukemia (CMML) by high-dimensional single-cell characterization of sorted CD34+ HSPCs from 39 patients at diagnosis. They identify differentiation trajectories with three biases: normal, megakaryocyte-erythroid, and monocytic. The latter associates with adverse outcomes and is phenotypically characterized by stem cell marker depletion, CD120b+, and increased diversity and activation of cytokine receptor signaling. The monocytic bias is diminished in CMML patients following hypomethylating therapy, and is instigated by inflammation in preclinical mouse models.See article, p. 536.Mutations in the splicing factor SF3B1 are commonly found in patients with myelodysplastic syndrome with ringed sideroblasts (MDS-RS), but it is unknown how the dysregulated downstream iron metabolism pathways disrupt erythroid differentiation. Adema, Ma et al. leverage single-cell transcriptomics in SF3B1-mutant MDS-RS patient samples to identify buildup of late-stage erythroid progenitor cell types, which upregulate EIF2AK1-associated heme deficiency stress response and downstream autophagy genes. Knockout of EIK2AK1 in SF3B1-mutant erythroblasts and MDS-RS cells, as well as response to hypomethylating therapy in patients, was associated with rescue of erythroid progenitor differentiation, reduced autophagy, and increased heme metabolism gene expression, suggesting targeting of the EIF2AK1 pathway as a therapeutic strategy for MDS-RS.See article, p. 554.

Abstract 5299: A therapeutic antibody against G-protein-coupled receptor class 5 member D to treat multiple myeloma

Abstract Multiple myeloma (MM) comprised 0.9% of all new cancer cases and 1.2% of cancer death in 2020 and is the second most common hematological neoplasm in adults. Primary refractory to treatment and relapse resulted in a high death rate of MM patients. Bispecific antibodies (BsAbs) are attracting candidate drugs against multiple myeloma by targeting CD3 on T cells and MM specific cell surface markers such as B cell maturation antigen (BCMA), CD38, and G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D). In the current study, we developed a humanized antibody targeting GPRC5D (clone number G-02-9D7). The antibody was derived from hybridoma of mouse immunized with GPRC5D overexpressed mouse cell line. Compared to the GPRC5D antibody in clinical (JNJ-64407564), this antibody has a higher affinity to 293T cells overexpressing GPRC5D (EC50=1.9nM vs. 3nM), MM cell lines, and MM patient samples. In addition, this antibody did not bind to GPRC5A, B, and C and had weaker non-specific binding to human serum albumin than JNJ-64407564. Bispecific antibody derived from the GRPC5D antibody and CD3 antibody (clone number: sp34) induced specific cytotoxicity of MM cells in the presence of T cells from PBMC. This bispecific antibody stimulated the release of cytokines from T cells such as INF-γ, IL-2, IL-8, and TNF-α. In NCI-H929 prophylactic xenograft mouse model with PBMC transplantation, tumor growth could be prevented by administration weekly with this bispecific antibody as low as 0.1ug per mouse. In the presence of T cells, the bispecific antibody also resulted in complete remission of established MM.1S tumor in xenografted NSG mouse with the injection of 1ug antibody weekly. At last, MM patient bone marrow samples cultured in vitro with this bispecific antibody increased cell death of the CD138+ population. In summary, the GPRC5D antibody could efficiently target Multiple myeloma tumor cells. Citation Format: Feng He, Kefei Wu, Jiabei Liang, Jianhui Li, Hao Peng, Guojin Wu, Jinying Ning. A therapeutic antibody against G-protein-coupled receptor class 5 member D to treat multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5299.

Abstract 408: Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay

Abstract Introduction: Multiple myeloma (MM) is a plasma cell disorder characterized by clonal expansion and accumulation of immature undifferentiated neoplastic cells in the bone marrow (BM). Recent advances in fluorochrome chemistries, flow cytometry (FACS) instrumentation and rapid tissue preservation methodologies have created opportunities for broader adoption of FACS in the clinical management of multiple myeloma patients. Methods: We have developed a 10 color, 2 tube FACS assay based on recommendations made by the European Myeloma Network for phenotyping plasma cell gammopathies to identify abnormal blasts in multiple myeloma patient bone marrow aspirates. The assay includes essential plasma cell markers CD138, CD38, CD56 and CD19 to differentiate abnormal blasts from reactive plasma cells. Use of a multi epitope fluorochrome conjugated anti-CD38 antibody enables CD38 detection in patients on anti-CD38 therapy. Results: Quantification of cell surface HLA-A2 and intracellular survivin protein was made possible using fluorochrome conjugated antibodies in combination with bead standards. The SmartTube™ proteomic stabilizer system for rapid preservation was used to freeze BM aspirate specimens for later analysis at locations with appropriate FACS equipment and trained staff. Consistent with previously published literature, patient survivin levels were elevated on abnormal MM blasts in comparison to survivin levels on non-blast immune cells. Conclusion: We have developed a robust FACS methodology that would be easy to implement in the clinical trial and development setting with minimal training. The assay can consistently identify abnormal blasts in BM samples of multiple myeloma patients and quantify intracellular and cell surface biomarkers related to MM disease or disease treatment responses. Citation Format: Tejaswini Hardas, Hyun-Jeong Ra, James Sheridan, Maria Kovalenko, Catherine Tribouley. Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 408.

TGFβ Inhibition Stimulates Collagen Maturation to Enhance Bone Repair and Fracture Resistance in a Murine Myeloma Model.

Multiple myeloma is a plasma cell malignancy that causes debilitating bone disease and fractures, in which TGFβ plays a central role. Current treatments do not repair existing damage and fractures remain a common occurrence. We developed a novel low tumor phase murine model mimicking the plateau phase in patients as we hypothesized this would be an ideal time to treat with a bone anabolic. Using in vivo μCT we show substantial and rapid bone lesion repair (and prevention) driven by SD-208 (TGFβ receptor I kinase inhibitor) and chemotherapy (bortezomib and lenalidomide) in mice with human U266-GFP-luc myeloma. We discovered that lesion repair occurred via an intramembranous fracture repair-like mechanism and that SD-208 enhanced collagen matrix maturation to significantly improve fracture resistance. Lesion healing was associated with VEGFA expression in woven bone, reduced osteocyte-derived PTHrP, increased osteoblasts, decreased osteoclasts, and lower serum tartrate-resistant acid phosphatase 5b (TRACP-5b). SD-208 also completely prevented bone lesion development in mice with aggressive JJN3 tumors, and was more effective than an anti-TGFβ neutralizing antibody (1D11). We also discovered that SD-208 promoted osteoblastic differentiation (and overcame the TGFβ-induced block in osteoblastogenesis) in myeloma patient bone marrow stromal cells in vitro, comparable to normal donors. The improved bone quality and fracture-resistance with SD-208 provides incentive for clinical translation to improve myeloma patient quality of life by reducing fracture risk and fatality. © 2019 American Society for Bone and Mineral Research.

Abstract 4972: Preclinical development of a first-in-class NKp30xBCMA NK cell engager for the treatment of multiple myeloma

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells. Although several new drugs for the treatment of MM have greatly improved survival, many patients are known to relapse and become refractory to all presently available therapies or experience treatment-related toxicities. Therefore, MM remains an unmet medical need and the development of additional novel therapies is required. NK cells play a crucial role in the control of multiple myeloma and accumulating evidence shows the presence of highly cytotoxic NK cells in the bone marrow of MM patients suggesting that targeting NK cells could provide a specific treatment modality to leverage NK cell cytotoxicity in myeloma. Furthermore, NK cells are the first lymphocytes population to reconstitute after autologous stem cell transplant (ASCT) providing an opportunity to target minimal residual disease (MRD) shortly after transplant. B-cell maturation antigen (BCMA) is an excellent target in MM because its restricted expression in normal and malignant plasma cells from untreated and relapsed myeloma patients, but absent in all other main bone marrow cell subsets. Our first-in-class NKp30xBCMA NK cells engager, CTX-4419, binds to BCMA on MM cells and to NKp30 and CD16A (FcγRIIIA) on NK cells, specifically redirecting NK cells towards tumor cells expressing BCMA. We demonstrate here that CTX-4419 retains activity in the presence of high levels of BCMA ligands and serum IgG and induces potent NK cytotoxicity against high and low BCMA expressing cell lines as well as patients (autologous) primary myeloma cells. Moreover, CTX-4419 does not require CD16A binding to kill tumor cells, a unique characteristic that overcomes the reduction or loss of activity of CD16A due to receptor shedding or downregulation in the tumor microenvironment. Furthermore CTX-4419 induces NK proliferation and cytokines/chemokines production by NK cells only in the presence of tumor cells providing sustainable anti-tumor specificity to NK cells. CTX-4419 activates in-vitro cynomolgus NK cells in presence of target cells expressing cynomolgus BCMA and, when given intravenously, CTX-4419 decreases plasma cell counts. CTX-4419 represents a novel class of NK-cell engagers that shows strong potency even in the absence of CD16A engagement and induces NK cell proliferation and lysis of tumor cells expressing low amount of antigen. CTX-4419 has strong activity in an autologous setting when tested in bone marrow samples of MM patients and shows efficacy in a non-human primate model of plasma-cell depletion. These data show that CTX-4419 is strongly differentiated from conventional therapeutic antibodies and is a promising candidate for MM treatment with the potential to be used as monotherapy or in combination with adoptive transfer of NK cells and/or other immuno-therapies. Citation Format: Monia Draghi, Jamie L. Schafer, Allison Nelson, Zach Frye, Amanda Oliphant, Sara Haserlat, jason Lajoie, Kenneth rogers, Francois Villinger, Michael Schmidt, Robert Tighe, Piotr Bobrowicz, Jennifer Watkins-Yoon, Thomas Schuetz. Preclinical development of a first-in-class NKp30xBCMA NK cell engager for the treatment 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 4972.

The WNT receptor ROR2 drives the interaction of multiple myeloma cells with the microenvironment through AKT activation.

Multiple myeloma is the second most frequent hematological cancer after lymphoma and remains an incurable disease. The pervasive support provided by the bone marrow microenvironment to myeloma cells is crucial for their survival. Here, an unbiased assessment of receptor tyrosine kinases overexpressed in myeloma identified ROR2, a receptor for the WNT noncanonical pathway, as highly expressed in myeloma cells. Its ligand, WNT5A is the most abundant growth factor in the bone marrow of myeloma patients. ROR2 mediates myeloma cells interactions with the surrounding bone marrow and its depletion resulted in detachment of myeloma cells from their niche in an in vivo model, triggering apoptosis and thus markedly delaying disease progression. Using in vitro and ex vivo 3D-culture systems, ROR2 was shown to exert a pivotal role in the adhesion of cancer cells to the microenvironment. Genomic studies revealed that the pathways mostly deregulated by ROR2 overexpression were PI3K/AKT and mTOR. Treatment of cells with specific PI3K inhibitors already used in the clinic reduced myeloma cell adhesion to the bone marrow. Together, our findings support the view that ROR2 and its downstream targets represent a novel therapeutic strategy for the large subgroup of MM patients whose cancer cells show ROR2 overexpression.

Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells.We first tried to define and characterize CD8+ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either “SLLMWITQC” or “LLLGIGILV”), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8+ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1+ myeloma-specific CD8+ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (EomeshiT-betlo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity.Consistent with the immunophenotypes of myeloma-specific CD8+ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14-CD19-CD138+CS1+CD56hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8+ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8+ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8+ T cells was only modestly improved in terms of proliferation and cytokine production.Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8+ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF-β signaling blockade.Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma DisclosuresNo relevant conflicts of interest to declare.

Systems Biology Analysis Identifies Targetable Vulnerability Networks to Proteasome Inhibitors in Multiple Myeloma

Proteasome inhibitors (PI) such as bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy, yet all MM patients eventually develop refractory disease. We developed a non-biased method to identify and validate dysregulated pathways associated with PI-resistance in myeloma by combining RNAseq data from 522 MM patient specimens obtained from our Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center with paired ex vivo sensitivity to PIs and kinase inhibitors (KI). Dimensionality reduction analysis (t-SNE) and Fuzzy C-means was used to identify 422 clusters of genes that co-express in individual patients, and Gene Set Enrichment Analysis (GSEA) was used to identify clusters with gene expression patterns that correlated with PI sensitivity. Using publicly curated databases and in silico integrative analyses, we built protein-protein interaction networks to identify putative transcription factors, corresponding master regulators (kinases), and candidate KIs to promote PI sensitization. This systems biology approach identified a Chk1-Cdk1-Plk1 circuit associated with PI-resistance and also found 21 additional kinases (of 501 expressed in our cohort's kinome) that could be targeted to re-sensitize PI-resistant MM, which we confirmed in cell lines, specimens from relapsed patients, and two in vivo models.A panel of paired isogenic PI-resistant and sensitive MM cell lines were differentially screened to find kinases associated with PI-resistance using activity-based protein profiling (ABPP) and KI activity measured by high-throughput viability assay. The MM cell lines 8226 and U266, along with their drug resistant counterparts 8226-B25 and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe. Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer) to identify 85 kinases out of a total of 715 proteins in 8226-B25 MM cells and 35 kinases out of a total of 688 proteins in U266-PR MM cells that were preferentially enriched by 2-fold change compared to parental cell lines. Twenty-four kinases were commonly activated among PI-resistant cell line pairs and were screened in PI-resistant myeloma lines using a label-free, high throughput viability assay that simulates the tumor microenvironment. Three KIs targeting Plk1 (volasertib and GSK461364) and Cdk1/5 (dinaciclib) consistently maintained LD50s in the low-nanomolar range and induced caspase-3 activation in four PI-resistant MM cell lines: 8226-B25, U266-PR, ANBL-6-V10R, and Kas6-V10R.Twenty-four kinases each were identified by RNAseq/ex vivo PI sensitivity of MM specimens and ABPP of PI-resistant/sensitive MM cell line pairs. Of these, 7 kinases were identified by both methods: Cdk1, Chk1, Plk1, ILK, Syk, PKA, and p70S6K. Several KIs targeting Cdk1, Plk1, ILK, DNAPK, Syk, MKK7, Nek2, and mTOR identified in patient specimen or cell-line screens showed single agent activity in MM patient bone marrow specimens purified by a CD138 affinity column. Among these, inhibitors to Cdk1, ILK, mTOR, and Plk1 showed the most activity in patient specimens with an average 96h LD50 of 25 nM (n=56), 2.4 uM (n=42), 2.7 uM (n=57) and 3.8 uM (n=53), respectively. Six KIs targeting Plk1, ILK, Syk, MKK7, Nek2 and MARK3 were synergistic with carfilzomib in 20 patient specimens and maintained or improved ex vivo activity in relapsed refractory MM (RRMM) specimens. Volasertib, which targets Plk1, was the most synergistic with carfilzomib of all KIs tested in patient specimens and was further validated in two in vivo models: a NSG/U266 xenograft model of PI resistance and the syngeneic C57BL/6-KaLwRij/5TGM1 immunocompetent model. Volasertib significantly increased survival and reduced tumor burden in both models as a single agent, and was more effective versus PI-resistant tumors compared to PI-sensitive counterparts. Our pharmaco-proteomic screen, coupled with rich gene expression data from patients identified Plk1 as a target critical to MM survival in the context of acquired PI resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. We anticipate that these data will also produce a critical path for the personalized allocation of therapy to maximize efficacy and minimize the use of ineffective therapies in RRMM. DisclosuresNo relevant conflicts of interest to declare.

PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients

Characterization of CD8+ T cells in the tumor microenvironment (TME) is important to predict responses to checkpoint therapy. The TME in multiple myeloma is the bone marrow, which also is an immune organ where immune responses are generated and memory cells stored. The presence of T cells with other specificities than the tumor in the bone marrow may affect the search for biomarkers to predict responses to immunotherapy in myeloma. Here, we found similar proportions of PD1+ CD8+ T cells and similar levels of PD1 expression on CD8+ T cells in the bone marrow of myeloma patients and healthy controls. PD1 expression on CD8+ T cells did not correlate with tumor load suggesting that at least some of the PD1+ CD8+ T cells were specific for non-myeloma antigens. Indeed, PD1+ EBV-specific CD8+ T cells were detected it the bone marrow of patients. Terminal effectors (Teff), effector memory (Tem) and central memory (Tcm) cells as well as exhausted T cells were all found in the myeloma bone marrow. However, myeloma patients had more terminal effectors and fewer memory cells than healthy controls suggesting that the tumor generate an immune response against myeloma cells in the bone marrow. The presence of CD8 EOMEShigh Tbetlow T cells with intermediate levels of PD1 in myeloma patients suggests that T cell types, that are known to be responsive to checkpoint therapy, are found at the tumor site.

Vγ9Vδ2 T Cells in the Bone Marrow of Myeloma Patients: A Paradigm of Microenvironment-Induced Immune Suppression

Vγ9Vδ2 T cells are non-conventional T cells with a natural inclination to recognize and kill cancer cells. Malignant B cells, including myeloma cells, are privileged targets of Vγ9Vδ2 T cells in vitro. However, this inclination is often lost in vivo due to multiple mechanisms mediated by tumor cells and local microenvironment. Multiple myeloma (MM) is a paradigm disease in which antitumor immunity is selectively impaired at the tumor site. By interrogating the immune reactivity of bone marrow (BM) Vγ9Vδ2 T cells to phosphoantigens, we have revealed a very early and long-lasting impairment of Vγ9Vδ2 T-cell immune functions which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even in clinical remission after autologous stem cell transplantation. Multiple cell subsets [MM cells, myeloid-derived suppressor cells, regulatory T cells, and BM-derived stromal cells (BMSC)] are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the redundant expression of multiple immune checkpoints (ICPs). This review will address some aspects related to the dynamics of ICP expression in the BM of MM patients in relationship to the disease status (MGUS, diagnosis, remission, and relapse) and how this multifaceted ICP expression impairs Vγ9Vδ2 T-cell function. We will also provide some suggestions how to rescue Vγ9Vδ2 T cells from the immune suppression operated by ICP and to recover their antimyeloma immune effector functions at the tumor site.

Quantitative and functional alterations of 6-sulfo LacNac dendritic cells in multiple myeloma

ABSTRACTMultiple myeloma (MM) results from expansion of abnormal plasma cells in the bone marrow (BM). Previous studies have shown that monocytes play a crucial role in MM pathophysiology. A 6-sulfo LacNAc-expressing population of dendritic cells (Slan-DCs) that overlaps with intermediate and non-classical monocytes in terms of phenotype has been described. Slan-DCs represent a circulating and tissue proinflammatory myeloid population which has been shown to play a role in different cancer contexts, and which exhibits a remarkable plasticity. Herein, we studied Slan-DCs from the BM and blood of MM patients. We performed quantitative and functional analyses of these cells from 54 patients with newly diagnosed, symptomatic MM, 21 patients with MGUS and 24 responding MM patients. We found that circulating Slan-DCs were significantly decreased in MM patients as compared to those of healthy donors or patients with MGUS, while CD14+CD16+ intermediate monocytes accumulate in the BM. Moreover, after activation with TLR7/8 ligand R848, IL-12-producing Slan-DCs from the BM or peripheral blood from MM patients were decreased as compared with healthy donors. We show that MM cell lines or MM cells isolated from patients at diagnosis were able to inhibit the production of IL-12 by Slan-DCs, as well as to shift the phenotype of Slan-DCs towards an intermediate monocyte-like phenotype. Finally, Slan-DCs that have been cultured with MM cells reduced their capacity to induce T cell proliferation and Th1 polarization. We conclude that Slan-DCs represent previously unrecognized players in MM development and may represent a therapeutic target.

Abstract IA06: Intratumor heterogeneity and its role in therapeutic escape in multiple myeloma

Abstract Multiple myeloma (MM) is a mature B cell neoplasm characterized by the accumulation of immunoglobulin-secreting plasma cells (PC) within the bone marrow. Despite treatment advances, MM remains incurable in the majority of patients and is over-represented in current cancer death rates. The lack of reliable cure in MM, despite the achievement of deep clinical responses with proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and myeloablative melphalan, implicates the presence of intratumoral heterogeneity and the existence of drug-resistant cells with tumorigenic capacity. However, the molecular and cellular basis of this intratumoral heterogeneity remains only partially understood. Using sequential FACS and single-cell immunofluorescence-FISH or genomics strategies, we have identified clonal tumor progenitor subpopulations within the bone marrow of MM patients that recapitulate the physiologic maturation stages between post-germinal center B cells and plasma cells. These clonal MM subpopulations include tumor cells that resemble CD20+ CD27+ CD38- CD138- Irf4- Xbp1s- memory B cells, CD38+/- CD138- Irf4- Xbp1s- pre-plasmablasts, CD38+ CD138- Irf4+ XBP1s+ plasmablasts, and tumor-bulk CD38+ CD138+ IRF4+ Xbp1s+ plasma cells. Importantly, compared to tumor bulk plasma cells, early Xbp1s- tumor progenitor cells appear innately resistant to mainstay treatment with proteasome inhibitors (PI). Mechanistically, absence of Ire1-Xbp1 signaling in these primitive MM cells is associated with secretory immaturity; as Xbp1s- MM cells produce less secretory immunoglobulin, an important determinant of endoplasmic reticulum (ER) load in PCs, they experience less ER stress than Xbp1s+ cells and are thus less susceptible to induction of cytotoxic ER stress when ER-associated degradation (ERAD) is inhibited by PIs. Significantly, small numbers of Xbp1s- tumor cells can be detected in most MMs and as these progenitor cells are innately insensitive to PI-induced cytotoxic ER stress, these observations can conceivably account for a universal low level resistance that is inherent in the pathogenesis and residual progenitor structure of MM and that prevents PI-induced cure. While these findings may account for some clinical features of MM, at present it remains unknown whether or not Xbp1s- MM tumor progenitors are fully malignant or if they possess the capacity required to reinitiate MM disease. Plausibly, Xbp1s- pre-plasma cells in MM may instead represent a counterpart to the premalignant preleukemic stem cells identified in AML, CLL and HCL, or may alternatively represent dedifferentiated PCs. Although this heterogeneity in tumor cell maturation may contribute to therapeutic evasion, MM tumors also commonly contain multiple genetic subclones that show tidal dominance under therapeutic pressure that may also facilitate treatment escape. Such genetic heterogeneity is not mutually exclusive with diversity in tumor cell maturation. When MM genetic subclones with common recurrent secondary chromosomal structural abnormalities (such as del 13, del 17p, +1q or del 1p) are identified among tumor bulk PCs, similar subclones may also be identified amongst non-PC Irf4- Xbp1s- MM subpopulations; this suggests that tumor progenitor cells may play a role in the genetic evolution of tumors and may represent a reservoir for subclonal relapse. In support of a model in which MM PCs can be replenished from MM progenitor cells, primary Xbp1s- MM progenitors possess the ability to differentiate in vitro into mature Xbp1s+ MM plasma cells. Notably, however, in contrast to a model in which MM PCs are continuously replenished by MM progenitors, we have also identified some genetic events that appear to show restricted representation among tumor PCs, consistent with MM PCs comprising a self-sustaining tumorigenic population in its own right. Overall, these data support a model in which Xbp1s- progenitor subpopulations in MM represent a reservoir of innately PI-resistant cells that, following eradication of tumorigenic PCs, may give rise to clonally related clinical relapse via MM progenitor cell differentiation. Overall these findings imply that treatment strategies in MM need to better address Xbp1s- IRF4- tumor stem/progenitor cells, while also eradicating MM-bulk PCs, to achieve deeper treatment responses and cure for patients. Citation Format: Rodger E. Tiedemann. Intratumor heterogeneity and its role in therapeutic escape in multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA06.

Selective inhibition of matrix metalloproteinase-2 in the multiple myeloma-bone microenvironment.

Multiple myeloma is a plasma cell malignancy that homes aberrantly to bone causing extensive skeletal destruction. Despite the development of novel therapeutic agents that have significantly improved overall survival, multiple myeloma remains an incurable disease. Matrix metalloproteinase-2 (MMP-2) is associated with cancer and is significantly overexpressed in the bone marrow of myeloma patients. These data provide rationale for selectively inhibiting MMP-2 activity as a multiple myeloma treatment strategy. Given that MMP-2 is systemically expressed, we used novel “bone-seeking” bisphosphonate based MMP-2 specific inhibitors (BMMPIs) to target the skeletal tissue thereby circumventing potential off-target effects of MMP-2 inhibition outside the bone marrow-tumor microenvironment. Using in vivo models of multiple myeloma (5TGM1, U266), we examined the impact of MMP-2 inhibition on disease progression using BMMPIs. Our data demonstrate that BMMPIs can decrease multiple myeloma burden and protect against cancer-induced osteolysis. Additionally, we have shown that MMP-2 can be specifically inhibited in the multiple myeloma-bone microenvironment, underscoring the feasibility of developing targeted and tissue selective MMP inhibitors. Given the well-tolerated nature of bisphosphonates in humans, we anticipate that BMMPIs could be rapidly translated to the clinical setting for the treatment of multiple myeloma.

Signatures of Mesenchymal Cell Lineages and Microenvironment Factors Are Dysregulated in High Risk Myeloma

IntroductionMesenchymal lineages including osteoblasts, adipocytes and pericytes derived from mesenchymal stem cells (MSCs) are key components of hematopoietic stem cell and myeloma (MM) niches in bone marrow (BM).The aim of the study was to define high risk signatures of mesenchymal cells in BM of MM patients at diagnosis and post-treatment, and their biological importance.MethodsUniquely we have generated and extracted RNA from random BM biopsy (BM-BX) and focal lesion (FL-BX) samples that contain all cellular populations and factors of the marrow, including malignant plasma cells. By comparison of global gene expression profiling (GEP) derived from cultured MSCs, purified MM cells, and buffy coat BM samples as a source of hematopoietic cells, we identified a set of 485 probe sets encoding 350 specific MSC genes.GEP were available from baseline BM-BX from healthy donors, patients with MGUS, SMM, and presenting cases of GEP70 low-risk (LR, n=467) and high-risk (HR, n=64) MM. Also available are paired BM-BX and FL-BX cases (n=77). To address the post-treatment role of signatures on outcome, BM-BX were taken during the first year of maintenance (n=161) and complete remission (n=132).Associations between expression of MSC genes and progression-free survival (PFS) was assessed in Total Therapy (TT) 3-5 patients of whom 2/3 were randomly selected for training set and 1/3 used for test set.Immunohistochemistry for EDNRA and IGFBP2 was performed in bone biopsy sections. Cell lysates and conditioned media from normal MSCs cultured alone or preconditioned with HR MM cells underwent GEP and proteomics analysis (n=4).ResultsOur analysis identified 61 and 178 MSC probe sets differentially expressed (FDR<0.05) between HR and LR patients and between random BM and FL sites, respectively. 43 probe sets were commonly changed in these two analyses highlighting the role of FL as a high risk niche. The top overexpressed MSC genes in BM-BX from HR patients were markers of pericytes and basement membrane ECM (EDNRA, COL4A1), whereas underexpressed genes were related to osteoblast (NPR3, ITGBL1, COL1A) and adipocyte (IGFBP2) activity. Expression of these genes in BM-BX gradually changed between healthy donors, MGUS/AMM, LR MM, HR MM and FL biopsies.We developed a gene-score signature combining expression of 3 MSC genes (COL4A1, NPR3, ITGBL1), which was highly associated with outcome. A high 3-MSC gene-score signature at baseline was associated with adverse clinical and molecular parameters, ISS stage, and with shorter PFS in training (p<0.0002) and test (p<0.02) sets. GEP70 LR patients with a high MSC-gene score had shorter PFS compared to GEP70 LR patients with low MSC gene-score in the training set (p<0.008), whereas the outcome of GEP70 HR patients was not impacted by the MSC gene-score. A high 3-MSC gene-score was also detected in 26% of MGUS/SMM patients which predicted a time to therapy in SMM patients (p<0.08).To define a favorable impact of post-treatment score we analyzed BM-BX taken during the 1st year of maintenance, a standardized landmark time point in our Total Therapy protocol for newly diagnosed patients. We identified 3 MSC genes with highest hazard ratio (RBPMS, TGM2 and NEXN); the first two genes are involved in activated TGFβ pathway. Maintenance-based 3-MSC gene-score was associated with disease progression from maintenance therapy landmark in training (p<0.0008) and test (p<0.017) sets and could effectively differentiate GEP70 LR MM patients who are at risk for early relapse in both sets of data (p<0.0001).We confirmed the gene expression data at the protein level using immunohistochemistry and identify EDNRA+ mesenchymal-like cells at a higher number in bones from MM vs. MGUS/SMM (p<0.0003) and between HR vs. LR (p<0.03). In contrast, IGFBP2 was highly expressed in immature adipocytes and its expression was lower in MM than SMM samples. In MSCs preconditioned with MM cells, EDNRA increased by 5 fold whereas NPR3, ITGBL1 and IGFBP2 decreased by 3 fold compared to control MSCs. Levels of the secreted factors, IGFBP2 and ITGBL1, were reduced in conditioned media from preconditioned MSCs compared to control MSCs.ConclusionMSC lineages and factors play a pivotal role in forming the MM niche and mediate the transition from asymptomatic to symptomatic stage and high risk. DisclosuresBarlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Next-Generation Sequencing Based Minimal Residual Disease Assessment in Peripheral Blood RNA from Multiple Myeloma Patients

Introduction: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow, and related organ or tissue impairment. MM patients are monitored during and after therapy using immunoglobulin, M-protein and free light chain assays. Minimal residual disease (MRD) assessment of bone marrow samples from MM patients in complete remission (CR) using next-generation sequencing (NGS)-based technology has been shown to have prognostic value, and previous studies have demonstrated evidence of circulating myeloma cells in peripheral blood. In this prospective study, we compared the sensitivity of RNA- and DNA-based MRD assays in peripheral blood (PB) and bone marrow (BM) samples collected from MM patients.Methods: Matched BM and PB samples were obtained from 50 MM patients in various stages of their disease. Diagnostic BM samples were used to identify clonal immunoglobulin sequences (i.e. clonotypes) unique to each MM patient. Matched BM and PB samples were then assessed for the presence of the MM clonotype. Using universal primer sets, we amplified immunoglobulin (IgH) variable (V), diversity (D), and joining (J) gene segments, the incomplete IgH-DJ rearrangement, and IgK receptors. Genomic DNA samples were assessed for all 3 receptors, while RNA samples were only assessed for the functional IgH-VDJ and IgK receptors. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype quantification. Myeloma-specific IgH, IgK, and IgH-DJ clonotypes were identified for each patient based on their high frequency at diagnosis. The presence of the myeloma clonotype was then assessed in BM cells (DNA), PB cells (DNA and RNA), and PB plasma (DNA) samples. Myeloma clonotype levels were calculated as previously described (Martinez-Lopez et al. 2014).Results: High-frequency myeloma clonotypes were identified in the BM of all 50 patients. We performed a sequence-level assessment of each clonotype to determine whether it was also evaluable in RNA. Non-functional clonotypes (e.g. IgH-DJ and kappa deleting element clones, frameshift/nonsense mutations, nonfunctional V or J segments, loss of cysteine in CDR3) were excluded from the analysis. 37 evaluable RNA clonotypes were identified in 28 of the 50 patients (56%).We compared the sensitivity of MRD assessment using DNA and RNA extracted from PB mononuclear cells. 20 clonotypes were qualitatively concordant in both DNA and RNA. 17 clonotypes were discordant, with all 17 clonotypes being detectable in RNA but not in DNA (Figure 1A). Only 4 of 21 (19%) clonotypes detectable in RNA were also detectable in DNA in the PB. Thus, RNA provides a clear sensitivity advantage over DNA analysis in PB cellular samples.We then investigated whether the sensitivity advantage conferred by RNA in PB cells was equivalent or superior to the sensitivity of DNA analysis in BM cells. On a patient level, 23 patients were qualitatively concordant in the PB and BM assays. 5 patients were discordant, with all patients being positive in BM and negative in PB. 4 of the discordant patients were positive at low levels in the BM (1-10 MM clonotype molecules per 1 million cells). On a clonotype level, 28 clonotypes were qualitatively concordant in BM DNA and PB RNA. 9 clonotypes were discordant, with all 9 clonotypes being positive in BM DNA and negative in PB RNA (Figure 1B). Nevertheless, PB RNA detected MRD in 21 of 30 with measurable disease in the BM DNA (ie, 70% sensitivity). These results demonstrate that MRD assessment using PB RNA is more informative than PB DNA in MM; however, despite the sensitivity improvement provided by RNA, MRD assessment in the BM DNA remains the superior sample source for MRD assessment, particularly when achievement of MRD negativity is the goal of therapy.Conclusions: NGS-based MRD assessment of BM in myeloma patients has been shown to have prognostic value. PB-based MRD assessment would improve clinical MRD assessment and monitoring paradigms. Our results demonstrate that RNA provides increased sensitivity for blood-based MRD assessment. Additional assay optimization to improve the fraction of clonotypes evaluable in RNA and to enhance sensitivity compared with BM is necessary before PB MRD monitoring in MM patients can be incorporated into routine clinical practice. [Display omitted] DisclosuresWolf:Telomere Diagnostics: Consultancy; Pharmacyclics: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Bayes:Adaptive Biotechnologies Corp: Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Martin:Sanofi: Research Funding; Amgen: Research Funding.