Human IL-6 fosters long-term engraftment of patient-derived disease-driving myeloma cells in immunodeficient mice.

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned human IL-6-transgenic (hIL-6-transgenic) NSG (NSG+hIL6) mice reliably support the engraftment of malignant and premalignant human plasma cells, including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and postrelapse myeloma, plasma cell leukemia, and amyloid light chain amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single-cell RNA sequencing showed nonmalignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma-engrafted mice given CAR T cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient-derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

Bone marrow plasma cells require P2RX4 to sense extracellular ATP.

Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.

Human IL-6 fosters long-term engraftment of patient derived disease-driving myeloma cells in immunodeficient mice.

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy.

ABSTRACT: Patients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9-mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.

Chimeric Antigen Receptor T Cells in the Treatment of Multiple Myeloma.

Chimeric antigen receptor T cells (CARTs) represent another powerful way to leverage the immune system to fight malignancy. Indeed, in multiple myeloma, the high response rate and duration of response to B cell maturation antigen-targeted therapies in later lines of disease has led to 2 Food and Drug Administration (FDA) drug approvals and opened the door to the development of this drug class. This review aims to provide an update on the 2 FDA-approved products, summarize the data for the most promising next-generation multiple myeloma CARTs, and outline current challenges in the field and potential solutions.

Combined epigenetic and immunotherapy for blastic and classical mantle cell lymphoma.

Classical MCL (cMCL) constitutes 6-8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870 CCND1 polymorphism is predictive of blastic disease, nuclear localization of cyclinD1 and response to SCR therapy. The major resistance mechanisms to SCR therapy are loss of CD20 expression and evasion of treatment by sanctuary in the CNS. These data indicate that administration of epigenetic agents improves efficacy of anti-CD20 immunotherapies. This approach is promising in the treatment of MCL and potentially other previously treatment refractory cancers.

Combination epigenetic and immunotherapy overcomes resistance to monoclonal antibodies in hematologic malignancies: A new therapeutic approach.

We recently reported that addition of epigenetic agents could overcome resistance of leukemic cells to monoclonal antibody-mediated anti-tumor effects in T-cell prolymphocytic leukemia. We also reported that epigenetic agents could induce expression of the CD30 gene, thus providing a therapeutic target for the antibody drug conjugate brentuximab vedotin. Here we discuss these findings and their generality to treatment of other hematologic and solid malignancies.

Epigenetic therapy overcomes treatment resistance in T cell prolymphocytic leukemia.

T cell prolymphocytic leukemia (T-PLL) is a rare, mature T cell neoplasm with distinct features and an aggressive clinical course. Early relapse and short overall survival are commonplace. Use of the monoclonal anti-CD52 antibody alemtuzumab has improved the rate of complete remission and duration of response to more than 50% and between 6 and 12 months, respectively. Despite this advance, without an allogeneic transplant, resistant relapse is inevitable. We report seven complete and one partial remission in eight patients receiving alemtuzumab and cladribine with or without a histone deacetylase inhibitor. These data show that administration of epigenetic agents can overcome alemtuzumab resistance. We also report epigenetically induced expression of the surface receptor protein CD30 in T-PLL. Subsequent treatment with the anti-CD30 antibody-drug conjugate brentuximab vedotin overcame organ-specific (skin) resistance to alemtuzumab. Our findings demonstrate activity of combination epigenetic and immunotherapy in the incurable illness T-PLL, particularly in the setting of previous alemtuzumab therapy.

Suberoylanilide hydroxamic acid (SAHA) and cladribine synergistically induce apoptosis in NK-LGL leukaemia.

Natural killer (NK) large granular lymphocyte (LGL) leukaemia features a clonal proliferation of CD3(-) NK cells that can be classified into either aggressive or chronic categories. The NKL cell line, derived from an aggressive Asian NK cell leukaemia, and patient samples from chronic NK-LGL leukaemia were used in our study to probe for synergistic efficacy of the epigenetic drugs vorinostat (SAHA) and cladribine in this disease. We demonstrate that histone deacetylases (HDACs) are over-expressed in both aggressive and chronic NK leukaemia. Administration of the HDAC inhibitor SAHA reduces class I and II HDAC expression and enhances histone acetylation in leukaemic NK cells. In vitro combination treatment with SAHA and cladribine dose-dependently exerts synergistic cytotoxic and apoptotic effects on leukaemic NK cells. Expression profiling of apoptotic regulatory genes suggests that both compounds led to caspase-dependent apoptosis through activation of intrinsic mitochondrial and extrinsic death receptor pathways. Collectively, these data show that combined epigenetic therapy, using HDAC and DNA methyltransferase inhibitors, may be a promising therapeutic approach for NK-LGL leukaemia.

Vorinostat downregulates CD30 and decreases brentuximab vedotin efficacy in human lymphocytes.

With an increasing number of clinical trials looking at combination therapies in cancer, potential drug-drug interactions require particular attention. One such instance is the treatment of CD30(+) tumors after previous vorinostat (SAHA; suberoylanilide hydroxyamic acid) failure with the anti-CD30 antibody-drug conjugate brentuximab vedotin. Using B-, T-, and natural killer (NK)-cell lines in vitro, we demonstrate that SAHA downregulates the expression of CD30 and lowers the efficacy of subsequent brentuximab vedotin treatment if baseline CD30 levels are reduced by 50% or more. Interestingly, low-dose SAHA treatment that maintained 50% or more of basal CD30 expression followed by subsequent treatment with brentuximab vedotin led to enhanced antitumor activity. The downregulation of CD30 was short lived upon SAHA removal, suggesting that allowing SAHA washout may circumvent any interactions with subsequent drug therapies. Our findings confirm the requirement of CD30 for brentuximab vedotin efficacy and suggest that combination treatment with SAHA in CD30(dim) tumors may decrease efficacy. Combination treatment in highly CD30(+) tumors, however, increases efficacy and warrants further consideration as a new treatment paradigm.

Does IL-15 have a causative role in large granular lymphocyte leukemia?

Evaluation of: Mishra A, Liu S, Sams GH et al. Aberrant overexpression of IL-15 initiates large granular lymphocyte leukemia through chromosomal instability and DNA hypermethylation. Cancer Cell 22(5), 645-655 (2012). There is increasing evidence identifying a link between inflammation and cancer. A potent proinflammatory cytokine, IL-15, stimulates the proliferation and maintenance of both NK and T cells, and it is therefore likely that it may play a prominent role in certain hematologic malignancies. Previous studies have demonstrated that IL-15 overexpression can initiate leukemic transformation in murine models and that both NK- and T-cell malignancies can develop; the mechanism is explored in this article. The authors illustrate that IL-15 can cause chromosomal instability and DNA hypermethylation in large granular lymphocytes. These aberrations led to an aggressive acute large granular lymphocyte leukemia. Through studying the affected pathways, the authors were able to identify potential therapeutic targets and induce remission in a murine model.