Systematic characterization of therapeutic vulnerabilities in Multiple Myeloma with Amp1q reveals increased sensitivity to the combination of MCL1 and PI3K inhibitors.

The development of targeted therapy for patients with Multiple Myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chr1q (Amp1q) is the most frequent arm-level copy number gain in patients with MM, and it is associated with higher risk of progression and death despite recent advances in therapeutics. Thus, developing targeted therapy for patients with MM and Amp1q stands to benefit a large portion of patients in need of more effective management. Here, we employed large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with Amp1q and showed increased sensitivity to the combination of MCL1 and PI3K inhibitors. Using single-cell RNA sequencing, we compared subclones with and without Amp1q within the same patient tumors and showed that Amp1q is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number for part of the chr1q arm, we showed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with Amp1q.

Clonal hematopoiesis is associated with adverse outcomes in multiple myeloma patients undergoing transplant.

Multiple myeloma (MM) is a plasma-cell neoplasm that is treated with high-dose chemotherapy, autologous stem cell transplant (ASCT) and long-term immunomodulatory drug (IMiD) maintenance. The presence of somatic mutations in the peripheral blood is termed clonal hematopoiesis of indeterminate potential (CHIP) and is associated with adverse outcomes. Targeted sequencing of the stem cell product from 629 MM patients treated by ASCT at the Dana-Farber Cancer Institute (2003-2011) detects CHIP in 136/629 patients (21.6%). The most commonly mutated genes are DNMT3A, TET2, TP53, ASXL1 and PPM1D. Twenty-one from fifty-six patients (3.3%) receiving first-line IMiD maintenance develop a therapy-related myeloid neoplasm (TMN). However, regardless of CHIP status, the use of IMiD maintenance associates with improved PFS and OS. In those not receiving IMiD maintenance, CHIP is associated with decreased overall survival (OS) (HR:1.34, p = 0.02) and progression free survival (PFS) (HR:1.45, p < 0.001) due to an increase in MM progression.

Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK+ memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14+ monocytes, which results in T cell suppression in vitro. These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.

Profiling of circulating exosomal miRNAs in patients with Waldenström Macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by disease progression from IgM MGUS to asymptomatic and then symptomatic disease states. We profiled exosomes from the peripheral blood of patients with WM at different stages (30 smoldering/asymptomatic WM, 44 symptomatic WM samples and 10 healthy controls) to define their role as potential biomarkers of disease progression. In this study, we showed that circulating exosomes and their miRNA content represent unique markers of the tumor and its microenvironment. We observed similar levels of miRNAs in exosomes from patients with asymptomatic (smoldering) and symptomatic WM, suggesting that environmental and clonal changes occur in patients at early stages of disease progression before symptoms occur. Moreover, we identified a small group of miRNAs whose expression correlated directly or inversely with the disease status of patients, notably the known tumor suppressor miRNAs let-7d and the oncogene miR-21 as well as miR-192 and miR-320b. The study of these miRNAs' specific effect in WM cells could help us gain further insights on the mechanisms underlying WM pathogenesis and reveal their potential as novel therapeutic targets for this disease.

Inhibition of microRNA-138 enhances bone formation in multiple myeloma bone marrow niche.

Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.

Safety and immunogenicity of conjugate quadrivalent meningococcal vaccination after hematopoietic cell transplantation.

Immunization with the conjugated quadrivalent (serogroups A, C, Y, and W-135) meningococcal vaccine (MCV4) after hematopoietic cell transplantation (HCT) is recommended. However, immune responses to MCV4 have not been prospectively studied after HCT. We conducted a vaccine response study among 67 adults who received 1 MCV4 dose a year after autologous or allogeneic HCT from January to September 2014. Pre- and postvaccination serogroup serum bactericidal antibody (SBA) titers were measured a median of 57 days after vaccination. Serogroup-specific responses were defined as a fourfold increase in SBA titer with postvaccination titers ≥1:8. Prior to vaccination, 44 (65.7%) patients had no protective titers (<1:8) to any meningococcal serogroup, and 3 (4.5%) patients had protective titers to all 4 serogroups. The median serogroup-specific postvaccination SBA titers were 1:2048 for A, 1:64 for C, 1:128 for W-135, and 1:128 for Y (P < .001 for all pre- and postvaccination pairwise comparisons; similar among serogroups, Spearman ρ 0.5-0.6, P < .0001). Among serogroup-specific nonimmune patients prior to vaccination, serogroup-specific response rates were 76.9%, 65.5%, 51.7%, and 65% to serogroups A, C, W-135, and Y, respectively. One dose of MCV4 elicited protective titers in the majority of patients. These data suggest that a second vaccine dose may be beneficial.

Antibody-Dependent Cellular Phagocytosis by Macrophages is a Novel Mechanism of Action of Elotuzumab.

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.

Blocking IFNAR1 inhibits multiple myeloma-driven Treg expansion and immunosuppression.

Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and ß receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.

Platelets Enhance Multiple Myeloma Progression via IL-1ß Upregulation.

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-1ß was upregulated in MM cell lines co-cultured with platelets, whereas IL-1ß knockout in MM cell lines abrogated the effects of platelets on MM cell proliferation and engraftment in vivoConclusions: Platelets from MM patients were highly activated with disease progression. IL-1ß 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.

Metformin Affects Cortical Bone Mass and Marrow Adiposity in Diet-Induced Obesity in Male Mice.

Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our objective was to determine how a high-fat diet with or without metformin treatment affects skeletal development. We also sought to characterize changes that occur in white adipose tissue, circulating metabolites, lipids, and gut microbiota. A diet-induced obesity C57BL/6J mouse model was used to test the effects of obesity and metformin on bone using bone histomorphometry and microcomputed tomography. Bone marrow adipose tissue was quantified with osmium tetroxide microcomputed tomography and histology. Dual-energy x-ray absorptiometry was used to analyze body composition. Hematoxylin and eosin staining was used to assess changes in white adipose depots, mass spectrometry was used for circulating lipids and protein metabolite analysis, and ribosomal RNA sequencing was used for gut microbiome analysis. Mice fed a high fat-diet since wean displayed increased medullary areas and decreased osteoblast numbers in the long bones; this phenotype was partially normalized by metformin. Marrow and inguinal adipose expansion was also noted in obese mice, and this was partially normalized by metformin. A drug-by-diet interaction was noted for circulating lipid molecules, protein metabolites, and gut microbiome taxonomical units. Obesity was not detrimental to trabecular bone in growing mice, but bone marrow medullary expansion was observed, likely resulting from inhibition of osteoblastogenesis, and this was partially reversed by metformin treatment.

A novel in vivo model for studying conditional dual loss of BLIMP-1 and p53 in B-cells, leading to tumor transformation.

The tumor suppressors B-lymphocyte-induced maturation protein-1 (BLIMP-1) and p53 play a crucial role in B-cell lymphomas, and their inactivation contributes to the pathogenesis of a wide spectrum of lymphoid malignancies, including diffuse large B-cell lymphomas (DLBCLs). Patients with activated B-cell-like (ABC) DLBCL may present with loss of BLIMP-1, c-Myc over-expression, decreased p53, and poor prognosis. Nevertheless, there is a lack of in vivo models recapitulating the biology of high-grade ABC DLBCL. We therefore aimed to develop an in vivo model aiming to recapitulate the phenotype observed in this cohort of patients. A Cre-Lox approach was used to achieve inactivation of both p53 and BLIMP-1 in murine B-cells. Contextual ablation of BLIMP-1 and p53 led to development of IgM-positive B-cell lymphoma with an aggressive phenotype, supported by c-Myc up-regulation, and accumulation of somatic mutations, as demonstrated by whole exome sequencing. Sensitivity of B-tumor cells to BTK inhibition was demonstrated. This model mirrors what reported in patients with ABC DLBLC, and therefore represents a novel model for studying the biology of ABC-DLBCL harboring the dual loss of BLIMP-1/p53 and c-Myc over-expression.

Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a frequently incurable hematological cancer in which overactivity of MYC plays a central role, notably through up-regulation of ribosome biogenesis and translation. To better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small-molecule library for anti-MM activity. The most potent hits identified were rocaglate scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates.

The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma.

The development of sensitive and non-invasive "liquid biopsies" presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs) is prognostic in multiple myeloma (MM), but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM) tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.

Prognostic role of circulating exosomal miRNAs in multiple myeloma.

Exosomes, secreted by several cell types, including cancer cells, can be isolated from the peripheral blood and have been shown to be powerful markers of disease progression in cancer. In this study, we examined the prognostic significance of circulating exosomal microRNAs (miRNAs) in multiple myeloma (MM). A cohort of 156 patients with newly diagnosed MM, uniformly treated and followed, was studied. Circulating exosomal miRNAs were isolated and used to perform a small RNA sequencing analysis on 10 samples and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) array on 156 samples. We studied the relationship between miRNA levels and patient outcomes, including progression-free survival (PFS) and overall survival (OS). We identified miRNAs as the most predominant small RNAs present in exosomes isolated from the serum of patients with MM and healthy controls by small RNA sequencing of circulating exosomes. We then analyzed exosomes isolated from serum samples of 156 patients using a qRT-PCR array for 22 miRNAs. Two of these miRNAs, let-7b and miR-18a, were significantly associated with both PFS and OS in the univariate analysis and were still statistically significant after adjusting for the International Staging System and adverse cytogenetics in the multivariate analysis. Our findings support the use of circulating exosomal miRNAs to improve the identification of patients with newly diagnosed MM with poor outcomes. The results require further validation in other independent prospective MM cohorts.

CXCR4 Regulates Extra-Medullary Myeloma through Epithelial-Mesenchymal-Transition-like Transcriptional Activation.

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines; identified that epithelial-to-mesenchymal transition (EMT) transcriptional patterns were significantly enriched in both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4 silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These results suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

Metabolic signature identifies novel targets for drug resistance in multiple myeloma.

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.

The cancer glycome: carbohydrates as mediators of metastasis.

Glycosylation is a frequent post-translational modification which results in the addition of carbohydrate determinants, "glycans", to cell surface proteins and lipids. These glycan structures form the "glycome" and play an integral role in cell-cell and cell-matrix interactions through modulation of adhesion and cell trafficking. Glycosylation is increasingly recognized as a modulator of the malignant phenotype of cancer cells, where the interaction between cells and the tumor micro-environment is altered to facilitate processes such as drug resistance and metastasis. Changes in glycosylation of cell surface adhesion molecules such as selectin ligands, integrins and mucins have been implicated in the pathogenesis of several solid and hematological malignancies, often with prognostic implications. In this review we focus on the functional significance of alterations in cancer cell glycosylation, in terms of cell adhesion, trafficking and the metastatic cascade and provide insights into the prognostic and therapeutic implications of recent findings in this fast-evolving niche.

Pyk2 promotes tumor progression in multiple myeloma.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumor growth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/ß-catenin signaling by destabilizing ß-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM.

Regulation of microRNAs in cancer metastasis.

Metastasis is a phenomenon of crucial importance in defining prognosis in patients with cancer and is often responsible for cancer-related mortality. It is known that several steps are necessary for clonal cells to disseminate from their primary tumor site and colonize distant tissues, thus originating metastatic lesions. Therefore, investigating the molecular actors regulating this process may provide helpful insights in the development of efficient therapeutic responses. Recent evidences have indicated the role of microRNAs (miRNAs) in modulating the metastatic process in solid tumors. miRNAs are small regulatory non-coding RNAs that bind to specific target mRNAs, leading to translational repression. miRNAs are known to act as negative regulators of gene expression and are involved in the regulation of biological processes, including cell growth, differentiation and apoptosis, both in physiological conditions and during diseases, such as tumors. In the specific field of tumorigenesis, miRNAs play an important role in mediating oncogenesis and favoring tumor progression, as a result of their ability to modulate epithelial-to-mesenchymal transition (EMT) and other series of events facilitating the formation of metastasis. The role of miRNAs in cancer development has been widely studied and has helped elucidate events such as the change in expression of oncogenes, tumor-suppressors and cancer-related proteins. This review focuses on the mechanisms underlying the role of miRNAs as part of the metastatic process.