Fotis Asimakopoulos

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line‐only control. Furthermore, the combined co‐culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.

LMP1-deficient Epstein-Barr virus mutant requires T cells for lymphomagenesis

Epstein-Barr virus (EBV) infection transforms B cells in vitro and is associated with human B cell lymphomas. The major EBV oncoprotein, latent membrane protein 1 (LMP1), mimics constitutively active CD40 and is essential for outgrowth of EBV-transformed B cells in vitro; however, EBV-positive diffuse large B cell lymphomas and Burkitt lymphomas often express little or no LMP1. Thus, EBV may contribute to the development and maintenance of human lymphomas even in the absence of LMP1. Here, we found that i.p. injection of human cord blood mononuclear cells infected with a LMP1-deficient EBV into immunodeficient mice induces B cell lymphomas. In this model, lymphoma development required the presence of CD4+ T cells in cord blood and was inhibited by CD40-blocking Abs. In contrast, LMP1-deficient EBV established persistent latency but did not induce lymphomas when directly injected into mice engrafted with human fetal CD34+ cells and human thymus. WT EBV induced lymphomas in both mouse models and did not require coinjected T cells in the cord blood model. Together, these results demonstrate that LMP1 is not essential for EBV-induced lymphomas in vivo and suggest that T cells supply signals that substitute for LMP1 in EBV-positive B cell lymphomagenesis.

Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies

B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. SIRT3 (sirtuin 3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. In mantle cell lymphoma patient samples, we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities, and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater sensitivity to inhibition of the hypoxia-inducible factor-1α pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but hypoxia-inducible factor-1α-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies.

Single-molecule analysis reveals widespread structural variation in multiple myeloma.

Significance In the last several years, we have seen significant progress toward personalized cancer genomics and therapy. Although we routinely discern and understand genomic variation at single base pair and chromosomal levels, comprehensive analysis of genome variation, particularly structural variation, remains a challenge. We present an integrated approach using optical mapping—a single-molecule, whole-genome analysis system—and DNA sequencing to comprehensively identify genomic structural variation in sequential samples from a multiple myeloma patient. Through our analysis, we have identified widespread structural variation and an increase in mutational burden with tumor progression. Our findings highlight the need to routinely incorporate structural variation analysis at many length scales to understand cancer genomes more comprehensively. Multiple myeloma (MM), a malignancy of plasma cells, is characterized by widespread genomic heterogeneity and, consequently, differences in disease progression and drug response. Although recent large-scale sequencing studies have greatly improved our understanding of MM genomes, our knowledge about genomic structural variation in MM is attenuated due to the limitations of commonly used sequencing approaches. In this study, we present the application of optical mapping, a single-molecule, whole-genome analysis system, to discover new structural variants in a primary MM genome. Through our analysis, we have identified and characterized widespread structural variation in this tumor genome. Additionally, we describe our efforts toward comprehensive characterization of genome structure and variation by integrating our findings from optical mapping with those from DNA sequencing-based genomic analysis. Finally, by studying this MM genome at two time points during tumor progression, we have demonstrated an increase in mutational burden with tumor progression at all length scales of variation.

Autologous Transplantation for Newly Diagnosed Multiple Myeloma in the Era of Novel Agent Induction: A Systematic Review and Meta-analysis

Importance The role of high-dose therapy with melphalan followed by autologous stem cell transplant (HDT/ASCT) in patients with multiple myeloma continues to be debated in the context of novel agent induction. Objective To perform a systematic review, conventional meta-analysis, and network meta-analysis of all phase 3 randomized clinical trials (RCTs) evaluating the role of HDT/ASCT. Data Sources We performed a systematic literature search of Cochrane Central, MEDLINE, and Scopus from January 2000 through April 2017 and relevant annual meeting abstracts from January 2014 to December 2016. The following search terms were used: “myeloma” combined with “autologous,” “transplant,” “myeloablative,” or “stem cell.” Study Selection Phase 3 RCTs comparing HDT/ASCT with standard-dose therapy (SDT) using novel agents were assessed. Studies comparing single HDT/ASCT with bortezomib, lenalidomide, and dexamethasone consolidation and tandem transplantation were included for network meta-analysis. Data Extraction And Synthesis For the random effects meta-analysis, we used hazard ratios (HRs) and corresponding 95% CIs. Main Outcomes and Measures The primary outcome was progression-free survival (PFS). Overall survival (OS), complete response, and treatment-related mortality were secondary outcomes. Results A total of 4 RCTs (2421 patients) for conventional meta-analysis and 5 RCTs (3171 patients) for network meta-analysis were selected. The combined odds for complete response were 1.27 (95% CI, 0.97-1.65; P = .07) with HDT/ASCT when compared with SDT. The combined HR for PFS was 0.55 (95% CI, 0.41-0.74; P < .001) and 0.76 for OS (95% CI, 0.42-1.36; P = .20) in favor of HDT. Meta-regression showed that longer follow-up was associated with superior PFS (HR/mo, 0.98; 95% CI, 0.96-0.99; P = .03) and OS (HR/mo, 0.90; 95% CI, 0.84-0.96; P = .002). For PFS, tandem HDT/ASCT had the most favorable HR (0.49; 95% CI, 0.37-0.65) followed by single HDT/ASCT with bortezomib, lenalidomide, and dexamethasone (HR, 0.53; 95% CI, 0.37-0.76) and single HDT/ASCT alone (HR, 0.68; 95% CI, 0.53-0.87) compared with SDT. For OS, none of the HDT/ASCT-based approaches had a significant effect on survival. Treatment-related mortality with HDT/ASCT was minimal (<1%). Conclusions and Relevance The results of the conventional meta-analysis and network meta-analysis of all the phase 3 RCTs showed that HDT/ASCT was associated with superior PFS with minimal toxic effects compared with SDT. Both tandem HDT/ASCT and single HDT/ASCT with bortezomib, lenalidomide, and dexamethasone were superior to single HDT/ASCT alone and SDT for PFS, but OS was similar across the 4 approaches. Longer follow-up may better delineate any OS benefit; however, is likely to be affected by effective postrelapse therapy.

Tumoricidal Effects of Macrophage-Activating Immunotherapy in a Murine Model of Relapsed/Refractory Multiple Myeloma.

Jensen and colleagues report that inhibition of innate immunity checkpoint TPL2 kinase signaling potentiates the efficacy of anti–CD40-based immunotherapy, which expands M1-polarized macrophages in the bone marrow, prolonging survival in an immunocompetent, transplant-based preclinical model of relapsed/refractory multiple myeloma. Myeloma remains a virtually incurable malignancy. The inevitable evolution of multidrug-resistant clones and widespread clonal heterogeneity limit the potential of traditional and novel therapies to eliminate minimal residual disease (MRD), a reliable harbinger of relapse. Here, we show potent anti-myeloma activity of macrophage-activating immunotherapy (αCD40+CpG) that resulted in prolongation of progression-free survival (PFS) and overall survival (OS) in an immunocompetent, preclinically validated, transplant-based model of multidrug-resistant, relapsed/refractory myeloma (t-Vκ*MYC). αCD40+CpG was effective in vivo in the absence of cytolytic natural killer, T, or B cells and resulted in expansion of M1-polarized (cytolytic/tumoricidal) macrophages in the bone marrow. Moreover, we show that concurrent loss/inhibition of Tpl2 kinase (Cot, Map3k8), a MAP3K that is recruited to activated CD40 complex and regulates macrophage activation/cytokine production, potentiated direct, ex vivo anti-myeloma tumoricidal activity of αCD40+CpG–activated macrophages, promoted production of antitumor cytokine IL12 in vitro and in vivo, and synergized with αCD40+CpG to further prolong PFS and OS in vivo. Our results support the combination of αCD40-based macrophage activation and TPL2 inhibition for myeloma immunotherapy. We propose that αCD40-mediated activation of innate antitumor immunity may be a promising approach to control/eradicate MRD following cytoreduction with traditional or novel anti-myeloma therapies. Cancer Immunol Res; 3(8); 881–90. ©2015 AACR.

Versican-Derived Matrikines Regulate Batf3–Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair–deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow–derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Extracellular matrix and the myeloid‐in‐myeloma compartment: balancing tolerogenic and immunogenic inflammation in the myeloma niche

The last 10–15 years have witnessed a revolution in treating multiple myeloma, an incurable cancer of Ab‐producing plasma cells. Advances in myeloma therapy were ushered in by novel agents that remodel the myeloma immune microenvironment. The first generation of novel agents included immunomodulatory drugs (thalidomide analogs) and proteasome inhibitors that target crucial pathways that regulate immunity and inflammation, such as NF‐κB. This paradigm continued with the recent regulatory approval of mAbs (elotuzumab, daratumumab) that impact both tumor cells and associated immune cells. Moreover, recent clinical data support checkpoint inhibition immunotherapy in myeloma. With the success of these agents has come the growing realization that the myeloid infiltrate in myeloma lesions—what we collectively call the myeloid‐in‐myeloma compartment—variably sustains or deters tumor cells by shaping the inflammatory milieu of the myeloma niche and by promoting or antagonizing immune‐modulating therapies. The myeloid‐in‐myeloma compartment includes myeloma‐associated macrophages and granulocytes, dendritic cells, and myeloid‐derived‐suppressor cells. These cell types reflect variable states of differentiation and activation of tumor‐infiltrating cells derived from resident myeloid progenitors in the bone marrow—the canonical myeloma niche—or myeloid cells that seed both canonical and extramedullary, noncanonical niches. Myeloma‐infiltrating myeloid cells engage in crosstalk with extracellular matrix components, stromal cells, and tumor cells. This complex regulation determines the composition, activation state, and maturation of the myeloid‐in‐myeloma compartment as well as the balance between immunogenic and tolerogenic inflammation in the niche. Redressing this balance may be a crucial determinant for the success of antimyeloma immunotherapies.

MAP3K8 kinase regulates myeloma growth by cell-autonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages

Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase‐dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches. In myeloma tumour cells, MAP3K8 acts as mitogen‐induced MAP3K in mitosis and is required for TNFα‐mediated ERK activation. Pharmacological MAP3K8 inhibition results in dose‐dependent, tumour cell‐autonomous apoptosis despite contact with primary stroma. MAP3K8 blockade may disrupt crucial macrophage‐tumour cell interactions within myeloma niches.

Versican Proteolysis Predicts Immune Effector Infiltration And Post-Transplant Survival In Myeloma

High-dose alkylator-based conditioning followed by autologous stem-cell transplantation (ASCT) is a therapeutic mainstay for eligible patients with multiple myeloma. However, post-transplant relapses are common and prognostic biomarkers are scarce. Relapses are characterized by the influx of regulatory myeloid cells and dysfunctional T effectors. We have shown that myeloma-infiltrating myeloid cells produce versican (VCAN), a large matrix proteoglycan with tolerogenic activities. VCAN proteolysis by a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs (ADAMTS) proteases generates versikine, a bioactive fragment (“matrikine”) that regulates Batf3-dendritic cells, known to control CD8+-attracting chemokine networks. Here we demonstrate that intense VCAN proteolysis predicts CD8+ infiltration post-transplant and paradoxically portends significantly inferior survival outcomes. Our data suggest that VCAN proteolysis promotes the influx of CD8+ effectors that are rendered overwhelmingly dysfunctional and/or frankly immunoregulatory (CD8+ Treg) at the tumor site. Thus, complex immunosuppressive circuits orchestrated through VCAN accumulation and turnover generate conditions favorable for myeloma tumor regrowth and point to a readily-assayed biomarker to identify the patients at risk for relapse and early death. The dismal outcomes associated with VCAN proteolysis may be rationally overcome through immunotherapies such as checkpoint inhibition (e.g., anti-TIGIT), tumor vaccines or anti-myeloid (e.g., anti-CSF-1R) approaches.