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The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies.Significance:ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Supplementary Figure 1 showing ROBO1 expression in primary MM cells versus normal donor bone marrow plasma cells via gene expression profiling and immunohistochemistry. Supplementary Figure 2 showing distribution of ROBO1 expression in the CoMMpass database; ROBO1 expression in CoMMpass database according to cytogenetics; ROBO1 expression in IFM170 database according to cytogenetics; ROBO1 expression in t(4;14) cell lines WT or KO for MMSET. Supplementary Figure 3 showing ROBO1 protein down regulation after ROBO1 knock down via shRNA. Supplementary Figure 4 showing effects of shRNA4- and shRNA-5 mediated ROBO1 KD on cell viability across a panel of MM cell lines with western confirming proper protein down regulation. Supplementary Figure 5 showing successful ROBO1 KO across several KMS11 monoclones, but not H929 monoclones. Supplementary Figure 6 showing expression of ROBO1 FL construct in polyclonally and monoclonally-transduced OPM2 cells. Supplementary Figure 7 showing expression of FL ROBO1 addback in distinct KMS11 ROBO1 KO monoclones. Supplementary Figure 8 showing radiographic images of WT versus KO ROBO1 mu-SCID mice. Supplementary Figure 9 showing adhesion defect in ROBO1 KO KMS11 cells that is fully rescued by FL ROBO1 addback. Supplementary Figure 10 showing Kaplan-Meier survival curve of disseminated mouse model injected with ROBO1 KO versus FL OPM2 cells. Supplementary Figure 11 showing cleaved cytosolic ROBO1 fragments in 293T cells transduced with FL ROBO1 tagged with C-LAP tag and in WT MM.1S cells. Supplementary Figure 12 showing interacting partners of ROBO1 cytosolic domain as deconvoluted via STRING database. Supplementary Figure 13 showing differentially expressed genes in ROBO1 WT versus KO plasmacytoma represented via Volcano plot. Supplementary Figure 14 showing gene set enrichment analysis of differentially expressed genes in ROBO1 WT versus KO plasmacytoma. Supplementary Figure 15 showing schema of patient-derived ROBO1 mutation and expression of G674 truncation in WT and KO ROBO1 OPM2 cells. Supplementary Table 1 showing clinical annotation of newly diagnosed MM parents used in Supplementary figure 1.

Supplementary Methods

Supplementary Figure from Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma

Supplementary Data from Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma

Supplementary Table from Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma

Purpose:Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model.Experimental Design:We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment.Results:Patients with >0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P 20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio >20, >2 g/dL M-protein, and >0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN− nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient’ stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years.Conclusions:This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.

Therapy-related myeloid neoplasm (t-MN) is a threatening complication of autologous stem cell transplantation (ASCT). Detecting clonal hematopoiesis (CH) mutations in cryopreserved cells before ASCT has been associated with a higher risk of t-MN, but the evolution of molecular abnormalities from pre-ASCT to t-MN, within the same patient, remains to be elucidated. We evaluated the mutational profile of 19 lymphoma/myeloma patients, at both pre-ASCT and t-MN diagnosis, using a targeted NGS approach; 26 non-developing t-MN control patients were also studied pre-ASCT. At ASCT, we found a higher frequency of CH in patients developing t-MN (58%) than in those who did not (23%) (P = 0.029); mutations in epigenetic (DNMT3A, TET2, and ASXL1) and DNA repair genes (PPM1D, RAD21, TP53, and STAG2) were the most represented. At t-MN, CH increased to 82% of patients. Cumulative mutational burden and variant allele frequency (VAF) also increased at t-MN. CH clones detected at ASCT were found at t-MN in eight out of 16 patients, mainly with stable VAF. Among the new driver mutations appeared at t-MN, TP53 increased from one to 13 mutations, in nine patients; being associated with complex karyotype. Mutations in transcription factor (RUNX1, CEBPA) and intracellular signaling genes (FLT3, RAS genes) also increased from three to 17 mutations in eight patients, presenting with a normal karyotype. Overall, we found that preexisting CH at ASCT rarely causes t-MN directly, but may rather facilitate the appearance of new mutations, especially those involving TP53, RUNX1, and RAS, that can drive the evolution to t-MN of at least two distinct types.

Supplementary Figures S1-S3.

PDF file, 77K, An anti-cMET neutralizing antibody leads to modulation of phospho(p)-proteins in MM cell lines.

PDF file, 121K, SU11274 inhibits p-cMET expression and chemotaxis toward fibroblasts' conditioned medium more intensely in R5 cells.

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Single file with supplemental figures, methods, tables, legends: Figure S1: Effects of FGF blockade in MM; Figure S2: FGF inhibition induces oxidative stress-mediated apoptosis in MM cells; Figure S3: Mitochondrial oxidative stress is responsible for the antitumor activity exerted in vivo by FGF inhibition; Figure S4: Oxidative stress-induced apoptosis following FGF inhibition is c-Myc-dependent; Figure S5: Western blot analysis for caspase 3 activation in PBMCs compared to NSC12-sensitive patient-derived MM cells treated with NSC12 for 12 hours; Figure S6: Effects of FGF inhibition in BTZ-resistant KMS-11 cells; Figure S7: High doses of BTZ hamper basal and NSC12-induced c-Myc degradation; Table S1: List of Log2 2-fold down- or upregulated genes in KMS-11 cells after 6 and 12 hours of NSC12 6μM treatment; Table S2: Patient-derived MM cells treated with NSC12; Reagent table.

Purpose: The TORC1 inhibitor everolimus has previously shown significant activity as a single agent in hematologic malignancies, with reported responses of 30% to 70% in Waldenstrom macroglobulinemia. However, the specific mechanisms by which this class of mTOR inhibitors exerts anti–Waldenstrom macroglobulinemia activity have not been fully investigated. We therefore sought to dissect the mechanisms of everolimus-dependent modulation of Waldenstrom macroglobulinemia cell survival.Experimental Design: We confirmed that everolimus targets mTOR in patients treated with everolimus and responding to therapy. We evaluated the effect of everolimus on proliferation and survival of primary Waldenstrom macroglobulinemia cells, as well as of other IgM-secreting lymphoma cell lines. Everolimus-dependent mechanisms of induced apoptosis and its effect on Waldenstrom macroglobulinemia cells in the context of bone marrow microenvironment have been also evaluated. miRNA-155 loss-of-function studies were conducted. Moreover, the combinatory effect of bortezomib and rituximab has been tested.Results: We showed that everolimus targeted mTOR downstream signaling pathways, ex vivo, in patients responding to everolimus treatment. Everolimus induced toxicity in primary Waldenstrom macroglobulinemia cells, as well as in other IgM-secreting lymphoma cells, supported by cell-cycle arrest and caspase-dependent and -independent induction of apoptosis. Importantly, everolimus targeted Waldenstrom macroglobulinemia cells even in the context of bone marrow milieu, where it affected migration, adhesion, and angiogenesis. Everolimus-dependent anti–Waldenstrom macroglobulinemia activity was partially driven by miRNA-155. Moreover, everolimus synergized with bortezomib and rituximab in targeting Waldenstrom macroglobulinemia cells, as shown by synergistic inhibition of p65/ and p50/NF-κB activities.Conclusions: These findings provide a better understanding of the mechanisms that are responsible for everolimus-induced anti–Waldenstrom macroglobulinemia activity. Clin Cancer Res; 18(24); 6609–22. ©2012 AACR.

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

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Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

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

Purpose: Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.Experimental Design: We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.Results: We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G1 cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.Conclusions: Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)

PDF file, 104K, Effect of SU11274 treatment on cMET/phospho(p)-cMET expression in R5 vs. RPMI8226 cells; and plasma cells (PCs) from relapsed/resistant vs. newly diagnosed MM patients.

Purpose: The aim of the study was to verify the hypothesis that the cMet oncogene is implicated in chemio- and novel drug resistance in multiple myeloma.Experimental Design: We have evaluated the expression levels of cMET/phospho-cMET (p-cMET) and the activity of the novel selective p-cMET inhibitor (SU11274) in multiple myeloma cells, either sensitive (RPMI-8226 and MM.1S) or resistant (R5 and MM.1R) to anti–multiple myeloma drugs, in primary plasma cells and in multiple myeloma xenograft models.Results: We found that resistant R5 and MM.1R cells presented with higher cMET phosphorylation, thus leading to constitutive activation of cMET-dependent signaling pathways. R5 cells exhibited a higher susceptibility to the SU11274 inhibitory effects on viability, proliferation, chemotaxis, adhesion, and to its apoptogenic effects. SU11274 was able to revert drug resistance in R5 cells. R5 but not RPMI-8226 cells displayed cMET-dependent activation of mitogen-activated protein kinase pathway. The cMET and p-cMET expression was higher on plasma cells from patients with multiple myeloma at relapse or on drug resistance than on those from patients at diagnosis, complete/partial remission, or from patients with monoclonal gammopathy of unknown significance. Viability, chemotaxis, adhesion to fibronectin or paired bone marrow stromal cells of plasma cells from relapsed or resistant patients was markedly inhibited by SU11274. Importantly, SU11274 showed higher therapeutic activity in R5- than in RPMI-8226–induced plasmocytomas. In R5 tumors, it caused apoptosis and necrosis and reverted bortezomib resistance.Conclusion: Our findings suggest that the cMET pathway is constitutively activated in relapsed and resistant multiple myeloma where it may also be responsible for induction of drug resistance, thus providing the preclinical rationale for targeting cMET in patients with relapsed/refractory multiple myeloma. Clin Cancer Res; 19(16); 4371–82. ©2013 AACR.

PDF file, 91K, Functional analysis of differentially expressed genes in RPMI8226 or R5 cells after SU11274 treatment.

Multiple myeloma, the second most common hematologic malignancy, frequently relapses because of chemotherapeutic resistance. Fibroblast growth factors (FGF) act as proangiogenic and mitogenic cytokines in multiple myeloma. Here, we demonstrate that the autocrine FGF/FGFR axis is essential for multiple myeloma cell survival and progression by protecting multiple myeloma cells from oxidative stress–induced apoptosis. In keeping with the hypothesis that the intracellular redox status can be a target for cancer therapy, FGF/FGFR blockade by FGF trapping or tyrosine kinase inhibitor impaired the growth and dissemination of multiple myeloma cells by inducing mitochondrial oxidative stress, DNA damage, and apoptotic cell death that were prevented by the antioxidant vitamin E or mitochondrial catalase overexpression. In addition, mitochondrial oxidative stress occurred as a consequence of proteasomal degradation of the c-Myc oncoprotein that led to glutathione depletion. Accordingly, expression of a proteasome-nondegradable c-Myc protein mutant was sufficient to avoid glutathione depletion and rescue the proapoptotic effects due to FGF blockade. These findings were confirmed on bortezomib-resistant multiple myeloma cells as well as on bone marrow–derived primary multiple myeloma cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from patients with high-risk multiple myeloma. Altogether, these findings dissect the mechanism by which the FGF/FGFR system plays a nonredundant role in multiple myeloma cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for patients with multiple myeloma with poor prognosis and advanced disease stage.Significance:This study provides new insights into the mechanisms by which FGF antagonists promote multiple myeloma cell death.

PDF file, 113K, Effect of SU11274 treatment on PCs of newly diagnosed MM patients.

PDF file, 97K, SU11274 impacts marginally on in vivo growth of RPMI8226 plasmocytomas. It reverts bortezomib-resistance in R5 plasmocytomas.

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

Purpose: Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.Experimental Design: We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.Results: We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.Conclusions: These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas. Clin Cancer Res; 17(7); 1753–64. ©2011 AACR.

PDF file, 93K, FACS analysis of cMET/phospho(p)-cMET expression in MM.1R vs. MM.1S cells.

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

DiD- and DiO-labeled MM.1S cells do not differ for their ability to migrate to the CHT niches.

Advancement of many solid tumors and hematologic malignancies is frequently characterized by dissemination and homing of cancer cells to the bone marrow (BM). Methods to quantitatively characterize these key steps of the metastatic cascade in mammalian models are currently limited and do not offer opportunities to perform rapid, large-scale genomic, or drug screening. Because of their optical clarity, we used zebrafish to develop an in vivo model of cancer cell dissemination and homing to the BM. We performed intracardiac injection of multiple myeloma (MM) cells derived from human BM or cell lines and monitored their migration to the caudal hematopoietic tissue (CHT), the region where hematopoiesis occurs in the zebrafish embryo, which recapitulates a BM-like niche. Transcriptomic analyses confirmed that MM cells homing to the CHT displayed gene-expression differences compared with MM cells outside of the CHT, including significant enrichment for genes known to regulate interleukin-6 (IL6) signaling, cell adhesion, and angiogenesis. Collectively, our findings point to the zebrafish as a valuable model in which to study cancer cell homing to the hematopoietic niche and to establish a screening platform for the identification of factors and mechanisms contributing to the early steps of bone metastasis. Cancer Res; 76(2); 463–71. ©2016 AACR.

Legends for Supplemental Table S1 and Supplemental Figures S1-S3.

Quality control of RNA after human exon enrichement.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB–mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell–to–T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell–Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.