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Melphalan prednisone was the mainstay of myeloma treatment in the 1960s with 50% of overall response, yet this treatment was associated with a short duration of response and a poor median overall survival (only 3 years). Thalidomide, initially used as an antiemetic and sedative agent, was demonstrated to have substantial antitumor activity in multiple myeloma and other solid cancers. The first studies established a promising response among patients with relapsed or refractory disease. Melphalan–prednisone–thalidomide is currently the treatment of choice for a large proportion of elderly myeloma patients ineligible for autologous stem cell transplantation. Melphalan prednisone is appropriate only for a minority of patients with poor performance status and/or significant comorbidities. Thalidomide has opened a complete new field of novel agents termed the IMiDs®class of immunomodulatory drugs; of which, lenalidomide (Revlimid®, Celgene Corporation, NJ, USA) and pomalidomide (Actimid™, Celgene Corporation, NJ, USA) are the next and last in line developed agents, respectively. These options could lead to more personalized treatment approaches, based on patient comorbidities, as the novel agents have somewhat different toxicity profiles. Questions regarding the relative efficacy of melphalan-based regimens versus dexamethasone-based regimens with low-dose dexamethasone will require randomized Phase III trials. Additionally, the important issue of maintenance treatment needs to be further investigated. These new and emerging therapies offer multiple effective treatment options for multiple myeloma patients and greatly enhance the treatment strategies for clinicians.

Marie-Dominique Venon2, Aldo M Roccaro1,3, Julie Gay2, Anne-Sophie Moreau1,2, Remy Dulery2, Thierry Facon2, Irene M Ghobrial1, Xavier Leleu1,21Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 2Service des Maladies du Sang, Hopital Huriez, CHRU, Lille, France; 3Units of Blood Diseases and Cell Therapies, University of Bresica, Medical School, Bresica, ItalyAbstract: Melphalan combined with prednisone (MP) has long been the historical treatment of reference for a large proportion of elderly myeloma (MM) patients ineligible for autologous stem cell transplantation, and is still the backbone of new regimens that include the new era of novel agents. Melphalan–prednisone–thalidomide (MPT) and melphalan–prednisone–bortezomib (Velcade®, MPV), proved superior to MP, currently appear to be the treatments of choice for this population. In the near future melphalan–prednisone–lenalidomide (Revlimid®, MPR) will also provide a third therapeutic option (MPT, MPV, and MPR), in elderly multiple myeloma, eventually. These options could lead to more personalized treatment approaches, based on patient comorbidities, as the three novel agents have somewhat different toxicity profiles. Dexamethasone-based regimen is another option and questions regarding the relative efficacy of melphalan-based versus low-dose dexamethasone-based regimens will require randomized phase III trials. More intensive approaches with new drug combinations or with the incorporation of polyethylene glycolated (PEGylated) liposomal doxorubicin will also require additional studies. Additionally, the important issue of maintenance treatment needs to be further investigated. These new and emerging therapies offer multiple effective treatment options for MM patients and greatly enhanced treatment strategies for clinicians.Keywords: multiple myeloma, elderly, bortezomib, thalidomide, revlimid, IMiDs, supportive care

Aldo M Roccaro1, Irene M Ghobrial1, Simona Blotta1, Steven P Treon1, Michele Malagola2, Kenneth C Anderson1, Paul G Richardson1, Domenico Russo21Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 2Unit of Blood Diseases and Cell Therapies, University of Brescia Medical School, Brescia, ItalyAbstract: The paradigm for the treatment of monoclonal gammopaties has dramatically changed: therapeutic options in multiple myeloma (MM) have evolved from the introduction of melphalan and prednisone in the 1960s, high-dose chemotherapy and stem cell transplantation in the late 1980s and 1990s, to the rapid introduction of small novel molecules within the last seven years. Based on the understanding of the complex interaction of the MM cells with the bone marrow microenvironment and the signaling pathways that are dysregulated in this process, a number of novel therapeutic agents are now available. Specifically, three novel agents with a specific-targeted anti-MM activity, have been FDA-approved for the treatment of this disease, namely Bortezomib, thalidomide, and lenalidomide which are now all playing a key role in the treatment of MM. The success of targeted therapy in MM has since led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias such as Waldenström’s macroglobulinemia and primary amyloidosis, both in the preclinical settings and as part of clinical trials.Keywords: monoclonal gammopaties, targeted therapies

Summary Waldenstrom macroglobulinaemia (WM) is an incurable lymphoplasmacytic lymphoma with secretion of serum monoclonal immunoglobulin M (IgM). We previously showed that patients receiving cholesterol-lowering statins, had the lowest IgM value in a large cohort of patients with WM. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19 + WM cells. Interestingly, those effects were reversed by addition of mevalonate and geranylgeranylpyrophosphate, demonstrating that simvastatin inhibited cell growth, survival and IgM secretion on BCWM.1 WM cells by inhibition of geranylgeranylated proteins. Furthermore, simvastatin overcame tumour cell growth induced by co-culture of WM cells with bone-marrow stromal cells. Simvastatin also decreased IgM secretion by BCWM.1 cells at an early time-point that had not affected cell survival. Simvastatin-induced cytotoxicity was preceded by a decrease in Akt (protein kinase B, PKB) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways at 18 h. In addition, simvastatin induced an increase in stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/ JNK) MAPK followed by caspase-8, -9, -3 and poly(ADP-ribose) polymerase (PARP) cleavages at 18 h, leading to apoptosis. Furthermore, simvastatin enhanced the cytotoxicity induced by bortezomib, fludarabine and dexamethasone. Our studies therefore support our earlier observation of statin-mediated anti-WM activity and provide the framework for future clinical trials testing simvastatin in WM.

The paradigm for the treatment of monoclonal gammopaties has dramatically changed: therapeutic options in multiple myeloma (MM) have evolved from the introduction of melphalan and prednisone in the 1960s, high-dose chemotherapy and stem cell transplan- tation in the late 1980s and 1990s, to the rapid introduction of small novel molecules within the last seven years. Based on the understanding of the complex interaction of the MM cells with the bone marrow microenvironment and the signaling pathways that are dysregulated in this process, a number of novel therapeutic agents are now available. Specifi cally, three novel agents with a specifi c-targeted anti-MM activity, have been FDA-approved for the treatment of this disease, namely Bortezomib, thalidomide, and lenalidomide which are now all playing a key role in the treatment of MM. The success of targeted therapy in MM has since led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias such as Waldenstrom's macroglobulinemia and primary amyloidosis, both in the preclinical settings and as part of clinical trials.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

OBJECTIVES: Waldenstrom Macroglobulinemia (WM) is a B-cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta-2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. METHODS: We assessed the serum levels of the total soluble HLA-I molecules and the HLA-Gs molecules in 105 patients with IgM-related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. RESULTS: We found higher levels of HLA-Is in WM, compared to IgM MGUS and healthy donors. HLA-Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA-Is at the cut-off of 1.8 microg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA-Is level was strongly associated with high serum beta2M level >3 mg/L [OR = 2, (CI 95% 1.1-5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5-4.1), P = 0.06] and haemoglobin < or =11.5 g/dL [OR = 3.3, (CI 95% 1.2-9.7); P = 0.03]. High levels of serum HLA-Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM-MGUS status. CONCLUSION: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.

Purpose: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. Experimental Design: MM cell lines were incubated with perifosine (5 and 10 μmol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. Results: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. Conclusions: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Waldenström's macroglobulinemia is a B-cell disorder characterized by bone marrow infiltration with lymphoplasmacytic cells and demonstration of an immunoglobulin M monoclonal gammopathy. Despite advances in therapy, Waldenström's macroglobulinemia remains incurable. As such, novel therapeutic agents are needed for the treatment of Waldenström's macroglobulinemia. In ongoing efforts, we and others have sought to exploit advances made in the understanding of the biology of Waldenström's macroglobulinemia so as to better target therapeutics for this malignancy. Importantly, as part of these efforts, we have prioritized the development of stem cell-sparing drugs because autologous stem cell transplantation remains a viable salvage option in Waldenström's macroglobulinemia. These efforts have led to the development of several novel agents for treating Waldenström's macroglobulinemia, including bortezomib; monoclonal antibodies and/or blocking protein targeting CD40, CD52, or CD70, a proliferation-inducing ligand and B-lymphocyte stimulator; the immunomodulator thalidomide as an enhancer of rituximab activity, as well as agents interfering with stem cell factor, phosphatidylinositol 3-kinase/Akt, phosphodiesterase, cholesterol, and protein kinase C beta signaling. This report provides an update on biologic studies and clinical efforts for the development of these novel agents in the treatment of Waldenström's macroglobulinemia.

Familial aggregation of Waldenstrom macroglobulinemia (WM) cases, and the clustering of B-cell lymphoproliferative disorders among first-degree relatives of WM patients, has been reported. Nevertheless, the possible contribution of inherited susceptibility to familial WM remains unrevealed. We performed whole exome sequencing on germ line DNA obtained from 4 family members in which coinheritance for WM was documented in 3 of them, and screened additional independent 246 cases by using gene-specific mutation sequencing. Among the shared germ line variants, LAPTM5(c403t) and HCLS1(g496a) were the most recurrent, being present in 3/3 affected members of the index family, detected in 8% of the unrelated familial cases, and present in 0.5% of the nonfamilial cases and in

The bone marrow (BM) microenvironment of multiple myeloma (MM) is reported to play a role in the biology of disease. In this study, we found that the extracellular BM microenvironment in MM contains a unique miRNA signature detectable by miRNA microarray and quantitative real-time PCR, which is partially represented in the peripheral blood. Eleven miRNAs were significantly decreased in both BM and serum of MM patients in comparison with controls. Evaluation of these miRNAs in plasma of a separate cohort of MM patients and controls confirmed significantly aberrant levels of let-7a, let-7b, let-7i, miR-15b, miR-16, and miR-20a in both serum and plasma. We then studied the myeloma precursor diseases and found that a subset of the MM miRNAs exhibited aberrant expression in monoclonal gammopathy of undetermined significance and smoldering myeloma. miRNA analysis of enriched CD138(+) plasma cells from MM and monoclonal gammopathy of undetermined significance found that most of the validated MM BM signature miRNAs were significantly decreased in MM plasma cells. Gene expression profiling indicated that multiple targets of the decreased miRNAs found increased expression in MM plasma cells, including ATF2, HRAS, HDAC4, TGFB1, TGFBR1, and mitogen-activated protein kinases. The findings suggest that these miRNAs are detectable in aberrant levels in the peripheral blood of patients with plasma cell proliferation and may play a role in aberrant plasma cell proliferation and disease progression.

Extramedullary disease (EMD), defined as an infiltrate of clonal plasma cells at an anatomic site distant from the bone marrow, is an uncommon manifestation of multiple myeloma. Six hundred and sixty-three consecutive patients with multiple myeloma who underwent stem cell transplantation between January 2005 and December 2011 were assessed for the presence of EMD. A cohort of 55 patients with biopsy-proven EMD was identified, comprising 8.3% of the total study population. EMD was present at the time of diagnosis in 14.5% of cases and at the time of relapse in 76% of patients. The most common EMD presentations at relapse were liver involvement and pleural effusions. EMD specimens had high expression of CD44 (92%) and moderate expression of CXCR4. The median overall survival from time of myeloma diagnosis was 4.1 years (95% confidence interval: 3.1, 5.1) and the median overall survival from time of EMD diagnosis was 1.3 years (95% confidence interval: 0.8, 2.3). This report demonstrates that the incidence of EMD has not increased with the introduction of novel agents and stem cell transplantation. The most common EMD presentations in the relapsed setting were liver and pleural fluid. The presence of CD44 and CXCR4 expression may represent new markers of EMD that warrant further investigation.

Bortezomib (Velcade, formerly PS-341) represents the first proteasome inhibitor to have shown anti-tumor activity in both solid and haematological malignancies. It blocks activation of nuclear factor-kappa B (NF-kB), resulting in increased apoptosis, decreased angiogenic cytokine production, and inhibition of tumor cell adhesion to stroma. Additional mechanisms of action include c-Jun N-terminal kinase activation, effects on growth factor expression and anti-angiogenic properties. Multiple myeloma is the prototype of cancer where bortezomib has shown marked in vitro activity, which was followed by rapid translation to phase I, II and III clinical trials, and resulted in accelerated approval by the FDA for the treatment of patients with relapsed refractory disease. Different clinical trials are currently ongoing in multiple myeloma as well as in many others haematologic and solid tumors (mantle cell and follicular non-Hodgkin's lymphoma; peripheral T-cell lymphoma; Waldenström's macroglobulinemia, chronic lymphocytic leukemia; head and neck / gastroesophageal junction / stomach /colo-rectal / prostate / non-small cell lung cancer). This reviews focuses on the proteasome inhibition exerted by bortezomib, the first proteasome inhibitor to have shown anti-cancer activity in both solid and haematologic malignancies.

Spontaneously arising tumor cells are not usually angiogenic at first. The phenotypic switch to angiogenesis is usually accomplished by a substet that induces new capillaries that then converge toward the tumor. The switch clearly involves more than simple upregulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators. Tumor growth is although to require disruption of this balance and hence this switch must turned on for cancer progression. Progenitor endothelial cells, the crosstalk between angiogenic factors and their receptors and the interaction between vasculogenesis and lymphangiogenesis are all factors that may contribute to the switch. Its promotion is also the outcome of genetic instability resulting in the emergence of tumor cell lines. This review describes the history of the angiogenic switch illustrated in the literature and with particular reference to the three transgenic mouse models, namely RIP1-TAG2, keratin-14 (K14) (human papilloma virus) HPV16 and papilloma virus, used for stage-specific assessment of the effects of antiangiogenic and antitumorigenic agents.

Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.

Honokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM. (Blood. 2005;106:1794-1800)

Cyclin-dependent kinase (CDK) inhibitors have the potential to induce cell-cycle arrest and apoptosis in cancer cells. Seliciclib (CYC202 or R-roscovitine) is a potent CDK inhibitor currently undergoing phase-2 clinical testing in lung and B-cell malignancies. Here we studied the in vitro cytotoxic activity of seliciclib against multiple myeloma (MM) cells. Our data demonstrate that seliciclib has potent cytotoxicity against MM cells that are both sensitive and resistant to conventional therapy as well as primary MM cells from patients. Cell-cycle and Western blot analysis confirmed apoptosis. Importantly, seliciclib triggered a rapid down-regulation of Mcl-1 transcription and protein expression independent of caspase cleavage. Adherence of MM cells to bone marrow stromal cells (BMSCs) induced increased Mcl-1 expression associated with signal transducer and activator of transcription 3 (STAT3) phosphorylation, which was inhibited in a time- and dose-dependent manner by seliciclib. Furthermore, seliciclib inhibited interleukin 6 (IL-6) transcription and secretion triggered by tumor cell binding to BMSCs. Up-regulation of Mcl-1 expression in cocultures was only partially blocked by neutralizing antibody to IL-6, suggesting alternative mechanisms of Mcl-1 modulation by seliciclib. Finally, combination studies of seliciclib with doxorubicin and bortezomib show in vitro synergism, providing the rationale for testing these drug combinations to improve patient outcome in MM.

In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex. (Blood. 2005;106:706-712)

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.

Thalidomide alone or in combination with steroids has significant activity in multiple myeloma (MM). However, given its teratogenic potential, analogs have been synthesized, retaining the anti-MM activity without these side effects. We examined the anti-MM activity of two thalidomide analogs, CPS11 and CPS49. Direct cytotoxicity of the drugs on myeloma cell lines and patient myeloma cells was examined using thymidine uptake. Tumor cell apoptosis was evaluated by flow cytometry as well as Western blotting for caspase and PARP cleavage. Cellular signaling events were examined by immunoblotting for phosphorylated proteins. Both drugs inhibit proliferation of several MM cell lines sensitive and resistant to conventional therapies. They decrease secretion of IL-6, IGF, and VEGF by marrow stromal cells. Importantly, they inhibit proliferation of MM cells adherent to stromal cells. These drugs induce caspase-mediated apoptosis in MM cell lines, as well as patient MM cells. They inhibit the PI3K/Akt and JAK/STAT (signal transducers and activators of transcription) pathways in MM cells and are antiangiogenic in matrigel-based assays. CPS11 and CPS49 have potent antimyeloma activity and can overcome protective effects of the tumor microenvironment. They have potent antiangiogenic activity and direct effect on bone marrow stroma. These encouraging preclinical data provide the basis for further evaluation in the clinic.

Despite advances in systemic and supportive treatments multiple myeloma remains an incurable disease, and approaches are needed to improve the quality of response to therapy and hopefully overall survival. The pathophysiology of multiple myeloma is complex and still poorly characterised. It entails features of the malignant clone and interactions with bone marrow microenvironment. Multiple myeloma begins when different pathways induce dysregulation of cyclin D and transformation of plasma cells. Evolving mutations and epigenetic mechanisms (angiogenesis, cytokines) lead to progression of disease.

The growth of solid tumors is certainly angiogenesis dependent. However, the role of angiogenesis in the growth and survival of leukemias and other hematological malignancies has only been rendered evident since 1994 in a series of demonstrations showing that the progression of several forms is clearly related to their degree of angiogenesis. Here, we present an overview of the literature concerning the relationship between angiogenesis and disease progression in several hematological malignancies and the recent advances in antiangiogenesis in these diseases and we describe the most important active substances, preclinical and clinical data, and future perspectives.

Multiple myeloma (MM) progresses from an avascular to a vascular phase (active MM) accompanied by a significant increase in microvessel density in the bone marrow. This article summarizes the literature concerning the specific role played by vascular endothelial growth factor (VEGF) in this process. Recent applications of antiangiogenic agents that interfere with VEGF signaling and block MM progression are also described.

Erythropoietin (Epo) is produced by the fetal liver and adult kidney and is an essential stimulator of erythropoiesis. It has, however, been shown to modulate host cellular signal transduction pathway to perform many other functions. New sites of Epo production have been found, such as the female reproductive organs and central nervous system. This review summarizes the involvement of Epo in the regulation of angiogenesis in both normal and pathological conditions.

Experimental studies have demonstrated that thalidomide (Thal), a drug developed as a sedative, has antitumoural properties. The possible antitumour mechanisms of action involve: inhibition of angiogenesis, cytokine-mediated pathways, modulation of adhesion molecules, inhibition of cyclooxygenase-2 and stimulation of immuno response. Therefore, Thal is under clinical evaluation in oncology. This paper provides an overview of the data currently available in literature regarding, in terms of activity and toxicity, the use of Thal in cancer patients. Multiple myeloma is so far the most responsive malignancy. A moderate activity has been documented in certain solid tumours: glioblastoma multiforme, renal cell carcinoma and malignant melanoma. Tolerability is generally satisfactory with peripheral neuropathy being the most relevant dose-dependent toxicity. The more frequent, but moderate side effects are: somnolence, constipation, dizziness and fatigue. More studies are needed to properly evaluate the anticancer activity of Thal alone or in combination with other anticancer treatments. Preliminary studies suggest promising results of Thal in combinations with corticosteroids and cytotoxic drugs as front-line therapy of multiple myeloma. Regarding therapy of solid tumours in the adult, combination with chemotherapy, radiation therapy and molecular-targeting compounds are under investigation.

Background Changes in angiogenesis and expression of extracellular matrix-degrading enzymes have been substantiated in tumour changeover and progression. Methods Tissues from 44 biopsies of stage III and IV ovarian endometriomas, and 10 biopsies of normal (control) endometrium were investigated immunohistochemically to count microvessels, and by in situ hybridization to assess the expression of mRNA of matrix metalloproteinase-2 (MMP-2) and MMP-9. Implants of the tissues were investigated in the chick embryo chorioallantoic membrane (CAM) assay to determine their angiogenic capacity. Results The endometriomas displayed significantly higher counts than normal endometria and the highest values were associated with the deepest invasion level (stage IV). Microvessels localized in close association with ectopic endometrial cells in the form of winding and arborized tubes, often dilated in microaneurysmatic segments. These were absent in normal endometrium. Expression of MMP-2 and MMP-9 mRNA, evaluated as percentages of positive biopsies and intensity of expression, was up-regulated in endometriomas and more pronounced in stage IV. MMP-2 and MMP-9 mRNA were also expressed by host stromal cells, including microvascular endothelial cells, fibroblasts and macrophages, whereas the control endometrium showed very little expression of MMP-2 mRNA in a few endothelial cells and no expression of MMP-9 mRNA. Implants from stage IV endometrioma induced a more intense vasoproliferative response than those from stage III, while no vasoproliferative response was induced by the normal endometrium. Conclusion These data suggest that angiogenesis and degradation of extracellular matrix occur together in endometriosis and are more pronounced in stage IV, and that endometriosis cells and some host stromal cell populations co-operate in disease progression.

The widespread involvement of the bone marrow with tumor cells indicates that there is continuous cell trafficking of WM cells in and out of the bone marrow leading to cell dissemination in the bone marrow and in the lymph nodes in many patients with WM. The interaction of the WM cells with the bone marrow is critical for the regulation of cell proliferation, cell cycle, drug resistance as well as cell dissemination and trafficking. Advances in understanding the interaction of the tumor clone with the BM microenvironment have led to the development of therapeutic agents that not only target the tumor clone but also regulate the bone marrow microenvironment. Here, we review the role of the cellular and liquid bone marrow compartments in the regulation of cell proliferation and dissemination in WM.

Epigenetics represent heritable changes in gene expression that are not due to any alteration in the DNA sequence. One of the best-known epigenetic markers is histone acetylation, which has been shown to be deregulated in neoplastic diseases, including B-cell malignancies, such as Waldenström's Macroglobulinemia (WM), a low-grade B-cell lymphoma characterized by the presence of lymphoplasmacytic cells in the bone marrow and a serum monoclonal immunoglobulin M in the circulation. It has been recently demonstrated that microRNAs may be responsible for modulating histone acetylation in WM cells, thus providing the preclinical evidences for using microRNA-based therapeutic strategies in this disease.

Disease assessment in Waldenstrom Macroglobulinemia (WM) is dependent on the percent involvement of B-cell neoplasm in the bone marrow and IgM paraprotein in the serum. A subset of patients also demonstrates extramedullary involvement, which is infrequently examined. The role of extramedullary involvement in the diagnosis and prognosis of WM is poorly understood. The purpose of this study is to report the characteristics of WM patients with extramedullary disease (EMD). Nine hundred and eight-five patients with WM were evaluated at one academic center and the presence of EMD was assessed in these patients. Forty-three (4.4%) patients were identified to have EMD. Nine (21%) patients presented with involvement at WM diagnosis, while 34 (79%) developed EMD post-therapy for WM. Most frequent EMD sites involved were pulmonary (30%), soft tissue (21%), cerebrospinal fluid (23%), renal (8%), and bone (9%). The median overall survival at 10 years was 79% (95% CI: 57-90%). This is the first study to describe the clinical characteristics, response and overall survival in patients with extramedullary WM. Further studies to define the molecular characteristics of this entity and mechanisms of its development are warranted.

Epigenetic changes frequently occur during tumorigenesis and DNA hypermethylation may account for the inactivation of tumor suppressor genes in cancer cells. Studies in Multiple Myeloma (MM) have shown variable DNA methylation patterns with focal hypermethylation changes in clinically aggressive subtypes. We studied global methylation patterns in patients with relapsed/refractory MM and found that the majority of methylation peaks were located in the intronic and intragenic regions in MM samples. Therefore, we investigated the effect of methylation on miRNA regulation in MM. To date, the mechanism by which global miRNA suppression occurs in MM has not been fully described. In this study, we report hypermethylation of miRNAs in MM and perform confirmation in MM cell lines using bisulfite sequencing and methylation-specific PCR (MSP) in the presence or absence of the DNA demethylating agent 5-aza-2'-deoxycytidine. We further characterized the hypermethylation-dependent inhibition of miR-152, -10b-5p and -34c-3p which was shown to exert a putative tumor suppressive role in MM. These findings were corroborated by the demonstration that the same miRNAs were down-regulated in MM patients compared to healthy individuals, alongside enrichment of miR-152-, -10b-5p, and miR-34c-3p-predicted targets, as shown at the mRNA level in primary MM cells. Demethylation or gain of function studies of these specific miRNAs led to induction of apoptosis and inhibition of proliferation as well as down-regulation of putative oncogene targets of these miRNAs such as DNMT1, E2F3, BTRC and MYCBP. These findings provide the rationale for epigenetic therapeutic approaches in subgroups of MM.

The phosphatidylinositol-3 kinase (PI3K) pathway is activated in multiple myeloma (MM) and Waldenstrom Macroglobulenima (WM), and plays a crucial role in tumor progression and drug resistance. In this study, we characterized the role of pan-class I PI3K inhibition on cell trafficking and survival of MM and WM cells. We tested the effect of pan-class I PI3K inhibition by siRNA silencing or pharmacologic inhibition with buparlisib on MM cell survival, apoptosis and cell cycle in vitro and tumor growth and mobilization of MM cells in vivo. We then evaluated buparlisib-dependent mechanisms of induced MM cell mobilization. Moreover, the effect of buparlisib on cell survival, apoptosis, and adhesion of WM cells to bone marrow stromal cells (BMSCs) has been evaluated. We showed that buparlisib induced toxicity in MM cells, supported by induction of apoptosis and cell cycle arrest. Buparlisib was also found to reduce tumor progression in vivo. Importantly, buparlisib enhanced MM cell mobilization in vivo which was driven by decreased adhesion of MM cells to BMSCs and increased chemotaxis via up-regulation of CXCR4 expression. Similar to its effects on MM cells, buparlisib also induced cell survival and apoptosis, and decreased adhesion in WM cells. These data highlight the critical contribution of class I PI3K signaling to the regulation of survival and cell dissemination in B-cell malignancies.

Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.

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.

Factor VIII-related antigen (FVIII-RA)-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM, and monoclonal gammopathies of undetermined significance (MGUS). A five-to sixfold larger area was found in patients with active MM compared to the other two groups. Aquaporin 1 (AQP1)-positive microvessel areas, measured with the same techniques on adjacent tissue sections, were also increased in active MM, and tended to be larger than and closely correlated with the FVIII-RA areas. Numerous mast cells were found in the bone marrow of active MM patients, and counts were strictly correlated with the microvessel density. The conditioned medium (CM) of bone marrow plasma cells from active MM patients stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis, and angiogenesis in vivo (assessed by the chick embryo chorioallantoic membrane [CAM] system) more strongly and frequently than the CM of patients with nonactive MM and MGUS. Immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 46% to 68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data indicate that patients with active MM represent the vascular phase of plasma cell tumors that is induced, at least partly, through FGF-2 and MMP-2. Mast cells possibly contribute to the vascular phase via angiogenic factors in their secretory granules. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells in patients with active MM.

Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19Cre/Cre) mice were crossed with either C57BL/6 BLIMPflox/flox or C57BL/6 p53flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19Cre/Cre BLIMPflox/flox mice were crossed with CD19Cre/Cre p53flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19Cre/Cre BLIMPflox/flox p53flox/flox), referred as CD19/Bl-/p53- mice. Transgenic experimental mice (CD19/Bl-/p53-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl-/p53-derived B220 cells. Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl-/p53- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl-/p53- showed worse survival compared to Bl-/p53- mice non-expressing the CD19/Cre recombinase, CD19/p53-, or CD19/Bl- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220+ cells selected from CD19/Bl-/p53--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220+ and IgM+ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220+ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220+ cells selected from lymph nodes harvested from CD19/Bl-/p53-mice were sensitive to ibrutinib treatment. Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development. Disclosures Ghobrial: Celgene: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Takeda: Other: Advisory Board; Janssen: Other: Advisory Board. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

Introduction: Recent data show that Multiple Myeloma (MM) always progresses from a precursor state (monoclonal gammopathy of undetermined significance [MGUS]/smoldering multiple myeloma [SMM]) to overt MM indicating that there is continuous dissemination/clonal evolution of tumor cells from the original stages of tumor development to the time of clinical presentation. A major challenge in understanding the progression and metastasis of MM is to distinguish alterations driving the tumor growth and evolution from passenger mutations. Genetic screens are powerful tools for assaying phenotypes and identifying causal genes in various hallmarks of cancer progression. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged as a powerful technology to efficiently and simultaneously perform genome editing of multiple genes. Here we report a genome-wide CRISPR/Cas9-mediated loss of function screen in a xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM. Methods: Lentiviral particles from 2 subpools of a human sgRNA library (Avana), each containing 1 sgRNA per gene were introduced into MM1.S (Cas9+/GFP+/Luc+) cell line with the pre-determined amount of virus to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5-1. Cells were selected with puromycin for 5-7 days following infection to remove uninfected cells. Selected cells were injected subcutaneously into SCID-Beige mice on both flanks. Genomic DNA from pre-transplantation cells, early primary tumors (~3 weeks post tumor cell injection), late stage primary tumors and metastatic bone marrow samples were extracted. gDNA was amplified following adaptor ligation and barcoding of the samples and PCR products were subsequently sequenced on a HiSeq2000 (Illumina). Results: To investigate the sgRNA library dynamics in different sample types (pre-transplantation cells, early primary tumor, late primary tumor, and bone marrow metastasis), we compared the overall distributions of sgRNAs from all sequenced samples. The early tumor sample replicates of both subpools on average retained 77.3% and 94.7% of the sgRNAs found in the pre-transplanted cell populations, while the late primary tumors retained 59.4% and 65.6% of the sgRNAs respectively, compared to early tumors. Interestingly, only a small fraction of sgRNAs (1.1% and 3.4% of sgRNAs in the pre-transplantation cells, 10.7% and 7.2% of sgRNAs in the late primary tumors for the 2 subpools respectively) were detected in the metastatic bone marrow samples. Using gene set enrichment analysis (GSEA), we found that the gene targets of the most enriched sgRNAs in the bone marrow samples were preferentially involved in important cellular processes, such as cell cycle regulation, protein translation, and several signaling pathways. Additionally we compared sgRNAs present in early primary tumor versus pre-transplantation cells and late primary tumor and found that many sgRNAs were depleted during tumor progression, indicating that their target genes were important for progression. These depleted sgRNAs in both stages mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors. Conclusion: We established a platform for future in vivo Cas9 screens using the genome-wide CRISPR screening libraries to explore potential new targets in regulating tumor dissemination, colonization and metastasis in MM. In addition, this in vivo screening could potentially be used to investigate essential genes of response to targeted therapies or/and immunotherapies. Thus, CRISPR/Cas9-based in vivo screening is a powerful tool for functional genomics discoveries. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria.

Introduction According to the clonal evolution model, tumor progression proceeds in a branching rather than in a linear manner, leading to substantial clonal diversity and coexistence of genetically heterogeneous sets of subclones. Unlike many cancers, in which the evolutionary history can only be inferred from the established disease, Multiple Myeloma (MM) has well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. In MM, multiple subclones can co-exist because they are of similar fitness, potentially interact with each other or with the surrounding microenvironment and further disseminate to spatially separate areas of the bone marrow (BM). Therefore, in order to accurately predict the course of the disease, we require methods to estimate clone-specific growth rates within the BM and define clones that have the propensity of dissemination. Methods We developed a MM metastatic xenograft model by performing tumor-bearing bone chip implantation to SCID-beige mice (SCID-murine model) and examining tumor clones that are present in the implanted bone chips (primary sites) compared to those present in the distant BM sites (metastatic sites). To obtain a perspective of clonal heterogeneity in vivo, we used the "rainbow" system by which fluorescent proteins were infected into cells using lentiviruses to label the cells with 15 distinctive fluorescence profiles (rainbow MM cells). Rainbow MM cells with equal proportion of all 15 colors were injected into donorfemurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. To further investigate the dynamics of heterogeneity at the single cell level, similar experiments were performed using a DNA-barcode library. For genomic and transcriptomic characterization, primary and metastatic tumor clones were purified by sorting and underwent whole exome and RNA sequencing. To identify key regulators of the metastatic process, we conducted in vivo CRISPR library screening of the most critical targets identified. Briefly, the MM cell library was prepared by transduction of sgRNAs targeted for 20 genes and control sgRNAs to MM.1S cells stably expressing Cas9. The cell library was used in SCID-murine model and the fractions of each sgRNA were calculated in the primary and metastatic sites to identify genes that facilitate tumor metastasis. Results We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Specific subclones (winner clones) had a greater advantage of growing in the metastatic site. Interestingly, the winner clones were similar between the bilateral femurs of most of the mice, suggesting the existence of potential metastatic subclones. Experiments using DNA-barcoding further demonstrated that single clones could become disproportionately present in the metastatic sites, even though they account for a smaller fraction of the primary tumors. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from these mouse models demonstrated a distinct gene expression profile of the metastatic tumors compared to the primary sites. By intersecting differentially expressed genes, we identified 110 shared up-regulated genes and 238 shared down-regulated genes, which we designated as the "metastatic signature". Gene Set enrichment analysis of the metastatic signaturein publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Finally, the CRISPR in vivo screening prioritized two transcription factors as the key regulatory molecules, namely EGR3 and ATF3. Conclusions Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. On the molecular level, a metastatic gene signature was found and two genes were identified as potential regulator of MM metastasis. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Hatake:Chugai: Research Funding; Meiji-Seika: Consultancy; Kyowa Kirin: Honoraria, Research Funding; Otsuka: Consultancy. Scadden:Dr. Reddy's: Consultancy; Bone Therapeutics: Consultancy; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlaxoSmithKline: Research Funding. Ghobrial:Amgen: Honoraria; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Introduction The bone marrow (BM) microenvironment in multiple myeloma (MM) plays a pivotal role in tumor growth and bone destructive process. Mesenchymal stromal cells (MSCs) in MM exhibit different genomic and cytokine secretion profiles that ultimately impair their osteogenic differentiation abilities compared to normal MSCs. However, the underlying molecular mechanisms are not fully understood. In the present study, we explored the role of miR-138 in MSCs derived from MM patients (MM-MSCs) and the potential for anti-miR-138 treatment to rescue impaired osteogenic differentiation in MM, both in vitro and in vivo using a human xenograft MM model. Materials and methods Primary BM aspirates were obtained from MM patients and normal healthy donors, after obtaining informed consent in accordance with the Declaration of Helsinki. MiR-138 expression in MM-MSCs was measured by quantitative real-time PCR. Publicly available microarray data sets (GSE17306 and E-TABM-508) were analyzed for miR-138 expression in MM cells compared to normal plasma cells. To test the effect of inhibiting miR-138 function, a high-affinity 15-mer locked nucleic acid (LNA)-modified anti-miR oligonucleotide and a corresponding scramble sequence control oligonucleotide were used (In collaboration with Dr. Kauppinen, Denmark). Anti-miR-138 oligonucleotides were transfected into MM-MSCs or normal MSCs co-cultured with MM cell lines and osteogenic differentiation in MSCs was assessed by alizarin red staining. For the in vivo studies, 6-week-old female SCID-beige mice (n=6, each group) were injected intravenously with anti-miR-138 or scramble control oligonucleotides (15 mg/kg) 2 times a week. 3 weeks later, GFP+Luc+ MM.1S cells (3 × 106) were injected into mice. Anti-miR-138 or control oligonucleotides were continued until day 28 after injection of myeloma cells. At day 28, the effect of anti-miR138 was assessed by the number of osteoblastic lineage cell (OBC: Lin-/CD45-/CD31-/CD51+/Sca-1-) from hematopoietic cell-depleted, collagenase-treated crushed bones of mice by flow cytometry. Results MiR-138 expression in MSCs from MM patients (n=10) was significantly higher than MSCs from normal donors (n = 4) (P Conclusions These findings indicate that miR-138 plays an important role in impaired osteogenic differentiation in MSCs in MM. Inhibition of miR-138 promotes osteogenic differentiation of MSCs in MM and anti-miR-138 treatment holds the potential to prevent MM induced bone loss and lytic lesions. Additional studies are ongoing to further understand the connection between MM cells and MSCs mediated by miR-138. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Novartis: Honoraria; Noxxon: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding.

Introduction: Recent data shows that multiple myeloma (MM) almost always arises from precursor states called Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), but not all patients with MGUS or SMM develop MM. Risk factors of progression for SMM patients are largely based on tumor load as represented by an M-protein ≥ 3 g/dL, a free light chain (FLC) ratio outside the range of 0.125 to 8, and ≥ 10% plasma cells in the BM. However, the genetic lesions that underlie progression, the molecular factors that cause rapid versus slow progression, and the factors that distinguish the relatively indolent MGUS from SMM are not well known. Further, the genomic landscape of SMM is not well characterized. One potential factor is MYC overexpression. Bergsagel et al. have found that MYC levels increase when comparing MGUS, SMM and overt MM. Other frequently altered pathways in MM are NF-kB, MAPK and DNA damage. In addition, limited studies of paired SMM and MM samples show that in many cases, the aggressive subclones can already be detected, in small cell fractions, before overt MM develops. However, the cause of progression to MM is unclear, in large part because sequential genomic studies of MGUS/SMM progression have yet to be undertaken. To address these questions, in this study we examine clinically-annotated samples from patients with SMM. Methods: We performed whole exome sequencing (WES) (mean target coverage 50X/100X) on 49 germline-tumor matched samples from patients with SMM (DNA from bone marrow CD138+ plasma cells matched with germline DNA from peripheral blood mononuclear cells). Libraries were constructed using Agilent SureSelect XT2 library prep kit, and hybridized to Agilent's whole exome V5+UTR capture probes and then sequenced on HiSeq 2500 (Illumina). We also performed targeted deep sequencing using a custom enrichment bait set on 25 samples of progressor (n=12) and non-progressor (n=13) SMM samples. Libraries were also constructed with Agilent SureSelect XT2 library prep kit and enriched by hybridizing to an in-house designed customized target bait, then sequenced on HiSeq 2500. Sequencing data were analyzed using previously established analytic pipelines including MuTect, RecapSeg, GISTIC, MutSig, and ABSOLUTE. Results: The number of Somatic Single Nucleotide Variants (SSNVs) seen in SMM ranged from 1 to 98 nonsilent mutations with an average of 1.14 mutations/Mb, which is slightly lower than MM (1.6 mutations/Mb) from previous studies (p-value=0.05). This large and varying range of mutational load among samples suggests that SMM is likely a heterogenous entity where some patients are closer to MGUS and others closer to MM. We identified likely drivers in SMM in about ~32% of the samples, including mutations in MM candidate driver genes such as NRAS, KRAS and PTPN11(overall 36 events were present in COSMIC). SMM also had somatic CNAs in about ~50% of SMM samples, such as hyperdiploidy, gain of chromosome 1q, deletion of 13p and 17p, which match the hallmark chromosome changes seen in MM. Comparing deep targeted sequencing of 100 genes (mean target coverage 361X) in samples from 12 SMM patients who progressed to myeloma vs. 13 SMM patients who did not, we found non-synonymous mutations exclusive to progressors, suggesting that with more samples we may find genetic alterations that predict progression in SMM. Conclusion: This study demonstrates that WES and targeted sequencing of SMM patients can detect MM candidate driver genes as well as hallmark CNAs seen in MM patients. Further, there may be potential different mutational features between progressors and non-progressors. Thus, this approach can be used to identify genetic drivers of clonal progression from MGUS/SMM to MM that may present opportunities for early therapeutic intervention and prevention of disease progression. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Background . Cell-free DNA (cfDNA) sequencing enables serial temporal sampling, which offers the possibility of following the dynamics of biomarkers and clonal evolution in Multiple Myeloma (MM) over time. The use of cfDNA in clinical practice as a molecular biomarker and for monitoring response/resistance is dependent on a comprehensive profile of matched cfDNA and tumor DNA (tDNA) samples. Here we performed Ultra-Low Pass Whole Genome Sequencing (ULP-WGS) followed by whole-exome sequencing (WES) and targeted deep sequencing of matched cfDNA/tDNA samples from MM patients. Methods. We performed next generation sequencing of matched cfDNA/tDNA samples for 63 patients with newly diagnosed or relapsed MM, SMM, or MGUS. Libraries were constructed using the Kappa Hyper kit and sequenced by ultra-low-pass whole-genome sequencing (ULP-WGS, 0.1x coverage) to quantify tumor fraction within cfDNA. WES was performed on 30 matched samples cfDNA/tDNA/germline DNA from 10 patients with more than 5% of tumor fraction. Libraries were hybridized to the Nextera Rapid Capture Exome kit (Illumina) and then sequenced on HiSeq 4000 (Illumina). Targeted deep sequencing was performed on 32 matched cfDNA/tDNA samples from 16 patients using the HaloPlex HS technology (Agilent), allowing for molecular barcoding. Libraries were constructed according to the manufacturer's instructions and sequenced on HiSeq 2500 (Illumina). Sequencing data were analyzed using the Firehose pipelines, including MuTect, ABSOLUTE, ReCapSeg, GISTIC and MutSig. Results. We first used a cost-effective approach to establish the tumor content of cfDNA in a large-scale manner by ULP-WGS. Among 63 tested samples (53 MM, 6 SMM and 4 MGUS patient samples), the tumor fraction within cfDNA ranged from 0 to 81% with a mean of 10%. About 43% of these samples had tumor fraction greater than 5% within cfDNA. To assess whether cfDNA can capture the genetic diversity of MM and inform clinical management, we performed WES of matched cfDNA/tDNA/germline DNA samples for 10 patients (mean target coverage 194x). Copy number alterations (CNAs) assessed by WES (ReCapSeg) were consistent between cfDNA and tumor DNA. Similarly, focal CNAs assessed by GISTIC were consistent between tDNA and cfDNA. We then examined the overlap of somatic single nucleotide variants (SSNVs) between WES of cfDNA and matched tDNA. We found, on average, 100% of the clonal and 96% of the subclonal (range 54-100%) SSNVs that were detected in the tumor were confirmed to be present in cfDNA. Similarly, for mutations detected in the cfDNA, we found, on average, 100% of the clonal and 99% of the subclonal (range 98-100%) SSNVs were confirmed in the tumor. To assess whether targeted deep sequencing of cfDNA could be a good proxy for tumor biopsy we used a targeted deep sequencing approach of known MM driver genes. Libraries were prepared using unique molecular barcodes to avoid duplication rates, for 32 matched cfDNA/tDNA samples from 16 patients with MM. The mean target coverage was 596x. We found similar frequencies of altered MM driver genes in both cfDNA and tDNA, including KRAS, NRAS, and TP53, indicating that cfDNA can be used for precision medicine. Conclusions. Our study demonstrates that both WES and targeted deep sequencing of cfDNA are consistently representative of tumor DNA alterations in terms of CNAs, focal CNAs and SSNVs. This approach could therefore be used to longitudinally follow clonal evolution across the course of the disease and precision medicine in patients with MM. Disclosures Palumbo: Takeda: Employment, Honoraria; Janssen Cilag: Honoraria. Kumar:Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; Onyx: Consultancy, Research Funding. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria. Facon:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy; Janssen: Consultancy, Speakers Bureau; Bristol: Consultancy; Millenium/Takeda: Consultancy; Celgene: Consultancy, Speakers Bureau; Karyopharm: Consultancy. Ghobrial:Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Introduction. Waldenström Macroglobulinemia (WM) is a low- grade B-cell lymphoma with a heterogeneous clinical presentation. In many patients, the diagnosis is preceded by an asymptomatic precursor state of IgM monoclonal gammopathy of undetermined significance (MGUS) and smoldering WM. However, little is known about the mechanisms involved in the progression from asymptomatic WM to symptomatic WM. Exosomes are small vesicles secreted by cells that enable the transfer of nucleic acids, proteins and lipids between distant cells in the organism. Because of the active role of exosomes in promoting tumor growth and metastasis, we investigated the microRNA content of circulating exosomes in patients at different stages of WM in order to identify possible markers of progression. Methods.We isolated exosomes by ultracentrifugation from the peripheral blood plasma of 101 patients with WM and 10 normal donors. The WM cases included 7 patients with IgM MGUS, 42 patients with smoldering WM and 52 patients with symptomatic WM. We first profiled microRNAs from the exosomes of 14 patients and 5 normal controls using the TaqMan Array Human MicroRNA A Card (Thermo Fisher Scientific) which enables the quantitation of 377 human microRNAs. Exosomes from an additional group of 14 individuals (2 normal donors, 8 patients with asymptomatic WM and 4 patients with symptomatic WM) were profiled with the Oncology Panel of the Firefly Multiplex Circulating miRNA Assay (Abcam), which measures the expression level of 68 different human microRNAs. Following these analyses, a group of 33 microRNAs deregulated between patients' groups was selected and a custom microRNA panel was built to allow quantitation of these microRNAs with the Firefly Multiplex Circulating miRNA Assay (Abcam). The level of expression of these 33 microRNAs was measured in a group of 80 individuals comprised of healthy controls (n=8), patients with asymptomatic WM including IgM MGUS and smoldering WM (n=34) and patients with previously untreated symptomatic WM (n=11), relapsed WM (n=21) and refractory WM (n=6). Additionally, exosomes were imaged using electron microscopy with immunogold labeling for the detection of the exosome-specific receptors CD63 and CD81. Results. Imaging with electron microscopy showed vesicles with a size ranging from 50 to 150 nm and expressing CD63 and CD81, thus validating our ultracentrifugation method for exosome isolation. Among the 33 microRNAs analyzed, 12 were significantly deregulated between WM patients and healthy controls (P Conclusion. The microRNA content of circulating exosomes differs at different stages of WM progression. This could potentially be used to further define prognostic markers of progression in patients with WM, specifically progression from asymptomatic WM to overt WM. The study of these deregulated microRNAs' effect on in vitro and in vivo WM models can help us gain insights on mechanisms of disease progression in WM. Disclosures Castillo: Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria; Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria. Hermine:Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Alexion: Research Funding; Celgene: Research Funding. Ghobrial:Noxxon: Honoraria; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria.

Background: Whole genome sequencing has recently shown the presence of recurrent mutations, such as MYD88, CXCR4 and ARID1A, in patients with Waldenstrom's Macroglobulinemia (WM) at diagnosis. Nevertheless, the contribution of each genomic aberration within the clonal evolution of the tumor during WM progression has not been reported. We therefore aimed to investigate whether sequential genomic events sustain WM disease progression by using whole exome sequencing (WES) and targeted sequencing of serial samples. In addition, we investigated if specific genomic alterations can change in response to treatment with samples before and after proteasome or BTK inhibitor treatments. Methods: We have sequenced 74 samples from 32 patients using WES or targeted sequencing technology. DNA was collected from bone marrow CD19-selected cells that were isolated from 6 patients with WM at different stages of disease (2-3 serial samples per patient, total 15 samples), and was subjected to library construction, followed by Agilent Sure-Select Human All Exon v2.0-based hybrid selection. Germline DNA was isolated from matched CD19-depleted peripheral blood samples. All libraries were sequenced with Illumina Hiseq 2500 instrument (New York Genome Center, Rockefeller University, New York, NY). Reads were aligned to GRCh37, and quality control, mutation calling, insertion and deletion identification, copy number variation detection, coverage calculations were accomplished via Firehose at Broad. Somatic Single Nucleotide Variations (SSNVs) were identified and annotated using MuTect and Oncotator, respectively. Insertions and deletions were detected using Strelka. In addition, the deep targeted sequencing was done with a customized bait set in 59 independent serial samples obtained from 26 patients who were treated with either proteasome inhibitor (Pi)- or BTK inhibitor (BTKi)-based therapies in 12 and 14 cases, respectively. Results: Whole exome sequencing, performed at a total average depth of 83X for germline and 85X for tumor samples, led to the identification of average of 14 non-silent mutations per sample (range 2-60). Base conversion signature showed dominant A>C/G transitions. Copy number analysis showed 6q deletion being the most prevalent one. MYD88 was the most recurrent somatic variants in WM patients (>93%), followed by several genes including IRS4, VCAN, CXCR4 and ALDH2, being detected in ~20-30% of the samples. Specifically, changes in the Cancer Cell Fraction (CCF) of these mutations, such as MYD88 and CXCR4, occurred in the serial samples depending on progression and response to therapy. The mutated genes mentioned above, including MYD88, CXCR4, LRG1 and VCAN, were also observed in targeted sequencing of 59 independent serial samples (mean target coverage 434X). CXCR4 was linked to disease progression exposed to Pi or BTK; but not detected in patients responding to therapies. MYD88 was present in patients with either progressive disease or PR/VGPRs. Specifically, different frequencies of MYD88 mutations were identified in patients with progression or PR/VGPR to BTKi-based therapies compared to Pi-based therapies. LRG1 was detected in a patient in response to Pi, while VCAN was observed in both patients showing progression or response to Pi regimens only. Conclusion: These findings reveal the occurrence of clonal variations in patients with WM during disease progression and response to therapies. MYD88 was confirmed to be the most prevalent somatic aberration, and was present in post-treatment samples of progressors and responders to Pi- or BTKi-based regimens. In contrast, mutations in CXCR4 were enriched in patients with WM progressing to either Pi or BTKi therapies. This study demonstrates that WES and targeted sequencing of serial samples of WM patients can detect clonal variations during disease progression. Disclosures Ghobrial: Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

Introduction Molecular mechanisms of multiple myeloma (MM) pathogenesis are unknown, but a role for bone marrow (BM) niche in supporting MM is well established. The transmembrane receptor Roundabout 1 (ROBO1) has been historically annotated as a tumor suppressor gene in solid cancer, and more recently myelodysplastic syndrome. High ROBO1 expression was identified as a poor prognostic factor in newly diagnosed MM; however, its function in MM is unknown. Material and Methods We analyzed ROBO1 and SLIT2 expression in human MM cell lines (HMCL) and a panel of human cancer cell lines via western blot (WB). ROBO1/SLIT2 expression in primary BM samples was assessed via immunohistochemistry and/or WB. We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD). We designed and cloned single guide RNA (sgRNA) into pSpCas9(BB)-2A-GFP vector and developed CRISPR-Cas9-mediated ROBO1 knock out (KO) 293T and OPM2 clones. WT and KO ROBO1 OPM2 cells were used for standard viability, proliferation, and adhesion assays. Full length (FL) and mutant ROBO1 isoforms devoid of extracellular or intracellular domain, with a C-terminus triple FLAG tag were designed and cloned into pCDH lentiviral vectors. WT and ROBO1 KO OPM2 were injected in the BM cavity of femoral bones harvested from donor SCID mice, and implanted subcutaneously into recipient SCID mice to study ROBO1 KO in vivo. Results HMCL, primary MM and BM stromal cells (MM-BMSC) from patients, but not healthy donor BM-resident plasma cells and BMSC, express ROBO1 and SLIT2. Across a panel of myeloid and lymphoid cancer cell lines, high ROBO1 expression was detected only in SU-DHL-4 and BCWM1 cell lines. In newly diagnosed MM patients enlisted in the IMF170 database, high ROBO1 portended worse overall survival compared to low ROBO1 expression (P=0.018). Sh-RNA mediated ROBO1 KD was profoundly and specifically cytotoxic to HMCL, with no cytotoxicity observed in high-ROBO1 expressing SH-SY5Y and HeLa cancer cell lines. Consistent with on target effect, ROBO1 shRNA was not cytotoxic for non-ROBO1 expressing cancer cell lines. To study the functional consequences of ROBO1 loss in MM, we performed CRISPR-Cas9-mediated ROBO1 KO in HMCL. We were unable to establish biallelic ROBO1 KO H929 or U266 clones, suggesting that biallelic loss may be lethal in these HMCL, but created two clones of ROBO1 KO OPM2 cells. In vitro, ROBO1 KO and WT OPM2 cells have comparable viability and proliferation rate, even in the presence of MM-BMSC. However, ROBO1 KO OPM2 cells have profound defects in adhesion to MM-BMSC and BM endothelial cell lines (BMEC). Lentiviral transduction of OPM2 ROBO1 KO cells with FL-ROBO1 addback lentiviral construct completely rescues the BMEC adhesion defect, confirming that loss of ROBO1 is responsible for the defective adhesive phenotype. Interestingly, treatment with heparin abolishes the rescue effect of FL-ROBO1, implying that surface heparan sulfate proteoglycans (HSPG) are necessary for ROBO1-mediated MM-BMEC adhesion. Consistent with a role for ROBO1 in MM-BM niche interaction, ROBO1 KO OPM2 cells show higher failure to engraft in the bone implants compared to ROBO1 WT OPM2 (bone implants without MM engraftment: 5/15 versus 1/15, respectively, P=0.067). PET-CT scan of study mice shows ROBO1 KO tumors to be significantly smaller compared to ROBO1 WT (mean volume 1.2 cm3 vs. 2.9 cm3, P=0.003), suggesting a profound in vivo proliferation/survival defect of ROBO1 KO OPM2. Summary and Conclusions Our data demonstrate that ROBO1 and SLIT2 are highly expressed in HMCL and in the BM of MM patients, but not healthy controls. ROBO1 expression correlates with worse OS in newly diagnosed MM patients; and ROBO1 KD is selectively cytotoxic for HMCL, but not high-ROBO1 expressing SH-SY5Y and HeLa cells. CRISPR-Cas9-mediated ROBO1 KO significantly impairs OPM2 cell adhesion to BMEC and MM-BMSC, which is completely reversed by adding back FL-ROBO1. In vivo, ROBO1 KO OPM2 cells show impaired engraftment to bone implants and significantly decreased growth rate compared to ROBO1 WT OPM2. Altogether, our data demonstrate that ROBO1 is a novel pro-survival protein in MM, mediating MM-BM niche interaction and suggest the therapeutic potential of targeting ROBO1/SLIT2 pathway in MM. Proteomic and RNA-sequencing studies on the downstream signaling effectors of ROBO1/SLIT2 are currently ongoing to elucidate the molecular mechanisms of pathway activation in MM. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Factor VIII-related antigen-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM and monoclonal gammopathies of undetermined significance (MGUS). A 5- to 6-fold larger area was found in patients with active MM compared to the other two groups. The conditioned medium (CM) of their bone marrow plasma cells stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis and angiogenesis in vivo [chick embryo chorioallantoic membrane (CAM) system] more strongly and frequently than the CM of patients with nonactive MM and MGUS. An immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 54–68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and gelatin zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data suggest that patients with active MM represent the vascular phase of plasma cell tumors that is triggered by bone marrow plasma cells, at least partly, through FGF-2 and MMP-2. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells during the active MM.

Tumor-associated neovasculature is a critical therapeutic target; however, despite significant progress made in the clinical efficacy of anti-vessel drugs, the effect of these agents remains transient: over time, most patients develop resistance, which inevitably leads to tumor progression. To develop more effective treatments, it is imperative that we better understand the mechanisms involved in tumor vessel formation, how they participate to the tumor progression and metastasis, and the best way to target them. Several mechanisms contribute to the formation of tumor-associated vasculature: i) neoangiogenesis; ii) vascular co-option; iii) mosaicism; iv) vasculogenic mimicry, and v) postnatal vasculogenesis. These mechanisms can also play a role in the development of resistance to anti-angiogenic drugs, and could serve as targets for designing new anti-vascular molecules to treat solid as well as hematological malignancies. Bone marrow-derived endothelial progenitor cell (EPC)-mediated vasculogenesis represents an important new target, especially at the early stage of tumor growth (when EPCs are critical for promoting the "angiogenic switch"), and during metastasis, when EPCs promote the transition from micro- to macro-metastases. In hematologic malignancies, the EPC population could be related to the neoplastic clone, and both may share a common ontogeny. Thus, characterization of tumor-associated EPCs in blood cancers may provide clues for more specific anti-vascular therapy that has both direct and indirect anti-tumor effects. Here, we review the role of vasculogenesis, mediated by bone marrow-derived EPCs, in the progression of cancer, with a particular focus on the role of these cells in promoting progression of hematological malignancies.

BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC-derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC-derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.