ALBERT

Description: Platelets play important roles in both physiological and pathological conditions. For instance, platelets may facilitate cancer metastasis by protecting circulating tumor cells (CTCs) from shear stress and immunological assault during their intravascular phase and by supporting CTCs extravasation. Moreover, soluble factors released from activated platelets enhance proliferation and migration of endothelial cells, thereby promoting tumor angiogenesis. Correlations between increased platelet counts and shorter survival time have been described for many solid tumors. However, the role of platelets/megakaryocytes (MKs) in regulating tumor progression and dissemination in Multiple myeloma (MM) has not been previously examined. We measured the platelet aggregation-inducing abilities of MM cell lines and found MM cell lines (MM.1S, OPM-2, KMS-11, U266, and H929) induced platelet aggregation. This was not observed using leukemia cells (K562) and primary healthy donor-derived peripheral blood mononuclear cells. We next investigated whether platelets interact with MM cells within the bone marrow (BM) niche, in vivo. In order to identify platelets/MKs, femurs were harvested from MM.1S GFP+-harboring mice and stained with a DyLight649-conjugated anti-GPIb-beta antibody. The femurs were rendered transparent by the CUBIC method as previously described (Cell 159, 911-24 (2014)) and examined using confocal microscopy; and found that platelets/MKs co-localized within MM.1S GFP+-infiltrated BM niches. Further confirmation of the co-localization of MM cells and MKs was performed using immunohistochemistry (CD138+ and GPIb-alpha+). We next investigated the effect of platelets on MM cell proliferation: MM cells, where co-cultured with platelets and found that platelets enhanced the proliferation rate of MM cells in a platelet number-dependent manner, as shown by using BrdU (p

Description: Introduction: Massive parallel sequencing of tumor cells obtained from the bone marrow (BM) of patients with multiple myeloma (MM) has demonstrated significant clonal heterogeneity with a median of five clones present in each sample. However, it could be envisioned that such clonal diversity may be even higher since single BM samples only represent a small fraction of the whole BM compartment, and the pattern of BM infiltration in MM is typically patchy. Accordingly, it remains unknown whether using liquid biopsies (i.e.: patients' genetic characterization performed in peripheral blood -PB- samples) can provide a more complete profile of MM clonal diversity. Moreover BM biopsies and cannot be repeated multiple times during the course of therapy, indicating a need for less invasive methods to genomically characterize MM patients. We aimed to determine the overall applicability of performing genomic characterization of MM patients non-invasively, and define if the mutation profile of circulating tumor cells (CTCs) reflected that of patient-paired BM clonal PCs. Methods: We performed CTC enumeration using multiparameter flow cytometry (MFC) in 50 newly-diagnosed patients with symptomatic MM who were prospectively enrolled on the Spanish clinical trial PETHEMA/GEM2010MAS65 as well as 64 patients with MM with relapsed disease or in remission/on maintenance therapy seen at the Dana-Farber Cancer Institute. For whole exome sequencing studies, we obtained 8 samples of newly-diagnosed untreated patients whose bone marrow, CTC and germline T lymphocytes were available and selected for exome sequencing. We sequenced the whole exome of BM clonal PCs and CTCs up to 200x, and germline cells up to 50x. Whole genome amplification (WGA) was performed for CTCs, and two independent libraries were constructed from the sample, followed by sequencing up to 100x for each duplicate. For samples with WGA, only single nucleotide variants (SNVs) shared in both parallel libraries were used. Results: Before investigating if CTCs could represent a reliable non-invasive alternative to perform genomic characterization of MM patients, we first aimed to define its true applicability at different disease stages. Using sensitive MFC, we showed that CTCs were detectable in 40/50 (80%) newly-diagnosed MM patients, and in 71/130 (55%) of multiple sequential samples from patients with relapsed disease or in remission/on maintenance. As for the prognostic value of CTC enumeration, 19 of the 40 newly-diagnosed cases displaying PB CTCs had relapsed (median time-to-progression of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed (median time-to progression not reached; P=.08). Afterward, we investigated whether dynamic changes in the kinetics of CTCs in sequential PB samples from patients with relapsed disease or in remission/on maintenance therapy was also predictive of outcome. Accordingly, increasing CTC counts were associated with poor overall survival (P= .01), indicating that both the absolute numbers of CTCs and trend of CTC are predictive of outcome in MM. After demonstrating that CTCs can be readily detected in the majority of MM patients, we then determined the mutational profile of CTCs and compared it to that of patient-paired BM clonal PCs. We identified a median of 223 and 118 SNVs in patient-paired BM clonal PCs and CTCs, respectively. The concordance of somatic variants found in matched BM clonal PCs and CTCs was of 79%. Noteworthy, upon investigating specific mutations implicated in MM (eg. KRAS, NRAS, BRAF) a total of 18 nonsynonymous SNVs (NS-SNVs) in 13 genes were identified in our cohort, and most of these NS-SNVs were simultaneously detected in matched BM clonal PCs and CTCs from the same patients. That notwithstanding, we also identified several unique mutations present in CTC or BM clonal PCs; of those, up to 39 NS-SNV were identified as CTC specific, and 6 NS-SNVs in 4 genes (CR1, DPY19L2, TMPRSS13, HBG1) were detected in CTC from multiple patient samples. A significant concordance for the pattern of copy number variations (CNVs) between matched BM and PB tumor cells was also observed. Conclusion: This study defines a new role for CTCs in the prognostic and molecular profiling of MM patients, and provides the rational for an integrated flow-molecular algorithm to detect CTCs in PB and identify candidate patients for noninvasive genomic characterization to predict outcomes. Disclosures Paiva: Sanofi: Consultancy; Millennium: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMAb AG: Consultancy. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Schlossman:Millennium: Consultancy. San Miguel:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Description: 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.

Description: Introduction: Multiple Myeloma (MM) is a genetically complex and evolutionary process with well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. A small number of detectable pre-malignant clones are present in early stage and continue to acquire more genomic abnormalities leading to overt disease. The interaction between cancer cells and their environment is reciprocal, multiple components in the tissue environment can influence cancer clonal evolution and cancer cells in turn can also remodel the microenvironment and further disseminate to spatially separated areas of BM. To accurately predict the course of disease with the presence of BM environment, we require methods to estimate clone-specific growth rates and define clones that have the propensity of dissemination. Methods: We developed a novel 'bone chip' MM metastatic xenograft model using fluorescent protein tagged 'rainbow' system which enables both molecular profiling and functional tracking of clonal dissemination of tumor cells by performing tumor-bearing bone chip implantation subcutaneously to SCID-beige mice (SCID-murine model). Rainbow MM cells with equal proportion of all 15 colors were injected into donor femurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. Tumor clones in the implanted bone chip (primary sites) and distant host BM (metastatic sites) were purified by sorting and underwent RNA sequencing. By intersecting differentially expressed genes, we identified a set of genes, the expression of which were altered during disease dissemination and designated this set of genes as 'metastatic signature'. In addition, we also performed genome-wide CRISPR/Cas9-mediated loss-of-function screen in a subcutaneous xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM. The cell library infected with human sgRNA library was injected subcutaneously into SCID-Beige mice on both flanks. When metastasis was established, the fractions of each sgRNA of the primary and metastatic tumors were calculated 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. 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 SCID-murine model demonstrated a distinct gene expression profile of the metastatic tumors. Gene Set Enrichment Analysis of the metastatic signature in publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature is significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Through genome-wide CRISPR screening in vivo, we found that the gene targets of the most enriched sgRNAs in the BM samples were preferentially involved in important cellular processes, such as cell cycle regulation and several oncogenic signaling pathways. Additionally, many sgRNAs that remained the implanted sites until late stage were depleted during dissemination, indicating their targeted genes were important for progression. These depleted sgRNAs 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. By using a network-based inference of protein activity method, we chose 4 genes (HMGA1, KLF6, TRIM28 and PA2G4) and validated in SCID-murine model using CRISPR mediated loss-of-function screen which prioritized HMGA1 as the key regulator in MM dissemination. 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. We then established a platform for future invivo CRISPR screens to investigate essential genes of response to targeted therapies and/or immunotherapies. Furthermore, a metastatic gene signature was identified and among these, HMGA1 was validated as potential regulator of MM metastasis. Disclosures Roccaro: AMGEN: Other: Advisory Board; GILEAD: Research Funding. Ghobrial:Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy; Janssen: Consultancy.

Description: Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P

Description: 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.