ALBERT

Description: Abstract 566 Bone destruction is a hallmark of multiple myeloma (MM). More than 80% of MM patients have osteolysis, which is characterized by pathological fractures, severe bone pain, spinal cord compression, and hypercalcemia. These symptoms can severely compromise a patient's quality of life and performance status. It has been proposed that MM cells activate osteoclast (OC)-mediated bone resorption and inhibit osteoblast (OB)-mediated bone formation. However, the mechanism underlying the association of MM cells with development of bone lesions remains poorly elucidated. Our previous studies showed that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in MM cells, is a master regulator of MM-mediated bone destruction. Knocking down or inhibiting p38 MAPK activity in MM cells prevented MM-induced bone destruction in vivo. In the present study, we further investigated the mechanism of MM cell p38 MAPK-induced bone destruction. We hypothesized that p38 MAPK activity in MM cells can regulate OB and OC differentiation and activity by upregulating cytokine production by MM cells. In a cytokine array analysis, we examined the expression and secretion of MM-derived cytokines that regulate OB and OC differentiation. Our results showed for the first time that either knockdown or inhibition of p38 MAPK activity by p38 MAPK short hairpin RNAs or inhibitors significantly downregulated the production of dickkopf-1 (DKK-1) and monocyte chemotactic protein-1 (MCP-1) by MM cells. Real-time PCR and ELISA quantified and confirmed the array analysis results. To determine the role of p38 MAPK-upregulated DKK-1 and MCP-1 production in bone destruction, we administered treatment with neutralizing antibodies to SCID mice injected intravenously with ARP-1 or MM.1S cells. Our results showed that neutralization of DKK-1 and MCP-1 led to fewer bone lesions in these mice. Furthermore, we examined the impact of MM cell p38 MAPK activity on OB and/or OC differentiation. Our results showed that knockdown or inhibition of MM cell p38 MAPK significantly downregulated osteoclastogenesis but upregulated osteoblastogenesis in vitro and in vivo. Although DKK-1 is well known to inhibit OB differentiation, we found that DKK-1, together with MCP-1, promoted OC differentiation and bone resorption. Mechanistic studies further showed that MCP-1 upregulated RANK expression in OC precursors and that DKK-1 increased RANKL secretion from stromal cells and mature OBs, all of which led to activation of the NF-kB and MAPK signaling pathways in OCs. Thus, our study uncovered a novel mechanism by which p38 MAPK signaling in MM cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction in patients with this disease. These findings strongly suggest that disrupting and targeting MM cell p38 signaling are effective approaches to treating osteolytic bone lesions in MM patients. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: XPO1 (CRM1, Exportin 1) is the sole transporter of many tumor suppressor proteins (including MYC, BCL2, BCL6, BTK, IkB) and is elevated in non-Hodgkin Lymphoma. Selinexor (Sel, KPT-330) is an oral covalent inhibitor of XPO1, the first clinical molecule of the Selective Inhibitors of Nuclear Export drug class. The phase I clinical trial of Sel in hematologic malignancies showed promising early single-agent efficacy with modest toxicity in relapsed Diffuse Large B-cell Lymphoma (DLBCL, Gutierrez et al, ASCO 2014). DLBCL, the most common lymphoid malignancy, is currently cured in only 10% of relapsed patients, and consists of 2 subtypes based on putative cell of origin (COO): activated B-cell (ABC) and germinal center B-cell (GCB). We performed preclinical studies of Sel, modeling its single-agent efficacy in frontline and relapsed DLBCL and its potential synergy with other clinically relevant therapeutics. Methods: To model drug resistant DLBCL, resistant subpopulations of 12 patient-derived DLBCL cell lines were created by in vitro intermittent exposure to active congeners of cyclophosphamide, doxorubicin, and vincristine (ivCHOP), approximating clinical practice. To determine if CHOP-resistant DLBCL is also resistant to other agents, we determined single-agent dose response curves and IC50 values for both parental and ivCHOP resistant (CHOP-res) subclones of 4 of these lines at submission (HBL1 & TMD8 of ABC subtype, OCI-Ly7 & HT of GCB subtype, with 8 lines in progress) with Sel, chemotherapy (CT, ivCHOP, DHAP, and ICE), and targeted therapy (TT, ibrutinib, ABT-199, idelalisib, everolimus, MLN0128, alisertib, lenalidomide, bortezomib, I-BET151, and ONC201). Viability was assessed with CellTiter-Glo (Promega) after a 3 day cell culture. IC50 values were determined using GraphPad Prism. Based on these results, we evaluated the ability of Sel to synergize with other agents or restore sensitivity in CHOP-res with a combination “checkerboard” (orthogonal dose titration for each drug). The Combination Index (CI) for pairs at all concentrations was calculated with ComboSyn, with CI values

Description: Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.

Description: Introduction. Targeting antigen-driven B-cell receptor (BCR) signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), but not germinal center B-cell (GCB) DLBCL. We have formally confirmed that GCB-DLBCL cell lines utilize tonic BCR signaling, by showing: 1) sensitivity (variable) to knockout (KO) of the BCR, SYK, and CD19; 2) dependence on CD79A ITAM phosphorylation; and 3) independence from BCR antigen specificity. However, uncertainty remains about molecular events in upstream parts of tonic BCR signaling, why dependence of GCB-DLBCL cells on tonic BCR signaling is variable, and their clinical relevance. Methods. We used CRISPR/Cas9 methods to modify selected genes by KO and/or knock-in (KI) of the cDNA of a fluorescent protein (FP; e.g., GFP), with the FP serving as a marker of cells with gene KO or modification, or as a gene-fused tag for localization or quantitation. Cells expressing a membrane-targeted Forster resonance energy transfer (FRET) based AKT activity reporter (Lyn-AktAR2) were used to measure AKT activity directly by flow cytometry (FCM). Results. The effect of KI of CD79A Y188F mutation alone was similar to complete BCR KO, implying that CD79A Y188 phosphorylation is essential for tonic BCR signal transduction. Western blot analysis of GCB-DLBCL cell lines after BCR KO showed variable decreases of AKT S473 phosphorylation (frequently used as surrogate measure of AKT activity), but these did not correlate well with the variable decreases in proliferation of GCB-DLBCL cell lines caused by BCR KO. Measuring AKT activity directly (Fig. 1), or by another indirect approach (surface expression of CXCR4, a target gene of FOXO1 inhibited by AKT activity), showed high correlation between decreases in AKT activity and proliferation after BCR KO. In contrast to the variable effect of BCR KO on growth, pan-AKT KO was uniformly growth-slowing in GCB-DLBCL lines (Fig. 2). Interestingly, baseline surface density of BCR units in GCB lines, quantified by FCM using CD79A-GFP KI cells or anti-CD79B staining, correlated highly with reduction in growth or AKT activity caused by BCR KO (Fig. 3). These findings lead us to conclude that the BCR contributes to AKT activation in GCB-DLBCL cell lines, to a variable degree determined by BCR surface density. We also conclude that BCR surface density is determined by cell line-specific factors, as well as immunoglobulin heavy (IgH) and light (IgL) hypervariable region (HVR) sequences, based on measurements of BCR surface levels after exchanging endogenous HVR sequences in OCI-Ly19 and OCI-Ly7 cell lines for HVRs derived from other GCB and ABC-DLBCL cell lines. Reduction of AKT activity after BCR KO (measured by FRET reporter) and baseline BCR surface density in GCB-DLBCL cell lines also correlated well with the sensitivity of GCB-DLBCL lines to the clinically-tested SYK inhibitor (P505-15, PRT062607) or FDA-approved PI3K p110d isoform specific inhibitor (idelalisib). Interestingly, isogenic GCB-DLBCL cell lines with KO of PTEN, a negative regulator of AKT activation, were substantially more resistant to both inhibitors. A crucial role of PTEN deletion in overcoming dependence on tonic BCR signaling in GCB-DLBCL is supported by evidence from two naturally PTEN-deficient cell lines: SUDHL10, which adjusts to BCR KO and resumes normal growth, and HT, which lacks BCR expression, due to a frameshifting deletion in its IgH HVR. Re-expression of the BCR in HT, by KI to correct the IgH sequence, does not affect HT cell line growth. Conclusion. Our findings suggest a biomarker-guided therapeutic strategy in GCB-DLBCL: targeting tonic BCR signaling in BCR-high patients, by inhibiting CD79A phosphorylation, SYK, or PI3K, and downstream targeting of AKT in BCR-low and/or PTEN-deficient patients. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Disclosures Burger: Pharmacyclics: Research Funding. Westin:Chugai: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL. Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting. Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells. GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO. Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways. Table A. B. Figure 1. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1B Figure 1B. Figure 2 Cell cycle changes with BCR KO in OCI-Ly7. Figure 2. Cell cycle changes with BCR KO in OCI-Ly7. Disclosures No relevant conflicts of interest to declare.

Description: Chemotherapy is the most effective treatment for multiple myeloma (MM). Several new drugs have been developed to prolong MM patient survival. However, application of these drugs, such as bortezomib (BTZ), usually induces drug resistance, and patients are prone to quick relapse. It is known that cell death or proliferation is regulated by the crosstalk between apoptosis and autophagy, and autophagy activation inhibits apoptosis by reducing caspase cleavage. Recent studies have shown that autophagy activation plays a role in chemotherapy drug resistance in cancer patients, in particular, BTZ treatment activates autophagy in MM, indicating that inhibition of autophagy could overcome BTZ-induced drug resistance. We have recently reported the generation of monoclonal antibodies (mAbs) against human beta2 microglobuline (b2M), and have found that high dose mAbs have strong apoptotic effects on MM. In this study, using a BTZ treatment-setting, we hypothesized that anti-β2M mAbs could overcome drug resistance and enhance chemotherapy efficacy in MM by inhibiting autophagy. To investigate the combined effects of mAbs and BTZ, MM cells were cultured with addition of BTZ (5 nM) or mAbs (clone D1; 10 mg/ml) or both for 24 hours. Results from Annexin-V binding assay indicated that combined treatment significantly enhanced apoptosis in MM cell lines, such as ARK, ARP-1, MM.1S, and U266, and in primary MM cells as compared with individual treatment, respectively. Next, BTZ-sensitive or -resistant MM cells KAS-6 WT/V10R or OPM-2 WT/V10R were used to determine the importance of BTZ in the synergistic effects, and such synergistic effects was only shown in apoptosis of BTZ-sensitive cells, but not in BTZ-resistant cells. Furthermore, combined treatment significantly increased apoptosis in β2M-overexpressing, but decreased in β2M-knocking down MM cells, which indicated that the synergistic effects is dependent on the surface β2M expression on MM cells. Mechanistic studies further showed that BTZ treatment resulted in an accumulation of cleaved-caspase 9/3 and PARP cleavage, suggesting that combined treatment enhanced caspase activation. In addition, Western blot analysis showed that BTZ treatment upregulated the expression of autophagy proteins, such as LAMP-1, Beclin 1, and LC3B, in a dose dependent manner, whereas combined treatment decreased their expression. Beclin 1, which is a key protein required for autophagy, has been reported to have a potential NF-kB p65 binding site on its promoter. Therefore, we checked if mAbs inhibited BTZ-induced autophagy via NF-kB signaling pathway. Our data showed that BTZ treatment increased p65 nuclear translocation and the levels of phosphorylated p65 in MM cells, while combined treatment significantly reduced both. ChIP assay has further verified that mAb treatment inhibited p65 binding to Beclin 1 promoter. To examine our hypothesis in vivo, MM cells ARP-1 or MM.1S were subcutaneously injected into SCID mice. The mice were then subcutaneously injected with mAbs (0.6 mg/kg) or intraperitoneally injected with BTZ (0.1 mg/kg) individually or in combination. Tumor burdens were assessed by measuring tumor volumes and serum M-protein levels by ELISA. The combination of anti-β2M mAb and BTZ treatment repressed tumor growth and prolonged the survival of tumor-bearing mice as compared with individual treatment. In conclusion, our study for the first time demonstrated that anti-β2M mAbs prevent BTZ resistance and enhance its anti-MM efficacy by reducing the expression of autophagy proteins via NF-kB signaling. Thus, our studies provide a new insight into clinical development of anti-β2M mAbs to overcome chemotherapy drug resistance and improve MM patient survival. Disclosures: No relevant conflicts of interest to declare.

Description: Currently, chemotherapy is the most effective treatment for multiple myeloma (MM). Although some new drugs have been shown to prolong survival in MM patients, these patients are prone to rapid relapse after high-dose treatment. Recent studies show that several bone marrow (BM) stromal cells are potentially involved in drug resistance. However, the role of other stromal cells is unclear. Adipocytes (ADs) are a major component of BM stromal cells. ADs have been shown to be involved in tumor rapid growth, metastasis, and apoptosis. Clinical studies suggest that BM ADs are associated with an increased risk of MM. Moreover, ADs isolated from patient BM biopsies were shown to support MM proliferation and migration. However, no published study has examined the importance of ADs in MM drug resistance. In addition, autophagy activation has been shown to induce drug resistance in cancer patients. We hypothesized that BM ADs protect MM cells from chemotherapy drug-induced apoptosis by autophagy activation. To examine the role of ADs in MM drug resistance, MM cells were cocultured with ADs at a ratio of 1:5 for 24 hours in medium with melphalan, dexamethasone, or bortezomib, the commonly used drugs for the treatment of MM. MM cells included primary MM cells isolated from BM aspirates of 5 MM patients and 6 MM cell lines. Human ADs were generated from mesenchymal stem cells derived from the BM mononuclear cells of healthy human fetal bones or BM aspirates of MM patients or healthy adult donors, cultured in AD medium for 2 weeks. ADs generated in vitro contained cytoplasmic Oil red O+ lipid droplets and produced triglycerol. Our results showed less drug-induced MM apoptosis in cocultures of MM cells and ADs compared with cultures of MM cells alone. Western blot analysis showed that treatment with melphalan upregulated the levels of cleaved caspase-9 and -3, but not -8, and PARP in MM cells. Compared with cultures alone, cocultures with ADs showed significantly lower levels of cleaved caspase-9, -3, and PARP in melphalan-treated MM cells. Mechanistic studies further showed that cocultures of ADs, compared with cultures alone, significantly upregulated the expression of autophagy proteins LC3B, Atg3, Atg5, and LAMP-1, but not Beclin-1. The addition of autophagy inhibitors 3-methyl adenine and chloroquine diphosphate to the cocultures remarkably enhanced apoptosis and caspase activation. Furthermore, we observed that cocultures of MM cells and ADs with either cell-cell contact or those separated by transwell inserts conferred similar protection from drug-induced apoptosis. We identified that AD-produced adipokines such as adiponection, leptin, adipsin, IL-6, MCP-1, TNF-a, and IGF-1, but not VEGF and CRP, were abundant in all examined ADs. Among these adipokines, adiponection, leptin, and adipsin were mainly produced from ADs and not from BM stromal cells, whereas other adipokines were produced from both cells. The addition of antibodies against these adipokines to the cocultures enhanced apoptosis and reduced autophagy, whereas addition of these adipokines to the cultures alone inhibited apoptosis and enhanced autophagy. In vivo studies validated these findings that injection of BM-derived ADs into the implanted human bones of SCID-hu mice bearing primary MM cells reduced response to treatment with melphalan and induced autophagy activation. Taken together, our findings elucidate a novel mechanism of MM drug resistance, through BM ADs. Our studies also provide evidence that targeting BM ADs may be a new approach to improve the efficacy of chemotherapy for the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points CD138+ MM cells are a major source of SHH. Autocrine SHH enhances MM drug resistance.

Description: Key Points The GCB subtype of DLBCL relies exclusively on tonic BCR signaling via CD79A Y188. PTEN protein expression and BCR surface density determine the contribution of tonic BCR signaling to AKT activity in GCB-DLBCL.

Description: Abstract 439 In multiple myeloma (MM), an incurable bone malignancy characterized by plasma cell accumulation in the bone marrow (BM), patients have increased osteoclast (OC) activity. As a result, more than 80% of MM patients develop osteolytic bone lesions during the course of the disease. Osteolytic bone lesions cause morbidity, such as pathological fractures, bone pain, and hypercalcemia, and therefore, severely affect the patients' quality of life. A better understanding of the mechanism of MM cell-induced OC activation could lead to a novel approach to treating MM bone disease. It is commonly accepted that MM cells are responsible for OC activation. Cocultures of MM cells with monocyte-derived OC precursors (preOCs) induce OC formation and bone resorption. In addition, previous studies have demonstrated the critical roles of receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) in aberrant OC activity upregulation in MM BM. Moreover, MM-derived cytokines, such as IL-3, IL-7, and MIP-1d, have been shown to enhance OC formation in a RANKL-dependent or -independent manner. When we cocultured MM cells with monocytes, but not preOCs, and treated them with RANKL, we made the novel observation that MM cells inhibited RANKL-induced OC differentiation. Specifically, human monocytes, isolated from five different healthy donor PBMCs or murine monocytic/macrophage cell line RAW264.7 cells, were transwell-cocultured with MM cells, either human MM cell lines or primary MM cells isolated from patients, in mediums with or without RANKL (50 mg/mL) for 14 days. Mature OCs, characterized as TRAP+ multinuclear cells, were detected by TRAP staining and further confirmed by quantitative RT-PCR for the expressions of mature OC marker genes, such as CTSK, CALCA, and TRAP. Our results showed that coculture with MM cells inhibited the development of mature OCs from monocytes, and suppressed RANKL-induced NFκB and JNK activation. By ELISA, we analyzed the levels of soluble cytokines in the conditioning medium of MM cells and found that MM cells produced large amounts of IL-10. Adding neutralizing antibody against IL-10 significantly abrogated MM inhibition of osteoclastogenesis, whereas adding IL-10 inhibited OC differentiation and downregulated the mRNA and protein levels of RANK on monocytes/preOCs. As MM cells grow in the BM, we wondered whether bone marrow stromal cells (BMSCs) could regulate the MM cell inhibitory effect on osteoclastogenesis. Our results showed that although BMSC itself did not affect osteoclastogenesis, cocultures of monocytes with both MM cells and BMSCs significantly restored RANKL-induced osteoclastogenesis. Analysis of soluble cytokines by ELISA showed that the levels of MCP-1, but not IL-10, were significantly upregulated in medium from MM/BMSC cocultures. Adding neutralizing antibody against MCP-1 highly inhibited OC activation, while adding MCP-1 enhanced OC differentiation and upregulated the expression of RANK on monocytes/preOCs. Overall, our findings are the first to elucidate a novel mechanism by which MM cells inhibit osteoclastogenesis by producing IL-10 and the presence of BMSCs suppresses MM inhibitory effects by up-regulating MCP-1. Regulation of RANK expression on monocytes/preOCs by MCP-1 and IL-10 determines osteoclastogenesis. Our studies provide evidence that targeting BM microenvironmental cells and/or factors may be a new approach to treating MM bone lesions. Disclosures: No relevant conflicts of interest to declare.