Walters, Denise K.

Abstract The introduction of novel immunomodulatory drugs (IMiDs), such as lenalidomide, has drastically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis and apoptosis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). Additionally, we examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its putative downstream target interferon regulatory factor 4 (IRF4), both of which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that response to lenalidomide did not correlate with specific cytogenetic translocations. There were, however, distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μM versus 0.005 μM, p=0.004), but did not have an effect on the IC50 of the non-responsive HMCL DP-6 (p>0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN or IRF4 expression levels. In summary, our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. Additionally, while CRBN and IRF-4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment. These results suggest that the development of a new treatment paradigm based on cytogenetic subtype of MM alone may be insufficient, and that additional markers may need to be incorporated. Furthermore, the disconnect between our findings of expression of CRBN and IRF-4 in lenalidomide-treated HMCLs and patient data highlights the need for further investigation into the role of the bone marrow microenvironment on the regulation of these previously demonstrated markers of lenalidomide response. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2817 Poster Board II-793 Abnormal plasma cells (PC) present in patients with multiple myeloma (MM) and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS), characteristically possess multiple chromosomal abnormalities. Moreover, both stages of disease exhibit considerable intratumor heterogeneity, which often becomes even more complex during disease progression. The precise mechanism(s) underlying this process remains unknown. However, we hypothesize that DNA double-strand breaks (DSBs) and compromised repair of these deleterious lesions may underlie intratumor heterogeneity and clonal evolution in the monoclonal gammopathies. In this regard, H2AX, a member of the H2A family of histones, plays a particularly important role in the DSB response and prevention of cancer. Immediately following DSB formation, one or more of the PI3K-like kinases become activated and rapidly phosphorylate H2AX on a conserved serine residue. Phosphorylated H2AX (γH2AX) is then rapidly recruited to the DSB site and is readily detectable as DNA damage foci by immunohistochemistry. The precise function of γH2AX has yet to be determined, however, it is hypothesized that γH2AX may recruit DNA repair proteins to the DSB site and may aid in keeping severed DNA ends in place in order to avoid erroneous end joining. Despite the functional uncertainty of γH2AX, the presence of γH2AX nuclear foci serves as an excellent indicator of DSBs. Therefore, the goal of our study was to assess MM cells for evidence of DSBs. We began our studies using a panel of 8 human MM cell lines. Of note, the number of foci was found to vary among the MM cell lines and to vary from cell to cell with the number of γH2AX foci per cell ranging from 0 to 28. The presence of γH2AX in these cells was also confirmed via flow cytometry and western blotting. We also wished to determine if primary MM and MGUS PCs displayed evidence of DSBs. Among primary patient samples, freshly isolated PCs from 13/18 MM patients and 1/3 MGUS patients exhibited evidence of γH2AX foci. Taken together with the MM cell line data, the number of γH2AX foci was found to increase across the disease spectrum of MGUS to MM patient sample to MM cell line. Endogenous γH2AX foci have previously been detected in a variety of tumor cell lines. Although these foci have been hypothesized to derive from multiple factors, the extent of phosphorylation has been shown to be associated with the number of chromosomal aberrations as well as the phase of the cell cycle. In general, S and G2/M phase cells tend to demonstrate higher levels of H2AX phosphorylation, which is most likely due to doubling of histone content during the cell cycle and the fact that chromatin condensation during DNA replication can also trigger H2AX phosphorylation. Thus, it remained possible that the γH2AX displayed by the cell lines simply reflected cells in the S phase of the cell cycle. To address this possibility, we labeled cells with BrdU and then measured levels of γH2AX in cells in the G1, S and G2/M phases of the cell cycle. However, we observed nearly equal levels of γH2AX in G1 and S phase cells suggesting some level of γH2AX foci was independent of DNA replication. These results were also consistent with our observation that there is no correlation between the plasma cell labeling index and the number of γH2AX foci in CD138+ plasma cells isolated from 18 MM patients. Thus, endogenous γH2AX in MM cells does not appear to be primarily attributed to cycling cells and may be indeed reflective of DSBs. Finally, to further demonstrate that the γH2AX foci genuinely reflected sites of DSBs, we performed double staining for γH2AX foci and 53BP1, a protein that is known to be recruited to DSB sites following DNA damage. Results revealed clear colocalization of γH2AX and 53BP1 in both MM cell lines and MM patient samples. Given that DSBs can lead to genomic instability and tumor progression, our observations that primary MGUS and MM PCs display evidence of DSBs at isolation are intriguing and suggest a mechanism whereby clonal evolution occurs in the monoclonal gammopathies. The presence of a higher frequency of γH2AX foci in MM cell lines is consistent with their derivation from MM patients with aggressive disease. Collectively, these studies suggest MGUS/MM PCs may display an impaired ability to repair DNA damage and studies designed to examine this possibility are underway. Disclosures: Dispenzieri: Celgene: Research Funding.

Abstract Abstract 470 Multiple myeloma (MM) is an incurable and fatal neoplasm characterized by the accumulation of clonal plasma cells (PC) that results in significant end organ and tissue damage. MM is preceded by either of two premalignant asymptomatic stages, monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), both of which are identified by the presence of clonally expanded abnormal PC populations. While MGUS and SMM patients' abnormal PC populations may remain stable for years, both have a life-long significantly increased risk of progressing to MM (1% and 10% per year, respectively). For this reason, a better understanding of the molecular events prompting malignant progression and increased accuracy in identifying those patients that have begun to transition to MM is urgently needed. In this study, we identified a novel MM marker, CD147 (also known as extracellular matrix metalloproteinase inducer (MMP), or EMMPRIN), that is not only over-expressed on MM PCs as compared to its premalignant counterparts, but whose increased expression correlates with the level of abnormal PC proliferation. To our knowledge, we are the first to demonstrate a role for CD147 in MM. CD147 has been shown by others to display a variety of activities and those with potential relevance to MM include stimulation of increased MMP production and angiogenesis, and playing a critical role in glycolysis via facilitation of excess cellular lactate transport. Our initial experiments revealed that MM PCs overexpress CD147 mRNA relative to MGUS PCs. Flow cytometric analysis corroborated these data and demonstrated variable expression of CD147 across the disease continuum ranging from no expression to bimodal or uniform expression. Indeed, there was a significant difference between CD147 expression on MGUS and SMM PCs compared to that on MM PCs (p=0.02 and 0.005, respectively). We next determined whether CD147 had a signaling role in these cells. Using the natural CD147 ligand, cyclophilin B (CypB), we showed that addition of CypB to either human MM cell lines (HMCLs) or CD138+ patient PCs resulted in increased PC proliferation as measured by [3H]-thymidine incorporation. In a complementary manner, addition of CD147 antibodies significantly inhibited proliferation without an effect on cell viability. By western blot analysis we further demonstrated that CypB-mediated CD147 activation leads to MAPK phosphorylation. Next, we isolated CD147+ and CD147- MM cells from patients whose tumor cells bimodally expressed this marker and assessed the response of each subset to IL-6 and CypB. The CD147+ subset was almost solely responsible for the proliferative response in all cases examined. In addition, we cultured bone marrow mononuclear cells from CD147 bimodally expressing MM patients overnight with bromodeoxyuridine before performing cell cycle analysis on the CD147+ and CD147- MM PC populations. Remarkably, the CD147+ PCs were greatly enriched for cells in the S and G2/M phases of the cell cycle, whereas the CD147- PCs resided almost entirely in the G0/G1 phase. In the final set of experiments, we employed siRNA knockdown strategies using HMCLs to definitively test the role of CD147 in MM cell proliferation. Indeed, IL-6 induced proliferation was significantly compromised following CD147 down regulation, which was not attributed to increased apoptosis. However, IL-6 mediated phosphorylation of MAPK remained robust suggesting that the IL-6 signaling pathway overall was not compromised in these cells. Finally, cell cycle analysis demonstrated that CD147 down regulation resulted in a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a decrease in the number of cells in the G2/M phase of the cell cycle, as compared to cells transfected with control siRNA. In conclusion, our data suggest that the CD147 molecule plays a critical role in the biology of malignant MM PCs, particularly as it concerns MM cell proliferation, and may thus serve as a useful and attractive target for reducing the proliferative compartment of this disease. Ongoing studies are investigating additional roles for MM cell CD147 expression, e.g., its role in MMP induction in the tumor microenviroment. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2917 Multiple myeloma (MM) is a cancer in the bone marrow (BM) characterized by the accumulation of transformed plasma cells (PCs). The pre-malignant form of the disease, monoclonal gammopathy of undetermined significance (MGUS), shares many of the genetic abnormalities found in MM, including chromosomal translocations, hyperdiploidy, and gene-specific mutations. Given this, we believe other factors within the tumor microenvironment must contribute to disease progression by influencing cell survival and/or proliferation. Eosinophils (Eos) are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states such as allergy and asthma. Recently, they were also shown to play a role in the regulation of murine BM PC homeostasis via their secretion of IL-6 and APRIL. Murine BM PCs and Eos both express CXCR4 and are believed to home to CXCL12 expressing stromal cells (SCs) in the BM. The goal of this study, therefore, was to investigate whether Eos, or soluble mediators released by Eos, have biological activity on MM cells. Thus, it is possible that this type of innate immune cell may be present in the tumor microenvironment in MM patients to support disease progression. To our knowledge, a potential role for Eos in MM has not been previously studied. We began our studies by assessing whether Eos are co-localized with normal BM PCs and/or MM PCs. Immunofluorescence analysis of BM core biopsies from normal subjects revealed occasional colocalization of PCs with Eos. Similarly, MM biopsies showed regions of MM cell clusters with increased Eos density, suggesting possible biological interactions. However, we also observed regions of MM cell clusters that lack Eos, which could indicate the liberation of these transformed cells from the requirement of Eos for survival/proliferation. Next, using Eos isolated from human BM aspirates and a panel of disease-relevant human MM cell lines (HMCLs) extensively characterized in our laboratory, we aimed to verify that MM cells and Eos could both migrate toward the chemokine CXCL12. Our data showed that indeed the KAS-6/1, ANBL-6, DP-6, and KP-6 HMCLs and human BM Eos all migrated toward this chemokine. We then determined if Eos had biological activity towards MM cells as revealed by enhanced DNA synthesis. Consistent with our interpretation of the immunofluorescence stained sections, the proliferation of some, but not all, HMCLs was enhanced when cocultured with Eos isolated from either human BM or peripheral blood. To address whether contact between Eos and HMCLs was required for this phenomenon, we assessed HMCL proliferation upon treatment with Eos culture supernatant (SN). Our data suggest that the effect of Eos on the Eos-inducible HMCLs can be contact-independent as treatment of these HMCLs with Eos SN increased their proliferation. Similarly, proliferation of primary CD138+ MM cells was also enhanced when treated with Eos SN. Because BM PCs reside in niches that include support cells such as SCs and that proliferation of MM PCs is enhanced in the presence of SCs, we next questioned whether Eos can substitute for SCs in this niche or if Eos and SCs support PC survival/proliferation through different mechanisms. We observed that Eos and BM SCs together stimulated more HMCL proliferation than either cell type did alone, indicating the presence of non-redundant roles for the two cell types. Finally, we began to investigate the mechanism by which Eos enhance HMCL proliferation. In contrast to prior reports that murine Eos express IL-6, mRNA transcripts of IL-6, a known proliferation-inducing cytokine for MM cells, were not detected in human Eos. Moreover, analysis of the Eos SN showed an absence of IL-6. Neutralization of IL-6 in the HMCL-Eos coculture did not abolish the induced proliferation. Altogether, these data suggest that human Eos utilize a yet to be identified, IL-6-independent mechanism to support malignant PC proliferation. Taken together, our data show that Eos and MM cells can colocalize in the BM via their migration toward a common source of CXCL12, i.e. BM SCs, to create a niche that promotes tumor cell growth. Eos can enhance malignant PC proliferation via soluble products, although additional contact-dependent effects may exist as well and have not been explored. Additional studies are currently underway to further characterize the mechanism by which Eos may influence the biology of MM in the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract Reliable model systems are critical for investigating the genetic and molecular drivers of disease pathogenesis. Although the number of cell line model systems for multiple myeloma is extensive, recent studies showed patient tumors and cell lines clustering separately based on gene expression profiles. Furthermore, the hyperdiploid karyotype, present in approximately 50% of myeloma patients, is also extremely underrepresented in established human myeloma cell lines (HMCLs): only one of 59 HMCLs contains this phenotype. These data suggests that the average HMCL does not accurately represent the average myeloma patient and questions the validity of using HMCLs as model systems. However, most HMCLs were established from patients with pleural effusions or plasma cell leukemia, which are highly progressed and rare forms of myeloma. To assess how cell lines correlate with patient material, we performed a copy number abnormality (CNA) comparison between four myeloma primary tumors and their corresponding HMCLs. Here, we show that established HMCLs maintain the majority of the CNAs, contain a ten-fold greater frequency of 7q gains compared to patients, and that KP-6 is the second hyperdiploid HMCL known to date. CNAs were identified using array comparative genomic hybridization (Agilent 244k) with data analyzed at a resolution of 26kb. On average 59% (46.7-77.3) of the CNAs were shared between the tumors and their cell lines, 17% (8.5-25.9) were unique to the patient samples, and 24% (13.6-36.6) were only detected in the HMCLs. To put these differences in context we also compared the diagnostic material from which JMW-1 was established with a progression sample collected 9 months later after 3 cycles of VAD followed by an autologous peripheral blood stem cell transplant. The patient diagnostic and relapse tumors showed 58% similarity based on CNAs while the diagnostic and relapse tumors contained 8% and 34% unique events respectively. These results indicate that established HMCLs are no more divergent from their primary cells as is the same patient's relapse material. Overall the HMCLs studied appear highly representative of the patient from which they were derived, with most differences attributed to an enrichment of subclonal populations present in the primary tumor admixture. Of particular interest, ubiquitous 7q gains were an unexpected unifying event across all patient tumors and their HMCLs. We then analyzed the 729 baseline myeloma tumors with copy number data from the MMRF CoMMpass study against our panel of 59 similarly characterized HMCLs. We observed that HMCLs contained a 10-fold increase in the occurrence of 7q gains with respect to non-hyperdiploid patients with 7q gains (35/59 in HMCLs and 19/344 in patients). Because 97% of HMCLs are non-hyperdiploid and 7q gains are extremely rare in this population, over-representation of 7q gains in cell lines suggests that this event may promote extra-medullary growth and the establishment of cell lines. Profiling of patient tumors for gains on 7q may therefore increase the probability of establishing a new HMCL and help expand our model systems for understanding this disease. Further studies are required to understand the potential biological significance of this event in vitro . We also identified the KP-6 cell line as a novel hyperdiploid HMCL established from an extramedullary site of an advanced hyperdiploid patient. The presence of trisomies for at least four of the odd-numbered chromosomes is characteristic of a hyperdiploid myeloma patient. From the patient's tumor, clear trisomies were observed on chromosomes 3, 7, 11, 15, 19, and 21 and the HMCL established from this hyperdiploid patient maintains full trisomies of chromosomes 15, 19, and 21, with partial p-arm trisomies on chromosomes 7, 11, and 17. Since this is only the second bona fied hyperdiploid myeloma cell line to date, KP-6 promises to play a major role in our ability to understand the pathogenesis and progression of this prominent patient population. Overall, our research indicates that, based on CNAs, HMCLs are as representative of the primary tumor as is the same patient's tumor at a later time point. Additionally, HMCLs contain a 10-fold overabundance of 7q gains. Finally, we showed that KP-6 is the second hyperdiploid HMCL known to date. Taken together, these results suggest that HMCLs are reliable model systems for investigating the underlying biology of multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Abstract Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Abstract Receptor transactivation, i.e., interaction between unrelated receptor systems, is a growing theme in cytokine and growth factor signaling. In this study we reveal for the first time the ability of IFN-α to transactivate gp130 in myeloma cells. An epidermal growth factor receptor/gp130 chimeric receptor previously shown by us to transactivate endogenous gp130, provided a complementary tool to study the underlying mechanisms of receptor cross-talk. Further analysis revealed that transactivation of gp130 by IFN-α did not require the extracellular or trans-membrane domain of gp130. Moreover, transactivation of gp130 was critically dependent upon Janus kinase activation by the initiating receptor and correlated with rapid and sustained Janus kinase 1 and tyrosine kinase (Tyk) 2 tyrosine phosphorylation. Finally, transactivation of gp130 may be a common theme in myeloma cells, perhaps providing a mechanism for enhanced or qualitatively distinct cellular responses to specific stimuli.

AbstractThe introduction of novel immunomodulatory drugs (IMiDs) has dramatically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib‐based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). We examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its downstream target interferon regulatory factor‐4 (IRF4), which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that lenalidomide response did not correlate with specific cytogenetic translocations. There were distinct groups of lenalidomide‐responsive and non‐responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC‐1 (IC50 = 2.6 μm vs. 0.005 μm, P < 0.0001), but did not have an effect on the IC50 of the non‐responsive DP‐6 HMCL (P > 0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN and IRF4 expression. Our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. While CRBN and IRF4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment.