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Activating BRAF mutations are found in a small subset of patients with newly diagnosed multiple myeloma, but prevalence increases in late-stage, refractory disease, and the mutations are associated with adverse outcome. This prospective single-arm, open-label, multicenter phase 2 trial assessed the efficacy and safety of combined BRAF/MEK inhibition, using encorafenib and binimetinib, in patients with relapsed/refractory multiple myeloma (RRMM) carrying a BRAFV600E mutation. Patients received 450 mg encorafenib once daily and binimetinib 45 mg twice daily. The primary end point was the overall response rate achieved within the first year after start of treatment according to International Myeloma Working Group criteria. Twelve RRMM patients with a median of 5 prior lines of therapy were enrolled. The overall response rate was 83.3%, with 10 patients achieving at least a partial response. The median progression-free survival was 5.6 months, and overall survival was 55% at 24 months. Emerging resistance to therapy was driven by RAS mutations and structural variants involving the BRAF locus. This is the first prospective clinical trial to demonstrate that combined BRAF/MEK inhibition is highly effective in patients with BRAFV600E-mutated RRMM, and it represents a successful targeted precision medicine approach in this disease. This trial was registered at www.clinicaltrials.gov as #NCT02834364.

Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.

Blinatumomab, the first-in-class CD3/CD19 bispecific T-cell engager antibody construct, has recently been approved for treating patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia. However, the clinical proof of concept of blinatumomab efficacy was initially demonstrated in patients with R/R B-cell non-Hodgkin lymphoma (B-NHL) in the MT103-104 phase 1 dose-escalation and expansion trial (NCT00274742), which defined 60 µg/m2 per day as the maximum tolerated dose (MTD). The clinically most relevant adverse effects were neurologic symptoms and cytokine release syndrome. Currently, there are no data on long-term outcomes and toxicity for B-NHL patients receiving blinatumomab treatment, so we performed a single-center, long-term follow-up analysis of 38 patients who participated in the MT103-104 phase 1 trial. We found no evidence for long-term toxicities, especially no blinatumomab-induced neurocognitive impairments. For the entire study population, the median overall survival (OS) was 4.6 years. Remarkably, patients who had received ≥60 µg/m2 per day and responded to blinatumomab achieved a median OS of 7.7 years. Of note, 6 of the surviving patients treated at the MTD have been treatment-free for more than 7 years. In contrast, patients who were treated at dose levels below the MTD had a median OS of only 1.1 years. These results indicate that 60 µg/m2 per day seems to represent the targeted dose level of blinatumomab required for durable remission in R/R B-NHL. Here, we provide the first clinical evidence that blinatumomab lacks long-term toxicity and has the potential to induce sustained remissions in patients with R/R B-NHL.

Introduction The CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape. Methods Primary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed. Results Of n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function. Conclusion Our study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

Introduction: The treatment of patients (pts) with rrMM remains challenging and response is often limited in depth and duration. In contrast to many other malignant diseases, targeted therapy in MM is hampered by the limited number of actionable targets. Activating mutations of BRAF have been found in 2-4% of newly diagnosed MM and in up to 8% of rrMM. Several case reports have reported clinical efficacy of downstream pathway inhibition in MM. This phase II trial is evaluating the safety and efficacy of combined BRAF/MEK inhibition in rrMM pts with BRAF V600E mutation. Aims: The primary objective of this study was to demonstrate the therapeutic efficacy of encorafenib in combination with binimetinib. The primary endpoint was the overall response rate (ORR), defined as best response within 1 year of treatment. Main secondary endpoints included progression-free survival (PFS) and duration of response. Safety analyses included all adverse events (AEs) with those of grade 1/2 only being assessed further if considered to be related to study medications by the investigator. Methods: A total of 15 pts with rrMM who have failed at least 2 prior lines of therapy, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMID), were planned to be enrolled in this open-label phase II multicenter trial. Key inclusion criteria were presence of a BRAF V600E/K mutation confirmed by both mutation-specific immunohistochemistry (IHC) and next-generation sequencing in more than 50% of MM cells. Exclusion criteria included plasma cell leukemia, CNS involvement, cardiac dysfunction, or a history of retinal vein occlusion. Pts received encorafenib 450mg p.o. daily and binimetinib 45mg p.o. twice daily. Responses were assessed using IMWG 2016 criteria. The primary endpoint was analyzed by testing the one-sided null hypothesis of ORR Results: As of July 2020, 12 pts have been enrolled and were evaluable for safety and 11 pts were evaluable for response as 1 pt had just completed cycle 1 with confirmation of response pending at the time of data lock. Subtypes of MM were IgG in 6 pts, IgA in 2 pts, and Bence Jones in 4 pts. Pts had received a median of 5 prior lines of therapy (range 2-14). All pts had failed previous treatment with both a PI and an IMID and in addition carfilzomib, pomalidomid, and/or anti-CD38 antibodies in 8/4/6 pts, respectively. The study already reached its primary endpoint. The ORR was 82% (lower limit of the 95% CI 56.4%, one-sided exact binomial test, p Adverse events of all grades, assessed to be at least possibly related to a study drug, occurred in 9/12 pts and included blurred vision, macula edema, cramps, arthralgia, diarrhea, skin rash, and decreased left ventricular function. Grade 3 or 4 AEs irrespective of causality occurring in more than 1 pt were reported in 8 pts with anemia, hypertension and thrombocytopenia (3/ 3/ 2 pts, respectively). SAEs were reported in 2 pts, respiratory tract infection in both pts and tooth decay in 1 pt that was not related to the study medication. No deaths occurred while on treatment or within 30 days of the end of treatment. Preliminary results of biomarker assessments reveal new RAS mutations and amplification of the BRAF locus at the time of relapse while on study treatment as potential markers of resistance. Analysis of pharmacodynamic markers by phospho-IHC reveals suppression of BRAF/MEK signaling at cycle 1 / day 28 and restoration of expression at the time of relapse. Conclusion: Targeting activating BRAF mutations in rrMM by combining the BRAF inhibitor, encorafenib, and the MEK inhibitor, binimetinib, induces rapid and deep responses in the majority of pts. No new safety signal has been observed when compared to prior reports on these compounds. The study reached its primary endpoint with fewer pts than expected. The primary efficacy and safety analysis will be presented at the conference. Disclosures Raab: Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Celgene: 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; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Scheid:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Besemer:GSK: Honoraria; Janssen: Honoraria. Metzler:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; GSK: Consultancy; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Trautmann-Grill:Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Takeda: Consultancy, Honoraria; GSK: Consultancy; BMS: Honoraria. Goldschmidt:Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp and Dohme (MSD): Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Molecular Partners: Research Funding; Incyte: Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:. Weisel:Abbvie: Consultancy, Honoraria; Adaptive: Consultancy, Honoraria; GlaxoSmithKline: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. OffLabel Disclosure: Encorafenib and Binimetinib in BRAF V600E mutant multiple myeloma

Introduction RAS-associated pathways are promising therapeutic targets in RAS-driven tumors because oncogenic RAS itself is not druggable. Alongside the pathways via PI3K/AKT and RAF/MAPK, the small GTPase RAL is considered to represent a third route for oncogenic RAS signaling, which mediates malignant transformation and tumor cell survival. We performed shRNA-mediated knockdown studies in multiple myeloma (MM) cells to investigate the functional role of RAL activity and to analyze the putative functional link between oncogenic RAS and RAL. Moreover, we used screening approaches to identify possible RAL effectors and interacting partners. Methods First, we analyzed expression of the isoforms RALA and RALB in bone marrow biopsies (n=24) and in CD138+ selected primary MM cell samples (n=10) by immunohistochemistry and Western blotting, respectively. Next, we generated two isoform-specific shRNA expression vectors for each of the RAL isoforms to perform RNAi-mediated RAL knockdown and to analyze the functional consequences of RAL abrogation. Experiments were also carried out in combination with clinical anti-myeloma drugs. Furthermore, we treated MM cells with a recently developed pharmacological RAL inhibitor. MM cell survival and apoptotic cells were measured by flow cytometry with annexin V/propidium iodide staining. Changes in RAF/MAPK, PI3K/AKT or RAL pathway activation were detected by Western analysis. We then combined RAL pulldown and knockdown of mutated RAS to investigate the functional link between RAL activation and oncogenic RAS signaling. In addition, we compared RAS- and RAL-mediated gene expression profiles by RNA sequencing. Last, we used mass spectrometry to identify potential RAL effectors and interaction partners. Results RAL protein was detected in all MM cells tested, with RALA showing ubiquitously strong expression and RALB showing more heterogeneous stainings. In contrast, RALA and RALB expression was weak or absent in MGUS or normal plasma cells. MM cell survival was strongly impaired by shRNA-mediated RALA or RALB knockdown, with cell survival rates of 25% or 40 % in L-363 cells and 32% or 52% in MM.1S cells, respectively. No cross-activation between RAL and PI3K/AKT or RAF/MAPK pathways could be detected in MM cell lines. Pharmacological RAL inhibition was achieved in an MM subgroup at concentrations of 20 µM. Combining RAL knockdown and treatment with carfilzomib, pomalidomide, or ixazomib led to enhanced cell death induction. Of note, knockdown of oncogenic RAS also strongly reduced MM cell survival, but did not change constitutive RAL activation as detected by pulldown assay. Moreover, RNA sequencing after RAS or RAL knockdown produced differential gene expression profiles (1473 KRAS-regulated genes versus 771 RALA-regulated genes, with an overlap of 235 genes), again suggesting that both targets represent distinct signaling pathways. Using mass spectrometry, we identified novel RAL interaction partners which are currently being evaluated. Conclusion Our data indicate that the RAL signaling pathway constitutes a promising therapeutic target in MM and mediates MM cell survival and apoptosis independently of oncogenic RAS. Clinical translation of novel pharmacological RAL inhibitors may therefore be a future therapeutic strategy to tackle MM. Disclosures No relevant conflicts of interest to declare.

Background: CD38 is a type II transmembrane glycoprotein widely expressed in many hematological malignancies including multiple myeloma. MOR202, a human IgG1 CD38 monoclonal antibody demonstrates cytotoxic activity. The main mode of action for MOR202-induced cell lysis is through antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis. MOR202 does not induce complement-dependent cytotoxicity, a suspected major contributor to infusion-related reactions (IRRs) observed with other monoclonal antibodies. MOR202 has demonstrated activity as monotherapy and synergy in combination with the immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) in preclinical models of multiple myeloma. Methods: The primary objectives of this multicenter, dose-escalation phase I/IIa study in patients with RRMM (NCT01421186) are to evaluate the safety, and to determine the maximum tolerated dose (MTD)/recommended phase II dose of MOR202. We present the primary analysis of safety and efficacy data from patient cohorts treated with MOR202 at doses of 4, 8 and 16 mg/kg q1w plus Dex (≤40 mg), or MOR202 at 8 or 16 mg/kg q1w plus LEN/Dex or POM/Dex. MOR202 infusion time was 2 hours for all three regimens, which was successfully reduced to 30 minutes for the majority of patients at the time of primary analysis (cutoff 31 Dec 2017). Results: As of 31 December 2017, 91 patients had been treated, 56 at the doses previously described: 18 patients were treated with MOR202 + Dex, 17 with MOR202 + LEN/Dex and 21 with MOR202 + POM/Dex. Before entering the study, patients had undergone a median of 3, 2 and 3 prior treatment lines, respectively. Notably all patients in the MOR202 + POM/Dex group were refractory to lenalidomide. The MTD of MOR202 was not reached. The combinations were generally well tolerated. In the 56 patients, grade ≥3 adverse events (AEs) were mainly hematological in nature. Two patients discontinued due to a MOR202-related AE (one a grade 4 thrombocytopenia and one a grade 3 bacterial infection complicated by acute kidney failure). IRRs to MOR202 (all grade 1 or 2) were observed in 4/56 (7%) patients. These mainly occurred during the first infusion. Twelve of 16 patients remaining on study at the time of primary completion received MOR202 as a 30 minute infusion without obvious safety concerns. In patients treated with MOR202 + Dex, 28% (5/18) showed a response: 3 partial responses (PRs) and 2 very good PRs (VGPRs). Higher response rates of 65% and 48% were observed in patients treated with MOR202 + LEN/Dex (11/17, 7 PRs, 2 VGPRs, 2 complete responses [CRs]) and MOR202 + POM/Dex (10/21, 5 PRs, 3 VGPRs and 2 CRs) respectively. Median time on study was 3.8 months for patients treated with MOR202 + Dex, 9 months for patients who received MOR202 + LEN/Dex and 4.7 months for those treated with MOR202 + POM/Dex. Longest response duration was 27.6 months (MOR202 + POM/Dex). Median progression-free survival was 8.4 months in patients treated with MOR202 + Dex, was not reached in those receiving MOR202 + LEN/Dex and was 17.5 months in those receiving MOR202 + POM/Dex. Conclusions: MOR202 administered as infusions as short as 30 minutes at doses up to 16 mg/kg with Dex or in combination with LEN/Dex or POM/Dex in heavily pretreated patients with RRMM showed a favorable safety profile, including good infusion tolerability. Promising efficacy and long-lasting tumor control were observed for MOR202 + Dex and in particular for MOR202 combined with LEN/Dex or POM/Dex. Disclosures Raab: Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Goldschmidt:Chugai: Honoraria, Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; ArtTempi: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnology: Consultancy. Agis:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Prothena: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Ferstl:Janssen Cilag: Honoraria, Other: Travel EHA. Gramatzki:Affimed: Research Funding. Rollig:Bayer: Research Funding; Janssen: Research Funding. Weisel:Amgen, Celgene, Janssen, and Sanofi: Research Funding; Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Jarutat:Morphosys AG: Employment. Weinelt:Morphosys AG: Employment. Winderlich:Morphosys AG: Employment. Peschel:Morphosys AG: Honoraria.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients with hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation. Current treatment of GVHD relies on immunosuppressive regimens, considerably increasing the incidence of opportunistic infections. As T cells mediate both GVHD as well as protection against viral infections and the malignant disease, strategies to selectively target host-reactive T cells without impairing pathogen- and disease-specific immunity are highly warranted. Activation of T cells is accompanied by increased expression of the chaperone heat shock protein of 90 kDa (Hsp90), which stabilizes several key signaling pathways crucial for T-cell activation. In this study, selective targeting of Hsp90 in activated T lymphocytes with pharmacologic inhibitors already applied successfully in anticancer therapy resulted in induction of apoptosis predominantly in activated cells. Moreover, if T cells were stimulated with allogeneic dendritic cells, alloreactive T cells were selectively eliminated. In contrast, third party reactions including antiviral T-cell immunity were quantitatively and functionally fully preserved. These data suggest that Hsp90 represents a novel target for selective depletion of alloreactive T cells, and provide the rationale for application of Hsp90 inhibitors as potential approach to selectively prevent and treat GVHD in hematopoietic stem cell transplantation recipients without impairing pathogen- and disease-specific T-cell immunity.

Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt–specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.

Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53–murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.

Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells and thereby control cell fate decisions and survival. We recently demonstrated that Notch signaling is involved in proliferation and survival of B cell-derived tumor cells of classic Hodgkin disease and described a novel mechanism for the oncogenic capacity of Notch. In this study we investigated whether Notch signaling is involved in the tight interactions between neoplastic plasma cells and their bone marrow microenvironment, which are essential for tumor cell growth in multiple myeloma (MM). Here we demonstrate that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts show low to undetectable levels of Notch. Functional data indicate that ligand-induced Notch signaling is a growth factor for MM cells and suggest that these interactions contribute to myelomagenesis in vivo. (Blood. 2004;103:3511-3515)

The interleukin 6/glycoprotein 130/signal transducer and activator of transcription 3 (IL-6/gp130/STAT3) pathway has been reported to play an important role in the pathogenesis of multiple myeloma (MM) and for survival of MM cells. However, most data concerning the role of IL-6 and IL-6–triggered signaling pathways were obtained from experiments performed with MM cell lines and without considering the bone marrow microenvironment. Thus, the precise role of IL-6 and its intracellular signaling pathways for survival of human MM cells is still unclear. Here we show that treatment of human MM cells (IL-6–dependent MM cell line INA-6 and primary MM cells) with the IL-6 receptor antagonist Sant7 or with an anti-gp130 monoclonal antibody (mAb) induced apoptosis if the cells were cultured in the absence of bone marrow stromal cells (BMSCs). In contrast, apoptosis could not be observed if the MM cells were cocultured with BMSCs. The analysis of intracellular pathways revealed that Sant7 and anti-gp130 mAb were effectively inhibiting the phosphorylation of gp130 and STAT3 in the absence and presence of BMSCs, whereas ERK1 and ERK2 (ERK1,2) phosphorylation was only slightly affected. In contrast, treatment with the farnesyl transferase inhibitor, FPT III, induced apoptosis in MM cells in the absence or presence of BMSCs and led to a complete inhibition of the Ras/mitogen-activated protein kinase pathway. These observations indicate that the IL-6/gp130/STAT3 pathway is not essential for survival of human myeloma cells if they are grown in the presence of cells from the bone marrow microenvironment. Furthermore, we provide evidence that farnesyl transferase inhibitors might be useful for the development of novel therapeutic strategies for the treatment of MM.

Introduction: Proteasome inhibition is the backbone of various Multiple Myeloma (MM) treatment regimens, leading to durable responses and high quality remissions. However, under prolonged therapy patients eventually develop drug resistance and the underlying mechanisms have been poorly understood. Proteasome inhibitor resistant cell lines were generated by continuous exposure to proteasome inhibiting drugs. In these cell line models a number of variants within the PSMB5 gene were observed. This includes single point mutations, leading to conformational changes of the _5 subunit within the 20S proteolytic core of the proteasome, impairing its chymotryptic catalytic function and the binding of inhibitory drugs. However, no PSMB5 mutations could, so far, be identified in human disease, leaving the functional relevance of such mutations to be determined. Methods: We applied the MM Mutation Panel (M3P) and used the Personal Genome Machine (Life Technologies) to sequence CD138+ purified bone marrow plasma cells and peripheral blood germ line control from a MM patient in third relapse that had been previously treated by various proteasome inhibitor containing therapies (VTD-PACE, VCD, PAD-Rev). This patient was subsequently treated with a pomalidomide, bortezomib, adriamycin and dexamethasone combination therapy (Pom-PAD) and achieved a partial remission after 4 cycles of therapy. When being exposed to VCD at earlier relapse a complete remission was induced within 6 cycles of therapy, demonstrating excellent response to proteasome inhibition at this earlier disease stage. Results: We identified clonal missense mutations in this patient at known NRAS hotspot location (p.Gln61Arg) and in MAX (p.Arg35His). Most notably we found and validated four subclonal single point mutations in PSMB5, each of them in subclonal frequencies with variant reads (VR) ranging from 1.9%- 5.9% (average read depth 750X). All PSMB5 mutations occurred in a highly conserved region in exon 2 of the gene (p.Cys122Tyr, p.Met104Ile, p.Ala86Pro, p.Ala79Thr), with three of them being located within the proteasome inhibitor drug binding site. Our 400bp amplicon design allowed us to observe that each mutation identified in PSMB5 is exclusively present on a different sequencing read and no reads are shared between the mutations. This implies that the mutations are present on different subclones of the tumor, which means that, despite low VR frequencies of the single mutation, in sum more than a quarter of the whole tumor might be affected by mutated PSMB5. At a disease stage when the patient was well responsive to proteasome inhibitor treatment (at diagnosis and at first relapse), of note, none of the mutations in PSMB5 is detectable. Conclusion: Here we report the first in human PSMB5 mutation in a MM patient. These mutations evolved in parallel within different subclones of the disease under the selective pressure of bortezomib- containing treatment regimens, representing branching evolution. It is to speculate whether previous investigations negating the existence of PSMB5 mutations in proteasome inhibitor treated patients might not have sequenced deep enough or did set up a sensitivity cut-off too rigid to detect such subclonal mutations. Our finding suggests that the mutations identified may contribute to the development of proteasome inhibitor resistance, emphasizing the need for more detailed genomic characterization of tumors, including minor subclonal mutation detection. Functional analysis of the mutations is ongoing and results will be presented at the meeting. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Introduction:CD38 is a type II transmembrane glycoprotein widely expressed in many hematological malignancies including multiple myeloma (MM). MOR202, a HuCAL-derived, human IgG1 CD38 monoclonal antibody, induces antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). MOR202 does not induce complement-dependent cytotoxicity, which is suspected to be a major contributor to infusion-related reactions (IRRs). Preclinical models of MM demonstrate high single-agent antitumor activity of MOR202 and synergy in combination with immunomodulatory drugs (IMiDs), lenalidomide (LEN) or pomalidomide (POM). Methods: This is an interim analysis of a multicenter, dose-escalation phase I/IIa study of MOR202 in relapsed or refractory (RR)MM. Preliminary safety and efficacy data from 3 patient cohorts treated with clinically relevant doses of MOR202 (administered as an IV 2-hour infusion), alone or in combination with an IMiD are presented: MOR202 4, 8 and 16 mg/kg weekly; MOR202 8 or 16 mg/kg weekly with either LEN or POM. All patients in these cohorts also received low dose dexamethasone. Primary objectives were to evaluate the safety, maximum tolerated dose (MTD) and recommended phase II dose of MOR202. Secondary objectives included an assessment of overall response rate, duration of response and progression-free survival. Results: As of July 12, 2016, a total of 66 patients had been treated; 31 in clinically relevant cohorts, including 18 patients receiving MOR202 alone, 8 receiving MOR202 + LEN and 5 receiving MOR202 + POM. Patients treated with MOR202 alone and MOR202 + POM had both received a median of 4 prior lines of therapy; 78% and 100% had been refractory to last prior treatment, respectively. Patients treated with MOR202 + LEN had received a median of 2 prior lines of therapy and 50% had been refractory to last prior treatment. Most of the patients had received bortezomib, LEN, cyclophosphamide, and melphalan alone or in combination with autologous stem cell transplant as part of their prior regimens. In this trial the MTD has not been reached yet. MOR202 alone or in combination with an IMiD was well tolerated, with mainly hematological toxicity reported. A 2-hour MOR202 infusion was feasible in all patients. In the clinically relevant cohorts only 1 patient discontinued due to an adverse event considered to be related to MOR202 (platelet count decreased) and no deaths related to any of the study drugs occurred. IRRs were seen in only 3/31 (10%) patients, all occurring during the first infusion. All IRRS were ≤ grade 2. So far, 28 patients were evaluable for response in the MOR202 clinically relevant cohorts. Of 16 evaluable patients in the MOR202 alone cohort, 3 partial responses (19%) and 2 very good partial responses (13%) were reported. In the MOR202 + LEN cohort 5/7 partial responses were seen, and 3/5 patients responded to MOR202 + POM treatment including 2 complete responses. Median time to response was 4 weeks, with responses tending to deepen over time. Most responses (10/13) are ongoing with the longest duration of response currently being 48 weeks. Preliminary analysis in 5 patients revealed preservation of high CD38 levels on MM cells under MOR202 therapy, with a mean decrease of only 10% from baseline to day 1 cycle 2 (4 weeks). Conclusions: In this analysis, a 2-hour infusion of MOR202 (up to 16 mg/kg) alone, or in combination with POM or LEN showed a very good safety profile, particularly an excellent infusion tolerability in heavily pretreated patients with RRMM. Promising preliminary efficacy and long-lasting tumor control was seen for MOR202 +/- IMiDs. The data suggest that CD38 expression on patient MM cells is preserved during treatment. Disclosures Raab: Novartis: Consultancy, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Amgen: Consultancy, Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Agis:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Einsele:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Engelhardt:Amgen: Research Funding; Janssen: Research Funding; MSD: Research Funding; Celgene: Research Funding. Ferstl:Novartis: Other: Case report presentation; Bristol Myers Squibb: Other: Advisory Board. Weisel:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Novartis: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Jarutat:MorphoSys AG: Employment. Weinelt:MorphoSys: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Peschel:MophoSys: Honoraria.

Background: CD38 is a type II transmembrane glycoprotein that is expressed at high levels on multiple myeloma cells. MOR202 is a HuCAL-derived, human, IgG1 anti-CD38 monoclonal antibody showing effective antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and high activity in preclinical models of multiple myeloma. Patients and Methods: Here we reportinterimsafety and preliminary efficacy data from this ongoing, multicenter, MOR202 dose-escalation, phase I/IIa study in patients with relapsed or refractory disease who had failed ≥2 prior therapies for multiple myeloma, including an immunomodulatory drug and a proteasome inhibitor. The objectives are to evaluate the safety, maximum tolerated dose (MTD)/recommended dose and preliminary efficacy of MOR202 when administered as monotherapy or in combination with dexamethasone (DEX); pomalidomide (POM) + DEX; and lenalidomide (LEN) + DEX. Patients received MOR202 as a 2-hour intravenous infusion every 2 weeks (q2w; dose levels 0.01-16 mg/kg), 4 mg/kg weekly (q1w) and 4, 8 and 16 mg/kg q1w + DEX. The combination cohorts receiving MOR202 8 mg/kg with LEN + DEX and POM + DEX have been opened, and the 16 mg/kg q1w with LEN + DEX or POM + DEX, as well as confirmation cohorts, are planned. Results: As of 26 June 2015, 44 patients have been treated; 31 and 13 patients in the q2w and q1w dose levels, respectively. Median age was 69 years (range 44-80); median number of prior therapy lines was 4 (2-11). The MTD has not been reached. The most common treatment-emergent adverse events (TEAEs) at any grade were anemia (15 patients, 34%), fatigue (14 patients, 32%), infusion-related reactions (IRRs) and leukopenia (13 patients, 30% each), lymphopenia and nausea (11 patients, 25% each). Grade ≥3 TEAEs were reported for 28 patients (64%); the most common included lymphopenia (8 patients, 18%), leukopenia (5 patients, 11%) and hypertension (4 patients, 9%). IRRs arose mainly during the first infusion; all were grade 1-2 except for one patient (grade 3); no IRRs occurred in patients receiving MOR202 in combination with DEX. Infections were commonly reported (26 patients, 59%) but in the majority of the cases were not considered to be treatment-related. There have been no treatment-related deaths. Pharmacokinetic (PK) data demonstrated a significant target-mediated drug disposition effect for most patients treated q2w. By contrast, patients treated q1w (4 or 8 mg/kg) showed constant or slightly accumulating MOR202 trough levels, suggesting the potential for full target occupancy at 16 mg/kg. Long-lasting tumor control has already been observed in early monotherapy cohorts, including one partial response and one very good partial response in the weekly cohorts; efficacy analyses are ongoing. First data from the dose escalation of the weekly cohorts with DEX and the combination cohorts with LEN + DEX and POM + DEX will be presented. Conclusions: At doses up to 16 mg/kg,MOR202 was safe and well tolerated. Encouraging preliminary activity of MOR202 was observed, especially with the weekly regimen. PK data show the potential for full target occupancy in patients receiving MOR202 16 mg/kg q1w. This dosing schedule of MOR202 is currently being tested in combination with DEX, LEN + DEX, and POM + DEX. Disclosures Raab: MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; BMS: Consultancy. Goldschmidt:Chugai: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Einsele:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Ferstl:Bristol-Myers Squibb: Other: Advisory board; Novartis: Other: Case report presentation. Weisel:Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Support; Novartis: Other: Travel Support; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; BMS: Consultancy, Honoraria, Other: Travel Support; Onyx: Consultancy, Honoraria; Noxxon: Consultancy. Klöpfer:MorphoSys: Employment. Weinelt:MorphoSys: Employment. Härtle:MorphoSys: Employment.

Background: HPSE is an endo-ß-d-glucuronidase that trims the heparan sulfate (HS) chains of proteoglycans, releasing biologically active fragments of HS. HPSE activity impacts cell signaling, gene expression and promotes extracellular matrix remodeling within the tumor microenvironment; high HPSE expression is associated with enhanced tumor growth, angiogenesis and metastases in several cancer types. As a result of its tumor promoting activities, HPSE is a promising new and unexploited target for anti-cancer therapy. There is a single enzymatically active HPSE in humans and HPSE knockout mice appear to be healthy, thus therapeutic neutralization of HPSE activity would likely have limited negative side effects. In MM preclinical models HPSE was shown to be a master regulator of aggressive tumor behavior. Preclinical evidence also indicates that HPSE promotes chemoresistance suggesting it plays a pivotal role in regulating myeloma response to therapy (Ramani VPC et al., AACR 2014, Abstract nr. 1708). In preclinical studies, bortezomib or melphalan were found to enhance HPSE expression and secretion. High HPSE expressing MM cells were less susceptible to the cytotoxic effects of those drugs. Likewise, a very significant increase in HPSE gene expression following chemotherapy was observed in patient-derived tumor samples, indicating a potential role for HPSE in regulating myeloma response to therapy. Roneparstat (SST0001), a 100% N-acetylated and glycol split heparin, is a potent HPSE inhibitor devoid of any significant anticoagulant activity. In an in vivo model of disseminated myeloma, Roneparstat in combination with either bortezomib or melphalan, significantly decreased both the number of animals with detectable tumor and the tumor burden when compared with animals treated with either of these drugs alone. In addition, studies in animal models of MM indicated that the mechanism of action of Roneparstat was consistent with it having anti-HPSE activity in vivo (reduced angiogenesis and diminished expression of HGF, VEGF and MMP-9 and diminished HPSE induced shedding of syndecan-1, a HS proteoglycan known to be a potent promoter of myeloma growth). Patients and Methods: A First in Man, multicenter, international, phase I clinical study is currently ongoing in advanced heavily pre-treated refractory MM patients (pts) who have exhausted all available anti-MM therapies. Roneparstat is administered subcutaneously, with a starting flat dose defined according to ICH S9 guidelines. A schedule DX5W1,W2 Q28D is being tested. Each cohort plans 3 + 3 pts. A direct fluorescence method (Heparin Red assay) is used in pharmacokinetic studies along with aPTT, used as a surrogate (indirect) measurement of Roneparstat plasma concentration. The pharmacodynamic effect of the drug on the coagulation cascade and any antitumor effect are also evaluated. Results: 15 pts have been enrolled to date. 5 cohorts (doses ranging from 25 to 200 mg/day) have been evaluated, while a 400 mg cohort has just been opened. Five pts have received 1 cycle of therapy, six pts 2 cycles, one 3 cycles, one 5 cycles, one 9 cycles; one patient is currently on treatment, one is not evaluable. Roneparstat administration was found to be safe with only minimal transient side effects. No DLTs and no bleeding complications have been observed. Roneparstat has been well tolerated both systemically and locally. The only side effect observed was minor reactions (redness, bruising) at the injection site (in 6 pts, all grade 1). A decrease > 50% in the serum monoclonal component was observed in one patient, lasting for 6 cycles. Conclusions: Preclinical studies in MM lines and animal models have demonstrated Roneparstat as a potent anti-myeloma compound, particularly when used in combination with other drugs. In the ongoing Phase I escalating dose study (n. pts = 15), Roneparstat administration (at a dose of up to 200 mg/day) was found to be safe with only minimal local side effects. Based on these results, Roneparstat, at a defined dose, in combination with other anti-myeloma agents, will be evaluated in relapsed/resistant MM pts. Disclosures Galli: sigma-tau Research Switzerland SA: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Barbieri:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Paoletti:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Pace:sigma-sau Industrie Farmaceutiche Riunite SpA, Pomezia (RM), Italy: Employment. Sanderson:Sigma-tau Research S.P.A.: Consultancy, Research Funding. Nagler:Novaratis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.

Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract 831 Introduction: Dysregulated signaling pathways contribute to cell growth, survival and drug resistance in multiple myeloma (MM). In about 50 percent of primary MM samples the protein kinase B (PKB)/Akt pathway appears to be constitutively active and its blockade strongly impairs survival, thus defining Akt-dependent versus Akt-independent MM. Because illegitimate activation of survival pathways can be a consequence of oncogenic Ras mutations, we analyzed the role of Ras in Akt-dependent primary MM samples. Methods: Primary MM samples from different patients (n=35) were tested for cell death induction with the small molecule Akt-inhibitor Akti 1/2 and the sensitivity was correlated to the mutation status of N- or K-Ras in these samples, which was obtained by RT-PCR. Immunohistochemistry was used to demonstrate the presence of phosphorylated Akt in corresponding patient biopsies. Apoptosis was measured by flow cytometry with Annexin V-APC or Annexin V-FITC and propidium-iodide staining. We further analyzed the consequences of Ras depletion with regard to Akt activation in a Ras wildtype (AMO-1) versus a K-Ras mutated (MM.1s) and an N-Ras mutated (INA-6) myeloma cell line using siRNA knockdown and Western blotting. Isoform specificity of the siRNAs was confirmed through the effects on ectopically expressed HA-tagged Ras proteins. Results: Of the 35 primary MM samples tested 17 (48.6%) showed sensitivity to pharmacologic inhibition of Akt and thus constituted the Akt-dependent myeloma subgroup. Activating mutations of either K-Ras (n=7) or N-Ras (n=7) were found in 14 samples (40%). However, we did not observe any obvious correlation between the mutation status of Ras and sensitivity to the Akt inhibitor, suggesting that oncogenic Ras is not a prerequisite for the constitutive activation of Akt. We extended our analysis to MM cell lines and performed isoform-specific siRNA mediated Ras knockdown. Depletion of K-Ras induced apoptosis selectively in K-Ras mutated MM.1s cells, while N-Ras mutated INA-6 cells were most sensitive to N-Ras knockdown. Contrarily, knockdown of either of these isoforms had no effect on survival in the Ras wildtype cell line AMO-1. Ras knockdown did not lead to a notable decrease in Akt(Ser473) phosphorylation, again indicating that oncogenic Ras signaling may not be mediated through the PKB/Akt-pathway in MM. Conclusion: Whereas MM cell survival might well be promoted by activating Ras mutations, oncogenic Ras does not appear to be instrumental in the constitutive activation of the PKB/Akt pathway in primary MM. Targeting Ras isoforms themselves can, however, lead to apoptosis in Ras mutated MM cell lines. Thus, even though oncogenic mutated Ras is currently not a druggable target, these experiments underscore that it is nonetheless desirable to develop ways to specifically block its function in MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 2829 Poster Board II-805 Introduction: We could recently show that the heat shock proteins (HSP) HSP90 and HSP70 are frequently overexpressed in multiple myeloma (MM), stabilize as molecular chaperones various oncogenic proteins and contribute to survival of MM cells. Currently, several clinical Phase I/II studies are under way to evaluate the concept of pharmacological HSP90 blockade in human cancer. Under cellular stress conditions the heat shock transcription factor 1 (HSF1) has a key regulatory role for the up-regulation of HSP. Importantly, it has been observed that treatment with the proteasome inhibitor bortezomib, a clinically effective anti-MM agent, induces up-regulation of HSP90, HSP70 and HSP27. Furthermore, it has recently been demonstrated that HSF1 can protect cells from oncogene-driven malignant transformation. We therefore analyzed the role of HSF1 for the malignant growth of MM cells. Methods: Western analyses were performed to determine HSF1 expression and regulation in different human MM cell lines. To examine the expression of HSF1 and different HSP like HSP90, HSP70 and HSP27 in situ, samples from 60 bone marrow biopsies obtained from MM patients were immunohistochemically stained. To analyze the role of HSF1 for the survival of MM cells, HSF1 was either selectively depleted by siRNA-mediated knockdown using a pSUPER-based siRNA expression vector or targeted by treatment with a novel pharmacological HSF1 inhibitor triptolide. In addition, pharmacological inhibition of HSF1 was combined with concomitant pharmacological inhibition of either HSP90 (with the novel inhibitor NVP-AUY922) or bortezomib. Furthermore, gene expression analyses with an Affymetrix GeneChip were performed to identify HSF1 target genes in MM cells. Results: Here, we show that HSF1 is frequently overexpressed in MM cell lines in vitro and in the majority of the analyzed MM biopsies in situ, but not in MGUS or in normal plasma cells. Blockade of HSF1 either by siRNA-mediated knockdown or treatment with the novel pharamacological HSF1 inhibitor triptolide led to a strong induction of apoptosis in cells of the MM cell lines INA-6 and MM.1s. Importantly, also primary MM cells showed apoptosis induction after triptolide treatment. HSF1 inhibition led to downregulation of HSP70, HSP27 and HSP90. Gene expression analysis revealed a number of additional molecular targets of HSF1 involved in apoptosis regulation. Furthermore, initial experiments indicated that the apoptotic effect of pharmacological HSF1 inhibition is enhanced by the concomitant pharmacological inhibition of either HSP90 or the proteasome. Conclusion: We demonstrate that HSF1 is overexpressed in MM, contributes to the survival of MM cells and controls the activity of oncogenic HSP like HSP90, HSP70 and HSP27. Targeting HSF1 may therefore represent an attractive potential therapeutic strategy in MM, in particular in combination with HSP90 or proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

We have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumor cell survival. Pharmacologic blockade of Hsp90 has consistently been shown to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumor cells is lacking. Furthermore, these investigations have so far exclusively relied on geldanamycin derivatives. Here, we analyzed the anti-tumor effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) in a large set of primary MM samples and in MM cell lines. The majority of cell lines (n = 8), as well as most primary samples (n = 20), displayed profound apoptotic responses and steep dose-effect curves with EC50 values in the range of 5–15 nM and EC90 values between 8 and 25 nM. This effect was not attenuated in coculture with cells from the bone marrow microenvironment. Some cell lines and about a quarter of primary MM samples displayed greater resilience to drug treatment, with EC50 values but not EC90 values reached at concentrations up to 50 nM. Sensitivity of MM cells to the Hsp90 inhibitor was not correlated with TP53 mutation or Hsp70 induction levels. Western analyses of MM cell lines and flow cytometric analyses of antibody-stained Hsp90 client proteins in primary tumor cells showed that NVP-AUY922-treatment entailed molecular effects and pharmacodynamic properties consistent with abrogation of Hsp90 function. Consequently, downregulation of multiple signaling and survival pathways was detectable through, for example, decreases in the phosphorylated (activated) forms of extracellular signal-regulated kinase (ERK) 1 and 2, signal transducer and activator of transcription (STAT) 3 and glycogen synthase kinase-3beta. All samples treated displayed strong upregulation of Hsp70. Importantly, peripheral blood mononuclear cells as well as primary bone marrow stromal cells were much less affected by high (50–100 nM) concentrations of NVP-AUY922, showing that a therapeutic window might be established for the treatment of multiple myeloma. Taken together, NVP-AUY922 could be a promising new drug for the treatment of a majority of myeloma patients.

The PI3K/Akt pathway has been reported to critically contribute to survival and malignant growth of multiple myeloma (MM). Because most of these data are based on pharmacologic inhibition it is not clear if the effects are due to Akt inhibition or off-target effects. Furthermore, the Akt family of kinases consists of three highly homologous isoforms, that may, nonetheless, display individual functional properties. We therefore conducted siRNA experiments to evaluate if any single isoform posesses a special role for the viability of MM cells. This was complemented with extensive analyses into the functional and signaling properties of the Akt pathway in primary MM cells (n = 30). Our knock-down experiments revealed that in MM.1S, an MM cell line with constitutive phospho-Akt signaling, Akt1 and Akt2 both contributed to MM cell survival whereas Akt3 seemed to be of less relevance. Conversely, survival of MM cell line AMO-1 which has no constitutive phospho-Akt signal was completely unaffected. Treatment of these MM cell lines with the Akt1 and Akt2 specific inhibitor Akti-1/2 showed that this drug totally abolished the phospho-Akt signal in MM.1S at a concentration of 10 microM. Again, MM.1S cells underwent apoptosis whereas AMO-1 cells were resistant. Next, we analyzed Akt signaling in a large panel of primary MM samples. Phosphorylated Akt was determined with intracellular staining and flow cytometry analysis in primary tumor samples and could be detected in about 60% of MM cases. This constitutive signal could be blocked with Akti-1/2 in the presence and absence of bone marrow stromal cells. Pharmacologic inhibition of Akt led to strong induction of cell death in 46% of primary MM samples, whereas the rest was largely resistant to Akt inhibition. The samples sensitive to Akt inhibition were mostly identical to those that displayed a constitutive phospho-Akt signal. Of interest, there was no correlation between Akt dependence and mutational inactivation of PTEN. Further inhibition of other signaling cascades implicated in growth and survival of MM cells, such as the MAPK or STAT3 pathways, had only minor additional effects on tumor cell viability of samples resistant to Akt inhibition. Our analysis indicates substantial heterogeneity in MM cells that defines Akt dependent and Akt independent MM subgroups. Akt1 and Akt2 proved relevant for the survival of subsets of MM cell lines and primary samples. Taken together, this is the first comprehensive functional and molecular signaling analysis of primary MM samples which led to the identification of novel functionally defined myeloma subgroups.

The combined blockade of the IL-6R/STAT3 and the MAPK signaling pathways has been shown to inhibit bone marrow microenvironment-mediated survival of multiple myeloma (MM) cells. Here, we identify the molecular chaperones heat shock protein (Hsp) 90alpha and beta as target genes of both pathways. Interestingly, in situ overexpression of both Hsp90 proteins was observed in the majority of MM, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of Hsp90 or treatment with the novel Hsp90 inhibitor 17-DMAG attenuated the levels of phospho-STAT3 and phospho-ERK and decreased the viability of MM cells. Although knockdown of Hsp90beta unlike knockdown of Hsp90alpha was sufficient to induce apoptosis, this effect was strongly increased when both Hsp90s were targeted, indicating a cooperation of both. Given the importance of the bone marrow microenvironment for drug resistance and MM cell survival, apoptosis induced by Hsp90 inhibition was not mitigated in the presence of bone marrow stromal cells, osteoclasts or endothelial cells. These observations suggest, that a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supports the survival of MM cells. Our results emphasize a role for Hsp90alpha and Hsp90beta in MM pathogenesis.

Introduction: The Y-box binding protein YB-1 is a member of the cold shock domain protein superfamily and represents one of the most evolutionary conserved nucleic-acid binding proteins. Yb-1 is involved in a wide variety of cellular functions, such as regulation of transcription and translation, but also in DNA-repair and stress response to extracellular signals. Furthermore, recent reports from our group could show that overexpressed YB-1 plays a role in drug resistance of breast cancer cells and might act as an oncogene. The goal of this study was to investigate a potential pathogenetic role of YB-1 in MM. Material and Methods: For the detection of YB-1 expression in vivo, bone marrow biopsies of MM patients were analyzed by immunohistochemistry. The expression of YB-1 protein in a number of MM cell lines was analyzed by Western Blotting. The regulation of YB-1 expression through major signaling pathways, e.g. IL-6R/STAT3, Ras/MAPK and PI3K/AKT, was analyzed using specific inhibitors of these pathways (Sant7, PD98059 and Ly294002). To determine the role of YB-1 for survival and proliferation, siRNA technology was exploited to transiently knockdown the expression of YB-1 in the MM cell lines INA-6 and MM.1s. In addition, these YB-1 knockdown cells were exposed to doxorubicin and melphalan to evaluate the influence of YB-1 on drug resistance. Results: Immunohistochemical analyses of bone marrow biopsies revealed that YB-1 is strongly expressed only in MM cells of samples that show a highly proliferative phenotype. This subgroup of MM patients was characterized by an aggressive clinical course. In contrast, MM cells of samples with a slow proliferative status did not show YB-1 expression. Interestingly, tumor cells of patients that responded to chemotherapy did not express YB-1. Furthermore, neither normal bone marrow plasma cells nor premalignant plasma cells of MGUS patients showed YB-1 expression. YB-1 was detected in all of the evaluated MM cell lines. YB-1 expression was not regulated by the IL-6R/STAT3, Ras/MAPK and PI3K/AKT pathways. Knockdown of YB-1 in INA-6 and MM1.s cells by siRNA resulted in a slow proliferation phenotype with a higher apoptotic cell fraction. In addition, we observed that YB-1 knockdown remarkably sensitized cells towards drug-induced apoptosis. Conclusions: YB-1 expression in MM appears to be correlated with a highly proliferative phenotype and with disease progression. YB-1 contributes to the malignant growth and drug resistance and might be therefore an attractive therapeutical target to circumvent aquired drug resistance in MM.

The p53 pathway is central to cellular defences against neoplastic transformation, and mutations that impair p53 function are prominent oncogenic events. However, certain malignancies, such as multiple myeloma (MM), rarely present with p53 defects except in late stages. MM might therefore be vulnerable to p53 induction therapy, but it is unknown if the p53 pathway remains actually functional in this disease, and if it can be sufficiently well triggered to induce tumor cell death. We have used nutlin-3, a newly developed small-molecule antagonist against the p53 suppressor murine double minute 2 (MDM2), to analyze the effect on myeloma cell lines and a large panel of patient tumor samples. Nutlin-3 specifically and exclusively induced p53 downstream targets in myeloma cells with wild-type p53, and a large majority of primary medullary myeloma samples was susceptible to nutlin-3-mediated apoptosis. Comparison with the clinically relevant genotoxic drugs melphalan and etoposide showed that nutlin-3 was as effective or better at inducing apoptosis of primary tumor cells, and that combinations of nutlin-3 with genotoxic cytostatics at low doses produced better than additive anti-tumor effects. Importantly, broad anti-tumor activity of nutlin-3 persisted even when primary tumor cells were kept in coculture with bone marrow stromal cells (BMSCs). However, primary tumor isolates often contain a fraction of cells apparently unaffected by drug treatment, and some primary samples were conspicuously less sensitive to nutlin exposure when the cells were kept in coculture with BMSCs, as compared to culture in medium supplemented with interleukin-6. We have begun to analyze to what extent heterogeneity for p53 induction within the tumor cell population may underlie these differences, and if the capacity to activate intracellular growth and survival pathways has an influence on the result of p53 pathway activation. Nutlin-3, as a non-genotoxic agent to specifically induce or strengthen p53-mediated apoptotic responses may be a promising agent to complement standard genotoxic regimens for the treatment of MM.