ALBERT

Description: Introduction: Daratumumab (DARA) is a first-in-class human CD38 IgG1κ monoclonal antibody that has demonstrated activity as monotherapy and in combination with standard of care regimens for multiple myeloma (MM). Population pharmacokinetics (PPK) analyses were conducted to describe the PK characteristics of DARA following its administration in combination therapies, to evaluate the influence of covariates on its disposition in patients with MM who had received ≥1 prior therapy, and to compare its PK in combination therapies with that of monotherapy. Exposure-efficacy/safety analyses were performed to investigate the relationship between DARA exposure and selected efficacy and safety endpoints. Methods: The PPK analysis primarily included data from two phase 3 studies in which DARA was combined with background regimens: MMY3003 (POLLUX; lenalidomide [R]/dexamethasone [d]) and MMY3004 (CASTOR; bortezomib [V]/d). Data from two phase 1/2 studies (GEN503 [Rd] and MMY1001 [pomalidomide/d; Vd; V-thalidomide-d; V-melphalan-d]) were also used. Most patients included in the analysis (684 of 694) received 16 mg/kg DARA intravenously. A PPK model based on previous monotherapy studies was used to fit the concentration-time data from combination studies. Subgroup analyses were conducted to evaluate the influence of patient and disease characteristics on exposure to DARA. Based on data from MMY3003 and MMY3004, the exposure-efficacy analyses investigated the relationship between maximal trough concentrations (Cpre-infusion,max) and progression-free survival (PFS), duration of response (DOR), and overall response rate (ORR), while the exposure-safety relationship was explored for infusion-related reactions (IRRs), thrombocytopenia, anemia, neutropenia, lymphopenia, and infections. Results: Exposure to DARA was similar between the monotherapy and combination therapies. Based on combination therapy data, the effects of the intrinsic and extrinsic factors (age, sex, race, renal and hepatic impairment, baseline albumin, type of MM, region, type of combination therapy, ECOG, refractory status, and number of prior lines of therapy) were similar to or smaller than those in the monotherapy studies. Consistent with the monotherapy studies, none of the investigated intrinsic and extrinsic factors had clinically important effects on the exposure to DARA as all the covariate effects were within 25%. Although the clearance and volume of distribution of DARA increased with increasing body weight, the exposure to DARA was relatively consistent across the range of body weights after administration on a mg/kg-basis. Despite the decreasing concentration of DARA over time due to less frequent dosing, the current dosing schedule was adequate to produce concentration levels that maintained target saturation during the Q4W dosing period in the dosing schedules for MMY3003 (QW for 8 weeks, Q2W for 16 weeks, and Q4W, thereafter) and MMY3004 (QW for 9 weeks, Q3W for 15 weeks, and Q4W, thereafter). The exposure-efficacy analyses on the data from combination therapies suggest that maximum clinical benefit to PFS, DOR, and ORR was attained for the majority of the patients with an acceptable safety profile at the recommended dose of 16 mg/kg. No apparent relationships between drug exposure and IRRs, thrombocytopenia, anemia, neutropenia, and lymphopenia were identified within the studied concentration range. Although the overall rate of infection (any grade) appeared to increase with drug exposure, this trend was not observed for grade ≥3 infections. These findings were consistent with results from the monotherapy studies. Conclusion: The PK of DARA was similar between monotherapy and combination studies. No clinically relevant demographic or clinical characteristics were identified. Therefore, no dose adjustment based on these factors is recommended. Population PK and exposure-response analyses for combination therapies support the recommended body weight-based 16 mg/kg dose and the dosing schedules for the MMY3003 and MMY3004 studies. Disclosures Xu: Janssen: Employment, Equity Ownership. Liao:Pharmax: Employment; Janssen Research & Development: Consultancy; Johnson & Johnson: Equity Ownership. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Amgen, Celgene, Janssen, Karyopharm, Takeda: Consultancy, Honoraria; Amgen, Celgene, Janssen, Karyopharm: Research Funding. Ho:Janssen, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Belch:Amgen, Celgene, Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Capra:Janssen: Speakers Bureau. Gomez:Janssen, Bristol Myers Squibb, Celgene, Amgen: Consultancy. Medvedova:Oregon Health & Science University: Employment. Iida:Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Ono Pharmaceuticals Co: Honoraria; Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Chugai Pharmaceuticals, Kyowa Hakko Kirin Co, Eli Lilli Japan, Novartis Pharma, Sanofi, Bayer Yakuhin, Toyama Chemical Co, Teijin Pharma, Astellas Pharma: Research Funding. Qi:Janssen: Employment. Schecter:Janssen: Employment, Equity Ownership. Khokhar:Janssen: Employment. Yan:Janssen: Employment; Johnson & Johnson: Equity Ownership. Zhang:Janssen: Employment, Equity Ownership. Clemens:Johnson & Johnson: Equity Ownership; Janssen: Employment.

Description: Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Description: Cell surface glycoprotein CS1 is universally expressed in the majority of MM patients. CS1 gene is localized on chromosome 1q which amplified in many MM patients and in MM cell lines (i.e., H929 and OPM2). We recently further detected CS1 protein in MM patient sera, but not in individuals with monoclonal gammopathy of undetermined significance (MGUS) or in healthy donors (Abstract # 950059). Since circulating CS1 levels correlate with active MM (Abstract # 950059), we postulated that CS1 may regulate MM cell growth and survival. We here defined a role of CS1 in MM cell growth and survival by generating CS1-null OPM2 MM cells using lentiviral CS1 short interfering RNA (CS1 siRNA). Four CS1-null OPM2 transfectants were derived using 4 CS1 siRNA targeting different regions in CS1 gene. Specific knockdown was confirmed by complete abrogation of CS1 mRNA and protein expression. In contrast, CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cnt-OPM2). Significantly, immunoblotting analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in all 4 CS1-null OPM2 cells, compared with OPM2 and cnt-OPM2 cells. We next examined growth and survival of these cells using MTT and Alamar Blue colorimetric/fluorescence assays. Serum deprivation markedly induced apoptosis at earlier time points in CS1-null OPM2 cells, but not in cnt-OPM2 cells. Earlier apoptosis in CS1-null OPM2 cells was mediated by earlier activation and cleavage of caspases, i.e. caspase 3 (〉 7-fold) and caspase 8 (〉 10-fold). Microarray gene expression profiling of cells under normal cultures showed altered cell cycle regulators (i.e., decreased CDK1, cyclin B1, cdc25b, cyclin D2, and increased CDK inhibitor p18), reduced survival/anti-apoptotic proteins (i.e., Mcl-1), and increased proapoptotic molecules (i.e., BINP3, BIM) in CS1-null OPM2 vs. cnt-OPM2 cells (〉4-fold alteration, p

Description: A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily that stimulates growth and survival of multiple myeloma (MM) cells, is mainly produced by MM patient bone marrow stromal cells (BMSCs) and osteoclasts in the BM microenvironment. Like BAFF, APRIL binds two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI). Since BCMA has significantly higher affinity for APRIL than for BAFF (nM vs. mM range), APRIL/BCMA signaling may be more important than BAFF/BCMA in MM. Specifically, BCMA but not TACI is unequivocally expressed at mRNA level in the majority of CD138-expressing MM cell lines and patient MM cells. To date, it is unclear how APRIL signaling through BCMA contributes to BM microenvironment in support of MM cells. We here studied the functional sequelae of APRIL/BCMA pathway in MM cells, and further identified molecular mechanisms regulating these processes. Using flow cytometric analysis, cell surface BCMA expression was confirmed in the majority of MM cell lines. Microarray gene expression analysis showed significant expression of BCMA mRNA in CD138+ patient MM cells (n=105). Higher BCMA mRNA levels were observed in patient MM cells than in normal plasma cells (n=27) (p〈 0.03). Importantly, CD138+ patient MM cells express BCMA protein on the cell surface (n=15), as confirmed by CD38+BCMA+ dual immunofluorescence staining followed by flow cytometric analysis and immunohistochemical (IHC) study. Next, H929 MM cell line that expresses only BCMA, but not TACI, and Daudi B cells transduced with huBCMA (huBCMA-Daudi) were stimulated with APRIL. These cells were subjected to mRNA extraction at different time points followed by microarray analysis to identify downstream transcriptional targets. Stimulation of these two cell lines by APRIL activated the NF-kB signaling in a dose-dependent manner. More than 6-fold increase in NF-kB activity was induced by APRIL (100 ng/ml) using a p65 enzyme-linked DNA/protein interaction assay. IL-6 and PI3K/AKT signaling cascades were concurrently induced. Additionally, APRIL upregulated chemotactic/osteoclast activating factors, i.e., MIP-1a and MIP1b, in a dose-dependent manner, which was further validated by specific ELISA. Multi-Analyte Profiles also confirmed that angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES, MDC (ccl22), were significantly induced upon APRIL stimulation. Conversely, a soluble BCMA-Fc protein inhibited APRIL binding to BCMA and blocked secretion of APRIL-induced chemokines, indicating specific functional interaction of BCMA with APRIL. Importantly, APRIL similarly induced these signaling cascades in CD138+ patient MM cells. Taken together, our studies are the first to demonstrate cell surface BCMA on the majority of CD138+ patient MM cells. Furthermore, APRIL induced osteoclasts-, migration-, and angiogenesis-associated factors in MM cells. These studies therefore establish a role of BCMA activation in the BM microenvironment that provides a niche for MM disease progression, supporting novel therapeutics specifically targeting APRIL/BCMA pathway in MM.

Description: Background: Immunosuppressive therapy is a known risk factor for hepatits B reactivation. The highest risk is reported in hematologic patients treated with anti-CD20 monoclonal antibodies, glucocorticoids and hematopoietic stem cell transplantation. Currently, treatment with tyrosine kinase inhibitors (TKIs) in the vast majority of chronic myeloid leukemia (CML) patients is life-long. Recently healthcare professionals were advised to test for hepatitis B infection before initializing therapy with TKIs and closely monitor HBV carriers. The risk is considered class effect with all TKIs and recommendations are based on case reports. Aim: The aim of the current study was to evaluate the risk of hepatitis B reactivation in patients with CML treated with TKIs. Methods: The records of patients with CML treated with TKIs from a single center were systematically reviewed for hepatitis B serology and serum biochemistry. Results: One hundred eighty one patients diagnosed with CML between the years 1983 and 2016 were evaluated. The median age at diagnosis was 49.7 years (range 18-89), 117/181 (65%) males, with median duration of follow up with TKI therapy of 5.3 years (range 0.4 to 33 years). Over a total of 1195 years of therapy with TKIs no cases of HBV reactivation were identified. Among 114 patients with hepatitis serology, 11 patients (10%) had evidence of prior resolved HBV infection (HBsAg negative/anti-HBc positive). Two of them had anti-HBs positive serology, one had negative PCR for HBV DNA and other two patients received lamivudine prophylaxis. Only one patient had HBsAg positive serology. None of the patients with positive hepatitis serology had clinical or biochemistry evidence of hepatitis B reactivation. The 67 patients without available hepatitis serology had normal liver transaminases at 6 months of TKI therapy and at last day of follow up, confirming that no overt hepatitis B reactivation occurred. Conclusions: We evaluated hepatitis B reactivation in a rather large cohort of CML patients, treated with TKIs. Although there were no cases of HBV reactivation during long term follow up, it should be emphasized that even a low incidence may exert a significant risk due to the long duration of treatment in a chronic disease with lifelong therapy. Our study infers that patients with serology of prior resolved HBV infection are at low risk for hepatitis B reactivation. Larger cohorts of patients with positive hepatitis B serology in a multicenter long term evaluation should be performed in order to address current recommendations for patients' safety and concerns. Disclosures No relevant conflicts of interest to declare.

Description: Activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade mediates human multiple myeloma (MM) growth and survival triggered by cytokines and adhesion to bone marrow stromal cells (BMSCs). Here, we examined the effect of AZD6244 (ARRY-142886), a novel and specific MEK1/2 inhibitor, on human MM cell growth in the bone marrow (BM) milieu. AZD6244 blocks constitutive and cytokine-stimulated ERK1/2 phosphorylation and inhibits proliferation and survival of human MM cell lines and patient MM cells, regardless of sensitivity to conventional chemotherapy. Importantly, AZD6244 (200 nM) induces apoptosis in patient MM cells, even in the presence of exogenous interleukin-6 or BMSCs associated with triggering of caspase 3 activity. AZD6244 sensitizes MM cells to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. AZD6244 down-regulates the expression/secretion of osteoclast (OC)–activating factors from MM cells and inhibits in vitro differentiation of MM patient PBMCs to OCs induced by ligand for receptor activator of NF-κB (RANKL) and macrophage-colony stimulating factor (M-CSF). Finally, AZD6244 inhibits tumor growth and prolongs survival in vivo in a human plasmacytoma xenograft model. Taken together, these results show that AZD6244 targets both MM cells and OCs in the BM microenvironment, providing the preclinical framework for clinical trials to improve patient outcome in MM.

Description: Abstract 4993 Introduction: Extramedullary organ impairment in patients with multiple myeloma (MM) is a very rare event, mostly occurring during disease relapse after high-dose chemotherapy with autologous or allogeneic stem cell transplantation. Recent data reported by different authors suggest a very unfavorable outcome and rapid clinical course for patients with extramedullary relapses. Often the extramedullary manifestations are associated with special biological features, such as loss of CD56 expression or plasmablastic cell morphology. In this context, only one FISH study of 9 MM patients reported that the incidence of TP53 deletions in the bone marrow of those MM patients with central nervous system (CNS) involvement was about 75% higher than in the MM patient without CNS involvement. However, there is a lack of genetic data for specific extramedullary manifestations. We herein report the first cytogenetic investigation of extramedullary organ infiltrations compared to bone lesions or consecutive soft tissue impairment of MM patients using cIg-FISH on paraffine embedded sections. Material and method: We investigated paraffin-embedded sections of different extramedullary organ manifestations of 13 MM patients and 11 MM patients with bone or soft tissue impairment originating from a bone lesion. Extramedullary organ involvements comprised biopsies from skin, pleura, pleural effusion, uterus, liver, CNS, subcutaneous soft tissue, lymph node, and thyroid gland, attained at different stages of the disease. The second group consisted of bone lesions or surrounding soft tissue, like muscle, which was infiltrated per continuitatem. For investigation of paraffine-embedded samples, we further developed the conditions of the well known cIg-FISH method to utilize all the advantages of this very sensitive method. We evaluated the most important prognostic chromosomal regions in MM using the following FISH-probes: 13q14 (D13S25), 17p13 (TP53), 8q24 (MYC), t(4;14) (FGFR3/MMSET;IGH). Results: The incidences in the group of extramedullary organ involvement vs. bone and soft tissue were as follows: deletion of TP53: 23% vs. 18% MYC-overrepresentation: 36% vs. 36% deletion of 13q14: 18% vs. 36% t(4;14) 42% vs. 30% The incidence of TP53 deletions in organ infiltrations by malignant plasma cells was similar to bone or soft tissue involvement. Our result for MYC-overrepresentation is in line with the findings of recent analysis of bone marrow aspirates from patients with advanced multiple myeloma (32%) and showed again no difference between the two groups. Incidences of 13q14 deletion and t(4;14) varied slightly between the two groups, but are in range with other cIg-FISH-analyses on bone marrow plasma cells. Discussion: In this first cytogenetic investigation of extramedullary manifestations in MM patients, we showed similar frequencies of the chromosomal regions analyzed in the group of organ infiltrations by malignant plasma cells compared to bone or soft tissue involvement. All in all we could not detect differences in the incidence of the most relevant genetic aberrations of the investigated tissue probes compared to already well known bone marrow cytogenetics. Interestingly, this is in contrast to the findings of bone marrow probes in the study with patients with MM and CNS involvement, who showed a higher incidence of TP53 deletions. Concerning the group of soft tissues, originating from bone lesions this is not astonishing, maybe even expected. However, this work analyzed bone marrow of the MM patients and not the extramedullary manifestation, as we did. Further investigations or larger patient samples are needed to proof if these or other genetic aberrations are associated to the aggressive course of extramedullary manifestations of MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2962 Background: Multiple myeloma (MM) is characterized by clonal proliferation of malignant plasma cells (PCs) in the bone marrow (BM) compartment. Interaction of plasma cells with the BM stromal cells (BMSCs) is critical for homing, growth and drug resistance acquisition of the malignant PCs. However, the functional significance of other cellular components of the MM milieu, which includes osteoclasts and immune effector cells, is less clear. Both MM-derived and stromal cell-produced factors, including cytokines and chemokines, are believed to participate in the cross-talk between the MM and stroma leading to disease progression. Aim and Results: We hypothesized an important role for CXCL12 (SDF-1) chemokine and its receptor CXCR4 in MM-stroma interactions and microenvironment formation. We now show that MM cell lines ARH77 and RPMI8226 and primary MM cells may produce high amounts of CXCL12 and co-express CXCR4 receptor. Co-culture of the MM cells with BMSCs significantly up-regulated both CXCR4 cell-surface expression and CXCL12 secretion by the MM cells. Enhanced CXCR4 signaling in the MM cells upon the interaction with BMSCs promoted the survival and proliferation of the cells in an autocrine way. Moreover, the paracrine effect of increased CXCL12 production on immune cell migration was tested. We found, that conditioned medium (CM) produced by MM cells cultured with BMSCs specifically attracted increased numbers of CXCR4-expressing PB CD14+ cells. Furthermore, CXCR4 inhibition, using neutralizing antibodies toward CXCR4, inhibited the MM-induced migration of CD14+ monocytes, suggesting the possible role of CXCR4/CXCL12 axis in monocyte recruitment to the site of the disease. We next examined the functional consequence of MM-macrophage interaction. We saw that PB-generated macrophages induced the proliferation of MM cells, even more effectively than BMSCs. Furthermore, co-culture with macrophages strongly increased the expression of various pro-inflammatory and pro-angiogenic factors by MM cells, including CCL2 (MCP-1), CCL4 (MIP1a), IL-1b, IL-8 and VEGF. Interestingly, expression of IL-10 by MM cells was also up-regulated following the interaction with macrophages, suggesting the possible reciprocal effect of MM-produced factors on macrophage phenotype polarization. Conclusion: Taken together, our findings demonstrate that interaction of MM with BM stromal cells positively regulates the expression of CXCR4 and CXCL12 by MM cells, affecting both MM proliferation and CXCR4-dependent monocyte recruitment. The migrated monocytes may in turn interact with MM cells, support their growth and activate cytokine release, therefore producing favorable pro-inflammatory and pro-angiogenic environment and promoting disease progression. Overall, our data provide the basis for future targeting MM-BMSCs and MM-macrophage interactions with anti-CXCR4 agents as a therapeutic strategy to improve the outcome of patients with MM. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Despite advances in treatment, multiple myeloma (MM) remains incurable due to development of drug resistance in the bone marrow microenvironment. Microtubules (MTs) are dynamic protein biopolymers formed through polymerization of heterodimers of α- and β-tubulins. Disruption of microtubules induces cell cycle arrest in G2-M phase and formation of abnormal mitotic spindles. MTs are also involved in many processes in interphase cells, including intracellular trafficking, cell motility and angiogenesis. The important functions of MT in the cells make them an attractive target for anti-myeloma drug discovery. Furan metotica is a novel class of anti-mitotic spindle drugs that inhibit kinetochore-microtubule binding and trigger a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We evaluated the activity of STK405759, a member of the furan metotica family, as a novel, potential antitubulin drug for MM treatment in preclinical models. Methods: Cytotoxic activity of STK405759 was evaluated by XTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. Tubulin polymerization inhibition was evaluatedusing a biochemical cell free assay and by testingthe levels of soluble and polymerized tubulin in MM-treated cells using Western blot analysis. Efficacy and toxicity of the drug were checked in a murine MM xenograft model. Histochemistry was used to assay tumor apoptosis. Results: STK405759 had a potent cytotoxic activity against a wide variety of MM cell lines and patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. In contrast, the viability of normal peripheral blood mononuclear cells derived from healthy donors and MM patients was not affected. Importantly, STK405759 induced cell death of RPMI MM cells co-cultured with HS-5 bone marrow stromal cells. STK405759 inhibited tubulin polymerizationin a cell free system anddecreased the level of polymerized tubulin in MM treated cells.The STK405759 anti-tubulin activity was supported by demonstration of MM cell cycle arrest followed by activation of an apoptotic default pathway. Activation of pro-caspase-8 and poly (ADP-ribose) polymerase in the cleaved forms, as well as down-regulation of the Mcl-1 anti-apoptotic protein was detected in RPMI treated cells. Combination studies of STK405759 with bortezomib, lenalidomide or dexamethasone showed significant synergistic and additive cytotoxicity in MM cells. In vivo studies revealed decreased MM tumor burden and prolonged survival of STK405759-treated mice compared to controls. STK405759 induced apoptosis of tumors cells from treated mice. Summary/Conclusion: STK405759 is an active, microtubule-targeting agent with potent anti-myeloma activity. These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM. Disclosures No relevant conflicts of interest to declare.

Description: Scientific background: Increasing evidence points to the crucial role of malignant lymphoma interaction with the tumor microenvironment. Our previous investigations indicated that the stromal cytokine activin A induces apoptotic death of myeloma cells due to its antagonism with the growth promoting effect of interleukin-6. Activin A is a homodimer of the ßA inhibin polypeptide chain. Like other transforming growth factor (TGF)ß superfamily members, it is a pleiotropic molecule, which is widely expressed. It has initially been studied in the reproductive system, but has also been implicated in the regulation of hemopoiesis; it is an erythroid differentiation factor and is expressed within the bone marrow microenvironment. Activin A functions are tightly regulated by the competitive inhibitor inhibin A (a heterodimer, /ßA) and the binding inhibitors, follistatins. We previously showed that abundance of activin A was restrictive for B cell production in vitro and that within human nasal polyps activin A expression was widespread but absent from foci of B lineage cells. We were therefore interested to find out whether activin A plays a role in the occurrence of BM (bone marrow) involvement in malignant lymphomas. Materials and methods: The patient population consisted of 17 patients with lymphoma and 3 patients without lymphoma served as controls. Paraffin embedded sections were prepared and immunohistochemical staining was performed using an antibody to the bA chain. The slides were reviewed by team of 5 investigators and graded separately. Results: Out of 17 lymphoma cases, 10 patients showed BM involvement. In patients with BM involvement the level of activin A was significantly decreased in the area surrounding the lymphoid infiltrate (Fig 1a). This was seen uniformly in all the patients except for one. The level of activin A in the rest of the BM was similar to the level seen in specimens of reactive BM. In all 7 patients that had no BM involvement we found a diffuse staining for activin A (similar to what we saw in patients with reactive BM) (Fig.1b). Discussion: It is interesting that only some of the patients with malignant lymphomas have BM involvement. This could stem from a difference in the migratory abilities of the lymphoid cells, which is unlikely, or from a difference in their ability to home and flourish in the BM microenvironment. We have demonstrated that activin A, is significantly down regulated in the vicinity of the lymphoid infiltrate in the BM, as opposed to what occurs in normal inflammatory BM. This suggests that an interaction between the lymphoma cell and the BM microenvironment leads to down-regulation of activin A expression and possibly promotes the survival of the lymphoid cells. Figure 1: low power view of activin staining in the vicinity of the involved BM (a) and uninvolved BM (b). Figure 1:. low power view of activin staining in the vicinity of the involved BM (a) and uninvolved BM (b).