ALBERT

Description: Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH 2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination. Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques. Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells. FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIMEL BIML, BIMS(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF. These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively). Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF. This work was funded in part by the company Array BioPharma. Disclosures Tunquist: Array BioPharma: Employment. Mateos: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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ocio:Jassen: Honoraria; Celgene: Honoraria, Research Funding; Pharmamar: Consultancy, Research Funding; MSD: Research Funding; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding.

Description: Introduction. EDO-S101 is a hybrid molecule of bendamustine plus vorinostat, new in its class. Our group has previously demonstrated that EDO-S101 is effective in vitro in MM cell lines independently of p53 state, and also in a murine plasmacytoma model where it decreases tumor growth and prolongs survival with respect to bendamustine and/or vorinostat treatment. The objective of this work was to gain further insights into the efficacy of EDO-S101, its mechanism of action and its combination with other drugs used in MM. Methods. The mechanism of action was assessed by western blot, comet assay, immunohistochemistry, and flow cytometry. Homologous recombination (HR) efficiency was calculated using chromosomally integrated green fluorescent protein reporter construct-based assay. The efficacy of different combinations was studied in vitro (HMCLs), in vivo (murine plasmacytoma model CB-17 SCID mice) and ex vivo (cells from patients). Results. In addition to the activity of EDO-S101 in MM cell lines we demonstrated that it was active ex vivo in cells isolated from 7 MM patients, with median IC50 of 5 µM (ranging from 1,8 to 8 µM), some of them previously exposed and resistant to alkylators such as melphalan. Interestingly, EDO-S101 could also overcome alkylators-resistance in vitro, as it was active in melphalan resistant cells (U266-LR7 and RPMI8226-LR5). EDO-S01 was also effective in the presence of factors that confer proliferative advantage to plasma cells, like IL-6, IGF or co-culture with mesenchimal cells hMSC-TERT. Regarding its mechanism of action, we found that the apoptosis induced by EDO-S101 was caspase-independent but calpain-dependent, since PD150606, an inhibitor of this protein could overcome EDO-S101-induced apoptosis, whereas the caspase inhibitor Z-VAD -FMK did not. This data was consistent with the finding that under treatment with EDO-S101, MM1S cells showed AIF (apoptotic inducing factor) translocation from the mitochondria into the nucleus. Interestingly, the release of this pro-apoptotic protein from the mitochondria could be mediated by calpains, as it has been described in literature. We subsequently demonstrated that EDO-S101 causes DNA damage, as revealed by the phosphorylation and subsequent activation of several components of the DNA Damage Response (DDR) such as ATM, H2AX, chk1, chk2 or p53, and the induction of DNA fragmentation, that was detected by the comet assay. EDO-S101 was also found to induce cell cycle arrest in different phases depending on the dose and cell line. It has previously been suggested that DACi may impair DNA repair by inhibiting homologous recombination (HR), a pathway related with genomic instability and progression, very active in MM. Therefore we next evaluated the efficiency of HR using a reported construct that was chromosomally integrated in two MM cell lines, JJN3 and U266. Treatment with EDO-S101 significantly reduced the efficiency of HR in both cell lines, by 50% and 20% of untreated controls respectively. Finally, we tested potential combinations with other antimyeloma agents like lenalidomide and thalidomide; and also with proteasome inhibitors (bortezomib, carfilzomib and oprozomib). EDO-S101 potentiated the activity of all these agents, but the most synergistic combination was that including Bortezomib + Dexamethasone (CI 0,4). This combination was also evaluated in vivo, where it significantly decreased tumor growth and prolonged survival compared to agents in monotherapy and in double combinations. We are currently deepening into the mechanism of action of this combination. Conclusions. EDO S101 is active ex vivo in cells isolated from patients and is able to overcome resistance to alkylators. It induces caspase-independent apoptosis, and cell cycle arrest in MM cell lines. These effects are due to the potent DNA damage which is enhanced by HR impairment induced by the hybrid molecule. Moreover, the combination with bortezomib and dexamethasone is especially attractive to be taken into the clinical setting. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy.

Description: Background and objectives. PIM kinases (PIM1, PIM2, PIM3) are proteins known to be overexpressed in several hematological malignancies. In particular, in chronic lymphocytic leukemia (CLL) they are involved in cell survival, resistance to apoptosis (especially PIM2 and PIM3) and interactions with the microenvironment (PIM1). The aim of this study was dual: I) to evaluate the preclinical efficacy of PIM447, a pan PIM kinase inhibitor, in CLL and to study potential synergies with other drugs; and II) to evaluate the expression of PIM-kinases in different stages of the disease and correlate it with the prognosis and the sensitivity to the drug. Methods. Peripheral blood samples from untreated patients with different stages of the disease (monoclonal B lymphocytosis (MBL), stable CLL not requiring treatment (sCLL), and active CLL requiring treatment (aCLL)) were collected after informed consent. The ex vivo efficacy of PIM447 was analyzed by flow cytometry with annexin V in these samples. Moreover, PIM447 efficacy was also analyzed in two cell lines (MEC-1 and JVM-2) by MTT assay. Synergy with other drugs effective in CLL (bendamustine and fludarabine) was evluated with the calcusyn software. Protein levels of PIM Kinase proteins were evaluated by capillary electrophoresis immunoassay (WESTM ProteinSimple) in monoclonal B cells purified by CD19 selection with anti-CD19 magnetic microbeads and the autoMacs Cell separator (both from Miltenyi Biotec) from a subset of patients. Results. The pan PIM inhibitor, PIM447 was active in both cell lines tested, MEC-1 (IC50 5μM) and JVM2 (IC50 7μM), and also in monoclonal B cells from freshly isolated patients samples (sCLL=11; aCLL=5), with no difference in sensitivity between the different stages of the disease (IC50 of 4,8 μM and 4,7 μM for sCLL and aCLL respectively). There was a clear therapeutic window as treatment with PIM447 at doses toxic for monoclonal B cells, preserved T lymphocytes (figure 1) (median % of apoptosis for B cells and T lymphocytes respectively of 23 vs 20 at 5μM and 87 vs 35 at 10 μM). Moreover, PIM447 demonstrated to potentiate the activity of both bendamustine and fludarabine, being especially synergistic with this last one (combination index 0.1-0.6). A second objective was to analyze PIM2 protein expression by western blot in monoclonal B cells from these samples and correlate it with clinical and biological features. Up to now, it has been evaluated in 18 samples (MBL=4; sCLL=8; aCLL=6,). All of them expressed PIM-2. Expression levels of this protein were significantly higher in active CLL as compared with indolent stages of the disease (p=0,012). Patients with an unmutated IGHV status also displayed higher levels of PIM2 (p=0,01). Finally, samples with high PIM2 levels were slightly more resistant to PIM447 as compared with samples with lower protein levels (IC50 of 7,7 μM vs 5 μM, respectively). We are currently completing the analysis of the PIM2 levels of remaining samples and we are also measuring the levels of PIM1 protein, what will be available at the meeting. Conclusions: PIM-Kinase inhibition with PIM447 is effective in vitro in CLL cell lines and ex vivo in samples from patients. It synergizes with other agents especially fludarabine. PIM2 protein levels correlated with the clinical activity of CLL and with the mutational state of IGHV. Although all patients appear sensitive ex vivo to PIM447, further work is required to define PIM2 expression as a marker of sensitivity. Figure 1. Figure 1. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Jassen: Honoraria.

Description: Abstract 3003 During the last years, several novel drugs combinations have changed the outcome of Multiple Myeloma (MM) patients; nevertheless, relapse is still the rule for most of these patients, and, therefore, other drugs with novel mechanisms of action are required. The current research in this topic is usually based on the addition of novel drugs to standard of care combinations in order to try to increase their efficacy. Bortezomib + steroids has turned out to be one of these standards, both for induction therapy before high dose melphalan and also for non-trasplant candidates. Zalypsis is a novel marine compound with remarkable preclinical activity in MM, which is currently being explored in a phase I/II trial. We explored the preclinical efficacy of the triple combination of zalypsis, bortezomib and dexamethasone in different models and investigated its mechanism of action. This combination showed high potency when compared to the respective single agents and double combinations, and, in fact, the analysis by the Calcusyn software, evidenced combination indexes in the highly synergistic range (CI 0.2–0.6). This effect was observed not only in the MM1S cell line, but also in freshly isolated cells from MM patients and, most importantly, in a subcutaneous xenograft of a human plasmocytoma in SCID mice. In this last model, we used suboptimal doses of bortezomib and dexamethasone (0.1 mg/Kg and 1 mg/Kg respectively ip, 5 days per week indefinitely) combined with an active dose of zalypsis (0,75 mg/Kg iv weekly for three total doses). Probably due to the low doses used, the double combination of bortezomib + dexamethasone did not show much effect as compared to the control group, and neither bortezomib nor dexamethasone alone could significantly improve the efficacy of Zalypsis. Nevertheless, when the three drugs were combined together, the tumor volume drastically shrunk with differences highly statistically significant (p

Description: Introduction: Filanesib (ARRY-520) is a highly selective inhibitor of kinesin spindle protein (KSP), a microtubule motor protein active in proliferating cells, which plays an essential role in assembly and maintaining of the bipolar spindle. In cells arrested by KSP inhibition, Mcl-1 is rapidly depleted resulting in cell death, and consequently cells that are dependent on this pro-survival protein, such as myeloma cells, are particularly sensitive to filanesib. We investigated the mechanisms underlying the antimyeloma effect of this agent, focusing on other Bcl-2 family members. Methods: In vitro action of filanesib, alone and in combination with calpain inhibitor PD150606, was evaluated in multiple myeloma (MM) cell lines by MTT assay, Annexin V staining and cell cycle profile analysis by flow cytometry. MM cells were transiently transfected with non- targeting control short interfering RNA (NT-siRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Expression levels of different proteins were analyzed by Western-Blot. Results: We previously showed that all 11 MM cell lines tested were sensitive to filanesib and that sensitivity to this agent correlated with Bax levels. For these experiments, we focused on 3 cell lines with different Bax expression and sensitivity to filanesib: OPM-2 and MM1S (sensitive and high Bax levels) and U266 (less sensitive and low Bax levels). Treatment of MM1S with this agent triggered the translocation to the mitochondria of several proapoptotic Bcl-2 family members such as Noxa, Bim and Bax with several downstream effects. The mitochondrial translocation and activation of Noxa is key in the degradation of Mcl-1 by mediating the translocation of this protein from the cytosol to the mitochondria promoting its degradation. Regarding Bim, filanesib also induced the early (12-24 hours) expression of several proapoptotic isoforms of Bim that also translocated to the mytochondria. As previously reported, Bax is the top determinant of sensitivity to filanesib. In the present study, remarkably, once translocated into the mitochondria, Bax was also cleaved into the very potent proapoptotic 18 kDa fragment. All these events triggered the mitochondrial release of cytochrome C (caspase dependent apoptosis) and AIF (caspase independent apoptosis). In order to confirm the role of Bax in filanesib-induced apoptosis, we knocked-down Bax in MM1S by using small interfering RNA. This approach clearly decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced only 26% apoptosis in the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib). Furthermore, treatment with filanesib also induced cleavage of effector caspases (3 and 7) in all cell lines studied (OPM2, MM1S, U266). PARP was also cleaved in these cells, but it was previous to caspase activation in the most sensitive cell lines (OPM2, MM1S) suggesting a caspase-independent mechanism of apoptosis. This was confirmed by pre-treatment with the pan caspase-inhibitor Z-VAD-FMK, which did not rescue OPM2 and MM1S cells from apoptosis. Interestingly, one potential mechanism that could link both effects is the activity of calpain, a cysteine protease involved in caspase-independent apoptosis. This protein is a well-known caspase-independent way of processing PARP into the 60 kDa fragment, and has also been described as being responsible for Bax cleavage into the 18-kDa fragment. Consistent with this hypothesis, pre-treatment with the calpain inhibitor PD150606 clearly reduced the activity of filanesib in these cells (35 % to 70 % of survival) as assessed by MTT. Finally, consistent with the previous hypothesis, the less sensitive U266 cell line contained undetectable Bax protein suggesting that filanesib was not able to trigger caspase-independent apoptosis. However, a secondary caspase dependent apoptosis mechanism was confirmed as the pan-caspase inhibitor ZVAD-FMK was able to almost completely abrogate the activity of filanesib. Conclusions: Our results show that filanesib primarily initiates apoptosis by activating Bax in a caspase-independent manner, probably via calpain, a powerful accelerator of the apoptotic process. In addition, Noxa and BIM appear to be crucial for modulating Mcl-1 proteasomal degradation and Bax activation. This work was funded in part by the company Array BioPharma. Disclosures Tunquist: Array BioPharma: Employment. Mateos:Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Ocio:Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding; MSD: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria.

Description: Abstract 4077 Several murine models resembling multiple myeloma have been developed in the last years: the subcutaneous plasmocytoma model and the disseminated model, based on the iv or intracardiac injection of MM cells, have been widely used to test the efficacy of novel agents. Nevertheless, it is well known the crucial role that the interaction between MM plasma cells and the bone marrow microenvironment plays in MM pathogenesis as well as in the proliferation of MM cells and drug resistance; however, unfortunately, most murine models do not recapitulate these interactions. In order to overcome this caveat, other models such as the SCIDhu or the SCIDrab have been developed. However, in these models, the induced microenvironmental milleu is derived from a fetus and a rabbit respectively, which can be relatively different from that found in an adult patient with MM. We have developed a novel model of a human disseminated MM with the presence of human adult microenvironment. For this purpose, CB17-SCID mice, which lack B and T lymphocytes, were anesthetized and a human bone chip obtained from a healthy adult patient undergoing a hip replacement due to a non-malignant fracture, was subcutaneously implanted in the back of the mice. In the same day 3×106 MM1S-Luc cells were iv injected through the tail vein. Two groups were also included as controls in the study, one with MM1S-Luc cells without the bone chip, and the other with only the human bone chip (without MM cells). Luciferase activity was measured three times per week using a Xenogen-IVIS 50 system after ip injection of 0,15 mg/Kg luciferin. Mice were sacrificed when they developed signs associated with advanced disease, such as body weight loss or back limb paralysis. Although the group of mice receiving MM1S-Luc without bone, did develop a disseminated myeloma, this was much more precocious in the group of mice with a human bone subcutaneous implant. In fact, in mice with human bone and MM1S-Luc cells, the luciferase activity was detectable a median of 9 days after the injection of the cells whereas it was necessary 16 days for obtaining the same effect in mice without the bone chip. This is pointing out that the bone chip in these mice induces a favourable environment for myeloma growth and could be appropriate model to study the interactions of MM cells with human stroma. The increase of tumour growth rate also correlated with a decrease in survival. Median survival (CI 95%) of 64 (47-80) days for mice without bone vs 48 (45-50) days for mice with human bone (Log Rank=0.004). Regarding the sites of infiltration, luminescence images showed a disseminated involvement in all skeletal regions, and this was confirmed by histopathology, as there was a general infiltration of spine, femora, tibia and cranium of these mice. By contrast we did not find infiltration of extra-skeletal regions such as heart, lung or spleen in any mice and only unclear infiltrates were observed in the kidney and liver of some mice. In order to validate the suitability of this model for testing the in vivo antimyeloma activity of novel drugs in the presence of a human microenvironment, 6 mice bearing this novel model were randomized to receive: bortezomib (0.5 mg/Kg ip five days per week) + dexamethasone (0.5 mg/Kg 2 days per week) or vehicle control. A second group of the same number of mice with MM1S-Luc but without the bone chip were randomized in the same way and used as control. Bortezomib + dex significantly decreased tumour growth in both groups of mice without any clear differences in activity between both models, suggesting that this combination overcomes the influence of the microenvironment. In summary we have developed a murine model of MM that may allows us to 1) gain insights into the role of the BM microenvironment interactions in MM pathogenesis. 2) Evaluate the in vivo activity of different anti-MM agents in the presence of human microenvironment. 3) Potentially, this model could also contribute to analyze the effect of different agents in the myeloma bone disease. Disclosures: No relevant conflicts of interest to declare.

Description: Background Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat. Objectives To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies. Methods In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals. Results In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours. In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo. Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors. EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6. Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8. Conclusions This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies. Disclosures Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.

Description: The identification of subclones in multiple myeloma (MM) could have clinical implications since the complete eradication of all clones is required to prolong patients’ survival. Hence, there is an unmet need to fully characterize such subclones. In the present study, we started by investigating using multidimensional (23-color) flow cytometry (MFC) the presence of ≥2 distinct bone marrow PC subclones in 116 newly-diagnosed (MM) patients included in the Spanish GEM2010 trial: sequential VMP (9 cycles) followed by Rd (9 cycles) vs alternating VMP with Rd (18 cycles). By principal component analysis (PCA) using the Infinicyt software, the immunophenotypic expression profile (based on the simultaneous evaluation of 23 antigens in single-clonal-PCs) showed the presence of two or more subclones in 35/116 (30%) newly-diagnosed MM patients. The most powerful antigens for the identification of subclonal heterogeneity were CD56, CD27, CD81, CD20, CD33, β7, CD138, CD49e, CD54, HLADR, CD19, CD44 and CXCR4. Then, we investigated the genetic profile of distinct phenotypic subclones (sorted by FACS with purity ≥97%) through high-density Cytoscan750K (n=5) arrays and FISH (n=8). In 2/5 (40%) patients, specific copy number abnormalities (CNA; only those with minimum of 25 consecutive imbalanced markers per segment and minimum 100 Kb length were considered) were present in one clone [del(13q), del(14q), del(20p), +(20q), +(21q), or del(22q)] but absent in the other. Similar results were observed by FISH while investigating those probes used as part of the clinical workout. Noteworthy, in three cases del(17p13) was detected in only one subclone, whereas del(13q14) was restricted to one subclone in two patients. To assess the clonogenic potential of distinct phenotypic subclones detected in the whole malignant PC compartment, these were sorted by MFC and seeded in coculture with the human mesenchymal cell line hTERT (with Methocult® supplemented with lymphocyte conditioned medium (10%) plus 20 ng/ml IL-6 plus 20 ng/ml IGF-1). In 2 out of 7 patients (29%) we observed different clonogenic potential between two patient-specific subclones (patient 1, 208 vs 0 colonies; patient 2, 5 vs 1 colony). Finally, we aimed to investigate if distinct phenotypic subclones display different chemoresistance. To address this question, we compared the immunophenotypic expression profile of paired baseline vs MRD clonal PC in 9 patients enrolled in the Spanish GEM2010 trial in which ≥2 subclones were identified at diagnosis. Our results show a phenomenon of clonal restriction in 7/9 (78%) patients after chemotherapy, indicating that among the initial tumor bulk, only specific subclones are primary chemoresistant (i.e.: MRD) in patients otherwise achieving a serological response. As an example, in one patient showing at diagnosis four clonal PC subsets (CD33-/CD117-, CD33+/CD117-, CD33+/CD117+ and CD33+/CD117+), only one subclone (CD33-/CD117-) persisted after 9 cycles of alterning Rd-VMP. In conclusion, our results show that distinct phenotypic subclones translate into a different cytogenetic profile and clonogenic potential. Since the balance between different subclones may change over the course of treatment, so may change the extent of specific cytogenetic abnormalities and the patient risk. Thus, careful monitoring of specific subclones may have implications towards an adapted therapy to overcome chemoresistant cells. This work was supported by funding from the RTICC-Hematology Group (RD12/0036/0058), the Asociación Española Contra el Cáncer (AECC) (GCB120981SAN) and Multiple Myeloma Research Foundation (MMRF) 2012 Research Fellow Award (B Paiva) Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Multiple myeloma (MM) is characterized by the presence of complex karyotypes and chromosome instability, suggesting that cell cycle checkpoints are defective. Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle proteins (KSP), which are required to establish mitotic spindle bipolarity, driving centrosome separation. Filanesib monotherapy has demonstrated clinical activity in heavily pretreated MM patients (Lonial et al, ASH 2013). In this work we aimed to explore the preclinical activity of filanesib alone and in combination with IMiDs. Methods: In vitro activity of filanesib alone and in combination with IMiDs (thalidomide, lenalidomide and pomalidomide) was evaluated in MM cell lines by MTT assay and Annexin V, Propidium Iodide and DiOC6 analysis by flow cytometry, Western Blot and immunofluorescence. Synergy was quantified with combination indices (CI) by Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice. Results: Filanesib demonstrated significant activity in a broad panel of 11 MM cell lines, with 48-hour IC50 values ranging between 0.3 and 5 nM. Interestingly, the highest activity was observed in drug-resistant cell lines such as OPM-2 and RPMI-LR5. We next evaluated whether the cell death MoA was dependent on apoptosis or blockade of proliferation. Time response experiments performed in three different cell lines with different sensitivity to filanesib (OPM-2, MM1S and U266) showed accumulation of cells in G2/M, followed by loss of the mitochondrial membrane potential and activation of apoptosis. Accordingly, Western blot analysis demonstrated an activation of the mitotic checkpoint indicated by an increase in Cyclin B1, and activation of apoptosis with PARP, and caspase-3 and -7 cleavage. Furthermore, filanesib activity was very rapid as 15 minutes of exposure was sufficient to exert all of the apoptotic and cell cycle effects observed at 48 hours. Immunofluorescence microscopy using alpha-tubulin demonstrated that filanesib induces monopolar spindle formation. The sensitivity of MM to filanesib has been previously correlated with the cell-dependency of the anti-apoptotic protein Mcl-1. We, therefore, studied the basal levels of six Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the 11 cell lines and observed a correlation between the basal levels of these proteins and drug sensitivity. In particular, we confirmed the relationship with the anti-apoptotic proteins Mcl-1 and Bcl-2 and demonstrated that conversely to what is observed with proteasome inhibitors, cells with high basal levels of pro-apoptotic Bax or Bak were more resistant to filanesib. Moreover, treatment with filanesib induced a clear decrease of Mcl-1 in the 3 cell lines analyzed (U266, MM1S and OPM-2) that coincided with a decrease of Bcl-2 in the most sensitive cell line, OPM-2. Finally, we evaluated the activity of filanesib in combination with IMiDs and dexamethasone. In vitro studies showed a synergistic effect of filanesib with dexamethasone (CI: 0.21), and with all IMiDs, being most pronounced with pomalidomide (CI: 0.09). Of note, this triple combination demonstrated the highest synergistic activity (CI: 0.06). These results were confirmed in vivo where the triple combination of filanesib, dexamethasone, and pomalidomide was also synergistic, with a significant reduction of tumor growth of up to 50 days, which correlated with a statistically significant survival improvement. Mechanistic studies on the combination are ongoing. Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. It was identified that this activity is dependent on the anti-apoptotic protein Mcl-1 and pro-apoptotic proteins Bax and Bak. Furthermore, in animal xenograft studies, filanesib exhibited robust synergism in combination with dexamethasone and any IMiD, pomalidomide being the most synergistic. These data are the rationale for the clinical trial "Pomdefil" which uses this combination in patients with relapsed refractory MM. The trial will begin soon as a collaboration of the Spanish Myeloma Group (GEM). This work was supported in part by Array BioPharma. Disclosures Humphries: Array Biopharma: Employment. Tunquist:Array Biopharma: Employment. Mateos:Array Biopharma: Honoraria. Ocio:Array Biopharma: Honoraria, Research Funding.

Description: Key Points Detailed characterization of myeloma circulating tumor cells shows that these represent a unique subpopulation of BM clonal PCs. Myeloma CTCs are clonogenic, quiescent, and may represent an ancestral clone potentially driven by circadian rhythms.