Search

Introduction: Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow (BM) and represents the second most common hematologic malignancy. Although therapeutic options have broadened over the years, the disease is challenged by frequent relapses. Relapsed/refractory MM (RRMM) often becomes non-responsive to previous lines of treatment and has significantly poorer survival outcome. Physicians are faced with a difficult task to choose a right treatment regimen. Thus, a precision medicine tool that predicts the clinical response of individual patients to therapy is greatly desired. We have previously developed a novel 3D tissue-engineered BM (3DTEBM) culture model, which is patient derived, closely recapitulates the pathophysiological conditions in the BM, and allows ex vivo proliferation of primary cells of various hematologic malignancies. In this study, we conducted a retrospective study that tests the ability of the ex vivo 3DTEBM platform to predict the clinical response in individual MM patients, to help decision-making process for RRMM. We hypothesized that the 3DTEBM will be able to predict clinical responses of RRMM patients. Methods: We first performed a literature search to examine the clinical efficacious concentrations (Css) for 10 MM drugs. We then experimentally determined the in vitro efficacious concentrations (IC50) of these drugs in MM cell lines, in both 2D and 3DTEBM cultures. The IC50 values were then correlated with their respective clinical Css values to evaluate how well each culture system reproduce drug efficacy. For the retrospective trial, we used viably frozen whole BM samples from 19 RRMM patients with known clinical responsiveness to the regimen they received. 3DTEBM cultures were developed for each patient with BM biopsies obtained prior to the start of clinical regimen. Cultures were treated ex vivo with the same treatment regimen each patient received clinically, at increasing concentrations (0X, 3X and 10X of Css of individual drugs). After 4 days, cultures were digested and cells were retrieved for flow cytometry analysis. Primary cells were stained Leukocyte-/CD38+ and counted against counting beads. Survival was determined as % of untreated control, and ex vivo responsiveness was analyzed by ANOVA. Finally, the clinical team correlated the ex vivo response with the clinical response for each patient. Results: To demonstrate this discrepancy between drug efficacy in laboratory settings and clinical outcomes, we compared the in vitro IC50 to the clinical Css of 10 drugs used for the treatment of MM. We found that there was no correlation between the IC50 in classic 2D culture systems and the clinical Css (R 2=0.019) (Figure 1A). In contrast, the IC50 in the 3DTEBM directly correlated with the clinical Css (R 2=0.993) (Figure 1B). We then conducted a retrospective clinical trial to determine if the 3DTEBM platform is able to predict each patient's clinical response by recreating the same treatment regimen ex vivo (Figure 1C). The 3DTEBM was able to predict the response in 89% of the MM patient cohort across multiple treatment regimens, with no false positives (Figure 1D). Conclusions: Our retrospective clinical trial demonstrated that the 3DTEBM technology is a feasible platform for predicting therapeutic responses in MM with a high predictive accuracy within a clinically actionable time frame. Such platforms can provide precise clinical insight about the efficacy of different treatment plans and assist physicians to propose the best choice of therapy for their individual patients. Future prospective studies are needed to validate these significant findings by testing prospective prediction ability of 3DTEBM to improve therapy response in hematologic malignancies. Figure 1 Figure 1. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Azab: Cellatrix LLC: Current Employment. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Azab: Cellatrix, LLC: Current Employment, Current holder of individual stocks in a privately-held company.

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo. Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O2) or hypoxia (1% O2) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478. For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice. Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival. Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM. Disclosures No relevant conflicts of interest to declare.

Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

Proteasome inhibitors (PIs) have improved the treatment of multiple myeloma (MM) and prolonged patient survival, but several challenges remain to overcome drug-resistance and toxicity. Bone marrow microenvironment (BMM) drives tumor progression and PIs-resistance in MM; and agents that inhibit the interaction between MM and BMM have been shown to re-sensitize MM cells to therapy. However, the synchronized in vivo delivery of BMM-targeting agents with PIs has been a challenge so far. Nanoparticles offer a valuable platform to encapsulate drugs, and if functionalized, they can facilitate specific delivery to tumor, thus improving treatment efficacy and reducing off-target effects. Within the BMM, the endothelium plays a relevant tumor promoting role. By analyzing the expression of an array of markers in normal and in MM-related endothelium, we found high levels of P-selectin expression on MM-activated endothelial cells (ECs) than normal cells and on ECs collected from the BM of either MM patients or MM-bearing mice compared to their respectively healthy BMMNCs. We next sought to develop lipid nanoparticles (LNPs) targeting the MM-related endothelium, loaded with both PI and BMM-targeting agent for synchronized delivery and reversal of the BMM-induced drug resistance. At this aim, we developed targeted LNPs towards P-selectin by decorating their surface with P-selectin-glycoprotein-ligand-1 (PSGL-1). PSGL-1-targeted LNPs showed specific binding to recombinant P-selectin than identically non-targeted particles, and to MM-associated endothelium compared to healthy endothelium, both in vitro and in vivo. To reverse BMM-induced resistance, LNPs were loaded with bortezomib (BTZ) together with a BMM disrupting agent, ROCK-inhibitor (Y-27632) that inhibits the downstream signaling of the RhoA GTPase pathway, known to be instrumental to the interaction of MM cells with BMM. Consequently, we tested the effect of synchronized delivery of BTZ and Y-27632 in the same LNP on MM cell survival in co-culture with the BMM in vitro. While Y-27632-loaded LNPs did not affect cell proliferation, LNPs loaded with both Y-27632 and BTZ enhanced responsiveness of MM cells to BTZ, compared to BTZ-loaded LNPs, thus overcoming the BMM-induced resistance. Mechanistically, we observed more significant inhibition of PI3K and MAPK signaling, decrease of pRb and up-regulation of p21 and induction of pro-apoptotic pathway (caspase-3, caspase-9 and PARP) by drug-loaded LNPs, compared to free drugs. In addition, drug-loaded LNPs were able to decrease adhesion and impair the migration of MM cells to ECs. We also investigated the in vivo efficacy of BTZ/Y-27632-loaded PSGL-1-targeted LNPs in a humanized murine model of MM. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls (vehicle, BTZ and Y-27632 alone or in combination as free drugs, or encapsulated in non-targeted or in PSGL-1-targeted LNPs) demonstrating that both P-selectin targeting and combination of Y-27632 with BTZ reverses the BMM-induced drug resistance and enhances the efficacy of therapy in vivo. Altogether, our data demonstrate the ability of PSGL-1-decorated LNPs to specifically target MM-BMM; to efficiently encapsulate and deliver drugs to tumor tissue; to overcome BMM-induced drug resistance in vitro and in vivo, to reduce tumor growth and prolong overall survival. This study provides the preclinical basis for future clinical trials using MM-BMM-targeted nanomedicine able to enhance the effect of PIs or other drugs for the treatment of MM. Disclosures Roccaro: GILEAD: Research Funding; AMGEN: Other: Advisory Board. Vij:Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Azab:Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding.

Introduction: The presence of a minimal residual disease (MRD) that remains after treatment is one of the main reasons for recurrence and relapse in multiple myeloma (MM) patients. Diagnosis of MRD is becoming vital to assess the effectiveness of the treatment as well as to predict survival among patients with complete remission. CD138 (syndecan-1) is the gold standard marker for detecting MM cells using multiparametric flow cytometry analysis or immunohistochemistry (IHC) staining of BM biopsies. However, the presence of highly clonogenic, drug resistant and stem-cell like CD138-negative MM cells have been previously demonstrated. Moreover, hypoxia which is known to drive MM progression, drug resistance and metastasis was shown to significantly decrease CD138 expression in MM cells. In this study, we utilized a novel set of biomarkers to detect MRD in the bone marrow as well as circulating tumor cells (CTCs) in the blood, in order to predict progression free survival (PFS) in MM patients defined as complete remission according to the detection of CD138+ cells in their bone marrow. Methods: To detect myeloma cells, we utilized a novel set of biomarkers, independent of CD138 expression and hypoxic state of MM cells (CD38+/CD3-/CD19-/CD14-/CD16-/CD123-) by two-color flow cytometry. We detected MM cells in the bone marrow of 25 patients with complete remission and very good partial response, whose bone marrow was defined as a CD138-negative (less than 0.5%). In order to retrospectively correlate the involvement of MM cells (%) with PFS, we used 24 months as a cut-off and compared the results acquired with the new method to traditional CD138-based flow or histology. In addition, based on the percentage of detected MM cells with the 2% cut-off, we analyzed the time to progression (months). Moreover, we detected MM cells in the peripheral blood from matching MM patients and performed analogous analysis. Furthermore, we also detected CTCs in 7 newly diagnosed patients and compared to 7 patients with progressive disease. Results: A study conducted on 25 patients with their bone marrow defined as a CD138-negative (less than 0.5%) showed that the novel strategy to detect MM cells was more precise than CD138-based flow or histology in predicting PFS. Patients who relapsed in less than 24 months had an average (±SEM) of 3.53±0.82%, while patients who relapsed later than 24 months had an average of 1.15±0.27% MM cells in the bone marrow when detected by the new method (p=0.004). In addition, the median PFS with Conclusions: The novel flow cytometry-based set of biomarkers provides an alternative strategy to detect MRD in the bone marrow and CTCs in the peripheral blood of MM patients, and both allowed prediction of PFS in MM patients. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Vij:Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Jazz: Consultancy; Shire: Consultancy; Karyopharma: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Cleave Bioscience: Research Funding; Verastem: Research Funding; Cell Works: Research Funding; Karyopharm: Research Funding; Vasculox: Research Funding; Glycomimetics: Research Funding; Cellatrix LLC: Other: Founder and owner; Selexys: Research Funding.

Introduction: Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA. Methods: Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0-10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions. We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions. Results: The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP. We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually. Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions. Conclusions: Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma. These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma. Disclosures Arbiser: ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.. Azab:Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding.

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy characterized by the continuous spread of MM cells in and out of the bone marrow (BM). Despite the introduction of novel therapies, cancer patients relapse due to the development of drug resistant cells, which are, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome drug resistance in MM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve the outcome of patients with solid tumors when combined with radiotherapy; however, it has not been tested in MM. We used TPZ for the first time in MM to target the drug resistant cancer cells and sensitize them to therapy. Methods: To test the effect of TPZ on tumor survival in vitro, MM cell lines (MM1.s, H929, OPM1, RPMI8226) were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 hours with different concentrations of TPZ in order to obtain an IC50, and cell survival was assessed using MTT assay. Also, a combination of bortezomib and carfilzomib with or without TPZ was tested on cell survival. For in vivo study, 5 x 106 MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. First, we tested the hypoxic status of mice treated with and without a high-dose bortezomib (1.5mg/kg). Pimonidazole (PIM) was injected intraperitoneally (IP) into mice and 4 hours later BM was harvested, stained with anti-PIM-APC antibody and followed by measuring PIM signal in MM1s-GFP+ cells using flow cytometry. Second, we developed drug resistant cells by treating mice with a high-dose bortezomib (1.5mg/kg), and then treated with (1) bortezomib only (0.5mg/kg; n=3), or (2) bortezomib and TPZ (40mg/kg; n=3), all administered IP sequentially twice a week. The number of residual MM1s-GFP+ cells was calculated by flow cytometry. Results: We found that TPZ was active in a dose-dependent manner only in hypoxic conditions in MM cell lines. We showed that residual MM cells in the BM after high-dose bortezomib are hypoxic, as demonstrated by PIM staining. The combination of TPZ with bortezomib and carfilzomib resensitized cancer cells to death in hypoxia, overcoming hypoxia-induced drug resistance in vitro. Moreover, TPZ-treatment in combination with bortezomib further decreased residual MM cells in vivo. Conclusions: We reported that MRD was hypoxic and that TPZ, which was cytotoxic for MM cells only in hypoxic conditions, overcame hypoxia-induced drug resistance in vitro and killed bortezomib-resistant residual MM cells in vivo. This is the first study to show the efficacy of TPZ in MM. This data provides a preclinical basis for future clinical trials testing efficacy of TPZ in MM. Disclosures Azab: Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Verastem: Research Funding.

Introduction: Diagnosis, responses to treatment, minimal residual disease (MRD) and circulating tumor cells (CTC) in multiple myeloma (MM) are all detected by the gold-standard marker CD138 flow cytometry-based method. However, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 downregulation were shown previously. In this study, we found that CD138 is significantly downregulated in MRD and CTC populations in MM, thus we developed a novel two-color flow cytometry-based set of biomarkers (independent of CD138) to detect MRD and CTC in MM. Methods: We created a MRD mouse model by treating MM-bearing mice with a high dose of bortezomib. We then tested the effect of hypoxia and drug treatment on the expression of different markers, including CD138. Therefore, to detect MM cells we utilized CD38-APC antibody, followed by the exclusion of non-MM CD38-expressing cells such as T cells, monocytes, NK cells, B cells and dendritic cells using (CD3, CD14, CD16, CD19 and CD123)-V450 antibodies, respectively. To verify the ability of the new method to selectively detect MM cells, mononuclear cells from peripheral blood (PB) from healthy patients and MM cell lines (hypoxic and normoxic) were stained by the antibody cocktail and analyzed by flow cytometry. Next, we compared the sensitivity of the traditional CD138 method to the new method in detecting (a) normoxic or hypoxic MM cells spiked into bone marrow (BM) cells in vitro, (b) normoxic or hypoxic MM cells spiked into PB in vitro, (c) MM1s-GFP+ cells in mice with different tumor burden in the BM in vivo, (d) circulating MM1s-GFP+ in the PB in vivo, (e) MRD cells in the BM samples from 16 patients with complete remission (CR) or very good partial response (VGPR), and (F) CTC in 12 progressive MM patients. We further aimed to predict time to progression (TTP) in 16 patients with complete remission based on the detection of MRD in these patients using the new method and compared with flow cytometry-based CD138 or histology. Results: In vivo, we found that bortezomib-treated MRD cells were hypoxic, compared to a progressive vehicle-treated cells. CD138 expression in these cells was significantly decreased, but CD38 expression was unchanged. In vitro expression of CD138 was decreased due to hypoxia and bortezomib treatment, whereas CD38 expression was unchanged. Furthermore, we developed a new method using an antibody cocktail, where the MM cell population is defined as CD38+/CD3-/CD14-/CD16-/CD19-/CD123- (APC+ and V450-). In vitro, the new method detected 100% of hypoxic and normoxic MM cells, and less than 0.5% of mononuclear cells from the PB or BM of healthy donors. In contrast, CD138+ cells failed to detect 50% of hypoxic MM cells and 10-25% of normoxic cells. In vivo, the amount of cells detected by the new method directly correlated with the number of MM1s-GFP+ cells detected in the BM with a range between 0-60%, with a correlation coefficient (slope) of 0.99 and R2 of 0.999. The CD138 detected only a fraction of the MM1s-GFP+ population ( In patients, the new method detected 0.5-8% MRD cells in the BM of 16 patients with CR or VGPR (which were defined as CD138-negative), and 0.1-1.8% CTC in the PB of 12 progressive MM patients. In contrast, CD138 marker detected less than 0.5% of MRD cells in the BM of the CR and VGPR patients, and less than 0.1% of CTCs in the PB of the progressive patients. Furthermore, we found that, while CD138 and histology failed to predict recurrence in CR patients, the new method successfully detected CD138-negative MM population whose prevalence in the BM inversely correlated with TTP in MM patients defined as CR based on CD138 and histology. Conclusions: We confirmed that CD138 expression is variable on MM cells, and that it is downregulated in MRD and CTC populations, and that it was not effective in detecting these particular populations in MM. Furthermore, we developed a novel two-color flow cytometry-based biomarker-set to detect MM cells independent of CD138. The new methods detected close to a 100% of all MM cells in vitro and in vivo, including MRD and CTC. Moreover, the new method detected a CD138-negative MRD and CTC in MM patients, and the prevalence of this population inversely correlated with TTP in MM patients. Disclosures Vij: Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) associated with lytic bone disease. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance. Low oxygenation (hypoxia) in the BM microenvironment, which was shown to decrease beyond physiologic BM conditions during the progression of MM, together with cellular and acellular components, plays a critical role in drug resistance. Increased levels of the nuclear exporter of tumor suppressors, exportin 1 (XPO1), was demonstrated in MM, and XPO1 inhibition showed a broad anti-tumor activity. In this study, we tested inhibition of XPO1 using the novel oral, covalent, slowly reversible inhibitor of nuclear export selinexor currently in Phase I/II clinical trials on sensitizing the hypoxia-inducible drug resistant MM cells both in vitro and in vivo. Methods: MM cell lines (MM1s, H929, OPM2 and U266) were treated with increasing concentrations of selinexor in normoxic (21% O2) and hypoxic (1% O2) conditions with or w/o bortezomib or carfilzomib. Assays included MTT proliferation Annexin V/PI staining for apoptosis, PI staining and immuniblots for cell cycle analysis. We tested the effects of selinexor as a single agent on tumor initiation in vivo, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and mice were treated instantaneously with selinexor (15mg/kg) or vehicle (PVP + F-68) three times a week. We also tested the effect of selinexor (15mg/kg) as a single agent on tumor progression and survival in mice with established MM tumor. Furthermore, we studied the effects of selinexor on sensitization to bortezomib-resistant MM cells in vivo. MM1s-Luc-GFP cells were injected IV into SCID mice and tumors were allowed to grow for three weeks. Animals were then treated with a high-dose of bortezomib (1.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were then randomly divided into 3 groups treated with (1) bortezomib (0.5mg/kg), (2) KPT-330 (10mg/kg), and (3) a combination of selinexor and bortezomib. Selinexor was administered by oral gavage three times a week (days 1, 3 and 5) and bortezomib was injected intraperitoneally twice a week (days 1 and 4). Tumor progression was imaged using bioluminescence imaging (BLI). Results: Selinexor, as a single agent, decreased proliferation, induced G1 cell cycle arrest and increased apoptosis of MM cells in both hypoxia and normoxia in a dose-dependent manner. Moreover, we found that the combination of selinexor with bortezomib or carfilzomib reversed the hypoxia-induced resistance in MM cells to proteasome inhibition and induced a synergistic effect on the inhibition of cell proliferation, G1 arrest and apoptosis, which was confirmed by response of respective cell signaling pathways as PI3K, MAPK and JNK. In vivo studies revealed that selinexor alone delayed tumor initiation and decreased by half existing tumor size as a single agent (p=0.0397), and significantly extended mice survival (53 days on vehicle versus 62 days on selinexor, p=000375). In addition, MM-bearing mice which developed resistance to bortezomib were resensitized by treatment of selinexor combined with bortezomib, decreasing tumor burden and showed considerably extended survival (half of the mice were alive at day 52) compared to mice treated with either selinexor (half of the mice were alive at day 49) or bortezomib alone (half of the mice were alive at day 40) (bortezomib versus selinexor p=0.0014, bortezomib versus combination treatment p=0.0004 as analyzed by Student t-test). Conclusions: The orally available inhibitor of nuclear export, selinexor, decreased survival of MM cells in vitro and delayed tumor initiation and tumor progression in vivo as a single agent. In addition, selinexor overcame hypoxia-induced resistance to proteasome inhibitors and resensitized MM cells to therapy both in vitro and in vivo, extending mice survival. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by inhibition of XPO1 with selinexor as well as combination studies of selinexor and proteasome inhibitors. A study of selinexor and carfilzomib is currently ongoing and early results that were published last year showed marked activity for this combination treatment in heavily pretreated MM patients. Disclosures Landesman: Karyopharm: Employment. Azab:Karyopharm: Research Funding; Selexys: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Cell Works: Research Funding.

Introduction: A plasma cell malignancy called multiple myeloma (MM) is an incurable cancer in which a majority of patients with refractory disease become resistant to therapy. The introduction of carfilzomib, a proteasome inhibitor, significantly improved the clinical outcome of the MM treatment. However, MM patients develop resistance to carfilzomib and relapse. In this study, we investigated the role of hypoxia and P-gp in the carfilzomib-resistance in MM cells in vitro and in vivo, and its therapeutic translational potential using a HIF inhibitor, PX-478. Methods: In vitro, MM cell lines were treated with carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions and cell survival was analyzed by MTT assay. The activity of P-gp was assessed by testing the efflux of a known P-gp substrate, RhodamineB (RhoB), in the hypoxic and normoxic conditions by measuring the intracellular RhoB content using flow cytometry. Moreover, we have tested the effect of HIF inhibition using PX-478 on the P-gp activity as well as the response to carfilzomib in hypoxic and normoxic cells. In vivo, we tested the effect of HIF inhibition on tumor initiation, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice, which were treated instantaneously with PX-478 (10mg/kg) three times a week. Furthermore, we tested the effect of PX-478 on MM tumor response to carfilzomib (measuring tumor size and mice survival). MM1s-Luc-GFP cells were injected IV into SCID mice, tumors grew for 3 weeks and the mice were then randomly divided into 4 groups treated with (1) vehicle (Captisol), (2) PX-478 (10mg/kg) alone, (3) carfilzomib (5mg/kg) alone, or (4) a combination of PX-478 (10mg/kg) and carfilzomib (5mg/kg). PX-478 or vehicle were administered by oral gavage three times a week (day 1, day 3, day 5), while vehicle and carfilzomib were injected IV twice a week (day 4 and day 5). Tumor size was imaged using bioluminescence imaging (BLI) and mice survival was followed for 70 days. Results: In vitro, we found that hypoxia induced resistance to carfilzomib in five MM cell lines. Moreover, hypoxia also increased activity of P-gp by causing decreased intracellular RhoB content in hypoxic MM cells. The HIF inhibitor, PX-478, as well as the P-gp inhibitor, tariquidar, reversed the activation of P-gp in hypoxic cells, while the combination of PX-478 and tariquidar did not induce further inhibition of P-gp activity. Furthermore, the combination of PX-478 or tariquidar with carfilzomib reversed the hypoxia-induced resistance in MM. However, tariquidar and other P-gp inhibitors have shown low selectivity and high toxicity in clinical trials; therefore, for our in vivo experiments we chose to inhibit HIF in order to reverse the hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells. In vivo, in the MM tumor-initiation model, our study revealed that the HIF inhibitor, PX-478, significantly delayed the tumor progression and extended survival in which all control mice died between 42-52 days, while the experiment was stopped at 70 days, with all PX-478-treated mice still alive. In the established in vivo tumor model, low dose carfilzomib alone delayed the progression by BLI but did not improve survival (vehicle and carfilzomib-treated mice died between 21-28 days after treatment). Despite the fact that PX-478 did not decrease tumor progression as shown by BLI compared to the vehicle-treated mice, it significantly extended the survival of the mice (animals died between 38-48 days). The combination of carfilzomib and PX-478 significantly decreased the proliferation of tumor shown by BLI (less than 5% of the growth at day 28), as well as considerable increase in survival (the experiment was stopped at 70 days with 100% of the group alive). Conclusions: We identified a novel resistance mechanism to carfilzomib in MM, in which hypoxia induces P-gp-mediated resistance to carfilzomib. Inhibition of the hypoxic response in MM cells by the HIF inhibitor reduced hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells in vitro, and delayed tumor progression significantly improving survival and response to carfilzomib in MM-bearing mice in vivo. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Introduction: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM). Despite the introduction of novel therapies more than 70% of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role on proliferation of MM and that its inhibition with small-molecule inhibitors sensitized MM cells to therapy. However, these small molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. In this study, we tested inhibition of P-selectin and PSGL-1 using functionally blocking monoclonal antibodies to sensitize MM cells to therapy. Methods: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals. Endothelial cells, stromal cells derived from MM patients, and MM cell lines (MM1s, H929, OPM1 and RPMI8226) were used for in vitro expression, proliferation, apoptosis and immuno-blotting assays. The expression of P-selectin and PSGL-1 were tested by the interaction of SelG1 (5-20µg/mL) and SelK2 (5-20µg/mL) antibodies with endothelial cells, stromal cells and MM cells for 1hr, followed by addition of a secondary-FITC antibody and flow cytometry analysis. For adhesion assay, cells were treated with increasing concentrations of SelG1 and SelK2 for 1hr; pre-labeled MM cells were then applied to unlabeled endothelial or stromal cells for 1hr, non-adherent cells were washed, and adherent cells were analyzed by a fluorescent reader. For proliferation, MM1s-GFP+ cells cultured alone, with stroma, or with endothelial cells; and were treated with, or without, bortezomib and carfilzomib, in the presence, or absence, of SelG1 and SelK2 antibodies, and proliferation was determined by flow cytometry. Protein expression associated with survival, apoptosis and cell cycle signaling was analyzed by western blotting. For in vivo, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumor progression followed for 4 weeks. Anti-mouse-P-selectin antibody was used to inhibit P-selectin in the mouse endothelial cells and stroma. Likewise, SelK2 and anti-mouse PSGL-1 were used to inhibit PSGL-1 on human MM cells in the mouse microenvironment, respectively. Mice were divided into 6 groups (1) vehicle treated control, (2) anti-mouse-P-selectin (5mg/kg) alone, (3) SelK2 (5mg/kg) and anti-mouse-PSGL-1 (5mg/kg) alone, (4) bortezomib (0.5mg/kg) alone, (5) a combination of anti-mouse-P-selectin and bortezomib, and (6) a combination of SelK2 (5mg/kg), anti-mouse-PSGL-1 (5mg/kg) and bortezomib. Anti-mouse-P-selectin, anti-mouse–PSGL-1, SelK2 and bortezomib were administered intraperitoneally twice a week. Results: The half-life of SelG1 in a Phase I clinical study was previously shown to be 363 hours while the half-life of SelK2 in a primate study was approximately 100 hours. P-selectin expression was detected on endothelial and stromal cells using the SelG1 antibody, while no expression was found on MM cells. PSGL-1 was highly expressed on MM cells as well as on endothelial cells and stromal cells as detected by SelK2 monoclonal antibody. Inhibition of P-selectin and PSGL-1 with SelG1 or SelK2, respectively, decreased MM cell adhesion to endothelial and stromal cells, and decreased the proliferation of MM cells induced by stromal and endothelial cells. Similarly, inhibition of the interaction between P-selectin and PSGL-1 sensitized MM cells to bortezomib and carfilzomib in vitro. In vivo results demonstrated that inhibition of the P-selectin or PSGL-1 as single agents delay tumor growth compared to non-treated mice and that it enhances the effect of bortezomib. Conclusions: These results demonstrate that inhibition of P-selectin and PSGL-1 by SelG1 and SelK2 antibodies, respectively, disrupts the interaction between MM cells and BM microenvironment and decreases adhesion and proliferation of MM cells. Moreover, inhibition of P-selectin and PSGL-1 increased the sensitivity of MM cells to bortezomib and carfilzomib in vitro, and to bortezomib in vivo. These data provides a basis for future clinical trials for sensitization of refractory MM patients to therapy by inhibition of P-selectin and PSGL-1 using SelG1 and SelK2 monoclonal antibodies. Disclosures Rollins: Selexys: Employment. Alvarez:Selexys: Employment. Kawar:Selexys: Employment. Azab:Selexys: Research Funding.

INTRODUCTION: Flow cytometry has been used extensively to detect MM cells in the bone marrow (BM), micro-residual disease and circulating myeloma cells. Accumulating literature defines MM cells as CD138+/CD38+ for the primary gating of plasma cells; however, several studies demonstrated the presence of clonogenic CD138-negative MM cells, and that hypoxia can decrease the expression of CD138 in MM cells. We propose a novel set of biomarkers to detect MM cells regardless of their CD138 expression or hypoxic status. METHODS and RESULTS: We have tested the effect of hypoxia on the expression of the MM markers, CD138 and CD38, and found that hypoxia decreased the expression of CD138, therefore it cannot be used as a universal marker for MM cells. Hypoxia did not alter the expression of CD38 in MM cells; however, CD38 is a general leukocyte marker and cannot be used alone to identify MM cells, since it is expressed on multiple other leukocytes including T-cells, B-cells, monocytes, NK cells, and dendritic cells. Therefore, we negatively selected these cells by flow cytometry using the specific markers for each of these populations including CD3, CD19, CD14, CD16, and CD123; respectively. Therefore, we detected MM cells as CD38+/CD3-/CD19-/CD14-/CD16-/CD123-. We used CD38 antibody conjugated with APC and FITC-conjugated antibodies for all the other markers, thus MM cells were defined as APC+/FITC- population. To compare traditional method (CD138-based) with our strategy to detect hypoxic and normoxic MM cells, MM cell lines were stained with a cocktail of CD38-APC, FITC-antibodies, and CD138-V450, and analyzed by flow cytometry. The use of CD138+ as a universal marker for MM cells detected 85-100% of the normoxic cells, and only 60-75% of the hypoxic MM cells. While APC+/FITC- strategy detected close to a 100% of the MM cells independent of the cells’ normoxic/hypoxic status or expression of CD138. The ability of the new strategy to detect hypoxic and normoxic MM cells in the peripheral blood was tested by staining hypoxic and normoxic MM cells with cell-tracker Calcein-Red-Orange (as a positive control), spiked 104 MM cells into 106 mononuclear cells from a healthy donor (1% MM in total), and the percentile of Calcein-Red-Orange+ (as a positive control), APC+/FITC-, and CD138+ populations, were analyzed by flow cytometry. Calcein-Red-Orange staining showed exact 1% of MM cells detected in total mononuclear cells for both hypoxic and normoxic MM cells; detection with CD138+ showed 0.95% for normoxic and 0.45% for hypoxic cells; and detection of MM cells using the APC+/FITC- strategy showed 1.05% for normoxic and 1.1% for hypoxic MM cells. Hence, the new strategy detects MM cells selectively and independently of their CD138 expression or hypoxic status. We have used the APC+/FITC- strategy to detect MM cells in BM CD138-negative fractions of 20 MM patients. The APC+/FITC- strategy was able to detect a range of 1.6-44% myeloma cells in the CD138-negative population of BM mononuclear cells isolated from MM patients. Moreover, we have assessed the clonality of this population using APC+/FITC- in the CD138-negative fractions of MM patients. We found that the clonality of the APC+/FITC- population was similar to the clonality of the original disease (CD138+ cells) in 70% of the cases. The other 30% of the cases showed very low involvement of myeloma population in the BM and showed Kappa/Lambda ratio within normal range. Analyzing the prevalence of circulating MM cells in the peripheral blood from 12 MM patients showed that all patients had a higher number of circulating MM cells as detected by APC+/FITC- strategy compared to CD138+, and the fold change ranged from 1.5 to 86 times. CONCLUSION: We found that CD138 cannot be used as a universal marker to detect MM cells. Moreover, we developed a novel strategy to detect MM cells independent of their CD138 expression or hypoxic status; and we used CD38+/CD3-/CD19-/CD14-/CD16-/CD123- population as an alternative set of biomarkers to detect MM cells. This strategy was able to detect a clonal MM cell population in the CD138-negative fraction of BM mononuclear cells isolated from MM patients, as well as in the peripheral blood. Currently, we are exploring the ability of this strategy to predict relapse in MM patients whose BM was defined a CD138-negative. More investigation to characterize this population and the role in tumor recurrence and drug resistance in MM is warranted. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: In vitro culturing of primary myeloma cells has been a major challenge because the lack of an in vitro technology capable of recreating the complicated bone marrow (BM) microenvironment which multiple myeloma (MM) cells depend on for its survival. While primary myeloma cells cannot grow ex vivo in SCID mice, they are able to growth in SCIDhu mice. However, animal models are expensive, time-consuming, and often have limited reproducibility. In addition, classic laboratory models cannot take into consideration the variability of disease in every patient, and the MM patient population is highly variable, both genetically and epigenetically, and the biological characteristics of patients are widely different, which demonstrates sensitivity of individual patients to different therapies. Typical two-dimensional (2D) models rely on a limited number of MM cell lines which cannot reflect the enormous heterogeneity and variations present in individual patients. The goal of this study is to create a patient derived three-dimensional tissue-engineered bone marrow (3DTEBM) culture system based on cross-linked MM-derived BM supernatant, including endogenous soluble growth factors and cytokines, and by incorporating mononuclear cells, including MM (CD138+ population) and accessory cells (CD138- population), from the same patient. METHODS: The 3DTEBMwas formed through calcium cross-linking of BM supernatants combined with the CD138+ and CD138- population from the same MM patient. We tested the growth of fresh primary CD138+ MM cells with CD138- cells in 2D vs patient-derived 3DTEBM cultures based on the original BM ratio (CD138-/CD138+) of the patient at 3 days by flow cytometry. We further selected a patient with not very aggressive tumor and evaluated the effect of CD138- increasing densities on CD138+ MM growth in 3DTEBM cultures at day 3 by flow cytometry. In addition, the effect of patient-derived 3DTEBM and 2D cultures with and without CD138- cells on CD markers expression of the CD138+ population was tested at day 3 by flow cytometry. Finally, the growth of frozen primary CD138+ MM cells with and without CD138- cells in 2D vs patient-derived 3DTEBM cultures was analyzed for 14 days by flow cytometry. RESULTS: We found that patient-derived 3DTEBM cultures support primary MM cell growth. CD138+ MM cells from patients with less aggressive tumors (high BM ratio CD138-/CD138+) showed better growth in 3DTEBM than in 2D systems at day 3. However, more aggressive tumors (low BM ratio CD138-/CD138+), in which MM cells are less dependent of accessory cells, the 3DTEBM cultures showed similar CD138+ MM growth as in 2D cultures. After that, we detected in the patient-derived 3DTEBM cultures a direct correlation between positive and negative fractions, with increased CD138- densities the CD138+ MM growth increased. Next, we analyzed the effect of 3D cultures in CD markers expression and we showed that CD138+ MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), reduction of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D cultures. Finally, patient-derived 3DTEBM supported the growth of frozen primary CD138+ MM cells better than 2D cultures, and the addition of CD138- cells enhanced even more CD138+ MM growth after 14 days in culture. CONCLUSIONS: Patient-derived 3DTEBM cultures support primary fresh and frozen CD138+ MM growth better than classic systems, and induced de-differentiation of MM cells while increased a stem-cell like phenotype. These results highlight the importance of the BM microenvironment (BM supernatant, including endogenous soluble growth factors and cytokines, and the CD138- population from the same patient) to facilitate primary MM cell growth in vitro. Therefore, patient-derived 3DTEBM cultures allowed long-term culture of primary fresh and frozen myeloma cells ex vivo, and these findings indicate that patient-derived 3DTEBM can be utilized for studying multiple myeloma biology and for testing patient-targeted therapy. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. The discrepancy between in vitro efficacy and clinical outcomes can be attributed to several limitations of the classic tissue culture drug screening models including: (1) most of the in vitro models use MM cell line cultures and neglect the vital role of the bone marrow (BM) microenvironment in MM progression, which promotes drug resistance. (2) The BM niche is a three-dimensional (3D) structure with a gradient of both oxygen and drug concentration as a function of distance from blood vessels. The classic two-dimensional (2D) in vitro tissue culture system cannot mimic oxygen and drug gradients in culture wells, making all cells highly oxygenated. Therefore, 2D cultures cannot accurately predict drug sensitivity in different parts of the BM niche due to lack of accurate effects throughout various tissue depths. The goal of this study is to develop an in vitro model that will allow for better evaluation of interactions of MM cells and their microenvironment in the BM niche in a 3D system and how these interactions may affect MM progression and drug resistance. METHODS: The 3D tissue-engineered bone marrow (3DTEBM)was formed through calcium cross-linking of BM supernatants from MM patients. MM cell lines (MM1s, H929 and RPMI) and BM microenvironment (BMM) components, including MM-derived BM stromal cells, HUVECs, and ECM component (fibronectin), were incorporated in 3DTEBM or 2D cultures. We tested by flow cytometry the growth of MM cell lines with and without BMM in 2D vs 3DTEBM cultures at 3 and 7 days. The effect of 3DTEBM and 2D cultures with and without BMM on cytokine expression was determined at day 3 by human cytokine antibody arrays. In addition, the effect of 3DTEBM and 2D cultures with and without BMM on CD markers expression was tested at day 3 by flow cytometry. Finally, drug uptake and hypoxia levels by MM cells in 3DTEBM of different depth of tissue and 2D cultures was determined using flow cytometry and immunohistochemistry, respectively. Then, drug resistance of MM cell lines with and without BMM in 2D vs 3DTEBM cultures after 24h drug treatment were analyzed by flow cytometry. RESULTS: We found that MM cells doubled within 3 days in 2D and 3DTEBM cultures with and without BMM, compared to day 0. In contrast, while in the 3DTEBM induced about 3-5-fold increase in 7 and 14 days, the 2D cultures showed lower growth. In addition, 3DTEBM expressed more cytokines than 2D media in absence of cells, and incorporation of MM cells and BMM induced higher cytokine expression in 3DTEBM than in 2D cultures of SDF-1, IL1-α, TNF-α, TNF-β, MIP-1-δ, PARC, angiopoietin, eotaxin 3 and osteoprotegin. MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), mildly reduced or no changes of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D without BMM. Finally, we found that MM cells in 3DTEBM exhibited 2-fold less drug uptake than MM cells grown in 2D cultures, and drug uptake of MM cells grown in the 3DTEBM was inversely correlated with the depth of the tissue; with increasing tissue depth the drug uptake by MM cells decreased. Accordingly, MM cells in the 3DTEBM exhibited higher hypoxia levels (MFI of PIM) compared to MM cells grown in 2D cultures, and anti-HIF-1α staining revealed that the cells at the lower area were more hypoxic that the cells at the upper area of the 3DTEBM. Therefore, we found that 3DTEBM cultures induced drug resistance in MM cells; for the same drug concentration about 50 and 35% of the MM cells were killed in 2D, and about 15 and 5% of the cells were killed in 3DTEBM without and with BMM, respectively. CONCLUSIONS: Our results suggest that 3DTEBM cultures promoted tumor growth, enhanced cytokine expression, and induced de-differentiation of MM cells, and that a stem-cell-like phenotype might be developing. The 3DTEBM recreated hypoxia and drug gradients by reproducing tissue-specific structural features, so the 3DTEBM cultures induced drug resistance in MM cells more accurately than 2D. Therefore, the 3DTEBM is a promising model for the study of multiple myeloma biology, for in vitro examination of anti-myeloma drugs, tumor microenvironment and interactions between myeloma cells and the BMM. Disclosures No relevant conflicts of interest to declare.

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and hyperproduction of immunoglobulin IgG. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance and micro-residual disease attributed to cancer stem cells. The hypoxic nature of the BM plays a critical role in MM cells acquiring a stem cell-like phenotype, and together with cellular and acellular components of the BM microenvironment contributes to drug resistance leading to minimal-residual disease. In this study, we tested inhibition of proline-rich tyrosine kinase 2 (PYK2), using VS-4718 and VS-6063, on reversing the hypoxia-inducible stem cell-like phenotype of MM cells and sensitizing them to therapy both in vitro and in vivo. Methods: VS-4718 and VS-6063 FAK/PYK2 inhibitors were obtained from Verastem, Inc. MM cell lines (MM1s, H929 and RPMI8226) were treated with bortezomib or carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the FAK/PYK2 inhibitors VS-4718 or VS-6063. MM cells were analyzed for cell proliferation by MTT assay; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with cell proliferation and apoptosis was analyzed by western blotting. Furthermore, we have tested the effect of the PYK2 inhibitors on sensitization of bortezomib-resistant MM cells in two models in vivo. In the first model, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumors were allowed to grow for four weeks; animals were then treated with a low-dose of bortezomib (0.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were divided into 6 groups treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) VS-6063 (50mg/kg) alone (4) bortezomib alone, (5) combination of VS-4718 and bortezomib, and (6) combination of VS-6063 and bortezomib, where VS-4718 and VS-6063 were administered by oral gavage twice a day every day. Mice survival was then followed for four more weeks. In the second model, H929 cells were injected subcutaneously into SCID mice. When tumors reached a mean of ~128 mm3 mice were randomized into five groups and treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) bortezomib (1mg/kg) alone, (4) combination of VS-4718 and bortezomib (concurrently), and combination of VS-4718 and bortezomib (sequentially to simulate minimum residual disease). VS-4718 was administered by oral gavage twice a day and tumor growth was measured twice weekly using calipers. Results: We found that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells in both hypoxia and normoxia as single agents. Moreover, we found that hypoxia induced drug resistance to bortezomib and carfilzomib in MM cells. The combination of the two PYK2 inhibitors with either of the proteasome inhibitors reversed the hypoxia-induced resistance in MM cells and induced a synergistic effect with proteasome inhibitors on inhibition of cell proliferation, apoptosis, and cell signaling related to proliferation and apoptosis. In vivo study revealed that mice that developed MM disease with resistance to bortezomib were resensitized by treatment with the PYK2 inhibitors and showed significantly extended survival compared to mice treated with bortezomib alone. Furthermore, in mice treated with bortezomib only, tumors progressively re-grew after bortezomib treatment ended. While, in the bortezomib and VS-4718 combination treated mice and mice treated with VS-4718 in minimal residual setting (MRD) tumor grow-back (relapse) was prevented after cessation of bortezomib treatment. Conclusions: We report that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells as single agents. Hypoxia induced resistance to proteasome inhibitors and the PYK2 inhibitors resensitized MM cell to therapy in vitro and in vivo. Moreover, FAK/PYK2 inhibitor VS-4718 was able to prevent relapse in in vivo MM model simulating minimal residual disease. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by FAK/PYK2 inhibitors and their use to reduce relapse post front line treatment in an MRD setting. Disclosures Trombino: Verastem: Employment. Padval:Verastem: Employment. Azab:Verastem: Research Funding.

Introduction Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and Results We analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. Conclusion This is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures: Vali: Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.

Introduction Waldenström Macroglobulinemia (WM) is a rare, low-grade B-cell lymphoma characterized by lymphoplasmacytic cells spread widely in the bone marrow (BM) and overproduction of monoclonal immunoglobulins M (IgM). Previous studies showed that tumor hypoxia develops in the BM of other hematologic malignancies and promotes dissemination. In this study, we tested the effect of hypoxia on cell proliferation, cell cycle and apoptosis; on egress and homing of WM cells from and into the BM; and on recovery and tumor colonization in the new BM niche. Methods We characterized the effect of tumor progression on generation of hypoxic conditions in the BM in vivo, by injecting BCWM1-mCherry cells to SCID mice, letting them grow for two weeks, analyzing the hypoxic state of the WM cells in the BM using pimonidazole, and testing the number of circulating cells. Moreover, we tested the effect of hypoxia on the homing of WM cells to the BM by injecting normoxic and hypoxic cells to mice and monitoring the number of the circulating WM cells in the blood at different time points by flow cytometry. Cancer cell colonization was assessed 1 and 3 days post IV injection of normoxic and hypoxic cells to mice; mononuclear cells were isolated from the BM, fixed, permeabilized and stained with antibodies for p-Rb and cyclin-E. The percentile of WM cells in the BM and the expression of cell cycle proteins were analyzed by flow cytometry. BCWM1 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) in vitro for 24hrs, and n some cases reoxygenated for 24hrs. The expression of E-cadherin, VLA-4 and CXCR4 was analyzed by western blot or flow cytometry. We tested the effect of hypoxia on adhesion of WM cells to BM stroma and fibronectin. We further tested the effect of hypoxia on chemotactic properties of WM cells towards SDF-1 using a transwell migration chamber. In addition, we tested the effect of hypoxia on WM cell survival (by MTT assay), apoptosis and cell cycle (by using AnnexinV-PI and PI, respectively), and signaling pathways associated with survival, apoptosis and cell cycle (by western blotting). Results Tumor progression was shown to increase hypoxic conditions in the BM in vivo. We found a direct correlation between the percent of WM cells in the BM to the level of hypoxia. The level of hypoxia was in a direct correlation with the number of circulating WM cells in vivo. Then we mimicked the hypoxic conditions in vitro and found that cell progression (MTT) and cell cycle (PI staining) were decreased, but apoptosis of WM cells was not affected (AnnexinV-PI staining). These results were confirmed by decreased activation of the PI3K signaling pathway (p-PI3K, p-AKT, p-GSK) and decreased expression of cell cycle proteins (p-Rb, CDK2, CDK4, cyclin-D1 and p-cyclin-E); however, no change was observed in apoptosis-related proteins (PARP, cleaved caspase-3, -8 and -9). Moreover, hypoxia decreased the expression of E-cadherin which contributed to reduction of adhesion of WM cells to the BM stromal cells. At the same time, hypoxic WM cells exhibited increased CXCR4 surface expression and augmented migratory abilities in the presence of SDF-1. Neither the expression of integrins (VLA-4) nor the adhesion of WM cells to fibronectin was affected by hypoxia. This data indicates the conservation of the homing machinery of the WM to the BM despite the hypoxic conditions accompanied by increased chemotactic ability. When hypoxic and normoxic cells were injected to naïve mice, hypoxic cells showed enhanced homing to the BM and tumor colonization. Similarly, hypoxic cells which were reoxygenated in vitro showed more proliferation, cell cycle and activation of proliferative signaling pathways compared to normoxic cells. Conclusions We report that WM tumor growth in the BM increases hypoxia, and that hypoxia induces cell cycle arrest, and less proliferation of cells with no apoptosis. At the same time, hypoxia induces egress of WM cells from the BM through reduction of E-cadherin expression and decreased adhesion. When in the circulation, previously hypoxic cells home more efficiently to the BM through increased expression of CXCR4 and chemotaxis, and through maintaining expression of integrins and adhesion to fibronectin. When in the new oxygenated BM niche, hypoxic WM cells recover and colonize the new niche better than normoxic cells, and reoxygenated hypoxic cells have faster cell cycle and proliferation rate. Disclosures: No relevant conflicts of interest to declare.

Introduction Multiple myeloma (MM) is the second most prevalent hematological malignancy, and it remains incurable with a median survival of 3 to 5 years. Despite the introduction of several novel drugs, only 60% of the patients respond to therapy, and more than 75% of patients relapse after 5 years. The interaction of MM cells with extracellular matrix (ECM) and stromal cells in the bone marrow (BM) milieu was shown to play a crucial role in MM progression and drug resistance. Therefore, an appropriate model that demonstrates the complex interactions between MM cells and their environment and predicts the response to treatment of MM patients is warranted. The goal of this study is to produce an in vitro model of BM microenvironment in MM, which will mimic the BM in the patient and have closest prediction of cell-behavior in vivo. Methods Scaffolds for the 3D tissue-engineered BM (3DBM) were prepared by crosslinking BM supernatants from MM patients with calcium chloride in the presence of the antifibrinolytic tranexamic acid. The concentration of the different components for scaffold formation and physico-chemical properties of the scaffold were optimized. Then, we incorporated cellular components (MM cell lines and stromal cell lines) and ECM components (fibronectin) to 3DBM scaffold. We tested the effect of different MM cell densities, fibronectin concentration, and presence of different densities of stromal cells in the scaffold on the proliferation of MM cells, which was analyzed by flow cytometry. After optimization of the cellular and ECM components, we tested the sensitivity of MM cells to chemotherapeutic agents used in MM, including melphalan, bortezomib and carfilzomib, in 3DBM scaffolds with and without the presence of stromal cells. We assessed the sensitivity of MM cells to the drugs in the 3DBM scaffolds compared with the sensitivity in two-dimensional (2D) classic in vitro tissue culture system, with or without the presence of stromal cells. Results We optimized the conditions for the production of a 3DBM from BM supernatants from MM patients. We found that calcium concentration was a critical factor for the formation of the 3DBM scaffold; and 1mg/ml calcium chloride was the optimal concentration for fastest and most stable scaffold formation. Moreover, we found that the presence of an antifibrinolytic agent was essential for the stabilization of the scaffold, and 4mg/ml of tranexamic acid was sufficient to stabilize the scaffold. We optimized the conditions for seeding of the cellular components and found that MM cells proliferated in the 3DBM scaffolds better than in regular 2D tissue culture plates, in which the expansion rate over 3 days was about 5-folds in the 3DBM, compared to about 2.4-folds in 2D classic in vitro tissue culture system. In addition, we optimized the co-culture condition of MM cells with stromal cells and found that the ratio of MM/stroma of 3/1 generated the highest proliferation of MM cells in the 3DBM scaffolds. Similarly, the addition of ECM components (fibronectin) increased the proliferation of MM in the 3DBM by about 30%. In addition, we tested the effect of the 3DBM scaffold on drug resistance of MM cells. In a 2D classic in vitro tissue culture system approximately about 40-50% of the MM cells were killed using the different chemotherapies, the culture of MM cells in 3DBM decreased the sensitivity of MM cells to treatment, and addition of stromal cells to the co-culture decreased the sensitivity of MM cells to treatment in both 3D and 2D. We found that the co-culture of MM cells with stromal cells in the 3DBM treatment and using the same concentrations of the different chemotherapies killed only 5-15% of the MM cells. Conclusions We produced and characterized a 3D scaffold made from supernatant of BM aspirates from MM patients. This 3DBM is all made from patient material (autologous), no synthetic scaffolds were used, and it could represent the closest in vitro model to the BM in MM. The 3DBM induced drug resistance in MM cells, especially when it contained stromal cells. The autologous 3DBM seems to be closer to the real BM microenvironment, therefore, a better drug screening method than 2D classic in vitro tissue culture systems. Disclosures: No relevant conflicts of interest to declare.

Introduction Multiple Myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and overproduction of immunoglobulin. The interaction of MM cells with the BM microenvironment was shown to play an important role in the drug resistance in MM. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we tested the effect of hypoxia on the proliferation, cells cycle, apoptosis and induction of drug resistance in MM cells. Methods MM cell lines were exposed to normoxia (21% O2) or hypoxia (1% O2) and the effect of hypoxia on cell survival (by MTT assay), apoptosis (by annexin-PI staining and analysis with flow cytometry) and cell cycle (by cell fixation and permeabilization, RNA degradation and DNA staining with PI, and analysis with flow cytometry) was tested. Moreover, the effect of hypoxia on the expression of PI3K pathway-associated proteins (p-PI3K, p-AKT and p-mTOR), cell cycle proteins (cyclin-D, cyclin-E, p-Rb and p27) and apoptosis proteins (cleaved PARP, caspase-3, Bcl-2, Bcl-Xl and Mcl-1) were studied. We tested the effect of hypoxia on the sensitivity of MM cells to different therapeutic agents using MTT assay after the MM cells were treated with increasing concentrations of bortezomib, carfilzomib and melphalan. Finally, we examined the effect of a HIF inhibitor on the sensitivity of MM cells to therapy. Results Hypoxia decreased the proliferation of MM cells and reduced phosphorylation of p-PI3K, p-AKT and p-mTOR. Similarly, MM cells exhibited G1 cell cycle arrest, decreased expression of cell cycle-associated proteins including cyclin-D3, cyclin-E, p-Rb, and increase in cell cycle inhibitory protein p27. However, hypoxia did not alter the apoptosis of MM cells, where neither apoptosis was detected in MM cells due to hypoxia using annexin-PI staining, nor pro-apoptosis proteins were activated due to hypoxia as shown by the unchanged levels of cleaved PARP, caspase-3, Bcl-2, Bcl-Xl, and Mcl-1 proteins. Moreover, we found that hypoxic cells displayed less sensitivity to bortezomib and carfilzomib, but it did not induce any change in melphalan activity. Treatment with carfilzomib or bortezomib (5nM for 48hrs) showed decreased survival of about 50% of normoxic cells, while no effect of the drugs was observed on survival in hypoxia. The same trends where observed in higher and lower concentrations of the drugs. Moreover, we found that treatment with a HIF inhibitor could partially rescue the sensitivity of MM cells to bortezomib and carfilzomib. Conclusions We report that hypoxia decreases the proliferation and cell cycle of MM cells without signs of apoptosis, and that hypoxia induces drug resistance to bortezomib and carfilzomib, an effect which was partially reversed by a HIF inhibitor. This data suggests the hypoxia signaling as a therapeutic target for sensitization of MM cells to therapy, and suggests the use of HIF inhibitors in combination with other drugs as a novel therapeutic strategy for treatment of MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.