Search

Chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) are hematological malignancies that remain incurable despite novel treatments. In order to improve current treatments and clinical efficacy, there remains a need for more complex

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM., The tumour microenvironment (TME) has a major role in chemoresistance in multiple myeloma. The authors show that a nanoparticle targeted to TME and loaded with bortezomib (BTZ) and Y27632 is more effective than free drugs, non-targeted and single-agent controls and reduces BTZ-related side effects.

Lack of efficacy and a low overall success rate of phase I-II clinical trials are the most common failures when it comes to advancing cancer treatment. Current drug sensitivity screenings present several challenges including differences in cell growth rates, the inconsistent use of drug metrics, and the lack of translatability. Here, we present a patient-derived 3D culture model to overcome these limitations in breast cancer (BCa). The human plasma-derived 3D culture model (HuP3D) utilizes patient plasma as the matrix, where BCa cell lines and primary BCa biopsies were grown and screened for drug treatments. Several drug metrics were evaluated from relative cell count and growth rate curves. Correlations between HuP3D metrics, established preclinical models, and clinical effective concentrations in patients were determined. HuP3D efficiently supported the growth and expansion of BCa cell lines and primary breast cancer tumors as both organoids and single cells. Significant and strong correlations between clinical effective concentrations in patients were found for eight out of ten metrics for HuP3D, while a very poor positive correlation and a moderate correlation was found for 2D models and other 3D models, respectively. HuP3D is a feasible and efficacious platform for supporting the growth and expansion of BCa, allowing high-throughput drug screening and predicting clinically effective therapies better than current preclinical models., Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health 5P20GM103548, 2018 LUSH Prize (Young Investigator Award)

Oxygen deprivation within tumors is one of the most prevalent causes of resilient cancer cell survival and increased immune evasion in breast cancer (BCa). Current in vitro models do not adequately mimic physiological oxygen levels relevant to breast tissue and its tumor-immune interactions. In this study, we propose an approach to engineer a three-dimensional (3D) model (named 3D engineered oxygen, 3D-O) that supports the growth of BCa cells and generates physio- and pathophysiological oxygen levels to understand the role of oxygen availability in tumor-immune interactions. BCa cells (MDA-MB-231 and MCF-7) were embedded into plasma-derived 3D-O scaffolds that reflected physio- and pathophysiological oxygen levels relevant to the healthy and cancerous breast tissue. BCa cells grown within 3D-O scaffolds were analyzed by flow cytometry, confocal imaging, immunohistochemistry/immunofluorescence for cell proliferation, extracellular matrix protein expression, and alterations in immune evasive outcomes. Exosome secretion from 3D-O scaffolds were evaluated using the NanoSight particle analyzer. Peripheral blood mononuclear cells were incorporated on the top of 3D-O scaffolds and the difference in tumor-infiltrating capabilities as a result of different oxygen content were assessed by flow cytometry and confocal imaging. Lastly, hypoxia and Programmed death-ligand 1 (PD-L1) inhibition were validated as targets to sensitize BCa cells in order to overcome immune evasion. Low oxygen-induced adaptations within 3D-O scaffolds validated known tumor hypoxia characteristics such as reduced BCa cell proliferation, increased extracellular matrix protein expression, increased extracellular vesicle secretion and enhanced immune surface marker expression on BCa cells. We further demonstrated that low oxygen in 3D-O scaffolds significantly influence immune infiltration. CD8+ T cell infiltration was impaired under pathophysiological oxygen levels and we were also able to establish that hypoxia and PD-L1 inhibition re-sensitized BCa cells to cytotoxic CD8+ T cells. Bioengineering the oxygen-deprived BCa tumor microenvironment in our engineered 3D-O physiological and tumorous scaffolds supported known intra-tumoral hypoxia characteristics allowing the study of the role of oxygen availability in tumor-immune interactions. The 3D-O model could serve as a promising platform for the evaluation of immunological events and as a drug-screening platform tool to overcome hypoxia-driven immune evasion.

Central nervous system atypical teratoid/rhabdoid tumors (ATRTs) are rare and aggressive tumors with a very poor prognosis. Current treatments for ATRT include resection of the tumor, followed by systemic chemotherapy and radiation therapy, which have toxic side effects for young children. Gene expression analyses of human ATRTs and normal brain samples indicate that ATRTs have aberrant expression of epigenetic markers including class I histone deacetylases (HDAC&rsquo, s) and lysine demethylase (LSD1). Here, we investigate the effect of a small molecule epigenetic modulator known as Domatinostat (4SC-202), which inhibits both class I HDAC&rsquo, s and Lysine Demethylase (LSD1), on ATRT cell survival and single cell heterogeneity. Our findings suggest that 4SC-202 is both cytotoxic and cytostatic to ATRT in 2D and 3D scaffold cell culture models and may target cancer stem cells. Single-cell RNA sequencing data from ATRT-06 spheroids treated with 4SC-202 have a reduced population of cells overexpressing stem cell-related genes, including SOX2. Flow cytometry and immunofluorescence on 3D ATRT-06 scaffold models support these results suggesting that 4SC-202 reduces expression of cancer stem cell markers SOX2, CD133, and FOXM1. Drug-induced changes to the systems biology landscape are also explored by multi-omics enrichment analyses. In summary, our data indicate that 4SC-202 has both cytotoxic and cytostatic effects on ATRT, targets specific cell sub-populations, including those with cancer stem-like features, and is an important potential cancer therapeutic to be investigated in vivo.

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.

Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

Multiple myeloma (MM) is a hematological malignancy that remains incurable, with relapse rates >90%. The main limiting factor for the effective use of chemotherapies in MM is the serious side effects caused by these drugs. The emphasis in cancer treatment has shifted from cytotoxic, non-specific chemotherapies to molecularly targeted and rationally designed therapies showing greater efficacy and fewer side effects. Traditional chemotherapy has shown several disadvantages such as lack of targeting capabilities, systemic toxicity, and side effects; low therapeutic index, as well as most anticancer drugs, has poor water solubility. Nanoparticle delivery systems (NPs) are capable of targeting large doses of chemotherapies into the target area while sparing healthy tissues, overcoming the limitations of traditional chemotherapy. Here, we review the current state of the art in nanoparticle-based strategies designed to treat MM. Many nanoparticle delivery systems have been studied for myeloma using non-targeted NPs (liposomes, polymeric NPs, and inorganic NPs), triggered NPs, as well as targeted NPs (VLA-4, ABC drug transporters, bone microenvironment targeting). The results in preclinical and clinical studies are promising; however, there remains much to be learned in the emerging field of nanomedicine in myeloma.

Despite significant improvements in research and development in the cancer field, about 95% of oncology drugs in clinical trials fail to receive FDA approval. Compelling evidence suggests that three-dimensional (3D) cell culture techniques provide useful advantages for modeling drug sensitivity; however, there are still challenges, including vast differences in cell growth rates, inconsistent use of drug metrics across different cellular platforms, costly and relatively time-consuming approaches, and findings that may not translate to humans due to the incorporation of non-native environments. A biochemically characterized human blood plasma matrix is used as the framework upon which the human-derived plasma 3D culture (huP3D) exists. Breast cancer (BCa) cell lines representing different molecular subtypes (MCF7, ZR-75-1, MDA-MB-453, SK-BR-3, and MDA-MB-231) were screened for commonly used chemotherapies (capecitabine, carboplatin, paclitaxel, docetaxel, epirubicin, methotrexate, and cyclophosphamide) in 2D and huP3D models after 7 days of treatment by flow cytometry. Relevant drug metrics for half maximal concentrations of inhibition (IC50), response (EC50), proliferation inhibition (GI50), and growth rate inhibition (GR50) were calculated. Literature searches of phase I/II studies for the clinical effective drug concentration (steady state concentration, Css) of the same chemotherapeutic drugs on patients, and preclinical IC50 on 2D or other 3D models, were performed to determine the ability of 2D, other 3D, and huP3D cultures to predict clinical efficacy. BCa cell lines in huP3D were able to synthetize relevant tumor microenvironment components including extracellular matrix proteins (collagens, laminin, and fibronectin), extracellular vesicles, and cytokines. Moderate (paclitaxel, capecitabine, methotrexate, cyclophosphamide) to strong (epirubicin, docetaxel, carboplatin) effects on BCa cell survival were found in 2D cultures, in addition to a heterogeneous response to the chemotherapeutic drugs in the BCa cell lines tested, with ZR-75-1 being the most resistant to the different drugs. 2D effective drug concentrations did not correlate with the literature search Css on patients (R2=0.05). On the contrary, BCa cells cultured in huP3D showed increased drug resistance to all the chemotherapeutic drugs and a better correlation with the clinical data than 2D and other 3D models. In particular, the GR50 drug metric showed the strongest correlation with clinical Css values (R2=0.87). Our results showed the feasibility and efficacy of the huP3D drug response metrics to predict clinically effective therapies better than current preclinical models. Our model presents a reproducible, human-derived, high-throughput in vitro 3D tissue culture system for modeling drug sensitivity and clinical efficacy. A critical value of the model would be to focus on the development of more personalized models using cells and plasma components from the same patient. Citation Format: Kristin Calar, Simona Plesselova, Somshuvra Bhattacharya, Pilar de la Puente. Feasibility and efficacy of a human-derived plasma 3D culture (huP3D) model to predict clinically effective drug treatment concentrations [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A22.

Introduction: Oxygen deprivation within tumors has been associated with enhanced cancer cell survival, altered extracellular matrix (ECM) production, and increased immune evasion in breast cancer (BCa). These outcomes collectively contribute to chemotherapeutic challenges. Current models do not adequately mimic physiologic oxygen levels relevant to breast tissue and tumor-immune interactions. Taking this into consideration, we developed a plasma-derived three-dimensional (3D) model that supports growth of BCa cells, generates physio- and pathophysiologic oxygen levels, and supports prevailing hypoxia-driven tumor outcomes such as immune evasion. Methods: BCa cells (MCF7 and MDA-MB-231) were embedded into 3D scaffolds formed through the cross-linking of patient-derived plasma. The oxygen content within these 3D matrices was engineered and further characterized to recapitulate physiologic blood and breast tissue (3D physiologic, gradient 14-10 kPa) or pathophysiologic (3D tumorous, gradient 9.6-2.4 kPa) levels by the manipulation of height, culture time, oxygen incubation environment, and cellular concentration. BCa cells grown within 3D physiologic and tumorous scaffolds were analyzed by flow cytometry, confocal imaging, or immunohistochemistry for the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), proliferation capabilities, and ECM production. Peripheral blood mononuclear cells were incorporated on the top of 3D physiologic and tumorous scaffolds, and the differences in the tumor-infiltrating capabilities of T cells into 3D physiologic and tumorous scaffolds were assessed by flow cytometry. Lastly, hypoxic inhibition was validated as a target to sensitize BCa cells in order to overcome immune surveillance. Results: BCa cells grown in oxygen-deprived 3D tumorous environments showed a significant increase in the expression of HIF-1 alpha and a reduced cell proliferation compared to the BCa cells in 3D physiologic scaffolds. Increased secretion of collagen I, collagen III, laminin, and fibronectin was demonstrated by BCa cells in 3D tumorous compared to 3D physiologic scaffolds. T-cell infiltration (especially CD8 T cells) was significantly impaired within the oxygen-deprived breast cancer tumor microenvironment (TME), and hypoxia inhibition was further confirmed to resensitize BCa cells to cytotoxic CD8 T cells. Conclusions: Modeling of the oxygen-deprived breast cancer TME in our engineered 3D physiologic and tumorous scaffolds supported known intratumoral hypoxia characteristics such as reduced BCa cell proliferation, increased ECM expression, and hindered immune infiltration. Furthermore, hypoxic inhibition was validated as a target to sensitize BCa cells to immune surveillance. These models could serve as a promising platform for the evaluation of immunologic events as well as a drug-screening platform tool to overcome hypoxia-driven immune evasion. Citation Format: Somshuvra Bhattacharya, Kristin Calar, Claire Evans, Pilar de la Puente. Modeling the oxygen-deprived breast cancer tumor microenvironment within a three-dimensional bioengineered platform that exhibits hypoxia-driven immune evasion [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B29.

Introduction: Endothelial progenitor cells (EPCs) are a subtype of stem cells capable of differentiating into mature endothelial cells. Tumor-derived paracrine signals activate the bone marrow-residing EPCs to home to the tumor to promote angiogenesis. The aim of this study was to utilize the EPCs and their specific pathophysiologic homing to tumors as a biologically targeted drug-delivery system, as a novel “Trojan horse” cellular immunotherapy in cancer. Methods: To test the specificity of EPCs’ homing to cancer, chemotaxis of EPCs towards conditioned media derived from a series of cancer cell lines or towards nonconditioned media was performed in vitro using Boyden Chamber. In vivo biodistribution of EPCs was studied in diverse tumor models (localized and disseminated) of different cancers (myeloma, lymphoma, leukemia, breast, lung, pancreatic and glioma) after injecting EPCs intravenously, by analysis of the accumulation of EPCs in the tumors and other organs. To enhance the ability of EPCs to home to tumors, EPCs were “primed” in vitro by exposing them to conditioned media from hypoxic (1% O2) tumor cells, followed by testing the homing of “primed” EPCs to tumors, both in vitro and in vivo. Next, EPCs were loaded with LS-542 (NIR imaging dye), and with Titanocene (Tc) (a nontoxic compound used in phototherapy that can be activated by positrons to become toxic) and tested for the effect of the loading on viability and functionality of EPCs, using MTT and migration assays, respectively. Also, we tested the effect of Tc-loaded EPCs on killing of cancer cells when activated by 18FDG/PET. Results: We found that EPCs specifically homed to tumors in vitro and in vivo, in tumor size-dependent and time-dependent manner, and that EPCs selectively accumulated in tumor tissues, with significantly negligible amounts in other organs. Moreover, EPCs homed to primary tumors and followed the metastatic pattern of the tumor. Hypoxia “priming” of EPCs significantly improved their homing to cancer in vitro and in vivo. Loading EPCs with LS-542 or with Tc did not affect their viability or functionality. Additionally, Tc-loaded EPCs, when activated by 18FDG, induced a dose-dependent killing of cancer cells. Conclusions: EPCs have a highly specific and efficient biologic machinery to target tumors in as a pan-cancer phenomenon, especially when “primed” with hypoxic tumor media. Loading EPCs with LS-542 or Tc did not change their viability and functionality; thus, both are promising molecules for imaging and therapy of cancer. These results provide a proof of concept that EPCs can be used as a novel cellular immunotherapy for specific and efficacious delivery of theranostic agents in cancer. Also, unlike engineered/targeted immunotherapies such as cellular (CAR-T cells), bispecific T cell engagers, or monoclonal antibodies that target specific molecules on a specific tumor type, EPCs can be used as a pan-cancer “Trojan horse” drug-delivery platform due to the universality of (angiogenesis) in all cancer types. Citation Format: Barbara Muz, Pilar de la Puente, Shruti Shah, Cinzia Federico, Feda Azab, Justin King, Mark Fiala, Rui Tang, Partha Karmakar, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Endothelial progenitor cells as drug-delivery Trojan horses for treatment and imaging of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A27.

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Chimeric antigen receptor (CAR)-T cells and bispecific T-cell engagers targeting BCMA, CS1, and CD38 are promising approaches for MM. However, using these targets individually limits the efficacy of these immunotherapy options due to the variable expression levels of these markers, leading to inefficient eradication of MM. Studies have attempted to develop CAR-T cells and T-cell engagers with multispecific targeting; however, these are still technically very challenging and are highly expensive. In this study, we developed nanoparticles with monovalent or multivalent specificity against MM targets and engage T cells, by chemical conjugation of monoclonal antibodies against each molecule to the surface of the liposome. Bi-specific nanoparticles with anti CD38/CD3, BCMA/CD3 or CS1/CD3 were termed nanoBiTEs, while the multispecific particles with anti BCMA/CS1/CD38/CD3 were termed nanoMuTEs. Methods: Liposomes were prepared using the thin-film hydration method followed by extrusion. nanoBiTEs and nanoMuTEs were developed by chemical conjugation of monoclonal antibodies against BCMA, CS1, and/or CD38; together with anti CD3 onto the liposomes. We have tested the expression of BCMA, CS1, and CD38 antigens in MM cell lines and primary MM patient samples, and we analyzed the binding of the CD38/CD3, BCMA/CD3, and CS1/CD3 nanoBiTEs, as well as the BCMA/CS1/CD38/CD3 nanoMuTEs to these cells by flow cytometry and confocal microscopy. Binding specificity was assessed by blocking the target with corresponding free antibodies. CD25 expression, a T-cell activation marker, was assessed for CD8 and CD4 T cells when incubated with or without nanoBiTEs and nanoMuTEs. Moreover, we tested the efficacy of the nanoBiTEs and nanoMuTEs against MM cells in vitro using the 3D tissue engineered bone marrow (3DTEBM) myeloma model. Finally, we tested the efficacy of the nanoBiTEs and nanoMuTE against MM tumors in vivo in xenograft MM models. Results: The expression of BCMA, CS1 and CD38 was variable in MM cell lines and patient samples, and so was the binding of each nanoBiTEs, while the binding of the nanoMuTE was higher and more universal than each nanoBiTE. Similar patterns were observed in activation of CD8 T cells (no significant activation was observed in CD4 T cells) and in eradication of MM cell lines and primary patient samples in vitro. In vivo, the nanoBiTEs extended the survival and reduced the tumor burden in the MM bearing mice compared to untreated and T cell alone groups, and nanoMuTE experiments are still ongoing. Conclusions: The nanoBiTEs and nanoMuTEs were bound specifically to MM cells and initiated CD8 T-cell activation that resulted in MM eradication in vitro and in vivo, while the effect of the nanoMuTEs was more universal and efficacious than each nanoBiTE. These nanoparticles are an inexpensive method of engaging T cells for the treatment of MM and represent a new class of immunotherapy that can be expanded to other tumors. Citation Format: Kinan Alhallak, Jennifer Sun, Barbara Muz, Christopher Egbulefu, Pilar de la Puente, Cinzia Federico, Justin King, Danny Kohnen, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Nanoparticle multispecific T-cell engagers for the treatment of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B01.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

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. 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 Tris (Dibenzylideneacetone) dipalladium (Tris DBA). Tris DBA reduces proliferation, induces G1 arrest and apoptosis in MM cells. Tris DBA showed additive effect with proteasome inhibitors reducing proliferation, cell cycle signaling, and increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to proteasome inhibitors 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.

Radiation-induced lymphopenia (RIL) is associated with treatment of different tumors (lung, colon, pancreas, breast, sarcomas, and glioblastoma). It is a significant clinical problem affecting the survival of cancer patients. The biologic mechanisms leading to RIL are not clearly understood. In this study, we established a mouse model of RIL representing therapeutic clinical regimen for lung cancer. Flow cytometry was used to analyze circulating levels of T and B cells and bone marrow (BM) stem cells. We found that fractionated radiation to the thorax significantly reduced circulating T and B cells as well as BM stem cells. Ex-vivo irradiation of blood and autologous reinjection to mice also significantly induced lymphopenia. Furthermore, we found that mobilization of stem cells from the BM and autologous stem cell transplant rescued RIL in mice. Overall, our results suggest that RIL has not only direct effect on circulating lymphocytes, but also has indirect effect on circulating lymphocytes as well as stem cells in the nonirradiated BM. These results open a new window for investigating the direct and indirect biologic mechanisms leading to RIL, and provide a preclinical basis to test the effect of stem cell transplantation for treatment of RIL in cancer patients. Cancer Res; 75(17); 3442–5. ©2015 AACR.

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority 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 in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interactionin vivoled to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. We have showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through BTZ diffusion from the surface and endocytosed NPs, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells when BTZ was administered through anti-CD38 chitosan NPs. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. The anti-CD38 chitosan NPs showed low toxicity profile allowing enhancement of proteasome-inhibitory activity and specificity of BTZ by endocytosis-mediated uptake of CD38 representing a promising therapy in MM.

Overall survival of patients with newly diagnosed glioblastoma (GBM) remains dismal at 16 months with state-of-the-art treatment that includes surgical resection, radiation, and chemotherapy. GBM tumors are highly heterogeneous, and mechanisms for overcoming tumor resistance have not yet fully been elucidated. An injectable chitosan hydrogel capable of releasing chemotherapy (temozolomide [TMZ]) while retaining radioactive isotopes agents (iodine, [ 131 I]) was used as a vehicle for localized radiation and chemotherapy, within the surgical cavity. Release from hydrogels loaded with TMZ or 131 I was characterized in vitro and in vivo and their efficacy on tumor progression and survival on GBM tumors was also measured. The in vitro release of 131 I was negligible over 42 days, whereas the TMZ was completely released over the first 48 h. 131 I was completely retained in the tumor bed with negligible distribution in other tissues and that when delivered locally, the chemotherapy accumulated in the tumor at 10-fold higher concentrations than when delivered systemically. We found that the tumors were significantly decreased, and survival was improved in both treatment groups compared to the control group. Novel injectable chemo-radio-hydrogel implants may potentially improve the local control and overall outcome of aggressive, poor prognosis brain tumors.

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib.

Brachytherapy is described as the short distance treatment of cancer with a radioactive isotope placed on, in, or near the lesions or tumor to be treated. The main advantage of brachytherapy compared with external beam radiation (EBR) is the improved localized delivery of dose to the target volume of interest, thus normal tissue irradiation is reduced. The precise and targeted nature of brachytherapy provides a number of key benefits for the effective treatment of cancer such as efficacy, minimized risk of side effects, short treatment times, and cost-effectiveness. Brachytherapy devices have yielded promising results in preclinical and clinical studies. However, brachytherapy can only be used in localized and relatively small tumors. Although the introduction of new delivery devices allows the treatment of more complex tumor sites, with wider range of dose rate for improving treatment efficacy and reduction of side effects, a better understanding about the safety, efficacy, and accuracy of these systems is required, and further development of new techniques is warranted. Therefore, this review focuses on the delivery devices for brachytherapy and their application in prostate, breast, brain, and other tumor sites.

Tumor hypoxia is a well-described phenomenon during the progression of solid tumors affecting cell signaling pathways and cell metabolism; however, its role in hematologic malignancies has not been given the same attention in the literature. Therefore, this review focuses on the comparative differences between solid and hematologic malignancies with emphasis on the role of hypoxia during tumorigenesis and progression. In addition, contribution of the bone marrow and angiogenic environment are also discussed. Insight is provided into the role of hypoxia in metastatic spread, stemness, and drug resistance in hematologic conditions. Finally, emerging therapeutic strategies such as small-molecule prodrugs and hypoxia-inducible factor (HIF) targeting approaches are outlined to combat hypoxic cells and/or adaptive mechanisms in the treatment of hematologic malignancies. Mol Cancer Res; 12(10); 1347–54. ©2014 AACR.

The phosphatidylinositide 3-kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K-α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs.

Blood vessel formation plays an essential role in many physiologic and pathologic processes, including normal tissue growth and healing, as well as tumor progression. Endothelial progenitor cells (EPC) are a subtype of stem cells with high proliferative potential that are capable of differentiating into mature endothelial cells, thus contributing to neovascularization in tumors. In response to tumor-secreted cytokines, EPCs mobilize from the bone marrow to the peripheral blood, home to the tumor site, and differentiate to mature endothelial cells and secrete proangiogenic factors to facilitate vascularization of tumors. In this review, we summarize the expression of surface markers, cytokines, receptors, adhesion molecules, proteases, and cell signaling mechanisms involved in the different steps (mobilization, homing, and differentiation) of EPC trafficking from the bone marrow to the tumor site. Understanding the biologic mechanisms of EPC cell trafficking opens a window for new therapeutic targets in cancer. Clin Cancer Res; 19(13); 3360–8. ©2013 AACR.

Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells.B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo.B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively.B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on hypoxic tumor metabolism. Further studies are warranted to evaluate B-381 and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma.

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.

Purpose Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. Experimental design: We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. Results 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. Conclusions 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

Current treatment options as well as clinical efficacy are limited for chronic myelogenous leukemia (CML), Ph+ acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). In response to the pressing need for more efficacious treatment approaches and strategies to override drug resistance in advanced stage CML, Ph+ ALL, and AML, we investigated the effects of inhibition of ILK as a potentially novel and effective approach to treatment of these challenging malignancies. Using the small molecule ILK inhibitor, Cpd22, and ILK knockdown, we investigated the importance of ILK in the growth and viability of leukemia. Our results suggest that the ILK inhibition may be an effective treatment for CML, Ph+ ALL, and AML as a single therapy, with ILK expression levels positively correlating with the efficacy of ILK inhibition. The identification of ILK as a novel target for leukemia therapy warrants further investigation as a therapeutic approach that could be of potential clinical benefit in both acute and chronic myeloid leukemias.

Boron neutron capture therapy (BNCT) is a promising cancer therapy modality that utilizes the nuclear capture reaction of epithermal neutrons by boron-10 resulting in a localized nuclear fission reaction and subsequent cell death. Since cellular destruction is limited to approximately the diameter of a single cell, primarily only cells in the neutron field with significant boron accumulation will be damaged. However, the emergence of BNCT as a prominent therapy has in large part been hindered by a paucity of tumor selective boron containing agents. While L-boronophenylalanine and sodium borocaptate are the most commonly investigated clinical agents, new agents are desperately needed due to their suboptimal tumor selectivity. This review will highlight the various strategies to improve tumor boron delivery including: nucleoside and carbohydrate analogs, unnatural amino acids, porphyrins, antibody-dendrimer conjugates, cationic polymers, cell-membrane penetrating peptides, liposomes and nanoparticles.

Waldenström macroglobulinemia, a rare and indolent type of non–Hodgkin lymphoma, is characterized by widespread lymphoplasmacytic B cells in the bone marrow. Previous studies have shown that hypoxic conditions play a key role in the dissemination of other hematologic malignancies. In this study, the effect of hypoxia was tested on the progression and spread of Waldenström macroglobulinemia. Interestingly, tumor progression correlated with hypoxia levels in Waldenström macroglobulinemia cells and other cells in the bone marrow and correlated with the number of circulating tumor cells in vivo. Mechanistic studies demonstrated that hypoxia decreased cell progression and cell cycle, did not induce apoptosis, and reduced the adhesion between Waldenström macroglobulinemia cells and bone marrow stroma, through downregulation of E-cadherin expression, thus explaining increased egress of Waldenström macroglobulinemia cells to the circulation. Moreover, hypoxia increased the extravasation and homing of Waldenström macroglobulinemia cells to new bone marrow niches in vivo, by increased CXCR4/SDF-1–mediated chemotaxis and maintaining the VLA4-mediated adhesion. Re-oxygenation of hypoxic Waldenström macroglobulinemia cells enhanced the rate of proliferation and cell cycle progression and restored intercellular adhesion between Waldenström macroglobulinemia cells and bone marrow stroma. This study suggests that targeting hypoxic response is a novel strategy to prevent dissemination of Waldenström macroglobulinemia. Implications: This study provides a better understanding of the biology of dissemination of Waldenström macroglobulinemia and opens new windows for investigation of new therapeutic targets in Waldenström macroglobulinemia based on tumor hypoxia mechanisms. Mol Cancer Res; 13(2); 263–72. ©2014 AACR.

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. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted.

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.

Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering.

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: Current treatment options as well as clinical efficacy are limited for acute myeloid leukemia (AML), Ph+ acute lymphoblastic leukemia (Ph+ ALL), and chronic myelogenous leukemia (CML). In response to the pressing need for more efficacious treatment approaches and strategies to override drug resistance in advanced stage AML, Ph+ ALL, and CML, we investigated the effects of inhibition of integrin-linked kinase (ILK) as a potentially novel and effective approach to treatment of these challenging malignancies. ILK is an intracellular adaptor and kinase that links the integrins, cell-adhesion receptors, and growth factors to a range of signaling pathways. It has been shown that inhibition of ILK expression and activity is anti-tumorigenic, which makes ILK an attractive target for cancer therapeutics. Compound 22 (Cpd22, N-Methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide) is a cell-permeable, tri-substituted pyrazol compound that acts as a potent and targeted ILK inhibitor. In the present study, we investigated ILK as a putative novel target for treatment of AML, Ph+ ALL, and CML, and Cpd22 as a potential novel anti-leukemia agent. Methods: Compound 22 (Cpd22) was purchased from EMD Millipore. The expression of ILK in AML (K052, NOMO-1, THP, MOLM14), CML (K562, Ku812F, LAMA84S and LAMA84R), and Ph+ ALL (SUP-B15) cell lines, primary patient cells, and human Bone Marrow Mononuclear Cells (BM MNCs) was analyzed by flow cytometry. AML, Ph+ ALL, CML cell lines and peripheral blood mononuclear cells (PBMCs) were cultured with ILK inhibitor, Cpd22 (0-1000 nM) for 3 days. Toxicity of Cdp22 (0-500 nM) toward BM MNCs and primary patient cells (CML, ALL, and AML) was also assessed. Cell lines and/or primary patient 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, ILK knockdown of AML and CML cell lines was evaluated. Results: Normal BM MNCs showed ILK expression, but expression was lower than in cell lines and primary samples from AML, CML, and ALL. Cpd22 inhibited the proliferation of AML, ALL, and CML cell lines, while none of normal PBMC and normal BM MNCs controls were affected by the same concentration range after three days of treatment with Cpd22 as a single agent. ILK expression in primary AML patient cells correlated with efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. ILK expression in primary ALL patient cells also correlated with the efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. We validated the target specificity of Cpd22 in CML cells by immuno-blotting and investigating inhibitor effects on signaling molecules downstream of ILK. Cpd22 potently suppressed the phosphorylation levels of Ser-473-Akt (pAKT) and another ILK substrate, pGSK-3. We observed cell cycle arrest in Cpd22-treated cells, specifically accumulation of apoptotic cells in subG1 and Annexin/PI staining showed a 3-fold increase in the fraction of apoptotic cells staining positive for Annexin and PI in Cpd22-treated cells. Immuno-blotting confirmed cell cycle arrest by decreased pRb and increased cell cycle inhibitor p27; and drug induction of apoptosis through the caspase intrinsic pathway by demonstrating increased cleavage of caspase-3 and caspase-9. The importance of ILK for the growth of leukemia cells was demonstrated by knockdown of ILK in AML and CML cell lines, which led to decreases in cell proliferation. These results suggest a partial dependency of acute and chronic myeloid leukemia cells on ILK for growth, and are consistent with results obtained with the ILK inhibitor, Cpd22. Conclusions: In conclusion, our results suggest that the ILK inhibition may be an effective treatment for AML, Ph+ ALL, and CML as a single therapy, with ILK expression levels positively correlating with the efficacy of ILK inhibition. The identification of ILK as a novel target for leukemia therapy warrants further investigation as a therapeutic approach that could be of potential clinical benefit in both acute and chronic myeloid leukemias. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; 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.

Hypoxia is a non-physiological level of oxygen tension, a phenomenon common in a majority of malignant tumors. Tumor-hypoxia leads to advanced but dysfunctional vascularization and acquisition of epithelial-to-mesenchymal transition phenotype resulting in cell mobility and metastasis. Hypoxia alters cancer cell metabolism and contributes to therapy resistance by inducing cell quiescence. Hypoxia stimulates a complex cell signaling network in cancer cells, including the HIF, PI3K, MAPK, and NFĸB pathways, which interact with each other causing positive and negative feedback loops and enhancing or diminishing hypoxic effects. This review provides background knowledge on the role of tumor hypoxia and the role of the HIF cell signaling involved in tumor blood vessel formation, metastasis, and development of the resistance to therapy. Better understanding of the role of hypoxia in cancer progression will open new windows for the discovery of new therapeutics targeting hypoxic tumor cells and hypoxic microenvironment.

Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM 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 cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2015-5468

Introduction: Despite the introduction of novel therapies, majority of multiple myeloma (MM) patients relapses. The major reason for the relapse in MM is the presence of a minimal residual disease (MRD) population that remains after treatment. MM cells are defined as CD138+; however, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 decrease in expression were previously shown. We propose a novel set of biomarkers (independent of CD138) to detect MRD in MM in order to predict patient relapse. Procedures: We tested the effect of hypoxia on CD markers expression in MM cell lines. We developed new method to detect MM cells by flow cytometry based on CD38(APC+) gating and negatively selecting other leukocytes including T cells, B cells, monocytes, NK cells, and dendritic cells using CD3, CD19, CD14, CD16, and CD123, respectively (FITC-conjugated), the MM cells were defined as APC+/FITC-. We compared traditional method (CD138-based) with the new set of biomarkers (1) to detect normoxic and hypoxic MM cells, (2) to detect MM cells in the peripheral blood (PB) and the bone marrow (BM) from MM patients, confirming cell clonality by internal staining with Kappa/Lambda light chain antibodies, and (3) to predict relapse in MM patients whose BM was defined a CD138-negative. Summary of data: We found that hypoxia decreased the expression of CD138 by ∼50%, while CD38 was not altered by hypoxia in MM cells. The use of CD138+ as a marker for MM detected only 60-75% of hypoxic MM cells, whereas APC+/FITC- strategy detected close to a 100% of the hypoxic MM cells in vitro. In patient samples, all patients had a higher number of BM-derived and circulating MM cells as detected by APC+/FITC- strategy compared to CD138+. In addition, the clonality of the APC+/FITC- population in BM negative fractions was the same as the original disease (CD138+ cells). Moreover, studies conducted on 16 patients with BM defined as a CD138-negative, showed that the novel APC+/FITC- strategy was more successful than CD138-based method or histology in predicting recurrence and time to relapse. The median time-to-progression with Conclusion: CD138 cannot be used as a universal marker to detect MM cells, especially when they are hypoxic. A novel set of biomarkers CD38+/CD3-/CD19-/CD14-/CD16-/CD123- (APC+/FITC-) was designed to detect MM cells independent of CD138 expression and hypoxic status. This strategy was able to detect a clonal CD138-negative MM cell population in the BM and PB from MM patients. Moreover, this strategy predicted tumor recurrence and relapse in MM patients more accurately compared to traditional methods such as CD138-based or histology. Citation Format: Barbara Muz, Feda Azab, Pilar de la Puente, Justin King, Micah Luderer, Ravi Vij, Abdel Kareem Azab. Predicting relapse using CD138-independent strategy to detect residual myeloma plasma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2015-5305

INTRODUCTION: Current methods to assess multiple myeloma (MM) cancer treatments are often inaccurate, in part, due to these methods cannot take into consideration the variability of every patient, and fail to recreate the bone marrow (BM) microenvironment. In this study we developed a patient-derived three-dimensional tissue-engineered BM (3DTEBM) niche as novel model that will allow better evaluation of interactions of MM cells and their microenvironment and how these interactions may affect MM drug resistance in the specific patient. METHODS & RESULTS: 3DTEBM cultures were made by crosslinking of patient-derived BM supernatants; the 3D-scaffold included MM cell lines or primary CD138+ MM cells and other accessory cells (stromal cells and endothelial cells, or CD138- cells). 3DTEBM cultures were studied for cell proliferation by flow cytometry, and compared with 2D cultures and other commercial 3D systems including PLGA microspheres, Algimatrix, and Matrigel, and imaged for 2 weeks using a confocal microscope. 3DTEBM cultures promoted the proliferation of MM cells more than 2D cultures and commercial 3D systems. 3DTEBM cultures supported the growth of fresh and frozen primary MM cells and allowed the use of these cells ex vivo at least 2 weeks. In addition, we found a natural development of polarization in the cellularity of the scaffold, with the bottom rich of stromal cells, top rich of endothelial cells, and MM cells through all the parts of the scaffold, reflecting the pathophysiological development of MM in the BM niche. The effect of 3DTEBM cultures on cytokine expression was determined by cytokine antibody arrays and 3D-tissue depth effects in drug gradients, oxygenation levels and proliferation (HIF-1α, PIM, Ki67, CXCR4, and CD138 expression) were determined using flow cytometry or IHC. 3DTEBM cultures showed that 23 out of 80 cytokines tested were highly expressed with at least 3-fold increase in the 3D cultures vs 2D cultures. Furthermore, 3DTEBM cultures recreated the BM niche drug gradients and oxygenation levels due to depth of tissue; MM cells showed lower drug uptake and higher hypoxia levels (PIM and HIF-1α) with depth of tissue, and hypoxia-associated decreased CD138 and increased CXCR4 expression. We also found that MM cells were quiescent (lower Ki67 expression) in the hypoxic niche. Finally, drug resistance of MM cell lines or primary MM cells in 3DTEBM cultures was analyzed by flow cytometry. 3DTEBM cultures induced drug resistance in MM cells more than 2D cultures and commercial 3D systems, and preliminary results indicate that drug resistance profile in the 3DTEBM matched the clinical manifestation of the disease in the specific patient, while 2D cultures did not. CONCLUSION: The patient-derived 3DTEBM is a more biologically relevant model for evaluation of interactions of MM cells and their microenvironment in the BM niche, especially in the context of drug resistance. Citation Format: Pilar de la Puente, Rebecca Gilson, Barbara Muz, Feda Azab, Justin King, Samuel Achilefu, Ravi Vij, Abdel Kareem Azab. 3D tissue-engineered bone marrow niche as novel method to study pathophysiology and drug resistance in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2015-5356

e22210 Background: Waldenstrom Macroglobulinemia (WM) is a rare and incurable lymphoma that can be can be managed by a wide number of available chemotherapy agents. In this study, we propose a nove...

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: 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 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.