ALBERT

Description: Introduction: Exportin 1 (XPO1) is a well characterized and essential nucleo-cytoplasmic transport protein in the karyopherin family, and is responsible for the nuclear export of over 200 cargo proteins, including the major tumor suppressor proteins (TSPs) p53, p21, FOXO and the translation regulator elF4E. XPO1 is overexpressed in numerous cancer types including solid and hematological malignancies, often correlating with poor prognosis. Recently, a novel class of Selective Inhibitors of Nuclear Export (SINE) compounds, selinexor (KPT-330) and the second generation KPT-8602, have been developed for the treatment of advanced cancers. We have previously shown that selinexor has marked activity in AML and DLBCL pre-clinical models. The BCL-2 family of anti-apoptotic proteins are deregulated and linked to maintenance and survival in AML and DLBCL. For its translation, the mRNA for BCL-2 is transported from the nucleus to the cytoplasm by forming a complex with XPO1 cargo, elF4E. Other important mRNAs exported from the nucleus via this mechanism include BCL6 and MYC. We hypothesize that SINE compounds inhibit XPO1/elF4E-mediated nuclear-cytoplasmic transport by covalently binding to the XPO1 cargo binding site and that in the absence of protein translation, BCL-2, BCL6 and MYC levels rapidly decline. Venetoclax (VEN; ABT-199) is a potent, selective inhibitor of BCL-2. In vitro, AML cells acquire resistance to VEN over time, often due to up-regulation of another BCL-2 family anti-apoptotic protein, MCL-1. MCL-1 is regulated by the anti-apoptotic transcription factor and XPO1 cargo NF-kB. We have previously shown that SINE compounds significantly decreased MCL-1 levels, presumably via inactivation of NF-kB. The goal of this study was to test whether SINE compounds will synergize with VEN via BCL-2 modulation and whether the combination would diminish MCL-1 mediated resistance to BCL-2 inhibition in DLBCL and AML models, respectively. Methods: BH3 profiling was performed in a sample of cell lines using a cytochrome c release assay to identify anti-apoptotic dependencies. The effects of SINE compounds and VEN as single agents or in combination on cell viability were performed in AML (K-562, MOLM-13, MV-4-11, and U-937) and DLBCL cell lines (SU-DHL-6, DoHH-2 and Toledo). Whole cell protein lysates were extracted 24 hours after treatment for immunoblot analysis. The activity of SINE compounds (5 mg/kg) and VEN (25 mg/kg) as single agents, or in combination were measured in AML (MV-4-11) and DLBCL (DoHH-2 and Toledo) xenografts in NSGS and nude mice, respectively. Tumor growth and survival were measured throughout these animal studies. Tumor tissue was collected at the end of treatment for flow cytometric analysis, western blotting and immunohistochemistry (IHC). Results: By employing BH3 profiling, we identified AML cell lines that were dependent (MV-4-11 and MOLM-13) and not dependent (U-937 and K-562) on MCL-1. Dose response analysis demonstrated that each of the AML cell lines was sensitive to the SINE compounds, while VEN only reduced viability in the MV-4-11 and MOLM-13 cells. Additionally, there was enhanced growth inhibition when the SINE compounds were combined with VEN in the MCL-1 dependent cells. SINE compound treatment synergistically decreased c-MYC protein levels in all 4 AML cell lines with the combination treatment (Figure 1), whereas PARP cleavage was only enhanced with the combination in the MV-4-11 and MOLM-13 cells. Likewise, MCL-1 is reduced in the presence of SINE compound or SINE compound-VEN combinations. In DLBCL xenograft studies (DoHH-2 and Toledo), combination of selinexor with VEN was synergistic for tumor reduction and increased animal survival when compared to either single agent alone. By IHC we observed a concomitant reduction in BCL-2 and BCL-6 and an increase in cleaved caspase 3 in DLBCL tumors after combination treatment. Conclusions: SINE compound-VEN combinations show enhanced antitumor effect, with reduction of oncogenic activity. SINE compounds reduce MCL-1 in VEN-resistant cells. As MCL-1 driven anti-apoptotic machinery is responsible for resistance to inhibition of BCL-2 in DLBCL and AML, SINE compound regulation of MCL-1 may lead to rescue of VEN resistance. SINE compounds and VEN are excellent candidate partners for combination therapies in AML and DLBCL. Disclosures Friedlander: Karyopharm Therapeutics: Employment. Chang:Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Savona:TG Therapeutics: Research Funding; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, is an anti-cancer drug that is currently being evaluated in Phase I and II clinical trials for the treatment of solid and hematological malignancies (clinicaltrials.gov). XPO1 (exportin 1/CRM1), the target of selinexor, is commonly overexpressed in cancer. XPO1 is the main nuclear export protein with over 200 different protein cargos which include tumor suppressor and growth regulatory proteins. Binding of selinexor to XPO1 inhibits the nuclear export of TSPs and GRPs and induces cell cycle arrest followed by cancer cell apoptosis.Certain cancers are more sensitive to selinexor than others. We hypothesized that part of the inherent differences in sensitivities are attributed to different levels of target inactivation upon selinexor binding. To predict patient response to treatment, we developed a binding assay based on Fluorescence Cross Correlation Spectroscopy (FCCS) to measure XPO1 target occupancy in cancer cells. Methods: Cells were treated in culture for 4 hours with 0-10 µM selinexor. Cells were harvested, lysed, and cleared by ultracentrifugation. In vivo, nude mice were inoculated in both flanks with Z-138 tumor cells. Once tumors reached approximately 250 mm3, mice were administered a single dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were harvested 6 hours post-dose, lysed, and cleared by ultracentrifugation. Cell or tumor supernatants were incubated with a fluorescently labeled antibody (Ab) against XPO1 and fluorescently labeled Leptomycin B (LMB). To allow binding of labeled Ab and LMB to XPO1, samples were measured by FCCS after 15, 120, and 1200 minutes. FCCS measures signal fluctuations induced by fluorescent molecules diffusing through an illuminated microscopic detection spot. The approach allows simultaneous monitoring of 2 different fluorescently labeled molecules and extracts information on particle concentration, size, binding state, and molecular brightness. Interacting particles are scored by codiffusion. FCCS measurements were performed to quantify the concentration of dually labeled XPO1 molecules in prepared lysates. In the absence of selinexor, complexes of XPO1, the labeled antibody (aXPO1ABATTO488), and the red labeled LMB (LMBDY647) are formed and indicate low target occupancy. XPO1 molecules occupied by selinexor cannot bind to the labeled tracer and decrease the number of dually labeled particles. Accordingly, loss of complex formation is a measure of target occupancy in cells and tumor samples. Results: Comparison of cytotoxicity and XPO1 binding in hematological cell lines treated with increasing concentrations of selinexor in vitro indicated that XPO1 occupancy by selinexor occurred to the same extent regardless of drug sensitivity (Table 1). However, there were two observations that distinguished the selinexor-resistant cell line THP-1 from the other 3 sensitive cell lines (Table 1): 1. Untreated THP-1 cells had the highest XPO1 occupancy of fluorescently labeled LMB compared to lower values in more sensitive cells, and 2. Treatment of THP-1 cells with very low dose selinexor (10 nM) demonstrated lower saturation of XPO1 protein, while treatment of MV-4-11, Z-138, and MM.1S with 10 nM selinexor resulted in marked XPO1 occupancy. These results suggest that high levels of XPO1 proteins might lead to resistance to XPO1 inhibition. We are currently testing whether these two observations are general predictors of resistance in other cell lines. Mouse studies showed that XPO1 target occupancy could be measured in tumors and that there was a dose-dependent effect of selinexor on complex formation with 〉90% target saturation at 10 mg/kg selinexor (estimated Cmax ~2400 ng/ml). Conclusions: For the first time, the FCCS method permits the direct measurement XPO1 occupancy by selinexor in cells and tumors following treatment. Here, we demonstrate XPO1 occupancy saturation at 10 mg/kg (30 mg/m2) in mice, a dose active against xenografts, and consistent with doses of selinexor that show anti-cancer activity in patients with heavily pretreated hematologic and solid tumors. Table 1. Cytotoxicity and FCCS values for cell lines. Cell Line MTT IC50 (nM) FCCS IC50 (nM) FCCS Complex without selinexor (nM) FCCS Complex with 10 nM selinexor (nM) THP-1 1060 35.5 3.95 3.19 MV-4-11 20 56.2 2.55 1.9 Z-138 40 24.0 0.72 0.66 MM.1S 20 20.4 2.25 1.64 Disclosures Crochiere: Karyopharm: Employment. Hannus:Intana: Employment. Hansen:Intana: Employment. Becker:Intana: Employment. Ellis:Karyopharm Therapeutics Inc: Employment. Lee:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm: Employment.

Description: Background: SINE compounds are a family of small-molecule drugs that inhibit XPO1 mediated nuclear export, resulting in nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB and subsequently in specific cancer cell death. Selinexor is a clinical stage SINE compound currently in human phase I/II clinical trials in patients with solid and hematological malignancies. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts as a GR agonist and inhibits NFκB activity. The combination of selinexor with Dex (Sel-Dex) shows enhanced anti-tumor potency in vitro and in vivo. The current study aimed at deciphering the molecular changes that contribute to the synergism of Sel-Dex. Methods: Total RNA and whole protein cell lysates from MM cell lines treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence microscopy. GR and NFκB transcriptional activity was analyzed using ELISA assays (Affimetrix and Thermo Scientific). RNA from naïve and drug treated cells was analyzed by deep sequencing (by Asuragen). Results: Dexamethasone, but not selinexor, induced phosphorylation of GR. Selinexor blocked nuclear export of phosphorylated GR, enhancing GR transcriptional activation. Deep sequencing revealed a set of genes, whose level of expression was synergistically modified by Sel-Dex. Those genes belong to several pathways including GPCR signaling, cell metabolism and ERK, TGF-β and PI3-AKT signaling. Among the genes that were synergistically up regulated by the combination were genes from the Early Growth Response family (EGR1, EGR3, EGR4). EGR1 is a tumor suppressor protein which down-regulates survivin and triggers Caspase signaling and cell death. Interestingly, EGR1 also mediates the cytotoxic effects of bortezomib and lenalidomide in multiple myeloma cells. The Glucocorticoid-Induced Leucine Zipper (GILZ) gene was also up regulated by Sel-Dex combination. It has been shown by others that silencing GILZ expression decreased the therapeutic effects of dexamethasone in multiple myeloma. Conclusions: Selinexor increased the nuclear retention of dexamethasone-activated-GR. The increased GR transcriptional activity induces expression of genes across different pathways leading to inhibition of cell proliferation and increase cancer cell death. We are currently testing the function of several of these genes in the context of Sel-Dex combination. The current finding supports the the previously reported anti cancer activity of Sel-Dex combination in patients with heavily pretreated relapsed/refractory multiple myeloma. Disclosures Kashyap: Pharma: Employment. Klebanov:Karyopharm Therapeutics Inc: Employment. Lee:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics: Employment.

Description: Abstract 2990 Adenosine A2A receptor agonists, in combination with standard of care Multiple Myeloma (MM) therapeutics, demonstrate potent and highly synergistic anti-proliferative effects in B-cell malignancies. We have previously reported that A2A agonists show significant anti-tumor synergy with established MM drugs in several anti-tumor models including: tumor cell lines, in vivo xenograft models and in primary human MM tumor cells ex vivo. While these initial studies robustly demonstrate the proof of concept for this unexpected synergistic interaction, they were conducted with research compounds at doses and routes of administration resulting in exposure above therapeutically relevant levels. Here we describe the preclinical evaluation of ATL313, a potent and highly selective adenosine A2A receptor agonist that synergizes with established anti-MM agents resulting in enhanced efficacy in pre-clinical models of MM. ATL313 shows binding affinity for the human A2A receptor in the low single digit nM range and shows at least 80-fold selectivity for A2A compared to other adenosine receptor subtypes. As with other agonists examined, the activity of ATL313 is dependent on expression of the A2A receptor in the target cell and demonstrates single agent inhibition of proliferation in MM cell lines with an IC50 of 0.5–1 nM in the MM.1S cell line. ATL313 potently synergizes with glucocorticoids (dexamethasone and prednisolone), bortezomib, lenalidomide, melphalan and doxorubicin as well as emerging drug classes including HDAC inhibitors and HSP90 inhibitors. Substantial increases in inhibition of proliferation and cell killing and 2 to 100 fold potency shifts are observed with ATL313 combinations in MM cell lines including those both sensitive and resistant to current MM agents. In MM.1S cells, addition of 0.5 nM ATL313 to 100 nM Dexamethasone results in 95% inhibition of proliferation as compared to approximately 45% and 70% inhibition with either ATL313 or dexamethasone alone respectively. This combination results in a 30 fold shift in the dexamethasone IC50 and a combination index of 0.1 indicating high levels of synergy. Furthermore, the combination results in nearly complete cell killing as compared to reductions in cell number of 10% and 70% by ATL313 or dexamethasone respectively. Importantly, we have observed that A2A agonists are effective in combination in cells resistant to dexamethasone. Evaluation of A2A agonist combinations in a panel of 83 cell lines including solid tumor types and hematological malignancies demonstrates that synergy is highly selective for B-cell malignancies with little to no activity in solid tumor cell lines. We have now translated these in vitro results with ATL313 to a mouse xenograft model where the ATL313 is delivered using continuous infusion osmotic pumps. Doses of 10, 33 and 100 ng/kg/min were evaluated (in addition to QD subcutaneous bolus dosing at 0.1 mg/kg) and demonstrate synergistic anti-proliferative effects with no significant body weight loss. Mice bearing subcutaneous MM.1S tumors show a 43% regression in tumor volume after treatment with the combination of dexamethasone (1 mg/kg, s.c QD) and ATL313 (100 ng/kg/min) for 24 days as compared to a 500% or 700% increase after treatment with either ATL313 or dexamethasone, respectively. Furthermore, treatment with ATL313 in combination with dexamethasone confers a statistically significant survival advantage as compared to either agent alone. In summary, we report the preclinical evaluation of ATL313, a potent and highly druggable A2A agonist as a novel, selective and synergistic drug candidate for the treatment of MM. Our preclinical data provides compelling evidence in support of the further development of ATL313 for use in multi-drug combination therapy of MM. Disclosures: Rickles: Zalicus, Inc. (formerly CombinatoRx Incorporated): Employment, Equity Ownership. Padval:Zalicus, Inc. (formerly CombinatoRx Incorporated): Employment, Equity Ownership. Giordanno:Zalicus, Inc. (formerly CombinatoRx Incorporated): Employment, Equity Ownership. Rieger:PGx Health, LLC, a division of Clinical Data, Inc.: Employment, Equity Ownership, Patents & Royalties. Lee:Zalicus, Inc. (formerly CombinatoRx Incorporated): Employment, Equity Ownership.

Description: Abstract 3762 Poster Board III-698 By using a high throughput combination screening strategy, we made the surprising discovery that adenosine A2A and beta-2 adrenergic receptor (b2AR) agonists have synergistic anti-proliferative activity in combination with dexamethasone, melphalan, lenalidomide, bortezomib and doxorubicin in preclinical multiple myeloma (MM) models. Both A2A and b2AR agonists are highly selective and demonstrate no single agent activity or synergy in normal cell types including human PBMCs, AoSMCs, HUVECs or HCAECs at concentrations 2-3 orders of magnitude greater than the IC50 in MM cell lines. To further examine the selectivity and breadth we have evaluated A2A and b2AR agonist combinations in a panel of 83 cell lines including solid tumor types and hematological malignancies. Single agents and combinations with dexamethasone and melphalan were systematically studied at multiple ratios of clinically relevant concentrations. Using a quantitative synergy score based on the Loewe model (Lehar et al. Nat Biotech 2009), we observe that combination activity for A2A or b2AR agonists is highly selective for hematologic malignancies with synergy observed most frequently in multiple myeloma and DLBCL cell lines. Synergy is also observed with the B-cell lines JM-1 (pre B-ALL) and GA-10 (Burkitt's lymphoma). Using a relative synergy cut-off (synergy score 〉1), we find that 13 of the 18 MM cell lines tested demonstrate a synergistic interaction between the A2A agonist CGS-21680 and dexamethasone and 11 demonstrate a synergistic interaction between CGS-21680 and melphalan. Using this same measure, 9 of 18 MM cell lines demonstrate synergy with combinations of the b2AR agonist salmeterol with either dexamethasone or melphalan. Nine and ten of the cell lines in this MM panel are insensitive or respond weakly to dexamethasone and melphalan as single agents respectively. All cell lines were treated with the same concentrations of dexamethasone or melphalan, pointing to A2A agonists having a higher breadth of activity across the MM cell line panel. An interesting observation is the strong synergy observed for A2A or b2AR agonists with dexamethasone in the glucocorticoid-insensitive cell lines EJM and ANBL-6, which suggests that these agents may help restore steroid sensitivity in refractory patients. In general, drug resistance is a recurrent problem for cancer drugs and development of resistance after chronic exposure can reduce drug efficacy and promote refractory disease. We therefore examined the effects of chronic exposure to either A2A or b2AR agonists in the MM.1S cell line. Exposure of cells to CGS-21680 or salmeterol for one month reduced single agent sensitivity 〉80%. Surprisingly, combinations of either agent with dexamethasone maintained similar amounts of synergy and cell killing as found with naïve untreated cells. As determined by Western blot analysis, the reduction in single agent activity after chronic exposure is not accompanied by a reduction in receptor levels. These data demonstrate that synergistic combinations of A2A and b2AR agonists are highly selective for B-cell malignancies and support the notion that synergistic drug combinations improve therapeutically relevant selectivity and circumvent drug resistance. This work further supports the rationalefor investigation of A2A and b2AR agonists in the treatment of B-cell malignancies and in particular, patients who have MM. Disclosures: Rickles: CombinatoRx, Inc.: Employment. Tam:CombinatoRx, Inc.: Employment. Necheva:CombinatoRx, Inc.: Employment. Giordano:CombinatoRx, Inc.: Employment. Borisy:CombinatoRx, Inc.: Employment. Lee:CombinatoRx, Inc.: Employment.

Description: Using a combination high-throughput screening technology, multiple classes of drugs and targeted agents were identified that synergize with dexamethasone (Dex) in multiple myeloma (MM) cells. Performing combination screening with these enhancers, we discovered an unexpected synergistic interaction between adenosine receptor agonists and phosphodiesterase (PDE) inhibitors that displays substantial activity in a panel of MM and diffuse large B-cell lymphoma (DLBCL) cell lines and tumor cells from MM patients. We have used selective adenosine receptor agonists, antagonists, and PDE inhibitors as well as small interfering RNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. The adenosine A2A receptor and PDE2, 3, 4, and 7 are important for activity. Drug combinations induce cyclic AMP (cAMP) accumulation and up-regulate PDE4B. We also observe rigorous mathematical synergy in 3-way combinations containing A2A agonists, PDE inhibitors, and Dex at multiple concentrations and ratios. Taken together, these data suggest that A2A agonist/PDE inhibitor combinations may be attractive as an adjunctive to clinical glucocorticoid containing regiments for patients with MM or DLBCL and confer benefit in both glucocorticoid-sensitive and -resistant populations.

Description: Context: The bone marrow microenvironment, including bone marrow stroma cells (BMSCs) attenuates response of multiple myeloma (MM) cells to various conventional and experimental agents. Development of novel agents for the treatment of MM depends on attenuating the protective interactions that occur between the tumor cells and its microenvironment. Utilizing a combination high throughput screening platform (cHTS™) and the compartment-specific bioluminescence imaging (CS-BLI) co-culture system we have identified 2 novel classes of agents, adenosine A2A receptor agonists and β2-adrenergic receptor (bAR) agonists, with increased activity in the context of the microenvironment. Methods/Results: We tested 10 human MM cell lines in both the cHTS and CS-BLI systems in the presence and absence of IL-6 and discovered that multiple compounds that agonize A2A and bAR receptors had potent anti-MM activity in MM.1S cells. To further explore this activity, we evaluated CRx-501, a potent selective A2A agonist and salmeterol, a prototypical bAR agonist, which were found to have IC50 values in MM.1S cells of 15 nM and 0.2 nM, respectively. Individual single agent activities of CRx-501 and salmeterol were enhanced in the presence of exogenous IL-6 (10ng/mL). We also observed enhancement of CRx-501 activity in the presence of HS-5 human BMSCs. CRx-501 and salmeterol were also observed to synergize with bortezomib, dexamethasone, lenalidomide, and doxorubicin both in the presence and absence of IL-6 or stromal cells. These interactions were highly synergistic, as determined by the Loewe additivity model, demonstrating an increase in efficacy with combination indices ranging from 0.17 to 0.74. To further explore the anti-tumor activity of A2A and bAR agonists, CRx-501 and salmeterol were evaluated in an MM.1S tumor xenograft model. SCID CB17 mice received subcutaneous inoculation of MM.1S cells and, upon tumors becoming palpable, were treated with vehicle, dexamethasone (1 mg/kg s.c.), salmeterol (10 mg/kg s.c), CRx-501 (3 mg/kg s.c.) as single agents or pair-wise combinations of salmeterol or CRx-501 with dexamethasone. After 34 days of treatment with dexamethasone, salmeterol or CRx-501, we observe 34%, 42% and 72% reduction in tumor volume compared to vehicle control, respectively. Interestingly, in vivo activity of dexamethasone was enhanced to 86% and 89% with the combination of CRx-501 or salmeterol, respectively, with minimal toxicity (e.g. 7% fluctuation in animal weight during the course of the study). Conclusion: The development of active agents and drug combinations for MM depends on the evaluation and modeling of microenvironmental factors that play a role in the pathophysiology of the disease. Utilizing a novel combination screening method and co-culture screening system we identified and evaluated 2 novel mechanisms that are active in the context of the microenvironment and synergize with many standard therapies. These studies provide the framework for further preclinical evaluation and possible trials for these combinations for the treatment of MM.