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Cystic fibrosis (CF) is an inherited, life-threatening disease caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter-class protein and ion channel that transports ions across epithelial cell membranes. The most common mutation leads to the deletion of a single phenylalanine, and the resulting protein, F508del-CFTR, shows reduced trafficking to the membrane and defective channel gating. The ideal therapeutic approach would address both of these defects and restore channel function at the same time. We describe here the application of a combination high-throughput screening to search for synergistic modulators of F508del-CFTR. With the adapted Fischer rat thyroid-yellow fluorescent protein halide flux assay to the combination high-throughput screening platform, we identified many interesting single agents as CFTR modulators from a library of approved drugs and mechanistic probe compounds, and combinations that synergistically modulate F508del-CFTR channel function in Fischer rat thyroid cells, demonstrating the potential for combination therapeutics to address the defects that cause CF.

We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of beta-catenin (beta-cat) signaling. Coexpression of mammalian beta-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both beta-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with beta-cat, reduces beta-cat binding to TCF4, and leads to increased cytoplasmic localization of beta-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to beta-cat and restores reporter gene transcription. Additional beta-cat signaling components, Axin and glycogen synthase kinase 3beta, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein beta-transducin repeat-containing protein reduces the stability of beta-cat and decreases reporter activation. Thus, we have reconstituted a functional beta-cat signal transduction pathway in yeast and show that beta-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 φι K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

The goal of this investigation was to establish methodology to determine and prevent phototoxic responses of normal skin to photodynamic therapy (PDT). The drug used was a second-generation photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA). The dependence of skin phototoxicity on drug dose (0.5-2.0 mg/kg), fluence (1.2-390 J/cm2), and wavelength (690 nm and 458 nm) was studied in the New Zealand albino rabbit in the first 5 h after injection. Skin responses were recorded for 2 wk after irradiation. Noninvasive measurements of drug fluorescence were made on unexposed skin sites during the first 5 h after drug injection. Immediate responses to PDT included erythema induced by 458 nm light and blanching induced by 690 nm light. Delayed reactions included edema on the day of exposure, purpura at 24 h, eschar by day 2 or 3, and scar by the end of follow-up. The threshold fluence for immediate responses correlated strongly with the threshold fluence for delayed reactions. The induction of threshold purpura on day 1 was a reliable index for skin phototoxicity that led to necrosis. The minimum purpura dose on day 1 after irradiation increased exponentially with the interval between drug injection and irradiation, independent of irradiation wavelength, for all drug doses. The action spectrum for threshold purpura mimics closely the absorption spectrum of BPD-MA. The in vivo drug fluorescence correlated with skin phototoxicity, thus allowing predictive dosimetry. This model system defines the safety limits for skin phototoxicity of PDT with BPD-MA.

The past year has been significant advances in our understanding of the mechanism of RNA movement between the nucleus and the cytoplasm. The emerging view is that proteins bind to and escort RNAs to their proper subcellular location. The discovery of peptide signals that target nuclear export and the identification of novel protein mediators of RNA export are examples of significant recent discoveries.

Background: XPO1 (Exportin-1/CRM1) is the non-redundant nuclear exporter of over 200 cargos including the major tumor suppressor proteins. Deregulated nuclear export by changes in XPO1 expression is a common characteristic for a broad range of cancers and may aid in the evasion of anti-neoplastic mechanisms. As a result, inhibition of XPO1 has emerged as a promising area of cancer treatment. The Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor, as well as a second generation, KPT-8602, bind to the XPO1 cargo binding pocket and disrupt XPO1-mediated nuclear export, resulting in cancer specific cell death. Although selinexor has been evaluated in >1,500 patients and has manageable tolerability, KPT-8602 may have improved tolerability and efficacy based on decreased brain penetration in animal models allowing more frequent dosing. Currently, the safety, tolerability and efficacy of KPT-8602 is being evaluated in a phase 1 trial of patients with relapsed/refractory multiple myeloma (MM; NCT02649790). Since selinexor synergizes with a broad array of anti-MM agents in patients, KPT-8602 is an excellent candidate for combination therapies. In this study, we investigated the use and mechanism of combining KPT-8602 with the pan-histone deacetylase (HDAC) inhibitor, panobinostat, in MM cell lines and in a xenograft mouse model of MM. Methods : MM.1S cells were treated with single agent KPT-8602, panobinostat or a combination of both. The effects of KPT-8602 and/or panobinostat on cell viability were examined using standard viability assays after 72 hours of treatment. Total RNA or protein levels were examined after 24 hours using quantitative PCR or immunoblots, respectively. Immune compromised mice were injected subcutaneously with MM.1S cells. Tumors were allowed to grow to ~150 mm3before treatment. The mice were treated with vehicle, sub-therapeutic doses of KPT-8602 (5 mg/kg PO QDx5) or panobinostat (5 mg/kg IP QDx5) alone or in combination. Tumor growth and animal weights was monitored to determine tumor growth inhibition (TGI), tumor regression, and tolerability to treatment. The tumors were then harvested for immunohistochemical (IHC) analysis. Results : The combination of KPT-8602 and panobinostat was highly effective against MM.1S cell viability. A synergistic anti-cancer effect was observed against MM.1S cells grown in culture and in mice. In cells, the MTT IC50of KPT-8602 was shifted from 50 to 23 nM by the addition of sub cytotoxic concentrations of panobinostat. In mice, single agent treatment with KPT-8602 led to 96.5% tumor growth inhibition whereas panobinostat resulted in 69.4% tumor growth inhibition within 22 days. Remarkably, in the combination of KPT-8602 and panobinostat, 3 out of 8 tumors totally disappeared and the overall tumor regression was 95%, (Figure 1). Both drugs, as single agents and in combination were well tolerated and no significant changes in weight were observed. Gene and protein expression studies revealed that although both compounds target independent proteins (e.g. HDACs or XPO1), the combination significantly enhances markers of cell death (cleavage of PARP-1, caspase-3, etc.). Curiously, KPT-8602 enhances the inhibitory effect panobinostat has on deacetylation as evidenced by histone acetylation. Moreover, DNA damage, as indicated by ϒ-H2AX, significantly increases in the presence of both compounds. Conclusion : KPT-8602 and panobinostat are dissimilar drugs with unique mechanisms of action, and individually affect a broad range of cellular processes. Here we show that the combination of these drugs can dramatically increase the already potent anti-cancer properties of these compounds in MM cell lines. In addition, KPT-8602 enhances the inhibitory effect exerted by panobinostat on histone deacetylation, which coincides with an increase induction of DNA damage. It should be noted that both panobinostat and SINE compounds have been shown to downregulate checkpoint and DNA damage response (DDR) proteins (e.g. RAD51 and Chk1). We hypothesize that the combination of KPT-8602 and panobinostat promotes significant chromatin remodeling in the presence of a compromised DDR pathway, which destabilizes genomic integrity in MM cells and leads to a synergistic effect on cell viability. Together, these data provide rational support for the study of KPT-8602 and panobinostat in clinical trials. Figure 1 Figure 1. Disclosures Argueta: Karyopharm Therapeutics: Employment, Equity Ownership. Chang:Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Elloul:Rubius Therapeutics: Employment. Friedlander:Karyopharm Therapeutics: Employment. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership.

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.

Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor has therapeutic benefit both pre-clinically and clinically (NCT01607892, NCT02336815) in multiple myeloma (MM). It has been previously demonstrated that the extent of NF-κB transcriptional inhibition is one of the critical mechanisms contributing to the efficacy and/or resistance to selinexor in cells. However, the mechanism leading to NF-κB inhibition after selinexor treatment is not fully understood. We hypothesized that the level of the cellular inhibitor of NF-κB, IκB-α, and its compartmental localization play an important role in NF-κB transactivation and response to selinexor. In this study, we investigate the effect of selinexor treatment on MM cells with low expression of IκB-α and high NF-kB activity in order to understand the mechanism of NF-κB inhibition by selinexor. Methods: IM9 and RPMI-8226cells were treated with selinexor in the presence or absence of 20 ng/mL tumor necrosis factor α (TNFα; inflammatory mimic) and whole protein lysates were analyzed by immunoblotting. Cytotoxic effects of selinexor were evaluated using standard viability assays. IκB-α knockdown was performed using transfection of specific siRNA duplexes. NF-κB transcriptional activity was analyzed using an ELISA assay. Results: Selinexor induces nuclear localization of IκB-α in MM cells. RNAi of IκB-α in MM cells reduced the cytotoxic effects of selinexor by 10-fold. In addition, knockdown of IκB-α reduces the synergy of the selinexor plus proteasome inhibitor (bortezomib or carfilzomib) combination. This data suggests that in MM, IκB-α plays a major role in cellular sensitivity to selinexor potentially through NF-κB activity. Selinexor inhibited NF-kB transcriptional activity in IM9 and RPMI-8226 cells with IC50 of 1079 nM and 591 nM respectively. Although the difference in NF-κB activity IC50 is only 2-fold between the two MM cell lines (MTT IC50s are ~100 nM), IM9 cells have a 100-fold higher basal NF-kB activity when compared to RPMI-8226 cells. Under TNFα stimulation NF-kB activity was induced by 1.5- and 35-fold in IM9 and RPMI-8226, respectively. We observed that selinexor treatment caused a dose dependent inhibition of IκB kinase (IKK)-mediated phosphorylation of serine 32/36 on IκB-α and serine 536 on the NF-κB p65 subunit (RelA) upon TNFα stimulation in both cell lines. In RPMI-8226 cells, selinexor reduced TNFα-induced IκB-α phosphorylation in a dose dependent manner and protected IκB-α from degradation. In IM9 cells that have high basal NF-κB activity, TNFα did not induce NF-kB activity or cause IκB-α degradation. However, selinexor treatment inhibited NF-kB activity below its basal level (70% reduction) which resulted in dose dependent reduction in the level of IκB-α protein perhaps through inhibition of NF-κB transcriptional control of IκB-α mRNA expression. Conclusions: IκB-α plays a major role in the cellular cytotoxicity of selinexor in cancer cells. Multiple myeloma cells lose sensitivity to selinexor treatment upon IκB-α silencing, which in turn reduces the cytotoxicity of selinexor. TNFα stimulation induces the phosphorylation of NF-κB p65 subunit and IκB-α through increased IKK activity resulting in IκB-α degradation and NF-κB activation. In RPMI-8226 cells, selinexor treatment blocked TNFα-induced degradation of IκB-α. However, in IM9 cells TNFα alone did not have any significant effect on IκB-α which might be due to the high basal NF-kB activity. Interestingly, IκB-α is also a transcriptional target of NF-kB. In IM9 cells, selinexor treatment reduces NF-kB activity below the high basal level in a dose dependent manner resulting in near complete inhibition of NF-kB-controlled IκB-α mRNA transcription and a loss of the IκB-α protein. Ultimately, selinexor treatment inhibits cell viability and NF-kB transcriptional activity regardless of basal NF-κB activity in MM cells. Because of this IκB-α/NF-kB transcriptional mechanism, selinexor treatment can inhibit both chronic (unresponsive to TNFα) and acute (TNFα-simulated) inflammatory signaling which makes selinexor an applicable therapy to cancer cells with a variety of aberrant signaling pathways. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta: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. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership.

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi-intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.

Nicotinamide adenine dinucleotide (NAD) is an essential metabolite and important cofactor of several biological processes (i.e. metabolism and genomic stability) that undergo significant alterations during malignant transformation. In cancer cells, NAD undergoes quick turnover through the high metabolic demands of rapidly proliferating cells and increased activity of NAD consuming enzymes, such as sirtuin 1 (SIRT1) and poly ADP ribose polymerase 1 (PARP1). NAD can be generated de novo from tryptophan or regenerated by nicotinamide phosphoribosyl transferase (NAMPT) or nicotinate phosphoribosyl transferase 1 (NAPRT1) in NAD salvage pathways. However, cancer cells do not utilize the de novo or NAPRT1 pathways effectively and instead rely on the NAMPT-dependent salvage pathway to generate NAD, making NAD depletion a promising anti-cancer therapy. We have previously described the PAK4 allosteric modulator, KPT-9274, which can also inhibit the enzymatic activity of NAMPT. KPT-9274 and other NAMPT inhibitors rapidly deplete cellular NAD levels, ultimately leading to ATP depletion and cell death. Similarly, several studies have shown that hyper activation of NAD consuming enzymes can lead to apoptosis. The purpose of this study is to determine whether NAD inhibition by KPT-9274 can synergize with NAD depleting enzymes to enhance the cytotoxic effects of KPT-9274 in cancer cells. Methods: Celltiter-Glo was used to measure ATP levels and viability of cells. NAD/NADH-Glo was used to measure total NAD levels in cells. Gene and protein expression was measured using quantitative PCR and western blot analysis, respectively. Protein knockdown was accomplished using RNAi. Results: We have identified an orally bioavailable dual inhibitor of PAK4 and NAMPT, which demonstrated potent anti-cancer activity in a variety of cell lines both in vitro and in vivo. We have identified a mechanistic combination that increase the antitumor activity of KPT-9274, through the activation of NAD consuming enzymes such as SIRT1 and PARP1. Specifically, we found that SRT1720 (an activator of SIRT1) synergizes with KPT-9274 to increase cancer cell death while RNAi of SIRT1 diminishes the efficacy of KPT-9274. In addition, activation of PARP1 by DNA damaging agents (e.g. gemcitabine) significantly enhances the effectiveness of KPT-9274 mediated cell death. Finally, we show that PARP activation through DNA-damaging agents enhances the cytotoxicity and anti-tumor properties of KPT-9274 in xenograft models. Conclusions: Here we report that KPT-9274 synergizes with NAD depleting enzymes to induce cancer cell death in vitro and in vivo. This noteworthy enhancement to the anticancer activity of KPT-9274, together with its previously described PAK4 inhibition, support the continued development of this orally bioavailable small molecule in combination with current therapies. Citation Format: Christian Argueta, Trinayan Kashyap, Margaret Lee, Yosef Landesman, Sharon Shacham, William Senapedis, Erkan Baloglu. KPT-9274 inhibits cellular NAD and synergizes with NAD depleting enzymes to induce cancer cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3016.

Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity. Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific). Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK. Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2899.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.

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.

Reactive oxygen species (ROS) are generated by cellular metabolism as well as by exogenous agents. While ROS can promote cellular senescence, they can also act as signaling molecules for processes that do not lead to senescence. Telomere homolog oligonucleotides (T-oligos) induce adaptive DNA damage responses including increased DNA repair capacity and these effects are mediated, at least in part, through p53.Studies were undertaken to determine whether such p53-mediated protective responses include enhanced antioxidant defenses.Normal human fibroblasts as well as R2F fibroblasts expressing wild type or dominant negative p53 were treated with an 11-base T-oligo, a complementary control oligo or diluents alone and then examined by western blot analysis, immunofluorescence microscopy and various biochemical assays.We now report that T-oligo increases the level of the antioxidant enzymes superoxide dismutase 1 and 2 and protects cells from oxidative damage; and that telomere-based gammaH2AX (DNA damage) foci that form in response to T-oligos contain phosphorylated ATM and Chk2, proteins known to activate p53 and to mediate cell cycle arrest in response to oxidative stress. Further, T-oligo increases cellular ROS levels via a p53-dependent pathway, and these increases are abrogated by the NAD(P)H oxidase inhibitor diphenyliodonium chloride.These results suggest the existence of innate telomere-based protective responses that act to reduce oxidative damage to cells. T-oligo treatment induces the same responses and offers a new model for studying intracellular ROS signaling and the relationships between DNA damage, ROS, oxidative stress, and cellular defense mechanisms.

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.

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.

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.

Abstract 3831 Poster Board III-767 Using a high-throughput combination screening strategy, we have demonstrated that adenosine A2A receptors (A2AR) or beta-2 adrenergic receptors (bAR) agonists, in low nano-molar range, exhibit highly synergistic anti-proliferative activity when combined with Multiple Myeloma drugs, such as dexamethasone, lenalidomide, bortezomib, melphalan and doxorubicin at clinically relevant concentrations. Synergy and selectivity have been observed in multiple myeloma (MM) cell lines and ex vivo using patient tumor cells. To understand this synergistic mechanism, we employed the Affymetrix U133 plus 2.0 cDNA microarray to investigate the differentially expressed genes in a multiple myeloma MM1.S cell line treated with A2AR agonists, bAR agonists, dexamethasone, or in combinations (A2AR/ bAR agonists and dex) for six hours. Using conventional hierarchical clustering, unsupervised analyses clearly separate the A2AR/ bAR agonists or the dexamethosone groups from the combination groups, suggesting a unique mechanism underlying the combination treatments. With SAM (Significance Analysis of Microarrays), we found 314 and 309 genes that showed statistically significant up-regulation or down-regulation respectively in the combination groups compared to the untreated control, with a false discovery rate= Disclosures: Tam: CombinatoRx, Inc.: Employment. Rickles:CombinatoRx, Inc.: Employment. Giordano:CombinatoRx, Inc.: Employment. Borisy:CombinatoRx, Inc.: Employment. Lee:CombinatoRx, Inc.: Employment.

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.

Using a combination high throughput screening technology, we have discovered an unexpected synergistic interaction between adenosine A2A receptor (A2A) agonism and phosphodiesterase (PDE) inhibition that displays substantial activity in preclinical Multiple Myeloma (MM) models. High throughput combination screening allows the systematic testing of combinations of approved drugs and other biologically active molecules in cell based assays of tumor cell proliferation and viability. In this approach we generate a dose matrix for each chemical combination, capturing the combined activity of two compounds over a broad range of single agent concentrations. Quantitative scoring of dose response matrices using models of drug interactions allows insight into the biological mechanism of action of drug combinations and the discovery of novel therapeutic applications. Using a panel of 4 MM cell lines (H929, MM.1S, MM.1R and RPMI-8226) we evaluated a defined set of combinations of approved drugs and molecular probes. A total of 2841 unique combinations were evaluated, 648 of which were assayed in all 4 cell lines. Multiple combinations were identified that exceeded the Loewe additivity model including enhancers of known anti-cancer drugs and combinations of targets not previously know in multiple myeloma. Multiple classes of drugs and targeted agents were identified that synergize with dexamethasone. Backcrosses of these active agents revealed multiple dexamethasone enhancers that also synergized with each other. Two of these classes, A2A agonists and PDE inhibitors demonstrated high levels of synergy and good breadth of activity across several MM cell lines. Interestingly, while the majority of the combinations screened had little or no synergistic effect in the glucocorticoid resistant MM.1R cell line, combinations of PDE inhibitors and A2A agonists demonstrated substantial efficacy (>90% inhibition of proliferation) and striking synergy (combination indices

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.

Using a high throughput combination screening strategy, we have discovered that agonism of either adenosine A2A receptors (A2A) or beta-2 adrenergic receptors (bAR) demonstrate significant, synergistic, anti-proliferative effects in preclinical Multiple Myeloma (MM) models. Using quantitative synergy analysis, we observe that A2A and bAR agonists have significant anti-proliferative effects in a broad panel of 10 MM cell lines when combined with each other or with standard MM agents. Individual A2A agonists CGS-21680 and HE-NECA inhibited proliferation 25–80% with EC50s ranging from 2–20 nM. Individual bAR agonists salmeterol and formoterol inhibited proliferation 35–75% with EC50s ranging from 10–30 pM. Potent, highly synergistic, inhibition of proliferation, up to 95%, was demonstrated with combinations of A2A or bAR agonists and multiple agents including dexamethasone, lenalidomide, bortezomib, melphalan, doxorubicin, HDAC inhibitors and HSP90 inhibitors at clinically relevant concentrations. These combinations exceeded Loewe additivity, and demonstrated both substantial increases in efficacy over maximal single agent levels as well as significant potency shifting with many combination indices (CIs) in the range of 0.1 to 0.3. Synergistic anti-proliferative effects were observed broadly across several MM cell lines and when using cell lines unresponsive to standard MM drugs, e.g. A2A agonists CGS-21680 and HE-NECA in combination with dexamethasone inhibited 75–85% of the proliferation of EJM, and MOLP-8 dexamethasone-insensitive cell lines as compared to 35–60% for the single agent A2A agonists. The selective A2A antagonist SCH58261 but not A1, A2B and A3 selective antagonists DPCPX, MRS1754 and MRS1523 blocked the synergy and antiproliferative activity of HE-NECA, demonstrating that the effect is mediated via the A2A receptor. siRNA directed against adenosine and adrenergic receptor isoforms, caused a concomitant reduction in the antiproliferative effects of HE-NECA and salmeterol. Synergy (CI