ALBERT

Description: Key Points High expression of PAK4 promotes myeloma cell proliferation through activation of MM antiapoptotic and survival pathways. Targeting PAK4 with a novel small molecule inhibitor, KPT-9274, has significant impact on MM cell growth and survival.

Description: Restoring nuclear localization of tumor suppressors by blocking exportin 1 (XPO1) holds promise as a new therapeutic paradigm in many cancers, including chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Oral selective inhibitor of nuclear export (SINE) compounds that covalently modify XPO1 were recently discovered and are exciting new compounds to implement this strategy. Selinexor, the clinical lead SINE, has made progress in Phase I/II clinical trials and is generally well tolerated, but limited to twice weekly dosing, with supportive care. The discovery of novel SINE compounds with improved tolerability is therefore of considerable clinical interest and represents a significant contribution beyond the targeted therapies currently available for hematologic malignancies and a variety of other cancers where upregulation of XPO1 is observed. Presented herein is the discovery of KPT-8602, the next generation SINE compound that shows lower brain penetration, improved tolerability allowing continuous dosing, and improved efficacy beyond any current XPO1 inhibitor. Results: Our crystallography data revealed that KPT-8602 binds covalently to XPO1 through a Michael acceptor that is activated by an electron withdrawing pyrimidyl moiety, allowing a 2-fold increased reversible interaction with XPO1 compared to earlier SINE compounds. The crystal structure of the KPT-8602-XPO1 complex showed interactions between XPO1 and the activating pyrimidyl group. In vitro pull-downs using immobilized GST-nuclear export sequences and purified recombinant XPO1 demonstrated greater reversible binding of KPT-8602 compared to KPT-330 (selinexor). In vivo toxicology studies demonstrated that KPT-8602 possesses lower brain penetration compared to KPT-330 allowing for continuous dosing and improved tolerability. Our results also showed that KPT-8602 induced a comparable level of cytotoxicity as well as inhibition of cell proliferation compared to KPT-330 in primary CLL tumors and in a representative panel of DLBCL cell lines. Furthermore, KPT-8602 inhibited proliferation and induced apoptosis in AML cell lines and primary AML blasts while inducing nuclear accumulation of p53 and NPM1. We hypothesized that these improved pharmacological parameters would allow daily KPT-8602 to abrogate disease progression in CLL and AML animal models. The Eµ-TCL1-C57BL/6 transplant model of CLL was used to evaluate the therapeutic benefit of continuous dosing of KPT-8602. Eµ-TCL1-engrafted mice were treated with KPT-8602 given daily or 2x/week. The KPT-8602 daily cohort had significantly improved survival with a median overall survival of 70 vs 50 days (vs vehicle 33 days), compared to those treated only 2x/week with KPT-8602 (p=0.001). Mice treated with KPT-330 2x/week showed a similar survival to mice treated with KPT-8602 2x/week. Mice given daily KPT-8602 had significantly smaller spleens and reduced circulating leukemic cells compared to all the other groups (p

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: Selective Inhibitor of Nuclear Export (SINE) compounds are a new class of oral anti-cancers drugs that block nuclear export by inhibiting the exportin XPO1 (also known as CRM1). The most advanced compounds in this class are selinexor and KPT-8602, which are being evaluated in clinical trials for the treatment of hematological malignancies (clinicaltrials.gov). XPO1 is commonly overexpressed in cancer and high levels of XPO1 are correlated with poor prognosis. Based on Phase I efficacy and safety studies, the recommended Phase 2 dose (RP2D) of selinexor is 60 mg twice weekly on days 1 and 3. We have previously reported the development of a pharmacodynamics (PD) assay to measure XPO1 target occupancy by selinexor in cancer cells based on Fluorescence Cross Correlation Spectroscopy (FCCS). Here we report proof of concept studies to determine the level of XPO1 occupancy associated with the selinexor RP2D regimen. Methods: Z138 cells in culture were treated with 1 µM selinexor either continuously for up to 32 hours or for 6 hours followed by drug removal and culture for up to 72 hours post-wash out. Cell were treated in culture for 4 hours with either 0-10 µM selinexor, KPT-8602, or Leptomycin B (LMB). Cells were harvested, lysed, and cleared by ultracentrifugation. Total protein was measured from lysates for Western blot with α-XPO1, α-GAPDH, and α-β-tubulin and for FCCS analysis. In vivo, nude mice were inoculated in both flanks with Z138 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 at 6, 24, 48, 72, and 96 hours post-dose, lysed, and cleared by ultracentrifugation. Cell line or tumor supernatants were incubated with a fluorescently labeled antibody (Ab) recognizing XPO1 and fluorescently labeled LMB. To allow binding of labeled Ab and LMB to XPO1, samples were measured by FCCS after 15, 120, and 1200 minutes. Results: XPO1 protein dynamics were evaluated in Z138 cells treated with selinexor either continuously or after drug removal. Selinexor treatment resulted in the reduction of XPO1 protein by 26% as early as 4 hours of treatment and reduced XPO1 levels by 93% with continuous drug exposure up to 32 hours. However, cells treated with selinexor for only 6 hours followed by drug washout showed nearly full restoration of XPO1 expression by 72 hours, indicating that continuous presence of drug is required to sustain reduced XPO1 protein levels. Measurements by FCCS showed that brief exposure to selinexor (6 hours) resulted in rapid XPO1 occupancy, and drug washout resulted in sustained occupancy that was not relieved until 72 hours post-selinexor wash out. To determine whether the second generation SINE compound clinical candidate, KPT-8602, had a similar XPO1 occupancy profile as selinexor, a panel of hematologic cell lines were treated with either compound and analyzed by FCCS. FCCS was also measured from cells treated with LMB, a natural product inhibitor of XPO1. FCCS analysis confirmed that KPT-8602 treatment resulted in a dose-dependent increase in drug-target occupancy with increasing drug concentration similar to that of selinexor, while LMB showed complete occupancy at very low levels of compound. Lastly, to confirm the level of target engagement in the RP2D regimen for selinexor, drug-target occupancy was measured in Z138 xenograft tumors from mice treated with a single oral dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were collected at 6, 24, 48, 72, and 96 hours post-single dose. The FCCS results showed that full occupancy occurred by 6 hours at all doses, but the duration of occupancy was dose-dependent. XPO1 occupancy was relieved by 24 hours at 5 and 10 mg/kg, 48 hours at 15 mg/kg, and 72 hours at 20 mg/kg selinexor. This finding supports the RP2D dosing regimen, where 10-15 mg/kg in mice (approximately equivalent to 50-75 mg flat dose in humans) is necessary to maintain XPO1 occupancy for up to 48 hours post-dose, thus requiring the twice weekly dosing for continuous target occupancy. Conclusions: Using an FCCS based PD assay developed to measure the dynamics of XPO1 target occupancy by selinexor, our studies support the selinexor RP2D dosing regimen for patients with heavily pretreated hematologic and solid tumors. Future studies are planned to measure XPO1 occupancy in CD138+ cells isolated from patients with advanced multiple myeloma following treatment with selinexor or KPT-8602. Disclosures Crochiere: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Hannus:Intana Bioscience GmbH: Consultancy. Hansen:Intanat Bioscience GmbH: Consultancy. Becker:Intana Bioscience GmbH: Consultancy. Baloglu:Karyopharm Therapeutics Inc: Employment, Other: stockholder. 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.

Description: Introduction - Inhibition of Exportin 1 (XPO1) is a novel treatment approach for multiple myeloma (MM). XPO1 mediates the nuclear export of cell-cycle regulators and tumor suppressor proteins leading to their functional inactivation. In addition, XPO1 promotes the export and translation of the mRNA of key oncoproteins (e.g. c-MYC, BCL-2, Cyclin D). XPO1 overexpression occurs in solid and hematological malignancies, including MM and is essential for MM cell survival. Selinexor, the first oral SINE compound, has shown promising anti-MM activity in phase 1 studies but has been associated with gastrointestinal and constitutional toxicities including nausea, anorexia and fatigue. KPT-8602 is a second generation oral SINE compound with similar in vitro potency to selinexor, however, has substantially reduced brain penetration compared with selinexor, and demonstrated markedly improved tolerability with minimal anorexia and weight loss in preclinical toxicology studies. In murine models of MM, KPT-8602 can be dosed daily (QDx5) with minimal anorexia and weight loss. We have therefore initiated a phase 1/2 first-in-human clinical trial. Methods - This phase 1/2 clinical trial was designed to evaluate KPT-8602 as a single agent and in combination with low dose dexamethasone (dex) in patients (pts) with relapsed / refractory MM (RRMM). KPT-8602 is dosed orally (QDx5) for a 28-day cycle with a starting dose of 5 mg. Low dose dex (20 mg, twice weekly) is allowed after cycle 1 if at least a minimal response (MR) is not observed. The primary objective is to evaluate the safety and tolerability including dose-limiting toxicity (DLT), determine the maximum tolerated dose (MTD), the recommended Phase 2 dose (RP2D), and evidence for anti-MM activity for KPT-8602 single agent and in combination with dex. The pharmacokinetic (PK) and pharmacodynamic (PDn; XPO1 mRNA) profile of KPT-8602 will also be determined. PDn predictive biomarker analysis and ex vivo drug response assays are underway using tumor cells from bone marrow aspirates before treatment, during and at relapse. These analyses include cell death pathway assays by flow and nuclear/cytoplasmic localization of XPO1, NF-ƙB, IƙBα, IKKα, NRIF and p53 by imaging flow and IHC. Results - As of 01-Aug-2016, 6 pts 2 M/4 F, (median of 6 prior treatment regimens, median age of 71) with RRMM have been enrolled. Common related grade 1/2 adverse events (AEs) include thrombocytopenia (3 pts), nausea (2 pts) and diarrhea (2 pts). Grade 3 AEs include neutropenia (1 pt) and dehydration (1 pt). No grade 4 or 5 AEs have been reported. No DLTs have been observed and the MTD has not been reached. 5 pts were evaluable for responses (1 pt pending evaluation): 1 partial response, 1 minimal response, and 3 stable disease; no pts have progressed on therapy with the longest on for 〉5 months. The PK properties following oral administration showed that 5 mg of KPT-8602 was rapidly absorbed (mean tmax= 1 hr, mean Cmax= 30.6 ng/mL). The mean AUCinf was calculated to be 141 ng•hr/mL. After tmax, KPT-8602 declined at an estimated mean t½ of 4 hr. At the same dose level, XPO1 mRNA expression was the highest (~2.5 fold) at 8 hr post dose. Conclusions - Oral KPT-8602 is well tolerated in heavily pretreated pts with RRMM. Gastrointestinal and constitutional toxicities observed with twice weekly selinexor have not been observed with 5x/week KPT-8602, including in pts on study for 〉4 months. PK was predictable and in line with selinexor. These early results show encouraging disease control with pts remaining on therapy. Enrollment is on-going. Disclosures Rossi: Takeda: Speakers Bureau; Janssen: Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Baz:Takeda/Millennium: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Signal Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Novartis: Research Funding. Hofmeister:Karyopharm Therapeutics: Research Funding; Arno Therapeutics, Inc.: Research Funding; Signal Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Incyte, Corp: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees. Shustik:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Richter:Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Jannsen: Speakers Bureau. Chen:Janssen: Honoraria, Research Funding; Takeda: Research Funding; Celgene: Honoraria, Research Funding. Vogl:Takeda: Consultancy, Research Funding; Celgene: Consultancy; GSK: Research Funding; Calithera: Research Funding; Teva: Consultancy; Karyopharm: Consultancy; Acetylon: Research Funding; Constellation: Research Funding. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Ellis:Karyopharm Therapeutics: Employment, Equity Ownership. Friedlander:Karyopharm Therapeutics: Employment. Choe-Juliak:Karyopharm Therapeutics: Employment. Sullivan:Karyopharm Therapeutics: Research Funding. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.