XPO1 Inhibitor Research Articles

Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down\u2010regulation

BackgroundDedifferentiated liposarcoma (DDLPS), a tumor that lacks effective treatment strategies and is associated with poor outcomes, expresses amplified MDM2 in the presence of wild-type p53. MDM2 ubiquitination of p53 facilitates its XPO1-mediated nuclear export, thus limiting p53 tumor suppressor functions. Consequently, nuclear export is a rational target in DDLPS. We directly compared the antitumor activity of the first-in class XPO1 inhibitor selinexor and doxorubicin, the standard front-line therapy in sarcomas, in DDLPS patient-derived xenografts (PDXs) and primary cell lines.MethodsDrug activity was assessed in three PDXs (and two corresponding cell lines) established from the dedifferentiated component of primary untreated retroperitoneal DDLPS with myogenic (N = 2) and rhabdomyoblastic (N = 1) differentiation from patients who underwent surgery. These models were marked by amplification of MDM2, CDK4 and HMGA2 genes.ResultsSelinexor was moderately active in the three PDXs but achieved greater tumor response compared to doxorubicin (maximum tumor volume inhibition: 46–80 % vs. 37–60 %). The PDX harboring rhabdomyoblastic dedifferentiation showed the highest sensitivity to both agents. PDX response to selinexor and doxorubicin was not associated with the extent of MDM2 and CDK4 gene amplification. Interestingly, the most chemosensitive PDX model showed the lowest extent of HMGA2 amplification. Selinexor was also more efficient than doxorubicinin in inducing an apoptotic response in PDXs and cell lines. Consistently, an increased nuclear accumulation of p53 was seen in all selinexor-treated models. In addition, a time-dependent decrease of survivin expression, with an almost complete abrogation of the cytoplasmic anti-apoptotic pool of this protein, was observed as a consequence of the decreased acetylation/activation of STAT3 and the increased ubiquitination of nuclear survivin.ConclusionsSelinexor showed a moderate antitumor activity in three DDLPS PDXs, which was, however, consistently higher than doxorubicin across all different models regardless the extent of MDM2 amplification and the histological differentiation. The depletion of survivin protein seems to significantly contribute to the induction of apoptosis through which selinexor exerts its antitumor activity.

KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPβ Signaling Pathway.

[Figure: see text].

The selective inhibitor of nuclear export (SINE) verdinexor exhibits biologic activity against canine osteosarcoma cell lines.

Verdinexor (KPT‐335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose‐dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21 to 74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50 = 21 μM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.

XPO1 inhibition synergizes with PARP1 inhibition in small cell lung cancer by targeting nuclear transport of FOXO3a.

Patient mortality rates have remained stubbornly high for the past decades in small cell lung cancer (SCLC) because of having no standard targeted therapies with confirmed advantages at present. Poly [ADP-ribose] polymerase (PARP) inhibitors have shown promise in preclinical models but have had unsatisfactory clinical results in SCLC. By RNA-seq and isobaric tags for relative and absolute quantification (ITRAQ), we revealed that PARP1 inhibition led to the relocalization of forkhead box-O3a (FOXO3a) from nuclear to cytoplasm. By performing co-Immunoprecipitation (co-IP) and CRISPR-Cas9-mediated knockout plasmid we showed that FOXO3a was subject to exportin 1 (XPO1)-dependent nuclear export. We demonstrated the effects of the PARP inhibitor BMN673 on apoptosis and DNA damage were markedly enhanced by simultaneous inhibition of XPO1 in vitro. The combination of BMN673 and the XPO1 inhibitor selinexor inhibited primary SCLC cell proliferation in mini-patient-derived xenotransplants (miniPDXs) and markedly inhibited tumor growth without significant toxicity in xenograft models. The efficacy was enhanced for more than 2.5 times, compared to the single agent. Based on these findings, we further designed a novel dual PARP-XPO1 inhibitor and showed its effectiveness in SCLC. In this work, we illustrated that combining a PARP inhibitor with an XPO1 inhibitor is associated with significantly improved efficacy and tolerability. Dual PARP-XPO1 inhibition restored the FOXO3a balance and activity in SCLC. Collectively, targeting PARP1 and XPO1 opens new avenues for therapeutic intervention against SCLC, warranting further investigation in potential clinical trials.

Exportin-1 (XPO1, CRM1) inhibitors for the management of viral infection

A therapeutic gap exists between new emerging infectious diseases and effective treatments. Targeting host factors involved in the replication strategies of evolutionarily diverse viruses may bridge this gap. Exportin-1 (XPO1, also termed chromosome region maintenance 1 [CRM1]) is a cellular nuclear export receptor for cellular and viral protein and ribonucleoprotein cargos harboring a nuclear export signal. XPO1 inhibition suppresses replication of numerous evolutionarily diverse viruses and one XPO1 inhibitor has entered clinical trials for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the etiologic agent of coronavirus disease-2019 (COVID-19). This review examines current and potential strategies and challenges in targeting XPO1-mediated nuclear export for treating established, emerging and future virus infections. © 2021 Clarivate Analytics.

Identifying the Mutational Profile of ABCB5 Upregulation in Colorectal Cancer: A Comprehensive Database Analysis

Colorectal cancer (CRC) is a leading cause of cancer related deaths worldwide and the third leading cause of cancer related deaths in the United States with a 5-year survival rate of 63%. Depending on the stage of disease, there are differing rates of overall survival: Stage I (90%) vs. Stage IV (14%). There is a clear discrepancy between early and late stage disease due to a variety of factors including acquired genetic and tumor mutations and lack of initial symptoms leading to late stage detection. It is clear that further understanding into the mechanisms of CRC need to be explored in depth to improve overall survival rates. ATP binding cassette subfamily B member 5 (ABCB5) has been found to be pathogenic in a variety of diseases including melanoma and colorectal cancer but there has been few studies looking at the cellular consequences of ABCB5 upregulation in CRC cells. Using publicly accessible databases, we explored ABCB5 positivity in CRC cells and found novel interactions with 64 differentially expressed genes involved in cellular processes like modulation of inflammation, (XCL1, TNFSF4), enhanced cell signaling via interaction with serine protease inhibitors (SERPINB4, WFDC6) and downregulation of anti-apoptotic proteins (SPIN2A). Although ABCB5 upregulation is not currently targetable with pre-clinical compounds, our analysis revealed the genetic effects induced by ABCB5 upregulation are targetable, including evidence of sensitivity to the XPO1 inhibitor XPOVIO (selinexor). It is clear further pre-clinical investigation is needed to validate these findings.

Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein

Nucleocytoplasmic transport of signaling modulators is essential for regulating cellular responses to extracellular stimulation and stress, as well as pathogen infection. Exportin 1 (XPO1), also known as chromosomal maintenance 1 (CRM1), mediates nuclear export of proteins, rRNAs, snRNAs, and some mRNAs. In this study, we have identified an essential role of XPO1 in regulating Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication during primary infection of primary human umbilical vein endothelial cells. Treatment with an XPO1 inhibitor KPT-8602 and short hairpin RNA (shRNA)-mediated knockdown of XPO1 reduced KSHV lytic replication but had no effect on KSHV entry and trafficking. XPO1 inhibition induced retention of autophagy adaptor protein p62 (SQSTM1) in the nucleus, which enhanced activation of TBK1 and IRF3. As a result, nuclear accumulation of p62 increased expression of innate immune-related genes including IRF7, ISG15, IFIT1, IFIT2, and IFIT3, leading to a reduction of KSHV lytic replication. These results illustrate a novel mechanism by which XPO1 mediates innate immune response and KSHV replication, and identify XPO1 as a potential therapeutic target and KPT-8602 as a promising therapeutic agent for KSHV infection.

The nuclear export protein XPO1 - from biology to targeted therapy.

Exportin 1 (XPO1), also known as chromosome region maintenance protein 1, plays a crucial role in maintaining cellular homeostasis via the regulated export of a range of cargoes, including proteins and several classes of RNAs, from the nucleus to the cytoplasm. Dysregulation of this protein plays a pivotal role in the development of various solid and haematological malignancies. Furthermore, XPO1 is associated with resistance to several standard-of-care therapies, including chemotherapies and targeted therapies, making it an attractive target of novel cancer therapies. Over the years, a number of selective inhibitors of nuclear export have been developed. However, only selinexor has been clinically validated. The novel mechanism of action of XPO1 inhibitors implies a different toxicity profile to that of other agents and has proved challenging in certain settings. Nonetheless, data from clinical trials have led to the approval of the XPO1 inhibitor selinexor (plus dexamethasone) as a fifth-line therapy for patients with multiple myeloma and as a monotherapy for patients with relapsed and/or refractory diffuse large B cell lymphoma. In this Review, we summarize the progress and challenges in the development of nuclear export inhibitors and discuss the potential of emerging combination therapies and biomarkers of response.

BIOM-26. MOLECULAR PREDICTORS OF RESPONSE TO SELINEXOR IN RECURRENT GLIOBLASTOMA (GBM)

Abstract The nuclear export protein exportin 1 (XPO1) is overexpressed in many cancers, including GBM. Selinexor is an inhibitor of XPO1 that crosses the blood-brain-barrier and targets cancer cells by sequestering tumor suppressor proteins and oncoprotein mRNAs in the nucleus, inducing cancer cell apoptosis. We previously reported encouraging results from a phase II trial of selinexor for molecularly unselected patients with recurrent GBM (ASCO 2019). Pre-treatment tumors from 57 patients were exome and RNA sequenced to explore molecular correlates of response, in a hypothesis generating, post-hoc, exploratory analysis. We compared overall survival (OS) and progression-free survival (PFS) between mutated and wild-type patients. Two mutated genes were associated with longer survival in selinexor treated patients: DOCK8 (n=7; PFS, P=0.013, hazard ratio [HR]=3.75 [1.32–10.62]; overall survival, P=0.009, HR=15.39 [2.00–118.34]) and PDX1 (n=5, PFS, P=0.014, HR=4.468 [1.361–14.670]). Other commonly mutated genes in glioma, including IDH1 (n=9) were observed but not associated with survival. RNAseq data were used to infer differential protein activities, which were input into a machine learning model that compared patients with selinexor sensitive disease (best response of partial or complete response, n=7) and resistant disease (best response of progressive disease, n=23). We found the inferred activities of three proteins emerged as the most associated with response and could be combined in a model to accurately predict benefit from selinexor treatment (area under the ROC curve from leave one out cross validation = 0.89 permutation test P< 0.04). The three proteins were ZC3H12A (also called MCPIP-1), a negative regulator of inflammation; RAB43, a member of the RAS family that binds GTP and regulates vesicle trafficking, and SOCS3, a suppressor of cytokine signaling that can antagonize JAK/STAT signaling. Together these data identified mutations and proteins activities for identifying patients most likely to benefit from selinexor treatment. Further studies are required for validation. ClinicalTrials.gov:NCT01986348.

Exportin-1 (XPO1) Inhibition Sequesters p53 from MDM2 and MDM4 and Is Highly Synergistic with MDM2 Inhibition in Inducing Apoptosis in Wild-Type p53 Acute Myeloid Leukemias

Exportin-1 (XPO1) Inhibition Sequesters p53 from MDM2 and MDM4 and Is Highly Synergistic with MDM2 Inhibition in Inducing Apoptosis in Wild-Type p53 Acute Myeloid Leukemias

XPO1 Relieves MYC-Induced Replication Stress Limiting the Immunogenicity of DLBCL Cells

The soluble nuclear pore component XPO1 is a therapeutic target in DLBCL and its selective inhibitor selinexor received FDA approval in relapsed/refractory DLBCL. XPO1 inhibitors block the nuclear export of a subset of cargo proteins. Since the spectrum of XPO1 cargos varies among cell types, the mechanism underlying the anti-lymphoma effect of selinexor has not been fully elucidated. Lack of this knowledge hampers the clinical use of XPO1 inhibitors in terms of patient selection and development of rational therapeutic combinations. We reported that XPO1 is amplified and overexpressed in DLBCL. At the molecular level, we described that XPO1 overexpression functionally remodels the nuclear pore to sustain the expression of DNA damage repair proteins increasing lymphoma cells' tolerance to genotoxic stress. Here, we investigated whether this XPO1 role is relevant for the anti-lymphoma effect of selinexor. Aberrant MYC expression induces DNA damage resulting from replication stress; thus, we speculated that selinexor kills MYC expressing (MYC+) DLBCL cells by limiting their ability to tolerate replication stress. Using B-cells expressing MYC in an inducible manner, we demonstrated that exposure to selinexor (6 h) caused G2-M cell cycle arrest upon MYC expression (p<0.0001, vs. vehicle-treated cells). Cell cycle arrest was associated with increased levels of DNA damage in S-phase as measured by yH2AX (p<0.0001). Furthermore, although MYC increased the expression of key replication stress repair proteins (i.e. RAD51, WEE and BRCA1), this effect was blunted by selinexor. To determine the clinical relevance of this mechanism, we interrogated publicly available datasets of DLBCLs and found that XPO1 amplification and overexpression was associated with MYC translocation (p<0.005), double-hit lymphoma signature (p<0.005), and elevated levels of chromosomal instability (p<0.005). Overall, these observations suggest that MYC-driven lymphomas sustaining high levels of genomic instability may critically depend on XPO1. To therapeutically capitalized this mechanism, we evaluated the effect of selinexor on MYC+ DLBCLs. Clinically achievable doses of selinexor induced cell cycle arrest and apoptosis in 83% (5/6) of cell lines investigated in vitro. In a patient-derived xenograft (PDX) MYC+ DLBCL model, a human-equivalent dose of selinexor impaired tumor growth (p< 0.05, vs. vehicle) without significant toxicities. Reduced tumor growth was secondary to apoptosis as determined by TUNEL (p<0.05). To characterize the biological effects of XPO1 inhibition, we compared the transcriptome of PDX from the previous experiment. Pathway analysis of differentially expressed genes revealed that selinexor induced pathways regulating innate immune signaling. The upregulation of key genes from this pathway (i.e. DDX58, DDX60, IRF7 and STAT1) was validated by qRT-PCR. Since unrepaired DNA damage elicits an innate immune response, we hypothesized that these effects could be mechanistically linked. To test this notion, we exposed MYC+ DLBCL cells to sub-lethal doses of the DNA damaging agent etoposide, selinexor, or their combination. Expression of innate immune genes was significantly higher in cells exposed to the combination compared to either drug alone (p<0.001, for both comparisons), indicating that selinexor increases the immunogenicity of MYC+ DLBCL cells and this effect is enhanced by concomitant replication stress. Furthermore, by analyzing primary DLBCLs we found that poorly immunogenic lymphomas, characterized by an immune deserted microenvironment, expressed higher levels of XPO1 and MYC compared to DLBCL with an immune rich microenvironment (P<0.001, for both genes). To determine a potential effect of selinexor in the function of immune effector cells, we performed single cells secretomic analysis (Isolight) of circulating CD8+ T cells obtained from two DLBCL patients before and after selinexor treatment and found that selinexor increases the secretory activity of CD8+ T cells in both patients. Overall, our study reveals a key oncogenic role for XPO1 in enabling DLBCL cells to tolerate MYC-induced replication stress while limiting their immunogenicity. This suggest that patients with MYC+ DLBCL and elevated genomic instability may benefit from therapeutic approaches based on XPO1 inhibitors in combination with immunomodulatory drugs, an approach we will test prospectively. Disclosures Rutherford: AstraZeneca: Consultancy; Celgene: Consultancy; Dova: Consultancy; Genentech/Roche: Research Funding; Heron: Consultancy; Juno: Consultancy; Karyopharm: Consultancy, Research Funding; Kite: Consultancy; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Seattle Genetics: Consultancy. Martin:Regeneron: Consultancy; Cellectar: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Morphosys: Consultancy; Kite: Consultancy; Incyte: Consultancy. Leonard:Miltenyi: Consultancy; Sutro: Consultancy; Roche/Genentech: Consultancy; BMS/Celgene: Consultancy; Regeneron: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; Epizyme: Consultancy; AstraZeneca: Consultancy. Cerchietti:Cellgene: Research Funding; Bristol Myers Squibb: Research Funding.

CRISPR/Cas9 Chemogenetic Profiling Identifies Candidate Biomarker Genes That Modulate Sensitivity to Selinexor

Introduction Selinexor (KPT-330) is a first-in-class selective inhibitor of XPO1-mediated nuclear export that has recently been approved by the US FDA for the treatment of relapsed/refractory multiple myeloma and relapsed diffuse large B-cell lymphoma. Additionally, the drug is being evaluated in ongoing clinical trials (phase I to III) on late-stage haematological and solid tumours. To improve outcomes and personalize therapeutics, specific biomarkers and underlying mechanisms of response to treatment need to be uncovered. Material and method We performed genome-wide CRISPR/Cas9-mediated loss-of-function genetic screening in two multiple myeloma (KMS-28-BM and SK-MM-1) and one chronic myeloid leukemia (HAP1) cell lines. After transduction with a knockout library, cells were divided and subsequently cultured into three parallel groups (control, IC20 and IC50) for at least 20 cell doublings. This setup allows monitoring of subtle relative changes between groups. After DNA extraction, sequencing of guide RNAs and statistical analysis, we identified genes that upon disruption confer either increased susceptibility or resistance to selinexor. Results and discussion For the multiple myeloma cell lines, the TGF-β/SMAD4 signalling pathway stood out as an important mediator of resistance to selinexor. Besides SMAD4 as a major hit in both cell lines, we identified five other genes from this pathway leading to resistance in KMS-28-BM cells, suggesting that downregulation of this pathway is a potential biomarker of response to SINE treatment in multiple myeloma. In addition, screens revealed significant variability across cell lines with hit genes involved in mRNA processing, proteasomal degradation and cell cycle regulation. Interestingly, we could simultaneously uncover resistance and sensitivity genes involved in the same pathway, suggesting that these have antagonistic effects. Notably, we identified knockout of the poorly characterized gene ASB8 as a strong, common sensitizer between the three cell lines. Conclusion Until now, most chemogenetic CRISPR/Cas9 knockout screens have focused on determining resistance genes, and few have employed the technique to screen for genes that enhance cell kill. We performed such screens with selinexor and found a number of general and cell type-specific hits. Genes identified to confer increased resistance are candidate biomarkers while genes inducing increased susceptibility represent new targets for drug combination therapies. Disclosures Kwanten: Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor). Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Daelemans:Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor).

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

Inhibiting the Nuclear Exporter XPO1 and the Antiapoptotic Factor BCL2 Is Synergistic in XPO1 Mutant and Wildtype Lymphoma

We have recently shown that XPO1 mutations are drivers of lymphomagenesis and occur across B-cell lymphomas, specifically in chronic lymphocytic leukemia (CLL), classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. The co-occurrence of other oncogenic events cooperating with XPO1 provides an opportunity for combined targeted therapy. Increased expression of the anti-apoptotic factor BCL2 has long been known to be a critical part of the pathophysiology of B-cell lymphomas. Recently, the oral BCL2 inhibitor, venetoclax, was approved in CLL and is currently being evaluated in clinical trials for other B-cell lymphomas. Selinexor is a potent, oral XPO1 inhibitor that was recently approved in multiple myeloma and diffuse large B-cell lymphoma. XPO1 inhibition exerts its antineoplastic effects by blocking key lymphomagenic pathways, such as NFκB, and decreasing the anti-apoptotic protein survivin. We therefore hypothesized that combining selinexor and venetoclax would have potential synergy and provide an oral precision combination therapy for relapsed, refractory lymphoma. We first set out to determine whether XPO1 mutant lymphoma cell lines showed differential response to either selinexor or venetoclax monotherapy. Five lymphoma cell lines; 3 diffuse large B-cell lymphoma (SU-DHL-6, SU-DHL-16, FARAGE) and 2 classical Hodgkin lymphoma (L428 and SUP-HD1), were subjected to next-generation sequencing (NGS) to assess for the presence or absence of XPO1 mutations. SUDHL-16 and SUP-HD1 were heterozygous for the XPO1 E571K hotspot mutation while SUDHL-6, FARAGE and SUP-HD1 were wildtype at the XPO1 locus. These 5 cell lines were used to assess sensitivity to Selinexor and/or Venetoclax. The CellTiter Glo assay was used to assess cell viability after 72 hours of treatment. Assays were performed in triplicate on 96-well plates that were read using a Spectramax plate reader. The XPO1 mutant cells showed increased sensitivity to selinexor (XPO1 mutant IC50 = 16-35nM; XPO1 WT IC50 = 41-231nM) as previously seen in conditional knockin mouse models of XPO1 mutant CLL (Figure A). Additionally, the XPO1 mutant cell lines showed increased sensitivity to single-agent venetoclax (XPO1 mutant IC50 = 2-13nM; XPO1 WT IC50 = 5-2853nM), an observation that has not previously been made (Figure B). Next, we tested the synergy of the combination of selinexor and venetoclax in the XPO1 mutant and wildtype cell lines. Increasing concentrations of the individual drugs were applied to each individual cell line in a 6x6 matrix. The cell viability percentage for each concentration was then entered into a synergy finder (www. synergyfinder.fimm.fi). The Bliss Independence model was used to calculate synergy of the Selinexor-Venetoclax combinations. As hypothesized, the combination of selinexor and venetoclax indeed showed synergy in both the wildtype and mutant XPO1 cell lines. Furthermore, the XPO1 mutant cell lines showed a higher degree of synergy compared to the wildtype cells (Figure C). Finally, a remarkable patient allowed us to test this combination ex vivo. This patient with CLL had undergone multiple therapies including chemoimmunotherapy, ibrutinib and venetoclax monotherapies. This patient had a founder XPO1 E571K mutation and also had acquired a BTK C481S ibrutinib resistance mutation and MYC amplification. These cells were unique in that they were easily able to be tested in ex-vivo culture to test sensitivity to different therapies. When tested with chemotherapy or ibrutinib they were completely resistant, and even with venetoclax they were fairly resistant; however, they remained sensitive to XPO1 inhibition with Selinexor. Selinexor and venetoclax showed remarkable synergy measured by a BLISS delta score of 18.78 (Figure D). In conclusion, inhibiting the nuclear exporter XPO1 and the anti-apoptotic factor BCL2 is synergistic in both XPO1 wildtype and mutant lymphoma. XPO1 mutant lymphomas show increased sensitivity to both selinexor and venetoclax. Additionally, selinexor and venetoclax showed a higher degree of synergism in XPO1 mutant lymphoma cell lines and were highly synergistic in primary XPO1 mutant CLL patient cells ex vivo. This combination is highly promising as an all oral alternative for relapsed, refractory lymphoma. Next steps include preclinical testing in mouse models in vivo using XPO1 mutant and wildtype mice crossed with mice overexpressing BCL2. Figure Disclosures No relevant conflicts of interest to declare.

Integration of Deep Multi-Omics Profiling Veals New Insights into the Biology of Poor-Risk Acute Myeloid Leukemia

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Frontline Selinexor and Chemotherapy Is Highly Active in Older Adults with Acute Myeloid Leukemia (AML)

Background: Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by resistance to treatment and dismal outcomes, especially in the elderly. Novel approaches are desperately needed. Exportin 1 (XPO1) is involved in the selective nuclear export of certain proteins and RNA species. It is overexpressed in a subset of AML, conferring an adverse prognosis. Selinexor is a small-molecule inhibitor of XPO1 with activity in AML. Selinexor sensitizes AML cells to anthracyclines by retaining topoisomerase II in the nucleus resulting in increased DNA strand breaks. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in AML. This abstract reports the ongoing results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML, 60 years of age or older and preclinical studies to assess the mechanisms of chemo-sensitization. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized 3:1 between 7+3+selinexor or 7+3. Responding patients could go on to high dose cytarabine consolidation with or without selinexor as per initial randomization. Patients in the selinexor arm who completed all consolidation could then move to maintenance therapy with selinexor alone. Induction consisted of cytarabine 100 mg/m2/d by continuous infusion for 7 days and daunorubicin 60 mg/m2 on days 1-3. Consolidation consisted of cytarabine at 1.5 gm/m2 given Q12 hours days 1-3 with G-CSF given 24 hours following the last dose of cytarabine. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days during maintenance. Preclinical studies were conducted with murine AML cell lines. Results: Twenty-seven of a planned twenty-eight patients were enrolled to date. Of the 27 evaluable patients, 20 were randomized to the selinexor arm and 7 to the control arm. Baseline demographics are listed in Table 1. In the standard arm, both 30- and 60-day mortality were 14% (1/7). In the selinexor arm, both 30- and 60-day mortality were 10% (2/20). In the standard arm, 43% (3/7) of patients achieved a complete remission (CR) or complete remission with incomplete count recovery (CRi). Of the 3 responders 1 has gone on to transplant. In the selinexor arm, 85% (17/20) of patients achieved a CR or CRi. Of the 17 responders, 4 have gone on to transplant. Progression free and overall survival both favor the selinexor arm with trends towards significance despite the small size of the trial (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. Selinexor induces retention of topoisomerase II in the nucleus increasing sensitivity to anthracyclines. To determine if selinexor sensitized to cytarabine viability assays using murine AML cell lines were done (Figure 2A). Selinexor significantly sensitized both cell lines to cytarabine. AML cells increase mitochondrial oxygen consumption in response to cytarabine leading to resistance. The ability of selinexor to interfere with this response was assessed using Seahorse flux analysis. AML cells treated with cytarabine for 16 hours showed a diminished mitochondrial oxygen response when co-treated with selinexor (Figure 2B). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older patients with de novo AML. Selinexor may increase response to cytarabine by interfering with nuclear-mitochondrial communication. Enrollment is ongoing. Disclosures Pardee: Rafael Pharmaceuticals: Consultancy; AbbVie: Consultancy; Genentech, Inc.: Consultancy; BMS: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Research Funding; Rafael: Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pharmacyclics: Speakers Bureau. Ellis:Rafael Pharmaceuticals: Consultancy. Howard:Jazz Pharmaceuticals: Consultancy. Powell:Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Genentech: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding. OffLabel Disclosure: Selinexor for the treatment of AML

A Six-Protein Activity Signature Defines Favorable Response to Selinexor Treatment for Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Selinexor is an oral selective inhibitor of XPO1 that was recently approved by the US FDA for treatment of adult patients with relapsed or refractory DLBCL, after at least two lines of systemic therapy. Approval was based on results of the Phase 2b SADAL study, which had an overall response rate of 29% in the primary analysis population (clinicaltrials.gov: NCT02227251). We herein investigated molecular markers of response to selinexor from patients treated on the SADAL study. Methods: Exome sequencing was performed on pre-selinexor treatment biopsies from 55 patients and used to compare mutation frequencies between 21 responder patients (best overall response of complete response [CR], 6; and partial response [PR], 15) and 34 non-responder patients (stable disease [SD], 8; and progressive disease [PD], 26). Additionally, RNA sequencing was performed on a subset of 33 patients and gene expressions were used to infer activities of regulatory proteins with the VIPER algorithm. Differences in inferred protein activities between responder and non-responder patients were assessed using four machine learning algorithms: linear regression (LR), linear discriminant analysis (LDA), ridge regression (RR) and random forest (RF). Model performance was estimated using leave-one-out cross-validation (LOOCV). A separate comparison was performed in the subset of 12 patients with germinal B-cell like (GCB) DLBCL. Results: Our analysis of genes commonly mutated in DLBCL revealed that non-responder patients more frequently harbored mutations in KMT2D (35% non-responders, 14% responders). Examination of the specific types of KMT2D mutations showed that the vast majority were loss-of-function frameshift or nonsense mutations (13 of 15 mutations), indicating they could have functional relevance to disease biology. The activities of 5,742 regulatory proteins were successful inferred from RNA sequencing performed on 33 patients. Unsupervised clustering identified two outlier samples that were removed from further analysis. The remaining 31 patients consisted of 16 responders (CR, 5; and PR, 11) that were compared to 15 non-responders (PD, 15). Dimension reduction of the 5,742 protein activities (filtering proteins with low variation, poor VIPER imputation, and strong linkage) resulted in 680 independent informative regulatory protein activities used for predicting selinexor response. Different numbers of regulatory proteins were iteratively input into the machine learning models to compare responders and non-responders. The best performance model was achieved using only six proteins (ASH1L, ZNF471, RRN3, CD248, ZNF750, INHBA) (Figure 1A), and had an area under the receiver operating characteristic curve (AUC) of 0.917, 0.925, 0.883, and 0.875, for the LDA, LR, RF and RR, models, respectively (p < 0.05, permutation test) (Figure 1B). A final integrated model combining the four methods achieved an AUC = 0.929 (p < 0.05, permutation test, AUC 95% CI: [0.831-1] DeLong non-parametric method) (Figure 1C). Similar results were obtained using 5-fold cross validation with the six-protein activity signature (integrated model AUC = 0.858, AUC 95% CI: [0.72-0.997]). Finally, we focused separately on 12 patients with germinal B-cell like (GCB) DLBCL, (6 responder patients, and 6 non-responder patients). Using LOOCV with the top three protein activities associated with selinexor response in GCB-DLBCL (COL1A1, INHBA, and CNOT2) resulted in remarkably high accuracy, with an integrated model AUC = .972 (p < 0.05, permutation test, AUC 95% CI: [0.895-1]). Discussion and Conclusions: The six proteins used for defining the DLBCL selinexor response signature are not typically associated with a role in DLBCL but have been implicated in cancer biology in other contexts. Notably, INHBA was found as predictor of response in the full set and also the GCB subtype patients, suggesting that activin/inhibin signaling could be important for response to selinexor in patients with DLBCL, especially the GCB subtype. Our results produced a protein activity signature that could be useful for identifying patients with DLBCL likely to respond well to selinexor treatment, which will be validated in a larger independent sample set. Figure 1 Disclosures Walker: Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Alvarez:DarwinHealth, Inc: Current Employment, Current equity holder in private company. Chang:Karyopharm Therapeutics Inc: Current Employment. Shah:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Shacham:Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Califano:DarwinHealth, Inc: Consultancy, Current Employment, Current equity holder in private company, Other: Founder. Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company.

Selinexor in Combination with R-CHOP for Frontline Treatment of Non-Hodgkin Lymphoma: Results of a Phase 1b Study

Selinexor in Combination with R-CHOP for Frontline Treatment of Non-Hodgkin Lymphoma: Results of a Phase 1b Study

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

73P Selinexor in combination with carboplatin and pemetrexed (CP) in patients with advanced or metastatic solid tumors: Results of an open label, single-center, multi-arm phase Ib study

Exportin 1(XPO-1) is overexpressed in various tumors and selinexor is a first-in-class oral potent selective inhibitor of XPO-1, leading to intranuclear accumulation of tumor suppressor proteins. In vivo studies have suggested that selinexor in combination with various chemotherapeutics exerts antitumor activity in multiple cancers. This was an open label, single-center, multi-arm phase Ib study utilizing a “3 + 3” design and a “basket type” expansion. CP + selinexor was employed as one of the 13 parallel arms. While carboplatin was dosed at AUC6 along with pemetrexed at 500 mg/m2 IV Q3W, selinexor was dosed at 60 mg twice weekly (BIW) or 40-60 mg once weekly (QW). Patients with advanced or metastatic solid tumors whose disease was refractory, had relapsed following prior systemic therapy or where the addition of selinexor to standard chemotherapy deemed appropriate and acceptable, were eligible. Six patients with the median age of 51 (range, 37–69 years) were treated, and 4 were evaluable for response. The cancer types were ovarian (n=2), and 1 each with thymoma, cervical, rectal, and non-small cell lung cancers. All patients had at least one treatment-emergent adverse events (TEAE) and the common TEAE were thrombocytopenia (6/6), neutropenia (5/6), fatigue (5/6), anemia (4/6), leukopenia (4/6), elevated AST or ALT (3/6), nausea (3/6), and vomiting (3/6). The most prevalent grade ≥3 TEAE were thrombocytopenia (4/6), anemia (3/6), neutropenia (3/6), leukopenia (2/6), and fatigue (2/6). A patient dosed at selinexor 40mg QW experienced DLT with grade 3 fatigue despite medical supportive care ≥5 days. The patient with lung adenocarcinoma who had 2 prior therapies including prior carbo-taxol achieved partial response (PR) whereas 2 patients with ovarian cancer and cervical cancer had stable disease (SD), contributing the clinical benefit rate (CR+PR+SD ≥4 months) of 50%. Albeit the number of patients in this study arm was small, once weekly oral selinexor in combination with CP was plausible and conferred some clinical activity and disease stabilization. The RP2D of selinexor was 40 mg QW in combination with CP.