E2F1 Is a Biomarker of Selinexor Resistance in Relapsed/Refractory Multiple Myeloma Patients
Authors: Lagana Alessandro , Park Seongjee , Edwards Donna , Leshchenko Violetta , Crochiere Marsha , Landesman Yosef , Melnekoff David , Beno Itai , Perumal Deepak , Madduri Deepu , Richter Joshua , Chari Ajai , Cho Hearn Jay , Barlogie Bart , Jagannath Sundar , Dudley Joel , Parekh Samir 
Published: October 2019

Abstract
Selinexor (KPT-330) is a selective inhibitor of nuclear export (SINE) which specifically targets XPO1 (Exportin 1)-mediated nuclear export, leading to increased nuclear retention of major tumor suppressor proteins and inducing selective apoptosis in cancer cells. Several phase I and II clinical trials demonstrate evidence of anti-cancer activity of Selinexor in solid tumors (i.e metastatic prostate cancer (PMID: 29487219), advanced refractory bone or soft tissue sarcoma (PMID: 27458288) and non-small cell lung cancer (PMID: 28647672); as well as, hematological malignancies, including non-Hodgkin lymphoma (PMID: 28468797), acute myeloid leukemia (PMID: 29304833) and multiple myeloma (MM) (PMID: 29381435). In the STORM (Selinexor Treatment of Refractory Myeloma) trial, the combination of Selinexor with dexamethasone in MM patients refractory to bortezomib, carfilzomib, lenalidomide and pomalidomide (quad-refractory), or in addition, to daratumumab (penta-refractory), has shown an overall response rate (ORR) of 21% (Vogl et al, JCO 2018). Our objective is to identify biomarkers for selection of patients at higher likelihood of clinical benefit from Selinexor salvage and understand mechanisms of Selinexor resistance.
We therefore analyzed transcriptional differences using RNA sequencing in CD138+ cells from bone marrow aspirates obtained prior to treatment from 32 MM patients enrolled in STORM. The raw data (fastq) was mapped by using the tool STAR and gene-level annotated by featureCounts. Patients were split in two groups based on their progression-free survival (PFS). Differential expression analysis was performed using the tool DESeq2, which enables a more quantitative analysis of comparative RNA-seq data using shrinkage estimators for dispersion and fold change. The results revealed significant up-regulation of 13 genes in patients with PFS < 120 days (n = 21, p < 0.05) versus patients with PFS > 120 days (n=11), including the transcription factor E2F1 and its targets MYBL2, FANCA, GINS3 and SLX4 (Fig. 1).
Next, we evaluated the expression of E2F1 in another set of 26 patients from the STORM trial by Affymetrix U133 gene expression microarrays. Data was analyzed using the Signal Space Transformation (SST)-Robust Multi-Chip Analysis (RMA) algorithm. Patients with PFS < 120 days (n = 19) exhibited significant up-regulation of E2F1 (p < 0.05) (Fig. 2).
E2F1 is a transcription factor that regulates cell cycle G1/S progression. At rest, E2F1 is complexed with its negative regulator retinoblastoma(RB) protein. Upon phosphorylation of RB by the Cyclin D1-CDK4/6 complex, pRB is inactivated allowing E2F1 to commence transcription of target genes allowing G1/S progression. E2F transcription factors are exported by XPO1 from the nucleus to the cytoplasm. We treated RPMI8226 (IC50=150nM) and MM1S (IC50=25nM) human myeloma cell lines with Selinexor at IC50 and examined nuclear vs cytoplasmic expression of E2F1 after 24 and 48 hours by western blotting. Our results demonstrated nuclear retention of E2F1 following treatment of HMCLs with Selinexor and suggest a model where overexpression of E2F1 overwhelms the nuclear export mechanism and may result in downstream gene programming that confers a proliferative advantage in cells, manifested by rapid progression (<120 days) in patients.
Our findings suggest a model where E2F1 expression may be a biomarker of Selinexor resistance. We are currently validating our findings in additional samples from patients with MM treated with Selinexor.

Disclosures
Crochiere: Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics Inc: Employment. Chari:Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Research Funding; Bristol Myers Squibb: Consultancy; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; The Binding Site: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cho:Janssen: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; J & J: Consultancy; Agenus Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Barlogie:Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Millenium: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Celgene: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend. Jagannath:Multiple Myeloma Research Foundation: Speakers Bureau; Merck: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medicom: Speakers Bureau.