Gene Expression and Transcription Factor (TF) Activation Profiling Identifies Suppression of Multiple Myeloma (MM) Cell Survival and Chemoresistance Pathways By Inhibition of XPO1/CRM1-Dependent Nuclear Export with Selinexor

INTRODUCTION

Despite improved response rates following the use of novel therapeutics, including proteasome inhibitors (PI) and immunomodulatory drugs, the majority of MM patients develop chemoresistance and relapse. Thus, it is essential to identify novel drugs that overcome resistance and promote remission. Our previous work detailed the synergistic activity of combined treatment with selective inhibitor of nuclear export (SINE) selinexor and PI carfilzomib (CFZ) in MM (Rosebeck et al, Blood 2013:122(21):279). In this study, we sought to characterize the underlying mechanism of selinexor, a novel drug being tested in multiple phase I/II clinical trials of advanced-stage cancers.

METHODS

Plasma cells (PC) were purified from MM patient bone marrow (BM) aspirates using EasySep (STEMCELL Tech). RPMI 8226, 8226/Dox40, and MM1S cells were cultured in RPMI1640/10% FBS. RNA was isolated with RNeasy minicolumns (Qiagen) from control or MM1S cells treated for 6 hrs with selinexor. Gene expression profiling was done on Illumina HumanHT12 microarray and InfernoRDN. Differentially expressed genes were input into Ingenuity Pathway Analysis software (Qiagen). Fisher’s exact test was used to calculate p-values for significant pathway enrichment. TF profiling plate array was from Signosis.

RESULTS

Selinexor treatment inhibited key MM pathway regulators including transforming growth factor beta 1 (TGFB1; p=1.65E-14), which is involved in paracrine BM signaling and bone formation, and the epidermal growth factor (EGF) receptor HER2 (p=4.84E-08), which deregulates EGF signaling and promotes survival. SINE also affected integrin-associated signaling (p=2.42E-03), which controls MM/BM stromal cell adhesion and contributes to chemoresistance.

We profiled SINE-dependent changes in TF activation and found increased DNA binding of cAMP response element-binding protein (CREB), which represses IL-6 production, an essential MM cell survival cytokine, and retinoid X receptor (RXR), which is associated with MM cell death. We also found impaired DNA binding of heat shock factors (HSF), which sensitizes MM cells to PI, Kruppel-like factor 4 (KLF4) and multidrug resistance (MDR) promoter-enhancing factors 1 and 2 (MEF-1/2), which are associated with chemoresistance in MM, nuclear respiratory factor 1 (NRF-1), which mediates a recovery pathway to compensate for reduced proteasome function, and paxillin 2 (Pax2), a transcriptional repressor of p53 and enhancer of Wilms tumor protein (WT1). In addition, TF associated with poor prognosis MM, including the pre-B cell homeobox (PBX) oncogene, soluble mucin 1 (SMUC), and WT1, were inhibited.

Next, we tested the ability of selinexor to overcome drug resistance in MM cells. 8226/Dox40 are insensitive to the effects of CFZ via upregulation of p-glycoprotein, however selinexor effectively killed these cells. Importantly, we demonstrate efficacy of SINE on PC from patients refractory to and/or relapsing (R/R) on PI-containing regimens. Additionally, we will present preliminary responses from CFZ-refractory patients enrolled in our newly-opened multi-site phase I clinical trial conducted in the MM Research Consortium combining selinexor and CFZ with dexamethasone in R/R MM (NCT02199665).

CONCLUSIONS

Our results suggest selinexor-dependent inhibition of pathways that promote intrinsic MM cell survival and contribute to chemoresistance in the BM milieu, which may be mediated by suppression of essential growth factors and genes involved in MM cell/BM stroma adhesion. We also found inhibition of TF that may influence MDR/PI resistance either directly (KLF4, MEF-1/2) or indirectly (HSF, NRF-1). Finally, we demonstrate the ability of SINE to overcome MDR in MM cells and PC from patients with R/R disease. Our studies provide evidence of the pleiotropic effects of SINE and underscore the potential of selinexor to be an effective therapeutic for advanced-stage cancers, including MM.