Selinexor in Combination with Chemotherapy or Idelalisib Elicits a Synergistic Cytotoxic Effect in Primary CLL Cells, Also Overcoming Intrinsic and Stromal Cells-Mediated Fludarabine Resistance

BACKGROUND: Despite the therapeutic efficacy of new target drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Disease aggressiveness can be ascribed to intrinsic features of the tumor cells such as immunoglobulin heavy chain (IGHV) mutational status and TP53 disruption, which are strong determinant of drug response. Many of the tumor suppressor and growth regulatory proteins with a known pathogenic role in CLL (i.e. p53, NF-kB, Akt, IkB) bind the nuclear export protein XPO1 (Chromosome Region Maintenance 1; CRM1) and are carried through the nuclear pore complex into the cell cytoplasm. Elevated protein levels of XPO1 and specific XPO1 mutations have been reported in various hematologic and solid tumors. In particular, XPO1 is overexpressed and recurrently mutated in CLL cells (Puente XS et al, Nature 2011; Lapalombella R et al, Blood 2012). Selinexor (KPT-330) an oral inhibitor of XPO1, is active as single agent in different hematologic malignancies including acute myeloid leukemia, non-Hodgkin lymphomas, CLL and multiple myeloma. The combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival of a CLL mouse model compared with ibrutinib alone (Hing ZA et al, Blood 2015).

AIM: The aim of this study is to evaluate the additive or synergistic in vitro cytotoxic effects of selinexor, used in combination with chemotherapeutic drugs or the new PI3k inhibitor idelalisib against primary CLL cells. Specifically, this study aims at identifying combination regimens that might overcome single agent resistance.

METHODS: 15 patients with CLL were included in the study, among these 9 with mutated (M) and 3 patients with unmutated (UM) IGHV; for 3 patients the mutational status was not available at the moment of data analyses. Purified CLL cells were exposed, alone or in presence of the murine stromal cell line M2-10B4, to selinexor (10 nM, 100 nM, 1 uM and 10 uM) in combination with fludarabine (F-ara-A, 10 nM, 100 nM, 1 uM and 10 uM), bendamustine (Ben, 3 mM, 10 mM, 30 mM and 50 mM) or idelalisib (Ide, 10 nM, 100 nM, 1 uM and 10 uM) for 24, 72 and 120 hours. Cell viability was analysed by Annexin-V/propidium Iodide (AnnV/PI) immunostaining and flow cytometry. Samples were considered resistant when the relative viability of F-ara-A treated CLL cells compared to untreated control was >0.5. Combination analysis was performed using Calcusyn software; combinations were considered synergistic when CI was <1.

RESULTS: Leukemic cells were cultured in the presence of increasing concentrations of selinexor, used alone or in combination with F-ara-A and Ben, and with the PI3Kδ inhibitor, Ide. After 72 hours of culture, the mean percentage of viable AnnV-/PI- CLL cells significantly decreased by 0,62-ratio following KPT-330 (100 nM), 0,42-ratio following F-ara-A (1 uM) and 0,24-ratio after dual treatment, compared to untreated controls. Combination analysis showed that selinexor and F-ara-A strongly synergize in inducing CLL cells apoptosis with a CI < 1. Similarly, we observed a synergistic interaction between selinexor (100 nM) and Ben (30 mM) that significantly enhanced the cytotoxic effect of the individual drugs (0,62-ratio for selinexor, 0,68-ratio for Ben and 0,41-ratio for selinexor + Ben) with a CI <1, at the same time point. The combination between selinexor (100 nM) and Ide (10 nM) at 72 hours resulted in a weaker, although significant, viability reduction (0,62-ratio for KPT-330, 0,5 for Ide and 0,38 for selinexor + Ide). We observed that IGHV UM CLL cells showed higher fold reduction values in cell viability when exposed to synergistic combinations, compared to IGHV M cells. Selinexor (100 nM) was also effective in impairing the viability of CLL cells that showed intrinsic resistance to F-ara-A (n=6, 0,61-ratio for selinexor, 0,74-ratio for F-ara-A and 0,4-ratio for selinexor + F-ara-A). Lastly, we exposed CLL-stromal cells co-coltures to the identified synergistic combinations and found that selinexor + F-ara-A or selinexor + Ide significantly reduced the viability of leukemic cells, effectively counteracting the protective effect exerted by stromal cells toward drug-induced apoptosis.

CONCLUSIONS: Our data demonstrate that the combination of selinexor with chemotherapy or Ide has synergistic cytotoxic effects, also counteracting intrinsic or stromal cells-mediated drug resistance.