The Synergistic Effect of Melphalan and XPO1 Inhibition in Pre-Clinical Models of Multiple Myeloma

Introduction:Multiple myeloma (MM) is an incurable cancer of plasma cells. It accounts for approximately 10% of all hematologic malignancies. In the US, it is estimated that there will be approximately 30,330 new cases and 12,650 deaths in 2016. In the past decade, responses/survivals have been significantly increased by newer therapies. However, almost all of the patients will eventually die from multi-drug resistant disease.

Materials and Methods:Weused XPO1 inhibitors (XPO1i) selinexor (300nM) or KPT-8602 (300nM) +/- melphalan (15 μM) to treat human MM parental RPMI8226 and U266 cells, and melphalan resistant LR5 and LR6 cell lines for 20 hours and then assayed for apoptosis and viability by flow cytometry. DNA damage was assayed by the comet assay and phospho-H2AX protein expression in H929 human myeloma cells. p53, NFkB, IKKα, FANCF, and FANCL were assayed by Western blot in H929 MM cells. We also treated cells from patients with newly diagnosed or relapsed/refractory MM with the XPO1i (300nM)/ melphalan (10μM) combination and assayed for apoptosis. In addition, selinexor/melphalan treated NOD/SCID-gamma mice with U226 MM tumors were assayed for tumor growth, survival, and toxicity.

Results:Cell viability of all tested MM cell lines was decreased synergistically and apoptosis increased by XPO1i/melphalan treatment (selinexor/melphalan, P = 2.2x10E-6 to 0.0032, KPT-8602/ melphalan, P = 1.2X10E-7 to 0.0031). Comet assays showed that the XPO1i/ melphalan drug combination increased DNA damage more than single agent melphalan or XPO1i alone. Phospho-H2AX expression also was increased (selinexor/ melphalan, P = 0.005 and KPT-8602/melphalan, P = 0.001). Western blot analysis showed that XPO1i treatment can increase p53 and decrease NFkB, IKKα, FANCF, and FANCL in MM cells. Apoptosis assays showed that both melphalan-resistant and parental MM cell lines were sensitized to melphalan by XPO1i. In addition, CD138+/light chain+ MM cells from newly diagnosed and relapsed/refractory MM patients were sensitized (20-fold and 5 to10-fold respectively) by XPO1i to melphalan. XPO1i/melphalan combination treatment demonstrated a strong synergistic anti-tumor effect when compared to single-agent melphalan (selinexor, P = 0.0024 and KPT-8602, P = 0.0030) in NOD/SCID-gamma mice challenged with U266 MM tumors. XPO1i/ melphalan treated mice had increased survival and no significant toxicity.

Conclusions:XPO1i's can improve the response of human MM cell lines and patient MM cells to melphalan both in vitro and ex vivo. The mechanism of this synergy reversing melphalan resistance may be due to increased nuclear p53, in combination with decreased NFkB and IKKα, and decreased DNA repair proteins FANCL and FANCF of the Fanconi Anemia/BRCA pathway. Our preliminary data suggest that the synergistic cell kill may be because XPO1i's increase melphalan-induced DNA damage and block the repair of the DNA damage. Thus using combination therapies of XPO1i, especially the clinical compounds selinexor and KPT-8602 +/- melphalan may have potential to improve the treatment outcomes of MM. Based on these promising pre-clinical data, we designed a phase 1/2 clinical trial evaluating the combination of selinexor and high-dose melphalan as a conditioning regimen for autologous hematopoietic cell transplantation in patients with multiple myeloma (NCT02780609).