XPO1 target occupancy measurements confirm the selinexor recommended phase 2 dose

Abstract

XPO1 (exportin 1) is the main nuclear export protein with over 200 different protein cargos. XPO1 is overexpressed in tumor cells and high levels are correlated with poor prognosis. Selective Inhibitor of Nuclear Export (SINE) compounds block nuclear export by inhibiting XPO1. The first SINE compound, selinexor, shows promising anti-cancer activity across hematological and solid tumors in Phase 2 and 3 clinical trials. The 2nd generation SINE compound KPT-8602 is being evaluated as an anti-cancer agent in a Phase 1 clinical trial. To predict patient response to treatment and confirm the selinexor recommended phase 2 dose (RP2D), an assay based on fluorescence cross correlation spectroscopy that measures XPO1 occupancy in cancer cells was developed. Studies comparing cytotoxicity and XPO1 occupancy in cell lines treated with selinexor or KPT-8602 indicated that XPO1 occupancy by both compounds could reach saturation regardless of drug sensitivity. However, higher levels of XPO1 protein correlated with lower sensitivity to SINE compound cytotoxicity. In vivo mouse studies showed XPO1 occupancy could be measured in tumors and was dose-dependent, with >90% target saturation at 10 mg/kg (∼50 mg flat dose in humans). Drug-target occupancy was measured in a dose-response time course and full occupancy occurred by 6 hours at all doses. The duration of occupancy was dose-dependent, where 10-15 mg/kg in mice (∼ 50-75 mg human flat dose) was necessary to maintain XPO1 occupancy up to 48 hours post-dose. These findings confirm the selinexor RP2D of 60 mg for achieving target occupancy and inhibition up to 48 hours.

Use of FCCS in drug discovery and development and presentation of a novel dedicated instrument for industrial FCCS applications

“…The data clearly indicate that 20mg/kg are necessary to obtain 50% TO after 48h. These data indicate the importance of the approach to find an optimal regimen for drug testing [12]. Patient samples drawn from a single person treated were analyzed for TO over a time course of 15 days as shown in figure 4C.…”

“…Lysates comprising respective proteins were incubated with increasing concentrations of fluorescently labeled NT(8-13) and analyzed by FCCS. Fig 2A: Dose dependent increase in complex formation of GFP fusions of NTR1 and its derivatives to Dy647-NT(8)(9)(10)(11)(12)(13). The stabilizing triple mutant TTM shows the highest affinity, followed by single mutant A85L.…”

The molecular mechanism and challenge of targeting XPO1 in treatment of relapsed and refractory myeloma

“…In addition, XPO1 is responsible for selectively transporting certain subsets of mRNAs, miRNA, and snRNA through exporting several RNA-binding proteins and adaptor proteins [ 119 , 120 ]. For example, XPO1: (1) selectively mediates the alternative export of both m 7 G-capped mRNAs and snRNA by binding to CBC20/80 [ 121 ]; (2) preferentially exports a subset of mRNAs encoding oncoproteins, such as Myc, CDC25A, BRD4, Bcl-2, Bcl-6, Mcl-1, Bcl-xL, cyclins, androgen receptor, and Pim1 by exporting several RNA-binding proteins, including LRPPRC, eIF4E, NXF3, and HuR; XPO1 thereby regulates the translation of these oncoproteins [ [121] , [122] , [123] , [124] , [125] , [126] , [127] , [128] , [129] ]; (3) exports U-snRNAs into the cytoplasm for modification and assembly, thus playing a critical role in the regulation of mRNA splicing through binding the adaptor protein PHAX; and (4) mediates the alternative export of both microRNAs and tRNAs.…”

Nuclear Export Inhibition for Pancreatic Cancer Therapy

“…One can argue that the pancreatic tumor-associated stroma may hinder appropriate delivery of selinexor to the tumor site. In this direction, using in vivo mouse studies, XPO1 occupancy could be measured in tumors (non-pancreatic model) and was found to be dose-dependent, with >90% target saturation at 10 mg/kg (which is equivalent to ~50 mg flat dose in humans) [ 91 ]. Drug-target occupancy was measured in a dose-response time course and full occupancy occurred by 6 h at all doses.…”