XPO1 Target Occupancy Measurements Using Fluorescence Cross Correlation Spectroscopy Support the Selinexor Recommended Phase 2 Dose
Authors: Crochiere Marsha L. , Hannus Stefan , Hansen Kerrin , Becker Frank , Baloglu Erkan , Kauffman Michael , Landesman Yosef 
Published: October 2019

Abstract
Introduction:
Selective Inhibitor of Nuclear Export (SINE) compounds are a new class of oral anti-cancers drugs that block nuclear export by inhibiting the exportin XPO1 (also known as CRM1). The most advanced compounds in this class are selinexor and KPT-8602, which are being evaluated in clinical trials for the treatment of hematological malignancies (clinicaltrials.gov). XPO1 is commonly overexpressed in cancer and high levels of XPO1 are correlated with poor prognosis. Based on Phase I efficacy and safety studies, the recommended Phase 2 dose (RP2D) of selinexor is 60 mg twice weekly on days 1 and 3. We have previously reported the development of a pharmacodynamics (PD) assay to measure XPO1 target occupancy by selinexor in cancer cells based on Fluorescence Cross Correlation Spectroscopy (FCCS). Here we report proof of concept studies to determine the level of XPO1 occupancy associated with the selinexor RP2D regimen.
Methods:
Z138 cells in culture were treated with 1 µM selinexor either continuously for up to 32 hours or for 6 hours followed by drug removal and culture for up to 72 hours post-wash out. Cell were treated in culture for 4 hours with either 0-10 µM selinexor, KPT-8602, or Leptomycin B (LMB). Cells were harvested, lysed, and cleared by ultracentrifugation. Total protein was measured from lysates for Western blot with α-XPO1, α-GAPDH, and α-β-tubulin and for FCCS analysis. In vivo, nude mice were inoculated in both flanks with Z138 cells. Once tumors reached approximately 250 mm3, mice were administered a single dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were harvested at 6, 24, 48, 72, and 96 hours post-dose, lysed, and cleared by ultracentrifugation. Cell line or tumor supernatants were incubated with a fluorescently labeled antibody (Ab) recognizing XPO1 and fluorescently labeled LMB. To allow binding of labeled Ab and LMB to XPO1, samples were measured by FCCS after 15, 120, and 1200 minutes.
Results:
XPO1 protein dynamics were evaluated in Z138 cells treated with selinexor either continuously or after drug removal. Selinexor treatment resulted in the reduction of XPO1 protein by 26% as early as 4 hours of treatment and reduced XPO1 levels by 93% with continuous drug exposure up to 32 hours. However, cells treated with selinexor for only 6 hours followed by drug washout showed nearly full restoration of XPO1 expression by 72 hours, indicating that continuous presence of drug is required to sustain reduced XPO1 protein levels. Measurements by FCCS showed that brief exposure to selinexor (6 hours) resulted in rapid XPO1 occupancy, and drug washout resulted in sustained occupancy that was not relieved until 72 hours post-selinexor wash out. To determine whether the second generation SINE compound clinical candidate, KPT-8602, had a similar XPO1 occupancy profile as selinexor, a panel of hematologic cell lines were treated with either compound and analyzed by FCCS. FCCS was also measured from cells treated with LMB, a natural product inhibitor of XPO1. FCCS analysis confirmed that KPT-8602 treatment resulted in a dose-dependent increase in drug-target occupancy with increasing drug concentration similar to that of selinexor, while LMB showed complete occupancy at very low levels of compound. Lastly, to confirm the level of target engagement in the RP2D regimen for selinexor, drug-target occupancy was measured in Z138 xenograft tumors from mice treated with a single oral dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were collected at 6, 24, 48, 72, and 96 hours post-single dose. The FCCS results showed that full occupancy occurred by 6 hours at all doses, but the duration of occupancy was dose-dependent. XPO1 occupancy was relieved by 24 hours at 5 and 10 mg/kg, 48 hours at 15 mg/kg, and 72 hours at 20 mg/kg selinexor. This finding supports the RP2D dosing regimen, where 10-15 mg/kg in mice (approximately equivalent to 50-75 mg flat dose in humans) is necessary to maintain XPO1 occupancy for up to 48 hours post-dose, thus requiring the twice weekly dosing for continuous target occupancy.
Conclusions:
Using an FCCS based PD assay developed to measure the dynamics of XPO1 target occupancy by selinexor, our studies support the selinexor RP2D dosing regimen for patients with heavily pretreated hematologic and solid tumors. Future studies are planned to measure XPO1 occupancy in CD138+ cells isolated from patients with advanced multiple myeloma following treatment with selinexor or KPT-8602.

Disclosures
Crochiere: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Hannus:Intana Bioscience GmbH: Consultancy. Hansen:Intanat Bioscience GmbH: Consultancy. Becker:Intana Bioscience GmbH: Consultancy. Baloglu:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.