Development of a Novel Small Molecule CRM-1 Inhibitor for Non Hodgkin's Lymphoma

Abstract 598

Recent evidence demonstrates that Non Hodgkin's Lymphoma (NHL) tumors have elevated expression of the XPO1 gene that codes for the nuclear exporter protein CRM-1 controlling the localization of critical tumor suppressors including p53 family members. For effective apoptosis and cell cycle regulation, p53/p73 nuclear localization and DNA binding are highly critical making CRM-1 an attractive therapeutic target for NHL that carries >90% wild type/functional p53 and only rare mutations in p73. We have identified novel small molecule inhibitors of CRM-1 (KPTs) that bind irreversibly and lock target proteins (including p53 and p73) in the nucleus leading to apoptosis of tumor cells. We demonstrate for the first time that KPTs can induce apoptosis in resistant NHL cell lines and corresponding xenograft models. The most potent CRM-1 inhibitor (KPT-185) induced growth inhibition and apoptosis in a panel of NHL cell lines with a median IC₅₀ ∼25 nM. Western blot and confocal microscopy analyses demonstrated that KPT-185 treatment resulted in nuclear localization of p53 in wt-p53 and p73 in mut-p53 cell lines. Additionally, we observed KPT-185 mediated activation of p21 and Bax, known downstream executioners of p53/p73 cell cycle control and apoptosis, respectively. Most significantly, siRNA knockdown of p53 in WSU-FSCCL and p73 in WSU-DLCL₂ abrogated the apoptotic potential of KPT-185, confirming that these were indeed p53/p73 dependent apoptotic events. KPT-185 showed a substantial enhancement in apoptosis when combined with genotoxic p53/p73 re-activating regimen CHOP. The KPTs selectively kill cancer cells with minimal toxicity to normal tissues, and possess clinically acceptable pharmacokinetic parameters. Using WSU-DLCL₂ SCID models, we further show that oral administration of related CRM-1 inhibitor KPT-276 (75 and 150 mg/Kg p.o) resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of KPT-251 (25 and 75 mg/Kg) resulted in 70 and 74% suppression of tumor growth with no observed toxicity to the host. Remnant tumor tissue analysis (histology and protein markers) fell in line with our in vitro results with clear activation of p73 pathway. Our study verifies CRM-1 as a potential therapeutic target in NHL irrespective of the functional status of p53. These results build a strong case for the clinical use of our novel CRM-1 inhibitors either as single agents or in combination with CHOP.