Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia

Abstract

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (e.g., p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500 nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

Figure 1

KPT-SINE significantly inhibits proliferation and induces cell-cycle arrest and apoptosis of AML cell lines and primary AML blasts. (A) WST-1 assays in MV4-11, Kasumi-1, MOLM-13, and OCI-AML-3. (B) Cell-cycle assessment using propidium iodine (PI) detection by flow cytometry at 24 hours. (C) Apoptosis as measured by annexin V/PI staining using FACS at 48 hours. (D) Colony assays in Kasumi-1, MV4-11, and OCI-AML3 cells after KPT-185 treatment at 24, 48, and 72 hours. (E) Apoptosis as measured by annexin V/PI staining using FACS at 48 hours in primary AML samples. (F) WST-1 assays in primary AML samples.

Figure 2

KPT-SINE treatment causes decrease of CRM1 protein level, accumulation of CRM1 cargo proteins in the nucleus, and down-regulation of FLT3 and KIT oncoproteins. (A) Top panel: CRM1 protein expression as measured by Western blotting in MV4-11, Kasumi-1, and OCI-AML3 cells after KPT-185 treatment or control (DMSO) after 24 hours. Loading control is actin. Bottom panel: CRM1 protein expression as measured by Western blotting in 3 primary AML blasts after KPT-185 treatment or control. (B) Confocal microscopy of p53 in MV4-11 and OCI-AML3 cells treated with KPT-185 or control at 24 hours. Left panel: DAPI staining (cell nucleus). Middle panel: p53 staining. Right panel: merge of p53 and DAPI staining. Note the increase in the p53 expression in the nucleolus. (C) Whole-cell p53 protein expression in MV4-11 and OCI-AML3 cells (top panel) or primary AML blasts (n = 3) after KPT-185 or control treatment at 2 and 24 hours. (D) Confocal microscopy of NPM1 in OCI-AML3 cells and in a primary AML blast from a patient with CN-AML and NPM1 mutation treated with KPT-185 or control at 24 hours. Left panel: DAPI staining (cell nucleus). Middle panel: NPM1 staining. Right panel: merge of NPM1 and DAPI staining. The arrows indicate the localization of NPM1 in the cytoplasm (NPMc⁺) in the untreated samples and the elimination of the cytoplasmic signal on treatment with the drug, being detected exclusively in the nucleus. (E) FLT3 protein expression in MV4-11, Kasumi-1, and OCI-AML3 cells as measured by Western blotting after KPT-185 treatment or control at 24 hours. (F) FLT3 protein expression in primary AML blasts as measured by Western blotting after KPT-185 treatment or control at 24 hours: patient 1, CN-AML NPM1 WT, FLT3 ITD⁺; patient 2, CN-AML, NPM1 mutated, FLT3 WT; and patient 3, CN-AML, NPM1 mutated, FLT3 WT. (G) c-KIT expression in Kasumi-1 and OCI-AML3 cells after KPT-185 treatment or control at 24 hours.

Figure 3

CRM1 inhibition induces differentiation of AML cell lines. (A) CD11b measurement by FACS in AML cell lines after KPT-185 treatment at 72 hours. (B) Giemsa stain of cytospins of Kasumi-1 and MV4-11 cells treated with KPT-185 or controls. Magnification, 40×. Arrows indicate nuclear condensation. (C) CEBPA protein expression in MV4-11 cells and OCI-AML3 cells after KPT-185 treatment or controls for 24 and 48 hours. Loading control is actin. (D) GCSFR and lysozyme mRNA expression after KPT-185 treatment or controls in MV4-11 cells and OCI-AML3 cells. Results are shown as fold change after normalization with 18s and 2ΔC_t calculations. (E) CEBPA protein expression after p53 siRNA or scramble oligonucleotide transfection in OCI-AML3 cells subsequently treated with KPT-185 or controls for 24 hours. Protein expression of p21 was measured as control for successful p53 inhibition. (F) CD11b expression in OCI-AML3 cells after transfection with scramble or p53 antisense oligonucleotides and treatment with DMSO or KPT-185 at 72 hours. (G) Apoptosis as measured by annexin V/PI staining using FACS at 48 hours after scramble or p53 antisense oligonucleotides and treatment with DMSO or KPT-185.

Figure 4

CRM1 inhibitor increases survival in a human leukemia xenograft model. (A) Survival of MV4-11 xenograft mice after treatment with KPT-276 150 mg/kg (n = 12) or vehicle control (n = 11). Survival comparison was made with log-rank test. (B) White blood cell count (in thousands) in KPT-treated mice versus vehicle control (n = 8) at 21 days. P values obtained using t test. (C) Spleen weights (mg) in KPT-treated mice versus vehicle control (n = 7). P values obtained using t test. (D) Spleen photographs of 3 representative cases (KPT-276 n = 3; vehicle n = 3) at 21 days. (E) FLT3 protein expression in mouse spleen cells as measured by Western blotting after KPT-276 treatment or vehicle control at 21 days.