Fig 2: XPO-1 inhibitor SXR resensitizes BT474 xenograft tumors to treatment with tamoxifen.Four- to 6-week-old BALB/C nude mice were ovariectomized and after 1 week were implanted with E2 pellets. The next day the animals were subcutaneously injected with 2.5 × 107 BT474 cells in 50% Matrigel. As tumors reached 200 mm3 in the animals, Veh or tamoxifen pellets were implanted subcutaneously. Starting from the next day, the animals were injected ip with Ctrl or 25 mg/kg SXR biweekly for 4 weeks (n = 5 animals per treatment group). A, Tamoxifen induces growth of BT474 xenografts in nude mice. Picture of representative tumors from Veh and tamoxifen-treated animals. B, Tamoxifen treatment increases XPO-1 expression in tumors. Tumors from Veh or tamoxifen-treated animals were harvested. RNA was extracted and cDNA was synthesized. Expression of XPO1 mRNA was assessed using qPCR assay. 36B4 primers were run as control. A t test was used to assess whether the expression of XPO1 mRNA in tumors from Veh or tamoxifen-treated mice were different. *, P < .05. C, Expression of XPO1 protein in tumors from Veh or tamoxifen-treated animals were assessed using immunofluorescence. XPO1 signal/cell intensity was calculated and normalized by dividing the total XPO1 signal by the cell number from each field. A t test was used to assess whether the expression of XPO1 protein in tumors from Veh or tamoxifen-treated mice were different. *, P < .05. D, SXR resensitizes BT474 xenografts to tamoxifen treatment. Tumor size was measured biweekly using a caliper. The tumor volume was calculated using the following: (length × width2) × 3.14/6. A two-way ANOVA model was fitted to assess the time-dependent contribution of ligand (Veh, tamoxifen) and inhibitor (Ctrl and SXR) treatment on tumor volume. When the main effects were statistically significant at a = .05, pairwise t tests with a Bonferroni correction were used to identify whether treatments were statistically different from each other. *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001. Ctrl, control.

Fig 3: HOX cluster region shows CRM1-dependent activation of RNA Pol II in LOUCY cells.(A) ChIP-qPCR analysis of active RNA Polymerase II (Pol II) (Ser2- or Ser5-phosphorylated) at HOX gene loci in LOUCY, K562, and HL60 cell lines cultured either in the presence of DMSO (vehicle control) or KPT-330 (1000 nM) for 24 hr. The primer set used was as follows: HOXA4A5 (intergenic region between HOXA4 and HOXA5); HOXA9 (promoter); HOXB4 (promoter); HOXB4 (enhancer); HOXB9 (promoter). Data are presented as mean values ± SEM of four independent experiments (n = 4). (B) The data in (A) were reanalyzed to obtain a ratio by comparing with the value for DMSO treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t-test; **p<0.01. (C–D) Binding profiles of Ser2- or Ser5-phosphorylated Pol II at HOXB (C) or HOXA (D) clusters cultured either in the presence of DMSO (vehicle control) or KPT-330 (1000 nM) for 24 hr. (E) The most affected genes (among CRM1-bound genes) by the treatment with KPT-330. S2P (left panel) and S5P (right panel).

Fig 4: CRM1 functions as a platform to recruit leukemogenic proteins to HOX cluster regions.CRM1 possesses two distinct functions: (1) Nuclear export of NES-cargo proteins, and (2) gene regulation. Leukemogenic proteins co-localize with CRM1 at HOX cluster regions either via (i) CRM1-nucleoporin (FG) interaction (Nup98HoxA9 and SET-Nup214) or (ii) CRM1-NES interaction (NPM1c). Note that both the binding of these leukemogenic proteins to HOX cluster(s) and HOX gene expression are dependent on CRM1. In the case of Nup98HoxA9 and SET-Nup214, the NES-containing protein(s) is/are most likely involved in the complex.

Fig 5: XPO1 inhibitor resensitizes tamoxifen-resistant breast cancer cells to tamoxifen.A, ERa (+) breast cancer cells are more sensitive to SXR treatment. Dose response for the SXR in ERa (+) MCF-7, T47D, and BT474 cells and ERa (-) MDA-MB-453 and MDA-MB-468 cells. IC50 values were calculated using a nonlinear regression analysis in GraphPad software. B, Impact of single and combination of 4-OH-TAM and SXR treatments in various ERa (+) cell lines. Cells were treated with increasing doses of 4-OH-TAM and/or SXR. Cell numbers were examined using the WST-1 assay at day 7. Values are the mean ± SEM from at least two independent experiments. C, Impact of XPO1 inhibitor on cell proliferation. Cells were treated with increasing doses of 4-OH-TAM in the presence or absence of 10-7 M SXR. Cell numbers were examined using the WTS-1 assay at day 7. Values are the mean ± SEM from at least two independent experiments.