Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma  Kejie Zhang, Michael Wang, Archito T. Tamayo, Sharon Shacham, Michael Kauffman, John Lee, Liang Zhang, Zhishuo Ou, Changping Li, Luhong Sun, Richard J. Ford, Lan V. Pham  Experimental Hematology  Volume 41, Issue 1, Pages 67-78.e4 (January 2013) DOI: 10.1016/j.exphem.2012.09.002 Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 CRM1 expression in normal B cells, MCL cell lines, and primary MCL cells and effect of CRM1 siRNA on MCL cell growth. (A) Nuclear, cytoplasmic, and whole-cell extracts from MCL cell lines (8), normal B cells, and primary MCL cells from three patients with relapsed/refractory MCL were analyzed by Western blotting to assess CRM1 expression. (B) Microarray data analyses of CRM1 mRNA expression in primary MCL and normal B cells. Stages: 1. B lymphocyte; 2. centroblast; 3. memory B lymphocyte; 4. naïve pregerminal center B-lymphocyte; 5. small cleaved follicle center cell; 6. mantle cell lymphoma. Boxes represent the 25th through 75th percentiles, horizontal lines represent the medians, whiskers represent the 10th and 90th percentiles, and asterisks represent the ends of the ranges. (C) Z-138 and Mino cells were transfected by electroporation with a CRM1-validated siRNA or negative control (NC) siRNA. After 48 hours of transfection, the cell lysates were analyzed by Western blot using anti-CRM1 and anti–β-actin antibodies. (D) Control and CRM1 siRNA transfected cells were also used for proliferation assays using 3H thymidine incorporation assays. Columns: mean of triplicate samples from three independent experiments; bars: standard deviation. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Effects of KPT-SINE compounds on the growth of MCL cells. Eight MCL cell lines, two blastoid-variant MCL (BV-MCL) (Z-138 and Rec-1), and six classic MCL (c-MCL) (JVM-2, Mino, Maver-1, NCEB-1, Jeko-1, and JVM-13) were treated with KPT-185 (A) or KPT-276 (B) in a dose-dependent manner. Cell proliferation was measured using 3H-thymidine incorporation assay after 48 hours incubation. Data shown are the means and ranges of triplicate samples relative to control samples of three independent experiments. Error bars: standard deviation. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 Effects of KPT-SINE compounds on apoptosis and the cell cycle in MCL cells. Z-138 MCL cells were treated with KPT-185 or KPT-276 (A) in a dose-dependent manner for 48 hours or (B) in a time-dependent manner (KPT-185, 200 nM; KPT-276, 400 nM) and apoptosis was measured using the Annexin V–binding assay. Z-138 MCL cells were treated with KPT-276 (0–400 nM) or KPT-185 (0–200 nM) for 48 hours and cell extracts were analyzed for (C) poly (ADP-ribose) polymerase cleavage by Western blot and (D) caspase-3 activity. (E) Effects of KPT-185 on the cell cycle profile. The percentages of cells in sub-G1, G0-G1, S phase, and G2-M phase are shown (n = 3). Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 Effect of KPT-SINE compounds on p53 and CRM1 localization and expression, and the role of p53 on KPT-185–induced MCL cell apoptosis. Z-138 and JVM-2 MCL cells were cultured in the presence of KPT-185 (100 nM) or KPT-276 (200 nM) for the indicated times and p53 and CRM1 localization and expression were assessed. (A) Nuclear, cytoplasmic, and whole-cell extracts were analyzed by Western blot. Densitometry was performed for nuclear CRM1 bands showing fold-increased or decreased over control. (B) Confocal images of CRM1 (green) and p53 (red) in control and KPT-185–treated Z-138 and JVM-2 MCL cells for 1 and 24 hours. Topro3 (blue) represents nuclear marker. (C) KPT-185–treated Z-138 and JVM-2 MCL cell extracts were analyzed by Western blot for Bax and Noxa. (D) MCL cells, Rec-1 (wild-type p53), Maver-1(missense mutation of p53), and Jeko-1 (truncating mutation of p53) treated with KPT-185 for 24 hours. Cell extracts were analyzed by Western blot for p53 protein levels and PARP cleavage. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 KPT-SINE compounds trap IκBα in the nucleus, inhibiting constitutive NF-κB activity in MCL cells. (A) Z-138 and Mino cells were treated with KPT-185 (100 nM) for 1, 3, 6, 12, and 24 hours. Nuclear extracts were analyzed by Western blot using anti–NF-κB and anti-IκBα antibodies. (B) Z-138 and Mino cells were cotransfected with the 6× NF-κB-luc reporter plasmid and β-gal reporter plasmid and then treated with KPT-185 or KPT-276 for the indicated times. Luciferase activity was measured and normalized according to β-gal activity. Columns: mean of triplicate samples of three independent experiments; bars: SD. (C) Nuclear extracts purified from transfected cells in Figure 1C were utilized for Western blotting for IkBa, p65, and Oct-1. (D) Z-138 MCL cells were cotransfected with the 6× NF-κB-luc reporter plasmid and negative control or CRM1 siRNA for 48 hours. Luciferase activity was measured and normalized according to β-gal activity. Columns: mean of triplicate samples of three independent experiments; bars: SD. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 6 KPT-276 inhibits growth of small and large Z-138 xenografts. Z-138 cells were grown as xenografts in an MCL SCID mouse model to 100 mm3 (small) or 600 mm3 (large, pink curve only). The mice were then treated with KPT-276 orally five times weekly or cyclophosphamide given intraperitoneally on days 1 to 3 and tumor growth was assessed for 30 days (for study groups see Supplementary Table E2; online only, available at www.exphem.org). Treatment with KPT-276 orally at 150 mg/kg or 75 mg/kg five times a week when tumors had reached ∼150 mm3 resulted in a statistically significant reduction in Z138 tumor growth (p < 0.001 for each group vs vehicle control). Treatment with KPT-276 at 150 mg/kg five times a week beginning on day 22 when tumors had reached a mean of ∼550 mm3 resulted in a statistically significant reduction in Z138 tumor growth (p < 0.001). After treatment with KPT-276, most xenografts were <200 mm3. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 7 Effect of KPT-276 in primary MCL cells. Freshly isolated MCL cells from three patients with relapsed/refractory MCL were treated with KPT-276 (1 μM) for 24 hours. (A) Cell extracts were analyzed by Western blot for CRM1 protein levels and PARP cleavage. (B) Apoptosis was measured using the Annexin V–binding assay. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 Nuclear expression of CRM1. Two MCL cell lines were utilized for nuclear cytoplasmic fractionation. Protein extracts were analyzed by Western blot for CRM1, actin (cytoplasmic protein), and Oct- (nuclear protein). Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E2 Effects of KPT-185 on the growth of MCL cells. Classic MCL cells (Jeko-1 and Maver-1) and blastoid-variant MCL cells (Z-138 and Rec-1) were treated with KPT-185 upon different times indicated. Cell proliferation was measured using 3H-thymidine incorporation assay. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E3 Effect of KPT-185 on p53 and CRM1 mRNA expression, Crm1 degradation. Z-138 and JVM-2 MCL cells were cultured in the presence of KPT-185 (100 nM) for the indicated times, harvested, and subjected to experiment to assess for p53 (A) and CRM1 (B) mRNA expression by real-time polymerase chain reaction analysis. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E4 Tumor histology. (A) Selected histological analyses of xenograft tissue from vehicle-treated mice and from mice with largest residual tumors after KPT-276 treatment. Size is based on caliper measurements, left- (L) and right- (R) sided xenografts, followed by mouse number. (B) In two cases (26L and 43L), only residual adipose and stromal tissue were observed, as shown. These data indicate that KPT-276 is inducing tumor cell death in vivo. Experimental Hematology 2013 41, 67-78.e4DOI: (10.1016/j.exphem.2012.09.002) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions