Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure

Summary Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Fig. S1 B A

Fig. S2 :
Fig. S2: Phosphoproteomics of SRSF2 WT/Mut cells and flow profiles of isogenic SRSF2 WT and P95L iPSC-derived HSPCs and engraftment of MOLM14 SRSF2 Mut cells in NSG mice.Phosphoproteomics was performed on SRSF2 WT/Mut MOLM14 and AML2 cells.Cells were treated with DMSO or RKI-1447 (0.5μM) for two hours and eight hours.Normalized intensity of phosphorylation level of several downstream targets of ROCKs, MYPT1 T696, MSN S407, SCRIB S1508, MYO9B T1492, SCRIB S1508 and EZR;MSN;RDX T558 were shown in AML2 cells (A) and MOLM14 cells (B).(C)Flow profiles Isogenic SRSF2 WT (D1-1) and P95L (Cre20) iPSC-derived HSPCs on day12 differentiation.CD34 and CD45 expression at days 12 were shown in far right column.Cells were then cultured with DMSO or RKI-1447 (3µM) for 48 hours and viability was measured with CellTiter-Glo (Fig. 1G).(B) NSG mice were injected with 0.2*10^6, 1*10^6, and 2*10^6 MOLM14 SRSF2 Mut cells by intravenous (IV) injection (N=3 for each group) to calibrate the number of cells for MOLM14 SRSF2 xenograft model.Mice were sacrificed after 8week transplantation to determine the amount of MOLM14 SRSF2 Mut cells for our in vivo model.(C) NSG mice were treated with vehicle (DMSO) or RKI-1447 (5mg/kg or 50mg/kg) by i.p. for 21 days after 5-week transplantation of one million SRSF2 Mut MOLM14 cells (IV) to calibrate the efficacious dosage of RKI-1447 in vivo.Mice were sacrificed and BM cells was flashed from tibia/femur.The percentage of human CD45+ (hCD45) cells in engrafted murine bone marrow is shown after staining for hCD45 and analyzed by flow cytometry.Mann−Whitney U test with FDR correction for multiple hypothesis testing, *P<0.05;**P<0.005;***P<0.0005.

Fig. S7 :
Fig.S7: F-actin staining of SRSF2 WT and Mut MOLM14 cells before and after exposure to RKI-1447.SRSF2 WT/Mut MOLM14 cells, as indicated, were either left untreated or treated with RKI-1447 (0.5µM) for 24 hours before fixation.(A) Cells were labeled with fluorescent phalloidin to visualize actin (shown in red).3D volumes were taken on the Leica SP8 scanning confocal microscope, represented slices corresponding to mid-plane of cells are shown.(B), Quantification of the nuclear vs cytoplasmic actin was performed, with N values as follows N = 36 for WT, N= 26 for mut, N=29 for WT + RKI-1447 and N=26 for Mut + RKI-1447.Note, that the nuclear-to-cytoplasmic actin ratio was higher in mutant compared to WT, and higher in RKI-1447-treated WT cells, compared to non-treated WT nuclei.

Fig. S10 :
Fig.S10: 3D rendering of nucleus and confocal images of SRSF2 WT/Mut OCI-AML2 cells before and after exposure to RKI-1447.SRSF2 WT/Mut OCI-AML2 cells, untreated or treated with RKI-1447 (0.5µM) for 24 hours before fixation, were acquired using the Leica SP8 scanning confocal microscope.(A) Nuclei were labeled with DAPI to visualize nuclear morphology imaged on the Olympus confocal microscope as described in Supplementary Methods.3D volumes were subjected to 3D rendering using IMARIS software.Rendered surfaces are shown in dark blue.Sphericity of the nucleus were measured and presented in Fig.4B.(B) Cells were labeled with anti-LaminB1 antibodies to outline nuclear membrane (shown in green) and anti-a-tubulin antibodies to label microtubules (shown in red), and nucleus were labeled with DAPI.Representative confocal slices corresponding to the middle plane of the cells are shown.

Fig. S11 :
Fig. S11: 3D rendering of nucleus of SRSF2 WT/Mut primary cells before and after exposure to RKI-1447.(A) One SRSF2 Mut primary AML sample (#830163) and one SRSF2 WT primary AML sample (#150279), as indicated, were either left untreated or treated with RKI-1447 (1µM) for 72 hours before fixation.Nuclei were labeled with DAPI to visualize nuclear morphology imaged on the Olympus confocal microscope as described in Methods, 3D volumes were subjected to 3D rendering using IMARIS software.Rendered surfaces are shown in dark blue.Note, that treatment with RKI-1447 causes statistically significant decrease nuclear sphericity in SRSF2 Mut primary AML sample, see Fig. 4B.The two-sample T-test with two-tailed distribution was used for evaluation of statistical significance.

Fig. S12 :
Fig. S12: TEM of SRSF2 WT/Mut MOLM14 cells before and after exposure to RKI-1447.(A-B) Showing low magnification images of TEM corresponding to the images shown in Fig. 4C.Higher magnification images with rectangles marking the enlarged areas, shown in A' and B', respectively.N and C mark nuclear and cytoplasmic areas, respectively.Arrows or arrowheads marked "Mt" point to microtubules, shown in longitudinal or cross sections, respectively.The arrow in A'right points to the inter-lobular sheet, connecting the two nuclear segments.The twinarrow in B'-left (NM) point to the attached nuclear membranes, and the arrow marked "L" point to the nuclear laminae.

Fig. S13 :
Fig. S13: TEM of SRSF2 WT /Mut OCI-AML2 cells before and after exposure to RKI-1447.(A-D) Showing lower magnification images of TEM in SRSF2 WT/Mut OCI-AML2 cells, either untreated or treated with RKI-1447 (0.5µM) for 24hours as indicated.(C'-D') Higher magnification images with rectangles marking the enlarged areas, shown in (C) and (D).N and C mark nuclear and cytoplasmic areas, respectively.Arrows marked "Mt" point to microtubules, shown in longitudinal.The arrow in C' and D' point to the centrioles or microtubules in the cytoplasm near the nucleus indentation.Note, that this phenomenon are often presented in OCI-AML2 cells after RKI-1447 treatment.

Fig. S15 :
Fig. S15: Confocal images of SRSF2 Mut/WT primary AML cells before and after exposure to RKI-1447.(A) SRSF2 WT/Mut primary AML cells, untreated or treated with RKI-1447 (1µM) for 72 hours before fixation, were acquired using the Leica SP8 scanning confocal microscope.Cells were labeled with anti-LaminB1 antibodies to outline nuclear membrane and anti-a-tubulin antibodies to label microtubules, nucleus were labeled with DAPI.Representative confocal slices corresponding to the middle plane of the cells are shown.Sphericity of the nucleus were measured and presented in Fig. 4B.

Fig. S16 :
Fig. S16: Representative confocal images of SRSF2 Mut primary AML cells after exposure to RKI-1447.SRSF2 Mut primary AML cells were treated with RKI-1447 (1µM) for 72 hours.Cells were labeled with anti-LaminB1 antibodies to outline nuclear membrane (shown in green) and anti-a-tubulin antibodies to label microtubules (shown in red), and nucleus were labeled with DAPI.(A) Representative confocal slice corresponding to the middle plane of the cells is shown.(B) Different Z-stacks of the highlighted cells in (A) were shown.Note, deep narrow folds or invagination of nuclear membrane that are presented upon RKI-1447 treatment.Red signal detected inside such invaginations points to the presence of microtubules (arrows).

Fig. S14: Confocal images of LaminB1 and microtubules staining of SRSF2 WT and Mut MOLM14 cells before and after exposure to RKI-1447.
SRSF2 WT/Mut MOLM14 cells were either untreated or 24-hour treated with RKI-1447 (0.5 µM), as indicated.LaminB1 labeling shown in green and microtubules in red.(A) Top to bottom rows: single confocal slices bottom to top direction extracted from 3D volume.The depth of the WT volume ranges from 0 to 11.4 μm, with confocal slices shown at intervals: 3.8 μm and 7.6 μm, the Mut volume ranges from 0 to 12.6 μm with confocal image intervals at 4.2 μm and 8.4 μm, WT + RKI-1447 is 12 μm deep with image intervals 4 μm and 8 μm, and Mut + RKI-1447 has a z-depth of 11.1 μm with image intervals at 3.7 μm and 7.4 μm.These are presented in order to give the view of depth of nuclear indentations and degree of lobulation.See also Movie S1. (B) Selected enlarged insets from images presented in panel (A).Cells in (A) are outlined with dotted white line, and presented in panel (B) according to numbers and treatment conditions.Note the presence of the microtubule organizing center at the base of nuclear deformation -indicated by bright microtubule labeling in first and third columns.Different focal planes reveal deep narrow indentations positive for LaminB1 and containing microtubules as indicated by red labeling (arrowheads in second and fourth columns).