Fig 2: SIAIS562055 combined with KRAS inhibitors overcomes drug resistance. A, Western blot analysis of SOS1 in Ba/F3 cells transformed with or without KRAS mutation after treatment with SIAIS562055 (1,000 nmol/L) or BI-3406 (1,000 nmol/L) for 24 hours. B, Antiproliferative effects of SIAIS562055 on Ba/F3 cells transformed with or without KRAS mutations for 72 hours. Histogram shows the IC50 values of compounds in two-dimensional (2D) cultured cells. C, Cell proliferative inhibition of MIA PaCa-2 and MIA PaCa-2/R cells treated with AMG510 and MRTX849 for 72 hours. Histogram shows the IC50 values of compounds and resistance factors (RF) in 2D cultured cells. D, Western blot analysis of SOS1 by SIAIS562055 in MIA PaCa-2 and MIA PaCa-2/R cells for 72 hours. MIA PaCa-2/R xenograft-bearing mice received vehicle, SIAIS562055, and MRTX849, alone or in combination, then, the relative tumor volumes (E) and mouse body weights (F) were tested. qd, once a day. G, Western blot analysis of SOS1, pERK, and cleaved caspase-3 in MIA PaCa-2/R xenograft tumor tissues. Data about relative tumor volumes and body weights are presented as mean ± SEM, n = 5, whereas other statistical data are presented as mean ± SD, n = 3. **, P < 0.01; ***, P < 0.001.

Fig 3: Combination of SIAIS562055 and TKIs synergistically inhibits the growth of CML xenografts in vivo and enhances cell death in primary samples from patients with CML. A–D, K562-xenografted mice received vehicle, SIAIS562055 (30 mg/kg/day), imatinib (100 mg/kg/day), or their combination; then, relative tumor volumes (A), tumor weights (B), photo of tumors (C), and mice body weights (D) were determined at indicated times. Data are presented as mean ± SEM, n = 5. E, Western blot analysis of SOS1, pERK, and pSTAT5 in K562 xenograft tumors after treatment with vehicle, SIAIS562055, imatinib, or their combination. Data are shown as mean ± SEM, n = 5. F, Cell viability of three primary BCR–ABL+ samples from patients with CML treated with varying doses of SIAIS562055 or imatinib, alone or in combination, for 72 hours was assessed by CellTiter-Glo assay. Data are presented as mean ± SD, n = 3. G, Western blot analysis of the indicated proteins in primary samples from patients with CML under 24-hour exposure to SIAIS562055 or imatinib, alone or in combination. Statistical significance was assessed using two-tailed unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. qd, once a day.

Fig 4: SIAIS562055 alone or coadministered with KRAS inhibitors suppresses tumor growth of KRAS-mutant xenografts in vivo. A-C, MIA PaCa-2–xenografted mice received vehicle, SIAIS562055 (20 or 40 mg/kg/day), or BI-3406 [50 mg/kg, twice a day (bid)], then relative tumor volumes (A), tumor weights (B), and mouse body weights (C) were determined at indicated times. D, Western blot analysis of SOS1, pERK, and cleaved caspase-3 in MIA PaCa-2 tumor tissues after treatment with SIAI562055 or BI-3406. E and F, MIA PaCa-2 xenograft-bearing mice received vehicle, SIAIS562055, and MRTX849, alone or in combination; the relative tumor volumes (E) and photos of tumors (F) are shown. G and H, GP2d xenograft-bearing mice received vehicle, SIAIS562055, and MRTX1133, alone or in combination; the relative tumor volumes (G) and photos of tumors (H) are shown. Data are presented as mean ± SEM, n = 5. Statistical significance was assessed using two-tailed unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. qd, once a day.

Fig 5: SIAIS562055 sensitizes KRAS-mutant cancer cells to KRAS inhibition in vitro. A, Western blot analysis of SOS1 in NCI-H358 (G12C), GP2d (G12D), and SW620 (G12V) cells treated with a concentration gradient of SIAIS562055 for 24 hours. B, Western blot analysis of SOS1 and pERK in NCI-H358 cells after the treatment of SIAIS562055 (1,000 nmol/L), SIAIS562092 (1,000 nmol/L), and BI-3406 (1,000 nmol/L) at different time points. C, Western blot analysis of SOS1 in MIA PaCa-2 (G12C), HPAF-II (G12D), and SW620 (G12V) cells after treatment with SIAIS562055 (1,000 nmol/L) for 12 hours, followed by wash-out and incubation with fresh media for another 0, 24, 48, 72, and 96 hours. D, Antiproliferative effects of SIAIS562055 and BI-3406 for 120 hours on KRAS-WT (293T, BxPC-3) and KRAS-mutant (NCI-H358, GP2d, HPAF-II, and SW620) cells. Histogram showed the IC50 values of compounds assessed in three-dimensional (3D) cultured cells. Data are shown as mean ± SD, n = 3. E, Inhibition rates of cell proliferation in NCI-H358 and GP2d cells treated with SIAIS562055 and KRAS inhibitors, alone or in combination, at different concentrations for 72 hours. CI values were calculated based on the data in red oval frames. F, Colony formation of NCI-H358 cells after treatment with SIAIS562055 (800 nmol/L), KRASG12C inhibitors (AMG510 or MRTX849, 5 nmol/L), or their combination for 7 days. G, Western blot analysis of SOS1 and pERK in NCI-H358 and GP2d cells after treatment with SIAIS562055 (1,000 nmol/L) combined with AMG510 (5 nmol/L), MRTX849 (5 nmol/L), or MRTX1133 (0.8 nmol/L) for 24 hours. Data are shown representatively from three independent repetitions.