SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma

Abstract

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Fig. 1.. Dasatinib inhibits pS6K and de novo protein synthesis independent of AKT/mTORC1 in IDHm ICC.

(A) Proliferation and survival curves of human IDH WT (black) versus IDHm (red) ICC cell lines treated with increasing doses of dasatinib. (B) Apoptosis assay measuring percentage of IDH WT and IDHm cells stained positive for annexin V by flow cytometry at 48 hours after dasatinib treatments. Data are shown as mean ± SEM between triplicates and are representative of three independent experiments (Student’s two-tailed t test). (C) IDH WT or IDHm ICC cell lines were treated with dasatinib (50 to 500 nM) for 6 hours, and protein lysates were probed for the indicated proteins in the mTOR, ERK, and STAT3 survival pathways by Western blot (WB). (D) Percent reduction in cell size of IDH WT or IDHm ICC cell treated with 100 nM dasatinib for 24 hours, as measured by flow cytometry. Representative flow cytometry forward scatterplots indicative of cell size shift comparing DMSO (solid) and dasatinib-treated (dotted) in RBE (red, top left) and HuCCT1 (black, bottom left). Percentage of average cell size change in IDHm and WT lines (right, one-way ANOVA). (E) Representative IDH WT and IDHm ICC cells were treated as in (C). Thirty minutes before harvest, cells were exposed to 1 μM puromycin. Lysates were probed for the indicated proteins or puromycin-labeled proteins by Western blot. **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. N.D., not defined.

Fig. 2.. Inhibition of SRC is both necessary and sufficient in killing IDHm ICC through inhibition of S6K/S6 axis.

(A) Proliferation curves of parental IDHm ICC cell lines (red) or isogenic lines harboring a genomic SRC T341I gatekeeper mutation rendering endogenous SRC dasatinib-resistant (blue) treated with increasing doses of dasatinib. (B) Parental SRC WT and SRC gatekeeper lines were treated with dasatinib at the indicated doses for 48 hours and assessed for the induction of apoptosis by measuring annexin V positivity by flow cytometry. (C) Cells were treated with increasing concentrations of dasatinib (50 to 500 nM) for 6 hours, and lysates were probed for the indicated proteins in mTOR, ERK, and STAT3 survival pathways by Western blot. (D) Cells were treated as in (C). Thirty minutes before harvest, cells were exposed to 1 μM puromycin. Lysates were probed for the indicated proteins or puromycin-labeled proteins by Western blot. (E) IDH WT (black) or IDHm (red) ICC cell lines were transduced with either a control shRNA or two independent shRNAs against SRC, and lysates were probed with antibodies of the indicated proteins by Western blot.

Fig. 3.. MAGI1 is a substrate of SRC and modulates downstream S6K signaling.

(A) Identification of SRC substrates by phosphoproteomic screen. Mutant IDH1 ICC SRC WT and SRC T341I gatekeeper mutant pairs were treated with 20 nM dasatinib for 1 hour, and phosphopeptides were extracted from the tryptic digests of the protein lysates, followed by mass spectrometry–based multiplexed quantitative phosphoproteomics. Phospho-tyrosine peptide, pMAGI1 Y373 (red dot), represented the top candidate that was inhibited by dasatinib in both SRC WT lines SNU-1079 (left) and RBE (right) but not their corresponding SRC gatekeeper lines. (B) Relative intensity of pMAGI1 Y373 (left) and total MAGI1 (right) signals in mutant IDH1 (SNU-1079 and SNU-1079 SRC T341I) and WT IDH1 (HuCCT1 and CCLP1) ICC lines treated with DMSO or dasatinib from phosphoproteomic screen. (C) Schematic of MAGI1 showing domain structure and the pMAGI1 Y373 site. (D) 293T cells were transfected with vector control, SRC, GFP-tagged MAGI1 WT, or GFP-MAGI1 Y373F, and lysates were probed with rabbit antisera against pMAGI1 Y373 or the indicated antibodies by Western blot. (E) 293T cells were transfected with vector control, SRC, myc-tagged MAGI1 full length, or myc-tagged MAGI1 deletion of GUK, WW, or GUK-WW domains, and lysates were probed with pMAGI1 Y373 antisera. (F) 293T cells were transfected with vector control, SRC, flag-tagged MAGI1 full length, MAGI1 GUK-deleted, MAGI1 WW-deleted, or MAGI1 GUK-WW–deleted truncation mutants or cotransfected with SRC, flag-tagged MAGI1 full length, and truncation mutants. Lysates were then immunoprecipitated (IP) with flag antibody and analyzed by WB along with 1% input and probed for SRC and flag antibodies. (G) IDHm ICC cells were treated with control siRNA or siRNA against MAGI1 and then exposed to increasing doses of dasatinib (5 to 50 nM) for 6 hours. Lysates were then probed with antibodies against the indicated proteins by Western blot. (H) IDHm cells RBE expressing Cas9 plus either control single guide RNA (sgRNA) or sgRNA targeting MAGI1 (clone 2 and clone 4) were then treated as in (G) and analyzed by Western blot. (I) The same control and MAGI1 knockout clones 2 and 4 as in (H) were treated with dasatinib at indicated doses for 48 hours and subjected to annexin V apoptosis assay (two-way ANOVA; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 4.. Dasatinib suppresses S6K signaling through induction of protein phosphatase 2A.

(A) IDHm ICC RBE, SNU-1079, and ICC5 cells were treated with dasatinib 100 nM for 6 hours and/or the 1 μM PP2A inhibitor okadaic acid for the indicated time points before harvest. Lysates were then analyzed by Western blot and probed for phospho- and total SRC, S6K, S6, and 4E-BP1. Quantification of pS6K and pS6 is presented below each blot and normalized to the corresponding total protein. (B) IDHm ICC cells were treated with control siRNA or siRNA against PPP2CA and then exposed to increasing doses of dasatinib (5 to 50 nM) for 6 hours. Protein lysates were probed for the indicated proteins in the SRC and mTOR pathways by Western blot. (C) mRNA expression of PPP2CA and PPP2CB by qRT-PCR in individual ICC lines confirms specific depletion of PPP2CA by siRNA. (D and E) Change of PP2A catalytic subunit posttranslational modification at pY307 in two IDH1 WT and IDHm ICC pairs (D) and two SRC WT and SRC T341I gatekeeper pairs in IDHm lines (E) upon increasing doses of dasatinib (50 to 500 nM). Lysates were then analyzed by Western blot as above.

Fig. 5.. SRC interacts with MAGI1-PP2A complex to regulate S6K.

(A) 293T cells were cotransfected with WT SRC and flag-tagged MAGI1 Y373 WT/Y307E/Y307F for 2 days and treated with DMSO or 50 nM dasatinib for 6 hours before lysis. Lysates were then immunoprecipitated with flag antibody and analyzed by Western blot along with 1% input and probed for SRC and flag antibodies. (B) 293T cells were transfected with vector control, HA-tag PP2A catalytic subunit (PP2AC), flag-tagged MAGI1 Y373 WT, flag-tagged MAGI1 Y373E, or flag-tagged MAGI1 Y373F or cotransfected with HA-PP2AC and flag-tagged MAGI1 Y373 WT/E/F mutants. Cells were treated with DMSO or dasatinib before harvesting for co-IP as in (A) and analyzed by Western blot with HA and flag antibodies. (C) 293T cells were transfected with HA-tagged PP2AC and flag-tagged MAGI1 full-length or without WW, GUK, or GUK-WW domains. Lysates were immunoprecipitated with flag antibody. (D) Flag-tagged MAGI1 full-length WT and HA-PP2AC Y307 WT/E/F mutants were cotransfected in 293T cells treated with DMSO or dasatinib for 6 hours and processed for immunoprecipitation as in (A). HA-tagged PP2AC and endogenous S6K pulled down were analyzed by WB. (E) 293T cells were cotransfected with HA-tagged S6K and flag-tagged MAGI1 Y373 WT/Y307E/Y307F and treated with DMSO or dasatinib before harvesting for co-IP as in (A).

Fig. 6.. 2-HG is required in dasatinib-induced cytotoxicity and inhibition of pS6K.

(A) Immunofluorescence staining of pS6 and total S6 in RBE, SNU1079, ICC5 (IDH1 mutant), and HuCCT1 (WT) cells. Scale bars, 50 μm. (B) Quantification of pS6 median staining intensity. Each data point represents one cell (one-way ANOVA test, ****P < 0.0001). (C) Immunohistochemistry staining of pS6 in samples from patients with IDH WT or IDH1 R132C ICC. Scale bars, 50 μm. (D) Quantification of pS6 IHC staining intensity by blinded histopathology scoring (one-way ANOVA test, *P < 0.05). (E) IC₅₀ curves of IDH1 mutant-specific inhibitor AG-120 in suppressing cellular 2-HG concentrations in three IDHm (SNU-1079, RBE, and ICC5) and IDH WT (RBE KI c5 and RBE KI c9) cell lines. (F) Proliferation curves of IDHm (red) and IDH WT (black) ICC cell lines with increasing doses of AG-120. (G to H) SNU-1079 cells were treated with regular media, DMSO, or 5 μM AG-120 for 1, 2, 3, or 4 days followed by dasatinib treatment for 72 hours at indicated doses and subjected to crystal violet staining (G), annexin V apoptosis assay (Student’s two-tailed t test, **P < 0.01; ****P < 0.0001) (H), and Western blot for apoptosis markers cleaved caspase-3 and PARP (I). (J) IDHm ICC cells were pretreated with either DMSO (black) or 5 μM AG-120 (red) for 4 days, followed by DMSO or increasing doses of dasatinib (5 to 50 nM) for 6 hours. Cells were then harvested, and lysates were probed for the indicated proteins by Western blot. (K) IDHm RBE cells and their isogenic WT knocked-in (KI) clone 9 were subjected to increasing doses of dasatinib (50 to 500 nM) treatment for 6 hours and analyzed for the indicated proteins by Western blot. (L) Baseline expression of pSRC, pS6K, and pS6 in a panel of human ICC cell lines (IDHm in red, WT in black, and AG-120 treated in gray). (M) Dasatinib-resistant clones generated through continuous dasatinib treatment were subjected to increasing doses of dasatinib treatment, and abundance of pS6 was analyzed by Western blot.

Fig. 7.. Combination treatment with SRC and S6K inhibitors suppresses IDHm ICC growth in patient-derived models.

(A) IDH WT and IDHM ICC cells were treated with increasing doses of dasatinib (50 to 500 nM) with or without S6K1/AKT inhibitor M2698 at 10 nM for 6 hours. Cells were then harvested, and lysates were probed for the indicated proteins by Western blot. (B to E) Patient-derived organoids ICC195, FHICC19 (WT), and FHICC17 (IDH1m) were treated with increasing doses of dasatinib (100, 500, and 1000 nM) for 16 hours, and the levels of pSRC, pS6K, and pS6 were analyzed by Western blot (B). FHICC17 IDH1m organoids were also treated with the combination of dasatinib and S6K1/AKT inhibitor M2698 at 20 nM for 16 hours, and the levels of pSRC, pS6K, and pS6 were analyzed by Western blot (C). Quantifications of pS6 levels in dasatinib-treated organoid lines ICC195, FHICC19, and FHICC17 are presented in (D), and quantifications of pS6 in dasatinib plus M2698-treated FHICC17 IDH1m organoids are presented in (E). (F to K) NSG mice with subcutaneously implanted IDH1 WT PDX (ICC 195) and IDH1 R132C mutant PDXs (PDX62) were treated with vehicle control, dasatinib (30 mg/kg), M2698 (10 mg/kg), or dasatinib (30 mg/kg) + M2698 (10 mg/kg) daily for 28 days by oral gavage (PDX62, n = 9, 10, 9, and 12 respectively; ICC195, n = 5 each arm). Part of the IDH1 mutant PDX cohort was harvested at day 28 of treatment (n = 3, 4, 3, and 4 for vehicle, dasatinib, M2698, and dasatinib + M2698 respectively), and the remaining mice were monitored for survival up to 70 days after treatment. Tumor volume fold change of IDH1 mutant PDX (F) and IDH1 WT (G), tumor volume change at day 29 compared with day 1 for IDH1 mutant PDX (H), and survival plot for IDH1 mutant PDX (Kaplan-Meier analysis and log-rank P values are shown between groups) (I). (J) Histological analysis of tumors from PDX62 vehicle, dasatinib, M2698, and combo groups. Left column: Hematoxylin and eosin (H&E) staining; middle column: IHC staining for Ki67 (proliferation marker); and right column: IHC staining for cleaved caspase-3 (cell death marker). Representative images of each experimental group are shown. Scale bar, 250 μm. (K) Quantification of Ki67 (top) and cleaved caspase-3 (bottom) IHC staining. Each dot represents the percentage of cells with positive staining in a randomly selected area. Five areas per slide were quantified. Data are mean ± SEM (one-way ANOVA multiple comparisons; *P < 0.05; ****P < 0.0001).