Figure 3. CRAF reactivation mediates resistance to MEK or ERK inhibitors.

(A) Western blot images of H/NRAS-mutant RMS cells following A/B/CRAF knockdown ± trametinib. SMS-CTR and RD cells were transduced with PLKO.1 lentiviral constructs expressing shRNA against GFP (CRL) or A/B/CRAF, selected with puromycin for 24 h, then treated with 2 nM trametinib for 72 h. (B) Western blots of FN-RMS cells treated with trametinib or ERKi LY3214996. FN-RMS cells were treated with indicated concentrations of trametinib or LY3214996 for 72 h and phosporylated CRAF (pCRAF, S289/296/301) and phosphorylated MEK1/2 (pMEK, Ser217/221) were detected by western blots. (C) Western blots of RMS cells expressing wild-type or phosphorylation deficient CRAF constructs and treated with trametinib or ERKi LY3214996. SMS-CTR and RD cells were transduced with retroviral vectors expressing wild-type CRAF (WT CRAF) or mutant CRAF deficient for phosphorylation at SER 29, 289, 296, 301 (4A CRAF). Control is empty vector (CRL). After puromycin selection for 2 days, the cells were treated with trametinib (T) or ERKi LY3214996 (L) for 72 h and cell lysates analyzed by western blots. (D) Crystal violet staining images of FN-RMS cells expressing empty vector, WT CRAF or 4A CRAF constructs and treated with increasing concentrations of trametinib or LSN3214996. Cells were plated at low density in 24-well plates and next day treated with increasing concentrations of trametinib or LY3214996. After two weeks, cell growth was visualized with crystal violet staining. (E) Top, growth curves of FN-RMS cells expressing doxycycline-inducible shRNA against CRAF. SMS-CTR or RD cells were transduced with pSMART lentiviral vector expressing doxycycline-inducible CRAF shRNA, selected with puromycin for 2 days and plated in 96 well plates. Next day, the cells were treated with DMSO, 200 ng/ml doxycycline, 2 nM trametinib +/− doxycycline or 100 nM LY3214996 +/− doxycycline. Percent confluency was determined by imaging with Incucyte every 2 h for 240 h. Bottom, western blot images of SMS-CTR and RD cells expressing pSMART CRAF shRNA vector and treated with DMSO or 200 ng/ml doxycycline for 72 h. (F) Immunofluorescence staining images for MHC of FN-RMS cell lines expressing dox-inducible shRNA against CRAF. Indicated cells were plated in 8-well slide chambers and treated with DMSO, 200 ng/ml doxycycline, 2 nM trametinib or both for 8 days. Scale bars, 200 μm. (G) Response to trametinib of SMS-CTR xenografts expressing dox-inducible CRAF shRNA. SMS-CTR cells transduced with pSMART vector expressing dox-inducible CRAF shRNA were subcutaeously injected in the flank area of SCID mice and indicated treatment cohorts were generated. N=5 mice per group. 1mg/kg trametinib was administered PO, 7 days a week for 28 days. Doxycycline was delivered in diet at 1 g/kg for 28 days. Error bars represent SEM. p-values were calculated using one-way ANOVA for single treatments vs combinatorial treatment at the end of the treatment (day 28).