Extended Data Fig. 6: The activity and requirement of Ras for complete Selinexor-induced AKT activation.
a) Immunoblot depicting active Ras following co-immunoprecipitation of Ras-GTP with GST-Raf1-Ras-binding domain (RBD) fusion proteins in a panel of selinexor-treated AML cell lines. Total Ras in input shown as control. b) Immunoblot depicting active Ras as in (c) in OCI-AML2 cells treated with AR-C 118925XX (2.5 μM) and Selinexor (200 nM), alone and in combination. Total Ras in input shown as control. c) Immunoblot depicting protein levels of immunoprecipitated GTP-bound Ras in OCI-AML2 cells with doxycycline-inducible shRNAs targeting P2RY2 versus scrambled shRNA control. Cells were exposed to doxycycline (75 ng/mL) for 48 hours and treated with either vehicle or selinexor for 36 hours. Total Ras in input shown as control. d) Immunoblot depicting protein levels of phosphorylated AKT at T308 and S473 in OCI-AML2 cells co-expressing doxycycline-inducible shRNAs against NRAS and KRAS versus scrambled shRNA control. Cells were exposed to doxycycline (75 ng/mL) for 48 hours and treated with either vehicle or selinexor for 36 hours. e) Immunoblot depicting protein levels of phosphorylated AKT at T308 and S473 in OCI-AML2 cells expressing doxycycline-inducible shRNAs against NRAS or KRAS. Cells were exposed to doxycycline (75 ng/mL) for 48 hours and treated with either vehicle or selinexor for 36 hours. f) Relative 72 h selinexor GI50 and dose–response curves in OCI-AML2 cells expressing GFP or Luciferase control or activating constructs of Ras or AKT. P values computed using one-way ANOVA with Tukey’s method for multiple comparisons; data are presented as mean + /-s.e.m. for n = 3 biological replicates. g) Genetic dependency of AML cell lines on XPO1 as defined by the DepMap dataset. P values computed using unpaired (two-sided) t-test. Extended Data Fig. 6a-e Representative immunoblots of n = 2−4 biologically independent experiments yielding similar results. B-actin included as loading control.
