Expanded analysis of primary CRLF2_r ALL samples demonstrates inhibition of JAK/STAT and PI3K pathway signaling. Analysis of JAK/STAT, PI3K/mTOR, and MAPK pathway STIs in 29 CRLF2_wt (A) and CRLF2_r (B) primary human leukemias. Data are displayed as whisker plots of 25th to 75th percentiles with means (central bars) and ranges (whiskers). The dashed horizontal line represents normalized basal phosphorylation for each phosphoprotein. Pink bars indicate TSLP; striped red, ruxolitinib, solid red, ruxolitinib + TSLP; striped green, rapamycin; solid green, rapamycin + TSLP; striped blue, PI103; solid blue, PI103 + TSLP; striped purple, PP242; solid purple, PP242 + TSLP; striped gray, PD901; solid gray, PD901 + TSLP. Minimal effects of TSLP stimulation or STIs were observed for the CRLF2_wt samples. TSLP stimulation induced pSTAT5, pAkt, pS6, p4EBP1, peIF4E, and pERK in the CRLF2_r samples (P < .05), and incubation with ruxolitinib resulted in continued inhibition of all aforementioned phosphoproteins (P < .05). PI103 inhibited TSLP-induced pAkt, pS6, and pERK (P < .05). PP242 abrogated TSLP-induced pAkt, pS6, and peIF4E (P < .05). PD901 inhibited TSLP-induced pS6, peIF4E, and pERK (P < .05).