Constitutive and TSLP-inducible JAK/STAT and PI3K/mTOR signaling occurs in CRLF2_r ALL. (A) Stimulation of MUTZ5 cells with IL-7 had no effect on JAK2, STAT5, S6, or ERK phosphorylation, as measured by phosphoflow cytometry. Stimulation with TSLP induced phosphorylation of pJAK2, pSTAT5, pS6, and, to a lesser extent, pERK. The JAK2-specific inhibitor XL019 inhibited pSTAT5 and pS6. Pervanadate was used as a positive control for measurement of maximal phosphorylation. Individual histograms are also displayed as a heat map for ease of comprehensive data visualization. Data were normalized to the basal phosphorylation level of each protein for colorimetric depiction of signaling changes. Blue indicates inhibition, and yellow is stimulation. (B) Basal levels of phosphorylation of JAK2, STAT5, S6, and ERK in primary CRLF2_wt JAK_wt (white bars, n = 27), CRLF2_r JAK_wt (light gray bars, n = 29), and CRLF2_r JAK_mut (dark gray bars, n = 14) were measured by phosphoflow cytometry, and median fluorescent intensity was calculated for each phosphoprotein. Data are displayed as whisker plots of 25th to 75th percentiles with means (central bars) and ranges (whiskers). PJAK2, pSTAT5, and pS6 were significantly elevated in the CRLF2_r JAK_wt and CRLF2_r JAK_mut samples in comparison with levels of the CRLF2_wt JAK_wt samples. ***P < .0005, **P < .005, *P < .05. Non-CRLF2–rearranged JAK wild-type = CRLF2_wt JAK_wt, CRLF2-rearranged JAK wild-type = CRLF2_r JAK_wt, CRLF2-rearranged JAK mutant = CRLF2_r JAK_mut. (C) Phosphoflow cytometry analysis of 41 primary human ALL samples without and with CRLF2 alterations and JAK mutations. 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. Stimulation with IL-7 (yellow bars) had no effect on phosphorylation in CRLF2_wt JAK_wt (n = 17), CRLF2_r JAK_wt (n = 12), or CRLF2_r JAK_mut (n = 12) ALL samples. TSLP stimulation (pink bars) induced pSTAT5 and pS6 in the CRLF2_r samples regardless of concomitant JAK mutations (P < .05), but had no effect on CRLF2_wt samples. XL019 (orange bars) inhibited phosphorylation below basal levels only for pS6 in the CRLF2_r JAK_mut subset (P = .013). Unlike in MUTZ5 cells, TSLP stimulation did not induce pERK significantly in these samples. *P < .05.