Figure 5.
STAT3 is activated independently of NPM1-ALK through the IL10/IL10R signaling pathway upon crizotinib inhibition. (A) Western blot analysis of differential JAK/STAT signaling activation in response to individual nontargeting (NT) sgRNA control or IL10RA sgRNA overexpression in SUP-M2 cells. Cells were treated with DMSO or 1000 nM crizotinib for 1 hour. Phosphorylated STAT3 (pSTAT3) levels normalized to STAT3 and relative to NT sgRNA (lane 5: 1, lane 6: 8.6, lane 7: 59.1, lane 8: 67.8). Blot is representative of 3 independent experiments. Lines indicate different blots. (B) Fold change in transcript level of the indicated STAT3 target genes relative to GAPDH and relative to NT sgRNA in SUP-M2 cells expressing sgRNAs targeting IL10RA and treated with 1000 nM crizotinib for 1 hour. Data are means ± standard deviation (SD) (n = 3). (C) Correlation between IL10RA and IL-10 mRNA expression levels in the Human Protein Atlas RNA-seq datasets, including nontransformed (red) and cancer (gray) cell lines. (D) Fold change in IL-10 mRNA expression levels in crizotinib-treated ALCL cell lines expressing sgRNAs inducing expression of IL10RA. Data are means ± SD (n = 3). (E) STAT3 ChIP-seq tracks near the IL10/IL10RB/IL10RA loci in ALCL cell lines treated for 3 hours with crizotinib (300 nM) or DMSO. (F) STAT3 ChIP-seq validation by ChIP, followed by qPCR, of the IL10/IL10RA/IL10RB and IRF4 TSSs in SUP-M2 cells treated for 3 hours with crizotinib (1000 nM) or DMSO. Data are means ± SD of technical replicates; experiment was performed independently 3 times. IRF4 served as a positive control. (G) Fold change in expression levels of STAT3 and IL-10 on STAT3 shRNA induction in the indicated ALCL cell lines compared with NT control shRNA and simultaneous expression of sgRNAs inducing overexpression of IL10RA. Data are means ± SD (n = 3). (H) Model summarizing the mechanism by which IL10RA overexpression leads to ALK TKI resistance.

STAT3 is activated independently of NPM1-ALK through the IL10/IL10R signaling pathway upon crizotinib inhibition. (A) Western blot analysis of differential JAK/STAT signaling activation in response to individual nontargeting (NT) sgRNA control or IL10RA sgRNA overexpression in SUP-M2 cells. Cells were treated with DMSO or 1000 nM crizotinib for 1 hour. Phosphorylated STAT3 (pSTAT3) levels normalized to STAT3 and relative to NT sgRNA (lane 5: 1, lane 6: 8.6, lane 7: 59.1, lane 8: 67.8). Blot is representative of 3 independent experiments. Lines indicate different blots. (B) Fold change in transcript level of the indicated STAT3 target genes relative to GAPDH and relative to NT sgRNA in SUP-M2 cells expressing sgRNAs targeting IL10RA and treated with 1000 nM crizotinib for 1 hour. Data are means ± standard deviation (SD) (n = 3). (C) Correlation between IL10RA and IL-10 mRNA expression levels in the Human Protein Atlas RNA-seq datasets, including nontransformed (red) and cancer (gray) cell lines. (D) Fold change in IL-10 mRNA expression levels in crizotinib-treated ALCL cell lines expressing sgRNAs inducing expression of IL10RA. Data are means ± SD (n = 3). (E) STAT3 ChIP-seq tracks near the IL10/IL10RB/IL10RA loci in ALCL cell lines treated for 3 hours with crizotinib (300 nM) or DMSO. (F) STAT3 ChIP-seq validation by ChIP, followed by qPCR, of the IL10/IL10RA/IL10RB and IRF4 TSSs in SUP-M2 cells treated for 3 hours with crizotinib (1000 nM) or DMSO. Data are means ± SD of technical replicates; experiment was performed independently 3 times. IRF4 served as a positive control. (G) Fold change in expression levels of STAT3 and IL-10 on STAT3 shRNA induction in the indicated ALCL cell lines compared with NT control shRNA and simultaneous expression of sgRNAs inducing overexpression of IL10RA. Data are means ± SD (n = 3). (H) Model summarizing the mechanism by which IL10RA overexpression leads to ALK TKI resistance.

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