Figure 1.
Cytotoxic CTCLs harbor targetable mutations that activate the JAK-STAT pathway. (A) Survival curves for PCAETCL and CTCL-NOS were generated and analyzed with Log-rank (Mantel-Cox) test. (B) Summary of all fusions and their known domains. *Transcripts detected by the Archer assay. The rest were identified by WGS and/or RNA-Seq. Breakpoints are denoted with bold, black junctions. Clathrin BD, Clathrin binding domain; DNA BD, DNA binding domain; Protein BD, binding domain; NES, nuclear export signaling domain; TET, tetramerization domain. (C) Western blots of pathway targets in CAPRIN1-JAK2 and SELENOI-ABL1–expressing Ba/F3. Cells were transduced with novel fusion genes or wild-type kinase controls and treated with either vehicle or indicated drug for indicated times. Antibodies used against JAK2 (#3230), pJAK2 (#3771), cABL (#2862), pABL (#2868), STAT3 (#9139), pSTAT3 (#9145), STAT5 (#94205), pSTAT5 (#4322), CRKL (#38710), and pCRKL (#3181) were obtained from Cell Signaling Technology. (D) Cell viability assay to determine IC50 values of ruxolitinib and imatinib in CAPRIN1-JAK2-expressing Ba/F3 and SELENOI-ABL1–expressing Ba/F3, respectively. We cultured 0.1 × 106 cells per mL with inhibitors or vehicle in a 384-well plate. After 48 hours, Cell-titer Glo (CTG) luminescent reagent (Promega) was added. Luminescence was read by EnVision Multilabel Reader (PerkinElmer). Each data point was quantified in quadruplicate. Cell line experiments were repeated at least in triplicate. Dose response curves were generated with GraphPad. (E) Gene set enrichment analysis of Molecular Signatures Database Hallmark IL-6–mediated JAK-STAT signaling in PCAETCL compared with unstimulated (unstim) CD8+ T cells and CTCL-NOS compared with unstimulated CD8+ T cells. P values were .005 and .00001, respectively. (F) Oncoplot of PCAETCL and CTCL-NOS. Fusion events were called using STAR-Fusion19 and confirmed via manual inspection of sequencing data. Splice site, frameshift, or nonsense mutations were called as damaging mutations in known tumor suppressors. Hotspot mutations were recurrent amino acid alterations. Copy number deletions for focal deletion (<5 Mb). DMSO, dimethyl sulfoxide; ENTH, epsin N-terminal homology domain; HetDel, heterozygous copy number deletion; HomDel, biallelic deletions; WT, wild type.

Cytotoxic CTCLs harbor targetable mutations that activate the JAK-STAT pathway. (A) Survival curves for PCAETCL and CTCL-NOS were generated and analyzed with Log-rank (Mantel-Cox) test. (B) Summary of all fusions and their known domains. *Transcripts detected by the Archer assay. The rest were identified by WGS and/or RNA-Seq. Breakpoints are denoted with bold, black junctions. Clathrin BD, Clathrin binding domain; DNA BD, DNA binding domain; Protein BD, binding domain; NES, nuclear export signaling domain; TET, tetramerization domain. (C) Western blots of pathway targets in CAPRIN1-JAK2 and SELENOI-ABL1–expressing Ba/F3. Cells were transduced with novel fusion genes or wild-type kinase controls and treated with either vehicle or indicated drug for indicated times. Antibodies used against JAK2 (#3230), pJAK2 (#3771), cABL (#2862), pABL (#2868), STAT3 (#9139), pSTAT3 (#9145), STAT5 (#94205), pSTAT5 (#4322), CRKL (#38710), and pCRKL (#3181) were obtained from Cell Signaling Technology. (D) Cell viability assay to determine IC50 values of ruxolitinib and imatinib in CAPRIN1-JAK2-expressing Ba/F3 and SELENOI-ABL1–expressing Ba/F3, respectively. We cultured 0.1 × 106 cells per mL with inhibitors or vehicle in a 384-well plate. After 48 hours, Cell-titer Glo (CTG) luminescent reagent (Promega) was added. Luminescence was read by EnVision Multilabel Reader (PerkinElmer). Each data point was quantified in quadruplicate. Cell line experiments were repeated at least in triplicate. Dose response curves were generated with GraphPad. (E) Gene set enrichment analysis of Molecular Signatures Database Hallmark IL-6–mediated JAK-STAT signaling in PCAETCL compared with unstimulated (unstim) CD8+ T cells and CTCL-NOS compared with unstimulated CD8+ T cells. P values were .005 and .00001, respectively. (F) Oncoplot of PCAETCL and CTCL-NOS. Fusion events were called using STAR-Fusion19 and confirmed via manual inspection of sequencing data. Splice site, frameshift, or nonsense mutations were called as damaging mutations in known tumor suppressors. Hotspot mutations were recurrent amino acid alterations. Copy number deletions for focal deletion (<5 Mb). DMSO, dimethyl sulfoxide; ENTH, epsin N-terminal homology domain; HetDel, heterozygous copy number deletion; HomDel, biallelic deletions; WT, wild type.

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