Primary cytotoxic T-cell lymphomas harbor recurrent targetable alterations in the JAK-STAT pathway

Abstract

Primary cytotoxic T cell lymphomas (CTCLs) are a rare subset of aggressive, poor-prognosis T-cell lymphomas targeting the skin; they include primary cutaneous γδT-cell lymphoma, primary cutaneous VD8⁺ aggressive epidermotropic T cell lymphoma (PCAETCL), and cytotoxic CTCL not otherwise specified. Lee et al report that all 3 subsets have JAK-STAT activation, but PCAETCL uniquely carries JAK2 gene fusions that may render them especially susceptible to JAK inhibitors.

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-Fusion 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.