Novel Gene Translocations Involving TYK2 in Cutaneous CD30-Positive Lymphoproliferative Disorders

Introduction: The CD30-positive cutaneous T-cell lymphoproliferative disorders (CD30-positive LPD) include lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large cell lymphoma (ALCL). Recurrent chromosomal translocations frequently underlie the pathogenesis of several hematologic malignancies and often define molecular subtypes with distinct biological behavior. The genetic events that contribute to the pathogenesis of CD30-positive LPD are largely unknown.

Goal: The goal of our study was to identify chromosomal translocations that result in the generation of oncogenic chimeric gene fusions that underlie the pathogenesis of

CD30-positive LPD.

Methods and Results: Paired end whole transcriptome sequencing (RNAseq) analysis was performed on the cutaneous T-cell lymphoma (CTCL) derived cell lines MyLa and HH. Bioinformatic analysis revealed an interchromosomal fusion between NPM1 (5q35) and TYK2 (19p13). The NPM1-TYK2 fusion was represented by 766 paired-end reads spanning the fusion junction of exon 9 of NPM1 and exon 16 of TYK2. Bi-directional Sanger sequencing confirmed predicted gene fusion sequence at the RNA level. SYBR Green I-based quantitative RT-PCR assays revealed specific expression of the NPM1-TYK2 fusion transcript only in MyLa and not in other CTCL (n = 2) or T-cell lymphoma-derived cell lines (n = 7). To establish that the NPM1-TYK2 gene fusion arose from a translocation event at the DNA level, we performed long range PCR which yielded a 1kb product containing the breakpoints and junctions in NPM1 (chr5:170,832,813) and TYK2 (chr19:10,469,817) introns that lead to juxtaposition of these genes by translocation. The fusion is predicted to encode an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. To determine if translocations targeting TYK2 were recurrent, we employed two independent fluorescence in situ hybridization (FISH) based assays, one using a TYK2 break-apart probe strategy to identify translocations targeting TYK2 and a second approach using an NPM1-TYK2 fusion probe strategy to specifically identify the NPM1-TYK2 translocations. These probes were used to screen patient-derived biopsies of CD30-positive LPD, CTCL and other T-cell neoplasms. FISH analyses revealed TYK2 translocations in 5 of 29 (17.2%) primary cases of CD30-positive LPDs (3/15 LYP; 2/14 cutaneous ALCLs) and was absent in other CTCL subtype (Mycosis fungoides, n = 44) and mature T-cell neoplasms (n = 107). Out of these 5 positive cases, one LYP case had an NPM1-TYK2translocation.

Since TYK2 is a member of Jak family kinases that are integral components of the

JAK-STAT signaling pathway, we investigated the downstream effects of NPM1-TYK2 fusion expression on TYK2 kinase activity and STAT protein pathway activation. Western blotting in MyLa, other cutaneous and mature T-cell lymphoma cell lines revealed hyperactivation of endogenous TYK2 kinase enzyme (pTYK2 levels) only in MyLa. Further, MyLa cells showed activation of downstream STAT signaling pathway proteins (pSTAT1, pSTAT3 and pSTAT5). Ectopic expression of NPM1-TYK2 wild type fusion gene in HEK293FT cells resulted in hyperactivation of TYK2 kinase enzyme and activation of STAT proteins, while a kinase-defective mutant NPM1-TYK2 K462R lacked TYK2 and STAT activity. Transcriptional activation assays for STAT proteins (STAT1, STAT3 and STAT5), showed more than two fold (P<0.01) elevation of reporter activity of the aforementioned STATs in NPM1-TYK2 wild type fusion protein expressing but not in kinase-defective mutant expressing cells. Silencing of TYK2 using a lentivirus-based shRNA approach resulted in decreased proliferation (P<0.01, 2.2 fold) of MyLa cells suggesting that NPM1-TYK2is an oncogenic-driver alteration.

Conclusions: Our study demonstrates for the first time recurrent TYK2 gene translocations in CD30-positive LPD or in any form of primary cancer. We identify NPM1 as one of the gene-fusion partners of TYK2 and provide functional support for

NPM1-TYK2 in mediating activation of STAT signaling to promote cell proliferation. Inactivation of TYK2 significantly diminishes proliferation, suggesting that TYK2 is an oncogenic driver kinase in a subset of CD30-positive LPD. Finally, our results raise the possibility that TYK2 may be a novel therapeutic target in a subset of CD30-positive LPDs.