C100 | IDENTIFICATION OF THE NOVEL FUSION GENE CCDC6::JAK2 IN A PATIENT WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASM

A significant amount of patients present with a Myeloproliferative Neoplasm (MPN) phenotype in absence of driver mutations. The identification of atypical alterations in these cases is crucial for refining diagnostic criteria, risk stratification, and treatment strategies. Here we present the discovery of a novel fusion gene causing MPN. Bone marrow (BM) samples were collected from a 34-year-old MPN patient with massive splenomegaly, leukocytosis (WBC:90x10⁹/L, 62% neutrophils), mild anemia (Hb:11g/dL) and thrombocytopenia (Plt:102x10⁹/L). BM biopsy showed grade II fibrosis and increased cellularity, with an increased myeloid:erythroid ratio. CD34+ blasts were <5%. No driver mutation or recurrent fusion event was found according to routine diagnostic. Working diagnosis was of TN PMF, and the patient started ruxolitinib. Conventional karyotyping identified t(9;10), but exact localization of breakpoint was not obtained. Therefore, we used Long-Read Sequencing (LRS) to define the fusion breakpoint. Both confirmed t(9;10) at cytobands p24 and q21.2, respectively. LRS mapped it to JAK2 exon 11 and CCDC6 exon 8. Sanger validated the CCDC6::JAK2 fusion at genomic and transcript levels. The transcript sequence was in frame, joining CCDC6 exons 1–8 and retained intron 8/9 to JAK2 exons 11–25. JAK2 was hyperactivated in patient's cells compared to healthy controls (+2.1-fold p-Tyr1007/8), whereas the mRNA expression level was unchanged. JAK2 hyperactivation was consistent with Endogenous Erythroid Colonies (EEC) growth from patient's CD34+ plated in absence of Epo (EEC:276±15.5 vs 3±1 in controls). Transcriptome of patient’s granulocytes at diagnosis showed upregulation of granule-related pathways compared to healthy donors, that returned to baseline levels upon ruxolitinib treatment. The CCDC6::JAK2 fusion was further modeled using an expression vector in UT7 cells, demonstrating an increased proliferation rate (+3.7-fold) compared to control UT7 transduced with an empty vector (p<0.001).In conclusion, we report a novel CCDC6::JAK2 fusion gene encoding a chimeric protein that retains the CCDC6 coiled-coil and JAK2 kinase domains. By analogy with other CCDC6 fusions, we hypothesize that dimerization via coiled-coil domain enables JAK2 autophosphorylation and constitutive activation, and provides explanation of the favorable effects of ruxolitinib. Further functional studies on the role of CCDC6::JAK2 fusion in MPN pathogenesis will be presented at the conference.