MOLECULAR PROFILE OF T-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA WITH CRLF2 OVEREXPRESSION

INTRODUCTION AND OBJECTIVES: CRLF2 overexpression is a potential biomarker of poor prognosis in T-cell acute lymphoblastic leukaemia (T-ALL) and also a candidate for targeted therapy. Recently, we demonstrated that CRLF2 overexpression (CRLF2-high) associates with mutations leading to the NOTCH1 intracellular protein stabilisation, however the mechanisms underlying CRLF2 deregulation in T-ALL remains unknown. Therefore, we aimed to evaluate the molecular profile of patients with CRLF2-high and delineate the mechanisms that contribute to this gene dysregulation. MATERIAL AND METHODS: We analysed data from 264 patients (Therapeutically Applicable Research to Generate Effective Treatments - TARGET) and 8 T-ALL cell lines (Cancer Cell Line Encyclopedia and Gene Expression Omnibus). Information of gene expression, mutations, copy number changes and gene fusions generated by RNA sequencing (RNA-seq), whole exome sequencing and microarrays were assessed to categorise the patients into CRLF2 subgroups and to delineate the molecular landscape of these leukaemias. We also analysed RNA-seq data from previous studies that investigated the Polycomb Repressive Complex 2 (PRC2) loss in LOUCY and JURKAT cell lines through BET proteins inhibition and EZH2 silencing, respectively. The CRLF2 region was investigated by chromatin immunoprecipitation (ChIP) followed by sequencing performed in T-ALL cell lines for H3K27ac, H3K4me1 and H3K4me3. Finally, we performed a quantitative ChIP (qChIP) assay to evaluate MEF2C binding in CRLF2 regulatory regions. RESULTS AND CONCLUSION: Initially, we observed an enrichment of early T-cell precursor ALL (ETP-ALL) samples in the CRLF2-high subgroup (23.08% vs 4.02%, p=7.579e-06) and these molecular entities showed similarities in their transcriptional profile. Likewise, JAK3, RUNX1 and EZH2 mutations and TCF7 deletions were associated with higher CRLF2 levels (p=0.0063, p=0.0009, p=0.0170 and p=0.0102, respectively). CRLF2 expression in LOUCY, an ETP-ALL cell line with CRLF2-high, was restored after BET inhibitors treatment (p=0.0095). However, in JURKAT, the EZH2 silencing did not upregulate CRLF2 expression. We observed active enhancer regions in LOUCY and identified 104 transcription factors (TFs) that recognize these regions. We filtered those TFs according to their association with ETP-ALL and the CRLF2-high subgroup and further investigated the MEF2C gene as a potential regulator of this dysregulation in T-ALL. Subsequently, through qChIP assay we demonstrated that MEF2C binds CRLF2 enhancer region. In the present study, we demonstrated the association of CRLF2 overexpression with the ETP-ALL subtype and with some molecular alterations. Furthermore, the PRC2 activity loss seems to be a mechanism underlying CRLF2 upregulation in ETP-ALL. Although, in other subtypes, PRC2 loss alone is unable to account for this gene dysregulation, suggesting that MEF2C activation may be a crucial trigger for this molecular event.