Evaluation of WNT pathway transcriptomes in T-cell Acute Lymphoblastic Leukemia by targeted RNA-Seq

Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. We previously described a recurrent rearrangement involving the WNT10B locus (WNT10BR), characterized by expression of the WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analyzed an italian cohort of patients (n=20) and detected the WNT10BIVS1 with a high prevalence (14/20). To address genes involved in WNT10B molecular response, we designed a Wnt targeted RNA sequencing panel. We identify Wnt agonists and antagonists, from which the expression of FZD6, PLCB4, and PROM1 stand out in WNT10BIVS1-positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as cell models, characterized by the expression of WNT10BIVS1, we found that WNT10B drives major Wnt activation through receipt of ligand to the FZD6 receptor complex. The WNT10B/FZD6 interaction were interfered with by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated inhibition of WNTs secretion. We found that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as new targets in the T-ALL treatment strategy. We used Ion AmpliSeq RNA WNT Signaling Pathway Research Panel targeting 178 genes that regulate the production of WNT signaling molecules, interactions with receptors on target cells, and signal transduction, to analyse mononuclear cells from T-ALL patients (n=5), healthy donors (n=2), MOLT4, and MUTZ2 cell lines.