LAUSR

Zebrafish (Danio rerio) has become a well-established and powerful in vivo model to study the molecular basis of Tcell acute lymphoblastic leukemia (T-ALL) [1]. Langenau et al. [2] were the first to generate a cancer model using transgenesis. Expression of the mouse Myc oncogene under the D. rerio lymphoblast-specific rag2 promoter resulted in rapid onset of leukemia [2]. However, since the development of this Myc model, the number of other oncogenedriven T-ALL models in zebrafish has remained limited. The TLX1-driven T-ALL subtype encompasses 5–10% of pediatric and 30% of adult T-ALL cases, and is caused by a translocation that juxtaposes the TLX1 transcription factor near the T-cell receptor α or β enhancer region [3]. TLX1 acts as an oncogene in T-ALL, mainly by binding DNA regulatory regions to repress a network of T-ALL tumor suppressor genes including BCL11B, RUNX1, WT1, TET1, EZH2, FBXW7, PTEN, PTPN2 and mitotic checkpoint genes, leading to genomic instability and aneuploidy [4, 5]. Notably, 43.5% of TLX1/3-positive T-ALL harbor mutations in PHF6 [6] when compared to 9.5% in TLX1/3negative cases [6], suggesting a functional cooperation between these genetic perturbations might drive leukemogenesis. PHF6 is amongst the most frequently affected genes in T-ALL due to loss-of-function mutations or deletions [6]. However, the exact functionality of PHF6 in TALL remains largely unclear. Nevertheless, recent studies in mice support a role of PHF6 in the regulation of leukemia stem cell activity [7, 8]. Interestingly, PHF6 has also been detected as a tumor promoting factor in B-ALL [9–11], suggesting a high degree of context specificity regarding the role of PHF6 in leukemia biology. Here, we used zebrafish to model TLX1-driven leukemia in vivo and investigate its putative cooperation with PHF6 deficiency in T-ALL leukemogenesis. We generated a stable Tg(rag2:hTLX1;rag2:EFGP) zebrafish line (henceforth called hTLX1 fish) by over-expressing human TLX1 and EGFP in developing lymphocytes (Supplementary Fig. 1a, b). Fish was screened by fluorescence microscopy for T-ALL development every 2 weeks for a total of 18 months. In total, 161 hTLX1 zebrafish were monitored; 2 fish (1.2%) developed T-ALL (samples named “T”) at 13 and 14 months of age (Fig. 1a). To assess functional cooperation with PHF6 loss during T-ALL formation, the hTLX1 stable line was crossed to a phf6 mutant line that harbors a 10 bp deletion in exon 2, leading to a frameshift that creates a premature stop codon (Supplementary Fig. 1c). No T-ALL tumors could be detected in the phf6 mutant (n= 104) only, suggesting the need for additional These authors contributed equally: Kaat Durinck, Frank Speleman