Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

T(17;19)(q21‐q22;p13), responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t(1;19)(q23;p13), producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t(17;19)‐ALL is extremely poor, while that of t(1;19)‐ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)‐ALL cases. Using 4 t(17;19)‐ALL and 16 t(1;19)‐ALL cell lines, drug response profiling was analyzed. t(17;19)‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)‐ALL cell lines. Sensitivities to three (Pred, VCR, and l ‐asparaginase [ l ‐Asp]), four (Pred, VCR, l ‐Asp, and DNR) and five (Pred, VCR, l ‐Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)‐ALL cell lines than for t(1;19)‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t(17;19)‐ALL cell lines than in t(1;19)‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t(17;19)‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t(17;19)‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t(17;19)‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t(17;19)‐ALL cell lines. In all four t(17;19)‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)‐ALL.