Targeted deep-sequencing of over 500 cancer-relevant genes in 57 HD-ALL patients revealed extensive tumor heterogeneity in the RTK/Ras/MAPK signaling pathway, with the majority of patients harboring clonal, subclonal, and/or microclonal mutations at hotspot loci in KRAS, NRAS, FLT3, or PTPN11. Further, we found a substantial proportion of patients with epigenetic regulatory gene mutations, including in the putative novel driver gene DOT1L, and these typically co-occurred with mutations in FLT3. Here, PTPN11 is linked to acute lymphoblastic leukemia.