Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1 BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1 CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1 blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1 patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1 and heterozygous RUNX1 BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1 BP-CML patients.