RUNX1 Mutations Identify an Entity of Blast Phase Chronic Myeloid Leukemia (BP-CML) Patients with Distinct Phenotype, Transcriptional Profile and Drug Vulnerabilities

Introduction: Genetic alterations of RUNX1 gene (mutations and fusion genes) are common in hematological malignancies including AML, ALL, MDS, and MPN. Mutations of RUNX1 occur in 10% of newly diagnosed AML patients and associate with inferior prognosis. Similarly, point mutations in RUNX1 are frequent in BP-CML, but little is known about their role in BP patients.

Methods: Bone marrow samples from four BP patients with RUNX1 mutations were collected from Helsinki University Hospital, and samples from four other BP patients without RUNX1 mutations were used as controls. Samples were analyzed by exome and RNA sequencing. For drug sensitivity and resistance testing (DSRT), a high-throughput platform comprising 195 approved and investigational oncology drugs was used. To further study the role of RUNX1 mutations in BP-CML, Baf3/BCR-ABL1+ RUNX1^-/- knock-out and RUNX1^-/mut heterozygous deletion mutant models were created using CRISPR-CAS9 technology. The effects of the mutations in the cell line models were characterized by flow cytometry, RNA sequencing, and DSRT analyses.

Results: The median age of RUNX1-mutated BP patients was 47 years (range 36-56 years) and included 3 patients with myeloid-BP and 1 with lymphoid-BP phenotype. RUNX1-mutated patients tended to have higher mutational load and co-occurrence with mutations to the BCOR, EZH2 and PHF6 genes in agreement with the mutational profile of RUNX1-mutated AML. Signature analysis of RUNX1-mutated BP patients showed dominance of signature 1 (age-related), signature 6, and 15 (DNA mismatch repair) correlating with the control group signature profiles. In addition, the RUNX1 mutated group showed specific enrichment of signature 9, related to somatic hypermutation and AID/RAG axis activity, that was not observed in the control group. RNA sequencing revealed that 388 genes were significantly differentially expressed between RUNX1-mutated and control BP patients (139 upregulated, 249 downregulated). Interestingly, the antigen processing and presentation pathway was among the top upregulated pathways in RUNX1 mutated patients with overexpression of CIITA and HLA-DR genes. Stem cell differentiation, complement, and hemostasis pathways were enriched in the downregulated gene set. Lymphoid-specific transcription factors (DNTT, PAX5 and VPERB1) in addition to CD19 and CD25 were significantly upregulated in the RUNX1-mutated group. DSRT data showed greater sensitivity to glucocorticoids, MEK-, mTOR- and VEGFR-inhibitors in RUNX1-mutated patients compared to the control group. Data from RUNX1^-/- and RUNX1^-/mut cell line models confirmed the induced expression of CD19 surface marker with RUNX1 mutation. DSRT results showed increased activity of MEK inhibitor and mTOR inhibitors in RUNX1^-/- knock-out and RUNX1^-/mut cell lines.

Conclusions:RUNX1 mutations in BP-CML patients associate with distinct phenotypic and transcriptional features. DSRT results together with transcriptional data support the potential benefits of glucocorticoids and MEK and mTOR inhibitors as well as immunotherapy targeting CD19 and CD25 in this group of BP-CML patients.