Cooperativity Of RUNX1 and CSF3R Mutations In The Development Of Leukemia In Severe Congenital Neutropenia: A Unique Pathway In Myeloid Leukemogenesis

Congenital neutropenia (CN) is a rare inherited disorder of hematopoiesis with a 20% risk of evolving into acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Using next-generation sequencing in 31 CN patients who developed leukemia we found that 20 of the 31 patients (64.5%) had mutations in RUNX1 (runt-related transcription factor 1). Of these 20 patients, 19 had inherited mutations associated with CN. Intriguingly, the majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R (colony stimulating factor 3 receptor) mutations. Other leukemia-associated mutations (EP300, FLT3-ITD, CBL, and SUZ12) were less frequent. In eight patients, we detected two distinct heterozygous RUNX1 mutations. These mutations were localized to the splice-acceptor site of intron 4, affecting splicing of exons 3 and 4, which encode the Runt homology/DNA binding domain (RHD) of RUNX1, or solely in the RHD or were present in both RHD and trans-activation domain (TAD). In two patients, we were able to perform allele-specific analysis of RUNX1 mutations. Patient #10 had an Phe13TrpfsX14 deletion on one allele of RUNX1 and an Arg139ProfsX47 deletion on the other allele. In Patient #14, two RUNX1 mutations were on the same allele; one of the mutations (Met240Ile) was inherited from the mother and was localized two amino acids before the TAD, and the second acquired mutation (Arg139Gly) was in the RHD of RUNX1. Ten patients with RUNX1 mutations developed monosomy 7 and six patients developed trisomy 21 at diagnosis of leukemia. In contrast to their high frequency in CN evolving into AML, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric AML. RUNX1 mutations were mainly found in pediatric AML patients with an adverse prognosis. A sequential analysis at stages prior to overt leukemia in ten CN/AML patients showed that RUNX1 mutation is a late event in leukemogenic transformation. In 6 of 10 patients, a CSF3R mutation occurred prior to RUNX1 mutations (24-192 months prior to CN/AML for CSF3R mutations vs. 1-36 months prior to CN/AML for RUNX1 mutations). Interestingly, monosomy 7 or trisomy 21 appeared after acquisition of RUNX1 mutations and no additional chromosomal aberrations were detected by array-CGH. Single-cell analyses in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Moreover, functional studies demonstrated elevated G-CSF-induced proliferation with diminished myeloid differentiation of hematopoietic CD34⁺ cells after co-transduction with mutated RUNX1 and CSF3R, in comparison to cells transduced with mutated RUNX1 or mutated CSF3R only. The importance of RUNX1 mutations in leukemogenic transformation was substantially strengthened by the analysis of a unique family with two siblings suffering from CN that subsequently transformed to AML. In both children, cooperating RUNX1 and CSF3Rmutations were detected that were not present in healthy family members.

Taken together, the high frequency and the time course of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia suggests a unique molecular pathway of leukemogenesis similar as has been reported in the Gilliland-Griffin two-hit hypothesis for AML development. The concomitant detection of RUNX1 and CSF3Rmutations represents a useful biomarker for identifying CN patients with a high risk of progressing to leukemia or MDS.