Germline Runx1 Mutations Induce Inflammation in Hematopoietic Stem and Progenitor Cells and Predispose to Hematologic Malignancies

Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 ^R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation.

Analysis of the bone marrow cells in Runx1 ^R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 ^R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 ^R188Q/+ CFU average = 45/85), indicating a myeloid bias.

The number and size of platelets were not altered in Runx1 ^R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 ^R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced.

Hematopoietic progenitor cells isolated from Runx1 ^R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 ^R188Q/+ nuclei, suggesting that Runx1 ^R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 ^R188Q/+ hematopoietic progenitor cells.

To understand underlying mechanism responsible for the observed myeloid bias in Runx1 ^R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 ^R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (> 1.5 FC; FDR<0.05), including 8 up- and 31 down-regulated genes. The expression of three repressed genes with important function in hematopoietic differentiation and malignancy (Cdh1, Gja1, and Fcer1a) were validated by qRT-PCR.

To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 ^R188Q/+ mice were monitored for 20 months. Although Runx1 ^R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 ^R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 ^R188Q/flmice over time. Unlike Runx1 ^R188Q/+mice, Runx1 ^R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 ^R188Q/Δ mice reduced the median latency to 14.7 weeks.

These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM.