RUNX1 TAD and RHD Mutations Found in SCN/AML Differentially Affect HSPC Expansion in Conjunction with Truncated CSF3 Receptors

Abstract

Severe congenital neutropenia (SCN) is characterized by a maturation arrest at the promyelocyte stage and consequently a severe reduction of peripheral neutrophils. Administration of colony stimulating factor 3 (CSF3) restores neutrophil levels in over 90% of SCN patients, leading to an improved survival rate. SCN patients have an increased risk to develop secondary MDS or AML. Leukemic progression of SCN frequently involves the acquisition of a mutation in the gene encoding CSF3 receptor (CSF3R) in the neutropenic phase, followed by a mutation in runt-related transcription factor 1 (RUNX1) prior to transformation to MDS/AML (Skokowa et al, Blood, 2014). CSF3R mutations in SCN truncate the CSF3R C-terminus. RUNX1 mutations in SCN/AML are predominantly single nucleotide variations in the Runt-homology domain (RHD). Frameshift/nonsense mutations in the transactivation domain (TAD) also occur, albeit less frequently.To investigate how the combination of CSF3R and RUNX1 mutations affects myelopoiesis, we isolated hematopoietic stem and progenitor cells (HSPCs) from mice expressing either wild-type (WT) or a truncated (d715) CSF3R, retrovirally transduced them with TAD (S291fsX9) or RHD (D171N) mutants of RUNX1, or an empty vector control (EV), and cultured the cells in the presence or absence of CSF3. FACS analysis was performed to assess numbers of hematopoietic stem cells (LSKs, Lin- Sca1+ Kit+), early myeloid progenitors (LKs, Lin- Sca1- Kit+), common myeloid progenitors (CMPs, LK CD34+ CD16/32low), granulocyte-monocyte progenitors (GMPs, LK CD34+ CD16/32hi), immature (CD11b+, Gr-1lo) and mature (CD11b+, Gr-1hi) neutrophils. Whereas a normal differentiation pattern was observed in the CSF3R-WT background, activation of CSF3R-d715 led to defective neutrophil differentiation. Introduction of the RUNX1 mutants further aggravated the differentiation block and resulted in a selective expansion of HSPC subsets. While the EV and CSF3R-WT controls showed a relatively equal distribution of LK and LSK cells over time, the combination of CSF3R-d715 and RUNX1-TAD led to a 10-fold increase in absolute LSK numbers relative to EV (LSKs day 9: TAD: 1.23x107, EV: 1.27x106, RHD: 4.86x105, n=3, p=0.05) and a 7:1 ratio of LSK over LK. In contrast, the combination of CSF3R-d715 and RUNX1-RHD predominantly expanded LKs (LKs day 9: RHD: 8.77x106, EV: 2.77x106, TAD: 1.69x106, n=3, p=0.027) resulting in a 18:1 ratio of LK over LSK. Further analysis showed that these LKs were blocked in differentiation at the CMP to GMP transition stage (CMP to GMP ratio: RHD: 111.5, EV: 9.3, TAD: 1.8).To interrogate which mechanisms are responsible for the distinct differentiation defects caused by the RUNX1-RHD and -TAD mutants, we performed RNA-Seq on FACS purified LSK and LK populations. CMP to GMP transition is controlled by CCAAT/enhancer-binding proteins (C/EBPs), mainly C/EBPα and C/EBPβ. The combination of CSF3R-d715 and RUNX1-RHD, blocking the transition from CMP to GMP, induced the expression of C/EBPγ, a strong antagonist of C/EBP transcriptional activity (FPKM: day 2: 107.2, day 5: 424.3, day 9: 801.3). A major function of CEBPs in driving CMP to GMP transition is to suppress E2F- and Myc-driven transcription of genes involved in cell cycling. Consistent with this, single sample gene set enrichment analysis with output of hallmark pathways (Broad Institute) showed de-repression of E2F (p=0.002) and Myc (p=0.036) pathways in CSF3R-d715/RUNX1-RHD LKs relative to CSF3R-d715/EV. Interestingly, the RUNX1-TAD mutant that selectively expanded LSKs in combination with CSF3R-d715 did not alter C/EBP expression and function but affected genes involved in ribosomal biogenesis.We conclude that RUNX1-TAD and RHD mutants differentially cooperate with the CSF3R truncating mutations that are frequently acquired in the neutropenic phase of SCN and take alternative routes for LSK versus LK expansion. How this affects the leukemogenic nature of the RUNX1 mutations in combination with CSF3R truncation and how we can functionally interfere with these mechanisms is currently under investigation in in vivo transplantation settings. DisclosuresNo relevant conflicts of interest to declare.