3080 – IL-3 SELECTIVELY RESCUES RUNX1-DEFICIENT HUMAN HSPCS WITH DYSREGULATED JAK/STAT SIGNALING

Abstract

Both germline and somatic loss-of-function mutations in RUNX1 are commonly found in hematopoietic diseases, ranging from thrombocytopenia and autoimmunity to myeloid malignancies. However, our understanding of the underlying mechanisms is lacking. Here, we established a faithful CRISPR/AAV6 model of RUNX1 knockout (KO) in human CD34+ hematopoietic stem and progenitor cells (HSPCs), demonstrating monocytic skew at the expense of erythro-megakaryocytic potential and increased inflammatory signaling. Importantly, RUNX1 KO severely limited in vitro proliferation and in vivo competitive engraftment potential. Using ATAC-seq and RNA-seq, we determined that RUNX1 KO cells displayed decreased JAK/STAT signaling and elevated expression of the negative regulator SOCS3. Knockdown of SOCS3 rescued the RUNX1 KO proliferative defect and both RUNX1 KO HSPCs and RUNX1 mutant primary patient samples showed increased sensitivity to treatment with JAK inhibitors, suggesting that RUNX1 loss represses JAK signaling. Next, we investigated whether cytokines signaling through JAK/STAT can reverse the RUNX1 KO proliferative defect. Exposure to IL-3, but not other cytokines, rescued STAT5 phosphorylation, proliferation, and competitive engraftment defects, and both RUNX1 KO HSPCs and RUNX1 mutant primary AMLs upregulated the IL-3 receptor IL3RA (CD123) at the protein level. Thus, RUNX1 loss sensitizes cells to IL-3 and overcome the JAK/STAT signaling deficiency by regulating IL3RA receptor expression. In summary, we established a human model of RUNX1 loss and found dysregulated IL-3/JAK/STAT signaling, which is achieved by modulated expression of negative regulators and the IL-3 receptor. Importantly, we identify conditions under which RUNX1 KO cells regain proliferative competitiveness and anticipate that targeting these pathways may be beneficial for a range of RUNX1-mutant diseases. Both germline and somatic loss-of-function mutations in RUNX1 are commonly found in hematopoietic diseases, ranging from thrombocytopenia and autoimmunity to myeloid malignancies. However, our understanding of the underlying mechanisms is lacking. Here, we established a faithful CRISPR/AAV6 model of RUNX1 knockout (KO) in human CD34+ hematopoietic stem and progenitor cells (HSPCs), demonstrating monocytic skew at the expense of erythro-megakaryocytic potential and increased inflammatory signaling. Importantly, RUNX1 KO severely limited in vitro proliferation and in vivo competitive engraftment potential. Using ATAC-seq and RNA-seq, we determined that RUNX1 KO cells displayed decreased JAK/STAT signaling and elevated expression of the negative regulator SOCS3. Knockdown of SOCS3 rescued the RUNX1 KO proliferative defect and both RUNX1 KO HSPCs and RUNX1 mutant primary patient samples showed increased sensitivity to treatment with JAK inhibitors, suggesting that RUNX1 loss represses JAK signaling. Next, we investigated whether cytokines signaling through JAK/STAT can reverse the RUNX1 KO proliferative defect. Exposure to IL-3, but not other cytokines, rescued STAT5 phosphorylation, proliferation, and competitive engraftment defects, and both RUNX1 KO HSPCs and RUNX1 mutant primary AMLs upregulated the IL-3 receptor IL3RA (CD123) at the protein level. Thus, RUNX1 loss sensitizes cells to IL-3 and overcome the JAK/STAT signaling deficiency by regulating IL3RA receptor expression. In summary, we established a human model of RUNX1 loss and found dysregulated IL-3/JAK/STAT signaling, which is achieved by modulated expression of negative regulators and the IL-3 receptor. Importantly, we identify conditions under which RUNX1 KO cells regain proliferative competitiveness and anticipate that targeting these pathways may be beneficial for a range of RUNX1-mutant diseases.