CSF3R T618I Collaborates with RUNX1-RUNX1T1 to Expand Human Haematopoietic Stem and Progenitor Cells
Abstract The cytokine receptor colony stimulating factor 3 (CSF3R) controls differentiation, proliferation and function of granulocytes as well as mobilization of hematopoietic stem cells upon activation via G-CSF. An oncogenic role of CSF3R mutations has been mainly described in chronic neutrophilic leukemia (CNL) and, more rarely, in atypical chronic myeloid leukemia and pediatric acute myeloid leukemia (AML) (Maxson et al., 2013, N Engl J Med; Maxson et al.,2016, Blood). CSF3R mutations are classified in two distinct categories: truncating mutations and proximal-membrane domain mutations. While truncating mutations lead to a higher sensitivity toward the ligand, the more common proximal-membrane mutations are ligand-independent in activating downstream signaling. Recently, we found CSF3R recurrently mutated in 5% (13/292) of patients with adult core-binding factor (CBF) leukemia (Opatz et al., 2020, Leukemia). Both CBF leukemia subgroups, characterized by either an inversion of chromosome 16 (inv(16)) or translocation t(8;21), were affected. The most prevalent mutation hotspot was T618 in the t(8;21) subgroup, localizing to the proximal-membrane region, recurrently affected also in CNL. We set out to study the role of CSF3R mutations in AML t(8;21). Using CD34+ hematopoietic stem cells from adult healthy donors, we observed that co-expression of the CSF3R mutant T618I (CSF3R T618I) but not CSF3R wildtype (WT)together with the t(8;21) related RUNX1-RUNX1T1 fusion led to a competitive outgrowth of cells (Figure 1A,B). In contrast, over-expression of either CSF3R WT or CSF3R T618Ialone did not lead to a competitive advantage. Furthermore, in a culture composed of >95% RUNX1-RUNX1T1 positive expanded cells, super-infection with the retroviral CSF3R T618I construct led to complete outgrowth of double positive cells over a period of 60 days. These data suggest an oncogenic cooperation between RUNX1-RUNX1T1 and CSF3R T618I. Comparing the cytomorphology of RUNX1-RUNX1T1 single positive cells and RUNX1-RUNX1T1/CSF3R T618I double positive cells, the single positive cells were heterogeneous with a mixture of mature and immature cells (Figure 1C), whereas double positive cells remained mostly undifferentiated, featuring a homogeneous blast-like morphology (Figure 1D). In accordance with these findings, limiting and serial dilution experiments clearly showed an increased self-renewal potential for double oncogene expressing cells compared to cells expressing RUNX1-RUNX1T1 only. This further supports a transforming potential of CSF3R T618I in combination with RUNX1-RUNX1T1. RNA sequencing of outgrown double oncogene expressing cells revealed upregulation of distinct pathways related to myeloid differentiation, in particular neutrophil cell activation and migration. In addition, we found GLI2 as potential downstream effector and putative pharmacological target in AML t(8;21) with CSF3R T618I (Figure 1E). GLI2 expression is a negative prognostic marker in AML, associated with significantly shorter event free and overall survival (Wellbrock et al., 2015, Clin Cancer Res). Acting in the hedgehog-signaling pathway, GLI2 is not expressed in healthy adult tissues. Of note, patient data and mouse models support a specific collaboration of upregulated GLI2 and FLT3-ITD in AML (Wellbrock et al., 2015, Clin Cancer Res; Lim et al., 2015, Sci Transl Med). Conducting further experiments in the granulocyte-macrophage colony stimulating factor (GM-CSF) dependent t(8;21)-positive cell line SKNO-1, we observed cytokine independent growth in the presence of CSF3R T618I but not CSF3R WT. GM-CSF withdrawal of SKNO-1 native cells, vector control or CSF3R WTexpressing cells led to cell cycle arrest that could be overcome by expression of CSF3R T618I. Subsequently, we investigated pharmacological counteraction of CSF3R T618I through inhibition of putative effectors. CSF3R T618I expressing SKNO-1 cells were significantly more sensitive towards treatment with the GLI inhibitor GANT61, as compared to vector or CSF3R WT controls (Figure 1F). Taken together, our results suggest a specific oncogenic collaboration between RUNX1-RUNX1T1 and CSF3R T618I with potential therapeutic implications. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Greif: AstraZeneca: Honoraria.
