Co-Acquisition of RUNX1 and CSF3R Mutations Transforms Hematopoietic Progenitor Cells of CN Patients into More Primitive Highly Proliferative Blasts: Evidence in CN Patients and in a Mouse Model

Abstract

Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a high risk of evolving into leukemia or myelodysplastic syndrome (MDS). Recently we demonstrated a very high frequency of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia or MDS (Skokowa, et al. Blood 2014). We proposed a novel molecular pathway of leukemogenesis: mutations in the cytokine receptor (G-CSFR) in combination with the second mutations in the hematopoietic transcription fator (RUNX1).In the majority of CN patients, CSF3R mutations were acquired prior to RUNX1 mutations. CSF3R mutations alone are unable to induce leukemia in CN patients or in mice expressing a transgenic d715 G-CSFR. Co-acquisition of RUNX1 mutations is an essential step in the leukemogenic transformation in CN. To characterize the expression signature of hematopoietic cells of CN/AML patients carrying CSF3R mutations prior to and after acquisition of RUNX1 mutations, we analyzed expression profiles of CD34+ hematopoietic cells of CN patient who developed AML. This patient acquired CSF3R mutation (p. Q718*) five years and RUNX1 mutation (p. R139G) 16 months prior to leukemia. We compared expression profiles of CD34+ cells harbouring CSF3R mutation only, or both CSF3R and RUNX1 mutations. Co-acquisition of RUNX1 and CSF3R mutations led to marked reduction of the expression of hematopoietic growth factors such as IL6 and NAMPT, inhibitors of cytokine signaling SOCS3, as well as of components of neutrophil granules OLFM4, DEFA4, MMP8, SLPI, CRISP3 and CTSG. At the same time expression levels of pro-proliferative downstream effectors of G-CSF such as STAT5A, STAT5B, SMAD1 and cyclin A1 (CCNA1) were dramatically elevated. Moreover, genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD109, HPGDS, PDLIM1, MLLT11 and FLT3 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations. Intriguingly, elevated expression of DNTT, BAALC, CD109 and FLT3 was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests rapid transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after acquisition of RUNX1mutation.To elucidate the role of cooperative CSF3R and RUNX1 mutations on the clonogenic capacity and myeloid differentiation of hematopoietic progenitors, we performed functional studies in mice. We transduced lineage negative (lin-) bone marrow hematopoietic progenitor cells of WT or transgenic d715 G-CSFR mice with lentiviral expression constructs containing either WT or mutated forms of RUNX1 cDNA. We used two different mutants of RUNX1 by introduction of mutations at amino acid positions 139 and 174. Acquired RUNX1 mutations in these amino acids were presented with high frequency in our cohort of CN/AML patients and in most of the cases were associated with acquired CSF3R mutations. We found that similar to the effect of CSF3R mutations, lin-hematopoietic cells of WT mice transduced with mutated RUNX1 alone did not show elevated clonogenic capacity in replating experiments. Interestingly, transduction of WT cells with RUNX1 mutants resulted in severely reduced numbers of CFU-G colonies but unaffected CFU-M and BFU-E colonies. Intriguingly, transduction of lin- hematopoietic cells from transgenic d715 G-CSFR mice with RUNX1 mutants resulted in a markedly elevated clonogenic capacity in replating experiments, as compared to cells transduced with WT RUNX1 or control vector: numbers of colonies after second replating were 7 and 8 times higher in RUNX1-R139G and RUNX1-R174X mutants, respectively, in comparison to RUNX1 WT transduced cells. Moreover, granulocytic differentiation of lin- cells from d715 G-CSFR mice transduced with RUNX1-R139G mutant was severely diminished, in comparison to cells transduced with WT RUNX1, as revealed by 5-fold reduction of CFU-G colonies.Taken together, co-acquisition of RUNX1 and CSF3R mutations shifted the hematopoietic differentiation program towards more primitive hematopoietic progenitors with elevated proliferative capacity and reduced myeloid differentiation, which ultimately lead to leukemia. DisclosuresNo relevant conflicts of interest to declare.