FoxM1 Haploinsufficiency Drives Clonal Hematopoiesis
Authors: Yu Chunjie , Sheng Yue , Qian Zhijian 
Published: February 2020

Hematopoiesis is an orchestrated process in which hematopoietic stem cells (HSCs) can self-renew and produce all lineages of blood cells. Majority of HSCs are in a quiescent state with a low growth rate. However, some genetic mutations that occur in HSCs impel HSCs to exit the quiescent state and to proliferate excessively, which enables mutant HSCs to outcompete normal HSCs and leads to clonal expansion of mutant HSCs. Myelodysplastic syndromes (MDSs) as a clonal disease, arise from the expansion of mutant HSCs and are characterized by morphologic dysplasia, ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia.
FoxM1 is one of transcription factors in the family of Fox ('Forkhead box') proteins. Analysis of public database revealed that the expression level of FOXM1 was decreased significantly in CD34 + cells from a subset of patients with MDS as compared to healthy individuals. Thus, we sought to determine whether haploinsufficiency of FOXM1 contributes to the development of MDS in mice. Our study showed that haploinsufficiency of Foxm1 led to an expansion of hematopoietic stem/progenitor cells in mice. Since FoxM1 has previously been implicated in regulation of cell cycle, we determined the cell cycle status of Foxm1 heterozygous HSCs. By BrdU incorporation assay, we showed that Foxm1 heterozygous HSCs have an increased S phase and G2/M phase as compared to control HSCs from wildtype mice. Additional analysis with Hochest33342/Pyronin-Y staining and Ki67/DAPI staining showed a significant decrease in the number of quiescent (G0) cells in Foxm1 heterozygous HSCs as compared to control HSCs. These results suggest that FoxM1 haploinsufficiency promotes HSCs to exit quiescence and to enter cell cycle, thereby leading to exhaustion of HSCs. To further assess the function of Foxm1 heterozygous HSCs in vivo, we performed competitive repopulation assay. We found that Foxm1 haploinsufficiency HSCs exhibited competitive repopulation advantage in the first and secondary recipient mice, but displayed significantly decreased capacity of repopulation in tertiary recipient mice as compared to control HSCs, suggesting that Foxm1 haploinsufficiency promoted clonal expansion of HSCs, which leads to an exhaustion of HSCs eventually. HSC proliferation can be induced by some specific immune effectors such as Toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) stimulates HSC proliferation by activating TLR4 signaling pathway. Low dose of LPS treatment over time accelerated the development of MDS in mice. Interestingly, low dose of LPS injection chronically induced defects in hematopoiesis in Foxm1 haploinsufficient mice but not the control wildtype mice. Recipient mice transplanted with Foxm1 heterozygous BM cells but not the control BM cells developed MDS-like disease with cytopenia and a decreased number of hematopoietic stem/progenitor cells after LPS stimulation. Moreover, we found that nearly half of aged Foxm1 haploinsufficient mice (20 months) developed splenomegaly. Analysis of histologic sections in Foxm1 haploinsufficient mice showed that the mice developed hematopoietic dysplasia including dysplastic megakaryocytes with bizarre-shaped nuclei in bone marrow and extramedullary hematopoiesis with accumulation of myeloid cells in spleen. RNA-seq analysis indicated that haploinsufficiency of Foxm1 perturbed multiple stem cell-maintenance mechanisms especially in metabolic processes. Taken together, our studies suggest that Foxm1 haploinsufficiency in mice causes clonal expansion of HSCs and promotes MDS-like disease, which underscores the significant role of FOXM1 downregulation in the initiation and development of human MDS.

Disclosures
No relevant conflicts of interest to declare.