Fat Mass Obesity-Associated (FTO) Driven Gene Regulatory Networks Control Hematopoietic Stem Cell Expansion and Homing

Hematopoietic stem cell (HSC) transplantation is the only known curative therapy for several hematological disorders, however high doses are required for long-term success. We previously optimized cocktails of stem cell agonists that promote greater expansion of HSCs compared to single agonists such as UM171 and StemReginin 1 (SR1). Limited dilution transplant assay revealed that the lead cocktail X2A promoted 15- and 4-fold expansions of scid repopulating cells, when compared to non-cultured cells or cells expanded with the combination of UM171 and SR1. RNA-seq experiments identified several genes of interest in human X2A-expanded HSPCs including the epitranscriptomic regulator FTO (Manesia et al., 2023). FTO is known to control global RNA stability via demethylation of N6-methyladenosine (m6A) on RNA. While FTO has been previously shown to contribute to oncogenesis, its role in normal human hematopoiesis has not been studied to date. The objective of the present study is to understand the role of FTO within human hematopoietic stem and progenitor cells.

Hematopoietic fate mapping analyses revealed that FTO expression is higher within HSC enriched populations and decreases with differentiation. Interestingly, chemical inhibition of FTO dramatically reduced the ability of X2A to expand HSPCs in cultures and increased global m6A levels over 2-fold (p<0.01, n=3). Furthermore, lentivirus shRNA mediated knock down (KD) of FTO within CD34+ cells led to a 2.5-fold increase in colony-forming units (CFU) (n=3, p<0.01). In contrast, overexpression of FTO increased HSPC expansion, but reduced the number of CFU colonies by 2-fold (n=3, p<0.01). Next, to assess the impact of loss of FTO on HSC’ engraftment properties, cord blood CD34+ cells transduced with either scramble or FTO KD shRNA lentiviruses were transplanted into NOD scid gamma mice. Peripheral blood analysis of human leucocyte and platelets 3 weeks post-transplantation revealed a 4-fold reduction in short-term engraftment in FTO KD recipients when compared to scramble cohort (p<0.05, 5-mice/cohort, n=2). This deficit in engraftment in FTO KD recipients also extended to long-term engraftment (p<0.05, 5 mice/cohort, n=2). At 20 weeks post transplantation bone marrow analysis revealed that mice transplanted with FTO deficient CD34+ cells had a 5-fold and 6-fold reductions in the percentage of human CD34+ cells and CD45+ cells, respectively (P<0.001, 5-mice/cohort, n=2). Secondary engraftment was also found blunted in FTO recipients.

To identify targets and gene regulatory pathways regulated by FTO that may be critical to the regulation of HSPC, we performed m6A sequencing and RNA sequencing on FTO KD or scramble KD CD34+ cells (n=2). Next, we drafted the gene regulatory networks (GRNs) of FTO, by integrating the m6Aseq and RNA seq data. The GRN contained 820 genes, and gene ontology analysis revealed that FTO drives several important pathways within HSPCs that play a role in, HSPC homing (CXCR4), RNA translation (mTOR), metabolism, and cell signaling (SRC).

In conclusion, we discovered for the first time that FTO driven m6A levels are critical for expansion of human HSC and for HSC’ engraftment properties. GRN analysis revealed that FTO controls several critical nodes important for HSC expansion and homing. Taken together, our results suggest that m6A targeting may provide a novel therapeutic area to enhance HSPC expansion and homing in patients receiving bone marrow transplants.