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1297 m6a-Son-CCL5 Axis Controls HSC Fate and Inflammation

Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, hematopoiesis, cell expansion, Biological Processes, Technology and Procedures, gene editing, omics technologies
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Hanzhi Luo, PhD1, Mariela Cortés-López, PhD2,3,4*, Cyrus L. Tam5,6*, Michael Xiao7*, Isaac Wakiro, BSc7*, Karen L. Chu7*, Aspen Pierson1*, Mandy Chan1*, Kathryn Chang7*, Xuejing Yang, PhD7*, Grace Han7*, Erin Ahn, PhD8, Quaid D. Morris, PhD5*, Dan A. Landau, MD, PhD2,9* and Michael G. Kharas, PhD10

1Memorial Sloan Kettering Cancer center, New York, NY
2New York Genome Center, New York
3Meyer Cancer Center, Weill Cornell Medicine, New York, NY
4Institute of Computational Biomedicine, Weill Cornell Medicine, New York
5Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York
6Tri-Institutional Training Program in Computational Biology and Medicine, Weill Cornell Medicine, New York
7Memorial Sloan Kettering Cancer Center, New York
8Bristol Myers Squibb, Princeton, NJ
9Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York
10Molecular Pharmacology Program, Center for Cell Engineering, Center for Stem Cell Biology, Center for Experimental Therapeutics, Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY

Blood stem cells intricately regulate their fate, balancing self-renewal and differentiation through symmetric and asymmetric divisions. When exposed to various stresses they can rapidly undergo symmetric commitment divisions to generate differentiated progenitors for immune regeneration or driving inflammation. In previous studies, we and others found that m6A RNA methylation controls symmetric commitment and inflammation in hematopoietic stem cells (HSCs) (Cheng and Luo et al., Cell Reports 2019). However, the precise molecular mechanisms underlying the role of m6A and its influence on the inflammatory program in stem cells remain elusive.

To uncover the dynamic RNA methylation during HSC commitment, we employed a recently developed tool called DART-seq, that fuses the YTH domain of the m6A reader protein YTHDF2 with the RNA editing protein APOBEC1. We identified 300 m6A sites and approximately 250-300 gene targets within HSCs and MPPs (padj beta binomial < 0.05). We found m6A sites in HSCs were enriched for: innate immune response pathways including NFκB and Toll-like receptor cascades, cellular signaling processes encompassing RhoGTPase and ERK/MAPK signaling targets, and metabolic pathways associated with amino acid transport and fatty acid oxidation. In contrast, the m6A targets enriched in MPPs were associated with lineage differentiation and myeloid programs such as MLL, GATA1, PU.1, and CEBPD.

By integrating our findings with two additional m6A mapping datasets, Son was a shared m6A target with increased m6A modification during the transition from HSCs to MPPs. SON, an RNA binding protein residing in nuclear speckles, has been implicated in a myriad of cellular processes including splicing control and transcriptional repression. Notably, de novo heterozygous loss-of-function variants in SON (ZTTK Syndrome) results in hematological symptoms, neurological impairments, and developmental delays.

The genetic deletion of METTL3 (Mettl3 conditional knockout mice; Mettl3 cKO) or METTL3 inhibitor STM2457 treatment, resulted in a twofold increase in Son transcripts in both HSCs and MPPs, but reduced SON protein abundance by 50%. Notably, SON exhibited asymmetric segregation during HSC division, with its abundance displaying a strong correlation with the commitment marker NUMB. Collectively, these findings suggest that m6A modification regulates the abundance of SON protein and implicates SON in HSC commitment fate.

Reintroduction of SON in m6A-deficient HSCs effectively rescued commitment defects (cKO EV 9.74% vs. cKO+SON 21.43%). Furthermore, SON overexpression partially restored in vivo engraftment defects in Mettl3 cKO LSKs (cKO EV 1.5% vs. cKO+SON 4.5%). Intriguingly, the functional rescue was accomplished using a fragment containing the RNA-binding domain of SON (cKO EV 1.2% vs. cKO+SON-RB 12.3%), highlighting the significance of SON's RNA-binding ability.

Additionally, deletion of SON in RosaCas9 WT LSKs using sgRNAs resulted in a substantial reduction in engraftment (WT EV 35.4% vs. WT+sgSON 11%), whereas SON overexpression in WT LSKs enhanced engraftment (WT EV 6% vs. WT+SON 39%). These results demonstrate that SON is a positive regulator of stem cell engraftment and function.

Moreover, SON overexpression effectively rescued specific clusters identified through scRNA-seq analysis in Mettl3 KO (KOsp1-1 and CCL5+) by 50%. In bulk RNA-seq analysis, we identified 257 upregulated genes and 227 downregulated genes between the Mettl3 cKO EV and Mettl3 cKO SON groups (padj<0.05). Remarkably, SON overexpression significantly downregulated inflammatory pathways that were aberrantly upregulated upon m6A loss. Furthermore, SON overexpression indirectly rescued dsRNA formation in Mettl3 cKO LSKs, aligning with the role of m6A in suppressing dsRNA formation in HSPCs.

SON overexpression reduced Ccl5 through binding to its transcript and decreasing nascent transcription. Strikingly, depletion of CCL5 significantly rescued Mettl3 cKO LSK engraftment (cKO 1.6% vs. cKO+shCCL5 5.3%), indicating that CCL5 is sufficient to mediate the effects downstream of SON. Moreover, co-culturing WT HSCs with CCL5 resulted in a symmetric commitment defect similar to that observed with m6A loss. In summary, our study delineates the critical m6A-SON-CCL5 axis governing HSC symmetric commitment fate and inflammation control.

Disclosures: Landau: Ultima Genomics: Research Funding; 10X Genomics: Research Funding; Pangea: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Alethiomics: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Illumina: Consultancy, Research Funding; AstraZeneca: Consultancy; C2i Genomics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Kharas: 858 Therapeutics, Inc.: Current equity holder in private company; Kumquat Biosciences: Consultancy; AstraZeneca: Consultancy.

*signifies non-member of ASH