-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4582 Mutant p53 Drives the Development of Myelodysplastic Syndromes Via Dysregulating Pre-mRNA Splicing in Hematopoietic Stem and Progenitor Cells

Program: Oral and Poster Abstracts
Session: 636. Myelodysplastic Syndromes—Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Diseases, Myeloid Malignancies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Sergio Barajas, BS1,2, Sisi Chen, PhD3*, Sasidhar Vemula2*, Yuxia Yang2*, Edward Simpson2*, Wenjie Cai, MS1,2, Shiyu Xiao, MS1, Hongxia Chen, MD/PhD1*, Stephanie Halene, MD4, Madina Sukhanova1*, Yunlong Liu, PhD2*, Omar Abdel-Wahab, MD5 and Yan Liu, PhD1

1Northwestern University, Chicago, IL
2Indiana University, Indianapolis, IN
3Memorial Sloan Kettering Cancer Center, New York, NY
4Section of Hematology, Yale Univ. School of Medicine, New Haven, CT
5Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY

Despite the clinical importance of TP53 mutations, to date there have been no studies on the functional impact of TP53 mutations on the initiation and progression of MDS. We found that heterozygous p53 mutant mice (p53R248W/+) show extended survival and died within 15-20 months after birth. p53 mutant mice had hypercellular bone marrow and displayed cytopenia, thrombocytopenia, and anemia. Approximately 60% of p53R248W/+ mice developed MDS based upon pathological analysis of bone marrow (BM) and peripheral blood smears, while the remaining p53R248W/+ mice developed lymphoma and sarcoma. We further showed that mutant p53-driven MDS are transplantable. Thus, we have generated a mouse model of MDS that has some features of human MDS with TP53 mutations.

To understand how mutant p53 drives MDS development, we performed RNA-seq studies to compare gene expression in hematopoietic stem and progenitor cells (HSPCs). GSEA analysis revealed that spliceosome genes were significantly downregulated in middle-aged p53R248W/+ HSPCs compared to age-matched p53+/+ HSPCs. We confirmed that the expression of splicing factors, such as Prpf3 and Prpf4, was significantly downregulated in middle-aged p53R248W/+ HSPCs. We conducted alternative splicing analysis utilizing MISO (Mixture of Isoforms) algorithm to identify differences in exon inclusion ratio between WT and mutant HSPCs. We observed differential splicing of all classes of alternative splicing events, including Cassette Exons (CE), Competing 5′ and 3′ splice sites (A5’ or A3’SS), Mutually Exclusive Exons (MXE), and Retained Introns (RI) in p53R248W/+ cells compared to that of the WT HSPCs. Notably, we found that murine p53 mutant HSPCs exhibited aberrant splicing of key regulators of NFκB activation, such as USP15. USP15 overexpression promotes NFκB expression through inhibiting its ubiquitination, whereas NFκB promotes USP15 expression. p53 mutant HSPCs tend to skip exon 7 and express high levels of short isoform of Usp15. The short isoform of Usp15, but not the long isoform of Usp15, activates NFκB in HSPCs, manifested by increased phosphorylation of p65. To determine the impact of mutant p53 on pre-mRNA splicing in human HSPCs, we ectopically expressed GFP or p53R248W in human cord blood CD34+ cells and performed RNA-seq in transduced cells (GFP+). Human HSPCs expressing mutant p53 displayed alterations in pre-mRNA splicing compared to HSPCs expressing GFP. We found that human HSPCs expressing mutant p53 display aberrant splicing in key regulator of NFκB such as IKBKE (Inhibitor of nuclear factor kappa‐B kinase subunit epsilon). Human HSPCs expressing mutant p53 tend to include exon 7 and express high levels of long isoform of IKBKE. The long isoform of IKBKE, but not the short isoform of IKBKE, activates NFκB in human HSPCs.

Many proinflammatory cytokine genes are NFκB targets and we observed increased levels of pro-inflammatory cytokines, including IL-1β and IL-6, in the BM of middle-aged p53 mutant mice. Notably, we observed increased levels of IL-1β and IL-6 in the BM of MDS or AML patients with TP53 mutations.

TP53 mutations co-occur with splicing factor mutations in MDS. To determine whether TP53 and SRSF2 mutations cooperate in pathogenesis of MDS, we have generated p53R248W/+SRSF2P95H/+ mice. We found that the survival of recipient mice repopulated with p53R248W/+SRSF2P95H/+ BM cells was significantly decreased compared to recipients repopulated with WT, p53R248W/+, or SRSF2P95H/+ BM cells. All recipient mice repopulated with p53R248W/+SRSF2P95H/+ BM cells died within 6 months and developed MDS or leukemia. Thus, we demonstrate that TP53 and SRSF2 mutations cooperate in MDS or leukemia development.

In summary, we discovered that mutant p53 dysregulates pre-mRNA splicing in key regulators of inflammatory response during aging, thereby generating a chronic inflammatory microenvironment to drive MDS pathogenesis.

Disclosures: Abdel-Wahab: AbbVie, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amphista Therapeutics: Consultancy; Nurix Therapeutics: Research Funding; Loxo/Lilly: Consultancy; AstraZeneca: Consultancy; Minovia Therapeutics: Research Funding; Harmonic Discovery: Current holder of stock options in a privately-held company.

Previous Abstract | Next Abstract >>
*signifies non-member of ASH