Somatic TP53 Mutations Rescue the Hematopoietic Stem and Progenitor Cell Defect at the Expense of Increased Risk for Myeloid Malignancies in ERCC6L2-Associated Inherited Bone Marrow Failure Syndrome
Authors: Schimmer Roman R. , Klemm Nancy , Topçu Ebru , Fullin Jonas , Zielinska Karolina , Doerdelmann Cyril , Lock Melissa , Treacy Milena , Caiado Francisco , Dietliker Nadja , Schwotzer Rahel , Manz Markus G. , Lopes Massimo , Boettcher Steffen 
Published: December 2024

Introduction
Recessively inherited loss-of-function mutations in Excision Repair Cross-Complementing 6 like 2 (ERCC6L2) cause a bone marrow failure syndrome (BMFS) characterized by moderate cytopenias but high frequencies of somatic TP53 mutations predisposing to myeloid malignancies. The pathophysiology and molecular mechanisms underlying the disease have not been elucidated yet. We therefore set out to investigate the hematopoietic stem and progenitor cell (HSPC) defect caused by biallelic ERCC6L2 mutations and the impact of TP53 mutations on the clinical phenotype and propensity for malignant transformation using novel pre-clinical in vitro and in vivo models.
Results
To systematically study the effects of ERCC6L2 (E6L2) mutations, we took advantage of a conditionally immortalized mouse HSPC cell line via estrogen-dependent homeobox protein B8 (HoxB8) overexpression. As &gt;90% of patients carry loss-of-function E6L2 mutations we introduced various deleterious E6L2 mutations at different exons into HoxB8 cells by CRISPR/Cas9-mediated knockout (KO). Under favorable in vitro growth conditions, no significant phenotypic and functional differences were observed between wildtype (WT) and E6L2-KO cells as measured by cell viabilities, proliferation rates, cell cycle distribution, apoptosis, and competitive cell growth assays. We therefore postulated that harsher environmental factors - likely present in the bone marrow - might be necessary to uncover the HSPC defect in vitro. To this end, we treated our cells with both naturally-occurring (nutrient deficiency, reactive oxygen species, or hypoxia) or iatrogenic (Etoposide, Bleomycin and Cisplatin) stressors, upon which E6L2-KO cells, indeed, showed hypersensitivity as demonstrated by a significant competitive disadvantage due to increased apoptosis. CRISPR/Cas9-mediated E6L2-KO in Lin-Sca1+c-kit+ (LSK) bone marrow cells revealed decreased erythroid clonogenic potential with reduced numbers of burst-forming unit-erythroid (BFU-E) colonies and delayed maturation with an increase in immature erythroid progenitors but decreased terminal erythroid differentiation. Furthermore, in vivo competitive transplantation assays demonstrated severely impaired engraftment and repopulation potential of E6L2-KO HSPCs and lower hemoglobin levels in recipient mice.
Transcriptional profiling of CRISPR-edited HoxB8 cells revealed hallmark gene sets associated with replication stress to be significantly upregulated in E6L2-KO cells. Accordingly, upon cellular stress, E6L2-KO cells accumulate DNA damage predominantly in the S-phase strongly suggesting replication stress as a potential molecular mechanism underlying the HSPC defect.
Acquisition of somatic TP53 mutations is a hallmark of E6L2-associated BMFS. Introduction of Trp53 mutations to E6L2-KO cells via CRISPR/Cas9 or Cre recombination fully rescued the HSPC defect in all observed phenotypes described above both in vitro,in vivo as well as in primary human and patient samples. Finally, to assess the potential for malignant transformation in vitro, we tested for estrogen-independent cell growth after induction of DNA damage by cytotoxic agents. While E6L2-KO alone is insufficient to promote malignant transformation, the presence of a double mutation (E6L2-Trp53-KO) results in transformation in 100% of times (n = 24/24), whereas a Trp53-KO alone only transforms cells in about 60% of times (n = 14/24). In line with this, we observed a sustained increase in DNA damage levels in E6L2-Trp53-KO cells. Experiments are ongoing testing for in vivo malignant transformation as well as replication stress and will be presented.
Discussion and Outlook
Using conditionally immortalized mouse HSPC cell lines together with CRISPR/Cas9-mediated genome editing, we developed an in vitro model system faithfully recapitulating major hallmarks of E6L2-associated BMF. By virtue of competitive cell growth assays, colony-forming unit assays, gene expression analysis, and in vivo competitive transplantation assay we demonstrate that E6L2 mutations result in an HSPC defect and identify increased replication stress as a potential underlying molecular mechanism. Furthermore, we show that the HSPC defect caused by E6L2 mutations is completely rescued upon acquisition of additional Trp53 mutations at the expense of increased potential for malignant transformation.