Correction of Heritable Epigenetic Defects Using Editing Tools

Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field.

The Prevalence of GNAS Deficiency-Related Diseases in a Large Cohort of Patients Characterized by the EuroPHP Network

Context: The term pseudohypoparathyroidism (PHP) was coined to describe the clinical condition resulting from end-organ resistance to parathormone (rPTH), caused by genetic and/or epigenetic alterations within or upstream of GNAS. Although knowledge about PHP is growing, there are few data on the prevalence of underlying molecular defects. Objective: The purpose of our study was to ascertain the relative prevalence of PHP-associated molecular defects. Design: With a specially designed questionnaire, we collected data from all patients (n = 407) clinically and molecularly characterized to date by expert referral centers in France, Italy, and Spain. Results: Isolated rPTH (126/407, 31%) was caused only by epigenetic defects, 70% of patients showing loss of imprinting affecting all four GNAS differentially methylated regions and 30% loss of methylation restricted to the GNAS A/B:TSS-DMR. Multihormone resistance with no Albright’s hereditary osteodystrophy (AHO) signs (61/407, 15%) was essentially due to epigenetic defects, although 10% of patients had point mutations. In patients with rPTH and AHO (40/407, 10%), the rate of point mutations was higher (28%) and methylation defects lower (about 70%). In patients with multihormone resistance and AHO (155/407, 38%), all types of molecular defects appeared with different frequencies. Finally, isolated AHO (18/407, 4%) and progressive osseous heteroplasia (7/407, 2%) were exclusively caused by point mutations. Conclusion: With European data, we have established the prevalence of various genetic and epigenetic lesions in PHP-affected patients. Using these findings, we will develop objective criteria to guide cost-effective strategies for genetic testing and explore the implications for management and prognosis.

Novel Myelodysplastic Syndromes-like Mouse Models By Cooperating Genetic/Epigenetic Mutations Reveal the Critical Role of HIF-1α for Disease Development

Myelodysplasitc syndromes (MDS) are associated with genetic and epigenetic defects in transcription factors and/or chromatin-modifying enzymes that regulate self-renewal, survival, and differentiation. Mixed lineage leukemia (MLL) is a transcription factor which regulates its downstream targets by an epigenetic manner and plays a critical role for regulation of normal hematopoiesis. Mutations in MLL have been identified in variety of hematopoietic malignancies. Internal partial tandem duplication (MLL-PTD) is a major aberration of MLL gene. MLL-PTD is often found in the patients with MDS, secondary AML (sAML), and de novo AML. We have previously shown that MLL-PTD knock-in (MLLPTD/WT) mice present increased self-renewal and apoptosis in hematopoietic stem/progenitor cells (HSPCs) and skewed myeloid differentiation, but never develop MDS or AML phenotypes. This suggests that additional genetic and/or epigenetic defects are necessary for the transformation. Interestingly, MLL-PTD is significantly associated with RUNX1 mutations in sAML and de novo AML. Thus, we hypothesized that combination of these commonly co-mutated genes might generate faithful mouse models for human diseases. To understand the impact of RUNX1 mutations in the MLL-PTD background, we retrovirally introduced MDS patient-derived RUNX1 mutants (RUNX1-D171N and RUNX1-291sX300) into BM cells obtained from 5-FU treated MLLPTD/WT mice. These transduced cells were transplanted into lethally irradiated recipient mice. Both MLLPTD/WT/D171N and MLLPTD/WT/291fsX300 BMT mice quickly developed macrocytic anemia and mild thrombocytopenia. Tri-lineage dysplasia was observed in BM from both groups. Anemia in MLLPTD/WT/291fsX300 BMT mice was more severe than that in MLLPTD/WT/D171N mice. MLLPTD/WT/D171N BMT mice presented hypo-cellular marrow with excess blasts (blasts< 5%), while MLLPTD/WT/291fsX300 BMT mice presented hyper-cellular marrow with higher percentage of blasts (blasts < 20%). Interestingly monocytes in PB from MLLPTD/WT/291fsX300 BMT mice were significantly increased compared to control and MLLPTD/WT/D171N BMT mice. MLLPTD/WT/291fsX300 BMT mice also developed BM fibrosis with slight splenomegaly. Interestingly, 20-30% of MDS patients, especially high-risk MDS with BM fibrosis, are also accompanied with splenomegaly. We have previously shown that MLL-PTD enhances self-renewal of HSPCs. To understand the mechanism of enhanced self-renewal, we performed gene expression array analysis and mRNA-sequencing, followed by KEGG pathway analysis. Interestingly, one of the top dysregulated pathways was Glycolysis/Gluconeogenesis pathway. HIF-1α is one well-known transcription factor for this pathway. Thus, we measured HIF-1α expression in HSPCs from MLLPTD/WT mice at both mRNA and protein levels. HIF-1α protein and mRNA of HIF-1α target genes in HSPCs from MLLPTD/WT mice were significantly increased compared with those from WT mice. To determine the direct interaction between MLL-PTD and HIF-1α, we introduced WT MLL or MLL-PTD together with HIF-1α cDNA into 293T cells and performed western blot (WB) and imuunoprecipitation-WB. We found the strong accumulation of HIF-1α in MLL-PTD co-transfectants and interaction between MLL-PTD and HIF-1α. To assess the significance of HIF-1α in MLL-PTD mediated pathogenesis, we performed CFU assay in the presence of a HIF-1α inhibitor, Echinomycin. MLLPTD/WT cells formed significantly fewer colonies than WT cells. RUNX1 mutants transduced MLLPTD/WT cells were hyper-sensitive to Echinomycin while WT HSPCs or AML cells derived from Cbfb/SMMHC knock-in mice showed less sensitivity. Echinomycin could induce myeloid differentiation and inhibit MDS-initiating cells. We further took a genetic approach and performed BMT assay. HIF-1α deletion significantly reduced reconstitution ability of MLLPTD/WT cells and MDS development in our mouse models. These results strongly suggest that HIF-1α is essential for MLLPTD/WT and MLLPTD/WT/RUNX1 mutant mediated self-renewal and MDS-like disease development. In conclusion, 1) we have established novel MDS-like mouse models which recapitulate human diseases by using genetic/epigenetic mutations (RUNX1/MLL-PTD); 2) we identified HIF-1α as a critical factor for MDS-like disease by using our robust MDS-like mouse models; 3) targeting HIF-1α could eradicate the MDS phenotypes and the MDS-initiation cells. Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding.