RESUMEN
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
Asunto(s)
Cromatina , Epigénesis Genética , Genotipo , Mutación , Análisis de la Célula Individual , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipaje , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Inflamación/genética , Inflamación/patología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Megacariocitos/patología , Proteínas de la Membrana/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , ARN/genética , Células Clonales/metabolismoRESUMEN
In acute myeloid leukemia (AML), p53 tumor suppressor activity can be reduced due to enhanced expression of MDM2 which promotes the degradation of p53. In TP53 wild-type malignancies, therapy with small molecule antagonists of MDM2 results in antileukemic activity. Current treatment strategies, however, have been limited by poor tolerability and incomplete clinical activity. We have developed a proteolysis-targeting chimera (PROTAC) MS3227 that targets MDM2 by recruiting the E3 ligase Von Hippel-Lindau, resulting in proteasome-dependent degradation of MDM2. In WT TP53 leukemia cell lines, MS3227 led to activation of p53 targets p21, PUMA, and MDM2 and resulted in cell-cycle arrest, apoptosis, and decreased viability. The catalytic PROTAC MS3227 led to more potent activation when compared to a stoichiometric inhibitor, in part by dampening the negative feedback mechanism in the p53 - MDM2 circuit. The effectiveness of MS3227 was also observed in primary patient specimens with selectivity towards leukemic blasts. The addition of MS3227 enhanced the activity of other anti-leukemic agents including azacytidine, cytarabine, and venetoclax. In particular, MS3227 treatment was shown to downregulate MCL-1, a known mediator of resistance to venetoclax. A PROTAC-based approach may provide a means of improving MDM2 inhibition to gain greater therapeutic potential in AML.
