RESUMEN
Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA.
Asunto(s)
División Celular , Cromatina/genética , ADN Satélite/genética , Expresión Génica Ectópica , Proteína 2 de Unión a Metil-CpG/metabolismo , ARN Nuclear/genética , Células HeLa , Humanos , Proteína 2 de Unión a Metil-CpG/genética , ARN Largo no CodificanteRESUMEN
The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Ratones Transgénicos , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteínas Nucleares/genética , Ftalazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Células U937 , Ensayos Antitumor por Modelo de Xenoinjerto , CohesinasRESUMEN
Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.