Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has ...suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later
. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains ...unresolved. We show that Stag2 deletion in hematopoietic stem and progenitor cells (HSPCs) results in altered hematopoietic function, increased self-renewal, and impaired differentiation. Chromatin immunoprecipitation (ChIP) sequencing revealed that, although Stag2 and Stag1 bind a shared set of genomic loci, a component of Stag2 binding sites is unoccupied by Stag1, even in Stag2-deficient HSPCs. Although concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to increased self-renewal and reduced HSPC commitment to the B cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.
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•Hematopoietic Stag2 loss enhances stem cell self-renewal and impairs differentiation•Stag1 can maintain TAD boundary integrity in the absence of Stag2•Stag2 is required for intra-TAD interactions at lineage genes (e.g., PU.1 targets)•Stag2 target expression, but not PU.1 overexpression, restores B cell differentiation
In murine hematopoietic Stag2 deletion, Stag1 rescues topologically associated domains in the absence of Stag2 but cannot restore the chromatin architecture required for hematopoietic lineage commitment. PU.1 target genes lose accessibility and expression. Induced target gene expression, but not PU.1 overexpression, is sufficient to restore differentiation in the altered chromatin state.
Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon-producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell ...neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.
Cell-type specific transcriptional programs are regulated by the activity of tissue-specific transcription factors and enhancer elements within structurally defined topologically associating domains ...(TADs). The coordinated and dynamic changes in chromatin architecture are highly regulated as transcriptional output is influenced by chromatin accessibility, histone modification, promoter enhance interactions, and recruitment of transcriptional co-activator complexes. The genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias, Ewing sarcoma, and glioblastoma multiforme. Despite this, the mechanistic role of STAG2 in gene regulation, hematopoietic function, and tumor suppression has not been delineated.
We show that somatic Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation across all three lineages consistent with myelodysplasia. Chromatin immunoprecipitation sequencing of Stag2-deficient HSPCs revealed that Stag2 and Stag1 have both shared and non-redundant cistromes with Stag1/2 common binding sites enriching at TAD boundaries with CTCF occupancy. This maintains TAD integrity in the setting of Stag2 loss of function which we confirmed with Hi-C chromosome capture. High resolution of the Hi-C data at 10kB resolution identified a specific role for Stag2, but not Stag1, in maintaining short-range chromatin interactions, specifically at genes with PU.1 and IRF8 motifs.
While co-deletion of Stag2 and Stag1 resulted in synthetic lethality, Stag2 loss alone resulted in decreased chromatin accessibility and reduced transcriptional output at key PU.1 target loci involved in lineage-specification, including reduced Ebf1 and Pax5 expression resulting in impaired B-lineage differentiation. We investigated whether expression of PU.1 could overcome the non-permissive chromatin state at these key downstream targets and rescue hematopoietic differentiation; however, PU.1 expression could not restore Ebf1 expression or B cell differentiation and did not attenuate the serial replating capacity of Stag2 deficient hematopoietic stem/progenitor cells (HSPCs). Given this transcriptional “choke-point” we investigated whether expression of Ebf1 would have a more significant impact on Stag2 deficient cells. Indeed Ebf1 rescue restored B cell colony formation/differentiation in vitro and in vivo and abrogated serial replating of Stag2 deficient HSPCs. These data highlight the non-hierarchical and non-redundant relationship between transcription factors and chromatin architecture and demonstrate a key role for Stag2-regulated local interactions in transcription factor output and hematopoietic differentiation.
Nonetheless, the mechanistic underpinnings of the structural basis for transcriptional regulation remain associative. We have recently been able to reduce the cell input for Hi-C assays such that we can now analyze the chromatin architecture of purified populations and model the structural transition from Lin- Sca-1+ Kit+ hematopoietic stem cells to committed granulocyte macrophage precursor cells both in normal hematopoiesis and in the Stag2 deficient setting. Previous studies using in vitro systems have shown that complete cohesin depletion results in the loss of structural loop components; however, cohesin levels are reduced, but not absent, in cancer cells. As such, our studies highlight a key role for locus-specific alterations in gene regulation and DNA interactions in Stag2 deficient cells, which results in altered gene expression and contributes to transformation.
Taken together, these data identify a key role for Stag2 loss in transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation. Moreover, we illustrate a critical link between cohesin, chromosomal contacts, and gene regulation that contributes to hematopoietic transformation.
Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Dekker:Arima Genomics: Membership on an entity's Board of Directors or advisory committees. Levine:Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees.
DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to ...poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
Stem Cell Therapy for COPD Glassberg, Marilyn K.; Csete, Isabelle; Simonet, Emmanuelle ...
Chest,
October 2021, 2021-10-00, Letnik:
160, Številka:
4
Journal Article
Recenzirano
COPD is a chronic inflammatory and destructive disease characterized by progressive decline in lung function that can accelerate with aging. Preclinical studies suggest that mesenchymal stem cells ...(MSCs) may provide a therapeutic option for this incurable disease because of their antiinflammatory, reparative, and immunomodulatory properties. To date, clinical trials using MSCs demonstrate safety in patients with COPD. However, because of the notable absence of large, multicenter randomized trials, no efficacy or evidence exists to support the possibility that MSCs can restore lung function in patients with COPD. Unfortunately, the investigational status of cell-based interventions for lung diseases has not hindered the propagation of commercial businesses, exploitation of the public, and explosion of medical tourism to promote unproven and potentially harmful cell-based interventions for COPD in the United States and worldwide. Patients with COPD constitute the largest group of patients with lung disease flocking to these unregulated clinics. This review highlights the numerous questions and concerns that remain before the establishment of cell-based interventions as safe and efficacious treatments for patients with COPD.
COPD is a chronic inflammatory and destructive disease characterized by progressive decline in lung function that can accelerate with aging. Preclinical studies suggest that mesenchymal stem cells ...(MSCs) may provide a therapeutic option for this incurable disease because of their antiinflammatory, reparative, and immunomodulatory properties. To date, clinical trials using MSCs demonstrate safety in patients with COPD. However, because of the notable absence of large, multicenter randomized trials, no efficacy or evidence exists to support the possibility that MSCs can restore lung function in patients with COPD. Unfortunately, the investigational status of cell-based interventions for lung diseases has not hindered the propagation of commercial businesses, exploitation of the public, and explosion of medical tourism to promote unproven and potentially harmful cell-based interventions for COPD in the United States and worldwide. Patients with COPD constitute the largest group of patients with lung disease flocking to these unregulated clinics. This review highlights the numerous questions and concerns that remain before the establishment of cell-based interventions as safe and efficacious treatments for patients with COPD.
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