Biology subjects

Link, D. C.

Publications and source records attributed to Link, D. C..

7 recordsLinked to original sources

bioRxiv2024

The Clonal Hematopoiesis-associated Gene Srcap Plays an Essential Role in Hematopoiesis

Somatic mutations arising in hematopoietic stem cells (HSCs) may provide the latter with a fitness advantage, allowing the mutant HSC to clonally expand. Such mutations have been recurrently identified in the chromatin modifier, SRCAP, in both non-malignant and leukemic clones, suggesting that this gene plays a significant role in hematopoiesis. We generated a conditional Srcap loss of function murine model and determined the consequences of hematopoietic-specific loss of this gene. We show that Srcap is essential for normal fetal liver erythropoiesis and monocytopoiesis. In Srcap deficient fetal livers, the number of phenotypic HSCs is similar to that of controls, but these HSCs exhibit a profound repopulating defect. Likewise, conditional deletion of Srcap during adult hematopoiesis results in a rapid loss of HSCs. Loss of Srcap is associated with evidence of increased DNA damage in HSCs and lineage-restricted progenitors as assessed by y-H2AX expression. Consistent with this finding, we observed strong transcriptional upregulation of the p53 pathway in Srcap deficient erythroid precursors. Collectively our data highlight the importance of Srcap in maintaining HSC function and supporting hematopoietic differentiation and suggests that it plays an essential role in maintaining genomic integrity. Key Points(1) Srcap plays an essential role in supporting normal hematopoietic differentiation. and in maintaining HSC function. (2) Loss of Srcap is associated with evidence of increased DNA damage and transcriptional upregulation of the p53 pathway.

cell biology↗

bioRxiv2022

IMC-Denoise: a content aware denoising pipeline to enhance Imaging Mass Cytometry

Imaging Mass Cytometry (IMC) is an emerging multiplexed imaging technology for analyzing complex microenvironments that has the ability to detect the spatial distribution of at least 40 cell markers. However, this new modality has unique image data processing requirements, particularly when applying this technology to patient tissue specimens. In these cases, signal-to-noise ratio for particular markers can be low despite optimization of staining conditions, and the presence of pixel intensity artifacts can deteriorate image quality and the subsequent performance of downstream analysis. Here we demonstrate an automated content-aware pipeline, IMC-Denoise, to restore IMC images. Specifically, we deploy a differential intensity map-based restoration (DIMR) algorithm for removing hot pixels and a self-supervised deep learning algorithm for filtering shot noise (DeepSNF). IMC-Denoise outperforms existing methods for adaptive hot pixel removal, and delivers significant image quality improvement and background noise removal to a diverse set of IMC channels and datasets. This includes a unique, technically challenging, human bone marrow IMC dataset; in which we achieve noise level reduction of 87% for a 5.6-fold higher contrast-to-noise ratio, and more accurate background noise removal with approximately two-fold improved F1 score. Our approach remarkably enhances both manual gating and automated phenotyping with cell-scale down-stream analysis on these complex data. We anticipate that IMC-Denoise will provide similar benefits in mass cytometry imaging domains to more deeply characterize the complex and diverse tissue microenvironment.

bioinformatics↗

bioRxiv2021

Immunosuppression and Outcomes in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T-cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T-cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T-cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

cancer biology↗

bioRxiv2021

Nonsense Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells with SF3B1 and U2AF1 Mutations

Nonsense-mediated RNA decay (NMD) is well recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTCs) for degradation; however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system that can accurately measure NMD activity in individual cells. By carrying out a genome-wide CRISPR/Cas9 knockout screen using this reporter system, we identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, we also found that cells with mutations in the U2 spliceosome genes SF3B1 and U2AF1--which are commonly found in myelodysplastic syndrome (MDS) and cancers--have overall attenuated NMD activity. Furthermore, we found that compared to wild type cells, SF3B1 and U2AF1 mutant cells are more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition could be rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, our findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel strategy for the treatment of MDS and cancers with spliceosome mutations.

cell biology↗

bioRxiv2021

Genome-wide analysis of focal DNA hypermethylation in IDH-mutant AML samples

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4,000 focal regions that were uniquely hypermethylated vs. normal CD34+ cells. These regions had modest, but significant, hypermethylation in AMLs with biallelic TET2 mutations, and 5-hydroxymethylation levels that were dependent on functional TET2, indicating that hypermethylation in these regions is caused by inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred in regions with low methylation in normal CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that methylation in these regions is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

cancer biology↗

bioRxiv2020

Organ-on-a-chip model of vascularized human bone marrow niches

Animal models of bone marrow have limited spatial and temporal resolution to observe biological events (intravasation and cellular egress) and are inadequate to dissect dynamic events at the niche level (100 microns). Utilizing microfluidic and stem cell technology, we present a 3D in vitro model of human bone marrow that contains perivascular and endosteal niches complete with dynamic, perfusable vascular networks. We demonstrate that our model can perform in vivo functions including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells (HSPC) for up to fourteen days, egress of myeloid progenitors, and expression of markers consistent with in vivo human bone marrow. The platform design enables the addition of tissue niches at a later timepoint to probe mechanisms such as tumor cell migration. Overall, we present a novel organ-on-a-chip platform that is capable of recapitulating the human bone marrow microenvironment to observe hematopoietic phenomena at high spatial and temporal resolution.

bioengineering↗

bioRxiv2019

Mutant p53 Drives Clonal Hematopoiesis through Modulating Epigenetic Pathway

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. We discovered that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Further, genetic and pharmacological inhibition of EZH2 decrease the repopulating potential of p53 mutant HSPCs. Thus, we have uncovered an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

cancer biology↗