Biology subjects

Harding, S. M.

Publications and source records attributed to Harding, S. M..

7 recordsLinked to original sources

bioRxiv2026

Longitudinal whole transcriptomic profiling of live cells through domain adaptation

Tracking transcriptomic profiles of cells over time in response to developmental cues and environmental stimuli can reveal critical insights into the fundamental mechanisms of development and disease. However, longitudinal molecular profiling at the global transcriptome level remains a major challenge, as RNA sequencing fundamentally alters or destroys cells. To overcome these limitations, we developed PENNE, a deep-learning framework that infers whole-transcriptomic profiles directly from live-cell images. Using gated attention mechanisms, PENNE trains on spatial transcriptomic datasets to align morphological features with gene expression. To enable inferences from images, our model performs domain adaptation to eliminate discrepancies between stained and unstained tissue images, effectively transferring molecular information from tissue sections to live-cell imaging. PENNE accurately identifies cell-type-specific and radiation-response markers via imputed expression. Furthermore, using only live-cell images stained with a G2/M cell cycle marker, our model captures temporal gene dynamics, evidenced by strong correlations between predicted expression and both ground-truth cellular confluency and cell-cycle progression. By bridging the gap between data-rich spatial transcriptomics and the practicality of live-cell imaging, PENNE provides a powerful new framework for monitoring molecular temporal dynamics directly through morphological information. This approach enables a paradigm-shifting workflow, fusing transcriptome-wide data with live-cell microscopy to fuel the discovery of novel gene programs via scalable, non-invasive, real-time interrogation of cellular states.

bioinformatics↗

bioRxiv2024

Deoxyuridine-rich cytoplasmic DNA antagonizes STING-dependent innate immune responses and sensitizes resistant tumors to anti-PD-L1 therapy

DNA damage and cytoplasmic DNA induce type-1 interferon (IFN-1) and potentiate responses to immune checkpoint inhibitors. Our prior work found that inhibitors of the DNA damage response kinase ATR (ATRi) induce IFN-1 and deoxyuridine (dU) incorporation by DNA polymerases, akin to antimetabolites. Whether and how dU incorporation is required for ATRi-induced IFN-1 signaling is not known. Here, we show that ATRi-dependent IFN-1 responses require uracil DNA glycosylase (UNG)-initiated base excision repair and STING. Quantitative analyses of nine distinct nucleosides reveals that ATRi induce dU incorporation more rapidly in UNG wild-type than knockout cells, and that induction of IFN-1 is associated with futile cycles of repair. While ATRi induce similar numbers of micronuclei in UNG wild-type and knockout cells, dU containing micronuclei and cytoplasmic DNA are increased in knockout cells. Surprisingly, DNA fragments containing dU block STING-dependent induction of IFN-1, MHC-1, and PD-L1. Furthermore, UNG knockout sensitizes cells to IFN-{gamma} in vitro, and potentiates responses to anti-PD-L1 in resistant tumors in vivo. These data demonstrate an unexpected and specific role for dU-rich DNA in suppressing STING-dependent IFN-1 responses, and show that UNG-deficient tumors have a heightened response to immune checkpoint inhibitors. STATEMENT OF SIGNIFICANCEAntimetabolites disrupt nucleotide pools and increase dU incorporation by DNA polymerases. We show that unrepaired dU potentiates responses to checkpoint inhibitors in mouse models of cancer. Patients with low tumor UNG may respond to antimetabolites combined with checkpoint inhibitors, and patients with high tumor UNG may respond to UNG inhibitors combined with checkpoint inhibitors.

cancer biology↗

bioRxiv2023

The proteomic landscape of genotoxic stress-induced micronuclei

Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signalling cascades. Here, we measure the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, with a core composition that is independent of the specific inciting stressor. Across stress conditions, MN are significantly depleted for spliceosome machinery and replication stress response proteins, but are enriched for a subset of the replisome. We find that the loss of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. This dataset represents a unique resource detailing the proteomic landscape of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.

cell biology↗

bioRxiv2022

cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status.

Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

cell biology↗

bioRxiv2020

Cell cycle checkpoints cooperate to suppress DNA and RNA associated molecular pattern recognition and anti-tumor immune responses

The DNA dependent pattern recognition receptor, cGAS mediates communication between genotoxic stress and the immune system. Mitotic chromosome missegregation is an established stimulator of cGAS activity, however, it is unclear if progression through mitosis is required for cancer cell intrinsic activation of immune mediated anti-tumor responses. Moreover, it is unknown if disruption of cell cycle checkpoints can restore responses in cancer cells that are recalcitrant to DNA damage induced inflammation. Here we demonstrate that prolonged cell cycle arrest at the G2-mitosis boundary from either CDK1 inhibition or excessive DNA damage prevents inflammatory stimulated gene expression and immune mediated destruction of distal tumors. Remarkably, DNA damage induced inflammatory signaling is restored in a cGAS-and RIG-I-dependent manner upon concomitant disruption of p53 and the G2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage associated molecular pattern recognition and have implications for the design of rational approaches to augment antitumor immune responses.

cell biology↗

bioRxiv2017

Cell cycle repression and DNA repair defects follow constricted migration

Cancer cell invasion into tissue or narrow capillaries often elongates the nucleus and sometimes damages it, but cell cycle effects are unknown and highly relevant to tumorigenesis. Here, nuclear rupture and DNA breaks caused by constricted migration are quantified in different phases of cell cycle - which is effectively repressed. Cancer lines with varying levels of contact inhibition and lamina proteins exhibit diverse frequencies of nuclear lamina rupture after migration, with prerupture dilation of gene-edited RFP-Lamin-B1 preceding DNA repair factor leakage in pressure-controlled distension. Post-migration rupture indeed associates with mis-localized DNA repair factors and increased DNA breaks as quantified by pan-nucleoplasmic foci of {gamma}H2AX, with foci counts always suppressed in late cell cycle. When contact-inhibited cells migrate through large pores into sparse microenvironments, cells re-enter cell cycle consistent with release from contact inhibition. In contrast, constricting pores effectively delay re-entry, but the excess DNA damage nonetheless exceeds any cell cycle dependence. Partial depletion of topoisomerase does not strongly affect cell cycle or the excess DNA damage, consistent with weak dependencies on replication stress. Constricted migration thus impacts cell cycle as well as DNA damage.

biophysics↗

bioRxiv2017

Mitotic progression following DNA damage enables pattern recognition within micronuclei

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumor responses to radiotherapy1. An enigmatic feature of this phenomenon is its delayed onset (days), in contrast to the acute DNA damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate limiting steps that are essential for DNA-damage induced inflammation. Here, we show that cell cycle progression through mitosis following DNA double-strand breaks (DSBs) leads to the formation of micronuclei, which precede activation of inflammatory signaling and are a repository for the pattern recognition receptor cGAS. Inhibiting progression through mitosis or loss of pattern recognition by cGAS-STING impaired interferon signaling and prevented the regression of abscopal tumors in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

cancer biology↗