Biology subjects

Gannon, D.

Publications and source records attributed to Gannon, D..

6 recordsLinked to original sources

bioRxiv2026

An iteratively curated CRISPR library reveals target-specific biological resistance landscapes across targeted protein degraders

Targeted protein degradation (TPD) has emerged as an increasingly powerful approach for therapeutic development and biological discovery. TPD compounds including proteolysis-targeting chimeras (PROTACs), molecular glues, and tag-targeting protein degraders (tTPD) enable rapid, selective and reversible degradation of proteins through recruitment of the ubiquitin-proteasome system (UPS). However, genome-wide CRISPR screens performed with targeted protein degraders are frequently dominated by resistance mechanisms that disrupt degrader activity, including loss of recruited E3 ligase components and broader UPS regulators. The strong selective advantage conferred by these perturbations can obscure less penetrant, biological genetic interactions that operate downstream of target degradation. To overcome this limitation, through iterative genome-wide screening and manual curation, we developed a TPD-compatible CRISPR knockout library that retains near-genome-scale coverage while excluding a focused set of genes recurrently associated with degrader failure. Across multiple degrader screens, this library reduced the dominance of UPS-associated resistance mechanisms and improved the detection and prioritization of genetic interactions linked to target biology. Using the RBM39 molecular glue degrader indisulam as a model, we identified ZMAT2 loss as a resistance mechanism that preserves RBM39 degradation but attenuates the transcriptional and splicing consequences of target depletion. Together, our work establishes a TPD-compatible CRISPR screening framework that improves the biological resolution of degrader resistance screens and facilitates the discovery of genetic dependencies operating downstream of targeted protein degradation.

genomics↗

bioRxiv2026

Termination dynamics set RNAPII elongation rate and gate the response to CDK12 inactivation

The transcriptional fidelity of RNA polymerase II (RNAPII) is governed by a tight equilibrium between elongation and termination activities, a balance frequently disrupted in human diseases such as cancer. The transcriptional cyclin-dependent kinase 12 (CDK12) maintains RNAPII elongation rate and processivity throughout the gene body. Inactivation of CDK12 disrupts this homeostatic balance and causes elongation stress, slowing RNAPII and triggering premature termination at intronic polyadenylation sites (IPAs). Despite this, the precise executors of intronic premature termination under CDK12-inactivation-induced elongation stress remain poorly understood. Using genome-wide CRISPR screening combined with chemical-genetic approach, we identified a pro-termination mechanism at intronic checkpoints; upon CDK12 inactivation, SCAF4 recruits the cleavage and polyadenylation (CPA) complex through its catalytic endonuclease CPSF3 to execute premature cleavage at IPAs. Disruption of SCAF4-CPA axis prevents early termination, restores full length transcription and confers resistance to CDK12/cyclin K inhibition. Genetic loss of SCAF4 restores the RNAPII elongation rate under CDK12 inhibition, revealing that termination dynamics actively shape the rate of transcription. Supporting this model, we uncovered an anti-termination mechanism driven by KHDRBS1/SAM68, whose depletion promotes proximal termination and sensitises cells to CDK12 targeting. Our findings mechanistically couple elongation and termination activities at intronic checkpoints as joint contributors to both the processivity and elongation rate of RNAPII. This establishes termination dynamics as an active and tractable axis of the cellular response to elongation stress.

molecular biology↗

bioRxiv2026

INTS12 Bridges Integrator and NELF to Prevent the Release of Non-processive RNA Polymerase II Complexes

Promoter-proximal RNA Polymerase II (RNAPII) pausing and the processivity are controlled by distinct modules of the Integrator complex, which together fine-tune transcription and protect against the accumulation of defective RNAPII complexes. Compromised activity of individual Integrator modules has been linked to human disease including cancer and developmental disorders, caused by defective transcription of protein-coding or small-nuclear RNAs. Despite extensive characterisation of the Integrator complex both genetically and structurally, the role of smallest member of the complex, INTS12, has remained enigmatic. Here, we uncover that INTS12 loss acts to stabilise the association between NELF and Integrator via its PHD domain and N-terminus, respectively, thus safeguarding against the release of defective RNAPII complexes. Acute degradation of INTS12 results in the selective dissociation of Integrator from the NELF-RNAPII complex which subsequently convert to their canonical paused form from which they can be released by CDK9. In the absence of INTS12 excess release of defective RNAPII via P-TEFb/SEC, loss of the ARMC5 salvage pathway and deletion of the catalytic and core Integrator subunits is toxic to cells. These findings demonstrate that there is interconversion between canonical paused RNAPII and paused-Integrator, and highlight the critical interplay between these processes and P-TEFb mediated pause-release to ensure that only transcription competent complexes are released into elongation. O_LIINTS12 degradation confers CDK9 inhibitor resistance and triggers cellular stress through a phosphatase module-independent mechanism. C_LIO_LIINTS12 stabilizes the Integrator-NELF complex through its N-terminus and PHD domain. C_LIO_LIAcute INTS12 degradation promotes aberrant release of promoter-proximal RNA polymerase II complexes. C_LIO_LIRNA polymerase II complexes released upon INTS12 loss exhibit defective elongation and reduced processivity. C_LIO_LIINTS12 loss removes Integrator from RNAPII resulting in aberrant paused-state from which it can be released by CDK9. C_LIO_LIExcess CDK9 activity and ARMC5 loss are synthetically lethal with INTS12 deficiency. C_LI

molecular biology↗

bioRxiv2025

AEBP2-Directed H3K27me2 Defines a Specific Vulnerability in EZH2-mutant Lymphoma

The catalytic subunit of Polycomb Repressive Complex 2 (PRC2), EZH2, is recurrently mutated in 25% of diffuse large B-cell lymphomas (DLBCL), causing increased H3K27me3 and decreased H3K27me2 levels. EZH2 inhibitors provide clinical benefit, but resistance frequently develops, highlighting the need for alternative therapeutic targets. Here, we identify the PRC2 accessory protein AEBP2 as a specific genetic dependency in EZH2-mutant DLBCL. While AEBP2 acts through PRC2, its essential role is surprisingly independent of canonical H3K27me3-mediated gene silencing. Instead, AEBP2 functions within a PRC2.2 complex lacking JARID2, using its zinc-finger domains to sample intergenic chromatin to sustain H3K27me2. Notably, loss of AEBP2 or NSD2 caused contrasting changes in intergenic H3K27me2 levels, driving sensitivity or resistance to PRC2 inhibitors, respectively. Our findings identify AEBP2-PRC2.2-maintained intergenic H3K27me2 as a therapeutic vulnerability in EZH2-mutant DLBCL and highlight dysregulated H3K27me2 as an underappreciated form of PRC2 dysfunction in cancer, with important therapeutic implications.

cancer biology↗

bioRxiv2025

A TAK1Cytokine Toxicity Checkpoint Controls Anti-Cancer Immunity

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFN{gamma} and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines. In BriefDjajawi et al. identify TAK1 as a cancer-intrinsic cytokine toxicity checkpoint that limits the efficacy of anti-cancer immune responses. Cancer cells exploit TAK1 activity to confers resistance to CD8+ T cell-derived TNF and IFN{gamma}-induced apoptosis, limiting both direct and bystander killing. Mechanistically, TAK1 loss acts through 1. cFLIP and RIPK1 to promote cell death, 2. The inactivation of pro-survival signals and 3. Via amplification of the IFNy response. Targeting of this TAK1 checkpoint enhanced anti-tumour immunity in vivo and improved adoptive cellular therapy. These findings identify a strategy employed by transformed cells to avoid destruction by inflammatory cytokines and provide new therapeutic vulnerabilities for enhancing immunotherapies. HighlightsO_LICRISPR screens identify TAK1 as a tumour-intrinsic survival checkpoint limiting destruction by CD8+ T cells. C_LIO_LITAK1 protects tumour cells from combined TNF and IFN{gamma}-induced apoptosis. C_LIO_LITAK1 loss destabilizes cFLIP, priming cells for STAT1 and RIPK1-dependent cell death. C_LIO_LITAK1 loss promotes tumour control in immune competent animals and enhances the efficacy of adoptive cell therapy. C_LI

cancer biology↗

bioRxiv2023

Specific cPRC1 complexes are co-opted to mediate oncogenic gene repression in diffuse midline glioma

Diffuse midline glioma (DMG) is a fatal childhood brain tumour characterised primarily by mutant histone H3 (H3K27M). H3K27M causes a global reduction in Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 by inhibiting PRC2 enzymatic activity. Paradoxically, PRC2 is essential in DMG tumour cells where residual complex activity is required for oncogenic gene repression, although the molecular mechanisms acting downstream of PRC2 in this context are poorly understood. Here, weve discovered this oncogenic gene repression is mediated by specific canonical PRC1 (cPRC1) formations. By combining CRISPR screening, biochemical and chromatin mapping approaches with functional perturbations we show that cPRC1 complexes containing CBX4 and PCGF4 drive oncogenic gene repression downstream of H3K27me3 in DMG cells. Remarkably, the altered H3K27me3 modification landscape characteristic of these tumours rewires the distribution of cPRC1 complexes on chromatin. CBX4 and PCGF4 containing cPRC1 accumulate at sites of H3K27me3 while other cPRC1 formations are displaced. Despite accounting for <5% of cPRC1 complexes in DMG, CBX4/PCGF4-containing complexes predominate as gene repressors. Our findings link the altered distribution of H3K27me3 with imbalanced cPRC1 function, promoting oncogenic gene repression in DMG cells, revealing new disease mechanisms and highlighting potential therapeutic opportunities in this incurable childhood brain tumour.

cancer biology↗