Biology subjects

Wuest, A. N.

Publications and source records attributed to Wuest, A. N..

4 recordsLinked to original sources

bioRxiv2025

A functional map of CDK-drug interactions at single amino acid resolution

Proteins that drive or support human disease phenotypes are attractive molecular targets for precision therapy, yet most are nominated by knockout studies and then targeted with drugs that inhibit core catalytic pockets. These strategies cannot resolve which residues are essential, whether non-catalytic sites offer better selectivity or potency, or identify on-target resistance mechanisms. We introduce a framework that integrates precision genome editing, mechanistically diverse therapeutics, and computational sequence-structure-function analysis to map protein essentiality and potential druggability at single amino acid resolution. Applying this framework across 9 cyclin-dependent kinases (CDKs) and 15 cancer therapeutics--including ATP-competitive inhibitors, PROTACs, and molecular glue degraders--we identify shared and CDK-specific residues critical for cell fitness and drug response, including known resistance mutations and dozens of new variants. The resulting functional maps resolve residue- and mechanism-specific differences in the resistance spectra among agents targeting the same protein. We show that this iterative strategy can also uncover higher order interactions by performing intra- and extragenic epistasis screens to identify residues that mediate on-target and within-family cell fitness and drug resistance. Finally, we find evidence of novel CDK6 mutations in breast cancer patients and concordance between experimental and clinical correlates of response to CDK4/6 inhibitors. By mapping residue-level essentiality and forecasting therapy resistance mutations, target-drug interaction maps could inform clinical treatment and guide design of more selective therapeutic molecules.

cancer biology↗

bioRxiv2025

Multiplexed in vivo base editing identifies functional gene-variant-context interactions

Human genome sequencing efforts in healthy and diseased individuals continue to identify a broad spectrum of genetic variants associated with predisposition, progression, and therapeutic outcomes for diseases like cancer1-6. Insights derived from these studies have significant potential to guide clinical diagnoses and treatment decisions; however, the relative importance and functional impact of most genetic variants remain poorly understood. Precision genome editing technologies like base and prime editing can be used to systematically engineer and interrogate diverse types of endogenous genetic variants in their native context7-9. We and others have recently developed and applied scalable sensor-based screening approaches to engineer and measure the phenotypes produced by thousands of endogenous mutations in vitro10-12. However, the impact of most genetic variants in the physiological in vivo setting, including contextual differences depending on the tissue or microenvironment, remains unexplored. Here, we integrate new cross-species base editing sensor libraries with syngeneic cancer mouse models to develop a multiplexed in vivo platform for systematic functional analysis of endogenous genetic variants in primary and disseminated malignancies. We used this platform to screen 13,840 guide RNAs designed to engineer 7,783 human cancer-associated mutations mapping to 489 endogenous protein-coding genes, allowing us to construct a rich compendium of putative functional interactions between genes, mutations, and physiological contexts. Our findings suggest that the physiological in vivo environment and cellular organotropism are important contextual determinants of specific gene-variant phenotypes. We also show that many mutations and their in vivo effects fail to be detected with standard CRISPR-Cas9 nuclease approaches and often produce discordant phenotypes, potentially due to site-specific amino acid selection- or separation-of-function mechanisms. This versatile platform could be deployed to investigate how genetic variation impacts diverse in vivo phenotypes associated with cancer and other genetic diseases, as well as identify new potential therapeutic avenues to treat human disease.

cancer biology↗

bioRxiv2024

Metastatic site influences driver gene function in pancreatic cancer

Driver gene mutations can increase the metastatic potential of the primary tumor1-3, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGF{beta} signaling - which is a hallmark of multiple gastrointestinal malignancies4,5. SMAD4 inactivation mediates TGF{beta}s remarkable anti-to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis6-14. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumors organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4s tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.

cancer biology↗

bioRxiv2022

Chromosome 9p21.3 Coordinates Cell Intrinsic and Extrinsic Tumor Suppression

Somatic chromosomal deletions are prevalent in cancer, yet their functional contributions remain ill-defined. Among the most prominent of these events are deletions of chromosome 9p21.3, which disable a cell intrinsic barrier to tumorigenesis by eliminating the CDKN2A/B tumor suppressor genes. However, half of 9p21.3 deletions encompass a cluster of 16 type I interferons (IFNs) whose co-deletions have not been functionally characterized. To dissect how 9p21.3 and other genomic deletions impact cancer, we developed MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements), a genome engineering strategy that enables flexible modeling of megabase-sized deletions. Generation of 9p21.3-syntenic deletions in a mouse model of pancreatic cancer revealed that concomitant loss of Cdkn2a/b and the IFN cluster led to immune evasion and metastasis compared to Cdkn2a/b-only deletions. Mechanistically, IFN co-deletion disrupted type I IFN signaling, altered antigen-presenting cells, and facilitated escape from CD8+ T cell surveillance in a cell extrinsic manner requiring loss of interferon epsilon (Ifne). Our results establish co-deletions of the IFN cluster as a pervasive route to tumor immune evasion and metastasis, revealing how deletions can disable physically linked cell intrinsic and extrinsic tumor suppression. Our study establishes a framework to dissect the functions of genomic deletions in cancer and beyond.

cancer biology↗