Hangai, S. | Search bioRxiv

bioRxiv2025

PKMYT1 inhibition induces DNA damage and synergizes with immune checkpoint blockade in CCNE1-amplified gastroesophageal adenocarcinoma

PurposeCCNE1 amplification, found in approximately 10% of gastroesophageal adenocarcinoma (GEA), drives chromosomal instability and is associated with an immune-cold tumor microenvironment. Recent studies suggest that PKMYT1 inhibition is synthetic lethal in CCNE1-amplified cancers; however, its role in GEA is largely uncharacterized. We investigated the therapeutic activity of PKMYT1 inhibition in CCNE1-amplified GEA and its potential combination with immunotherapy. Experimental DesignWe evaluated lunresertib (RP-6306), a selective first-in-class PKMYT1 inhibitor, in CCNE1-amplified and wild-type GEA cell lines to define its effects on cell viability, DNA damage, and gene expression changes. To investigate the role of PKMYT1 blockade on immune microenvironment modulation, we used a syngeneic murine model of CCNE1;Trp53-/- GEA xenografts treated with lunresertib, anti-PD-1, or the combination. ResultsLunresertib was selectively cytotoxic in CCNE1-amplified GEA cell lines in cell viability and clonogenic assays. Mechanistically, treatment with lunresertib induced DNA damage, evidence by increased {gamma}H2AX expression and DNA micronuclei formation, and activated pro-inflammatory signaling pathways by gene set enrichment analysis. In a CCNE1-amplified GEA murine model, PKMYT1 inhibition promoted T cell infiltration, reduced myeloid cells, and synergized with anti-PD-1 therapy to induce tumor regression. ConclusionsOur results suggest that CCNE1 amplification is a potentially actionable target in GEA and support the development of combination therapeutic strategies utilizing PKMYT1 inhibition and PD-1 blockade. Translational RelevanceGastroesophageal adenocarcinoma (GEA) is an aggressive malignancy with poor prognosis and few actionable molecular alterations. CCNE1 amplification is present in approximately 10% of patients with GEA and represents a potential therapeutic vulnerability through synthetic lethality with PKMYT1 inhibition. We demonstrate that targeting CCNE1-amplified GEA through PKMYT1 inhibition with lunresertib is selectively cytotoxic, induces DNA damage, and activates inflammatory signaling pathways. In a syngeneic murine model of CCNE1-amplified gastric cancer, we further identified that PKMYT1 inhibition induced T cell infiltration and tumor microenvironment remodeling, and synergized with anti-PD-1 therapy to drive tumor regression. Together, these results support further development of PKMYT1 inhibition in combination with PD-1 blockade for CCNE1-amplified GEA.

cancer biology↗

bioRxiv2023

CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

genomics↗