Clonal lineage tracing and parallel multiomics profiling reveal transcriptional diversification induced by ARID1A deficiency

Phenotypic heterogeneity among genetically identical cancer cells underpins tumor progression and therapy resistance. However, how epigenetic dysregulation drives such divergence remains unclear. Here, we introduce IMPACH, a scalable platform that integrates lineage tracing, genetic perturbation, and multimodal single-cell analysis. Applying IMPACH, we demonstrate that loss of epigenetic regulators—particularly ARID1A—enhances transcriptomic and epigenetic heterogeneity under controlled conditions. ARID1A deficiency promotes stochastic chromatin opening and induces an atypical gene expression program associated with poor clinical outcomes. Despite increased epigenetic randomness, chromatin changes remain localized to biologically meaningful regulatory elements, partially linked to SMARCA4 binding sites. These findings reveal that chromatin remodeling defects promote clonal diversification through both stochastic and constrained mechanisms. Given its scalability and versatility, IMPACH provides a powerful framework for dissecting how epigenetic perturbations shape cellular heterogeneity in cancer. Overall design: shPseuMO-Tag constructs targeting ARID1A (two independent shRNAs: ARI3# and ARI4#) or a nontargeting control (SCR#) were introduced into BxPC3 cells. Following single-cell cloning and approximately 20 cell divisions, 28 clonal subpopulations were established (eight controls and 20 ARID1A knockdowns). Subsequently, all 28 clones were pooled into a single cellular mixture, and scRNA-seq and scATAC-seq were performed to minimize the batch effect while evaluating transcriptional variations within and across clones.