Research Projects

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with ~50% survival rate for primary patients in India. Relapse is the principal cause of treatment failure and mortality in 20-25% of cases. Relapsed B-ALL shows a distinct molecular and transcriptomic profile, reflecting adaptive mechanisms acquired under therapeutic pressure. Next-generation sequencing (NGS) has revealed these changes, yet their functional relevance is mostly unexplored. The mentor’s lab has generated RNA-seq data from paired primary and relapsed B-ALL patients. Preliminary analysis revealed ~300 relapse-enriched genes with potential roles in cell survival, immune evasion, epigenetic regulation, and lineage plasticity (unpublished). This provides a novel starting point for functional genomics. Testing these candidates individually would be laborious; pooled CRISPR-Cas9 dropout screens enable systematic interrogation of all candidates in a single experiment. Here, B-ALL cells are transduced with a sgRNA library; sgRNAs that knock out essential genes leading to cell death deplete (“dropout”) over time and can be detected by NGS and bioinformatic tools. CRISPR-Cas9 screens have been widely used to identify cancer vulnerabilities, but most focus on genome-wide or canonical pathways. Relapse-specific datasets have not yet been leveraged for functional screening. This proposal aims to address that gap with a focused CRISPR-Cas9 screen to uncover relapse-specific vulnerabilities. We hypothesize that some genes upregulated at relapse are not merely passive markers but functionally relevant for leukemic cell survival in the post-therapy environment. Knocking them out will identify targets whose loss impairs viability or sensitizes cells to chemotherapy. The proposed objectives are: 1) design and perform a CRISPR-Cas9 pooled knockout screen targeting relapse-enriched genes in B-ALL cell models, 2) functionally characterize the top screen hits in cell survival, proliferation and chemoresistance, 3) identify mechanisms of action by RNA-seq and pathway analysis of knockout cells. For this, Cas9-expressing B-ALL cell lines will be transduced with a pooled sgRNA library targeting the top 100 upregulated genes identified from RNA-seq data. After culture, sgRNA abundance will be measured using NGS and analysed using MAGeCK to identify depleted sgRNAs (i.e., essential genes). The top 5 hits will be validated by individual knockouts and phenotypic assays. Rescue experiments with sgRNA-resistant cDNA will confirm on-target effects. RNA-seq of selected knockouts will be done to identify downstream transcriptional changes and key pathways controlled by each gene. This project combines transcriptomic insights with functional genomics to uncover critical drivers of relapsed B-ALL. The proposed approach could significantly contribute to identification of novel targeted therapies for relapsed B-ALL, a critical unmet clinical need in pediatric oncology.