Regulation of RNA biogenesis and function by RNA modifications

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Understanding how cancer cells resist therapy and evade immune surveillance is central to developing more effective treatments. Recent studies from our group have focused on uncovering molecular pathways that connect metabolism, RNA regulation, and immune evasion in aggressive kidney cancers. These efforts have revealed novel insights into mechanisms of drug resistance, the impact of metabolic rewiring on RNA modifications, and how RNA-modifying enzymes help tumors escape immune-mediated destruction. We examined how the accumulation of the oncometabolite fumarate, resulting from loss of fumarate hydratase (FH) in hereditary leiomyomatosis and renal cell carcinoma (HLRCC), affects RNA demethylation. Using a combination of genomic and biochemical methods, we assessed the impact of fumarate on ALKBH1, ALKBH5, and FTO, which regulate f5C, m6A, and m6Am RNA modifications. While fumarate accumulation did not affect f5C levels in mitochondrial tRNA, it led to elevated m6A levels, suggesting selective inhibition of m6A demethylation. These findings highlight a differential sensitivity of RNA-modifying enzymes to metabolic perturbations and point to a metabolic-epitranscriptomic axis in cancer biology. Building on these discoveries, we explored how RNA regulation affects the interaction between cancer cells and the immune system, particularly Natural Killer (NK) cells. FH-deficient tumors exhibit mitochondrial dysfunction and a chronic inflammatory phenotype, accompanied by increased immune cell infiltration, including NK cells. Despite the presence of NK cells, these tumors persist and metastasize, underscoring a disconnect between immune recognition and effective tumor clearance. We hypothesized that RNA post-transcriptional modifications may influence how tumor cells respond to immune stress. To systematically explore this, we conducted a CRISPR-Cas9 screen targeting writers, readers, and erasers of RNA modifications in a co-culture system with HLRCC cells and IL-2-activated NK-92 cells. This screen identified several candidate regulators that dampen NK cell cytotoxicity. Further characterization suggested that the top hits identified in our screen do not affect general viability or expression of NK ligands on the cell surface but instead acts as negative regulators of interferon responses. To dissect their functions, we are deploying genetic silencing of interferon pathway components and conducting CRISPR screens targeting interferon-stimulated genes. At the molecular level, single-cell RNA-seq showed increased expression of genes involved in leukocyte activation and immune effector function after co-culture of knock-out cell lines with NK-92 cells. We are now mapping interactions between our genes of interest and their RNA and protein interactors using CLIP, STAMP, SLAM-seq, Ribo-seq, and APEX-based proteomics. We also plan to evaluate whether RNA modification pathways play a role in how tumors respond to therapy in the context of immune surveillance. Since drug treatment can induce stress signals that engage the immune system, we will expose cancer cells to kidney cancer therapies and innate immune agonists while probing the contribution of RNA regulatory genes to NK cell responses. Larger CRISPR libraries targeting all RNA-binding proteins or interferon-stimulated genes will allow us to expand this effort and identify additional modulators of drug response and immune evasion. In a complementary line of investigation, we are addressing how non-coding mutations in tumor suppressor genes impair mRNA splicing and protein expression in hereditary kidney cancers. In collaboration with the Urologic Oncology Branch, we identified an intronic variant in FH that leads to the inclusion of a cryptic exon introducing a premature stop codon. Using mini-gene reporters and patient-derived fibroblasts, we demonstrated that antisense oligonucleotides (ASOs) and CRISPR tools can restore correct splicing and rescue protein production. Ultimately, we aim to determine whether partial restoration of FH expression is sufficient to re-establish cellular homeostasis and restrain disease progression. To this end, we are creating cell models where expression of these tumor suppressors depends solely on the pathogenic allele. We will also explore whether partial correction across a tumor population can yield therapeutic benefit. In vivo xenograft models will allow us to test the physiological relevance of our splicing correction strategies and assess their potential for future clinical translation.