Biology subjects

Sharan, S. K.

Publications and source records attributed to Sharan, S. K..

7 recordsLinked to original sources

bioRxiv2026

A single BRCA2 BRC repeat supports viability while multiple repeats ensure resilience under stress

BRC repeats are integral to BRCA2 function and mediate RAD51 loading during homologous recombination (HR). Their number varies across species, but mammals, including mice and humans, harbor eight repeats. Prior studies have suggested functional redundancy among these repeats, but the biological significance of maintaining multiple repeats remains unresolved. Here, we demonstrate that the presence of a single BRC repeat, either BRC2 or BRC4, is sufficient for mouse embryonic stem cell (mESC) viability, RAD51 loading, PARP inhibitor resistance and protection of stalled replication forks. Consistent with these findings, we show that knock-in mice with a single BRC repeat 2 or 4 are viable and exhibit normal growth and fertility. In contrast, embryonic fibroblasts from these mice display genomic instability and impaired RAD51 recruitment. Notably, this defect is rescued under low oxygen culture conditions, which mimics physoxic levels, whereas exposure to oxidative stress impairs RAD51 recruitment in mESCs harboring a single BRC repeat. Together, our findings indicate that while a single BRC repeat is sufficient under physiological conditions, the evolutionary retention of multiple BRC repeats likely ensures robust genome stability under extreme oxidative stress.

genetics↗

bioRxiv2024

FIGNL1 inhibits homologous recombination in BRCA2 deficient cells by dissociating RAD51 filaments

Homologous recombination (HR) deficiency upon BRCA2 loss arises from defects in the formation of RAD51 nucleoprotein filaments. Here, we demonstrate that loss of the anti-recombinase FIGNL1 retains RAD51 loading at DNA double-stranded breaks (DSBs) in BRCA2-deficient cells, leading to genome stability, HR proficiency, and viability of BRCA2-deficient mouse embryonic stem cells. Mechanistically, we directly show that strand invasion and subsequent HR defects upon BRCA2 loss primarily arises from the unrestricted removal of RAD51 from DSB sites by FIGNL1, rather than from defective RAD51 loading. Furthermore, we identify that the MMS22L-TONSL complex interacts with FIGNL1 and is critical for HR in BRCA2/FIGNL1 double-deficient cells. These findings identify a pathway for tightly regulating RAD51 activity to promote efficient HR, offering insights into mechanisms of chemoresistance in BRCA2-deficient tumors.

genetics↗

bioRxiv2024

5hmC enhances PARP trapping and restores PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells

Mutations in BRCA1 and BRCA2 genes are the leading cause of hereditary breast and ovarian cancer. BRCA1/2-mutant cells are defective in repairing damaged DNA by homologous recombination and are characterized by hypersensitivity to PARP inhibitors. PARP inhibitors can trap PARP proteins on the chromatin, a mechanism that can contribute to the death of BRCA1/2-deficient cells. The FDA has approved multiple PARP inhibitors for the treatment of metastatic breast and ovarian cancers, but despite the success of PARP inhibitors in treating BRCA1/2-mutant cancers, drug resistance is a major challenge. Here, we report that 5hmC enhances PARP1 trapping on the chromatin in olaparib-treated cells. Elevated PARP trapping generates replication gaps, leading to the restoration of PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells. Our findings suggest that combining 5hmC with olaparib can restore the sensitivity of chemoresistant BRCA1/2-deficient cells.

cancer biology↗

bioRxiv2023

AVENGERS: Analysis of Variant Effects using Next Generation sequencing to Enhance BRCA2 Stratification

Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). To determine the functional impact of VUSs, here we develop AVENGERS: Analysis of Variant Effects using NGs to Enhance BRCA2 Stratification, utilizing CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain of BRCA2. We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessing BRCA2 VUSs in patients.

genetics↗

bioRxiv2023

Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that IFN promotes replication fork stability, cell proliferation and survival in BRCA1/2-defective cancer cells and rescues the lethality of BRCA2-deficient mouse embryonic stem cells. Although IFN activates hundreds of genes, these effects are specifically mediated by the ubiquitin-like modifier ISG15 (IFN-stimulated gene 15). Inactivation of ISG15 or of the enzymes promoting its conjugation, referred as ISGylation, completely suppresses the impact of IFN on the replication process. Depletion of ISG15 significantly reduces cell proliferation rates whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in human BRCA1-mutated triple-negative and mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, representing a novel, in-built mechanism of drug resistance linked to BRCAness.

cancer biology↗

bioRxiv2022

PKM2-mediated epigenetic reprogramming regulates hypoxic expression of PFKFB3 to promote breast cancer progression

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms existing between the key glycolytic enzymes in hypoxic cancer cells are largely unexplored. In particular, M2 isoform of pyruvate kinase (PKM2) - the ratelimiting enzyme of glycolysis, is well-known to confer adaptive advantages under hypoxia. Herein, we report a non-canonical role of PKM2 in functioning as a co-activator of HIF-1 to govern the transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Nuclear PKM2 enhances HIF-1 and p300 occupancy at PFKFB3 hypoxia responsive elements (HREs) resulting in its upregulation. Consequently, absence of nuclear PKM2 fails to recruit HIF-1 which activates an opportunistic occupancy of HIF-2 at PFKFB3 HREs. Enhanced binding of HDAC3 also occurs in the absence of PKM2 which prevents HIF-2 from efficiently inducing PFKFB3 to hamper proliferation of hypoxic breast cancer cells. In addition, clinical relevance of the study has been investigated by demonstrating that Shikonin blocks nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, MCF7 cells-derived xenograft tumors in mice exhibited substantial tumor growth inhibition when treated with shikonin, highlighting the vitality of targeting PKM2. Taken in concert, this work provides novel insights into contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported molecular axis exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression essential for achieving high glycolytic flux. Statement of significanceNuclear PKM2 orchestrates the binding of histone modifiers to epigenetically alter PFKFB3 promoter and affects the binding of HIF-2. Notably, targeting this axis attenuates proliferation of hypoxic breast cancer cells.

cancer biology↗

bioRxiv2019

Multi-model preclinical platform predicts clinical response of melanoma to immunotherapy

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrates durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models representing the main molecular and phenotypic subtypes of human melanomas and exhibiting their range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify new mechanisms and treatment strategies to improve patient care.

cancer biology↗