Biology subjects

Mittal, K.

Publications and source records attributed to Mittal, K..

7 recordsLinked to original sources

bioRxiv2025

CHD1 is a synthetic lethal vulnerability in MYC-driven breast cancer

The MYC transcription factor is a key regulator of growth during development and a potent cancer driver when its expression is dysregulated. Strategies to inhibit MYC oncogenic activity would mark a significant advance, but decades of efforts to target MYC directly have not been fruitful. Understanding how MYC drives transformation and tumor growth may provide new therapeutic avenues in a variety of cancers. By intersecting two independent genome-wide screens, we identified loss of the chromatin remodeler Chromodomain-Helicase DNA-binding 1 (CHD1) as a potential synthetic lethal target in MYC-driven breast cancer. Knockdown of CHD1 in a xenograft model of MYC-driven breast cancer suppresses tumor growth in vivo. In tissue culture models, we found that knockdown of CHD1 suppresses cell proliferation and induces cell death, specifically when MYC is overexpressed. Mechanistically, we found that CHD1 is required to maintain an open chromatin landscape and a transcriptional program associated with cancer progression in MYC overexpressing breast cells. Follow-up experiments indicate that this synthetic lethality may arise from nucleolar stress and p53 activation. These findings provide new insights on the chromatin-level regulation of MYC-driven breast cancer and uncover CHD1 as a novel synthetic vulnerability and potential therapeutic target.

cancer biology↗

bioRxiv2025

Alpha satellite RNA marks the perinucleolar compartment and represses ribosomal RNA expression in naïve human embryonic stem cells

While most newly synthesized RNA is exported to the cytoplasm, a portion of non-coding RNA is retained in the nucleus and remains highly associated with chromatin. The strong binding of this RNA fraction to insoluble chromatin impairs its recovery in standard transcriptomic studies. Therefore, the landscape and potential functions of chromatin-associated RNAs are poorly understood. Recent studies indicate that chromatin-associated transcripts can have regulatory roles, particularly during mammalian development. Here we compare the dynamics of cytoplasmic versus chromatin-bound transcriptomes of naive and primed human embryonic stem cells (hESCs), as well as fibroblasts. We found a remarkable enrichment for RNA transcribed from alpha satellite repeat (ALR) in the chromatin fraction of naive hESCs, compared to primed hESCs. The co-localization and interaction of ALR RNA with Polypyrimidine Tract Binding Protein 1 (PTBP1) protein indicate that ALR RNA foci mark the perinucleolar compartment (PNC), a nuclear sub-compartment previously thought to be exclusive to cancer cells. Knockdown of ALR RNA leads to dispersion of PTBP1 foci, up-regulation of ribosomal RNA and global hypertranscription in naive hESCs. In contrast, loss of PTBP1 does not disturb ALR RNA localization, indicating that ALR is upstream in the hierarchy of organization of the PNC in hESCs. These results reveal a role for ALR RNA in nuclear compartmentalization and tuning rRNA synthesis in naive hESCs. Moreover, this study opens new avenues to dissect the function of ALR RNA and the PNC in cancer contexts.

genomics↗

bioRxiv2024

LINE1 promotes nuclear compartmentalization to repress the 8-cell state in embryonic stem cells

The family of LINE1 transposable elements underwent a massive expansion in mammalian genomes. While traditionally viewed as a mutagenic selfish element, recent studies point to roles for LINE1 in early mouse development, T cell quiescence and neurogenesis. Here we show that human LINE1 RNA is essential for self-renewal and identity of human embryonic stem cells (hESCs). Silencing of LINE1 using either antisense oligonucleotides or CRISPR interference in naive hESCs leads to a strong induction of 8C-like cells (8CLCs). We found that genes derepressed upon LINE1 KD are not uniformly distributed across the genome, with an enrichment for chromosome 19, which includes key markers of the 8C state such as TPRX1. Silencing of TPRX1, but not other putative 8C regulators p53 or H3.XY, suppresses the induction of the 8C program in LINE1 KD hESCs. We found that LINE1 RNA is preferentially localized to the lamina and periphery of the nucleolus in hESCs. Sequencing of Lamina-Associated Domains (LADs) and Nucleolus-Associated Domains (NADs) reveals a preferential association of chromosome 19 with NADs in hESCs. However, 8CLCs have a distinct nucleolar morphology and a lower association of chromosome 19 and TPRX1 loci with the nucleolus relative to naive and primed hESCs, suggesting a role for nucleolar dynamics in the 8CLC-hESC transition. In agreement, LINE1 KD leads to disruption of nucleolar architecture with signs of nucleolar stress. Independent perturbations of the nucleolus induce the 8C program in hESCs. Genes induced by LINE1 KD are enriched for targets of Polycomb Repressive Complex (PRC2), and inhibition of PRC2 leads to a strong induction of 8C genes. Our results indicate that LINE1 coordinates nuclear compartmentalization and chromatin-mediated gene repression to prevent developmental reversion of hESCs. HighlightsO_LIKnockdown of LINE1 induces TPRX1-dependent emergence of 8C-like cells in hESCs. C_LIO_LIGenes de-repressed upon LINE1 KD are enriched for Chr 19 and PRC2 targets. C_LIO_LI8CLCs display dissociation of Chromosome 19 from the nucleolus. C_LIO_LIDisruption of the nucleolus or inhibition of PRC2 strongly induce the 8C program. C_LI

developmental biology↗

bioRxiv2023

Towards establishing a 24-hour, microplate-based, transcriptomics assay for rainbow trout embryos

There is interest in the development of early-life stage (ELS) tests with fish embryo models that are high-throughput and can generate transcriptomics point of departure (tPOD) values. The objective of this study was to establish a method in rainbow trout (Oncorhynchus mykiss) hatchlings that could satisfy both of these interests. We based our pilot method on recent efforts by U.S. EPA researchers to establish a larval fathead minnow high throughput transcriptomics assay. Here, 1-2 day post hatch trout were assayed in 24-well plates in which they were exposed for 24 hours to 12 different concentrations of test chemicals, including a negative control (DMSO, culture water). Test concentrations were anchored with a chemicals LC50 data from the US EPA ECOTOX database and EnviroTox database, and from this, concentrations were spaced on a half-log basis that spanned 6-7 orders of magnitude. In pilot study 1 we tested 3,4-dichloroaniline, CuSO4 (0.34 mg/L), and ethinylestradiol. In pilot study 2 we tested 3,4-dichloroaniline (58.5 mg/L), CuSO4 (0.34 and 0.41 mg/L), ethinylestradiol (>10 {micro}g/L), permethrin (>10 {micro}g/L), malathion (0.61 mg/L), 6PPD quinone (5.6 {micro}g/L), acetaldehyde (41.2 mg/L), 4-fluoroaniline (242.7 mg/L), glyphosate ([~]150 mg/L), ethanol (>1 g/L), thiamethoxam (>300 mg/L), and allyl alcohol (>30 mg/L). In both pilot studies derived LC50 values are provided in parentheses. Repeated studies of CuSO4 yielded consistent LC50 values (0.34, 0.34, 0.41 mg/L). The correlation between LC50s from the current study for rainbow trout embryos versus those from the literature on adult rainbow trout for 7 chemicals was r2 = 0.91. Work is underway to optimize transcriptomics assays from these samples using EcoToxChips and UPXome, with the ultimate goal to be able to derive transcriptomics points of departure. Taken together these results provide a foundation towards establishing a novel testing platform for chemical and environmental risk assessment that is much quicker (24 hrs), ethical (non-protected life stages), resource efficient (e.g., microplate-based, small volumes of chemicals), and more informative (molecular clues into MOA) than traditional bioassay approaches.

pharmacology and toxicology↗

bioRxiv2022

Targeting TRIP13 in Wilms Tumor with Nuclear Export Inhibitors

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

cancer biology↗

bioRxiv2022

Resource requirements for ecotoxicity testing: A comparison of traditional and new approach methods

Toxicity testing is under transformation as it aims to harness the potential of New Approach Methods (NAMs) as alternative test methods that may be less resource intensive (i.e., fewer animals, cheaper costs, quicker assays) than traditional approaches while also providing more data and information. While many stakeholders are of the opinion that this unfolding transformation holds significant promise as a more efficient and ethical way forward, few studies have compared the resources required for NAMs versus those needed for traditional animal-based toxicity tests, particularly in the field of ecotoxicology. The objective was to compare resources needed for traditional animal-based ecotoxicity tests versus alternative tests using emergent NAMs. From a bibliometric review, we estimate that traditional tests for a single chemical cost $118,000 USD, require 135 animals, and take 8 weeks. In comparison, alternative tests cost $2,600, require 20 animals (or none), and take up to 4 weeks to test 16 (to potentially hundreds of) chemicals. Based on our analysis we conclude that NAMs in ecotoxicology can be more advantageous than traditional methods in terms of resources required (i.e., monetary costs, number of animals needed, and testing times). We note, however, that the evidence underpinning these conclusions is relatively sparse. Moving forward, groups developing and applying NAMs should provide more detailed accounts of the resources required. In addition, there is also a need for carefully designed case studies that demonstrate the domain of applicability of NAMs (and make comparisons to traditional tests) to ultimately build confidence among the user community.

pharmacology and toxicology↗

bioRxiv2016

Mapping Autosomal Recessive Intellectual Disability: Combined Microarray and Exome Sequencing Identifies 26 Novel Candidate Genes in 192 Consanguineous Families

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations (ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7, and USP44), and missense mutations include the first reports of variants in BDNF or TET1 associated with ID. The genes identified also showed overlap with de novo gene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

genetics↗