Biology subjects

Bandopadhayay, P.

Publications and source records attributed to Bandopadhayay, P..

11 recordsLinked to original sources

bioRxiv2026

Phenotypic screens identify biologic regulators of nanoparticle uptake in diffuse midline glioma

Nanoparticle drug delivery systems hold considerable promise for locoregional administration to central nervous system tumors, yet the biological determinants of nanoparticle-cancer cell interactions remain poorly understood. Using patient-derived histone-mutant diffuse midline glioma (DMG) models, we performed a pooled CRISPR-Cas9 perturbation screen to systematically identify regulators of liposomal nanoparticle delivery. The screen identified candidate genes spanning endocytosis, vesicle transport, and metabolic signaling, revealing that nanoparticle delivery is governed by a broader landscape than previously appreciated. Among these, CTNNB1, or {beta}-catenin, emerged as a common negative regulator across two independent DMG models and two distinct nanoparticle surface chemistries. Transcriptomic profiling of CTNNB1-depleted DMG cells revealed upregulation of membrane remodeling and extracellular matrix gene programs, accompanied by reduced cell stiffness measured by a microfluidic acoustic scattering assay. This resulted in a shift in endocytic activity characterized by decreased bulk-phase macropinocytosis and increased receptor-mediated endocytosis. We further identified MAPK and mTOR pathway members as nanoparticle trafficking modulators, and demonstrated concordance between genetic and pharmacologic perturbations in modulating the liposomal nanoparticle interactions in pediatric DMG cells. These findings establish a biology-first screening approach for identifying previously unappreciated regulators with potential relevance to nanoparticle-based therapeutic strategies in pediatric brain tumors.

bioengineering↗

bioRxiv2026

Standard treatment against paediatric BRAF-V600E glioma promotes senescence and sensitizes tumours to BCL-xL inhibition

BackgroundChemo-, radio-, and targeted therapies can induce senescence in tumour cells, a process called therapy-induced senescence (TIS), which can be exploited therapeutically using compounds that kill senescent cells (senolytics). Children with BRAF-V600E activated brain tumours are commonly treated with Dabrafenib, Trametinib or Vinblastine (alone or in combination) with good responses. However, a subset of patients experiences tumour regrowth upon treatment withdrawal. Here, we explore the role of TIS and senolytic therapies in preclinical models of paediatric BRAF-V600E driven brain tumours. MethodsHuman BT-40 tumour cells derived from pleomorphic xanthoastrocytoma are used in vitro or in vivo orthotopically transplanted into mice and treated with either Dabrafenib, Trametinib or Vinblastine. Senescence is assessed by immunostaining and RNA sequencing. Sensitivity to senolytics is determined in vitro in dose response assays and in vivo. ResultsTrametinib treatment induces a senescent phenotype in BT-40 cells in vivo with activation of a senescence-associated secretory phenotype (SASP) and expansion of IBA-1 microglia. Senescence is induced in BT-40 cells in vitro when treated not only with Trametinib, but also with Dabrafenib and Vinblastine. In vitro testing identifies Navitoclax as a potent senolytic against BT-40 senescent cells, mostly through inhibition of the anti-apoptotic protein BCL-xL. In BT-40 tumour bearing mice, combination of Trametinib and Navitoclax causes the ablation of senescent BT-40 cells with significant reduction in tumour regrowth after treatment withdrawal and increased mouse survival. ConclusionsThis study shows that Navitoclax enhances the anti-tumour efficacy of Trametinib and limits tumour regrowth following treatment withdrawal. Key PointsO_LITrametinib, Dabrafenib and Vinblastine induce senescence in BT-40 cells in vitro C_LIO_LISenescent BT-40 cells are sensitive to BCL-xL inhibition C_LIO_LICombination of Trametinib and Navitoclax reduces rebound tumour growth in vivo C_LI Importance of the StudyChildren with BRAF-V600E activated glioma are frequently treated with Dabrafenib, Trametinib or Vinblastine with good responses. However, a subset of patients shows tumour regrowth when the treatment is stopped. To prevent this rebound growth, patients must continue treatment with potential aggravation in quality of life and risk of malignant progression. Our research demonstrates that these therapies induce BT-40 tumour cells to enter a non-proliferative state that shows features of cellular senescence. We identify that senescent tumour cells can be killed using Navitoclax, and potent inhibitor of the anti-apoptotic protein BCL-xL and a well-studied senolytic. We demonstrate that the combination of Trametinib and Navitoclax reduces tumour regrowth after treatment withdrawal. Our research provides a strong rationale supporting the combined use of senolytics with current conventional and targeted therapies against BRAF-V600E mutated brain tumours.

cancer biology↗

bioRxiv2025

N-terminal modification of Histone H3 inhibits H3K27M-mediated loss of H3K27 trimethylation

The use of short epitope tags is widespread in the study of histone H3 biology as they allow for the antibody-mediated detection and pulldown of particular histone post-translational modifications (PTM) or exogenous histone transgenes. However, H3 is particularly sensitive to sequence modification and the addition of epitope tags may interfere with the native function of H3. Here, we use the known relationship between lysine-to-methionine K27M mutations and the loss of trimethylation at the H3 K27 residue to test whether the addition of epitope tags to the N or C terminus of H3 affects the levels of histone H3 PTMs. We find that all tested N-terminal tags abrogate the H3K27M-mediated loss of H3K27 trimethylation. These results suggest that the addition of epitope tags to the N-terminus of H3 should be performed with caution, and these findings may be of particular interest for the study of H3-driven cancers, like diffuse midline gliomas.

cell biology↗

bioRxiv2024

Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

Structural variants (SVs) represent a mechanism by which cancer cells activate oncogenes or disrupt the function of genes with tumor suppressor roles. A recent study by the PCAWG Consortium investigated structural variants in 30 tumor types, identifying focal rearrangements in the oncogenic BRD4 gene in ovarian, endometrial and breast cancers. These rearrangements resulted in decreased BRD4 expression despite increased copy number, suggesting a novel mechanism to finetune gene over-expression. In this study, we show that focal deletions of BRD4 disrupt genomic regulatory regions and impact gene isoform expression in breast and ovarian tumors when compared to their expression across normal tissues. To determine the functional impact of these concomitant amplification and focal deletion events, we first leveraged open-reading-frame (ORF) screen data from 16 cancer cell lines, where we observed that overexpression of BRD4-long and BRD4-short isoforms is toxic for cancer cell growth. We confirmed these results in OVSAHO ovarian cancer cells, where overexpression of both isoforms significantly reduced tumor growth. Next, we mimicked the focal deletions occurring in BRD4 regulatory regions by CRISPR-Cas9 technology, and observed that their depletion functionally ablates tumor cell growth. We finally show that these focal deletions rescue ovarian carcinoma cells from the toxicity effects associated with gene overexpression, suggesting that global BRD4 gene expression levels must be fine tuned to ensure proper cancer cell proliferation. Our study provides experimental evidence for BRD4 deletions constituting the first example of a driver SV alteration reducing toxicity in cancer, therefore expanding the landscape of cancer progression mechanisms.

cancer biology↗

bioRxiv2023

A compendium of Amplification-Related Gain Of Sensitivity (ARGOS) genes in human cancer

Chromosomal gains are among the most frequent somatic genetic alterations occurring in cancer. While the effect of sustained oncogene expression has been characterized, the impact of copy-number gains affecting collaterally-amplified "bystander" genes on cellular fitness remains less understood. To investigate this, we built a comprehensive map of dosage compensations across human cancers by integrating expression and copy number profiles from over 8,000 TCGA tumors and CCLE cell lines. Further, we analyzed the effect of gene overexpression across 17 human cancer ORF screens to provide an overview of genes that prove toxic to cancer cells when overexpressed. Combining these two independent approaches we propose a class of Amplification-Related Gain Of Sensitivity (ARGOS) genes. These genes are located in commonly amplified regions of the genome, have lower expression levels than expected by their copy-number status, and are toxic to cancer cells when overexpressed. We experimentally validated CDKN1A and RBM14 as high-confidence pan-cancer ARGOS genes in lung and breast cancer cell line models. We additionally suggest that RBM14s mechanism of toxicity involves altered DNA damage response and innate immune signaling processes following gene overexpression. Finally, we provide a comprehensive catalog of compensated, toxic, and ARGOS genes as a community resource.

cancer biology↗

bioRxiv2023

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets. HighlightsO_LIRibo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma C_LIO_LIHigh-resolution CRISPR tiling reveals uORF functions in medulloblastoma C_LIO_LIASNSD1-uORF controls downstream pathways with the prefoldin-like complex C_LIO_LIASNSD1-uORF is necessary for medulloblastoma cell survival C_LI

cancer biology↗

bioRxiv2022

Aberrant DNA repair is a vulnerability in histone H3.3-mutant brain tumors

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent oncogenic mechanism, which fosters genome instability and tumor cell growth in H3.3 mutant pHGG, thus opening new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme Polynucleotide Kinase 3-Phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

cancer biology↗

bioRxiv2022

Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic Signaling in Glioblastoma

Glioblastoma develops an immunosuppressive microenvironment that fosters tumorigenesis and resistance to current therapeutic strategies. Here we use multiplexed tissue imaging and single-cell RNA-sequencing to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioblastoma. We show that glioblastoma exhibit strong expression of CD39 and CD73 ectoenzymes, correlating with increased adenosine levels. Microglia are the predominant source of CD39, while CD73 is principally expressed by tumor cells, particularly in tumors with amplification of EGFR and astrocyte-like differentiation. Spatially-resolved single-cell analyses demonstrate strong spatial correlation between tumor CD73 and microglial CD39, and that their spatial proximity is associated with poor clinical outcomes. Together, this data reveals that tumor CD73 expression correlates with tumor genotype, lineage differentiation, and functional states, and that core purine regulatory enzymes expressed by neoplastic and tumor-associated myeloid cells interact to promote a distinctive adenosine-rich signaling niche and immunosuppressive microenvironment potentially amenable to therapeutic targeting.

cancer biology↗

bioRxiv2019

CloneRetriever: retrieval of rare clones from heterogeneous cell populations

BackgroundMany biological processes, such as cancer metastasis, organismal development, and development of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. However, identifying causal drivers of clonal fitness remains challenging. Population-level analysis has limited resolution to characterize clones prior to selection, while high-resolution single-cell methods are destructive and challenging to scale across large populations, limiting further functional analysis of relevant clones.\n\nResultsHere, we develop CloneRetriever, a methodology for tracking and retrieving rare clones throughout their response to selection. CloneRetriever utilizes a CRISPR sgRNA-barcode library that allows isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrated that CloneRetriever can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1,883 in a heterogeneous cell population.\n\nConclusionsCloneRetriever provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.

genomics↗

bioRxiv2018

Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. Through direct-to-patient outreach, we developed genomically faithful patient-derived models of RMC. Using whole genome sequencing, we identified intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele, confirming that SMARCB1 loss occurs in RMC. Biochemical and functional characterization of these RMC models revealed that RMC depends on the loss of SMARCB1 for survival and functionally resemble other cancers that harbor loss of SMARCB1, such as malignant rhabdoid tumors or atypical teratoid rhabdoid tumors. We performed RNAi and CRISPR-Cas9 loss of function genetic screens and a small-molecule screen and identified UBE2C as an essential gene in SMARCB1 deficient cancers. We found that the ubiquitin-proteasome pathway was essential for the survival of SMARCB1 deficient cancers in vitro and in vivo. Genetic or pharmacologic inhibition of this pathway leads to G2/M arrest due to constitutive accumulation of cyclin B1. These observations identify a synthetic lethal relationship that may serve as a therapeutic approach for patients with SMARCB1 deficient cancers.

cancer biology↗

bioRxiv2017

Genome-wide detection of structural variants and indels by local assembly

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at-scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABAs performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs, and substantially improved detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (< 1,000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types, and found that templated-sequence insertions occur in ~4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized SVs.

genomics↗