Biology subjects

Venkitaraman, A. R.

Publications and source records attributed to Venkitaraman, A. R..

12 recordsLinked to original sources

bioRxiv2024

KRAS G12 mutant alleles differentially control glutamine metabolism via FOXO1

Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming is unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we demonstrate distinctive features of each allele. We also demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy. In briefBer et al. reveal common and distinct features of oncogenic KRAS mutant isogenic cell lines. Mutant lines upregulate different nitrogen-recycling metabolic pathways and show sensitivity to combinatorial drugging of glutamine metabolism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/618135v1_ufig1.gif" ALT="Figure 1"> View larger version (101K): org.highwire.dtl.DTLVardef@1459558org.highwire.dtl.DTLVardef@14fcd43org.highwire.dtl.DTLVardef@2d22ccorg.highwire.dtl.DTLVardef@147b068_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDifferent KRAS mutations trigger distinct metabolic phenotypes in colorectal cell lines C_LIO_LIGlutamine metabolism and nitrogen recycling are upregulated in KRAS mutant cells C_LIO_LIFOXO1 is a key regulator of KRAS-induced rewiring of glutamine and nitrogen metabolism C_LIO_LIInhibition of glutamine synthetase and glutaminase selectively kills mutant KRAS lines C_LI

cancer biology↗

bioRxiv2024

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2is FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

molecular biology↗

bioRxiv2023

Genome-wide analysis of promoter contacts identifies novel regulators of late-stage adipogenesis

Adipogenesis is a multi-stage process essential for healthy fat storage and metabolic regulation. While early regulatory mechanisms are well characterized, the control of late-stage adipocyte differentiation remains poorly understood. Integrating CAGE-seq, promoter capture Hi-C, and a high-throughput siRNA screen of druggable genes, we report here that chromatin architecture rewiring promotes gene regulation changes essential for terminal adipogenesis. We identified nine clusters of dynamic promoter-anchored chromosomal interactions, many involving distal enhancers. Functional screening of genes engaged in these interactions revealed 19 novel regulators of late adipogenesis, including proteins with peptidase and ubiquitin ligase activity. Human genetic variant-to-gene mapping, coupled with cross-species chromatin interaction and synteny analyses, highlighted new gene-trait associations relevant to lipid traits (FXYD5, LAP3, SGPP1) and type 2 diabetes (FBXO17, FN3KRP, ZFAND6, TTC3). Our findings define the 3D gene regulatory landscape of late adipogenesis. The molecular links uncovered here provide mechanistic insight into metabolic disease risk and potential interventions.

genetics↗

bioRxiv2023

Selective inhibitors of the Aurora A-TPX2 protein-protein interaction exhibit in vivo efficacy as targeted anti-mitotic agents

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. Our biophysically, structurally and phenotypically validated lead compound, CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells, inhibition of Aurora A-TPX2 PPI synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

biochemistry↗

bioRxiv2023

Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia

The dependency of cancer cells on glucose can be targeted with high-fat low- carbohydrate ketogenic diet (KD). However, hepatic ketogenesis is suppressed in IL-6 producing cancers, which prevents the utilization of this nutrient source as energy for the organism. In two IL-6 associated murine models of cancer cachexia we describe delayed tumor growth but accelerated onset of cancer cachexia and shortened survival when mice are fed KD. Mechanistically, we find this uncoupling is a consequence of the biochemical interaction of two simultaneously occurring NADPH-dependent pathways. Within the tumor, increased production of lipid peroxidation products (LPPs) and, consequently, saturation of the glutathione (GSH) system leads to ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impairs the biosynthesis of corticosterone, the main regulator of metabolic stress, in the adrenal glands. Administration of dexamethasone, a potent glucocorticoid, improves food intake, normalizes glucose homeostasis and utilization of nutritional substrates, delays onset of cancer cachexia and extends survival of tumor-bearing mice fed KD, while preserving reduced tumor growth. Our study highlights that the outcome of systemic interventions cannot necessarily be extrapolated from the effect on the tumor alone, but that they have to be investigated for anti{-}cancer and host effects. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/528937v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@613bb6org.highwire.dtl.DTLVardef@19746a4org.highwire.dtl.DTLVardef@9ef652org.highwire.dtl.DTLVardef@3db934_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIKetogenic diet delays tumor growth but accelerates cancer cachexia and shortens survival C_LIO_LIIn the tumor, accumulation of lipid peroxidation products results in saturation of the GSH detoxifying pathway and ferroptotic death of cancer cells C_LIO_LIIn the host organism, systemic redox state imbalance causes NADPH depletion, GDF-15 elevations, and relative corticosterone deficiency C_LIO_LIDexamethasone coadministration with ketogenic diet delays onset of cancer cachexia by improving food intake, glucose homeostasis and utilization of nutritional substrates C_LI

cancer biology↗

bioRxiv2021

Inhibition of HIV-1 immune modulation by small molecules targeting viral Nef-host CD80 interface

HIV-1 causes diverse immunomodulatory responses in the host, including the down-regulation of co-stimulatory proteins CD80/86, mediated by HIV-1 protein Nef, blunting T-cell activation. Using a screening cascade of biochemical and cell-based assays, we identified potent small molecules representing three chemical scaffolds namely amino pyrimidine, phenoxy acetamide and bi-aryl heteroaryl carbamate which target the protein-protein interaction interface of CD80/86 and Nef with sub-micromolar potency. These molecules restore CD80/86 surface levels in HIV-1-Nef infected antigen presenting cells and T-cell activation. Nef-CD80 interface and small molecule binding sites were mapped by using computational docking and structural studies, followed by validation by mutational analysis. This analysis resulted in the identification of two key residues, K99 and R111, which were associated with down-modulation of CD80 surface levels by Nef and important for small molecule binding. Targeting these interacting residues disabled Nef-mediated down-modulation of CD80 surface levels, consequently restoring T-cell activation. Thus, we validate a new target, the Nef-CD80/86 protein-protein interaction interface, with a potential to develop new inhibitors to counteract the immunomodulatory consequences of HIV-1.

microbiology↗

bioRxiv2021

Early neutrophilia marked by aerobic glycolysis sustains host metabolism and delays cancer cachexia

An elevated neutrophil-to-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, we show using murine model systems of colorectal and pancreatic cancer that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, alike to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

cancer biology↗

bioRxiv2020

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer biology↗

bioRxiv2019

Structural features underlying the activity of benzimidazole derivatives that target phosphopeptide recognition by the tandem BRCT domain of the BRCA1 protein

The tandem BRCT (tBRCT) domains of BRCA1 engage pSer-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, a benzimidazole that represents a first selective small molecule inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domains, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features that underlie the biochemical and cellular activities of Bractoppin. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing single residue substitutions that alter K1702, a key residue mediating phosphopeptide recognition (K1702A), or alter hydrophobic residues (F1662R or L1701K) that adjoin the pSer-recognition site. However, mutation of BRCA1 tBRCT residue M1775R, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding. Collectively, these findings confirm a binding mode for Bractoppin that blocks the phosphopeptide-binding site via structural features distinct from the substrate phosphopeptide. We explored these structural features through structure-guided chemical elaboration of Bractoppin, synthesizing analogs bearing modifications on the left and right hand side (LHS/RHS) of Bractoppins benzimidazole ring. Characterization of these analogs in biochemical assay reveal structural features underlying potency. Analogs where the LHS phenyl is replaced by cyanomethyl (2091) and 4-methoxyphenoxypropyl (2113) conceptualized from structure-guided strategies like GIST and dimer interface analysis expose the role of phenyl and isopropyl as critical hydrophobic anchors. Two Bractoppin analogs, 2088 and 2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the biochemical and cellular activity of a novel benzimidazole inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target the protein-protein interactions of this previously undrugged family of protein domains.

cancer biology↗

bioRxiv2018

Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

cancer biology↗

bioRxiv2018

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Two decades of high-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution combined with the low-invasiveness and specificity characteristic of optical microscopy. However, fluorescence microscopy also enables scientists and clinicians to map and quantitate the physico-chemical properties (e.g., analyte concentration, enzymatic activities and protein-protein interactions) of biological samples. But the optimization of the biochemical resolving power in fluorescence microscopy is not as well-developed compared to its spatial resolution. Typical techniques rely on the observation of individual properties of fluorescence thus limiting the opportunities for sensing and multiplexing. Aiming to overcome existing limitations, we demonstrate a new imaging paradigm -- Hyper Dimensional Imaging Microscopy (HDIM) -- that enables the orthogonal properties of fluorescence emission (excited state lifetime, polarization and spectra) in biological samples to be quantified simultaneously and efficiently. Therefore, akin to how multi-dimensional separation in mass-spectroscopy and multi-dimensional spectra in NMR impacted proteomics and structural biology, we envisage that HDIM spectra of unprecedented dimensionality will impact the fields of systems biology and medical diagnostics by maximizing the biochemical resolving power of fluorescence microscopy.

biophysics↗

bioRxiv2018

Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage

DNA damage in proliferating mammalian cells causes death1, senescence2 or continued survival, via checkpoints that monitor damage and regulate cell cycle progression, DNA repair and fate determination3. Cell cycle checkpoints facilitate tumour suppression by preventing the generation of proliferating mutated cells4, particularly by blocking passage of DNA lesions into replication and mitosis5. While checkpoint non-fidelity permits cells to carry genomic aberrations into subsequent cell cycle phases6, its long-term consequences on lineages descendant from damaged cells remains poorly characterised. Devising methods for microscopy-based lineage tracing, we unexpectedly demonstrate that transient DNA damage to single living cells bearing a negligent checkpoint induces heterogenous cell-fate outcomes in their descendant generations removed from the initial insult. After transiently damaged cells undergo an initial arrest, pairs of descendant cells without obvious cell-cycle abnormalities either divide or die in a seemingly stochastic way. Progeny of transiently damaged cells may die generations afterwards, creating considerable variability of lineage fitness that promotes overall persistence in a mutagenic environment. Descendants of damaged cells frequently form micronuclei, activating immunogenic signalling. Our findings reveal previously unrecognized, heterogenous effects of cellular DNA damage that manifest long afterwards in descendant cells. We suggest that these heterogenous descendant cell-fate responses may function physiologically to ensure the elimination and immune clearance of damaged cell lineages, but pathologically, may enable the prolonged survival of cells bearing mutagenic damage.

cancer biology↗