Biology subjects

Radhakrishnan, S. K.

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

10 recordsLinked to original sources

bioRxiv2026

Tumor-selective Mcl1 degradation by AUTAC uncouples antitumor efficacy from cardiotoxicity

Mcl1 is a major driver of therapeutic resistance across hematologic malignancies, but direct Mcl1 inhibition has been limited by on-target cardiotoxicity. Here, building on our development of an Mcl1-targeting autophagy-targeting chimera (AUTAC), we show that AUTAC-mediated degradation creates a tumor-selective therapeutic window that spares the heart. AUTAC induced robust cytotoxicity and Mcl1 degradation in multiple myeloma models, while showing minimal toxicity in cardiac cell lines, primary cardiomyocytes, and murine heart tissue. In vivo, AUTAC reduced tumor Mcl1 without measurably affecting cardiac Mcl1. Mechanistically, this selectivity was associated with lower expression of the p62/SQSTM1, TRAF6, and UBC13 machinery required for AUTAC activity in cardiac cells, together with lower intracellular AUTAC accumulation relative to tumor cells. AUTAC also enhanced the antitumor activity of carfilzomib and venetoclax, including in resistant models, without worsening cardiotoxicity or promoting cardiac Mcl1 loss. Compared with classical Mcl1 inhibitors, AUTAC caused markedly less cardiomyocyte death, mitochondrial depolarization, and apoptotic signaling. These findings identify AUTAC-mediated Mcl1 degradation as a cardiac-sparing strategy to target an otherwise clinically constrained vulnerability and support tumor-selective lysosomal degradation as a path to safer Mcl1-directed therapy.

pharmacology and toxicology↗

bioRxiv2025

A metabolic hierarchy directs cell cycle transition and morphogenesis

To optimize survival, cells must align differentiation and proliferation with metabolic status. Yet, how metabolic cues fine-tune cell cycle programs and morphogenesis remain unclear. Here, we show that cytosolic redox dynamics critically govern cell cycle transitions in Caulobacter crescentus. By integrating evolutionary genetics with fluxomics, we uncover temporally orchestrated shifts in core metabolic pathways that remodel cytosolic redox across the cell cycle. Early stages channel carbon flux toward unsaturated fatty-acid synthesis and a reverse-TCA to drive cytosolic oxidation-coupled G1-S transition. The later stages depend on an enhanced forward-TCA cycle that promote cytosolic reduction-driven proliferation. Strikingly, perturbing fatty-acid synthesis or reverse/forward-TCA uncouples growth from proliferation. Intriguingly, a biphasic glucose uptake program dictates the cell cycle-stage-specific metabolism. These findings reveal an unprecedented metabolic-redox circuitry coordinating morphogenesis with cell cycle progression in a carefully choreographed menage a trois.

microbiology↗

bioRxiv2025

Cell cycle-dependent variation in endocytosis drives phenotypic diversity in M. tuberculosis

Cell-to-cell heterogeneity is a hallmark of biology, yet how host variability shapes intracellular pathogen phenotypes is unclear. Using single cell approaches and redox-sensitive Mycobacterium tuberculosis reporters, we reveal that interphase-driven shifts in endocytic capacity create distinct intracellular niches. Bacilli residing in high-endocytic G2 cells adopt more oxidized redox states, whereas those in low-endocytic G1 cells remain reduced, generating phenotypic diversity within a single infected population. Experimental manipulation of host cell cycle stage reprograms endocytic capacity and intrabacterial redox, establishing a causal link between host state and pathogen diversification. Notably, the finding that the cell cycle regulates endocytic capacity constitutes a fundamental cell-biological discovery with broad implications. This variability persists in post-differentiated macrophages, indicating that proliferative history imprints functional diversity onto innate immune cells. Together, these results identify interphase-regulated endocytosis as a host-intrinsic mechanism that shapes Mycobacterium tuberculosis phenotypes and suggest new host-directed avenues to influence infection trajectories and persistence.

cell biology↗

bioRxiv2025

Proteasome Inhibition Enhances Lysosome-mediated Targeted Protein Degradation

Proteasome inhibitor drugs are currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. These inhibitors cause accumulation of undegraded proteins, thus inducing proteotoxic stress and consequent cell death. However, cancer cells counteract this effect by activating an adaptive response through the transcription factor Nuclear factor erythroid 2-related factor 1 (NRF1, also known as NFE2L1). NRF1 induces transcriptional upregulation of proteasome and autophagy/lysosomal genes, thereby reducing proteotoxic stress and diminishing the effectiveness of proteasome inhibition. While suppressing this protective autophagy response is one potential strategy, here we investigated whether this heightened autophagy could instead be leveraged therapeutically. To this end, we designed an autophagy-targeting chimera (AUTAC) compound to selectively degrade the anti-apoptotic protein Mcl1 via the lysosome. Our results show that this lysosome-mediated targeted degradation is significantly amplified in the presence of proteasome inhibition, in a NRF1-dependent manner. Mechanistically, AUTAC-driven Mcl1 clearance requires K63-linked ubiquitination by UBC13 and TRAF6 and recognition by the cargo receptor p62/SQSTM1. The combination of the proteasome inhibitor carfilzomib and Mcl1 AUTAC synergistically promoted cell death in both in vitro models, including wild-type and proteasome inhibitor-resistant multiple myeloma and lung cancer cells, and in mouse tumor xenografts. Thus, our work offers a novel strategy for enhancing proteasome inhibitor efficacy by exploiting the adaptive autophagy response. More broadly, our study establishes a framework for amplifying lysosome-mediated targeted protein degradation, with potential applications in cancer therapeutics and beyond.

biochemistry↗

bioRxiv2025

A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp

Living cells should coordinate vital events such as DNA replication with the availability of nutrients. For example, when cells encounter starvation, to maintain genomic integrity they should harbour robust mechanisms to stop DNA replication. Mechanisms regulating the progression of DNA replication when bacterial cells encounter starvation remain largely unclear. Herein, we identify the role of IncA -a AAA+ ATPase homologous to the prokaryotic RarA and eukaryotic WRNIP1/Mgs1- in inhibiting the progression of chromosome replication in nutrient-starved stationary phase cells of Caulobacter crescentus. We show that the starvation-induced alarmone (p)ppGpp ensures the confinement of IncA production to the stationary phase cells. At the mechanistic level, IncA directly interacts with the {beta}-sliding clamp protein DnaN and disassembles DnaN from the replisome, thereby stalling the progression of DNA replication. Furthermore, we reveal the requirement of IncAs ATPase activity for disassembling DnaN. Remarkably, we demonstrate that the IncA homolog from E. coli is capable of inhibiting DNA replication in Caulobacter. We propose that IncA homologs serve a stress-dependent role in inhibiting DNA replication across diverse domains of life.

microbiology↗

bioRxiv2024

Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein. and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

cancer biology↗

bioRxiv2024

Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis

Non-small cell lung cancer (NSCLC) cells with oncogenic mutant p53 alleles (Onc-p53) exhibit significantly higher levels of proteasome activity, indicating that Onc-p53 induces proteotoxic stress which may be leveraged as a therapeutic vulnerability. Proteasome inhibitors (PIs), such as bortezomib (BTZ), can induce toxic levels of oxidative stress in cancer cells and thus we investigated whether PIs exhibit preferential cytotoxicity in Onc-p53 NSCLC cells. Indeed, BTZ and other PIs exhibited the IC50 6-7-fold lower in Onc-p53 cells vs. wild-type (WT) p53 cells. BTZ cytotoxic effects in Onc-p53 cells were nearly completely rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ-dependent cytotoxic effects in Onc-p53 cells. Importantly, we observed oxidative stress-dependent transcriptional induction of the pro-apoptotic NOXA with downstream cleaved caspase-3, consistent with apoptotic cell death in Onc-p53 but not in WT p53 cells treated with BTZ, and BTZ-generated oxidative stress was linked to nuclear translocation of NRF2 and transcriptional activation of ATF3, which in turn was required for NOXA induction. Validating BTZs translational potential in Onc-p53 NSCLC, BTZ and carboplatin or the BH3-mimetic navitoclax were synergistically cytotoxic in Onc-p53 but not WT p53 cells in vitro, and BTZ effectively limited growth of Onc-p53 NSCLC xenografts when combined with either carboplatin or navitoclax in vivo. Our data therefore support further investigation of the therapeutic utility of PIs combined with carboplatin or BH3-mimetics in Onc-p53 human NSCLC as novel therapeutic strategies. SignificanceNon-small cell lung cancer (NSCLC) is the leading cause of cancer death due, in part, to a lack of active therapies in advanced disease. We demonstrate that proteasome inhibitor/BH3-mimetic combination therapy is an active precision therapy in NSCLC cells and tumors expressing oncogenic mutant p53 alleles (Onc-p53).

cancer biology↗

bioRxiv2023

Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor erythroid derived 2-related factor 1 (Nrf1, also called NFE2L1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here we report that under similar circumstances, multiple components of the autophagy lysosomal pathway (ALP) are transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells (a model for NGLY1 disease) where Nrf1 is known to be non-functional. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

cell biology↗

bioRxiv2018

Proteotoxic stress-induced Nrf1 transcriptional program requires a functional TIP60 complex

In response to inhibition of the cellular proteasome, the transcription factor Nrf1 (also called NFE2L1) induces transcription of proteasome subunit genes resulting in the restoration of proteasome activity and thus enabling the cells to mitigate the proteotoxic stress. To identify novel regulators of Nrf1, we performed an RNA interference screen and discovered that the AAA+ ATPase RUVBL1 is necessary for its transcriptional activity. Given that RUVBL1 is part of different multi-subunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Our study provides a framework for manipulating the Nrf1-TIP60 axis to alter proteasome function in various human diseases including cancer.

molecular biology↗

bioRxiv2018

Sensory domain of the cell cycle kinase CckA in Caulobacter crescentus regulates the differential DNA binding activity of the master regulator CtrA

Sophisticated signaling mechanisms allow bacterial cells to cope with environmental and intracellular challenges. Activation of specific pathways facilitates the cells to overcome cellular damage and thereby warrant integrity. Here we demonstrate the pliability of the CckA-CtrA two component signaling system in the freshwater bacterium, Caulobacter crescentus. Our forward genetic screen to analyse suppressor mutations that can negate the chromosome segregation block induced by the topoisomerase IV inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in increased levels of phosphorylated CtrA (CtrA[~]P), the master cell cycle regulator. Surprisingly, this increase in CtrA[~]P levels did not translate into a genome-wide increase in the DNA occupancy of CtrA, but specifically enriched its affinity to the chromosomal origin of replication, Cori, and a very small sub-set of CtrA regulated promoters. We show that through this enhanced binding of CtrA to the Cori, cells are able to overcome the toxic defects rendered by stable NstA through a possible slow down in the chromosome cycle. Taken together, our work opens up an unexplored and intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis because of the highly conserved nature of CckA-CtrA pathway in alphaproteobacteria.

microbiology↗