Biology subjects

Topisirovic, I.

Publications and source records attributed to Topisirovic, I..

14 recordsLinked to original sources

bioRxiv2026

eIF4A inhibition disrupts resistance-associated translational and metabolic programs in BRAF-mutant melanoma

Acquired resistance to mitogen-activated protein kinase (MAPK) pathway inhibitors remains a major barrier to durable control of BRAF-mutant melanoma. Although resistance mechanisms are heterogeneous, they converge on adaptive programs that support survival, phenotypic plasticity, and metabolic fitness. We asked whether eukaryotic translation initiation factor 4A (eIF4A)-dependent mRNA translation represents a shared vulnerability of kinase inhibitor-resistant melanoma. Using matched BRAFV600E A375 and BRAF inhibitor-resistant A375R cells together with additional melanoma models, we integrated pharmacological and functional assays with polysome-associated RNA sequencing, quantitative proteomics, bioenergetic profiling, metabolomics, [U-13C5]glutamine tracing, and xenograft studies. Melanoma cells remained sensitive to multiple eIF4A inhibitors regardless of their responsiveness to BRAF inhibition. The eIF4A inhibitor CR-1-31-B rapidly reduced nascent protein synthesis when used alone in A375 cells and when added to the BRAF inhibitor PLX4032 in A375R cells; it also reduced BCL-2, CDK4, and cyclin D3 abundance, suppressed clonogenic growth, and induced apoptosis. Integrated analysis showed that acquired resistance involved broad RNA-abundance remodeling with superimposed changes in translational efficiency and buffering, affecting survival, extracellular-matrix and plasticity programs, and mitochondrial and metabolic functions. In resistant cells, CR-1-31-B induced early transcript-selective translational changes, accompanied at later time points by RNA-abundance and proteome remodeling. Publicly annotated 5' untranslated regions (5'UTRs) of CR-1-31-B-sensitive transcripts were enriched for purine-rich sequence architecture and local structural complexity. eIF4A inhibition preferentially attenuated the expression of proteins acquired during resistance and imposed a lower-output metabolic state in sensitive and resistant cells, reducing tricarboxylic-acid-cycle and pentose-phosphate-pathway metabolite pools and restricting intracellular glutamine-carbon transfer downstream of uptake. In A375 xenografts, CR-1-31-B delayed tumor growth, while its combination with PLX4720 produced deeper and more sustained tumor control and prolonged tumor endpoint-free survival compared with PLX4720 alone. These findings show that multiple resistance-associated programs spanning signaling, cell survival, and metabolism share a dependency on eIF4A-dependent translation and provide a preclinical rationale to test whether adding eIF4A inhibition can prolong responses to MAPK-targeted therapy in melanoma.

cancer biology↗

bioRxiv2026

Lactate-fueled hydride transfer metabolon drives breast cancer metastasis

Metabolic plasticity of cancer cells plays a major role in metastasis. Use of alternative carbon fuels (e.g., lactate) boosts metabolic plasticity, but the underlying mechanisms remain obscure. We show that lactate dehydrogenase B (LDHB) cooperates with the hydride ion transfer complex (HTC) comprised of pyruvate carboxylase (PC), malic enzyme (ME1) and malate dehydrogenase (MDH1) to form a metabolon that confers metabolic flexibility through lactate assimilation under physiological conditions. HTC/LDHB metabolon assembles in the aggressive breast cancer subtypes and reprograms nicotinamide adenine dinucleotide (NAD) metabolism to promote migration, invasion and escape from anoikis - thereby driving metastasis. Altogether, this work identifies a lactate-fueled metabolon that propels metastatic dissemination of breast cancer.

Cancer Biology↗

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2024

Targeting eIF4G1-dependent translation in melanoma

Expression of components of the translation initiation complex (eIF4F) is frequently elevated in cancer, resulting in enhanced synthesis of oncogenic proteins. We thus set out to limit eIF4F pro-oncogenic activity, a notable challenge given its essential role in normal tissues. CRISPR-Cas9-based functional screen using tiling sgRNAs identified the eIF4G1 MA3 domain, a subunit of the eIF4F complex, as a target for developing small molecule inhibitors. Combination of structure-guided in silico modeling and chemical library screening led to the identification of small molecule candidates M19 and its analog M19-6 that binds to the MA3 domain of eIF4G1 and disrupts eIF4F complex. M19-6 treatment reprograms the melanoma translatome, limiting synthesis of factors that promote cell proliferation and neoplastic growth, as well as reducing translation of mRNAs encoding ferroptosis suppressors. Whole genome CRISPR screen indentified ferroptosis activators to augment M19-6 activity, which was confirmed in cultured melanoma cells. M19-6 alleviates melanoma resistance to BRAF and MEK inhibitors, while eliciting anti-tumor and anti-metastatic effects in preclinical mouse models. Among several biomarkers found in M19-6 sensitive cell lines, THBS1 and TGF{beta}I expression were elevated in patients who are non-responders to PD-1 therapy as in patients with metastasis. Our studies identify M19-6 as a therapeutic candidate, offering a novel insights into targeting the eIF4F complex to overcome melanoma resistance to therapy and metastasis. SignificanceWe identify M19-6 as a small molecule that disrupts the eIF4F, translation initiation complex, by targeting the MA3 domain of eIF4G1, resulting in elimination of melanoma cells in culture, overcoming therapy resistance while inhibiting melanoma growth and metastasis in vivo. As M19-6 causes minimal toxicity to melanocytes, it offers a therapeutic modality for melanoma.

cancer biology↗

bioRxiv2024

Reduction in ETFDH expression optimizes cancer cell bioenergetics

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

cancer biology↗

bioRxiv2023

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR) which is a pivotal regulator of cellular metabolism, and epigenetic modifications remains poorly understood. We thus explored the impact of modulating mTOR signaling on histone methylation, a well-known epigenetic modification. Our results showed that mTORC1 activation caused by abrogation of TSC2 increased H3K27me3 but not H3K4me3 or H3K9me3. This appeared to be mediated via the induction of EZH2 protein synthesis, downstream of 4EBPs. Surprisingly, mTOR inhibition also induced H3K27me3 independently of TSC2. This coincided with reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the ability of mTOR inhibitors to induce H3K27me3 influences their anti-proliferative effects. HighlightsO_LIParadoxically, both mTOR activation and inhibition induce H3K27me3. C_LIO_LIThe effect of mTOR inhibitors on H3K27me3 are not secondary to cell cycle arrest. C_LIO_LIH3K27me3 triggered by mTOR suppression coincides with perturbations in EZH1/2 ratio. C_LIO_LIH3K27me3 impacts on the anti-proliferative effects of mTOR inhibitors. C_LI

molecular biology↗

bioRxiv2023

Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways is not yet understood. Herein, we report the cellular responses to acute peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered accumulation of its substrates, alterations in peroxisome size and numbers, and luminal protein import failure. Gene expression changes and lipidomic analysis revealed striking cell specific differences in the response to siLONP2. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway, and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight for our understanding of the tissue-specific responses seen in peroxisomal disorders.

cell biology↗

bioRxiv2021

Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

The precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3UTR. We previously reported the important diversity of 3UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

biochemistry↗

bioRxiv2021

Regulatory start-stop elements in 5' untranslated regions pervasively modulate translation

Sequence elements within the 5 untranslated region (UTR) of eukaryotic genes, e.g. upstream open reading frames (uORFs), control translation of eukaryotic genes. We describe an element consisting of a start codon immediately followed by a stop codon which is distinct from uORFs in the lack of an elongation step. Start-stops have been described for specific cases, but their widespread impact has been overlooked. Start-stop elements occur in the 5UTR of 1, 417 human genes and are more often occupied with a ribosome than canonical uORFs or control sequences. Start-stops efficiently halt ribosomes without evidence for accelerated RNA turnover, therefore acting as a barrier for the scanning of the small ribosomal subunit and repressing downstream translation. Our results suggest a model by which the ribosome undergoes repeated cycles of termination and partial ribosomal recycling, during which the large subunit detaches, but the 40S subunit with the Met-tRNAiMet remains associated with the mRNA to be rejoined by the 60S subunit. Start-stop elements occur in many transcription factors and signaling genes, and affect cellular fate via different routes. We investigate the start-stop element in several genes, i.e. MORF4L1, SLC39A1, and PSPC1, and in more detail in ATF4.

systems biology↗

bioRxiv2020

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress

Mammalian cells have to adapt to environmental challenges that range from mild to severe stress. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe stress conditions, cells induce a transient hibernation-like mechanism that anticipates recovery. We demonstrate that this Adaptive Pausing Response (APR) is a coordinated cellular response that limits ATP supply and consumption though mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery from severe stress. We further show that recovery from severe stress involves adaptive ISR (Integrated Stress Response) signaling that in turn permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress through the APR, a mechanism that preserves vital elements of cellular function under harsh environmental conditions.

molecular biology↗

bioRxiv2019

Translational control of breast cancer plasticity

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5 Untranslated Regions (5UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and \"fate-switching\" toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2 phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

cancer biology↗

bioRxiv2018

Estrogen receptor alpha controls gene expression via translational offsetting

Estrogen receptor alpha (ER) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ER on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ER coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ER largely manifest as \"translational offsetting\" of the transcriptome, whereby amounts of translated mRNA and protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome-association, are reduced following ER depletion lack features which limit translational efficiency including structured 5UTRs and miRNA target sites. In contrast, mRNAs induced upon ER depletion whose polysome-association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ER regulates levels of U34-modification enzymes, whereas altered expression of U34-modification enzymes disrupts ER dependent translational offsetting. Altogether, we unravel a hitherto unprecedented mechanism of ER-dependent orchestration of transcriptional and translational programs, and highlight that translational offsetting may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

systems biology↗

bioRxiv2017

Translational and HIF1α-dependent metabolic reprograming underpin oncometabolomeplasticity and synergy between oncogenic kinase inhibitors and biguanides.

There is heightened interest to devise therapies that target the oncometabolome. We show that kinase inhibitors (KIs) and biguanides synergistically target melanoma, leukemia, and breast, colon and renal cancer cells, but not non-transformed cells. Metabolic profiling confirmed opposing effects of KIs and biguanides on glycolysis, but this was insufficient to explain the observed synergy between the drugs. Rather, we define a critical role for the synthesis of non-essential amino acids (NEAA) aspartate, asparagine and serine as well as reductive glutamine metabolism, in determining the sensitivity of cancer cells to KI - biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine and serine synthesis by modulating translation of mRNAs encoding PC, ASNS, PHGDH and PSAT1. Ablation of 4E-BP1 and 2 results in a dramatic increase in serine, aspartate and asparagine levels and a substantial decrease in sensitivity of breast cancer and melanoma cells to KI - biguanide combinations. In turn, efficacy of KI - biguanide combinations is impeded by HIF1 and sustained reductive glutamine metabolism. These findings identify hitherto unappreciated translational reprograming of NEAA synthesis and HIF1-dependent stimulation of reductive glutamine metabolism as critical metabolic vulnerabilities of cancer that underpin synergy between KIs and biguanides.

cancer biology↗

bioRxiv2017

Genome-wide analysis of differential translation and differential translational buffering using anota2seq

mRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated results in various disorders. Optimal and universally applicable analytical methods to study transcriptome-wide changes in translational efficiency are therefore critical for understanding the complex role of translation regulation under physiological and pathological conditions. Techniques used to interrogate translatomes, including polysome- and ribosome-profiling, require adjustment for changes in total mRNA levels to capture bona fide alterations in translational efficiency. Herein, we present the anota2seq algorithm for such analysis using data from ribosome- or polysome-profiling quantified by DNA-microarrays or RNA sequencing, which outperforms current methods for identification of changes in translational efficiency. In contrast to available analytical methods, anota2seq also allows capture of an underappreciated mode for regulation of gene expression whereby translation acts as a buffering mechanism which maintains constant protein levels despite fluctuations in mRNA levels (\"translational buffering\"). Application of anota2seq shows that insulin affects gene expression at multiple levels, in a largely mTOR-dependent manner. Moreover, insulin induces levels of a subset of mRNAs independently of mTOR that undergo translational buffering upon mTOR inhibition. Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes and enables studies of translational buffering which represents an unexplored mechanism for regulating of gene expression.

bioinformatics↗