Translation elongation is highly sensitive to amino acid availability, with deprivation causing ribosome pausing at cognate codons, suppression of the mammalian target of rapamycin (mTORC1) signaling pathway, and GCN2-mediated phosphorylation of eIF2α. However, cell-type heterogeneity in these responses remains unclear. Integrating ribosome profiling datasets across over ten human cell lines and multiple starvation conditions, we uncover translational resistance in breast cancer cells specifically under leucine and glutamine deprivation. Unlike non-breast cancer cells, breast cancer cells maintain ribosome occupancy on mRNAs with a 5' terminal oligopyrimidine tract (5'TOP mRNAs), indicating sustained mTORC1 activity, and exhibit attenuated codon-specific pausing. GCN2-eIF2α pathway activation varies among breast cancer lines. Downregulation of the cystine/glutamate transporter SLC7A11 correlates with this resistance, and its overexpression restores sensitivity by reducing S6K phosphorylation while enhancing eIF2α phosphorylation. Our findings reveal that breast cancer cells adaptively reshape translation regulation to withstand amino acid starvation, highlighting a potential metabolic vulnerability.

The eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor in eukaryotes, functioning as a central hub that integrates signals from cellular stress, metabolism, and developmental pathways to regulate mRNA translation. Recent advances have uncovered subunit-specific roles of eIF3 that extend beyond canonical cap-dependent translation to include specialized mechanisms such as selective mRNA recruitment, noncanonical cap recognition, and translation elongation. This review summarizes the current mechanistic understanding of the contribution of aberrant eIF3 activity to diverse disease processes, including oncogenesis, neurodevelopmental and neurodegenerative disorders, muscle pathology, and infectious disease. We evaluate therapeutic strategies aimed at modulating eIF3 function, including subunit-selective small molecules, RNA-based therapeutics, and CRISPR-based interventions. We discuss the therapeutic promise of both inhibitory approaches-targeting oncogenic or pathogen-hijacked eIF3-and restorative strategies to correct genetic loss-of-function in neurological disease. Finally, we outline key challenges and opportunities for clinical translation, including tissue-specific delivery, subunit selectivity, and the identification of predictive biomarkers. eIF3 emerges as a versatile and druggable node in translational control with broad relevance across human disease.

SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.

Co-translational folding of nascent polypeptides is essential for protein function and cellular homeostasis. Ribosome-associated chaperones assist in this process, but coordination between their recruitment and translation initiation remains poorly understood. We report here that specific binding sites for eukaryotic translation initiation factor eIF3 within the 5 ' UTRs of mRNAs promote its retention on 80S ribosomes during the synthesis of select endoplasmic reticulum (ER) membrane proteins. Disruption of these eIF3 binding sites leads to misfolding and sequestration of newly synthesized membrane proteins into ER whorls. Sequestration into ER whorls is exacerbated by HSP70 inhibition but can be rescued by overexpressing HSPA1 and HSPA8. Cross-linking assays reveal that 5 ' UTR binding sites stabilize eIF3-80S interactions during early elongation, facilitating recruitment of HSPA1 and HSPA8 to ribosomes. These findings indicate that genetic instructions within 5 ' UTRs direct eIF3-mediated chaperone recruitment, ensuring proper co-translational folding of ER membrane proteins.

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR-Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.

Purpose Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration. Methods We developed and applied capture-based target enrichment from patient RNA samples combined with Oxford Nanopore long-read sequencing for the analysis of 123 hereditary cancer transcripts (capture and ultradeep long-read RNA sequencing (CAPLRseq)). Results Validating CAPLRseq, we confirmed 17 cases of hereditary non-polyposis colorectal cancer/Lynch syndrome based on the demonstration of splicing defects and loss of allele expression of mismatch repair genes MLH1 , PMS2 , MSH2 and MSH6 . Using CAPLRseq, we reclassified two variants of uncertain significance in MSH6 and PMS2 as either likely pathogenic or benign. Conclusion Our data show that CAPLRseq is an automatable and adaptable workflow for effective transcriptome-based identification of disease variants in a clinical diagnostic setting.

∼30% of clear cell renal cell carcinoma (ccRCC) patients present with metastatic disease at the time of diagnosis, causing a dire 5-year survival rate of 13%. Although anti-PD-1 immunotherapy has improved survival, a strong need remains for new therapeutic options. Using integrated network analysis, we identified the mitotic regulator NDC80 as a predictor of ccRCC progression. Overexpression of NDC80 fosters the malignant phenotype by promoting cell cycle progression through S phase as well as boosting glycolysis and mitochondrial respiration. Despite high levels of immune infiltration, particularly derived from tumor resident CD8+T cells with an exhausted phenotype, NDC80 defines a class of ccRCCs that poorly respond to immune checkpoint blockade. Instead, bioinformatics identified NDC80-high ccRCCs as sensitive to inhibitors of mitotic kinases, PLK1 and AURK, therapeutic approaches we validated in cell lines and mouse xenograft studies. Thus, NDC80 status pinpoints mitotic kinase inhibitors as promising therapeutic options in difficult-to-treat ccRCCs.

eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multiomic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5'-UTR of RSP15A mRNA negated them. eIF3k and eIF3l are selectively downregulated in response to endoplasmic reticulum and oxidative stress. Supported by mathematical modeling, our data uncover eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content, possibly to secure spare translational capacity that can be mobilized during stress.

eIF3 is a 13-subunit protein complex with essential functions in mRNA translation, including translation initiation, elongation, and termination. eIF3 appears to fulfill these multiple functions through distinct modules, but the interaction of these modules with partner proteins and their mRNA selectivity remains purely defined. Whereas eIF3 subunits are frequently overexpressed in cancer, their specific pro-oncogenic functions are unknown. Using multi-omics profiling upon acute depletion, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and mRNA-selective translation, they were each essential for cell proliferation and tumor growth in mice. Whereas eIF3a globally affects the translation of all mRNAs, eIF3e and eIF3f form a module that is specifically required for early translation elongation of mRNAs encoding proteins with membrane-associated functions. In contrast, depletion of eIF3k promoted cell proliferation, tumor growth, and global translation. This was pinpointed to a suppressive effect of eIF3k on the synthesis of ribosomal proteins, in particular RPS15A. Our studies define eIF3 as a multi-functional complex imposing positive as well as negative regulation on mRNA-selective translation that are differentially required for cell proliferation and tumor growth. We propose that targeting of specific eIF3 subunits – for example by inducing their selective degradation - may be a powerful therapeutic approach in cancer.

In 2019, non-parametric linkage analyses and genome sequencing revealed that biallelic AAGGG expansions in the replication factor C subunit 1 (RFC1) gene are a frequent cause of late-onset ataxia [1]. Subsequent studies described the phenotypic spectrum of patients with pathological RFC1 expansion: they mainly presented in their fifth decade of life with a triad of cerebellar dysfunction (i.e., gait ataxia, dysarthria, ocular motor disorders), sensory neuropathy with concomitant sensory ataxia, and vestibular areflexia bilaterally, denoted by the acronym CANVAS [1]. Quite recently, a multicentre observational study has shown that RFC1 expansion comprises a multisystemic disease with a chronic dry cough, dysautonomia, and bradykinesia as additional clinical features of variable degree [2]. It is still an outstanding issue, whether biallelic AAGGG expansion in RFC1 are not associated with an even broader phenotypic spectrum of neurodegenerative diseases. A 64-year-old male presented with a 1-year history of progressive and painless weakness of both hands. Neurological examination revealed generalized polytopic muscle fasciculations in 4/4 levels, muscle paresis for finger adduction/ abduction (r: MRC 4–5, l: MRC 4), finger extension (r: MRC 4–5, l: MRC 4), thumb opposition (r: MRC 4–5, l: MRC 4), wrist extension/flexion (r: MRC 4–5, l: MRC 4) and hip flexion (r/l: MRC 4–5). A split hand sign was conspicuous on both sides. Muscle reflexes were brisk on the left upper limb with decreased ankle jerks bilaterally. There were neither relevant sensory/proprioceptive deficits nor clinical signs of ataxia/vestibulopathy. Clinical suspicion of a degenerative motor neuron disease was confirmed by electromyography, muscle ultrasound and transcranial magnetic stimulation (for details see Table 1). Sural and superficial peroneal nerve potentials and sensory evoked potentials of the tibial nerves (P40) were absent bilaterally. Finally, amyotrophic lateral sclerosis (ALS) was diagnosed according to the current diagnostic criteria (see Table 1). Additionally, regarding sensory nerve conduction studies and evoked potentials subclinical sensory neuropathy/neuronopathy was diagnosed. Acquired conditions for sensory neuropathies/neuronopathies were excluded (see Table 1). The patient did not consent to a recommended additional CSF analysis. Genetic analysis by CRISPR/Cas9 target enrichment and Oxford Nanopore long-read sequencing [3], revealed biallelic AAGGG repeat expansions (~ 400) of the RFC1 locus. Negative results of all genetic testing are listed in Table 1. Due to the detected biallelic RFC1 repeat expansions we post-hoc performed vestibular testing by inner ear calorics and video-assisted head-impulse-test, which revealed isolated bilateral presbyvestibulopathy in the low-frequency range (see Table 1). This case with a diagnosis of ALS, additional subclinical sensory neuro(no)pathy and bilateral presbyvestibulopathy in the low-frequency range in association with a biallelic RFC1 expansion raises the following noteworthy future question: is ALS/motor neuron disease within the phenotypic spectrum of biallelic RFC1 repeat expansions? Florian Schoeberl and Angela Abicht have contributed equally to this work.

PURPOSE:Approximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms. METHODS:We applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years. RESULTS:We identified a complex 3.9 Mb rearrangement involving 14 fragments from chromosome 5q22.1q22.3 of which three were lost, 1 reinserted into chromosome 5 and 10 inserted into chromosome 10q21.3 in a seemingly random order and orientation thus fulfilling the major criteria of chromothripsis. The rearrangement separates APC promoter 1B from the coding ORF (open reading frame) thus leading to allele-specific downregulation of APC mRNA. The rearrangement also involves three additional genes implicated in the APC-Axin-GSK3B-β-catenin signalling pathway. CONCLUSIONS:Based on comprehensive genomic analysis, we propose that constitutional chromothripsis dampening APC expression, possibly modified by additional APC-Axin-GSK3B-β-catenin pathway disruptions, underlies the patient's clinical phenotype. The combinatorial approach we deployed provides a powerful tool set for deciphering unsolved familial polyposis and potentially other tumour syndromes and monogenic diseases.

Exposure to heat stress triggers a well-defined acute response marked by HSF1-dependent transcriptional upregulation of heat shock proteins. Cells allowed to recover acquire thermotolerance, but this adaptation is poorly understood. By quantitative proteomics, we discovered selective upregulation of HSP70-family chaperone HSPA1 and its co-factors, HSPH1 and DNAJB1, in MCF7 breast cancer cells acquiring thermotolerance. HSPA1 was found to have dual function during heat stress response: (i) During acute stress, it promotes the recruitment of the 26S proteasome to translating ribosomes, thus poising cells for rapid protein degradation and resumption of protein synthesis upon recovery; (ii) during thermotolerance, HSPA1 together with HSPH1 maintains ubiquitylated nascent/newly synthesized proteins in a soluble state required for their efficient proteasomal clearance. Consistently, deletion of HSPH1 impedes thermotolerance and esophageal tumor growth in mice, thus providing a potential explanation for the poor prognosis of digestive tract cancers with high HSPH1 and nominating HSPH1 as a cancer drug target. We propose dual roles of HSPA1 either alone or in complex with HSPH1 and DNAJB1 in promoting quality control of nascent/newly synthesized proteins and cellular thermotolerance.