Oscar Fernandez-Capetillo

Phosphatidylinositol 3-kinase–related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

Intercellular propagation of pathological TDP-43 drives the progression of ALS and FTD, yet the mechanisms enabling transmission remain elusive. Here, we demonstrate that Activity-regulated cytoskeleton associated protein (ARC/Arg3.1) is pathologically subverted to act as a retroviral-like capsid vehicle for TDP-43 spreading. In a Drosophila model, we reveal that massive Arc1 upregulation drives glia-to-neuron TDP-43 seeding, while its genetic ablation halts pathological transfer, improves motor function, and extends survival. In parallel human cellular models, stress-induced ARC colocalizes with TDP-43 to orchestrate its intercellular transmission. Guided by ARC’s capsid architecture, we reproposed Lenacapavir, an FDA-approved HIV-1 capsid modulator, as a stable binder of ARC/Arg3.1 capsid interfaces. Lenacapavir treatment effectively blocks TDP-43 propagation in vitro and rescues disease phenotypes in vivo. Our findings establish ARC as a conserved vehicle for pathological protein transmission and deliver an immediately translatable pharmacological strategy to arrest disease progression in ALS and FTD.

Summary: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of poor prognosis, for which age is the strongest risk factor. Despite significant progress in the discovery of ALS-associated mutations, no model explains how such a diversity of mutations converges in a common pathology. In addition, most ALS cases are sporadic and lack known genetic drivers. We recently reported that arginine-rich peptides arising from the C9ORF72 mutation trigger a widespread accumulation of orphan ribosomal proteins (oRP). Here, we show that oRP accumulation is also observed upon expression of other RNA-related ALS mutations, such as hnRNPA2 ^D290V and TDP-43 ^A315T , as well as upon exposure to the ALS-related neurotoxin β-N-methylamino-L-alanine (BMAA). Furthermore, the transcriptional signature of patients with sporadic ALS resembles that of Diamond-Blackfan anemia (DBA), a known ribosomopathy. Supporting the usefulness of our in vitro data, a transcriptional signature defined from these models provides diagnostic and prognostic value in ALS patients. We propose that the accumulation of oRPs due to dysfunctional ribosome biogenesis is a molecular hallmark of ALS that can contribute to the progressive loss of motor neurons in the disease.

Summary: C9ORF72 intronic repeat expansions are the most frequent mutation found in Amyotrophic Lateral Sclerosis (ALS), producing toxic arginine-rich dipeptides (DPR) that disrupt RNA metabolism and trigger the accumulation of orphan ribosomal proteins (RP). Through a large phenotypic chemical screen, we identified “SALSa”, a novel compound that mitigates DPR toxicity. Mechanistically, SALSa acts as a chemical inducer of ribophagy, a specialized form of autophagy that promotes RP clearance. Interestingly, this effect is unrelated to mTOR inhibition, the main regulator of autophagy. In contrast, this is due to an effect of the drug in ribosome biogenesis, which triggers a protective response to clear defective ribosomes. Accordingly, SALSa accumulates in nucleoli and perturbs the final steps of rRNA maturation. SALSa reduces DPR toxicity in differentiated neurons and significantly extends lifespan in a Drosophila melanogaster model of C9ORF72 ALS. These findings suggest that stimulating ribophagy could be beneficial for pathologies associated to dysfunctional ribosome biogenesis, including C9ORF72 ALS.

Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca ²⁺ and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca ²⁺ influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca ²⁺ ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

SUMMARY: SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53-deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, confirming that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibitors correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.