A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.

Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.

Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis.

Lysine lactylation is a post-translational modification that links cellular metabolism to protein function. Here, we find that AARS1 functions as a lactate sensor that mediates global lysine lacylation in tumor cells. AARS1 binds to lactate and catalyzes the formation of lactate-AMP, followed by transfer of lactate to the lysince acceptor residue. Proteomics studies reveal a large number of AARS1 targets, including p53 where lysine 120 and lysine 139 in the DNA binding domain are lactylated. Generation and utilization of p53 variants carrying constitutively lactylated lysine residues revealed that AARS1 lactylation of p53 hinders its liquid-liquid phase separation, DNA binding, and transcriptional activation. AARS1 expression and p53 lacylation correlate with poor prognosis among cancer patients carrying wild type p53. ß-alanine disrupts lactate binding to AARS1, reduces p53 lacylation, and mitigates tumorigenesis in animal models. We propose that AARS1 contributes to tumorigenesis by coupling tumor cell metabolism to proteome alteration.

Enhanced DNA repair through droplet formation and p53 oscillations.

Living organisms are constantly exposed to DNA damage, and optimal repair is therefore crucial. A characteristic hallmark of the response is the formation of sub-compartments around the site of damage, known as foci. Following multiple DNA breaks, the transcription factor p53 exhibits oscillations in its nuclear concentration, but how this dynamics can affect the repair remains unknown. Here, we formulate a theory for foci formation through droplet condensation and discover how oscillations in p53, with its specific periodicity and amplitude, optimize the repair process by preventing Ostwald ripening and distributing protein material in space and time. Based on the theory predictions, we reveal experimentally that the oscillatory dynamics of p53 does enhance the repair efficiency. These results connect the dynamical signaling of p53 with the microscopic repair process and create a new paradigm for the interplay of complex dynamics and phase transitions in biology.

p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR).

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma.

Mutational processes giving rise to lung adenocarcinomas (LADCs) in non-smokers remain elusive. We analyzed 138 LADC whole genomes, including 83 cases with minimal contribution of smoking-associated mutational signature. Genomic rearrangements were not correlated with smoking-associated mutations and frequently served as driver events of smoking-signature-low LADCs. Complex genomic rearrangements, including chromothripsis and chromoplexy, generated 74% of known fusion oncogenes, including EML4-ALK, CD74-ROS1, and KIF5B-RET. Unlike other collateral rearrangements, these fusion-oncogene-associated rearrangements were frequently copy-number-balanced, representing a genomic signature of early oncogenesis. Analysis of mutation timing revealed that fusions and point mutations of canonical oncogenes were often acquired in the early decades of life. During a long latency, cancer-related genes were disrupted or amplified by complex rearrangements. The genomic landscape was different between subgroups-EGFR-mutant LADCs had frequent whole-genome duplications with p53 mutations, whereas fusion-oncogene-driven LADCs had frequent SETD2 mutations. Our study highlights LADC oncogenesis driven by endogenous mutational processes.

Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation.

As the most frequent genetic alteration in cancer, more than half of human cancers have p53 mutations that cause transcriptional inactivation. However, how p53 modulates the immune landscape to create a niche for immune escape remains elusive. We found that cancer stem cells (CSCs) established an interleukin-34 (IL-34)-orchestrated niche to promote tumorigenesis in p53-inactivated liver cancer. Mechanistically, we discovered that Il34 is a gene transcriptionally repressed by p53, and p53 loss resulted in IL-34 secretion by CSCs. IL-34 induced CD36-mediated elevations in fatty acid oxidative metabolism to drive M2-like polarization of foam-like tumor-associated macrophages (TAMs). These IL-34-orchestrated TAMs suppressed CD8+ T cell-mediated antitumor immunity to promote immune escape. Blockade of the IL-34-CD36 axis elicited antitumor immunity and synergized with anti-PD-1 immunotherapy, leading to a complete response. Our findings reveal the underlying mechanism of p53 modulation of the tumor immune microenvironment and provide a potential target for immunotherapy of cancer with p53 inactivation.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

Alternative polyadenylation determines the functional landscape of inverted Alu repeats.

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches.

Inactivation of the transcription factor p53, through either direct mutation or aberrations in one of its many regulatory pathways, is a hallmark of virtually every tumor. In recent years, screening for p53 activators and a better understanding of the molecular mechanisms of oncogenic perturbations of p53 function have opened up a host of novel avenues for therapeutic intervention in cancer: from the structure-guided design of chemical chaperones to restore the function of conformationally unstable p53 cancer mutants, to the development of potent antagonists of the negative regulators MDM2 and MDMX and other modulators of the p53 pathway for the treatment of cancers with wild-type p53. Some of these compounds have now moved from proof-of-concept studies into clinical trials, with prospects for further, personalized anticancer medicines. We trace the structural evolution of the p53 pathway, from germ-line surveillance in simple multicellular organisms to its pluripotential role in humans.

p53 engages the cGAS/STING cytosolic DNA sensing pathway for tumor suppression.

Tumor suppression by TP53 involves cell-autonomous and non-cell-autonomous mechanisms. TP53 can suppress tumor growth by modulating immune system functions; however, the mechanistic basis for this activity is not well understood. We report that p53 promotes the degradation of the DNA exonuclease TREX1, resulting in cytosolic dsDNA accumulation. We demonstrate that p53 requires the ubiquitin ligase TRIM24 to induce TREX1 degradation. The cytosolic DNA accumulation resulting from TREX1 degradation activates the cytosolic DNA-sensing cGAS/STING pathway, resulting in induction of type I interferons. TREX1 overexpression sufficed to block p53 activation of the cGAS/STING pathway. p53-mediated induction of type I interferon (IFNB1) is suppressed by cGAS/STING knockout, and p53's tumor suppressor activities are compromised by the loss of signaling through the cGAS/STING pathway. Thus, our study reveals that p53 utilizes the cGAS/STING innate immune system pathway for both cell-intrinsic and cell-extrinsic tumor suppressor activities.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner.

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest.

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Thymine DNA glycosylase regulates cell-cycle-driven p53 transcriptional control in pluripotent cells.

Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis.

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

SPARCLE, a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage.

p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a ∼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.