Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element.

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis.

Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein.

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation.

The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses.

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

PTEN.

The importance of PTEN in cellular function is underscored by the frequency of its deregulation in cancer. PTEN tumor-suppressor activity depends largely on its lipid phosphatase activity, which opposes PI3K/AKT activation. As such, PTEN regulates many cellular processes, including proliferation, survival, energy metabolism, cellular architecture, and motility. More than a decade of research has expanded our knowledge about how PTEN is controlled at the transcriptional level as well as by numerous posttranscriptional modifications that regulate its enzymatic activity, protein stability, and cellular location. Although the role of PTEN in cancers has long been appreciated, it is also emerging as an important factor in other diseases, such as diabetes and autism spectrum disorders. Our understanding of PTEN function and regulation will hopefully translate into improved prognosis and treatment for patients suffering from these ailments.

A protein synthesis brake for hematopoietic stem cell maintenance.

Bmi1 is essential for normal and leukemic hematopoiesis, but its target genes in hematopoietic stem cells (HSCs) are incompletely understood. In this issue of Genes & Development, Burgess et al. (pp. 887-900) demonstrate a novel role of Bmi1 in regulating ribosome biogenesis and protein synthesis. Bmi1-deficient HSCs exhibited reduced transplantability, with the up-regulation of ARX and genes involved in ribosome biogenesis. However, depletion of ARX or its known targets, p16 Ink4a /p19 Arf , only partially rescues Bmi1 loss-induced hematopoietic defects. They further demonstrate an increased protein synthesis rate and resultant proteostatic stress in Bmi1 -/- HSCs, indicating a novel mechanism by which Bmi1 controls HSC maintenance.

Bmi1 suppresses protein synthesis and promotes proteostasis in hematopoietic stem cells.

The polycomb complex component Bmi1 promotes the maintenance of stem cells in multiple postnatal tissues, partly by negatively regulating the expression of p16Ink4a and p19Arf, tumor suppressors associated with cellular senescence. However, deficiency for p16Ink4a and p19Arf only partially rescues the function of Bmi1-deficient stem cells. We conditionally deleted Bmi1 from adult hematopoietic cells and found that this slowly depleted hematopoietic stem cells (HSCs). Rather than inducing senescence, Bmi1 deficiency increased HSC division. The increased cell division was caused partly by increased Aristaless-related homeobox (ARX) transcription factor expression, which also increased ribosomal RNA expression. However, ARX deficiency did not rescue HSC depletion. Bmi1 deficiency also increased protein synthesis, protein aggregation, and protein ubiquitylation independent of its effects on cell division and p16Ink4a, p19Arf, and ARX expression. Bmi1 thus promotes HSC quiescence by negatively regulating ARX expression and promotes proteostasis by suppressing protein synthesis. This highlights a new connection between the regulation of stem cell maintenance and proteostasis.

YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.

The Hippo-YAP signaling pathway plays a critical role in development, homeostasis, regeneration, and tumorigenesis by converging on YAP, a coactivator for the TEAD family DNA-binding transcription factors, to regulate downstream transcription programs. Given its pivotal role as the nuclear effector of the Hippo pathway, YAP is indispensable in multiple developmental and tissue contexts. Here we report that the essentiality of YAP in liver and lung development can be genetically bypassed by simultaneous inactivation of the TEAD corepressor VGLL4. This striking antagonistic epistasis suggests that the major physiological function of YAP is to antagonize VGLL4. We further show that the YAP-VGLL4 antagonism plays a widespread role in regulating Hippo pathway output beyond normal development, as inactivation of Vgll4 dramatically enhanced intrahepatic cholangiocarcinoma formation in Nf2-deficient livers and ameliorated CCl4-induced damage in normal livers. Interestingly, Vgll4 expression is temporally regulated in development and regeneration and, in certain contexts, provides a better indication of overall Hippo pathway output than YAP phosphorylation. Together, these findings highlight the central importance of VGLL4-mediated transcriptional repression in Hippo pathway regulation and inform potential strategies to modulate Hippo signaling in cancer and regenerative medicine.

Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

XPO7 is a tumor suppressor regulating p21CIP1-dependent senescence.

Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21CIP1 expression to control senescence and tumorigenesis.

p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.

p53 tetramerization: at the center of the dominant-negative effect of mutant p53.

The p53 tumor suppressor functions as a tetrameric transcription factor to regulate hundreds of genes-many in a tissue-specific manner. Missense mutations in cancers in the p53 DNA-binding and tetramerization domains cement the importance of these domains in tumor suppression. p53 mutants with a functional tetramerization domain form mixed tetramers, which in some cases have dominant-negative effects (DNE) that inactivate wild-type p53. DNA damage appears necessary but not sufficient for DNE, indicating that upstream signals impact DNE. Posttranslational modifications and protein-protein interactions alter p53 tetramerization affecting transcription, stability, and localization. These regulatory components limit the dominant-negative effects of mutant p53 on wild-type p53 activity. A deeper understanding of the molecular basis for DNE may drive development of drugs that release WT p53 and allow tumor suppression.

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/ß-catenin signaling.

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/ß-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/ß-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/ß-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/ß-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/ß-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

A molecular switch from tumor suppressor to oncogene in ER+ve breast cancer: Role of androgen receptor, JAK-STAT, and lineage plasticity.

Cancers develop resistance to inhibitors of oncogenes mainly due to target-centric mechanisms such as mutations and splicing. While inhibitors or antagonists force targets to unnatural conformation contributing to protein instability and resistance, activating tumor suppressors may maintain the protein in an agonistic conformation to elicit sustainable growth inhibition. Due to the lack of tumor suppressor agonists, this hypothesis and the mechanisms underlying resistance are not understood. In estrogen receptor (ER)-positive breast cancer (BC), androgen receptor (AR) is a druggable tumor suppressor offering a promising avenue for this investigation. Spatial genomics suggests that the molecular portrait of AR-expressing BC cells in tumor microenvironment corresponds to better overall patient survival, clinically confirming AR's role as a tumor suppressor. Ligand activation of AR in ER-positive BC xenografts reprograms cistromes, inhibits oncogenic pathways, and promotes cellular elasticity toward a more differentiated state. Sustained AR activation results in cistrome rearrangement toward transcription factor PROP paired-like homeobox 1, transformation of AR into oncogene, and activation of the Janus kinase/signal transducer (JAK/STAT) pathway, all culminating in lineage plasticity to an aggressive resistant subtype. While the molecular profile of AR agonist-sensitive tumors corresponds to better patient survival, the profile represented in the resistant phenotype corresponds to shorter survival. Inhibition of activated oncogenes in resistant tumors reduces growth and resensitizes them to AR agonists. These findings indicate that persistent activation of a context-dependent tumor suppressor may lead to resistance through lineage plasticity-driven tumor metamorphosis. Our work provides a framework to explore the above phenomenon across multiple cancer types and underscores the importance of factoring sensitization of tumor suppressor targets while developing agonist-like drugs.

PML isoforms: a molecular basis for PML pleiotropic functions.

PML is a stress-responsive protein that coordinates assembly of phase-separated nuclear aggregates, known as PML nuclear bodies (PML-NBs), where a large number of protein interactors and chromatin processes are finely regulated. Tampering with the PML gene produces a variety of phenotypic consequences that include promoting or interfering with tumor progression but the molecular underpinnings of PML pleiotropy are still elusive. In this review, we explore the contribution of PML splicing isoforms to PML-NB assorted activities. We describe recent literature indicating that distinct PML isoforms drive formation of specialized PML-NBs and perform unique functions and we suggest that future research efforts should delve into the contribution of isoform specificity to help elucidate the complex functionality of the PML gene.

Elucidating the chain of command: our current understanding of critical target genes for p53-mediated tumor suppression.

TP53 encodes a transcription factor that is centrally-involved in several pathways, including the control of metabolism, the stress response, DNA repair, cell cycle arrest, senescence, programmed cell death, and others. Since the discovery of TP53 as the most frequently-mutated tumor suppressor gene in cancer over four decades ago, the field has focused on uncovering target genes of this transcription factor that are essential for tumor suppression. This search has been fraught with red herrings, however. Dozens of p53 target genes were discovered that had logical roles in tumor suppression, but subsequent data showed that most were not tumor suppressive, and were dispensable for p53-mediated tumor suppression. In this review, we focus on p53 transcriptional targets in two categories: (1) canonical targets like CDKN1A (p21) and BBC3 (PUMA), which clearly play critical roles in p53-mediated cell cycle arrest/senescence and cell death, but which are not mutated in cancer, and for which knockout mice fail to develop spontaneous tumors; and (2) a smaller category of recently-described p53 target genes that are mutated in human cancer, and which appear to be critical for tumor suppression by p53. Interestingly, many of these genes encode proteins that control broad cellular pathways, like splicing and protein degradation, and several of them encode proteins that feed back to regulate p53. These include ZMAT3, GLS2, PADI4, ZBXW7, RFX7, and BTG2. The findings from these studies provide a more complex, but exciting, potential framework for understanding the role of p53 in tumor suppression.

Sequence-to-expression approach to identify etiological non-coding DNA variations in P53 and cMYC-driven diseases.

Disease risk prediction based on genomic sequence and transcriptional profile can improve disease screening and prevention. Despite identifying many disease-associated DNA variants, distinguishing deleterious non-coding DNA variations remains poor for most common diseases. In this study, we designed in vitro experiments to uncover the significance of occupancy and competitive binding between P53 and cMYC on common target genes. Analyzing publicly available ChIP-seq data for P53 and cMYC in embryonic stem cells showed that ~344-366 regions are co-occupied, and on average, two cis-overlapping motifs (CisOMs) per region were identified, suggesting that co-occupancy is evolutionarily conserved. Using U2OS and Raji cells untreated and treated with doxorubicin to increase P53 protein level while potentially reducing cMYC level, ChIP-seq analysis illustrated that around 16 to 922 genomic regions were co-occupied by P53 and cMYC, and substitutions of cMYC signals by P53 were detected post doxorubicin treatment. Around 187 expressed genes near co-occupied regions were altered at mRNA level according to RNA-seq data analysis. We utilized a computational motif-matching approach to illustrate that changes in predicted P53 binding affinity in CisOMs of co-occupied elements significantly correlate with alterations in reporter gene expression. We performed a similar analysis using SNPs mapped in CisOMs for P53 and cMYC from ChIP-seq data, and expression of target genes from GTEx portal. We found significant correlation between change in cMYC-motif binding affinity in CisOMs and altered expression. Our study brings us closer to developing a generally applicable approach to filter etiological non-coding variations associated with common diseases.