Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability.

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.

Proteome-scale discovery of protein degradation and stabilization effectors.

Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3-5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.

Mutational processes of tobacco smoking and APOBEC activity generate protein-truncating mutations in cancer genomes.

Mutational signatures represent a genomic footprint of endogenous and exogenous mutational processes through tumor evolution. However, their functional impact on the proteome remains incompletely understood. We analyzed the protein-coding impact of single-base substitution (SBS) signatures in 12,341 cancer genomes from 18 cancer types. Stop-gain mutations (SGMs) (i.e., nonsense mutations) were strongly enriched in SBS signatures of tobacco smoking, APOBEC cytidine deaminases, and reactive oxygen species. These mutational processes alter specific trinucleotide contexts and thereby substitute serines and glutamic acids with stop codons. SGMs frequently affect cancer hallmark pathways and tumor suppressors such as TP53, FAT1, and APC. Tobacco-driven SGMs in lung cancer correlate with smoking history and highlight a preventable determinant of these harmful mutations. APOBEC-driven SGMs are enriched in YTCA motifs and associate with APOBEC3A expression. Our study exposes SGM expansion as a genetic mechanism by which endogenous and carcinogenic mutational processes directly contribute to protein loss of function, oncogenesis, and tumor heterogeneity.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages.

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

Developmental priming of cancer susceptibility.

DNA mutations are necessary drivers of cancer, yet only a small subset of mutated cells go on to cause the disease. To date, the mechanisms that determine which rare subset of cells transform and initiate tumorigenesis remain unclear. Here, we take advantage of a unique model of intrinsic developmental heterogeneity (Trim28+/D9) and demonstrate that stochastic early life epigenetic variation can trigger distinct cancer-susceptibility 'states' in adulthood. We show that these developmentally primed states are characterized by differential methylation patterns at typically silenced heterochromatin, and that these epigenetic signatures are detectable as early as 10 days of age. The differentially methylated loci are enriched for genes with known oncogenic potential. These same genes are frequently mutated in human cancers, and their dysregulation correlates with poor prognosis. These results provide proof-of-concept that intrinsic developmental heterogeneity can prime individual, life-long cancer risk.

A designer peptide against the EAG2-Kvß2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma.

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvß2, which are predominantly expressed by GBM cells at the tumor-brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvß2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvß2-dependent manner. Genetic knockdown of the EAG2-Kvß2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2-Kvß2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2-Kvß2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer.

How the genetic landscape governs a tumor's response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by KrasG12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies.

The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma.

Receptor-interacting serine/threonine protein kinase 4 (RIPK4) and its kinase substrate the transcription factor interferon regulatory factor 6 (IRF6) play critical roles in the development and maintenance of the epidermis. In addition, ourselves and others have previously shown that RIPK4 is a NOTCH target gene that suppresses the development of cutaneous and head and neck squamous cell carcinomas (HNSCCs). In this study, we used autochthonous mouse models, where the expression of Pik3caH1047R oncogene predisposes the skin and oral cavity to tumor development, and show that not only loss of Ripk4, but also loss of its kinase substrate Irf6, triggers rapid SCC development. In vivo rescue experiments using Ripk4 or a kinase-dead Ripk4 mutant showed that the tumor suppressive function of Ripk4 is dependent on its kinase activity. To elucidate critical mediators of this tumor suppressive pathway, we performed transcriptional profiling of Ripk4-deficient epidermal cells followed by multiplexed in vivo CRISPR screening to identify genes with tumor suppressive capabilities. We show that Elovl4 is a critical Notch-Ripk4-Irf6 downstream target gene, and that Elovl4 loss itself triggers SCC development. Importantly, overexpression of Elovl4 suppressed tumor growth of Ripk4-deficient keratinocytes. Altogether, our work identifies a potent Notch1-Ripk4-Irf6-Elovl4 tumor suppressor axis.

In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

Cross-talk between mutant p53 and p62/SQSTM1 augments cancer cell migration by promoting the degradation of cell adhesion proteins.

Missense mutations in the p53 tumor suppressor abound in human cancer. Common ("hotspot") mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junction­associated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.

CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities.

Progression through G1/S phase of the cell cycle is coordinated by cyclin-dependent kinase (CDK) activities. Here, we find that the requirement for different CDK activities and cyclins in driving cancer cell cycles is highly heterogeneous. The differential gene requirements associate with tumor origin and genetic alterations. We define multiple mechanisms for G1/S progression in RB-proficient models, which are CDK4/6 independent and elicit resistance to FDA-approved inhibitors. Conversely, RB-deficient models are intrinsically CDK4/6 independent, but exhibit differential requirements for cyclin E. These dependencies for CDK and cyclins associate with gene expression programs that denote intrinsically different cell-cycle states. Mining therapeutic sensitivities shows that there are reciprocal vulnerabilities associated with RB1 or CCND1 expression versus CCNE1 or CDKN2A. Together, these findings illustrate the complex nature of cancer cell cycles and the relevance for precision therapeutic intervention.

A CRISPR Path to Finding Vulnerabilities and Solving Drug Resistance: Targeting the Diverse Cancer Landscape and Its Ecosystem.

Cancer is the second leading cause of death globally, with therapeutic resistance being a major cause of treatment failure in the clinic. The dynamic signaling that occurs between tumor cells and the diverse cells of the surrounding tumor microenvironment actively promotes disease progression and therapeutic resistance. Improving the understanding of how tumors evolve following therapy and the molecular mechanisms underpinning de novo or acquired resistance is thus critical for the identification of new targets and for the subsequent development of more effective combination regimens. Simultaneously targeting multiple hallmark capabilities of cancer to circumvent adaptive or evasive resistance may lead to significantly improved treatment response in the clinic. Here, the latest applications of functional genomics tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing, to characterize the dynamic cancer resistance mechanisms, from improving the understanding of resistance to classical chemotherapeutics, to deciphering unique mechanisms that regulate tumor responses to new targeted agents and immunotherapies, are discussed. Potential avenues of future research in combating therapeutic resistance, the contribution of tumor-stroma signaling in this setting, and how advanced functional genomics tools can help streamline the identification of key molecular determinants of drug response are explored.

Pan-cancer analysis of non-coding transcripts reveals the prognostic onco-lncRNA HOXA10-AS in gliomas.

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer and powerful biomarkers; however, most remain uncharacterized. Here, we analyze 5,592 prognostic lncRNAs in 9,446 cancers of 30 types using machine learning. We identify 166 lncRNAs whose expression correlates with survival and improves the accuracy of common clinical variables, molecular features, and cancer subtypes. Prognostic lncRNAs are often characterized by switch-like expression patterns. In low-grade gliomas, HOXA10-AS activation is a robust marker of poor prognosis that complements IDH1/2 mutations, as validated in another retrospective cohort, and correlates with developmental pathways in tumor transcriptomes. Loss- and gain-of-function studies in patient-derived glioma cells, organoids, and xenograft models identify HOXA10-AS as a potent onco-lncRNA that regulates cell proliferation, contact inhibition, invasion, Hippo signaling, and mitotic and neuro-developmental pathways. Our study underscores the pan-cancer potential of the non-coding transcriptome for identifying biomarkers and regulators of cancer progression.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.

Prevalence of the Brazilian TP53 Founder c.1010G>A (p.Arg337His) in Lung Adenocarcinoma: Is Genotyping Warranted in All Brazilian Patients?

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of TP53, c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine TP53 c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan® assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic EGFR mutations were more frequent in TP53 c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if TP53 c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of EGFR and germline TP53 c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if TP53 c.1010G>A screening in all Brazilian LUAD patients is justified.

AJUBA: A regulator of epidermal homeostasis and cancer.

The epidermis, outermost layer of the skin, is constantly renewing itself through proliferative and differentiation processes. These processes are vital to maintain proper epidermal integrity during skin development and homeostasis and for preventing skin diseases and cancers. The biological mechanisms that permit this balancing act are vast, where individual pathway regulators are known, but the exact regulatory control and cross-talk between simultaneously turning one biological pathway on and an opposing one off remain elusive. This review explores the diverse roles the scaffolding protein AJUBA plays during epidermal homeostasis and cancer. Initially identified for its role in promoting meiotic progression in oocytes through Grb2 and MAP kinase activity, AJUBA also maintains cytoskeletal tension permitting epidermal tissue development and responds to retinoic acid committing cells to initiate development of surface epidermal layer. AJUBA regulates proliferation of skin stem cells through Hippo and Wnt signalling and encourages mitotic commitment through Aurora-A, Aurora-B and CDK1. In addition, AJUBA also induces epidermal differentiation to maintain appropriate epidermal thickness and barrier function by activating Notch signalling and stabilizing catenins and actin during cellular remodelling. AJUBA also plays an imperative context-dependent tumor-promoting and tumor-suppressive role within epithelial cancers. AJUBA's abundant roles within the epidermis signify its importance as a molecular switchboard, vetting multiple signalling pathways to control epidermal biology.