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Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations 1 , 2 . Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models 3 – 8 . To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database 9 , 10 , we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations. Large-scale loss-of-function screens and TP53 saturation mutagenesis screens in human cancer cell lines suggest that mutational processes combine with phenotypic selection to shape the landscape of somatic mutations at the TP53 locus.

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer. As the human body develops, countless cells change from one state into another. Two important cell states are known as epithelial and mesenchymal. Cells in the epithelial state tend to be tightly connected and form barriers, like skin cells. Mesenchymal state cells are loosely organized, move around more and make up connective tissues. Some cells alternate between these states via an epithelial-to-mesenchymal transition (EMT for short) and back again. Without this transition, certain organs would not develop and wounds would not heal. Yet, cancer cells also use this transition to spread to distant sites of the body. Such cancers are often the most aggressive, and therefore the most deadly. The epithelial-to-mesenchymal transition is dynamically regulated in a reversible manner. For example, the genes for some proteins might only be active in the epithelial state and further reinforce this state by turning on other ‘epithelial genes’. Alternatively, there might be differences in the processing of mRNA molecules – the intermediate molecules between DNA and protein – that result in the production of different proteins in epithelial and mesenchymal cells. Li, Choi et al. wanted to know which of the thousands of human genes can endow epithelial state cells with mesenchymal characteristics. A better understanding of the switch could help to prevent cancers undergoing an epithelial-to-mesenchymal transition. From a large-scale experiment in human breast cancer cells, Li, Choi et al. found that a group of proteins that bind and modify mRNA molecules are important for the epithelial-to-mesenchymal transition. Two proteins in particular promoted the transition, most likely by binding to the mRNA of a third protein called FLNB and removing a small piece of it. FLNB normally works to prevent the epithelial-to-mesenchymal transition, but the smaller protein encoded by the shorter mRNA promoted the transition by turning on ‘mesenchymal genes’. This switching between different FLNB proteins happens in some of the more aggressive breast cancers, which also contain mesenchymal cells. Finding out which FLNB protein is made in a given cancer may provide an indication of its aggressiveness. Also, looking for drugs that can target the mRNA-binding proteins or FLNB may one day lead to new treatments for some of the most aggressive breast cancers.

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D. In this work, the authors report a sophisticated combination of genetic, biophysical, and biochemical analyses to identifies the cycling conformational states of PPM1D. The findings reveal how an allosteric inhibitor locks the protein into a conformationally inactive state, and explain the distribution of PPM1D activating mutations in cancer.

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure. To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118-310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118-310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118-310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118-310 treated mice were unable to seed the growth of secondary tumors after transplant. These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single–amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings. The functional impact of somatic mutations in cancer genes is determined by pooled Perturb-seq.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.

Chromatin remodeling complexes, such as the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, play key roles in regulating gene expression by modulating nucleosome positioning. The core subunit SMARCB1 is essential for these functions, as it anchors the complex to the nucleosome acidic patch, enabling effective chromatin remodeling. While biallelic inactivation of SMARCB1 is a hallmark of several aggressive pediatric malignancies, the functional implication of missense mutations is not fully understood. Current diagnostic approaches focus on detecting the presence or absence of SMARCB1 by immunohistochemistry often without consideration of mutation status. Here, we present a comprehensive deep mutational scanning of SMARCB1 , encompassing 8418 alterations, to assess their functional impact. We show that RPT2 missense mutations disrupt SMARCB1 antiproliferation function by destabilizing the SWI/SNF complex and impairing chromatin remodeling and transcriptional regulation comparable to nonsense mutations. These functional defects occur despite maintaining detectable protein expression thereby challenging current diagnostic reliance on IHC. These findings provide deeper understanding of the role of SMARCB1 in chromatin remodeling and cancer biology, highlighting limitations of mutation classification approaches.

Protein mutational landscapes are sculpted by the impacts of the resulting amino acid substitutions on the protein's stability and folding or aggregation kinetics. These properties can, in turn, be modulated by the composition and activities of the cellular proteostasis network. Heat shock factor 1 (HSF1) is the master regulator of the cytosolic and nuclear proteostasis networks, dynamically tuning the expression of cytosolic and nuclear chaperones and quality control factors to meet demand. Chronic increases in HSF1 levels and activity are prominent hallmarks of cancer cells. One plausible explanation for this observation is that the consequent upregulation of proteostasis factors could biophysically facilitate the acquisition of oncogenic mutations. Here, we experimentally evaluate the impacts of chronic HSF1 activation on the mutational landscape accessible to the quintessential oncoprotein p53. Specifically, we apply quantitative deep mutational scanning of p53 to assess how HSF1 activation shapes the mutational pathways by which p53 can escape cytotoxic pressure conferred by the small molecule nutlin-3, which is a potent antagonist of the p53 negative regulator MDM2. We find that activation of HSF1 broadly increases the fitness of dominant-negative substitutions within p53. This effect of HSF1 activation was particularly notable for non-conservative, biophysically unfavorable amino acid substitutions within buried regions of the p53 DNA-binding domain. These results indicate that chronic HSF1 activation profoundly shapes the oncogenic mutational landscape, preferentially supporting the acquisition of cancer-associated substitutions that are biophysically destabilizing. Along with providing the first experimental and quantitative insights into how HSF1 influences oncoprotein mutational spectra, these findings also implicate HSF1 inhibition as a strategy to reduce the accessibility of mutations that drive chemotherapeutic resistance and metastasis.