Tuft cells and fibroblasts promote thymus regeneration through ILC2-mediated type 2 immune response.

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response.

Computational optimization of antibody humanness and stability by systematic energy-based ranking.

Conventional methods for humanizing animal-derived antibodies involve grafting their complementarity-determining regions onto homologous human framework regions. However, this process can substantially lower antibody stability and antigen-binding affinity, and requires iterative mutational fine-tuning to recover the original antibody properties. Here we report a computational method for the systematic grafting of animal complementarity-determining regions onto thousands of human frameworks. The method, which we named CUMAb (for computational human antibody design; available at http://CUMAb.weizmann.ac.il ), starts from an experimental or model antibody structure and uses Rosetta atomistic simulations to select designs by energy and structural integrity. CUMAb-designed humanized versions of five antibodies exhibited similar affinities to those of the parental animal antibodies, with some designs showing marked improvement in stability. We also show that (1) non-homologous frameworks are often preferred to highest-homology frameworks, and (2) several CUMAb designs that differ by dozens of mutations and that use different human frameworks are functionally equivalent.

Clonal heterogeneity in ER+ breast cancer reveals the proteasome and PKC as potential therapeutic targets.

Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

Resistance of Lung Cancer to EGFR-Specific Kinase Inhibitors: Activation of Bypass Pathways and Endogenous Mutators.

Epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) have changed the landscape of lung cancer therapy. For patients who are treated with the new TKIs, the current median survival exceeds 3 years, substantially better than the average 20 month survival rate only a decade ago. Unfortunately, despite initial efficacy, nearly all treated patients evolve drug resistance due to the emergence of either new mutations or rewired signaling pathways that engage other receptor tyrosine kinases (RTKs), such as MET, HER3 and AXL. Apparently, the emergence of mutations is preceded by a phase of epigenetic alterations that finely regulate the cell cycle, bias a mesenchymal phenotype and activate antioxidants. Concomitantly, cells that evade TKI-induced apoptosis (i.e., drug-tolerant persister cells) activate an intrinsic mutagenic program reminiscent of the SOS system deployed when bacteria are exposed to antibiotics. This mammalian system imbalances the purine-to-pyrimidine ratio, inhibits DNA repair and boosts expression of mutation-prone DNA polymerases. Thus, the net outcome of the SOS response is a greater probability to evolve new mutations. Deeper understanding of the persister-to-resister transformation, along with the development of next-generation TKIs, EGFR-specific proteolysis targeting chimeras (PROTACs), as well as bispecific antibodies, will permit delaying the onset of relapses and prolonging survival of patients with EGFR+ lung cancer.

Regulation of nucleo-cytosolic 26S proteasome translocation by aromatic amino acids via mTOR is essential for cell survival under stress.

The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasm-a response regulated by newly identified mTOR-agonistic amino acids-Tyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.

TAZ facilitates breast tumor growth by promoting an immune-suppressive tumor microenvironment.

The core Hippo pathway module consists of a tumour-suppressive kinase cascade that inhibits the transcriptional coactivators Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ). When the Hippo pathway is downregulated, as often occurs in breast cancer, YAP/TAZ activity is induced. To elaborate the roles of TAZ in triple-negative breast cancer (TNBC), we depleted Taz in murine TNBC 4T1 cells, using either CRISPR/Cas9 or small hairpin RNA (shRNA). TAZ-depleted cells and their controls, harbouring wild-type levels of TAZ, were orthotopically injected into the mammary fat pads of syngeneic BALB/c female mice, and mice were monitored for tumour growth. TAZ depletion resulted in smaller tumours compared to the tumours generated by control cells, in line with the notion that TAZ functions as an oncogene in breast cancer. Tumours, as well as their corresponding in vitro cultured cells, were then subjected to gene expression profiling by RNA sequencing (RNA-seq). Interestingly, pathway analysis of the RNA-seq data indicated a TAZ-dependent enrichment of 'Inflammatory Response', a pathway correlated with TAZ expression levels also in human breast cancer tumours. Specifically, the RNA-seq analysis predicted a significant depletion of regulatory T cells (Tregs) in TAZ-deficient tumours, which was experimentally validated by the staining of tumour sections and by quantitative cytometry by time of flight (CyTOF). Strikingly, the differences in tumour size were completely abolished in immune-deficient mice, demonstrating that the immune-modulatory capacity of TAZ is critical for its oncogenic activity in this setting. Cytokine array analysis of conditioned medium from cultured cells revealed that TAZ increased the abundance of a small group of cytokines, including plasminogen activator inhibitor 1 (Serpin E1; also known as PAI-1), CCN family member 4 (CCN4; also known as WISP-1) and interleukin-23 (IL-23), suggesting a potential mechanistic explanation for its in vivo immunomodulatory effect. Together, our results imply that TAZ functions in a non-cell-autonomous manner to modify the tumour immune microenvironment and dampen the anti-tumour immune response, thereby facilitating tumour growth.

L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab.

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP-FC , comprising the ligand-binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0 ) expressing TRAP-FC ubiquitously under the control of the chicken-beta-actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc-1. Ablation of each ERBB family member, especially the loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration, and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.

Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis.

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies.

Background: Antitumor therapies targeting HER1/EGFR and HER2, such as monoclonal antibodies (MAbs) and tyrosine-kinase inhibitors (TKIs), have demonstrated a significant clinical benefit, but the emergence of resistance limits long-term efficacy. While secondary HER1 mutations confer tolerance to TKI, compensatory upregulation of HER2 drives resistance to anti-HER1 MAbs, which identifies MAb combinations targeting both receptors as an attractive therapeutic strategy. Nevertheless, toxicity hampers the clinical validation of this approach. Alternatively, cancer vaccines may induce antibodies directed against several antigens with less concern about induced toxicity. Methods: Polyclonal antibodies (PAbs) targeting HER1 and HER2 were induced in mice or rabbits through immunization. Recognition of different epitopes on targets by PAbs was validated by phage-display technology. Receptor downregulation was evaluated by flow cytometry, immunofluorescence, and Western blot. MTT assays assessed cytotoxicity, while the antitumor effect of PAbs was assayed in nude mice. Results: PAbs promoted degradation of HER1 and HER2 regarding clinical MAbs or their combinations. As a result, inhibition of cytotoxicity on tumor cell lines was improved, even in the presence of oncogenic mutations in HER1, as well as in cetuximab-insensitive cells. Accordingly, the antitumor effect of vaccination-induced PAbs was observed in lung tumor lines representative of sensitivity or resistance to HER1 targeting therapies. Conclusions: Immunization against HER1 and HER2 receptors offers an alternative to passive administration of combinations of MAbs, since vaccination-induced PAbs promote the downregulation of both receptors and they have a higher impact on the survival of tumor cells.

AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer.

Anticancer therapies have been limited by the emergence of mutations and other adaptations. In bacteria, antibiotics activate the SOS response, which mobilizes error-prone factors that allow for continuous replication at the cost of mutagenesis. We investigated whether the treatment of lung cancer with EGFR inhibitors (EGFRi) similarly engages hypermutators. In cycling drug-tolerant persister (DTP) cells and in EGFRi-treated patients presenting residual disease, we observed upregulation of GAS6, whereas ablation of GAS6's receptor, AXL, eradicated resistance. Reciprocally, AXL overexpression enhanced DTP survival and accelerated the emergence of T790M, an EGFR mutation typical to resistant cells. Mechanistically, AXL induces low-fidelity DNA polymerases and activates their organizer, RAD18, by promoting neddylation. Metabolomics uncovered another hypermutator, AXL-driven activation of MYC, and increased purine synthesis that is unbalanced by pyrimidines. Aligning anti-AXL combination treatments with the transition from DTPs to resistant cells cured patient-derived xenografts. Hence, similar to bacteria, tumors tolerate therapy by engaging pharmacologically targetable endogenous mutators. SIGNIFICANCE: EGFR-mutant lung cancers treated with kinase inhibitors often evolve resistance due to secondary mutations. We report that in similarity to the bacterial SOS response stimulated by antibiotics, endogenous mutators are activated in drug-treated cells, and this heralds tolerance. Blocking the process prevented resistance in xenograft models, which offers new treatment strategies. This article is highlighted in the In This Issue feature, p. 2483.

5'isomiR-183-5p|+2 elicits tumor suppressor activity in a negative feedback loop with E2F1.

BACKGROUND: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. METHODS: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. RESULTS: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. CONCLUSIONS: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.

Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patients.

The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.

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.

Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as ß-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-ß/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

Coordinated regulation of WNT/ß-catenin, c-Met, and integrin signalling pathways by miR-193b controls triple negative breast cancer metastatic traits.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs' ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes. METHODS: To identify miRNAs regulating WNT/ß-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients' datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients. RESULTS: We identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/ß-catenin and c-Met signalling pathways in TNBC patients. CONCLUSIONS: Integrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.