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.

Eukaryotic Ribosome Assembly.

Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo-electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

The Oncogenic Helicase ALC1 Regulates PARP Inhibitor Potency by Trapping PARP2 at DNA Breaks.

The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.

Circadian disruption: from mouse models to molecular mechanisms and cancer therapeutic targets.

The circadian clock is a timekeeping system for numerous biological rhythms that contribute to the regulation of numerous homeostatic processes in humans. Disruption of circadian rhythms influences physiology and behavior and is associated with adverse health outcomes, especially cancer. However, the underlying molecular mechanisms of circadian disruption-associated cancer initiation and development remain unclear. It is essential to construct good circadian disruption models to uncover and validate the detailed molecular clock framework of circadian disruption in cancer development and progression. Mouse models are the most widely used in circadian studies due to their relatively small size, fast reproduction cycle, easy genome manipulation, and economic practicality. Here, we reviewed the current mouse models of circadian disruption, including suprachiasmatic nuclei destruction, genetic engineering, light disruption, sleep deprivation, and other lifestyle factors in our understanding of the crosstalk between circadian rhythms and oncogenic signaling, as well as the molecular mechanisms of circadian disruption that promotes cancer growth. We focused on the discoveries made with the nocturnal mouse, diurnal human being, and cell culture and provided several circadian rhythm-based cancer therapeutic strategies.

A benchmarking framework for the accurate and cost-effective detection of clinically-relevant structural variants for cancer target identification and diagnosis.

BACKGROUND: Accurate clinical structural variant (SV) calling is essential for cancer target identification and diagnosis but has been historically challenging due to the lack of ground truth for clinical specimens. Meanwhile, reduced clinical-testing cost is the key to the widespread clinical utility. METHODS: We analyzed massive data from tumor samples of 476 patients and developed a computational framework for accurate and cost-effective detection of clinically-relevant SVs. In addition, standard materials and classical experiments including immunohistochemistry and/or fluorescence in situ hybridization were used to validate the developed computational framework. RESULTS: We systematically evaluated the common algorithms for SV detection and established an expert-reviewed SV call set of 1,303 tumor-specific SVs with high-evidence levels. Moreover, we developed a random-forest-based decision model to improve the true positive of SVs. To independently validate the tailored 'two-step' strategy, we utilized standard materials and classical experiments. The accuracy of the model was over 90% (92-99.78%) for all types of data. CONCLUSION: Our study provides a valuable resource and an actionable guide to improve cancer-specific SV detection accuracy and clinical applicability.

Elongin B promotes breast cancer progression by ubiquitinating tumor suppressor p14/ARF.

Elongin B (ELOB), a pivotal element in the ELOB/c-Cullin2/5-SOCS-box E3 ubiquitin-protein ligase complex, plays a significant role in catalyzing the ubiquitination and subsequent degradation of a broad spectrum of target proteins. Notably, it is documented to facilitate these processes. However, the regulatory role of ELOB in breast cancer remains ambiguous. In this study, through bio-informatic analysis of The Cancer Genome Atlas and Fudan University Shanghai Cancer Center database, we demonstrated that ELOB was over-expressed in breast cancer tissues and was related to unfavorable prognosis. Additionally, pathway enrichment analysis illustrated that high expression of ELOB was associated with multiple cancer promoting pathways, like cell cycle, DNA replication, proteasome and PI3K - Akt signaling pathway, indicating ELOB as a potential anticancer target. Then, we confirmed that both in vivo and in vitro, the proliferation of breast cancer cells could be significantly suppressed by the down-regulation of ELOB. Mechanically, immunoprecipitation and in vivo ubiquitination assays prompted that, as the core element of Cullin2-RBX1-ELOB E3 ligase (CRL2) complex, ELOB regulated the ubiquitination and the subsequent degradation of oncoprotein p14/ARF. Moreover, the anticancer efficacy of erasing ELOB could be rescued by simultaneous knockdown of p14/ARF. Finally, through analyzing breast cancer tissue microarrays and western blot of patient samples, we demonstrated that the expression of ELOB in tumor tissues was elevated in compared to adjacent normal tissues. In conclusion, ELOB is identified to be a promising innovative target for the drug development of breast cancer by promoting the ubiquitination and degradation of oncoprotein p14/ARF.

Anti-Cancer Roles of Probiotic-Derived P8 Protein in Colorectal Cancer Cell Line DLD-1.

A novel probiotics-derived protein, P8, suppresses the growth of colorectal cancer (CRC). P8 can penetrate the cell membrane via endocytosis and cause cell cycle arrest in DLD-1 cells through down-regulation of CDK1/Cyclin B1. However, neither the protein involved in the endocytosis of P8 nor the cell cycle arrest targets of P8 are known. We identified two P8-interacting target proteins [importin subunit alpha-4 (KPNA3) and glycogen synthase kinase-3 beta (GSK3ß)] using P8 as a bait in pull-down assays of DLD-1 cell lysates. Endocytosed P8 in the cytosol was found to bind specifically to GSK3ß, preventing its inactivation by protein kinases AKT/CK1ε/PKA. The subsequent activation of GSK3ß led to strong phosphorylation (S33,37/T41) of ß-catenin, resulting in its subsequent degradation. P8 in the cytosol was also found to be translocated into the nucleus by KPNA3 and importin. In the nucleus, after its release, P8 binds directly to the intron regions of the GSK3ß gene, leading to dysregulation of GSK3ß transcription. GSK3ß is a key protein kinase in Wnt signaling, which controls cell proliferation during CRC development. P8 can result in a cell cycle arrest morphology in CRC cells, even when they are in the Wnt ON signaling state.

NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP).

C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multiangle light scattering (MALS), we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H)-linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog-specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge that connects the substrate with coenzyme-binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with cotranscriptional activity of CtBP in cancer.

De Novo design of a humanized antiCD33 antibody-oridonin conjugate for acute myeloid leukemia therapy.

Acute myeloid leukemia (AML) is the most common blood cancer in adults. Patients' 5-year overall survival is less than 30% thus having a poor prognosis. To date, the development of novel target therapies is still necessary to ameliorate patients' survival. Antibody-drug conjugates (ADCs) represent a promising class of drugs for the treatment of AML. CD33 is highly expressed on AML cells, and the FDA-approved CD33-targeted ADC drug-gemtuzumab ozogamicin (GO) has proved the feasibility of CD33-targeted ADC drug design. In this study, we constructed a novel CD33-targeted ADC drug composed of a humanized anti-CD33 antibody and oridonin as a payload with a cleaved chemical linker. Oridonin is a natural product that has great cancer therapy potential while its poor bioavailability and targeting ability limited its clinical use. Herein, we demonstrated that antiCD33-oridonin specifically delivered oridonin in AML cells improved AML cells killing ability of oridonin. Meanwhile, it did not show any non-specific toxicity on CD33 negative cells. In summary, we developed a novel AML targeting ADC with clinical application potential, and therefore provided a new solution for the druggability improvement of oridonin.

Cancer Target Gene Screening: a web application for breast cancer target gene screening using multi-omics data analysis.

Breast cancer comprises several molecular subtypes with distinct clinical features and treatment responses, and a substantial portion of each subtype remains incurable. A comprehensive analysis of multi-omics data and clinical profiles is required in order to better understand the biological complexity of this cancer type and to identify new prognostic and therapeutic markers. Thus, there arises a need for useful analytical tools to assist in the investigation and clinical management of the disease. We developed Cancer Target Gene Screening (CTGS), a web application that provides rapid and user-friendly analysis of multi-omics data sets from a large number of primary breast tumors. It allows the investigation of genomic and epigenomic aberrations, evaluation of transcriptomic profiles and performance of survival analyses and of bivariate correlations between layers of omics data. Notably, the genome-wide screening function of CTGS prioritizes candidate genes of clinical and biological significance among genes with copy number alteration, DNA methylation and dysregulated expression by the integrative analysis of different types of omics data in customized subgroups of breast cancer patients. These features may help in the identification of druggable cancer driver genes in a specific subtype or the clinical condition of human breast cancer. CTGS is available at http://ctgs.biohackers.net.

Mechanistic insights on cytotoxicity of KOLR, Cinnamomum pauciflorum Nees leaf derived active ingredient, by targeting signaling complexes of phosphodiesterase 3B and rap guanine nucleotide exchange factor 3.

Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. In the current study, a natural product Kaempferol-3-O-(3'',4''-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves. KOLR exhibited higher cytotoxic effects against BxCP-3 pancreatic cancer cell line. In BxPC-3 cells, the KOLR could enhance the formation of RAPGEF 3/ PDE3B protein complex to inhibit the activation of Rap-1 and PI3K-AKT pathway, thereby promoting cell apoptosis and inhibiting cell metastasis. Mutation of RAPGEF3 G557A or low expression of PDE3B inactivated the binding action of KOLR resulting in KOLR resistance. The findings of this study show that PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.

Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.

The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.

Non-cytomembrane PD-L1: An atypical target for cancer.

Programmed death ligand 1 (PD-L1) has conventionally been considered as a type I transmembrane protein that can interact with its receptor, programmed cell death 1 (PD-1), thus inducing T cell deactivation and immune escape. However, targeting the PD-1/PD-L1 axis has achieved adequate clinical responses in very few specific malignancies. Recent studies have explored the extracellularly and subcellularly located PD-L1, namely, nuclear PD-L1 (nPD-L1), cytoplasmic PD-L1 (cPD-L1), soluble PD-L1 (sPD-L1), and extracellular vesicle PD-L1 (EV PD-L1), which might shed light on the resistance to anti-PD1/PDL1 therapy. In this review, we summarize the four atypical localizations of PD-L1 with a focus on their novel functions, such as gene transcription regulation, therapeutic efficacy prediction, and resistance to various cancer therapies. Additionally, we highlight that non-cytomembrane PD-L1s are of significant cancer diagnostic value and are promising therapeutic targets to treat cancer.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Targeting Protein Neddylation for Cancer Therapy.

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.

Assembly of human C-terminal binding protein (CtBP) into tetramers.

C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD+- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.

Combination of Vaccine-Strain Measles and Mumps Viruses Enhances Oncolytic Activity against Human Solid Malignancies.

Oncolytic measles and mumps viruses (MeV, MuV) have a potential for anti-cancer treatment. We examined the anti-tumor activity of MeV, MuV, and MeV-MuV combination (MM) against human solid malignancies (HSM). MeV, MuV, and MM targeted and significantly killed various cancer cell lines of HSM but not normal cells. MM demonstrated a greater anti-tumor effect and prolonged survival in a human prostate cancer xenograft tumor model compared to MeV and MuV. MeV, MuV, and MM significantly induced the expression of immunogenic cell death markers and enhanced spleen-infiltrating immune cells. In conclusion, MM combination significantly improves the treatment of human solid malignancies.

Inhibitors of apoptosis: clinical implications in cancer.

Inhibitor of apoptosis (IAP) family comprises a group of endogenous proteins that function as main regulators of caspase activity and cell death. They are considered the main culprits in evasion of apoptosis, which is a fundamental hallmark of carcinogenesis. Overexpression of IAP proteins has been documented in various solid and hematological malignancies, rendering them resistant to standard chemotherapeutics and radiation therapy and conferring poor prognosis. This observation has urged their exploitation as therapeutic targets in cancer with promising pre-clinical outcomes. This review describes the structural and functional features of IAP proteins to elucidate the mechanism of their anti-apoptotic activity. We also provide an update on patterns of IAP expression in different tumors, their impact on treatment response and prognosis, as well as the emerging investigational drugs targeting them. This aims at shedding the light on the advances in IAP targeting achieved to date, and encourage further development of clinically applicable therapeutic approaches.

De Novo Computational Design for Development of a Peptide Ligand Oriented to VEGFR-3 with High Affinity and Long Circulation.

The overexpression of VEGFR-3 is correlated with a worse prognosis in lung cancer and has been regarded as a rational target for specific drug delivery. Here, VEGFR-3 homing peptide library was efficiently established by computational design. Strong fluorescent signals of selected peptides were observed in A549 cells, but much weaker in other cells. The positive immunostaining overlapped with VEGFR-3 confirmed high affinity and selectivity of one novel peptide (CP-7). In addition, cell uptake of FITC-CP-7 peptide was significantly blocked by coinjection of excess CP-7 peptide. After labeled with 131I, the profile of pharmacology and biodistribution could be traced in vivo. The 131I-radiolabeled CP-7 peptide conjugates were >85% stable in serum over 4 h and exhibited a specific uptake of 18.04 ± 2.04% ID/g at 0.5 h after injection to high VEGFR-3 expressing A549 tumor mice. More importantly, lower uptake concentration in heart (1.06 ± 0.15% ID/g) after 2 h demonstrated the safety of peptide in vivo. The high uptake in the kidneys revealed that renal clearance was the main route of 131I-CP-7 peptide elimination from the body. Lower accumulation of 131I-CP-7 peptide in VEGFR-3 negative HeLa tumor mice further indicated that CP-7 peptide exhibited a higher tumor-homing efficiency. These studies provided a straightforward analytical access to design and screen bioactive peptide based on protein structure and revealed that CP-7 peptide represented a promising homing peptide of VEGFR-3-positive cancer in vitro and in vivo which could be used as a novel target molecule to achieve efficient drug delivery.