Tumor Suppressor Transcription Factor Research Articles

The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin.

Toxicity and the emergence of resistance are the main challenges in cancer treatment. The optimal dose of cisplatin, one of the most widely used chemotherapeutic anticancer drugs, is currently being widely debated. Furthermore, the dose-dependent molecular mechanisms of its action are poorly understood. To assess the role of protein kinase JNK (cJun N-terminal kinase) signaling in lung cancer treatment, we combined small-molecule JNK inhibitors and cisplatin. Wild-type p53 (tumor suppressor transcription factor TP53) and mutated RAS-bearing lung adenocarcinoma cell line A549 was used as a model in our studies. Here, we demonstrate cisplatin concentration-dependent opposing roles of JNK in killing cancer cells: a cell-protective role at low cisplatin concentrations and an apoptosis-promoting (or neutral) role at high concentrations. Time- and dose-dependent activation of pro-survival protein kinase AKT and TP53 was shown, with similar activation dynamics in cells exposed to different (low and high) cisplatin concentrations. Selective inhibition of AKT and activation of TP53 (expression and phosphorylation) led to a decrease in cell survival, indicating their involvement in cisplatin-induced cell death regulation. The activation levels of TP53 and AKT in cisplatin-treated A549 cells after cotreatment with the JNK inhibitor SP600125 correlated with their role in regulating cell death. TP53 and AKT were proposed as signaling proteins mediating the outcome of JNK inhibition in A549 cells exposed to different concentrations of cisplatin. Our findings suggest that a combination of stress kinase JNK inhibition and low-dose cisplatin, together with manipulation of drug-induced signaling, could be considered as a promising treatment strategy for certain lung cancers.

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

Abstract 1706: Investigating the interaction between ETS family members and mutant p53

Abstract Cancer cases are on the rise globally requiring a deeper understanding of the disease and identification of novel therapeutic targets. Mutations in genes often result in tumor formation. TP53 encodes p53, a tumor suppressive transcription factor, and is mutated in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. p53 mutations disrupt direct p53 binding to DNA and promotes p53 binding to regulatory regions of oncogenes via protein-protein interactions with other transcription factors. One of these interacting partners is ETS2, a member of the ETS transcription factor family. ETS binding sites are present in 50% of all mutant p53 occupied regulatory elements. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed weak (ETS1) or have yet to be identified as direct (ERG). It is my aim to determine the scope of interaction between the ETS family and mutant p53 and to elucidate whether this interaction is required for the observed oncogenic phenotypes of ETS proteins and mutant p53.To determine which ETS proteins interact with mutant p53 purified ETS proteins were used for affinity pull-down of purified mutant p53. The entire ETS family interacted with mutant p53 to some degree, and several ETS proteins had stronger interactions than ETS2. Truncation studies were used to determine interaction domains. One interacting region was the conserved DNA binding domain, which may explain interaction across the entire family. ERG and ETS2, both strong mutant p53 interactors, had two interaction interfaces. I hypothesize that the second interaction interface defines strong interactors. To determine if interacting ETS are responsible for targeting mutant p53 to the genome, chromatin immunoprecipitation sequencing measured mutant p53 binding in the presence or absence of different ETS factors. These data indicate that each of the conditions resulted in different p53 binding patterns and that there is a requirement for ETS in mutant p53 binding. Lastly, I wanted to determine if there was a correlation between p53 mutation and expression of strong mutant p53 interactors in certain cancer types. Analysis of TCGA samples showed that in ovarian cancers with p53 mutations, ETS factors that are strong mutant p53 interactors were upregulated in a mutually exclusive pattern, while weak interactors tended to be downregulated, or unchanged. Similar trends were observed among some other cancer types. My studies have shown that several ETS proteins interact with mutant p53 in vitro, recruit p53 to the genome, and that this interaction pattern correlates with expression in mutant p53 driven cancers. Additionally, studies are ongoing to determine phenotypes related to this interaction. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenesis, these will be attractive targets for drug development. Citation Format: Stephanie Metcalf, Peter Hollenhorst. Investigating the interaction between ETS family members and mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1706.

The Presence of Somatic Mutations on Specific Pathways Such As Chromatin Modifier, Spliceosome, and Myeloid Transcription Factor at Diagnosis Was Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplantation (HCT) in 1,228 Patients with Acute Myeloid Leukemia in First Complete Remission (CR1)

Introduction Allogeneic stem cell transplant (HCT) brings survival benefit to certain subgroups of acute myeloid leukemia (AML) patients, although it may not be suitable for all cases. Decision for HCT referral is based on the ELN risk grouping, which classifies pts into favorable, intermediate, and adverse risk categories. While HCT is recommended for pts in the intermediate and adverse risk, those in the favorable risk are deferred to receive HCT. However, currently, there is a lack of data directly demonstrating benefits of HCT in specific mutation groups. Patients and Method In this retrospective study, out of 1477 newly diagnosed AML pts 1,228 pts achieved CR1 and had undergone baseline next-generation sequencing (NGS) at diagnosis from January 1997 to January 2020. The data were collected from five different centers located in Toronto, Vancouver, and Montreal, Canada, as well as Hwasun and Seoul, South Korea. NGS was performed in each institution with each institution's standard targeted sequencing panel. To assess the effectiveness of HCT, particular attention will be given to the time from achieving complete remission 1 (CR1) to HCT. To handle immortal time bias, the Mantel-Byar method was used to depict outcomes during the follow-up period. Kaplan-Meier curves were avoided as they can introduce immortal bias, considering the period of time before treatment as part of the treatment arm. Instead, the MBT and landmark methods were employed to address this issue. Allogeneic HCT was treated as a time-dependent variable to prevent immortal bias. Gene mutations were grouped into 6 biologic pathways, including DNA methylation, chromatin modifiers, cohesion complex, activated signaling, tumor suppressor, spliceosome, and myeloid transcription factor (TF) mutations. In the multivariate (MVA) analysis, any mutations with a frequency of >5% were included as well as TP53 which has very grave prognosis. The primary objective of this study is to identify specific groups of AML pts who can derive significant benefits from receiving HCT in CR1, treating HCT as a time-dependent (td) covariate in the analysis. Statistical analyses were conducted using EZR version 1.41. Results The median duration of follow-up among survivors after achieving CR1 was 22.6 months (range: 1-217.9 months). Among the 593 pts, who underwent allogeneic HCT in CR1, the HCT group was composed of younger pts, with a median age of 50 years compared to 56 years in the non-HCT group (p < 0.001). Also, there were fewer pts with NPM1 mutation in the HCT group, accounting for 20.7% compared to 32.4% in the non-HCT group (p < 0.001). Apart from these characteristics, the two groups were similar in other parameters. The 2-year overall survival (OS) was higher in the HCT group, at 66.4%, compared to 54.6% in the non-HCT group (p < 0.0001). Similarly, the 2-year RFS was higher in the HCT group, with a rate of 64.8%, compared to 52.1% in the non-HCT group (p =1.82e-15). Furthermore, HCT as a time-dependent covariate was found to be favorable (Hazard ratio [HR] 0.76, 95% C.I. [0.63-0.91]; p=0.003), as demonstrated in the Simon-Makuch plot (p = 0.003). In the MVA, HCT as a time-dependent covariate retained its favorable impact on RFS (HR 0.67, [0.56-0.82]; p<0.0001). The MVA showed several prognostic factors besides HCT such as NPM1 and CEBPA mutations as favorable factors for RFS, being classified under the European Leukemia Network (ELN) favorable risk group. Conversely, FLT3-ITD, DNMT3A, IDH1, KIT, and TP53 mutations, monosomal karyotype, age > 60 years, and white blood count (WBC) at diagnosis > 55,600/mm^3 were associated with worse RFS. In terms of RFS, the greatest benefit of HCT was observed in the pts having mutations in ASXL1 (HR 0.34 [0.15-0.78]), BCOR (HR 0.29 [0.14-0.61]), IDH1/2 (HR 0.62 [0.42-0.92]), and RUNX1 (HR 0.17 [0.08-0.40]) but no significant benefit in those with KRAS (HR 1.59), NRAS (HR 1.21), PTPN11 (HR 1.23), and WT1 (HR 1.30). According to the biological pathway of somatic mutations, the greatest benefit of HCT in terms of RFS was observed in pts with mutations in the chromatin modifier (HR 0.37 [0.22-0.62]), spliceosome (HR 0.32 [0.16-0.65]), and myeloid TF pathway (HR 0.52 [0.34-0.79]). Conclusion Allogeneic HCT exerts a greater benefit on RFS in AML pts with mutations in the chromatin modifier, spliceosome, and myeloid TF pathways. Further study is warranted to replicate this finding.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established.MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten−/−/Cxcr2−/− and NRasQ61R/INK4a−/−/Cxcr2−/− melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten−/− and NRasQ61R/INK4a−/− mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA).ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models.ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Next-Generation Sequencing–Based Genomic Profiling of Children with Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML BFM protocols. Targeted next-generation sequencing (NGS) of 54 genes revealed 17 genes that were recurrently mutated in more than 5% of patients. Considerable differences were observed in the mutational profiles compared to previous studies as BCORL1, CUX1, KDM6A, PHF6 and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, WT1) mutations were found to be associated with induction failure and shorter event-free survival suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% vs. 44.4%) and transcription factors (35.1 vs. 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identified previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Assessing p53 aggregates over a lifetime: Studies on young, adult, and aged mice.

p53 is a tumor-suppressing transcription factor critical to cellular health and function. The importance of intact p53 has been well established over the forty-plus years since its discovery. However, the role of amyloid-like aggregation in p53-related pathologies remains a significant knowledge gap. A growing body of knowledge over the last two decades has connected several mutations prevalent in cancer to the amyloid-like aggregation of p53 into both cross beta-sheet stacked fibrils and non-fibrillar aggregates. Here, non-fibrillar aggregates encompass all non-native p53-p53 (homomeric) complexes currently off-pathway to highly structured low-energy endpoint fibrils. p53 aggregation has reasonably been considered a cancer-centric phenomenon; however, it is unknown whether fibrillar-like structures form throughout the organismal lifespan, analogous to amyloid plaques. Furthermore, it is unclear if these structural states are observable as end-point or transient intermediates, including those either on or off-pathway to fibrillization. Notably, it has been shown that tau aggregates in the frontal cortex of Alzheimer's disease (AD) patients and AD mouse models contained aggregated p53, suggesting p53 aggregation may be more prevalent in aging-related diseases than previously thought. Here, I use immunoprecipitation to isolate p53 from young, adult, and aged mice and compare the aggregation state and propensity using native-page, fluorescence spectroscopy, and electron microscopy. Further, I will compare these data to the aggregation state typically observed in immortalized cell lines. I hypothesize that in mice, WT and mutant p53 may form aggregates over time, although those observed with mutant p53 will appear more fibrillar than WT, and may be distinct from those observed in immortalized cell lines. Together, these experiments help broaden our understanding of p53 aggregation related to age and health in vitro and in vivo.

Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer-associated death worldwide with a dismal overall 5-year survival rate of less than 20%. The standard first-line therapy for advanced ESCC is concomitant chemo-radiation therapy (CCRT); however, patients usually develop resistance, resulting in unfavorable outcomes. Therefore, it is urgent to identify the mechanisms underlying CCRT resistance and develop effective treatment strategies.MethodsPatients’ endoscopic biopsy tumor tissues obtained before CCRT treatment were used to perform RNA-seq and GSEA analysis. Immunohistochemical (IHC) staining, chromatin immunoprecipitation (ChIP), and promoter reporter analyses were conducted to investigate the relationship between SOX17 and NRF2. Xenograft mouse models were used to study the role of SOX17/NRF2 axis in tumor growth and the efficacy of carboxymethyl cellulose-coated zero-valent-iron (ZVI@CMC).ResultsIn this study, a notable gene expression signature associated with NRF2 activation was observed in the poor CCRT responders. Further, IHC staining of endoscopic biopsy of 164 ESCC patients revealed an inverse correlation between NRF2 and SOX17, a tumor-suppressive transcription factor with low expression in ESCC due to promoter hypermethylation. Using ChIP and promoter reporter analyses, we demonstrated that SOX17 was a novel upstream transcriptional suppressor of NRF2. In particular, SOX17low/NRF2high nuclear level significantly correlated with poor CCRT response and poor survival, indicating that the dysregulation of SOX17/NRF2 axis played a pivotal role in CCRT resistance and tumor progression. Notably, the in-house developed nanoparticle ZVI@CMC functioned as an inhibitor of DNA methyltransferases to restore expression of SOX17 that downregulated NRF2, thereby overcoming the resistance in ESCC. Additionally, the combination of ZVI@CMC with radiation treatment significantly augmented anticancer efficacy to inhibit tumor growth in CCRT resistant cancer.ConclusionThis study identifies a novel SOX17low/NRF2high signature in ESCC patients with poor prognosis, recognizes SOX17 as a transcriptional repressor of NRF2, and provides a promising strategy targeting SOX17/NRF2 axis to overcome resistance.

The Antagonism between the Murine Gammaherpesvirus Protein Kinase and Global Interferon Regulatory Factor 1 Expression Shapes the Establishment of Chronic Infection.

Gammaherpesviruses infect most vertebrate species and are associated with B cell lymphomas. Manipulation of B cell differentiation is critical for natural infection and lymphomagenesis driven by gammaherpesviruses. Specifically, human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) drive differentiation of infected naive B cells into the germinal center to achieve exponential increase in the latent viral reservoir during the establishment of chronic infection. Infected germinal center B cells are also the target of viral lymphomagenesis, as most EBV-positive B cell lymphomas bear the signature of the germinal center response. All gammaherpesviruses encode a protein kinase, which, in the case of Kaposi's sarcoma-associated herpesvirus (KSHV) and MHV68, is sufficient and necessary, respectively, to drive B cell differentiation in vivo. In this study, we used the highly tractable MHV68 model of chronic gammaherpesvirus infection to unveil an antagonistic relationship between MHV68 protein kinase and interferon regulatory factor 1 (IRF-1). IRF-1 deficiency had minimal effect on the attenuated lytic replication of the kinase-null MHV68 in vivo. In contrast, the attenuated latent reservoir of the kinase-null MHV68 was partially to fully rescued in IRF-1-/- mice, along with complete rescue of the MHV68-driven germinal center response. Thus, the novel viral protein kinase-IRF-1 antagonism was largely limited to chronic infection dominated by viral latency and was less relevant for lytic replication during acute infection and in vitro. Given the conserved nature of the viral and host protein, the antagonism between the two, as defined in this study, may regulate gammaherpesvirus infection across species. IMPORTANCE Gammaherpesviruses are prevalent pathogens that manipulate physiological B cell differentiation to establish lifelong infection. This manipulation is also involved in gammaherpesvirus-driven B cell lymphomas, as differentiation of latently infected B cells through the germinal center response targets these for transformation. In this study, we define a novel antagonistic interaction between a conserved gammaherpesvirus protein kinase and a host antiviral and tumor suppressor transcription factor. The virus-host antagonism unveiled in this study was critically important to shape the magnitude of gammaherpesvirus-driven germinal center response. In contrast, the virus-host antagonism was far less relevant for lytic viral replication in vitro and during acute infection in vivo, highlighting the emerging concept that nonoverlapping mechanisms shape the parameters of acute and chronic gammaherpesvirus infection.

TF-DUBTACs Stabilize Tumor Suppressor Transcription Factors.

Targeted protein degradation approaches have been widely used for degrading oncogenic proteins, providing a potentially promising therapeutic strategy for cancer treatment. However, approaches to targeting tumor suppressor proteins are very limited, and only a few agonists have been developed to date. Here, we report the development of a platform termed TF-DUBTAC, which links a DNA oligonucleotide to a covalent ligand of the deubiquitinase OTUB1 via a click reaction, to selectively stabilize tumor suppressor transcription factors. We developed three series of TF-DUBTACs, namely, FOXO-DUBTAC, p53-DUBTAC, and IRF-DUBTAC, which stabilize FOXO3A, p53, and IRF3 in cells, respectively, in an OTUB1-dependent manner. These results suggest that TF-DUBTAC is a generalizable platform to achieve selective stabilization of tumor suppressor transcription factors as a therapeutic means to suppress tumorigenesis.

Dynamic Chromatin States Coupling with Key Transcription Factors in Colitis-Associated Colorectal Cancer.

Inflammation is one of the critical risk factors for colorectal cancer (CRC). However, the mechanisms for transition from colitis to CRC remain elusive. Recently, epigenetic changes have emerged as important regulatory factors for colitis‐associated cancer. Here, a systematic epigenomic study of histone modifications is performed, including H3K4me1, H3K4me3, H3K27ac, H3K27me3 and H3K9me3, in an AOM‐DSS‐induced CRC mouse model. In combination with transcriptomic data, the authors generate a dataset of 105 deep sequencing files and illustrate the dynamic landscape of chromatin states at five time points during inflammation‐cancer transition. Functional gene clusters are identified based on dynamic transcriptomic and epigenomic information, and key signaling pathways in the process are illustrated. This study's results reveal that enhancer state regions play important roles during inflammation‐cancer transition. It predicts novel transcription factors based on enhancer information, and experimentally proves OTX2 as a critical tumor suppressive transcription factor. Taken together, this study provides comprehensive epigenomic data and reveals novel molecular mechanisms for colitis‐associated cancer.

Activation of p53 in anoxic freshwater crayfish, Faxonius virilis.

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis and autophagy. Here, we studied the stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response were measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 were assessed using immunoblotting at sites known to be phosphorylated (serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phosphorylated p53 (p-p53) was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low-oxygen stress were studied. The results showed an increase in p-p53 levels during anoxia in both hepatopancreas and tail muscle. Increased transcript levels of ei24 support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser15 phosphorylated p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low-oxygen stress. Understanding how anoxia-tolerant organisms are able to protect themselves against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.

Prognostic and Immunotherapeutic Roles of KRAS in Pan-Cancer

KRAS is one well-established tumor-driver gene associated with cancer initiation, development, and progression. Nonetheless, comparative studies of the relevance of KRAS across diverse tumors remain sparse. We explored the KRAS expression and prognostic values in diverse cancer types via multiple web-based bioinformatics tools, including cBioPortal, Oncomine, PrognoScan, Kaplan–Meier Plotter, etc. We found that KRAS is highly expressed in various malignancies compared to normal cohorts (BRCA, CHOL, ESCA, HNSC, LIHC, LUAD, LUSC, and STAD) and less expressed in COAD, KIRC, READ, and THCA than in normal samples. We observed the dysregulation of the DNA methylation of KRAS in cancers and discovered that numerous oncogenic and tumor-suppressive transcription factors bind the KRAS promoter region. Pan-cancer analysis also showed that a high level of KRAS is associated with poor outcomes. Additionally, KRAS is remarkably correlated with the level of immune cell infiltration and tumorigenic gene signatures. In conclusion, our findings reveal novel insights into KRAS expression and its biological functions in diverse cancer types, indicating that KRAS could serve as a prognostic biomarker and is associated with immune infiltrates.

Modulating undruggable targets to overcome cancer therapy resistance

Many cancer patients frequently fail to respond to anti-cancer treatment due to therapy resistance which is the major obstacle towards curative cancer treatment. Therefore, identification of the molecular mechanisms underlying resistance is of paramount clinical and economic importance. The advent of targeted therapies based on a molecular understanding of cancer could serve as a model for strategies to overcome drug resistance. Accordingly, the identification and validation of proteins critically involved in resistance mechanisms represent a path towards innovative therapeutic strategies to improve the clinical outcome of cancer patients. In this review, we discuss emerging targets, small molecule therapeutics and drug delivery strategies to overcome therapy resistance. We focus on rational treatment strategies based on transcription factors, pseudokinases, nuclear export receptors and immunogenic cell death strategy. Historically, unliganded transcription factors and pseudokinases were considered undruggable while blocking the nuclear export e.g., through inhibition of the nuclear export receptor CRM1 was predicted as highly toxic. Recent success inhibiting Gli-1, HIF-1α, HIF-2α and reactivating the tumor suppressor transcription factors p53 and FOXO illustrates the feasibility and power of this targeting approach. Similarly, progress has been made in modulating the activity of pseudokinase proteins implicated in therapy resistance including members of the Tribbles protein family. On the other hand, the recent clinical approval of Selinexor, a specific inhibitor of CRM-1, a protein that mediates the transport of cargos with leucine-rich nuclear export signals and known to be a driver of drug resistance, represents the proof-of-concept for inhibiting the nuclear export as a feasible strategy to overcome therapy resistance. The ever-growing capacity to target resistance mechanisms with judiciously selected small molecules, some of which are being formulated within smart nanoparticles, will pave the way towards the improvement of the clinical outcome and realize the full potential of targeted therapies and immunotherapies.

P53 partial loss-of-function mutations sensitize to chemotherapy

The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational hotspot residues, resulting in a complete loss of transcriptional activity and mutant p53-driven chemotherapy resistance. Of the remaining 70% of non-hotspot mutants, many are partial loss-of-function (partial-LOF) mutants with residual transcriptional activity. The therapeutic consequences of a partial-LOF have remained largely elusive. Using a p53 mutation engineered to reduce DNA binding, we demonstrate that partial-LOF is sufficient to enhance oncogene-driven tumorigenesis in mouse models of lung and pancreatic ductal adenocarcinoma and acute myeloid leukemia. Interestingly, mouse and human tumors with partial-LOF mutations showed mutant p53 protein accumulation similar as known for hotspot mutants. Different from the chemotherapy resistance caused by p53-loss, the partial-LOF mutant sensitized to an apoptotic chemotherapy response and led to a survival benefit. Mechanistically, the pro-apoptotic transcriptional activity of mouse and human partial-LOF mutants was rescued at high mutant protein levels, suggesting that accumulation of partial-LOF mutants enables the observed apoptotic chemotherapy response. p53 non-hotspot mutants with partial-LOF, therefore, represent tumorigenic p53 mutations that need to be distinguished from other mutations because of their beneficial impact on survival in a therapy context.

Identification of Prognostic Gene Biomarkers in Non-Small Cell Lung Cancer Progression by Integrated Bioinformatics Analysis.

Simple SummaryNon-small cell lung cancer (NSCLC) is a major contributor to cancer related deaths worldwide. The progression of NSCLC is linked to epithelial-mesenchymal transition (EMT), a biologic process that enables tumor cells to acquire an invasive phenotype and resistance to therapies. Discovery of novel biomarkers in NSCLC progression is essential for improved prognosis and pharmacological interventions. We performed an integrated bioinformatics analysis on available gene expression datasets of transforming growth factor β (TGF-β) induced EMT in NSCLC cell lines aiming to establish new prognostic biomarkers in the disease. The retrieved candidate genes were involved in protein modifications, regulation of cell death and cell adhesions, oxidation-reduction reactions of aerobic respiration and mitochondrial translation. Out of these genes, we identified ten prognostic gene biomarkers, mostly involved in protein modifications, whose expressions correlated with patient survival in NSCLC. This ten-gene prognostic signature will be useful to improve risk prediction and guide treatment strategies in NSCLC. Deciphering the exact functions of the biomarker genes previously not linked with NSCLC will also lead to a better understanding of the pathomechanism of NSCLC progression, revealing novel therapeutic targets in the disease.The progression of non-small cell lung cancer (NSCLC) is linked to epithelial-mesenchymal transition (EMT), a biologic process that enables tumor cells to acquire a migratory phenotype and resistance to chemo- and immunotherapies. Discovery of novel biomarkers in NSCLC progression is essential for improved prognosis and pharmacological interventions. In the current study, we performed an integrated bioinformatics analysis on gene expression datasets of TGF-β-induced EMT in NSCLC cells to identify novel gene biomarkers and elucidate their regulation in NSCLC progression. The gene expression datasets were extracted from the NCBI Gene Expression Omnibus repository, and differentially expressed genes (DEGs) between TGF-β-treated and untreated NSCLC cells were retrieved. A protein-protein interaction network was constructed and hub genes were identified. Functional and pathway enrichment analyses were conducted on module DEGs, and a correlation between the expression levels of module genes and survival of NSCLC patients was evaluated. Prediction of interactions of the biomarker genes with transcription factors and miRNAs was also carried out. We described four protein clusters in which DEGs were associated with ubiquitination (Module 1), regulation of cell death and cell adhesions (Module 2), oxidation-reduction reactions of aerobic respiration (Module 3) and mitochondrial translation (Module 4). From the module genes, we identified ten prognostic gene biomarkers in NSCLC. Low expression levels of KCTD6, KBTBD7, LMO7, SPSB2, RNF19A, FOXA2, DHTKD1, CDH1 and PDHB and high expression level of KLHL25 were associated with reduced overall survival of NSCLC patients. Most of these biomarker genes were involved in protein ubiquitination. The regulatory network of the gene biomarkers revealed their interaction with tumor suppressor miRNAs and transcription factors involved in the mechanisms of cancer progression. This ten-gene prognostic signature can be useful to improve risk prediction and therapeutic strategies in NSCLC. Our analysis also highlights the importance of deregulation of ubiquitination in EMT-associated NSCLC progression.

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif.

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.

Deletion of the Wilms' Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development.

Expression of Wilms’ tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20–25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2Cre) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development.

Identification of consistent post-translational regulatory triplets related to oncogenic and tumour suppressive modulators in childhood acute lymphoblastic leukemia.

BackgroundAcute lymphoblastic leukemia (ALL) is the most common type of childhood cancer. It can be caused by mutations that turn on oncogenes or turn off tumour suppressor genes. For instance, changes in certain genes including Rb and p53 are common in ALL cells. Oncogenes and TSGs may serve as a modulator gene to regulate the gene expression level via their respective target genes. To investigate the regulatory relationship between oncogenes, tumour suppressor genes and transcription factors at the post translational level in childhood ALL, we performed an integrative network analysis on the gene regulation in the post-translational level for childhood ALL based on many publicly available cancer gene expression data including TARGET and GEO database.MethodsWe collected 259 childhood ALL-related genes from the latest online leukemia database, Leukemia Gene Literature Database. These 259 genes were selected from a comprehensive systematic literature with experimental evidences. The identified and curated genes were also associated with patient survival cases and we incorporated this pediatric ALL-related gene list into our analysis. We extracted the known human TFs from the TRRUST database. Among 259 childhood ALL-related genes, 101 unique regulators were mapped to the list of oncogene and tumour suppressor genes (TSGs) from the ONGene and the TSGene databases, and these included 74 TSGs, 62 oncogenes and 46 TF genes.ResultsThe resulted regulation was presented as a hierarchical regulatory network with transcription factors (TFs) as intermediate regulators connecting the top modulators (oncogene and TSGs) to the common target genes. Cross-validation was applied to the results from the TARGET dataset by identifying the consistent regulatory motifs based on three independent ALL expression datasets. A three-layer regulatory network of consistent positive modulators in childhood ALL was constructed in which 74 modulators (40 oncogenes, 34 TSGs) are considered as the most important regulators. The middle layer and the bottom layer contain 34 TFs and 176 target genes, respectively. Oncogenes mostly participated in positive regulation of gene expression and the transcription process of RNA II polymerase, while TSGs were mainly involved in the negative regulation of gene expression. In addition, the oncogene-specific targets were enriched with regulators of the MAPK cascade while tumour suppressor-specific targets were associated with cell death.ConclusionThe results revealed that oncogenes and TSGs possess a different functional regulatory pattern with regard to not only their biological functions but also their specific target genes in childhood ALL cancer progression. Taken together, our findings could contribute to a better understanding of the important regulatory mechanisms and this method could be used to analyse the targeted genes at the post-translational level in childhood ALL through integrative network analysis.