Tumor Suppressor Transcription Factor Research Articles

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.

The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.

AnnoFuse: an R Package to annotate, prioritize, and interactively explore putative oncogenic RNA fusions

BackgroundGene fusion events are significant sources of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy.ResultsWe developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling. We validated annoFuse using a random cohort of TCGA RNA-Seq samples (N = 160) and achieved a 96% sensitivity for retention of high-confidence fusions (N = 603). annoFuse uses FusionAnnotator annotations to filter non-oncogenic and/or artifactual fusions. Then, fusions are prioritized if previously reported in TCGA and/or fusions containing gene partners that are known oncogenes, tumor suppressor genes, COSMIC genes, and/or transcription factors. We applied annoFuse to fusion calls from pediatric brain tumor RNA-Seq samples (N = 1028) provided as part of the Open Pediatric Brain Tumor Atlas (OpenPBTA) Project to determine recurrent fusions and recurrently-fused genes within different brain tumor histologies. annoFuse annotates protein domains using the PFAM database, assesses reciprocality, and annotates gene partners for kinase domain retention. As a standard function, reportFuse enables generation of a reproducible R Markdown report to summarize filtered fusions, visualize breakpoints and protein domains by transcript, and plot recurrent fusions within cohorts. Finally, we created shinyFuse for algorithm-agnostic interactive exploration and plotting of gene fusions.ConclusionsannoFuse provides standardized filtering and annotation for gene fusion calls from STAR-Fusion and Arriba by merging, filtering, and prioritizing putative oncogenic fusions across large cancer datasets, as demonstrated here with data from the OpenPBTA project. We are expanding the package to be widely-applicable to other fusion algorithms and expect annoFuse to provide researchers a method for rapidly evaluating, prioritizing, and translating fusion findings in patient tumors.

The Epigenetic Modification of Epigallocatechin Gallate (EGCG) on Cancer.

Among the major components of green tea, epigallocatechin-3-gallate (EGCG) is the most effective for its anti-cancer characteristics. The bulk of studies provide the mechanisms of suppressive function of EGCG are involved in alteration of cancer cell cycle, development, and apoptosis through activation/inhibition of several signal pathways. Another mechanism that explains the multiple effects exerted by EGCG in cancer is the epigenetic change by DNA methylation or methyltransferases, histone acetylation or deacetylases, and no coding RNAs (micoRNAs). Furthermore, decontrolled expression of miRNA transcription has been tested to be directly regulated by oncogenic and tumor-suppressor transcription factors. Recently, several proteins have been identified as miRNA direct interactors by EGCG. However, the mechanisms explaining the action of miRNA being modulated by EGCG have not been completely understood yet. This review summarizes the state of epigenetic change being modulated by EGCG in a variety of cancers and oncogenic and tumor-suppressor transcription factors.

Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains

Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function.

Impact of the STAT1 N-terminal domain for fibrosarcoma cell responses to ɣ-irradiation

AbstractType I/II interferons (IFNα,β/IFNɣ) are cytokines that activate signal-transducer-and-activator-of-transcription-1 (STAT1). The STAT1 N-terminal domain (NTD) mediates dimerization and cooperative DNA-binding. The STAT1 DNA-binding domain (DBD) confers sequence-specific DNA-recognition. STAT1 has been connected to growth inhibition, replication stress and DNA-damage. We investigated how STAT1 and NTD/DBD mutants thereof affect fibrosarcoma cells. STAT1 and indicated mutants do not affect proliferation of resting and IFNα-treated cells as well as checkpoint kinase signaling, and phosphorylation of the tumor-suppressive transcription factor p53 ensuing ɣ-irradiation. Of the STAT1 reconstituted U3A cells those with STAT1 NTD mutants accumulate the highest levels of the replication stress/DNA-damage marker S139-phosphorylated histone H2AX (ɣH2AX). This is similarly seen with a STAT1 NTD/DBD double mutant, indicating transcription-independent effects. Furthermore, U3A cells with STAT1 NTD mutants are most susceptible to apoptotic DNA fragmentation and cleavage of the DNA repair protein PARP1. These data provide novel insights into the relevance of the STAT1 NTD.

Inferring Leading Interactions in the p53/Mdm2/Mdmx Circuit through Live-Cell Imaging and Modeling.

The tumor-suppressive transcription factor p53 is a master regulator of stress responses. In non-stressed conditions, p53 is maintained at low levels by the ubiquitin ligase Mdm2 and its binding partner Mdmx. Mdmx depletion leads to a biphasic p53 response, with an initial post-mitotic pulse followed by oscillations. The mechanism underlying this dynamical behavior is unknown. Two different roles for Mdmx have been proposed: enhancing p53 ubiquitination by Mdm2 and inhibiting p53 activity. Here, we developed a mathematical model of the p53/Mdm2/Mdmx network to investigate which Mdmx functions quantitatively affect specific features of p53 dynamics under various conditions. We found that enhancement of Mdm2 activity was the most critical role of Mdmx under unstressed conditions. The model also accurately predicted p53 dynamics in Mdmx-depleted cells following DNA damage. This work outlines a strategy for rapidly testing possible network interactions to reveal those most impactful in regulating the dynamics of key proteins.

Neural Stem Cells of the Subventricular Zone as the Origin of Human Glioblastoma Stem Cells. Therapeutic Implications.

Human glioblastoma is the most aggressive type of primary malignant brain tumors. Standard treatment includes surgical resection followed by radiation and chemotherapy but it only provides short-term benefits and the prognosis of these brain tumors is still very poor. Glioblastomas contain a population of glioma stem cells (GSCs), with self-renewal ability, which are partly responsible for the tumor resistance to therapy and for the tumor recurrence after treatment. The human adult subventricular zone contains astrocyte-like neural stem cells (NSCs) that are probably reminiscent of the radial glia present in embryonic brain development. There are numerous molecules involved in the biology of subventricular zone NSCs that are also instrumental in glioblastoma development. These include cytoskeletal proteins, telomerase, tumor suppressor proteins, transcription factors, and growth factors. Interestingly, genes encoding these molecules are frequently mutated in glioblastoma cells. Indeed, it has been recently shown that NSCs in the subventricular zone are a potential cell of origin that contains the driver mutations of human glioblastoma. In this review we will describe common features between GSCs and subventricular zone NSCs, and we will discuss the relevance of this important finding in terms of possible future therapeutic strategies.

MiR-638 represses the stem cell characteristics of breast cancer cells by targeting E2F2.

The miR-638 acted as a tumor suppressor and E2F transcription factor 2 (E2F2) was a critical regulator in some cancers, while the role of them on stemness of breast cancer stem cells (BCSCs) was rarely detailed. Hence, we focused on exploring the effects of miR-638 and E2F2 on BCSCs stemness. The proportion of CD24 -/CD44 + cells of BCSCs was detected by flow cytometry. The target relationship of miR-638 and E2F2 was explored using luciferase assays. The ability of self-renewal, proliferation, and invasion of BCSCs were determined by Mammosphere forming, Cell Counting Kit-8 (CCK-8), colony formation, and transwell assays. Xenograft tumor was established to detect the influence of miR-638 on tumor growth. miR-638 was down-regulated, while E2F2 was elevated in breast cancer. The E2F2 level was negatively correlated with miR-638. The BCSCs represented higher proportion of CD24 -/CD44 + cells and levels of sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4). The miR-638 was down-regulated and E2F2 was increased in BCSCs. MiR-638 could target to E2F2 and decreased the level of E2F2 in BCSCs cells. Overexpression of miR-638 decreased the proportion of CD24 -/CD44 + cells and the levels of SOX2 and OCT4 by inhibiting E2F2. The overexpression of miR-638 also inhibited the abilities of self-renewal, proliferation, and invasion of BCSCs by inhibiting E2F2. The miR-638 overexpression inhibited the breast tumor growth. MiR-638 represses the characteristics and behaviors of BCSCs by targeting E2F2. MiR-638 may be a potential target for breast cancer therapy.

Abstract 4400: Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC16 is one of several subunits of the anaphase-promoting complex (APC) with crucial role in cell division. Here, we show evidence that expression of CDC16 in T-ALL is modulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a tumor suppressor protein and transcription factor. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both cell lines and primary human ALL, shows that Ikaros binds to the promoter of the CDC16 gene. Ikaros functions as a tumor suppressor protein and its deletion is associated with development of T-ALL. Ikaros binding to CDC16 promoter was confirmed via quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. The role of Ikaros in regulating CDC16 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA resulted in increased transcription of CDC16 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strong reduction in transcription of CDC16. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of CDC16. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC16 expression. Since Ikaros function in T-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of CDC16 in T-ALL. Overexpression of CK2 via retroviral transduction resulted in increased CDC16 gene transcription as measured by qRT-PCR, as well as increased overall expression of CDC16, as analyzed by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the CDC16 gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor led to reduced expression of CDC16 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC16. Ikaros knock-down restored high expression of CDC16 in T-ALL cells that were treated with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are major transcriptional regulators of CDC16 transcription in T-ALL and that inhibition of CK2 represses CDC16 transcription via Ikaros-mediated repression. In summary, these results provide evidence that expression of the CDC16 gene in T-ALL is regulated by the CK2 signaling pathway which modulates Ikaros funtion. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in preclinical models of T-ALL. Presented data reveals a novel mechanism of therapeutic action of CK2 inhibitors – repression of CDC16 expression via Ikaros. These results provide a rationale for the use of novel CK2 inhibitors in T-ALL to target CDC16. Citation Format: Pavan Kumar Dhanyam Raju, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jonathan Payne, Soumya Iyer, Nathalia Moreno Curry, Mary McGrath, Yevgeniya Bamme, Bihua Tan, Mario Soliman, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4400.

Styryl quinazolinones and its ethynyl derivatives induce myeloid differentiation.

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.

A novel proviral function for IL-17 during gammaherpesvirus infection

Abstract Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with B cell lymphomas. These viruses infect naïve B cells and induce a polyclonal germinal center (GC) response to establish long-term infection in memory B cells. We showed that Interferon Regulatory Factor 1 (IRF-1), an antiviral and tumor suppressor transcription factor, selectively restricts the GC reaction driven by murine gammaherpesvirus 68 (MHV68). Further, latent MHV68 infection was poorly controlled in IRF-1−/− mice, including in the peritoneal cavity where the latency is not regulated by the GC response. In our search for the mechanism underlying the observed phenotypes, we have unexpectedly found a significant increase in Th17 cells in MHV68 infected IRF-1−/− mice. Herpesvirus saimiri (HVS), a related simian gammaherpesvirus, encodes a viral IL-17, posing an intriguing possibility that IL-17 is proviral. MHV68 does not encode an IL-17 homologue, suggesting that MHV68 may rely on a host derived IL-17. Intriguingly, latent MHV68 infection was significantly attenuated in IL-17RA−/− mice establishing, for the first time, a proviral role of host IL-17 signaling during gammaherpesvirus infection. Further, there was a significant decrease in the GC B cell response in IL-17RA−/− mice following MHV68, but not LCMV infection, indicating that IL-17 signaling is selectively required to support the GC response during gammaherpesvirus infection. Altogether, our study establishes IRF-1 as a suppressor of Th17 differentiation and reveals a novel proviral role of IL-17 during gammaherpesvirus infection.

Lung cancer deficient in the tumor suppressor GATA4 is sensitive to TGFBR1 inhibition

Lung cancer is the leading cause of cancer-related deaths worldwide. Tumor suppressor genes remain to be systemically identified for lung cancer. Through the genome-wide screening of tumor-suppressive transcription factors, we demonstrate here that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vivo. Ectopic GATA4 expression results in lung cancer cell senescence. Mechanistically, GATA4 upregulates multiple miRNAs targeting TGFB2 mRNA and causes ensuing WNT7B downregulation and eventually triggers cell senescence. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGF-β2 level and is significantly associated with poor prognosis. TGFBR1 inhibitors show synergy with existing therapeutics in treating GATA4-deficient lung cancers in genetically engineered mouse model as well as patient-derived xenograft (PDX) mouse models. Collectively, our work demonstrates that GATA4 functions as a tumor suppressor in lung cancer and targeting the TGF-β signaling provides a potential way for the treatment of GATA4-deficient lung cancer.

Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.

Ginger: A Novel Strategy to Battle Cancer through Modulating Cell Signalling Pathways: A Review.

Numerous studies have been performed in understanding the development of cancer. Though, the mechanism of action of genes in the development of cancer remains to be explained. The current mode of treatment of cancer shows adverse effects on normal cells and also alter the cell signalling pathways. However, ginger and its active compound have fascinated research based on animal model and laboratories during the past decade due to its potentiality in killing cancer cells. Ginger is a mixture of various compounds including gingerol, paradol, zingiberene and shogaol and such compounds are the main players in diseases management. Most of the health-promoting effects of ginger and its active compound can be attributed due to its antioxidant and anti-tumour activity. Besides, the active compound of ginger has proven its role in cancer management through its modulatory effect on tumour suppressor genes, cell cycle, apoptosis, transcription factors, angiogenesis and growth factor. In this review, the role of ginger and its active compound in the inhibition of cancer growth through modulating cell signalling pathways will be reviewed and discussed.

Intra-Tumor Heterogeneity in Acute Myeloid Leukemia (AML): Results from a Real Life Cohort

Context. AML is an oligoclonal disease. Intratumoral genetic heterogeneity (ITH) is pervasive in cancers and has often been linked to poor outcome. Except in patients (pts) with complex karyotypes (Bochtler, J Clin Oncol 2013), the clinical relevance of ITH remains unknown in AML. We assessed ITH in a real-life AML cohort.Methods. Since Nov 2015, all newly diagnosed AML pts at our institution were analyzed by targeted sequencing with a 60-gene myeloid panel at an average depth of 815X. Variant Allele Frequencies (VAF) were adjusted for copy number variation to estimate Cancer Cell Fractions (CCFs) and rank mutations by decreasing CCFs. Mutations were segregated into different clones when distant by >10% CCF. Ancestral (as opposed to secondary) mutations are defined as those belonging to the first clone (Figure). We could next derive for each pt the total number of clones and the ITH index, defined as the ratio of secondary to ancestral mutations (according to Turajlic, Cell 2018).Results. We studied 201 consecutive AML pts (median age 67 years, secondary AML 24%). The median number of mutations was 4 (range 0-12). Clonal heterogeneity (>1 clone) was found in 71% of pts with a median of 2 clones per pt (range 1-5). 140 pts received intensive chemotherapy (IC), 50 hypomethylating agents (HMA) and 11 best supportive care (BSC) only.Signaling (FLT3, NRAS, KRAS, KIT, JAK2, CBL, CSF3R, BRAF, NF1, PTPN11, RIT1, CALR and MPL, 60%), DNA methylation (DNTM3A, TET2, IDH1/2, 54%), NPM1 (29%), spliceosome (U2AF1, SRSF2, SF3B1, ZRSR2, PRPF8, 27.9%) and tumor suppressors (PHF6, TP53, WT1, 27.4%) were the most frequently mutated pathways. ELN 2017 risk category was favorable, intermediate, adverse and not evaluable in 35%, 27%, 35% and 3% of pts respectively. Increasing ITH was associated with a higher number of clones (p=0.001) and of mutations (p<0.001). ITH was greater in pts with at least one mutation in histone modifiers (ASXL1, ASXL2, EZH2, KMT2A-PTD KMT2D, KDM6A, p=0.0004), cohesin (STAG2, RAD21, SMC3, SMC1A, CTCF, p=0.0018) or signaling (p=0.002) genes but was not affected by the presence of mutations in DNA methylation or spliceosome, tumor suppressor genes, transcription factors (RUNX1, CEBPA, ETV6, GATA2) or co-activators (BCOR, BCORL1, EP300, CREBBP), all p>0.05. ITH was not different among ELN 2017 risk categories (p=0.15). ITH was independent of age, WBC count and type of AML (secondary vs de novo), all p>0.2.There was no significant difference in the number of mutations (p=0.12), clones (p=0.58) or ITH indexes (p=0.38) according to IC/HMA/BSC treatment strategies. We next performed a preliminary analysis of the prognostic impact of ITH in the 140 pts treated with IC (median age 58y), with a median follow-up of only 12.5 months. 117 (84%) pts achieved CR and 32 (23%) were transplanted in first CR. Median event-free survival (EFS) was 18.7 vs 8.0 months in pts with adv and non-adv ELN risk, respectively (p=0.016). In univariate analyses, DNA methylation (p=0.04) and spliceosome (p=0.007) mutations were the two pathways predicting adverse outcome. The number of mutations (p=0.6) or of clones (p=0.8) had no impact on EFS. Pts with fewer secondary than ancestral mutations (ITH <1, ‘ITH low’ group, n=64 [46%]) had a median EFS of 13.6 months vs 19.8 months in the 76 (54%) pts with ITH index ≥1 (‘ITH high’ group, p=0.067). In a multivariate Cox model accounting for age, log(WBC), ELN risk, AML type (secondary vs de novo), presence of DNA methylation and spliceosome mutations and ITH, ITH-high was identified as a significant predictor of improved EFS (HR=0.54, p=0.03), independently of older age (HR=1.02, p=0.07), adv ELN risk (HR=2.4, p=0.005) and higher WBC count (HR=1.27, p=0.004). Censoring at transplant in first CR did not affect this result. In 14 pts who relapsed after a median of 12.9 months since diagnosis with paired NGS at relapse, the number of clones significantly decreased at relapse (p=0.046).Conclusion. Our results confirm that most AMLs are oligoclonal. Surprisingly, higher ITH is not related to older age or secondary AML. Our preliminary results suggest that lower ITH translates into shorter EFS in pts treated with IC. Longer follow-up and/or increased pt numbers are warranted to determine whether NGS-based assessments of clonal heterogeneity can improve AML risk stratification. [Display omitted] DisclosuresFenaux:Otsuka: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

PAX3: A Molecule with Oncogenic or Tumor Suppressor Function Is Involved in Cancer

Metastasis is the most deadly aspect of cancer and results from acquired gene regulation abnormalities in tumor cells. Transcriptional regulation is an essential component of controlling of gene function and its failure could contribute to tumor progression and metastasis. During cancer progression, deregulation of oncogenic or tumor suppressive transcription factors, as well as master cell fate regulators, collectively influences multiple steps of the metastasis cascade, including local invasion and dissemination of the tumor to distant organs. Transcription factor PAX3/Pax3, which contributes to diverse cell lineages during embryonic development, plays a major role in tumorigenesis. Mutations in this gene can cause neurodevelopmental disease and the existing literature supports that there is a potential link between aberrant expression of PAX3 genes in adult tissues and a wide variety of cancers. PAX3 function is tissue-specific and could contribute to tumorigenesis either directly as oncogene or as a tumor suppressor by losing its function. In this review, we discuss comprehensively the differential role played by PAX3 in various tissues and how its aberrant expression is implicated in disease development. This review particularly highlights the oncogenic and tumor suppressor role played by PAX3 in different cancers and underlines the importance of precisely identifying tissue-specific role of PAX3 in order to determine its exact role in development of cancer.

Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking

Arginine methylation has emerged as a widespread and reversible protein modification with the potential to regulate a multitude of cellular processes, but its function is poorly understood. Endolysosomes play an important role in Wnt signaling, in which glycogen synthase kinase 3 (GSK3) becomes sequestered inside multivesicular bodies (MVBs) by the process known as microautophagy, causing the stabilization of many proteins. Up to 20% of cellular proteins contain three or more consecutive putative GSK3 sites, and of these 33% also contain methylarginine (meArg) modifications. Intriguingly, a cytoskeletal protein was previously known to have meArg modifications that enhanced subsequent phosphorylations by GSK3. Here, we report the unexpected finding that protein arginine methyltransferase 1 (PRMT1) is required for canonical Wnt signaling. Treatment of cultured cells for 5-30 min with Wnt3a induced a large increase in total endocytic vesicles which were also positive for asymmetric dimethylarginine modifications. Protease protection studies, both biochemical and in situ in cultured cells, showed that many meArg-modified cytosolic proteins became rapidly translocated into MVBs together with GSK3 and Lys48-polyubiquitinated proteins by ESCRT-driven microautophagy. In the case of the transcription factor Smad4, we showed that a unique arginine methylation site was required for GSK3 phosphorylation and Wnt regulation. The enzyme PRMT1 was found to be essential for Wnt-stimulated arginine methylation, GSK3 sequestration, and canonical Wnt signaling. The results reveal a cell biological role for PRMT1 arginine methylation at the crossroads of growth factor signaling, protein phosphorylation, membrane trafficking, cytosolic proteolysis, and Wnt-regulated microautophagy.

How mutations shape p53 interactions with the genome to promote tumorigenesis and drug resistance.

The tumor suppressive transcription factor p53 regulates a wide array of cellular processes that confer upon cells an essential protection against cancer development. Wild-type p53 regulates gene expression by directly binding to DNA in a sequence-specific manner. p53 missense mutations are the most common mutations in malignant cells and can be regarded as synonymous with anticancer drug resistance and poor prognosis. The current review provides an overview of how the extraordinary variety of more than 2000 different mutant p53 proteins, known as the p53 mutome, affect the interaction of p53 with DNA. We discuss how the classification of p53 mutations to loss of function (LOF), gain of function (GOF), and dominant-negative (DN) inhibition of a remaining wild-type allele, hides a complex p53 mutation spectrum that depends on the distinctive nature of each mutant protein, requiring different therapeutic strategies for each mutant p53 protein. We propose to regard the different mutant p53 categories as continuous variables, that may not be independent of each other. In particular, we suggest here to consider GOF mutations as a special subset of LOF mutations, especially when mutant p53 binds to DNA through cooperation with other transcription factors, and we present a model for GOF mechanism that consolidates many observations on the GOF phenomenon. We review how novel mutant p53 targeting approaches aim to restore a wild-type-like DNA interaction and to overcome resistance to cancer therapy.

Design, in silico prioritization and biological profiling of apoptosis-inducing lactams amenable by the Castagnoli-Cushman reaction

Five lactam chemotypes amenable by the Castagnoli-Cushman reaction of imines and cyclic anhydrides have been investigated for their ability to activate p53 tumor suppressing transcription factor thus induce apoptosis in p53+ cancer cells. A virtual library of 1.07 million chemically diverse compounds based on these scaffolds was subjected to in silico screening first. The compounds displaying high docking score were visually prioritized to identify the best-fitting compounds, i. e. the ones which adequately mimic the interactions of clinical candidate inhibitor Nutlin-3a. These 38 compounds were synthesized and tested for apoptosis induction in p53+ H116 cancer cells to identify 9 potent apoptosis-inducers (two of them exceeding the activity of Nutlin-3a) which belonged to four different chemotypes. The activation of p53 involved in the proapoptotic activity observed was supported by effective induction of EGFP expression in human osteocarcinoma U2OS-pLV reporter cell line. Moreover, the two most potent apoptosis inducers displayed antiproliferative profile identical to several known advanced p53 activators: they inhibited the growth of p53+/+ HCT116 cells in much lower concentration range compared to p53−/− HCT116 cells.