Mutations In DNA-binding Domain Research Articles

Generation of THRB-GS(E125G_G126S) and THRB-KO human iPSC lines to study noncanonical thyroid hormone signalling

THRB is a nuclear receptor, regulating gene expression dependent on thyroid hormone (TH) binding. The same receptor mediates signaling pathway activation in the cytosol. The challenge is to distinguish which of the two mechanisms is responsible for physiological effects of TH. We established an iPSC cell line with two mutations (E125G_G126S) in the THRB DNA-binding domain, which abrogates nuclear action and, thus, allows to study signaling pathway activation exclusively. We also generated a THRB knockout cell line to abolish all THRB effects. Comparison of WT and these two cell lines allows attribution of thyroid hormone effects to the underlying mechanism.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer

PurposeHow to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored.MethodBlood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen.ResultsOverall, the median PFS was 8.3 months (95% CI 5.7–11.1 months). The median cfDNA was 38.5 ng (range 4.4–1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA.ConclusionAfter adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

The human Stat1 gain-of-function T385M mutation causes expansion of activated T-follicular helper/T-helper 1-like CD4 T cells and sex-biased autoimmunity in specific pathogen-free mice.

Humans with gain-of-function (GOF) mutations in STAT1 (Signal Transducer and Activator of Transcription 1), a potent immune regulator, experience frequent infections. About one-third, especially those with DNA-binding domain (DBD) mutations such as T385M, also develop autoimmunity, sometimes accompanied by increases in T-helper 1 (Th1) and T-follicular helper (Tfh) CD4 effector T cells, resembling those that differentiate following infection-induced STAT1 signaling. However, environmental and molecular mechanisms contributing to autoimmunity in STAT1 GOF patients are not defined. We generated Stat1T385M/+ mutant mice to model the immune impacts of STAT1 DBD GOF under specific-pathogen free (SPF) conditions. Stat1T385M/+ lymphocytes had more total Stat1 at baseline and also higher amounts of IFNg-induced pStat1. Young mutants exhibited expansion of Tfh-like cells, while older mutants developed autoimmunity accompanied by increased Tfh-like cells, B cell activation and germinal center (GC) formation. Mutant females exhibited these immune changes sooner and more robustly than males, identifying significant sex effects of Stat1T385M-induced immune dysregulation. Single cell RNA-Seq (scRNA-Seq) analysis revealed that Stat1T385M activated transcription of GC-associated programs in both B and T cells. However, it had the strongest transcriptional impact on T cells, promoting aberrant CD4 T cell activation and imparting both Tfh-like and Th1-like effector programs. Collectively, these data demonstrate that in the absence of overt infection, Stat1T385M disrupted naïve CD4 T cell homeostasis and promoted expansion and differentiation of abnormal Tfh/Th1-like helper and GC-like B cells, eventually leading to sex-biased autoimmunity, suggesting a model for STAT1 GOF-induced immune dysregulation and autoimmune sequelae in humans.

Disease-related p63 DBD mutations impair DNA binding by distinct mechanisms and varying degree

The transcription factor p63 shares a high sequence identity with the tumour suppressor p53 which manifests itself in high structural similarity and preference for DNA sequences. Mutations in the DNA binding domain (DBD) of p53 have been studied in great detail, enabling a general mechanism-based classification. In this study we provide a detailed investigation of all currently known mutations in the p63 DBD, which are associated with developmental syndromes, by measuring their impact on transcriptional activity, DNA binding affinity, zinc binding capacity and thermodynamic stability. Some of the mutations we have further characterized with respect to their ability to convert human dermal fibroblasts into induced keratinocytes. Here we propose a classification of the p63 DBD mutations based on the four different mechanisms of DNA binding impairment which we identified: direct DNA contact, zinc finger region, H2 region, and dimer interface mutations. The data also demonstrate that, in contrast to p53 cancer mutations, no p63 mutation induces global unfolding and subsequent aggregation of the domain. The dimer interface mutations that affect the DNA binding affinity by disturbing the interaction between the individual DBDs retain partial DNA binding capacity which correlates with a milder patient phenotype.

Abstract NG03: Multiomics in disparities research: Identifying African ancestry-specific mechanisms in the triple negative breast cancer tumor microenvironment

Abstract NG03: Multiomics in disparities research: Identifying African ancestry-specific mechanisms in the triple negative breast cancer tumor microenvironment

Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities

Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.

Elucidation of the DNA-Binding Activity of VirF from Shigella flexneri for the icsA and rnaG Promoters and Characterization of the N-Terminal Domain To Identify Residues Crucial for Dimerization.

A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid β-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.

42P Efficacy of maintenance with PARPi in advanced ovarian cancer according to the location of BRCA mutation

Currently little is known about correlations between the type and the location of the BReast CAncer genes (BRCA) 1/2 mutations and response to Poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian cancer (OC). Clinical data from PAOLA1 study suggest that patients with mutations in DNA binding domain (DNA-BD) of BRCA1 are more sensitive to the combination olaparib/bevacizumab and those in the DNA-BD of BRCA2 confer excellent outcomes. We investigated if the type and the location of the BRCA 1/2 mutations are related with PARPi sensitivity. This multicenter and retrospective study evaluated the efficacy of PARPi according to the mutation type and location in the functional domain (FD) of BRCA1 (RING, DNA-BD or BRCA1 C terminus [BRCT]) and BRCA2 (PALB2, RAD51-BD; DNA-BD). BRCA1/2 germline mutated OC patients receiving PARPi as maintenance, after a response to platinum-based first line chemotherapy were enrolled. Baseline characteristics and survival data were collected from 3 academic centers. Of 202 patients treated between December 2008 and October 2021, 122 (60.4%) received PARPi in the first-line setting and 80 (39.6%) at recurrence. Among the first group, 73 (60%) and 49 (40%) harbored BRCA1 and BRCA2 mutations, respectively. BRCA1 mutations in FDs of RING, DNA-BD and BRCT were found in 3 (2.5%), 8 (6.6%) and 21 (17.2%) patients, respectively. BRCA2 mutation were detected in FDs of RAD51-BD and DNA-BD in 25 (20.5%) and 5 (4.1%) patients, respectively. Median follow-up was of 20 (4-100) months. In patients with DNA-BD mutation of BRCA1/2, 2-yr Progression Free Survival PFS was 100%, compared with 80% in mutation in the other domains (log rank p= 0.079). With regard to the mutation type, patients harboring a missense BRCA1 mutation had a longer PFS (2-yr PFS 100%), compared with 50% of those with a splicing mutation (log rank p= 0.021) and 75% of those with a nonsense mutation (log rank p= 0.049). Mutation types in BRCA2 were not analyzed due to the small numbers. The results confirmed that mutations in the DNA-BD region in the BRCA 1/2 genes confer longer PFS respect to other location of mutation. Besides, missense mutation has the best prognosis, compared with splicing and nonsense mutation.

Unraveling the Structural Changes in the DNA-Binding Region of Tumor Protein p53 (TP53) upon Hotspot Mutation p53 Arg248 by Comparative Computational Approach

The vital tissue homeostasis regulator p53 forms a tetramer when it binds to DNA and regulates the genes that mediate essential biological processes such as cell-cycle arrest, senescence, DNA repair, and apoptosis. Missense mutations in the core DNA-binding domain (109-292) simultaneously cause the loss of p53 tumor suppressor function and accumulation of the mutant p53 proteins that are carcinogenic. The most common p53 hotspot mutation at codon 248 in the DNA-binding region, where arginine (R) is substituted by tryptophan (W), glycine (G), leucine (L), proline (P), and glutamine (Q), is reported in various cancers. However, it is unclear how the p53 Arg248 mutation with distinct amino acid substitution affects the structure, function, and DNA binding affinity. Here, we characterized the pathogenicity and protein stability of p53 hotspot mutations at codon 248 using computational tools PredictSNP, Align GVGD, HOPE, ConSurf, and iStable. We found R248W, R248G, and R248P mutations highly deleterious and destabilizing. Further, we subjected all five R248 mutant-p53-DNA and wt-p53-DNA complexes to molecular dynamics simulation to investigate the structural stability and DNA binding affinity. From the MD simulation analysis, we observed increased RMSD, RMSF, and Rg values and decreased protein-DNA intermolecular hydrogen bonds in the R248-p53-DNA than the wt-p53-DNA complexes. Likewise, due to high SASA values, we observed the shrinkage of proteins in R248W, R248G, and R248P mutant-p53-DNA complexes. Compared to other mutant p53-DNA complexes, the R248W, R248G, and R248P mutant-p53-DNA complexes showed more structural alteration. MM-PBSA analysis showed decreased binding energies with DNA in all five R248-p53-DNA mutants than the wt-p53-DNA complexes. Henceforth, we conclude that the amino acid substitution of Arginine with the other five amino acids at codon 248 reduces the p53 protein's affinity for DNA and may disrupt cell division, resulting in a gain of p53 function. The proposed study influences the development of rationally designed molecular-targeted treatments that improve p53-based therapeutic outcomes in cancer.

Pathogenic RUNX1 C-Terminal Truncation Mutations Contribute to Differentiation Block and Result in Unique Changes in Transcription and Chromatin Organization

The transcription factor RUNX1 is one of the most frequently mutated genes in hematological disorders, including familial platelet disorder (FPD), predisposition to myeloid dysplastic syndromes (MDS) and acute myeloid leukemia (AML). In addition to a large number of mutations in the N-terminal DNA binding domain (RHD) of RUNX1, 27% of all hematopoietic mutations lie within the C-terminal region beyond the RHD. Previous studies have mainly focused on loss of function RHD mutations while C-terminal mutations are not well characterized and lack "hotspot" mutations. The majority of mutations in the C-terminal region are frameshift and nonsense mutations rather than missense. Some of these mutations are thought to be subject to degradation by nonsense-mediated decay (NMD), emulating loss of RUNX1. Here we demonstrate that RUNX1 C-terminal truncation mutations represent an alternative means of RUNX1 pathogenesis beyond loss of RUNX1 and RHD mutations. To examine the prevalence of RUNX1 C-terminal mutations we first analyzed publicly available data (COSMIC - Sanger Institute) and identified 247 RUNX1 nonsense/frameshift mutations C-terminal of the RHD in patients with blood disorders. Importantly, 76% of these mutations are predicted to escape Nonsense Mediated Decay (NMD) and produce protein products. To study this class of RUNX1 mutation, we introduced the pathogenic RUNX1R320* mutation into the endogenous RUNX1 locus of the human blood cell line K562 using CRISPR-Cas9. We experimentally validated that the RUNX1R320* transcript is not subject to NMD and is expressed at significantly higher levels than the wild-type at both the RNA and protein level. The resulting truncated protein retains the DNA binding RHD but lacks C-terminal domains for binding to other cofactors. To identify biological changes caused by the RUNX1R320* mutation we examined effects on proliferation, differentiation, and DNA damage. Although no significant change in proliferation was observed, megakaryocytic differentiation was impaired. After TPA treatment RUNX1R320* cells failed to fully differentiate and retained high levels of the erythroid marker CD235a. Furthermore, RUNX1R320* showed sensitivity to DNA damage after treatment with etoposide while cell death via apoptosis was not significantly affected. Together these data suggest that C-terminal truncation RUNX1 contributes to differentiation block and an increase in DNA damage sensitivity while evading apoptotic cell signaling pathways. As RUNX1 functions as a master transcriptional regulator of hematopoiesis, we examined how RUNX1 truncations alter transcription and if the effects emulate RUNX1 loss of function mutations by performing RNA-seq with parental, RUNX1 knockdown, and RUNX1R320* knock-in cells. We first identified 2880 significantly differentially expressed genes (DEGs) between wild-type and RUNX1R320*. When compared to RUNX1 knockdown data, we found that the majority of RUNX1R320* DEGs (2052) were unique to RUNX1R320*. This demonstrates that RUNX1R320* results in different transcriptional perturbations than the loss of RUNX1. We next asked whether changes in chromatin confirmation attributed to the observed transcriptional alterations. To study this the newly developed technology Global RNA Interactions with DNA by deep sequencing (GRID-seq) was performed on wild-type and RUNX1R320* cells. Combining these data with H3K27ac and H3K4me3 ChIP-seq data to identify active enhancers and promoters, respectively, we detected 2,891 significantly altered enhancer-promoter (E-P) pairs. Focusing on DEGs with GRID-seq detected enhancer RNA (eRNA), we conducted Gene Ontology (GO) analysis and found enrichment in pathways of chronic myeloid leukemia, regulation of hematopoietic stem cell differentiation, and cell division, revealing the effects of RUNX1R320* on key hematopoietic and myeloid pathways. In summary, we show that RUNX1 C-terminal truncation mutants can escape NMD, contribute to differentiation block, and increase sensitivity to DNA damage. Furthermore, RUNX1R320* affects transcription in a manner distinct from RUNX1 loss. In our GRID-seq analysis, we identify changes in enhancer-promoter interactions that affect key hematological pathways. Our findings demonstrate that pathogenic RUNX1 C-terminal mutations disrupt hematopoietic cell function, transcription, and chromatin in a distinctive fashion.

Cardioprotection by Hypothyroidism Is Not Mediated by Favorable Hemodynamics-Role of Canonical Thyroid Hormone Receptor Alpha Signaling.

Hypothyroidism has been shown to reduce infarct size in rats, but the underlying mechanisms are unclear. We used isolated pressure-constant perfused hearts of control, hypothyroid and hyperthyroid mice and measured infarct size, functional parameters and phosphorylation of key molecules in cardioprotective signaling with matched heart rate. Compared with controls, hypothyroidism was cardioprotective, while hyperthyroidism was detrimental with enlarged infarct size. Next, we asked how thyroid hormone receptor α (TRα) affects ischemia/reperfusion (IR) injury. Thus, canonical and noncanonical TRα signaling was investigated in the hearts of (i) mice lacking TRα (TRα0), (ii) with a mutation in TRα DNA-binding domain (TRαGS) and (iii) in hyperthyroid TRα0 (TRα0hyper) and TRαGS mice (TRαGShyper). TRα0 mouse hearts were protected against IR injury. Furthermore, infarct size was reduced in the hearts of TRαGS mice that lack canonical TRα signaling but maintain noncanonical TRα action. Hyperthyroidism did not increase infarct size in TRα0 and TRαGS mouse hearts. These cardioprotective effects were not associated with increased phosphorylation of key proteins of RISK, SAFE and eNOS pathways. In summary, chronic hypothyroidism and the lack of canonical TRα signaling are cardioprotective in IR injury and protection is not due to favorable changes in hemodynamics.

Recruitment of the CoREST transcription repressor complexes by Nerve Growth factor IB-like receptor (Nurr1/NR4A2) mediates silencing of HIV in microglial cells.

Human immune deficiency virus (HIV) infection in the brain leads to chronic neuroinflammation due to the production of pro-inflammatory cytokines, which in turn promotes HIV transcription in infected microglial cells. However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression after viral reactivation to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts directly to restrict HIV expression. HIV silencing following activation by TNF-α, or a variety of toll-like receptor (TLR) agonists, in both immortalized human microglial cells (hμglia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG) was enhanced by Nurr1 agonists. Similarly, overexpression of Nurr1 led to viral suppression, while conversely, knock down (KD) of endogenous Nurr1 blocked HIV silencing. The effect of Nurr1 on HIV silencing is direct: Nurr1 binds directly to the specific consensus binding sites in the U3 region of the HIV LTR and mutation of the Nurr1 DNA binding domain blocked its ability to suppress HIV-1 transcription. Chromatin immunoprecipitation (ChIP) assays also showed that after Nurr1 binding to the LTR, the CoREST/HDAC1/G9a/EZH2 transcription repressor complex is recruited to the HIV provirus. Finally, transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, further promoting HIV latency and microglial homoeostasis. Nurr1 therefore plays a pivotal role in modulating the cycles of proviral reactivation by potentiating the subsequent proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and ultimately for the amelioration of the neuroinflammation associated with HIV-associated neurocognitive disorders (HAND).

Clinical Characteristics of Patients With HNF1-alpha MODY: A Literature Review and Retrospective Chart Review.

The clinical manifestation of hepatocyte nuclear factor-1-alpha (HNF1-alpha) maturity-onset diabetes of the young (MODY) is highly variable. This study aims to investigate the clinical characteristics of patients with HNF1-alpha MODY in general, by geographical regions (Asian or non-Asian), HNF1-alpha mutations, and islet autoantibody status. A literature review and a chart review of patients with HNF1-alpha MODY were performed. The means and proportions from studies were pooled using the inverse variance method for pooling, and subgroup analyses were performed. A total of 109 studies involving 1,325 patients [41.5%, 95% confidence interval (CI): 35.2, 48.1; male] were identified. The mean age of diagnosis was 20.3 years (95% CI: 18.3–22.2), and the mean glycated hemoglobin was 7.3% (95% CI: 7.2–7.5). In comparison, Asian patients exhibited significantly higher HbA1c (p = 0.007) and 2-h post-load C-peptide (p = 0.012) levels and lower levels of triglyceride (TG) (p < 0.001), total cholesterol (TC) (p < 0.001), and high-density lipoprotein cholesterol (HDL-c) (p < 0.001) and less often had macrovascular complications (p = 0.014). The age of diagnosis was oldest in patients with mutations in the transactivation domain (p < 0.001). The levels of 2-h post-load C-peptide (p < 0.001), TG (p = 0.007), TC (p = 0.017), and HDL-c (p = 0.001) were highest and the prevalence of diabetic neuropathy was lowest (p = 0.024) in patients with DNA-binding domain mutations. The fasting (p = 0.004) and 2-h post-load glucose (p = 0.003) levels and the prevalence of diabetic neuropathy (p = 0.010) were higher among patients with positive islet autoantibodies. The study demonstrated that the clinical manifestations of HNF1-alpha MODY differed by geographical regions, HNF1-alpha mutations, and islet autoantibody status.

Comparative genomic analysis and its prognostic impact on survival between viral hepatitis-related and non-viral hepatitis intrahepatic cholangiocarcinoma.

4120 Background: Intrahepatic cholangiocarcinoma (IHCC) is hard-to-treat cancer with a high mortality rate worldwide. Hepatitis B virus (HBV) or hepatitis C virus (HCV) is involved in the development of IHCC, especially in Asian countries. Despite rapidly growing genomic profiling studies of IHCC in Western and Eastern populations in recent years, less on genomic heterogeneity of viral hepatitis-related and non-viral hepatitis IHCC been reported. This study aims to provide a comprehensive genomic analysis of IHCC and its prognostic value in IHCC populations with or without viral hepatitis infection. Methods: FFPE tissues from 157 patients with IHCC were subject to next-generation sequencing using the FoundationOne CDx (n = 52) or ACTOnco + comprehensive genomic profiling panels (n = 105). Genomic alterations and features, including single nucleotide variations (SNVs), short insertions and deletions (InDels), copy-number variations (CNVs), fusion genes (only FoundationOne CDx), tumor mutations burden (TMB), and microsatellite instability (MSI) status, were analyzed. The prevalence of genetic mutations and their prognostic values were compared between patients with a history of hepatitis B/C infection (BC; n = 71, 45.2%) and those without hepatitis B/C infection (NBNC; n = 79, 50.3%). Seven patients had no medical record of HBV or HCV infection. Results: The most frequently mutated genes in BC- and NBNC-related IHCC populations were TP53, KRAS, and IDH1. The genetic alternations related to PI3K/AKT/mTOR signaling pathway and copy number gain of receptor tyrosine kinase were relatively dominant in BC-related IHCC. In contrast, the Ras/Raf/MAPK pathway alterations were frequently common in NBNC-related IHCC. In addition, we investigated the correlation between commonly altered genes or pathways and overall survival (OS). Interestingly, the prognostic biomarkers in BC- and NBNC-related IHCC were significantly different. The results showed that TP53 DNA-binding domain (DBD) and TET2 mutations were associated with poor prognosis in BC-related IHCC. On the contrary, CDKN2A deletion and Ras/Raf/MAPK pathway alteration were associated with inferior prognosis in NBNC-related IHCC. However, neither the PI3K/AKT/mTOR signaling alteration nor IDH1/2 mutation affected the OS in BC- or NBNC-related IHCC. Notably, we found that SWI/ SNF complex involved in ARID1A, ARID1B, ARID2, PBRM1, and SMARCA4 alterations exhibited a beneficial prognosis in BC-related IHCC patients, which has not been discussed in previous studies. Conclusions: This study provides comparative genomic profiling between BC- and NBNC-related IHCC and shows genomic heterogeneity and different dominant prognostic biomarkers and activated signaling pathways. Different treatment strategies should be considered in these two subpopulations based on these results.

Mutations in DNA binding domain of p53 impede RSL1D1-p53 interaction to escape from degradation in human colorectal cancer cells

Different from the nucleolus-specific localization in some types of cancer cells, ribosomal L1 domain-containing protein 1 (RSL1D1) distributes throughout the nucleus in human colorectal cancer (CRC) cells. RSL1D1 directly interacts with DNA binding domain (aa 93–292) of wild-type p53 (p53-WT) and thereby recruits p53 to HDM2. The ensuing formation of RSL1D1/HDM2/p53 complex enhances p53 ubiquitination and decreases the protein level of p53 in CRC cells. In this study, we investigated the interaction between RSL1D1 and mutant p53 proteins. We first corroborated that aa 93–224 of p53 is a more precise domain for RSL1D1 binding and mutation in either aa 93–224 or aa 225–292 domain of p53 affects RSL1D1-p53 interaction. R175H mutated p53 does not interact with RSL1D1, whereas R273H mutated p53 still can bind to RSL1D1 but showing a remarkably decreased affinity than p53-WT. Although p53-R273H retains a weakened binding affinity with RSL1D1, it can hardly be recruited to HDM2 by RSL1D1 in HCT116 CRC cells. Accordingly, RSL1D1 loses its capacity to negatively regulate either R175H or R273H p53 mutant via directly interaction in HCT116 cells, thereby facilitating p53 mutants to accumulate and gain oncogenic function. Our findings help explain why mutant p53 proteins are more stable than p53-WT in CRC cells.

Versatility of berberine as an effective immunomodulator and chemo sensitizer against p53 mutant cell

Cancer is the leading cause of death among individuals due to its poor prognosis. Various therapeutics treatments are available in form radiation therapy, chemotherapy, or immunotherapy but major point of concern is the treatment of cancer resistant cell lines. Homozygous loss of the p53 gene is virtually present in every type of cancer. Mutation in DNA binding domain of p53 leads to formation of mutant forms having altered amino acid sequence which lacks DNA binding activity. Berberine is chemo-sensitizing isoquinoline quaternary alkaloid molecule obtained from Berberis vulgaris. Berberine has the capability to suppress the growth of broad range of tumors. It exhibits pharmacological, biochemical and anticancer properties which can potentiate the activities of the existing therapeutics available in a way that it can re-sensitize the cancer resistant clones. Berberine has an immanent potential to bind with DNA and can communicate with several cellular targets, further it also shows hormetic effect which refers to biphasic dose response curve in order to determine dose dependent stimulatory and inhibitory effect. Mode of action involved is yet not well understood but mechanistic pathway involved are autophagy, up-regulation of tumor-suppressor gene (p53) and epigenetic alterations in the viral DNA. In this review, versatility of berberine can be utilized ideally or in combination with chemotherapeutics drugs to potentiate chemo sensitization of the resistant cancer cell line. Further, cancer cell specific receptor targeting can also be employed in combination with berberine for therapeutic treatment of metastasizing cancer cells.

A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2.

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.

DNA-Binding domain mutations confer severe outcome at an early age among STAT1 gain-of-function patients.

STAT1gain-of-function (GOF) is an immune dysregulatory disorder with poorly studied genotype-phenotype correlation, impeding prognostication and early intervention. Given previous mechanistic studies, as well as anecdotal clinical reports, we sought to systematically determine whether DNA-binding domain (DBD) mutations in STAT1 result in a different phenotype than mutations in other gene domains. Negative prognostic features previously identified by the International STAT1 GOF Study Group (invasive infections, intracranial aneurysms, and malignancy), as well as other clinical features and mortality, were compared within a cohort of 30 patients with STAT1 GOF diagnosed at our center, consisting of 9 patients with DBD mutations and 21 patients with non-DBD mutations. We subsequently re-analyzed mortality data from a large, previously-published 274-patient cohort by the International STAT1 GOF Study Group. While no differences were noted with respect to malignancy or symptomatic aneurysms, invasive /opportunistic infections were substantially more common among DBD patients, as were sinopulmonary infections, bronchiectasis, enteropathy, endocrinopathies, lymphoproliferative manifestations, and recurrent fevers/HLH. DBD patients also had a lower probability of survival and younger age of mortality compared with non-DBD patients. Our re-evaluation of the published data from the International STAT1 GOF Study Group revealed a similar finding of earlier mortality among patients harboring DBD mutations. We report that STAT1 GOF patients with DBD mutations may be regarded as a unique subgroup, impacted more by early-onset profound combined immunodeficiency and with earlier mortality. These findings may impact clinical decision making with respect to early intervention, and in particular hematopoietic stem cell transplant considerations, in such patients.

P53 Frameshift Mutations Couple Loss-of-Function with Unique Neomorphic Activities.

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss. IMPLICATIONS: p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Associated with Congenital Heart Defects

Congenital Heart Defects (CHDs) is the most common neonatal disease in the U.S. with over 100,000 new reported cases each year. CHDs are characterized by malformations in the heart's chambers, walls and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) NKX2-5 and TBX5 play a critical role in developmental and physiological processes of the heart by regulating gene expression through DNA-protein interactions. Genetic analyses have identified various non-synonymous mutations within the DNA-binding domain (DBD) of NKX2-5 (Homeodomain, HD) and TBX5 (T-box) in association with CHDs. We hypothesize that non-synonymous mutations in cardiac TFs DNA-binding domain (DBD) will alter their DNA-binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we study how the cardiac TFs DNA-protein interactomes are affected by non-synonymous mutations found in CHD patients. We have cloned, over-expressed and purified functional DBDs of wild type (wt) NKX2-5 (HD) and TBX5 (T-box). In addition, we generated 8 non-synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R189P, R190C, Y191C) of NKX2-5 HD and 6 non-synonymous mutations (I54T, M74V, G80R, I101F, R113K, R237Q) of TBX5 T-box identified in CHD. Functional analysis of NKX2-5 HD mutants A148E, R161P, T178M, Q181H, R189P, Y191C showed retention of DNA binding. Similarly, TBX5 T-box mutants I54T, M74V, G80R, I101F, R113K, R237Q displayed DNA binding activity. This loss in TF DNA binding activity and recognition suggests a link of non-synonymous mutations deleterious effects in TF functions, which can lead to CHD. To comprehensively measure the protein-DNA interactomes of NKX2-5 HD and TBX5 T-box non-synonymous mutants, we performed High-Throughput Systematic Evolution of Ligands and Exponential Enrichment sequencing (SELEX-seq) and bioinformatics tools to predict changes in gene targets between wt and mutant TFs. Interestingly, SELEX-seq on TBX5 T-box wt and its mutant I54T reveals that both TF variants bind to similar AGGTGTCG and GGAGGTGT motifs. Further bioinformatic analysis will determine underlining differences in TF DNA binding activity that impact cardiac TFs’ crucial functions, ultimately revealing molecular mechanisms in CHD.