Mutations In DNA-binding Domain Research Articles

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.

Functional mechanisms of MYRF DNA-binding domain mutations implicated in birth defects

Myrf is a pleiotropic membrane-bound transcription factor that plays critical roles in diverse organisms, including in oligodendrocyte differentiation, embryonic development, molting, and synaptic plasticity. Upon autolytic cleavage, the Myrf N-terminal fragment enters the nucleus as a homo-trimer and functions as a transcription factor. Homo-trimerization is essential for this function because it imparts DNA-binding specificity and affinity. Recent exome sequencing studies have implicated four de novo MYRF DNA-binding domain (DBD) mutations (F387S, Q403H, G435R, and L479V) in novel syndromic birth defects involving the diaphragm, heart, and the urogenital tract. It remains unknown whether and how these four mutations alter the transcription factor function of MYRF. Here, we studied them by introducing homologous mutations to the mouse Myrf protein. We found that the four DBD mutations abolish the transcriptional activity of the Myrf N-terminal fragment by interfering with its homo-trimerization ability by perturbing the DBD structure. Since the Myrf N-terminal fragment strictly functions as a homo-trimer, any loss-of-function mutation has the potential to act as a dominant negative. We observed that one copy of Myrf-F387S, Myrf-Q403H, or Myrf-L479V, but not Myrf-G435R, was tolerated by the Myrf N-terminal homo-trimer for structural and functional integrity. These data suggest that F387S, Q403H, and L479V cause birth defects by haploinsufficiency, while G435R does so via dominant negative functionality.

Expression of GM-CSF Is Regulated by Fli-1 Transcription Factor, a Potential Drug Target.

Friend leukemia virus integration 1 (Fli-1) is an ETS transcription factor and a critical regulator of inflammatory mediators, including MCP-1, CCL5, IL-6, G-CSF, CXCL2, and caspase-1. GM-CSF is a regulator of granulocyte and macrophage lineage differentiation and a key player in the pathogenesis of inflammatory/autoimmune diseases. In this study, we demonstrated that Fli-1 regulates the expression of GM-CSF in both T cells and endothelial cells. The expression of GM-CSF was significantly reduced in T cells and endothelial cells when Fli-1 was reduced. We found that Fli-1 binds directly to the GM-CSF promoter using chromatin immunoprecipitation assay. Transient transfection assays indicated that Fli-1 drives transcription from the GM-CSF promoter in a dose-dependent manner, and mutation of the Fli-1 DNA binding domain resulted in a significant loss of transcriptional activation. Mutation of a known phosphorylation site within the Fli-1 protein led to a significant increase in GM-CSF promoter activation. Thus, direct binding to the promoter and phosphorylation are two important mechanisms behind Fli-1-driven activation of the GM-CSF promoter. In addition, Fli-1 regulates GM-CSF expression in an additive manner with another transcription factor Sp1. Finally, we demonstrated that a low dose of a chemotherapeutic drug, camptothecin, inhibited expression of Fli-1 and reduced GM-CSF production in human T cells. These results demonstrate novel mechanisms for regulating the expression of GM-CSF and suggest that Fli-1 is a critical druggable regulator of inflammation and immunity.

Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants

Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concentration. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodynamic stability, or both. Biophysical, cell-based, and murine xenograft experiments demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Simple SummaryThe risk of developing colorectal cancer at a younger age has increased, but current therapies are either risky or limited. We aim to demonstrate that the combination treatment of artemisinin derivatives and TRAIL could be a potential therapy to kill colon cancer cells. We found that artemisinin derivatives increase death receptor production and further sensitize colon cancer cells to TRAIL-induced apoptosis. Furthermore, we explored the role of P53 in response to artemisinin derivatives, which transactivates Death Receptor 5 (DR5) and the cyclin-dependent kinase inhibitor P21. Finally, a 3D tumor spheroid model also confirmed the efficacy of the combination treatment.Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single Ser241Phe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links.

DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

Targeting codon 158 p53-mutant cancers via the induction of p53 acetylation

Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.

Autosomal dominant hyper IgE syndrome from a single centre in Chongqing, China (2009-2018).

Autosomal dominant hyper IgE syndrome (AD-HIES) caused by STAT3 gene mutation is a rare primary immunodeficiency disease. To better understand the disease, we described the clinical characteristics of 20 AD-HIES patients in Chongqing, China and explored the effect of mutations in different domains of STAT3 gene on the function of STAT3 protein by Western blot and confocal microscopy. The mean age at onset was 0.12years. The mean age at diagnosis was 5.31years. The most common presentation was eczema, pneumonia, skin abscesses and chronic mucocutaneous candidiasis. Seven patients suffered from BCG complications. R382W/Q were identified in 12 patients, V637M mutation in three patients. Three patients have died. The phosphorylated STAT3 was expressed more in wild-type(WT) and R382W mutant STAT3 in the cytoplasm of COS7 cells with epidermal growth factor(EGF) stimulation, less in the V637M mutation and T620S mutation. Dynamic observation showed that STAT3 cytoplasmic accumulation and nuclear translocation occurred rapidly after EGF stimulation in WT-STAT3-GFP, the time of accumulation and nuclear translocation was later and the expression was less in R382W-STAT3-GFP compared with WT-STAT3-GFP, followed by V637M and T620S mutation. These results suggested that our patients had earlier onset, diagnostic age and higher rate of BCG complications. However, our patients had higher incidence of mortality though the earlier diagnostic age. We did not find a significant genotype/phenotype correlation, but Src homology 2 domain mutations (V637M and T620S) had a greater effect on STAT3 phosphorylation and nuclear translocation than DNA-binding domain mutation (R382W) in vitro.

Non-disruptive mutation in TP53 DNA-binding domain is a beneficial factor of esophageal squamous cell carcinoma.

BackgroundTP53 is frequently altered in esophageal squamous cell carcinoma (ESCC). However, the landscape of TP53 mutation and its effects on patients remain controversial.MethodsSomatic mutations of TP53 in 161 patients with resectable ESCC were identified by next-generation sequencing (NGS) and verified by immunohistochemistry (IHC). Patients were stratified into seven TP53 mutations, and depending on the extent of the effect on the encoded protein, it was divided into “disruptive” and “non-disruptive” types. The association of TP53 mutation with clinicopathological properties and disease outcome was investigated.ResultsTP53 mutations were discovered in 85.7% patients, of which 68.9% carried mutations in the DNA-binding domain (DBD). A total of 47.8% and 37.9% patients had disruptive and non-disruptive TP53 mutations, respectively. Most patients carried only one TP53 mutation, but 15.5% had double mutations. TP53 mutations were dominant in exons 5 to 8. Missense mutation was the most frequent (97/163, 59.5%), and the top five frequently occurring variations included R273X, Y220X, H193, H179X, and R175H. Multivariable analysis revealed non-disruptive mutation in TP53 DBD as the independent prognostic predictor for progression-free survival (PFS) and overall survival (OS). The expression of p53 positively correlated with non-disruptive mutation in DBD. Patients with high p53 protein expression showed better outcomes.ConclusionsNon-disruptive mutation in TP53 DBD serves as an independent beneficial prognostic factor of prolonged survival in resectable ESCC.

Abstract P6-08-18: Breast cancer in Li-Fraumeni syndrome and risk-reduction mastectomy in TP53 p.R337H carriers

Abstract Li-Fraumeni syndrome (LFS) is a hereditary genetic condition that predisposes to a wide variety of tumors. Early-onset breast cancer is the most prevalent tumor in female carriers, with a 49% risk by age 60. The risk of developing a second primary breast cancer in LFS remains unknown but it is estimated to be 33%. The Toronto protocol was proposed by Villani et al. (2011) and defined effective strategies for cancer screening in carriers. Risk-reduction mastectomy (RRM) may reduce over 90% breast cancer risk. Due to a founder mutation, TP53 p.R337H, LFS has been observed in a higher-than-expected prevalence in Brazil. This mutation occurs in the oligomerization domain and seems to have a milder lifetime penetrance than regular DNA binding domain mutations. Due to its estimated high frequency in the Brazillian population, this is a public health problem and implementation of effective strategies in breast cancer risk-reduction are needed. In this study, we have evaluated risk-reduction mastectomy in women who carried the founder TP53 p.R337H mutation. A total of 106 women from the Brazilian LFS TP53 p.R337H cohort were included in this study. Imaging surveillance was offered and RRM was discussed. All participants received genetic counseling and signed informed consent. The study was approved by the local ethics committee (HSL-02433418.5.0000.5461). A total of 65.1% (69/106) did not develop breast cancer at the time of this study. Among those, four women opted to perform bilateral prophylactic mastectomy (5.8% - 4/69). Overall, 34.9% (37/106) developed breast cancer prior to enrollment and 12 women were lost to follow-up. A total of 25 women opted to continue surveillance. Eleven (44%) opted to undergo RRM and 14 (66%) chose to continue annual breast magnetic resonance imaging (MRI), according to the Toronto protocol. A total of 32% (8/25) had been diagnosed with a second primary breast cancer prior to the study enrollment. The most common breast cancer histopathological subtype was invasive ductal carcinoma, present in 64% (16/25) in our study population. Only one woman was diagnosed with invasive lobular carcinoma (4%). Twelve percent (3/25) were in situ carcinomas. Estrogen and progesterone receptors stained positive in 76% (19/25). HER-2 was negative in 52% (13/25) of all cases. Our findings show that BPM was not a common procedure in carriers (5.8%). Nevertheless, RRM was performed in 44% of all women who have been previously diagnosed with breast cancer. Ductal invasive carcinoma is the most common cancer in TP53 p.R337H women and most of them have hormone receptor status positive. Unlike other previously published studies on LFS breast can cancer, HER-2 gene was non-amplified in most cases (52%). This is the first study to evaluate the frequency of RRM in a cohort of women with breast cancer carrying the same germline pathogenic variant, TP53 p.R337H. Citation Format: Mariana Cartaxo Alves, Renata Lazari Sandoval, Janina Pontes Pisani, Carla Vanessa Quirino, Elizabeth Santana Santos, Maria Isabel Achatz. Breast cancer in Li-Fraumeni syndrome and risk-reduction mastectomy in TP53 p.R337H carriers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-18.

C-MAF deletion in adult C57BL/6J mice induces cataract formation and abnormal differentiation of lens fiber cells.

The transcription factor c-MAF is a member of the large MAF family, members of which possess transactivation and bZIP domains. c-MAF plays an important role in lens formation, T-lymphocyte differentiation, hypertrophic chondrocyte differentiation, and kidney development in mouse embryos. However, because homozygous deletion of c-Maf in C57BL/6J mice causes embryonic lethality, the functions of c-MAF in adult mice remain largely uninvestigated. To address this issue, we generated c-Maf floxed (c-Maffl/fl) C57BL/6J mice and established tamoxifen-inducible c-Maf knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice, c-MafΔTAM). After tamoxifen injection, adult c-MafΔTAM mice showed successful deletion of c-Maf protein and developed severe cataracts; cataracts are also seen in human patients who have mutations in the c-MAF DNA binding domain. Furthermore, adult c-MafΔTAM mice exhibited abnormal lens structure and impaired differentiation of lens fiber cells. In summary, we established c-Maffl/fl and c-MafΔTAM C57BL/6J mice, which can be useful animal models for the investigation of c-MAF function in various developmental stages and can also be used as a disease model for cataracts.

High-throughput custom capture sequencing identifies novel mutations in coloboma-associated genes: Mutation in DNA-binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma.

Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high‐throughput screening of 38 known coloboma‐associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand‐binding domain, was a missense change in the highly conserved DNA‐binding domain predicted to affect the protein's DNA‐binding ability. In vitro studies revealed lower steady‐state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild‐type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA‐binding domain could result in human disease.

Abstract AP12: FOXM1 INDUCES DNA REPLICATION STRESS AND PROMOTES GENOMIC INSTABILITY DOWNSTREAM OF CYCLIN E1 IN HIGH-GRADE SEROUS OVARIAN CANCER

Abstract PURPOSE: Genomic instability (GI) is a hallmark of high-grade serous ovarian cancer (HGSC), and induction of the FOXM1 transcription factor regulatory network is the second most frequent genetic alteration in HGSC. FOXM1 expression is associated with cancer GI, but the mechanistic link is unknown. DNA replication stress (DRS), which involves altered replication fork dynamics, is a key contributor to cancer GI. FOXM1 and cyclin E1 (CCNE1) are both expressed in STIC lesions in the fallopian tube epithelium (FTE), and CCNE1-CDK2 enhances FOXM1 transcriptional activity. Based on these observations, we hypothesized that: 1) FOXM1 is linked to cancer GI through the induction of DRS, and 2) FOXM1 promotes GI downstream of CCNE1, by promoting unscheduled M phase entry in cells that have not completed DNA synthesis. METHODS: We determined associations between FOXM1 and markers of DRS and GI in TCGA HGSC data. We overexpressed FOXM1c and a DNA binding domain mutant (DBD) in immortalized FTE cells and characterized the impact on DRS, DNA replication, and DNA damage. We determined the impact of CCNE1 on FOXM1 expression in immortalized FTE cells. We determined the combined effect of CCNE1 and FOXM1 on GI in FTE cells. RESULTS: FOXM1 was significantly associated with markers of DRS (CHK1-Ser345) and GI (fractional copy number altered genome (CNA)) in TCGA HGSC data. FOXM1 expression, but not its DBD mutant, induced biomarkers of DRS and DNA damage in hTERT/mutant Tp53 immortalized FTE cells. Unexpectedly, FOXM1c increased DNA replication fork rate in FTE cells. Increased fork rate is a novel and recently recognized cause of DRS. CCNE1 expression in FTE cells induced and activated FOXM1, promoting mitotic entry. FOXM1 and CCNE1 co-expression in FTE cells induced an expression signature of GI, and TCGA HGSC tumors with FOXM1 and CCNE1 co-expression showed significantly increased GI. CONCLUSIONS: We demonstrate that FOXM1 induces DRS and DNA damage in FTE cells, providing an explanation for the known link between FOXM1 and GI. Notably, FOXM1 induces a novel form of DRS involving increased replication fork rate. We show that CCNE1 activates FOXM1, which in turn promotes mitotic entry and GI. These data reveal a potentially major form of oncogenic collaboration in HGSC, and has implications for HGSC treatment approaches that promote DRS or inhibit the cellular DRS response. . Citation Format: Carter J Barger, Kajal Biswas, Linda Chee, Ronny Drapkin, Shyam Sharan, Adam R. Karpf. FOXM1 INDUCES DNA REPLICATION STRESS AND PROMOTES GENOMIC INSTABILITY DOWNSTREAM OF CYCLIN E1 IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP12.

Comparison the Distinct Clinical Outcome with TP53 Mutation Status in MDS and AML :an Retrospective Study

Background: The TP5 gene is by far the most frequently mutated gene in human cancer. Some studies have shown that TP53 should be used as an independent prognostic biomarker in the prognostic stratification of myelodysplastic syndromes(MDS) and acute myeloid leukemia (AML). In this study, we want to explore the effects of different TP53 mutation status on clinical outcomes in AML and MDS. Method and Results: We retrospectively collected and analyzed 51 patients with TP53 mutation from Jan 1st , 2015 to May 1st , 2019 in West China Hospital, Sichuan University, China. Among 51 patients, 27 patients were diagnosed as MDS,11 were diagnosed as AML. Then average age is 55.7(15-77). According to IPSS-R of MDS, there are 18 out of 27 patients in the high-risk and very high-risk groups, only 1 patient in the intermediate risk group and 8 patients lost follow up. 10 out of 11 AML patients were in the high risk group according to the NCCN guideline. In our analysis, the incidences of complex karyotype and additional gene mutations were 86.67% and 36.84% in MDS and AML patients. AML evolution was 40.74% in MDS patients. The median follow-up time was 16 months (4-51 months) and the median overall survival was 7.61 months (range, 2-17 months). So far, 28.95% patients are still being treated, of which 9 have been treated with hypomethylating agents. To compare the clinical outcome with different TP53 mutations status, firstly, we identified the mutation types and various mutants activity. We counted 62 TP53 mutations in 51 patients and marked their sites at the transcriptional level in Fig1 . Codons 248, 273, 175, 282, 245, and 249 are most common somatic mutations in TP53. Herein, the TP53 mutation hot spots found in 38 AML or MDS patients are clustered in the DNA-binding domain. Then we used the luciferase reporting system to test the p53 mutant activity with missense mutations in the DNA-binding domain as a transcription factor. The results in Fig2 showed that with the exception of R158H, M237V , V143M , Q136E and H178Q, almost all the other mutation types have no activity. And we demonstrated these TP53 hotspot mutations in the structure ( Figure 3). The overall survival time of patients with residual P53 mutant activity (except for the compound mutation) far exceeded the median overall survival(7.61months). For the other patients with mutants which almost completely lost the activity of p53 protein as a transcription factor, two patients were treated with hypomethylating agents followed by hematopoietic stem cell transplantation have survived for 14 and 11 months after diagnosis, respectively. As shown in Fig4 and Fig 5, we compared the OS of these two group patients with different P53 mutant activity and different function areas for 6 months and 12 months, respectively. The results showed that the patients with residual P53 activity survived better Conclusion: Patients with different TP53 mutations do have different clinical outcomes. Patients with residual P53 mutant activity do respond better to treatment, and the final clinical outcome of patients who lose P53 activity completely is poor, but hypomethylating agents and hematopoietic stem cell transplantation can also ppropriately prolong survival time. Legends to the figures Figure1: 62 TP53 mutations in 51 patients with hematological neoplasms. The TP53 mutation hot spots found in 38 AML or MDS patients are clustered in the DNA-binding domain. There were 9 patients with multiple TP53 mutations. The black label represents somatic mutation, and the red label represents germline mutation. Missense mutations account for 61.70%(29/47) of all the TP53 mutations in 38 patients. Figure 2: The p53 mutant activity with missense mutations in DNA binding domain tested by the luciferase reporting system. We compared the normalized RLU on PUMA of P53 mutant with wild type. Figure 3: The structure of the p53 core domain bound to DNA. We marked the residues affected by the TP53 hotspot mutations . The deeper the red, the higher the mutation frequency and the more the mutation clustered Figure 4 : The overall survival of 30 follow-up patients. We use a bar chart to show the clinical outcome(OS) of 30 patients with different mutation types and P53 structure affected by different p53 domains. Figure 5 : The probability of overall survival of 30 follow-up patients. They are divided in two groups: patients with residual P53 activity VS patients with inactive P53 mutant. Disclosures No relevant conflicts of interest to declare.

Functional Classification of TP53 Mutations in Acute Myeloid Leukemia

In more than 50% of human cancers, somatically acquired aberrations of the tumor suppressor gene TP53 are encountered. Approx. 10% of patients with acute myeloid leukemia (AML) reveal TP53 mutations with higher incidences in therapy-related subtypes and erythroleukemias. These mutations are regarded early events of leukemogenesis. In contrast to mere deletions at the TP53 locus, TP53 mutations confer an exceedingly adverse prognosis in AML even when occurring at a subclonal level. However whether different TP53 mutations in AML exhibit a different functional impact on disease progression or outcome remains unknown. In the present study, we have investigated this issue using four TP53 specific, functional scoring systems in a large cohort of patients of the German-Austrian AML study group (AMLSG). The AMLSG cohort consisted of a total of 1537 patients with newly diagnosed AML who were intensively treated within three multicenter, clinical trials. A total of 108 TP53 mutations were detected in 98 patients using targeted amplicon sequencing - 88 (81.4%) missense, 8 (7.4%) nonsense and 6 (5.6%) splice site mutations as well as 6 small insertions and deletions. For each of the four functional TP53 scores, we have assessed their impact on overall survival (OS) and event-free survival (EFS). First, we compared the impact of TP53 missense mutations in 84 patients with all other types of mutations (n= 14). In a next approach, TP53 mutations were grouped into disruptive (n=42) and non-disruptive (n=56) ones, based on the impact of the particular mutations on the protein structure predicted from the crystal structure of p53-DNA complexes (Poeta et al. New Engl J Med 2007). We then classified missense TP53 mutations (n=84) based on their "Evolutionary Action Scores (EAp53)" (Neskey et al. Cancer Res 2015). This algorithm takes evolutionary sensitivity and amino acid conservation into account and scores missense TP53 mutations from 0 to 100. Mutations with the high EAp53 score are considered high risk whereas wild type TP53 has an EAp53 score of zero. We extracted the EAp53 score of those AMLSG patients showing missense mutations from the respective server (http://mammoth.bcm.tmc.edu/EAp53) and used the threshold of 75 from the initial publication to divide the patients into low-risk (<75, n=49) and high-risk groups (≥75, n=35). However, with these three functional scoring systems, no difference regarding OS and EFS could be shown between the mutational groups. The "Relative Fitness Score (RFS)" was recently developed for the TP53 DNA binding domain (DBD) mutants as an indicator of their functional impact (Kotlar et al. Molecular Cell 2018). A catalogue of 9833 TP53 DBD mutants were generated and their selective growth was assessed in p53 null cancer cell lines. The RFS score for each mutant is the median of its relative enrichment or depletion in culture, calculated at 3 time points and depicted as a log (base 2) value. A high RFS indicates selective growth of the mutant corresponding to its higher fitness. We extracted the RFS for the TP53 DBD mutations of the AMLSG cohort (n=83) using the online data resource (GSE115072) and performed a receiver-operating characteristics (ROC) analysis to calculate the optimal threshold of RFS separating deceased from survivors most efficiently. Thereby, the best RFS cut-off value according to the Youden index was -0.135. Applying this threshold (low-risk RFS ≤ -0.135, n=25; high-risk RFS > -0.135, n= 58) we demonstrated a significantly better OS (P=0.009) and EFS (P=0.037) for patients with a low-risk RFS in multivariable analyses adjusting for age, white blood cell count, cytogenetics and type of AML. Using the AML-specific TP53 RFS score, we could show that more than 30% of patients with TP53 mutations (25/83) reveal a significantly better survival indicating that this score is of prognostic value. Based on these findings, we propose that - along with clinical parameters - RFS values of TP53 mutations should also be considered for a comprehensive risk assessment of TP53 mutated AML patients. Disclosures Zebisch: Celgene: Honoraria; Novartis: Honoraria, Other: Advisory board; AbbVie: Other: Advisory board; Roche: Honoraria. Bullinger:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Menarini: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria. Döhner:Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Sill:Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board; Astex: Other: Advisory board.

Studying the Role of ETV6 in Megakaryopoiesis and Thrombopoiesis Using a Novel CRISPR-Cas9 Halotag Genome Editing Strategy

Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

TP53 DNA Binding Domain Mutations Predict Progression-Free Survival of Bevacizumab Therapy in Metastatic Colorectal Cancer.

(1) Background: Bevacizumab-based regimens are a standard treatment for metastatic colorectal cancer (mCRC) patients, however meaningful clinical biomarkers for treatment benefit remain scarce. (2) Methods: Tumor samples from 36 mCRC patients treated with bevacizumab-based chemotherapy underwent comprehensive genomic profiling. Alterations in frequently altered genes and important signaling pathways were correlated with progression-free survival (PFS). (3) Results: Overall genetic alteration analysis of investigated genes and pathways did not identify promising new predictors of PFS. However, when considering mutation subtypes, TP53 DNA binding domain (DBD) missense mutations were associated with prolonged PFS (HR, 0.41; 95% CI, 0.13−0.65; p = 0.005). In contrast, TP53 truncating mutations were associated with short PFS (HR, 2.95; 95% CI, 1.45−27.50; p = 0.017). Importantly, neither TP53 mutation subtype was associated with overall response rate. In multivariate analysis, TP53 DBD missense mutations remained an independent PFS predictor (HR, 0.31; 95% CI, 0.13–0.77; p = 0.011). The other genetic factor independently associated with PFS were PTPRT/PTPRD deleterious alterations, which we previously identified in a screen for biomarkers of bevacizumab response. (4) Conclusions: TP53 DBD missense mutations may predict prolonged PFS in mCRC patients treated with bevacizumab-based therapy. Analyses of TP53 mutations as clinical biomarkers should take the biological impact of different mutation subtypes into consideration to improve patient stratification.