Frequent Target Of Mutations Research Articles

The role of AdhE on ethanol tolerance and production in Clostridium thermocellum

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

The seasonal behaviour of COVID-19 and its galectin-like culprit of the viral spike.

Seasonal behaviour is an attribute of many viral diseases. Like other ‘winter’ RNA viruses, infections caused by the causative agent of COVID-19, SARS-CoV-2, appear to exhibit significant seasonal changes. Here we discuss the seasonal behaviour of COVID-19, emerging viral phenotypes, viral evolution, and how the mutational landscape of the virus affects the seasonal attributes of the disease. We propose that the multiple seasonal drivers behind infectious disease spread (and the spread of COVID-19 specifically) are in ‘trade-off’ relationships and can be better described within a framework of a ‘triangle of viral persistence’ modulated by the environment, physiology, and behaviour. This ‘trade-off’ exists as one trait cannot increase without a decrease in another. We also propose that molecular components of the virus can act as sensors of environment and physiology, and could represent molecular culprits of seasonality. We searched for flexible protein structures capable of being modulated by the environment and identified a galectin-like fold within the N-terminal domain of the spike protein of SARS-CoV-2 as a potential candidate. Tracking the prevalence of mutations in this structure resulted in the identification of a hemisphere-dependent seasonal pattern driven by mutational bursts. We propose that the galectin-like structure is a frequent target of mutations because it helps the virus evade or modulate the physiological responses of the host to further its spread and survival. The flexible regions of the N-terminal domain should now become a focus for mitigation through vaccines and therapeutics and for prediction and informed public health decision making.

RNF43 truncations trap CK1 to drive niche‐independent self‐renewal in cancer

Wnt/β‐catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt‐hypersensitive tumors that are susceptible to anti‐Wnt‐based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce β‐catenin‐mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin‐independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing β‐catenin turnover and propelling ligand‐independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche‐independent program for self‐renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti‐Wnt‐based therapy. Our data demonstrate the relevance of studying patient‐derived mutations for understanding disease mechanisms and improved applications of precision medicine.

Abstract LB-286: ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

Abstract Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are collectively mutated in ~20% of all human cancers, but the mechanism by which these mutations promote tumorigenesis is unclear. In this study, we investigate the tumor suppressor role of ARID1A, the subunit of SWI/SNF complexes that is the most frequent target of mutations. We develop an inducible Arid1a-knockout mouse model and find that mice develop invasive ARID1A-deficient colon adenocarcinoma. These cancers show prominent mucinous differentiation and tumor-infiltrating lymphocytes, features associated particularly with microsatellite-instable (MSI) human colorectal cancer - the subtype with the highest frequency of inactivating ARID1A mutations (37-39%). Although deregulation of Wnt signaling components (APC/β-catenin) is commonly utilized to model intestinal tumorigenesis, Arid1a-knockout mice reflect human colorectal cancer with greater accuracy. These mice do not show deregulation of Wnt signaling components, thus demonstrating a novel pathway to colon tumorigenesis that is independent of established models. Chromatin immunoprecipitation sequencing reveals that SWI/SNF complexes are targeted primarily to enhancers, where they function in coordination with transcription factors to activate gene expression. ARID1A loss impairs the targeting of SWI/SNF complexes to thousands of enhancers, which subsequently lose activity - showing reduced levels of H3K27ac and expression of nearest genes. Residual SWI/SNF complexes in ARID1A-deficient cells bind enhancers that remain active; these complexes contain ARID1B, the subunit that is mutually exclusive with ARID1A and has been identified as a therapeutic target in ARID1A-deficient cancers. Enhancers associated with developmental gene expression programs are most affected by ARID1A loss, both in the mouse colonic epithelium and in human colon cancer cells. Collectively, these findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as the principal tumor suppressor function of ARID1A. Citation Format: Radhika Mathur, Burak H. Alver, Adrianna K. San Roman, Boris G. Wilson, Xiaofeng Wang, Agoston T. Agoston, Peter Park, Ramesh Shivdasani, Charles W. Roberts. ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2017-LB-286

ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice.

Genes encoding subunits of SWI/SNF chromatin remodeling complexes are collectively mutated in ~20% of all human cancers1–2. Although ARID1A is the most frequent target of mutations, the mechanism by which its inactivation promotes tumorigenesis is unclear. Here, we demonstrate that Arid1a functions as a tumor suppressor in the mouse colon, but not the small intestine, and that invasive ARID1A-deficient adenocarcinomas resemble human colorectal cancer (CRC). These tumors lack deregulation of APC/β-catenin, crucial gatekeepers in common forms of intestinal cancer. ARID1A normally targets SWI/SNF complexes to enhancers, where they function in coordination with transcription factors (TFs) to facilitate gene activation. ARID1B preserves SWI/SNF function in ARID1A-deficient cells, but defects in SWI/SNF targeting and control of enhancer activity cause extensive dysregulation of gene expression. These findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as a principal tumor suppressor function of ARID1A.

Alterations in Mc1r gene expression are associated with regressive pigmentation in Astyanax cavefish.

Diverse changes in coloration across distant taxa are mediated through alterations in certain highly conserved pigmentation genes. Among these genes, Mc1r is a frequent target for mutation, and many documented alterations involve coding sequence changes. We investigated whether regulatory mutations in Mc1r may also contribute to pigmentation loss in the blind Mexican cavefish, Astyanax mexicanus. This species comprises multiple independent cave populations that have evolved reduced (or absent) melanic pigmentation as a consequence of living in darkness for millions of generations. Among the most salient cave-associated traits, complete absence (albinism) or reduced levels of pigmentation (brown) have long been the focus of degenerative pigmentation research in Astyanax. These two Mendelian traits have been linked to specific coding mutations in Oca2 (albinism) and Mc1r (brown). However, four of the seven caves harboring the brown phenotype exhibit unaffected coding sequences compared to surface fish. Thus, diverse genetic changes involving the same genes likely impact reduced pigmentation among cavefish populations. Using both sequence and expression analyses, we show that certain cave-dwelling populations harboring the brown mutation have substantial alterations to the putative Mc1r cis-regulatory region. Several of these sequence mutations in the Mc1r 5' region were present across multiple, independent cave populations. This study suggests that pigmentation reduction in Astyanax cavefish evolves through a combination of both coding and cis-regulatory mutations. Moreover, this study represents one of the first attempts to identify regulatory alterations linked to regressive changes in cave-dwelling populations of A. mexicanus.

Abstract 2076: RNF43 is a frequent mutational target in serrated colorectal neoplasia

Abstract BACKGROUND. The Wnt signalling pathway is altered in the majority of colorectal cancers by APC mutation. Cancers arising via the serrated neoplasia pathway are hallmarked by BRAF mutation and microsatellite instability (MSI), but do not commonly mutate APC. RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that are localised to intestinal stem cells where they cooperate to suppress Wnt signaling. Mutation of these genes may be an alternate mechanism to bypass the Wnt signal. RNF43 / ZNRF3 double knockout mice develop intestinal neoplasia and germline mutation of RNF43 has been reported in serrated polyposis. We therefore hypothesised that somatic mutation of RNF43 and/or ZNRF3 would be important in the development of serrated colorectal neoplasia. METHODS. RNF43 and ZNRF3 mutations were assessed in a pilot series of 24 BRAF mutant cancers representing the serrated neoplasia pathway and 10 control BRAF wild type cancers using Agilent V4+UTR whole exome capture followed by sequencing on an Illumina HiSeq 2000. An extended cohort of 80 BRAF mutant cancers (48 MSI, 32 MSS) and 67 BRAF wild type cancers was examined by Sanger sequencing. The frequency of a common hotspot mutation in RNF43 was validated in a second series of 42 BRAF mutant and 22 BRAF wild type colorectal cancers. Transcript expression for RNF43 and ZNRF3 was assayed using Illumina HT-12v4 BeadChip expression arrays in 29 BRAF mutant cancers, 187 BRAF wild type cancers and a subset of 32 matched normal mucosa samples. RESULTS. RNF43 mutations were detected in whole exome sequencing data in 11/24 BRAF mutant but no BRAF wild type cancers (P<0.01). Three of these 11 cancers also had a ZNRF3 mutation. RNF43 and ZNRF3 mutations were also frequently concordant in a larger series of cancers. RNF43 mutations were associated with MSI (P<0.0001), being identified in 43/48 BRAF mutant / MSI cancers (90%), 6/32 (19%) BRAF mutant / MSS cancers and 6/44 BRAF wild type / MSS cancers (14%). The high frequency of a G7 frameshift mutation at position 659 of RNF43 was similarly present in 25/33 (76%) BRAF mutant MSI cancers but only 1/34 MSS cancers from a second, validation cohort. Transcript levels of RNF43 and ZNRF3 were positively correlated (r2 0.47, P<0.0001) and were significantly lower in BRAF mutant compared to BRAF wild type cancers (P<0.0001). CONCLUSIONS. RNF43 is a frequent mutational target in serrated neoplasia. The high frequency of both substitution and frameshift mutations support the functional relevance of these alterations. We hypothesise that RNF43 is mutated as an alternative to APC in serrated lesions as a mechanism of bypassing control of the Wnt signal. These data support our current model of serrated neoplasia whereby BRAF is mutated early in serrated polyp development and RNF43 is subsequently mutated to remove constraints on the Wnt signal and allow progression to cancer. Citation Format: Vicki L. Whitehall, Catherine Bond, Diane McKeone, Jonathan Ellis, Mark Bettington, Sally Pearson, Barbara Leggett. RNF43 is a frequent mutational target in serrated colorectal neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2015-2076

Immunohistochemical study of p53 and proliferating cell nuclear antigen expression in odontogenic keratocyst and periapical cyst

Introduction:p53 protein is a product of p53 gene, which is now classified as a tumor suppressor gene. The gene is a frequent target for mutation, being seen as a common step in the pathogenesis of many human cancers. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and plays a critical role in initiation of cell proliferation.Aim:The aim of this study is to assess and compare the expression of p53 and PCNA in lining epithelium of odontogenic keratocyst (OKC) and periapical cyst (PA).Materials and Methods:A total of 20 cases comprising 10 OKC and 10 PA were included in retrospective study. Three paraffin section of 4 μm were cut, one was used for routine hematoxylin and eosin stain, while the other two were used for immunohistochemistry. Statistical analysis was performed using Chi-square test.Results:The level of staining and intensity were assessed in all these cases. OKC showed PCNA expression in all cases (100%), whereas in perapical cyst only 60% of cases exhibited PCNA staining. (1) OKC showed p53 expression in 6 cases (60%) whereas in PA only 10% of the cases exhibited p53 staining. Chi-square test showed PCNA staining intensity was more significant than p53 in OKC. (2) The staining intensity of PA using p53, PCNA revealed that PCNA stating intensity was more significant than p53.Conclusion:OKC shows significant proliferative activity than PA using PCNA and p53. PCNA staining was more intense when compared with p53 in both OKC and PA.

Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

IntroductionPancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and MethodsDiagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years).Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer's protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. ResultsExome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27).When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. ConclusionWhole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures:No relevant conflicts of interest to declare.

A miracle if restored, p53

The tumour suppressor function of p53 is due to its highly regulated activity as a transcriptional factor. This very important player is a frequent target of mutation in majority of human cancers. p53 activation is a valid anticancer approach that can result in development of new cancer treatments. New discoveries are emerging rapidly about the functional control of p53 that attempts to exploit the system for developing better therapeutics and diagnostics. This article is an attempt to explore the recent advancements in p53 biology for its restoration in human tumors. We have highlighted some of the potential molecules targeting the p53 pathway for tumor suppression and it is hoped that they will become effective and powerful weapons against tumors/cancers. Key words: p53, tumour suppressor function, human tumors, cancers.

RUNX1/AML1 mutant collaborates with BMI1 overexpression in the development of human and murine myelodysplastic syndromes

AbstractRUNX1/AML1 mutations have been identified in myelodysplastic syndromes (MDSs). In a mouse bone marrow transplantation model, a RUNX1 mutant, D171N, was shown to collaborate with Evi1 in the development of MDSs; however, this is rare in humans. Using enforced expression in human CD34+ cells, we showed that the D171N mutant, the most frequent target of mutation in the RUNX1 gene, had an increased self-renewal capacity, blocked differentiation, dysplasia in all 3 lineages, and tendency for immaturity, but no proliferation ability. BMI1 overexpression was observed in CD34+ cells from the majority of MDS patients with RUNX1 mutations, but not in D171N-transduced human CD34+ cells. Cotransduction of D171N and BMI1 demonstrated that BMI1 overexpression conferred proliferation ability to D171N-transduced cells in both human CD34+ cells and a mouse bone marrow transplantation model. Stepwise transduction of D171N followed by BMI1 in human CD34+ cells resulted in long-term proliferation with a retained CD34+ cell fraction, which is quite similar to the phenotype in patients with higher-risk MDSs. Our results indicate that BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.

Abstract 3105: Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells.

Abstract LKB1 (also known as STK11) is a frequent target for mutations in lung cancer, however, the role of LKB1 loss in tumorigenesis is undefined. NR4A2, together with NR4A1, and NR4A3, constitute the nuclear receptor (NR) subfamily 4A (NR4A), which are orphan NRs lacking identified ligands. NR4A family has been implicated in cell cycle regulation, apoptosis, inflammation, metabolism and more recently in carcinogenesis. However, there are conflicting data for tumorigenesis as NR4A1 and NR4A3 act as tumor suppressors in leukemia mouse models, while NR4A2 appears to stimulate tumor cell growth in vitro. Our laboratory discovered that the CRTC1 gene was aberrantly activated in LKB1-null cancer cells, stimulating the transcription of cAMP/CREB targets including NR4A gene members. The aim of this study is to examine NR4A2 as a potential oncogene and therapeutic target under the regulation of CRTC1 in LKB1 wildtype and null lung cancer cells. We demonstrated here that somatic loss of LKB1 in tumor cells was associated with underphosphorylation and nuclear localization of CRTC1, this resulted in upregulated expression and stronger transcriptional activity of NR4A2 in LKB1 mutant lung cancer cells and clinical primary lung cancer samples. We confirmed that forskolin, which can activate adenylate cyclase then catalyze the transformation of ATP to cAMP, could induce CRTC1 dephosphorylation resulting in enhanced NR4A2 expression. We identified that activation of the CRTC gene family induced NR4A2 expression, whereas knockdown of CRTCs decreased the expression of NR4A2. Furthermore, short hairpin RNA-mediated down-regulation of NR4A2, results in attenuated lung cancer cell proliferation and colony formation in vitro and decreased the tumor sizes in vivo in nude mice. In summary, our data suggested a CRTC-mediated transcriptional regulatory mechanism for NR4A2 activity, and proposed an oncogenic role of NR4A2 in LKB1 null lung cancer cell lines. Citation Format: Chunxia Cao, Min Zhang, Ruli Gao, Douglas Cress, Zirong Chen, Lizi Wu, Maria Zajac Kaye, Frederic Kaye. Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2013-3105

P53 Immunohistochemical Staining Patterns in Benign, Premalignant and Malignant Lesions of the Oral Cavity: A Study of 68 Cases

Context: p53 tumor suppressor gene is a frequent target for mutations in a variety of tumors. The mutated gene is more stable than the normal gene and can be detected by immunohistochemical methods. Aims: 1) To determine the expression of p53 immunohistochemically in a spectrum of benign, premalignant lesions and in various histological grades of oral squamous cell carcinoma 2) To verify if any correlation exists, between the localization and intensity of p53 staining pattern, and the degree of dysplasia. Methods and Material: p53 expression was studied in 68 cases of oral lesions using immunohistochemistry (Dako cytomation). The location and intensity of staining was noted. Suprabasal positivity was considered as abnormal. Results: All the benign lesions and low grade dysplasia were negative or showed only basal positivity. Suprabasal positivity increased from premalignant lesions (50%) to oral carcinomas (73.8%). In the premalignant category, intensity of p53 staining increased with increasing grades of dysplasia. In the malignant category, intensity of staining was stronger in the less differentiated tumors (68.4% vs 47.8%), while positivity was higher in better differentiated tumors (78.3% vs 68.4%). A small percentage of malignant tumors (21.4%) were negative. In 31% cases of SCC, the epithelium adjacent to the tumor which showed just hyperplasia/mild dysplasia on light microscopy, revealed suprabasal positivity. Conclusions: Expression of p53 above the basal layer is an early event in oral carcinogenesis. Stronger staining (increasing grades of dysplasia) had greater risk of progressing into malignancy. p53 positivity is an early indicator of a developing carcinoma preceding morphological tissue alterations.

RUNX1/AML1 Mutants Collaborate with BMI1 in the Development of Myelodysplastic Syndromes (MDS) / Acute Myeloid Leukemia (AML) in a Mouse BMT Model.

Abstract 2820RUNX1/AML1 mutations have been frequently detected in patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). RUNX1 mutations are rarely detected in lower-risk MDS, whereas approximately 20% patients with higher-risk MDS (H-MDS) have the mutations. The mutations were distributed throughout the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative suppression. In mouse transplantation systems D171N-transduced mice exhibited AML with multilineage dysplasia in collaboration with Evi1 overexpression. However, EVI1 overexpression was very rare in patients. Instead, most of H-MDS patients with RUNX1 mutations displayed a high expression of BMI1.RUNX1 D171N mutant showed an increased self-renewal capacity, differentiation block, dysplasia in all 3 lineages, slightly increased immature cells and no proliferation ability using enforced expression in human CD34+ cells, and the D171N-transduced cells showed low expression level of BMI1. Both D171N and BMI1 transduced cells displayed long-term proliferation ability. When BMI1 transduced later into D171N cells, the cells expanded with a retained CD34+ cell fraction, suggesting that BMI1 have a potential to boost the D171N cells to H-MDS.To confirm the collaboration of BMI1 overexpression with D171N mutant in vivo, we performed mouse BMT using BM cells transduced with both D171N and BMI1. Ly-5.1 murine BM mononuclear cells infected with retrovirus harboring D171N/BMI1 or control vectors were transplanted into sublethally irradiated syngeneic Ly-5.2 mice.Mice that received transplants of BMI1-transduced cells remained healthy over the observation period (n=12/12), as well as those that were transplanted with empty vectors-transduced cells (n=4/4). Most of the mice that received transplants of D171N -transduced cells developed MDS/AML mainly 6–8 months after transplantation (n=6/11, P<0.0001), as observed in the previous report. Of note, mice that received transplants of BM cells expressing D171N/BMI1 developed MDS/AML with significantly shorter latencies (mainly 3–5 months) compared with the D171N group (n=12/12, P=0.001). Morbid mice with D171N or D171N/BMI1 exhibited similar phenotypes, characterized by leukocytosis, anemia, and marked splenomegaly, while the mice with BMI1 or empty vectors, sacrificed 8 months after BMT, showed none of these phenotypes. In the leukemic mice with D171N or D171N/BMI1, BM and spleen were occupied by immature myeloid cells including myeloid blasts. More myeloblasts in BM were observed in D171N/BMI1 mice than in D171N ones. The leukemic cells displayed similar morphological abnormalities and surface markers: leukemic cells were CD11blow to high, Gr-1low, B220low and c-kitlow to high. The normal structure of the spleen was completely destroyed with massive blast and immature myeloid cell infiltration, and these cells also invaded into the hepatic portal areas in the liver. Meanwhile, BMI1-transduced BM cells did not become dominant in vivo and myeloid cells showed normal differentiation. Collectively, BMI1 overexpression has a strong potential to induce MDS/AML in concert with D171N in a mouse BMT model, although BMI1 overexpression by itself does not result in maturation block or leukemogenesis.BMI1 is well-known to be essential for self-renewal of HSCs, in part via repression of genes involved in senescence, and self-renewal of HSCs is enhanced by BMI1 expression. To address the mechanisms by which BMI1 would contribute to MDS/AML development, we analyzed gene expressions involved in BMI1 downstream signaling pathways. It is suggested that BMI1 overexpression may act as one of the partner abnormalities collaborating with master gene mutations for MDS-genesis.RUNX1 D171N mutant showed no proliferation ability using enforced expression in human CD34+ cells, and D171N-transduced mice exhibited MDS/AML in collaboration with Evi1 overexpression. In addition, co-transduction of D171N and BMI1 into BM cells resulted in faster induction of MDS/AML in BMT mice. Taken together, the RUNX1 mutant may require collaborating genes such as EVI1 and BMI1 to develop MDS/AML. We confirmed that BMI1 have a potential to boost the D171N cells to MDS/AML in vivo. Disclosures:No relevant conflicts of interest to declare.

Exome Sequencing Identifies Recurring DNMT3A Mutations in Adult ETP-ALL

AbstractAbstract 1377 Introduction:Early T-cell precursor (ETP) ALL accounting for 10% of all T-ALL cases is of special interest because of its proposed origin from early thymic progenitors with multilineage differentiation potential. ETP-ALL is associated with a poorer outcome in pediatric and adult patients. On the molecular level, ETP-ALL is characterized by a specific immunophenotype (CD1-, CD5weak, CD8-, co-expression of stem cell and/or myeloid antigens) and distinct molecular features (expression of stem cell genes, high frequency of FLT3 mutations with absence of NOTCH1 mutations). Whereas a highly heterogeneous genetic pattern was revealed by whole genome sequencing in pediatric patients, the genetic background of adult ETP-ALL remains largely unknown. Here we investigated genetic alterations in adult ETP-ALL by whole exome sequencing and subsequently analyzed specific target genes. Patients and methods:We performed whole exome sequencing of five paired (diagnosis/remission) adult ETP-ALL patients enrolled in German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) trials. Using exon capturing from genomic DNA, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform, we generated at least 5 Gb of exome sequence from each ETP-ALL and remission samples. Somatic mutations were identified by comparing the ETP-ALL with the remission exome sequence, excluding all annotated polymorphisms (dbSNP130), non-coding positions and positions with evidence of a variant in the corresponding remission samples. Candidate variants were confirmed by capillary sequencing of genomic DNA. The DNMT3A mutations status was analyzed by Sanger sequencing of exons 11–23 in additional 68 adult ETP-ALL (55 male, 13 female, median age: 38 years) as well as the mutation status of the polycomb repressor complex (PRC) genes EZH2 and SUZ12. For 52 of 68 patients clinical follow-up data were available. Results:Using whole exome sequencing we found a total of 56 non-synonymous somatic mutations or indels in the five ETP-ALL patients (range: 6 to 16 per patient). Eleven mutations/indels affected cancer genes. DNMT3A (2/5) and FAT3 (2/5) were recurrently mutated in the five patients. The DNA-methyl-transferase DNMT3A is a frequent mutational target in acute myeloid leukemia (AML; 20%), whereas FAT3 (FAT, tumor suppressor homolog 3) mutations were recently reported in ovarian carcinoma (TCGA, Nature 2011). Novel mutations identified in adult ETP-ALL involved genes in epigenetic regulation (e.g. MLL2, MLL3, BMI1), and in genes previously reported to be mutated in ETP-ALL (e.g. in JAK1, ETV6, NOTCH1, DNM2).By Sanger sequencing, we screened for DNMT3A mutations in a larger cohort of adult ETP-ALL. DNMT3A mutations were present in 11 of the 68 (16%) patients, a mutation rate similar to AML. Amino acid R882 (exon 23), the most frequently mutated amino acid in AML, was mutated in five ETP-ALL. The remaining six mutations occurred in single spots, with one exception in the ZNF or the MTF domain. Patients with a DNMT3A mutation were significantly older (median: 63 vs 37 years, P=0.016). No correlation was found between DNMT3A and FLT3 mutations (27% in DNMT3A mut pts. vs. 37% in DNMT3A wt pts., P=0.41) or NOTCH1 mutations (10% in DNMT3A mut pts. vs. 16% in DNMT3A wt pts., P=0.47).In addition, we investigated genetic alterations in epigenetic regulators including members of the polycomb repressor complex (PRC). Mutations were seen in EZH2 in 4/68 (6%), SUZ12 in 1/68 (1%) and SH2B3 in 4/69 (6%) of ETP-ALL. Interestingly, patients with at least one mutation in an epigenetic regulator gene (DNMT3A, SUZ12, SH2B3, MLL2, or EZH2) showed a trend towards an inferior survival (one-year-survival: 50% vs. 85%, P=0.08). Conclusion:Adult ETP-ALL patients display a heterogenous spectrum of mutations, particularly affecting genes involved in epigenetic regulation. The spectrum is different to pediatric patients with a lower rate of polycomb repressor complex and a higher rate of DNMT3A mutations. The higher rate of DNMT3A mutations in older patients might point to a different pathogenesis compared to pediatric ETP-ALL. Like in AML, DNMT3A mutations in adult ETP-ALL show a similar frequency, within the same hot spots and are correlated with an adverse prognostic value, underscoring the myeloid character of ETP-ALL. Thus, these data may provide a rationale to use epigenetic therapy in ETP-ALL. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

RUNX1 mutations in cytogenetically normal acute myeloid leukemia are associated with a poor prognosis and up-regulation of lymphoid genes

The RUNX1 (AML1) gene is a frequent mutational target in myelodysplastic syndromes and acute myeloid leukemia. Previous studies suggested that RUNX1 mutations may have pathological and prognostic implications. We screened 93 patients with cytogenetically normal acute myeloid leukemia for RUNX1 mutations by capillary sequencing of genomic DNA. Mutation status was then correlated with clinical data and gene expression profiles. We found that 15 out of 93 (16.1%) patients with cytogenetically normal acute myeloid leukemia had RUNX1 mutations. Seventy-three patients were enrolled in the AMLCG-99 trial and carried ten RUNX1 mutations (13.7%). Among these 73 patients RUNX1 mutations were significantly associated with older age, male sex, absence of NPM1 mutations and presence of MLL-partial tandem duplications. Moreover, RUNX1-mutated patients had a lower complete remission rate (30% versus 73% P=0.01), lower relapse-free survival rate (3-year relapse-free survival 0% versus 30.4%; P=0.002) and lower overall survival rate (3-year overall survival 0% versus 34.4%; P<0.001) than patients with wild-type RUNX1. RUNX1 mutations remained associated with shorter overall survival in a multivariate model including age and the European Leukemia Net acute myeloid leukemia genetic classification as covariates. Patients with RUNX1 mutations showed a unique gene expression pattern with differential expression of 85 genes. The most prominently up-regulated genes in patients with RUNX1-mutated cytogenetically normal acute myeloid leukemia include lymphoid regulators such as HOP homeobox (HOPX), deoxynucleotidyltransferase (DNTT, terminal) and B-cell linker (BLNK), indicating lineage infidelity. Our findings firmly establish that RUNX1 mutations are a marker of poor prognosis and provide insights into the pathogenesis of RUNX1 mutation-positive acute myeloid leukemia.

Special issue Heidelberg Heart II: Abstracts of oral and poster presentations

s of oral presentations A 1 — Desmosomal molecules in and out of junctions Kathleen J. Green, Adi Dubash, Lisa M. Godsel Departments of Pathology and Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA kgreen@northwestern.edu Desmosomes are intercellular junctions that anchor the intermediate filament (IF) cytoskeleton to sites of strong intercellular adhesion, and play a critical role in ensuring mechanical integrity of the skin and the heart. Desmosome building blocks come primarily from three protein families. Transmembrane members of the cadherin family, the desmogleins and desmocollins, cooperate to form the adhesive interface. Within the junctional plaque, the cytoplasmic tails of the cadherins provide a scaffold for armadillo family members including plakoglobin and plakophilins (PKPs) 1-3 and the IF-binding protein, desmoplakin (DP), which in turn anchors the strain-bearing IF cytoskeleton to the plaque. In cardiac muscle, desmosome molecules are major architectural components of the intercalated discs, highly organized regions of the plasma membrane comprising components of adherens junctions, gap junctions, and desmosomes that together coordinate mechanical and electrochemical signaling between adjacent cardiac myocytes. In vertebrates, postnatal remodeling of these specialized regions of the plasma membrane occurs, giving rise to the area composita in which desmosomal building blocks are intermixed with components of adherens junctions. The crucial functions of desmosome molecules in epithelial and cardiac tissues are highlighted by the discovery of mutations that cause skin and heart disease. While interference with the architectural roles of desmosome molecules has been assumed to make an important contribution to tissue responses that lead to disease pathogenesis, functions that transcend their well-established roles in adhesion and IF-anchorage are emerging. Desmosome molecules have recently been shown to guide the remodeling of microtubules during epidermal morphogenesis, and also govern actin remodeling by regulating Rho GTPases during junction assembly. Of particular interest is the desmosomal armadillo protein plakophilin 2 (PKP2), which is reported as the most frequent target for mutation in arrhythmogenic right ventricular cardiomyopathy (ARVC), a leading cause of sudden cardiac death in the young. We previously showed that in epithelial cells, PKP2 regulates the localization and activity of RhoA to locally control actomyosin contractile signaling important for cell junction formation, while keeping global RhoA signaling in check. In the heart, signaling through RhoA and its downstream effectors is critical for normal cardiac development and physiology. Indeed, our data show that PKP2 silencing results in elevated RhoA and disruption of actin organization in cardiac myocytes. Further, loss of PKP2 or its fellow armadillo protein plakoglobin leads to elevated expression of target genes of the transcriptional regulator SRF (serum response factor), which is activated in a RhoA-dependent fashion, and along with Rho plays essential roles in cardiac development, hypertrophy and fibrosis. These data suggest a new cellular pathway through which desmosome deficiency could contribute to pathogen-

P53 immunoprofiling of potentially malignant oral disorders: A case series analysis

p53 tumor suppressor gene which is a frequent target for mutations in a high percentage of oral cancer is regarded as an early event in carcinogenesis. The role of p53 was assessed in potentially malignant oral disorders (PMOD) to ascertain its prognostic significance. Retrospective case series analysis was carried out on 30 paraffin-embedded tissue blocks of confirmed oral leukoplakia with dysplasia. 10 cases of each of mild, moderate, and severe dysplasia were immunohistochemically analyzed for p53 expression. The intensity of staining, intracellular localization, and basal and/or suprabasal distribution were assessed. The intensity of p53 staining and its distribution were analyzed by the Chi-square test. The intracellular localization of p53 in different grades of dysplasia was subjected to one way ANOVA. P<0.05 was considered significant. 21/30 cases of epithelial dysplasia were positive for p53 immunopositivity. Intensity of p53 expression was strong in 12 cases and weak in 9 cases (P<0.05). p53 positivity was confined to basal cells in mild dysplasia, while severe dysplasia showed both basal and suprabasal staining (P<0.05). Nuclear and cytoplasmic staining between and within the groups were F=9.027 and F=6.465 respectively with high significance noted between mild dysplasia and severe dysplasia. Increased p53 expressivity and greater cellular localization with increase in the severity of dysplasia indicated a direct association between the degree of epithelial dysplasia and p53 accretion, which occurs as an early event in oral carcinogenesis.

Mutant p53 Disrupts Mammary Tissue Architecture via the Mevalonate Pathway

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent ofacinar structures found invivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells toa more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.