Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancer uncovers rare somatic mutations in TNK2 and DDR1

Background: Endometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets. Methods: In a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines. Results: Overall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2P761Rfs*72 frameshift mutation was recurrent in EC, and the DDR1R570Q missense mutation was recurrent across tumor types. Conclusions: This is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

Lck SH3 Domain Function Is Required for T-Cell Receptor Signals Regulating Thymocyte Development

Thymocyte development is shaped by signals from the T-cell antigen receptor. The strength of receptor signaling determines developmental progression as well as deletion of self-reactive T cells. Receptor stimulation of the extracellular signal-regulated kinase (ERK) pathway plays an important regulatory role during thymocyte development. However, it is unclear how differences in receptor signaling are translated into distinctive activation of the ERK pathway. We have investigated the potential role of the Lck tyrosine kinase in regulating intracellular signaling during thymocyte development. While Lck is known to be critical for initial T-cell receptor signaling events, it may have an independent role in regulating intracellular signaling through the function of its SH3 domain. To determine whether such a regulatory mechanism functions during thymocyte development, we generated mice in which the normal lck allele is replaced with an lck SH3 domain mutant. Analysis of these mice revealed that both early thymocyte development and maturation of CD4(+) and CD8(+) lineages is impaired. Investigation of thymocyte responses to antigen receptor stimulation showed a significant reduction in proliferation and ERK pathway activation, although initial signaling events were intact. These findings indicate that Lck SH3 domain function may provide a means to independently couple receptor signaling to regulation of the ERK pathway during thymocyte development

Sequencing of Candidate Chromosome Instability Genes in Endometrial Cancers Reveals Somatic Mutations in <i>ESCO1</i>, <i>CHTF18</i>, and <i>MRE11A</i>

<div><p>Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in <i>ESCO1</i>, <i>CHTF18,</i> and <i>MRE11A</i> in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in <i>ESCO1</i> and <i>CHTF18</i> was statistically significant (<i>P</i> = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within <i>ESCO1</i> and <i>CHTF18</i> in endometrial tumors and of <i>MRE11A</i> mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.</p></div

Nonsynonymous, somatic mutations in <i>ESCO1</i>, <i>CHTF18</i> and <i>MRE11A</i>, in ECs.

†<p>Case no T3 is also known as OM-1323.</p><p>n/a Not applicable.</p>§<p>Transcript accession numbers: <i>ESCO1</i> (NM_052911.1), <i>CHTF18</i> (NM_022092.1), <i>MRE11A</i> (NM_005591).</p>¶<p>Protein accession numbers: ESCO1 (NP_443143.2), CHTF18 (NP_071375.1), MRE11A (NP_005582.1).</p

Genes resequenced in the mutation discovery screen.

1<p>Gene analyzed because it is somatically mutated in colorectal cancer.</p>2<p>Implicated in meiotic specific cohesion.</p

Co-occurrence of <i>ESCO1</i> mutations with <i>CHTF18</i> or <i>ATAD5</i> mutations in EC.

§<p>Two-tailed Fisher's exact test.</p

Oncoprint displaying nonsynonymous somatic mutations in <i>ESCO1</i>, <i>CHTF18</i>, <i>MRE11A</i>, and <i>ATAD5</i> in eight primary endometrial cancers.

<p>Individual tumors (T) are indicated by vertical gray bars. Tumors consist of NEECs (T3, T51, T62, T68, T77, T79, T113) and an EEC (T88). Genes (left) and nonsynonymous somatic mutations (orange boxes) are indicated. <i>ESCO1</i>, <i>CHTF18</i>, and <i>MRE11A</i> were analyzed in this study; *<i>ATAD5</i> mutations, <i>MSH6</i> mutations, and microsatellite instability (MSI) have previously been described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Bell1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-LeGallo1" target="_blank">[52]</a>.</p