Genetic Variation in DNA Repair Pathways and Risk of Non-Hodgkin's Lymphoma

Molecular and genetic evidence suggests that DNA repair pathways may contribute to lymphoma susceptibility. Several studies have examined the association of DNA repair genes with lymphoma risk, but the findings from these reports have been inconsistent. Here we provide the results of a focused analysis of genetic variation in DNA repair genes and their association with the risk of non-Hodgkin's lymphoma (NHL). With a population of 1,297 NHL cases and 1,946 controls, we have performed a two-stage case/control association analysis of 446 single nucleotide polymorphisms (SNPs) tagging the genetic variation in 81 DNA repair genes. We found the most significant association with NHL risk in the ATM locus for rs227060 (OR = 1.27, 95% CI: 1.13–1.43, p = 6.77×10−5), which remained significant after adjustment for multiple testing. In a subtype-specific analysis, associations were also observed for the ATM locus among both diffuse large B-cell lymphomas (DLBCL) and small lymphocytic lymphomas (SLL), however there was no association observed among follicular lymphomas (FL). In addition, our study provides suggestive evidence of an interaction between SNPs in MRE11A and NBS1 associated with NHL risk (OR = 0.51, 95% CI: 0.34–0.77, p = 0.0002). Finally, an imputation analysis using the 1,000 Genomes Project data combined with a functional prediction analysis revealed the presence of biologically relevant variants that correlate with the observed association signals. While the findings generated here warrant independent validation, the results of our large study suggest that ATM may be a novel locus associated with the risk of multiple subtypes of NHL

Next-Generation Sequencing Suggests Complex, Heterogeneous Pathogenesis In Peripheral T-Cell Lymphoma Unspecified

Abstract Peripheral T-cell lymphoma (PTCL) makes up about 12 percent of non-Hodgkin lymphoma, comprising 18 diseases that are poorly understood and carry a generally worse prognosis than B-lymphomas. PTCL not otherwise specified (PTCL-NOS), a diagnosis of exclusion, is most common, making up 25-30 percent. Gene-expression studies suggest a heterogeneous origin of this diagnosis, with overlap to other PTCL types, but the genetic factors underlying its pathogenesis are undefined. Current therapy for PTCL-NOS is empiric and ultimately ineffective for most patients. Identification of specific therapeutic targets is therefore a high priority. We have sought better understanding of pathogenesis through next-generation sequencing of PTCL-NOS tumor DNA. Whole-exome sequencing revealed candidate genes but low availability of fresh-frozen samples limited our ability to draw conclusions by this method alone. We therefore sequenced the coding regions of 237 candidate genes in a collection formalin-fixed paraffin-embedded samples. We used Nimblegen Sequence Capture for PCR amplification of exons and Illumina hiSeq for raw sequence generation. Results were aligned to hg19 and compared to dbSNP and the 1,000 genomes data to exclude germline variants. Analysis, including comparison to the COSMIC database of cancer-specific mutations, revealed high-confidence mutations affecting more than 60 known cancer-related genes in 25 PTCL-NOS cases. Recurrent mutations pointed to frequent activation of three key signaling pathways: NF-kB (TNFAIP3), WNT/B-Catenin (APC, CHD8, CELSR2), and NOTCH (NOTCH1, FBXW7). Recurrent deregulation of epigenetic processes was indicated by mutations in genes affecting histone acetylation (EP300, CREBBP), histone methylation (MLL2, KDM6A), and DNA methylation (TET2, DNMT3A). In addition, components of core tumor suppressor pathways showed evidence of frequent inactivation (TP53, ATM, RB1, CUL9, PRKDC). In all, 22 of 25 cases had mutations in at least one of these 17 recurrently mutated genes. Multiple additional candidate disease mechanisms also were suggested by lower-confidence mutations but require confirmation studies, which are under way. In sum, analysis of the coding region of PTCL-NOS tumor DNA suggests a complex and heterogeneous pathogenesis, in line with gene-expression profiling. This work provides an opportunity to better sub-classify entities within the diagnosis of PTCL-NOS and identify specific therapeutic targets and their associated biomarkers. Disclosures: Horwitz: Seattle Genetics, Inc.: Consultancy, Research Funding; Millennium: Consultancy, Research Funding

Cell‐free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience

Purpose The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell‐free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. Methods Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra‐ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA‐cleared tumor sequencing assay, MSK‐IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. Results Tumor‐guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. Conclusion Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow‐up schemes, and risk of metastasis.ISSN:2045-763

Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

<div><p>Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an <em>in vitro</em> HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC₅₀ 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8–27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.</p> </div

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition

Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation are insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacological disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling, and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability

NU9056 reduces PSA and p53 protein levels.

<p>To confirm the effects of Tip60 on androgen receptor activity we used 2.5 nM siRNA specifically targeted against Tip60 in LNCaP cells, or non-silencing control. Knockdown was achieved after 48 hours in steroid depleted medium after which time 10 nM DHT was applied to induce androgen receptor activity and PSA expression. RNA was collected after 24 hours DHT stimulation, reverse transcription and real-time PCR performed. Expression of (A) PSA and (B) Tip60 was normalised relative to HPRT1 expression. (C) LNCaP cells were treated with 24 µM NU9056 over 48 hours and protein samples were collected in SDS sample buffer. Protein analysis was carried out by SDS PAGE and Western blotting for p53, p21, AR, PSA and alpha tubulin. (D) Densitometry was performed on Western blots. All experiments were performed twice and the mean ± standard deviation is shown.</p

NU9056 reduces LNCaP cell survival by inducing apoptosis.

<p>(A) LNCaP cells were seeded onto 6 well plates for 24 hours, then increasing doses of NU9056 were applied for (i) 24 hours, (ii) 96 hours or (iii) GI₂₅ (17 µM) or (iv) GI₅₀ (24 µM) was applied over 4 days. All cells were collected and fixed with cytofix/cytoperm (BD) then caspase 3 and caspase 9 assay kits (BD) were used to assess their activity by flow cytometry. Fluorescence was detected on the FL-1 channel of the FACSCAN. (B) Analysis of the SubG1 population was performed on these same cells using propidium iodide to stain cellular DNA. LNCaP cells were seeded onto 6 well plates for 24 hours, then NU9056 was applied for (C) 1 or (D) 4 days. (E) LNCaP, LNCaP-AI and LNCaP-CdxR cells were seeded out onto 6 well plates and NU9056 was applied for 24 hours. Analysis of SubG1 was performed as described above. All cells were collected and fixed with cytofix/cytoperm (BD) then cell cycle analysis was performed using propidium iodide to stain cellular DNA. All FACS data was analysed using WinMDI. All experiments were performed 3 times and the mean ± standard error is shown. *p-value <0.05; **p-value <0.005; ***p-value <0.001.</p

IC₅₀ values of 1,2-bis(isothiazol-5-yl)disulfane (7) and related compounds towards HATs.

<p>To assess the activity against Tip60 HAT, <i>in vitro</i> HAT assays using ³H acetyl-CoA were carried out using histones as substrates. Assays were performed in quadruplicate and repeated twice. Individual IC₅₀ values are presented.</p