Radiosensitization In Vivo by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity.

As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and bladder, for such patients. We developed methods to determine normal tissue toxicity severity in intestine and bladder in vivo, using novel radiotherapy techniques on a small animal radiation research platform (SARRP). The effects of panobinostat on in vivo tumor growth delay were evaluated using subcutaneous xenografts in athymic nude mice. Panobinostat concentration levels in xenografts, plasma, and normal tissues were measured in CD1-nude mice. CD1-nude mice were treated with drug/irradiation combinations to assess acute normal tissue effects in small intestine using the intestinal crypt assay, and later effects in small and large intestine at 11 weeks by stool assessment and at 12 weeks by histologic examination. In vitro effects of panobinostat were assessed by qPCR and of panobinostat, TMP195, and mocetinostat by clonogenic assay, and Western blot analysis. Panobinostat resulted in growth delay in RT112 bladder cancer xenografts but did not significantly increase acute (3.75 days) or 12 weeks' normal tissue radiation toxicity. Radiosensitization by panobinostat was effective in hypoxic bladder cancer cells and associated with class I HDAC inhibition, and protein downregulation of HDAC2 and MRE11. Pan-HDAC inhibition is a promising strategy for radiosensitization, but more selective agents may be more useful radiosensitizers clinically, resulting in fewer systemic side effects. Mol Cancer Ther; 17(2); 381-92. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

E3 Ligase cIAP2 Mediates Downregulation of MRE11 and Radiosensitization in Response to HDAC Inhibition in Bladder Cancer.

The MRE11/RAD50/NBS1 (MRN) complex mediates DNA repair pathways, including double-strand breaks induced by radiotherapy. Meiotic recombination 11 homolog (MRE11) is downregulated by histone deacetylase inhibition (HDACi), resulting in reduced levels of DNA repair in bladder cancer cells and radiosensitization. In this study, we show that the mechanism of this downregulation is posttranslational and identify a C-terminally truncated MRE11, which is formed after HDAC inhibition as full-length MRE11 is downregulated. Truncated MRE11 was stabilized by proteasome inhibition, exhibited a decreased half-life after treatment with panobinostat, and therefore represents a newly identified intermediate induced and degraded in response to HDAC inhibition. The E3 ligase cellular inhibitor of apoptosis protein 2 (cIAP2) was upregulated in response to HDAC inhibition and was validated as a new MRE11 binding partner whose upregulation had similar effects to HDAC inhibition. cIAP2 overexpression resulted in downregulation and altered ubiquitination patterns of MRE11 and mediated radiosensitization in response to HDAC inhibition. These results highlight cIAP2 as a player in the DNA damage response as a posttranscriptional regulator of MRE11 and identify cIAP2 as a potential target for biomarker discovery or chemoradiation strategies in bladder cancer. Cancer Res; 77(11); 3027-39. ©2017 AACR.

High-throughput DNA Sequencing Identifies Novel CtIP (RBBP8) Variants in Muscle-invasive Bladder Cancer Patients.

BACKGROUND: Germline mutations in DNA damage signalling and repair genes predispose individuals to cancer. Rare germline variants may also increase cancer risk and be predictive of outcomes following cancer treatments, but require high-throughput sequencing (HTS) for detection in large cohorts. OBJECTIVE: To use a dual indexing system on a HTS platform to detect novel variants in CtIP (RBBP8) which may be associated with clinical outcomes following radiotherapy treatment for bladder cancer. METHODS: All exons and flanking introns of CtIP were amplified from germline DNA from bladder cancer patients using seven primer pairs by automated long-range PCR. Amplicons were pooled, fragmented and ligated to adaptor sequences. One of 96 tag sequences was introduced at each end by PCR. Sequencing was performed on a single flow cell of an Illumina MiSeq. Reads were mapped by Stampy and variants called by Platypus. For phasing experiments, target regions were amplified and cloned for Sanger sequencing. RESULTS: Of 201 samples, 160 were successfully amplified. Eleven CtIP variants were called, within the exons and 15 bp adjacent intronic DNA, including eight known variants from the 1000 Genomes project, plus three previously unreported variants now confirmed by Sanger sequencing. In two individuals, phasing experiments showed two variants of interest to be on separate alleles, likely to result in stronger impairment of gene function. CONCLUSIONS: We have demonstrated proof of principle for dual indexing on 160 samples on one MiSeq flow cell sequencing surface, and show that for the CtIP gene multiplexing of up to 720 samples would provide sufficient coverage to achieve >98% detection power for rare germline variation, reducing HTS costs substantially.

Post-transcriptional regulation of MRE11 expression in muscle-invasive bladder tumours.

Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.