Marja Nieuwland

Tumor Exome Analysis Reveals Neoantigen-Specific T-Cell Reactivity in an Ipilimumab-Responsive Melanoma

The evidence for T-cell–mediated regression of human cancers such as non–small-cell lung carcinoma, renal cell carcinoma, and—in particular—melanoma after immunotherapy is strong. Anti-CTLA4 (ipilimumab) treatment has been approved for treatment of meta-static melanoma,1 and antibody-mediated blockade of PD-1, a second inhibitory receptor on T cells, has shown highly encouraging results in early clinical trials.2,3 Although the clinical activity of these treatments is apparent, it is still unknown which T-cell reactivities are involved in immunotherapy-induced cancer regression.4 T-cell reactivity against nonmutated tumor-associated self-antigens has been analyzed in patients treated with ipilimumab or with autologous tumor-infiltrating T cells, but the magnitude of the T-cell responses observed has been relatively modest.5,6 In part on the basis of such data, recognition of patient-specific mutant epitopes (hereafter referred to as neoantigens) has been suggested to be a potentially important component.7 A potential involvement of mutated epitopes in T-cell control would also fit well with the observation that the mutation load in sun-exposed melanomas is particularly high.8-10 Intriguingly, on the basis of animal model data, it has recently been suggested that (therapy-induced) analysis of T-cell reactivity against patient-specific neoantigens may be feasible through exploitation of cancer genome data.11,12 However, human data have thus far been lacking. Here we report a case of a patient with stage IV melanoma who exhibited a clinical response to ipilimumab treatment. Cancer exome–guided analysis of T-cell reactivity in this patient revealed reactivity against two neoantigens, including a dominant T-cell response against a mutant epitope of the ATR (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exome–guided analysis to dissect the effects of melanoma immunotherapy.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX–MDC1–RNF8–RNF168–53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Interactions among Polycomb Domains Are Guided by Chromosome Architecture

Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.

Drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin

DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.

Gene expression profiles from formalin fixed paraffin embedded breast cancer tissue are largely comparable to fresh frozen matched tissue.

Background and Methods Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material. Methodology and Principal Findings We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results. Conclusions and Significance We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.

Glucosylated Hydroxymethyluracil, DNA Base J, Prevents Transcriptional Readthrough in Leishmania

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (β-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.

High-throughput identification of antigen-specific TCRs by TCR gene capture

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen–reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

ERBB2 Mutations Characterize a Subgroup of Muscle-invasive Bladder Cancers with Excellent Response to Neoadjuvant Chemotherapy

UNLABELLED A pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum is a strong prognostic determinant for patients with muscle-invasive bladder cancer (MIBC). Despite comprehensive molecular characterization of bladder cancer, associations of molecular alterations with treatment response are still largely unknown. We selected pathologic complete responders (ypT0N0; n=38) and nonresponders (higher than ypT2; n=33) from a cohort of high-grade MIBC patients treated with NAC. DNA was isolated from prechemotherapy tumor tissue and used for next-generation sequencing of 178 cancer-associated genes (discovery cohort) or targeted sequencing (validation cohort). We found that 9 of 38 complete responders had erb-b2 receptor tyrosine kinase 2 (ERBB2) missense mutations, whereas none of 33 nonresponders had ERBB2 mutations (p=0.003). ERBB2 missense mutations in complete responders were mostly confirmed activating mutations. ERCC2 missense mutations, recently found associated with response to NAC, were more common in complete responders; however, this association did not reach statistical significance in our cohort. We conclude that ERBB2 missense mutations characterize a subgroup of MIBC patients with an excellent response to NAC. PATIENT SUMMARY In this report we looked for genetic alterations that can predict the response to neoadjuvant chemotherapy (NAC) in bladder cancer. We found that mutations in the gene ERBB2 are exclusively present in patients responding to NAC.

High-resolution mapping of heterochromatin redistribution in a Drosophila position-effect variegation model

BackgroundPosition-effect variegation (PEV) is the stochastic transcriptional silencing of a gene positioned adjacent to heterochromatin. white-mottled X-chromosomal inversions in Drosophila are classic PEV models that show variegation of the eye color gene white due to its relocation next to pericentric heterochromatin. It has been suggested that in these models the spreading of heterochromatin across the rearrangement breakpoint causes the silencing of white. However, the extent of this spreading and the precise pattern of heterochromatin redistribution have remained unclear. To obtain insight into the mechanism of PEV, we constructed high-resolution binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes.ResultsWe find that HP1 invades euchromatin across the inversion breakpoints over ~175 kb and ~30 kb, causing de novo association of HP1 with 20 genes. However, HP1 binding levels in these regions show substantial local variation, and white is the most strongly bound gene. Remarkably, white is also the only gene that is detectably repressed by heterochromatin. Furthermore, we find that HP1 binding to the invaded region is particularly sensitive to the dosage of the histone methyltransferase Su(var)3-9, indicating that the de novo formed heterochromatin is less stable than naturally occurring constitutive heterochromatin.ConclusionOur molecular maps demonstrate that heterochromatin can invade a normally euchromatic region, yet the strength of HP1 binding and effects on gene expression are highly dependent on local context. Our data suggest that the white gene has an unusual intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive to PEV than most other genes.

Volatile Anesthetics Modulate Gene Expression in Breast and Brain Tumor Cells

Gene expression is increasingly used for diagnostic, prognostic, and therapeutic purposes in clinical practice. We tested the hypothesis that volatile anesthetics (VA) affect gene expression of tumor cells. Cells from the neuronal cell line SH-SY5Y and from the breast cell line MCF-7 were exposed ex vivo to enflurane, isoflurane, desflurane, halothane, sevoflurane, or nitrous oxide. Microarray gene expression profiles were studied. We observed significant differences in gene expression levels of cell cultures and response in time when exposed to different VA. Some genes used for predictive genetic fingerprints for breast cancer were affected by VA. Our findings suggest that VA modulate gene expression in breast and brain tumor cell cultures in a unique and time-dependent manner.