Plasma cell-free DNA hydroxymethylation profiling reveals anti-PD-1 treatment response and resistance biology in non-small cell lung cancer.

BACKGROUND: Treatment with immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) can yield durable antitumor responses, yet not all patients respond to ICIs. Current approaches to select patients who may benefit from anti-PD-1 treatment are insufficient. 5-hydroxymethylation (5hmC) analysis of plasma-derived cell-free DNA (cfDNA) presents a novel non-invasive approach for identification of therapy response biomarkers which can tackle challenges associated with tumor biopsies such as tumor heterogeneity and serial sample collection. METHODS: 151 blood samples were collected from 31 patients with non-small cell lung cancer (NSCLC) before therapy started and at multiple time points while on therapy. Blood samples were processed to obtain plasma-derived cfDNA, followed by enrichment of 5hmC-containing cfDNA fragments through biotinylation via a two-step chemistry and binding to streptavidin coated beads. 5hmC-enriched cfDNA and whole genome libraries were prepared in parallel and sequenced to obtain whole hydroxymethylome and whole genome plasma profiles, respectively. RESULTS: Comparison of on-treatment time point to matched pretreatment samples from same patients revealed that anti-PD-1 treatment induced distinct changes in plasma cfDNA 5hmC profiles of responding patients, as judged by Response evaluation criteria in solid tumors, relative to non-responders. In responders, 5hmC accumulated over genes involved in immune activation such as inteferon (IFN)-γ and IFN-α response, inflammatory response and tumor necrosis factor (TNF)-α signaling, whereas in non-responders 5hmC increased over epithelial to mesenchymal transition genes. Molecular response to anti-PD-1 treatment, as measured by 5hmC changes in plasma cfDNA profiles were observed early on, starting with the first cycle of treatment. Comparison of pretreatment plasma samples revealed that anti-PD-1 treatment response and resistance associated genes can be captured by 5hmC profiling of plasma-derived cfDNA. Furthermore, 5hmC profiling of pretreatment plasma samples was able to distinguish responders from non-responders using T cell-inflamed gene expression profile, which was previously identified by tissue RNA analysis. CONCLUSIONS: These results demonstrate that 5hmC profiling can identify response and resistance associated biological pathways in plasma-derived cfDNA, offering a novel approach for non-invasive prediction and monitoring of immunotherapy response in NSCLC.

Epigenomic Blood-Based Early Detection of Pancreatic Cancer Employing Cell-Free DNA.

BACKGROUND & AIMS: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma. METHODS: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs. RESULTS: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). CONCLUSIONS: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals.

The Cell Type-Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. SIGNIFICANCE: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265.

Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA.

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

Proteomic Analyses Identify a Novel Role for EZH2 in the Initiation of Cancer Cell Drug Tolerance.

Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance.

Human DNA Helicase B as a Candidate for Unwinding Secondary CGG Repeat Structures at the Fragile X Mental Retardation Gene.

The fragile X syndrome (FXS) is caused by a CGG repeat expansion at the fragile X mental retardation (FMR1) gene. FMR1 alleles with more than 200 CGG repeats bear chromosomal fragility when cells experience folate deficiency. CGG repeats were reported to be able to form secondary structures, such as hairpins, in vitro. When such secondary structures are formed, repeats can lead to replication fork stalling even in the absence of any additional perturbation. Indeed, it was recently shown that the replication forks stall at the endogenous FMR1 locus in unaffected and FXS cells, suggesting the formation of secondary repeat structures at the FMR1 gene in vivo. If not dealt with properly replication fork stalling can lead to polymerase slippage and repeat expansion as well as fragile site expression. Despite the presence of repeat structures at the FMR1 locus, chromosomal fragility is only expressed under replicative stress suggesting the existence of potential molecular mechanisms that help the replication fork progress through these repeat regions. DNA helicases are known to aid replication forks progress through repetitive DNA sequences. Yet, the identity of the DNA helicase(s) responsible for unwinding the CGG repeats at FMR1 locus is not known. We found that the human DNA helicase B (HDHB) may provide an answer for this question. We used chromatin-immunoprecipitation assay to study the FMR1 region and common fragile sites (CFS), and asked whether HDHB localizes at replication forks stalled at repetitive regions even in unperturbed cells. HDHB was strongly enriched in S-phase at the repetitive DNA at CFS and FMR1 gene but not in the flanking regions. Taken together, these results suggest that HDHB functions in preventing or repairing stalled replication forks that arise in repeat-rich regions even in unperturbed cells. Furthermore, we discuss the importance and potential role of HDHB and other helicases in the resolution of secondary CGG repeat structures.

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells.

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Human DNA helicase B interacts with the replication initiation protein Cdc45 and facilitates Cdc45 binding onto chromatin.

The chromosomal DNA replication in eukaryotic cells begins at replication initation sites, which are marked by the assembly of the pre-replication complexes in early G1. At the G1/S transition, recruitment of additional replication initiation proteins enables origin DNA unwinding and loading of DNA polymerases. We found that depletion of the human DNA helicase B (HDHB) inhibits the initiation of DNA replication, suggesting a role of HDHB in the beginning of the DNA synthesis. To gain insight into the function of HDHB during replication initiation, we examined the physical interactions of purified recombinant HDHB with key initiation proteins. HDHB interacts directly with two initiation factors TopBP1 and Cdc45. In addition we found that both, the N-terminus and helicase domain of HDHB bind to the N-terminus of Cdc45. Furthermore depletion of HDHB from human cells diminishes Cdc45 association with chromatin, suggesting that HDHB may facilitate Cdc45 recruitment at G1/S in human cells.

Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a "one-pot" hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.