Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory double-stranded RNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T-cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi-resistant ovarian tumor growth in vivo, and promotes antitumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

Telomerase misbehaves after a breakup.

Suppressing telomerase action at broken DNA preserves genome integrity.

Meet the authors: Nausica Arnoult.

Molecular Cell talks to Nausica Arnoult about her paper "The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining," the lab environment she looks to create, and barriers faced by women pursuing careers in science.

The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining.

Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.

Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models.

Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.

Classical non-homologous end joining (cNHEJ) and homologous recombination compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

Complex interactions between the DNA-damage response and mammalian telomeres.

Natural chromosome ends resemble double-stranded DNA breaks, but they do not activate a damage response in healthy cells. Telomeres therefore have evolved to solve the 'end-protection problem' by inhibiting multiple DNA damage-response pathways. During the past decade, the view of telomeres has progressed from simple caps that hide chromosome ends to complex machineries that have an active role in organizing the genome. Here we focus on mammalian telomeres and summarize and interpret recent discoveries in detail, focusing on how repair pathways are inhibited, how resection and replication are controlled and how these mechanisms govern cell fate during senescence, crisis and transformation.

ALT telomeres borrow from meiosis to get moving.

Telomere clustering is required for the homologous recombination events that maintain chromosome ends in cells relying on alternative lengthening of telomeres (ALT). New data demonstrate that damage signaling at telomeres, a likely step in activating maintenance mechanisms, induces directional movement and synapsis driven by the machinery responsible for recombination in meiosis.

Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin.

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/ß/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.

Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin.

Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway.

Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.

The mechanism of activation of the alternative lengthening of telomeres (ALT) pathway of mammalian chromosome-end maintenance has been unclear. We have now discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT-associated PML (promyelocytic leukemia) bodies (APBs), the presence of extrachromosomal telomeric DNA species, an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and intertelomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We determined that ALT induction is positively regulated by the proteins RAD17 and BLM and negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly supports the hypothesis that ALT is a consequence of histone management dysfunction.

Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators.

Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)-based proteomics to obtain a high-confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtained many candidates reported in these two studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knockdown of 10 genes encoding candidate proteins significantly affected total cellular levels of TERRA, and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/ARS2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knockdown of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation.

The heterochromatic chromosome caps in great apes impact telomere metabolism.

In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species.

Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1α.

Gene silencing by the repressive telomeric chromatin environment, referred to as telomere position effect (TPE), has been well characterized in yeast and depends on telomere length. However, proof of its existence at native human chromosome ends has remained elusive, mainly owing to the paucity of genes near telomeres. The discovery of TERRAs, the telomeric noncoding RNAs transcribed from subtelomeric promoters, paved the way to probing for telomere-length impact on physiological TPE. Using cell lines of various origins, we show that telomere elongation consistently represses TERRA expression. Repression is mediated by increased trimethylated H3K9 density at telomeres and by heterochromatin protein HP1α, with no detectable spreading of the marks beyond the telomeric tract, restricting human TPE to telomere transcription. Our data further support the existence of a negative-feedback mechanism in which longer TERRA molecules repress their own transcription upon telomere elongation.

Cancer-linked satellite 2 DNA hypomethylation does not regulate Sat2 non-coding RNA expression and is initiated by heat shock pathway activation.

Epigenetic dysfunctions, including DNA methylation alterations, play major roles in cancer initiation and progression. Although it is well established that gene promoter demethylation activates transcription, it remains unclear whether hypomethylation of repetitive heterochromatin similarly affects expression of non-coding RNA from these loci. Understanding how repetitive non-coding RNAs are transcriptionally regulated is important given that their established upregulation by the heat shock (HS) pathway suggests important functions in cellular response to stress, possibly by promoting heterochromatin reconstruction. We found that, although pericentromeric satellite 2 (Sat2) DNA hypomethylation is detected in a majority of cancer cell lines of various origins, DNA methylation loss does not constitutively hyperactivate Sat2 expression, and also does not facilitate Sat2 transcriptional induction upon heat shock. In melanoma tumor samples, our analysis revealed that the HS response, frequently upregulated in tumors, is probably the main determinant of Sat2 RNA expression in vivo. Next, we tested whether HS pathway hyperactivation may drive Sat2 demethylation. Strikingly, we found that both hyperthermia and hyperactivated RasV12 oncogene, another potent inducer of the HS pathway, reduced Sat2 methylation levels by up to 27% in human fibroblasts recovering from stress. Demethylation occurred locally on Sat2 repeats, resulting in a demethylation signature that was also detected in cancer cell lines with moderate genome-wide hypomethylation. We therefore propose that upregulation of Sat2 transcription in response to HS pathway hyperactivation during tumorigenesis may promote localized demethylation of the locus. This, in turn, may contribute to tumorigenesis, as demethylation of Sat2 was previously reported to favor chromosomal rearrangements.

Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear localization.

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN.

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.