From weakness comes strength Pfister, Sophia X.
Science (American Association for the Advancement of Science),
07/2022, Letnik:
377, Številka:
6604
Journal Article
Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a ...role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.
Display omitted
•A role for SETD2 in DSB resection and homologous recombination repair•Histone H3K36me3 is required for homologous recombination•SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining•Mutations affecting H3K36me3 levels may promote tumorigenesis
The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.
Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which ...H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.
Display omitted
•WEE1 inhibition selectively kills H3K36me3-deficient cancer cells•These cells are killed through dNTP starvation because of RRM2 depletion•RRM2 is regulated by H3K36me3 through transcription and WEE1 via degradation•WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumors in vivo
Pfister et al. show that WEE1 inhibition selectively kills H3K36me3-deficient cancer cells through dNTP starvation resulting from RRM2 depletion. Pfister et al. further show that H3K36me3 facilitates RRM2 transcription whereas WEE1 inhibition promotes RRM2 degradation via CDK activation.
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end ...joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting ...from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.
SET-ting the stage for DNA repair Jha, Deepak K; Pfister, Sophia X; Humphrey, Timothy C ...
Nature structural & molecular biology,
08/2014, Letnik:
21, Številka:
8
Journal Article
Recenzirano
Mechanisms of DNA damage repair within actively transcribed genes are poorly understood. Five new reports shed light on the contributions of chromatin to this process by uncovering roles for histone ...H3 Lys36 methylation, a post-translational modification previously linked to transcription elongation, in the control of DNA-damage signaling and double strand break repair.
Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that ...are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage.
Abstract
Background: Loss of the histone mark H3 Lysine 36 trimethylation (H3K36me3) is highly prevalent and is associated with poor prognosis. However, there is currently no therapy targeting ...H3K36me3-deficient cancers. Recently we found a novel synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to inhibition of WEE1 (a checkpoint kinase). Here we provide our latest and detailed understanding of the underlying mechanism and report our findings in biomarker development.
Methods: SETD2 is the sole methyltransferase responsible for generating H3K36me3. We used CRISPR-Cas9 methodology to knockout expression of SETD2 in order to create isogenic cancer cell lines that are negative for H3K36me3. Survival of these isogenic cell lines was measured after treatment with the WEE1 inhibitor AZD1775. Recruitment of transcription factors to the chromatin was analyzed by chromatin immunoprecipitation (ChIP). Rescue experiments were performed by expressing a mutant ribonucleotide reductase (RRM2) that cannot be phosphorylated by CDK (Cyclin-dependent kinase) and is resistant to subsequent degradation by SCF(CyclinF). Immunohistochemistry (IHC) was performed on patient tissue microarrays (>100 samples per cancer type) using a monoclonal antibody against H3K36me3.
Results: We showed that SETD2 CRISPR knockout cells are 12-fold more sensitive to the WEE1 inhibitor AZD1775 than parental cells (IC50 = 10 nM vs. 128 nM, p<0.0001) and established that RRM2 (a ribonucleotide reductase subunit that produces deoxynucleotides (dNTPs)) is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways in this context: first, H3K36me3 facilitates RRM2 transcription by recruiting transcription initiation factors to the promoter of RRM2; second, WEE1 inhibition degrades RRM2 through activation of CDK and untimely phosphorylation of RRM2. Therefore WEE1 inhibition in H3K36me3-deficient cells results in critical depletion of RRM2 and dNTPs (70% reduction in dNTP, p<0.01), collapsed replication forks and apoptosis. Accordingly, lethality in SETD2 knockout cells is suppressed by increasing RRM2 transcription or inhibiting RRM2 degradation. Encouraged by a robust regression in xenograft tumors, we tested the clinical potential of treating H3K36me3-deficient cancers with the WEE1 inhibitor. To this end we developed an immunohistochemistry assay for patient tissue microarrays, and found that 46% of renal, 16% of colorectal and 10% of non-small cell lung cancers exhibited markedly reduced levels of H3K36me3.
Conclusions: Our study has uncovered the mechanism for the selective killing of H3K36me3-deficient cancers by inhibition of WEE1. We identified novel roles for histone H3K36me3 in promoting gene transcription and DNA replication, and for WEE1 in maintaining nucleotide pools by controlling RRM2 degradation. Our proposed treatment of H3K36me3-deficient cancers is based on synthetic lethality, which provides a less toxic and more effective treatment as it specifically targets cancer cells. Our biomarker analyses revealed that H3K36me3 loss is highly prevalent in renal, colorectal and lung cancers, suggesting that it could be an important therapeutic target in these cancers. H3K36me3 loss can be used as a predictive biomarker for WEE1 inhibitor treatment, and may enable patient selection through immunohistochemistry. Finally, as the WEE1 inhibitor AZD1775, for which we describe a new target, is in clinical trials, we anticipate that these findings will be of immediate clinical relevance.
Citation Format: Sophia X. Pfister, Enni Markkanen, Yanyan Jiang, Sovan Sarkar, Mick Woodcock, Lykourgos-Panagiotis Zalmas, Giulia Orlando, Neele Drobnitzky, Grigory Dianov, Songmin Ying, Nicholas B. La Thangue, Vincenzo D'Angiolella, Anderson Ryan, Timothy C. Humphrey. WEE1 inhibition selectively kills histone H3K36me3-deficient cancers by dNTP starvation. abstract. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B40.
PDF
