PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies.

Expression of protein arginine methyltransferase 5 (PRMT5) is highly positively correlated to DNA damage repair (DDR) and DNA replication pathway genes in many types of cancer cells, including ovarian and breast cancer. In the current study, we investigated whether pharmacologic inhibition of PRMT5 downregulates DDR/DNA replication pathway genes and sensitizes cancer cells to chemotherapy and PARP inhibition. Potent and selective PRMT5 inhibitors significantly downregulate expression of multiple DDR and DNA replication genes in cancer cells. Mechanistically, PRMT5 inhibition reduces the presence of PRMT5 and H4R3me2s on promoter regions of DDR genes such as BRCA1/2, RAD51, and ATM. PRMT5 inhibition also promotes global alternative splicing changes. Our data suggest that PRMT5 inhibition regulates expression of FANCA, PNKP, and ATM by promoting exon skipping and intron retention. Combining C220 or PRT543 with olaparib or chemotherapeutic agents such as cisplatin demonstrates a potent synergistic interaction in breast and ovarian cancer cells in vitro. Moreover, combination of PRT543 with olaparib effectively inhibits the growth of patient-derived breast and ovarian cancer xenografts. Furthermore, PRT543 treatment significantly inhibits growth of olaparib-resistant tumors in vivo. These studies reveal a novel mechanism of PRMT5 inhibition and suggest beneficial combinatorial effects with other therapies, particularly in patients with tumors that are resistant to therapies dependent on DNA damage as their mechanism of action. SIGNIFICANCE: Patients with advanced cancers frequently develop resistance to chemotherapy or PARP inhibitors mainly due to circumvention and/or restoration of the inactivated DDR pathway genes. We demonstrate that inhibition of PRMT5 significantly downregulates a broad range of the DDR and DNA replication pathway genes. PRMT5 inhibitors combined with chemotherapy or PARP inhibitors demonstrate synergistic suppression of cancer cell proliferation and growth in breast and ovarian tumor models, including PARP inhibitor-resistant tumors.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and B-Cell Growth Transformation Induce Lipogenesis through Fatty Acid Synthase.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkin's and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitt's lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1- and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids, and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could be used in the treatment of LMP1+ EBV-associated malignancies by targeting an LMP1-specific dependency on lipogenesis.IMPORTANCE Despite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational, and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of virus-associated malignancies. Noncancerous cells preferentially obtain fatty acids from dietary sources, whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis is an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

Poly(ADP-ribose) polymerase 1 is necessary for coactivating hypoxia-inducible factor-1-dependent gene expression by Epstein-Barr virus latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt's lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic 'Warburg' metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers.

IGH/MYC Translocation Associates with BRCA2 Deficiency and Synthetic Lethality to PARP1 Inhibitors.

Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC-positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with diminished HR activity and hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary Burkitt lymphoma xenografts. In conclusion, IGH/MYC-positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC-induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpgMol Cancer Res; 15(8); 967-72. ©2017 AACR.

PARP1 restricts Epstein Barr Virus lytic reactivation by binding the BZLF1 promoter.

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation.

MLL-AF9 leukemias are sensitive to PARP1 inhibitors combined with cytotoxic drugs.

PARP1 is required for the maintenance of MLL-AF9 leukemias.PARP1 inhibitors enhance the therapeutic effect of cytotoxic drugs against MLL-AF9 leukemias.

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

An implemented MRI program to eliminate radiation from the evaluation of pediatric appendicitis.

BACKGROUND: Recent efforts have been directed at reducing ionizing radiation delivered by CT scans to children in the evaluation of appendicitis. MRI has emerged as an alternative diagnostic modality. The clinical outcomes associated with MRI in this setting are not well-described. METHODS: Review of a 30-month institutional experience with MRI as the primary diagnostic evaluation for suspected appendicitis (n=510). No intravenous contrast, oral contrast, or sedation was administered. Radiologic and clinical outcomes were abstracted. RESULTS: MRI diagnostic characteristics were: sensitivity 96.8% (95% CI: 92.1%-99.1%), specificity 97.4% (95% CI: 95.3-98.7), positive predictive value 92.4% (95% CI: 86.5-96.3), and negative predictive value 98.9% (95% CI: 97.3%-99.7%). Radiologic time parameters included: median time from request to scan, 71 minutes (IQR: 51-102), imaging duration, 11 minutes (IQR: 8-17), and request to interpretation, 2.0 hours (IQR: 1.6-2.6). Clinical time parameters included: median time from initial assessment to admit order, 4.1 hours (IQR: 3.1-5.1), assessment to antibiotic administration 4.7 hours (IQR: 3.9-6.7), and assessment to operating room 9.1 hours (IQR: 5.8-12.7). Median length of stay was 1.2 days (range: 0.2-19.5). CONCLUSION: Given the diagnostic accuracy and favorable clinical outcomes, without the potential risks of ionizing radiation, MRI may supplant the role of CT scans in pediatric appendicitis imaging.

Peritoneal hemorrhage due to a ruptured ovarian cyst in ITP.

Immune thrombocytopenic purpura in children rarely causes severe bleeding. The incidence of intracranial hemorrhage is approximately 0.2% to 1.0%, and severe bleeding (defined as persistent epistaxis, melena, menorrhagia, gastrointestinal bleeding, etc, requiring hospitalization or transfusion) occurs in only 5% of patients. Epstein-Barr virus (EBV) associated idiopathic thrombocytopenic purpura (ITP) tends to behave similarly to non-EBV - associated ITP with no increase in hemorrhagic complications and only a small increase in time to remission. Immune thrombocytopenic purpura diagnosed in adolescence is more likely to be chronic then in childhood ITP, but has a higher rate of spontaneous resolution than in adults. However, females in this age group are in their early childbearing years and present a unique set of possible hemorrhagic complications not seen in younger patients. We present the case of an 18-year-old female with EBV-associated ITP, who developed a severe intra-abdominal bleed secondary to a hemorrhagic ovarian cyst.