Poly ADP-ribose Polymerase Inhibitor ABT-888 Research Articles

Ethanol- and PARP-Mediated Regulation of Ribosome-Associated Long Non-Coding RNA (lncRNA) in Pyramidal Neurons.

Although, by definition, long noncoding RNAs (lncRNAs) are not translated, they are sometimes associated with ribosomes. In fact, some estimates suggest the existence of more than 50 K lncRNA molecules that could encode for small peptides. We examined the effects of an ethanol and Poly-ADP Ribose Polymerase (PARP) inhibitor (ABT-888) on ribosome-bound lncRNAs. Mice were administered via intraperitoneal injection (i.p.) either normal saline (CTL) or ethanol (EtOH) twice a day for four consecutive days. On the fourth day, a sub-group of mice administered with ethanol also received ABT-888 (EtOH+ABT). Ribosome-bound lncRNAs in CaMKIIα-expressing pyramidal neurons were measured using the Translating Ribosome Affinity Purification (TRAP) technique. Our findings show that EtOH altered the attachment of 107 lncRNA transcripts, while EtOH+ABT altered 60 lncRNAs. Among these 60 lncRNAs, 49 were altered by both conditions, while EtOH+ABT uniquely altered the attachment of 11 lncRNA transcripts that EtOH alone did not affect. To validate these results, we selected eight lncRNAs (Mir124-2hg, 5430416N02Rik, Snhg17, Snhg12, Snhg1, Mir9-3hg, Gas5, and 1110038B12Rik) for qRT-PCR analysis. The current study demonstrates that ethanol-induced changes in lncRNA attachment to ribosomes can be mitigated by the addition of the PARP inhibitor ABT-888.

Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons.

We report on the effects of ethanol (EtOH) and Poly (ADP-ribose) polymerase (PARP) inhibition on RNA ribosomal engagement, as a proxy for protein translation, in prefrontal cortical (PFC) pyramidal neurons. We hypothesized that EtOH induces a shift in RNA ribosomal-engagement (RE) in PFC pyramidal neurons, and that many of these changes can be reversed using a PARP inhibitor. We utilized the translating ribosome affinity purification (TRAP) technique to isolate cell type-specific RNA. Transgenic mice with EGFP-tagged Rpl10a ribosomal protein expressed only in CaMKIIα-expressing pyramidal cells were administered EtOH or normal saline (CTL) i.p. twice a day, for four consecutive days. On the fourth day, a sub-group of mice that received EtOH in the previous three days received a combination of EtOH and the PARP inhibitor ABT-888 (EtOH + ABT-888). PFC tissue was processed to isolate both, CaMKIIα pyramidal cell-type specific ribosomal-engaged RNA (TRAP-RNA), as well as genomically expressed total-RNA from whole tissue, which were submitted for RNA-seq. We observed EtOH effects on RE transcripts in pyramidal cells and furthermore treatment with a PARP inhibitor "reversed" these effects. The PARP inhibitor ABT-888 reversed 82% of the EtOH-induced changes in RE (TRAP-RNA), and similarly 83% in the total-RNA transcripts. We identified Insulin Receptor Signaling as highly enriched in the ethanol-regulated and PARP-reverted RE pool and validated five participating genes from this pathway. To our knowledge, this is the first description of the effects of EtOH on excitatory neuron RE transcripts from total-RNA and provides insights into PARP-mediated regulation of EtOH effects.

Modulation of Poly ADP Ribose Polymerase (PARP) Levels and Activity by Alcohol Binge-Like Drinking in Male Mice

Binge drinking is a frequent pattern of ethanol consumption within Alcohol Use Disorders (AUDs). Binge-like ethanol exposure increases Poly(ADP-ribose) polymerase (PARP) expression and activity. PARP enzymes have been implicated in addiction and serve multiple roles in the cell, including gene expression regulation. In this study, we examined the effects of binge-like alcohol consumption in the prefrontal cortex (PFC) of adult C57BL/6J male mice via a 4-day Drinking-in-the-Dark (DID) paradigm. The role of PARP in associated gene expression and behavioral changes was assessed by administering the PARP inhibitor ABT-888 on the last DID day. We then conducted an RNA-seq analysis of the PFC gene expression changes associated with DID-consumed ethanol or ABT-888 treatment. A separate cohort of mice was inoculated with an HSV–PARP1 vector in the PFC and subject to a DID experiment to verify whether overexpressed PARP1 increased ethanol drinking. We confirmed that alcohol increases Parp1 gene expression and PARP activity in the PFC. RNA-seq showed significantly altered expression of 41 genes by DID-consumed ethanol, and of 48 genes by ABT-888. These results were confirmed by qPCR in 7 of the 10 genes validated, 4 of which have been previously associated with addiction. ABT-888 reduced, and overexpression of PFC PARP1 increased DID ethanol consumption. In our model, alcohol binge drinking induced specific alterations in the PFC expression of genes potentially involved in addiction. Pharmacological PARP inhibition proved effective in reversing these changes and preventing further alcohol consumption. Our results suggest an involvement of ethanol-induced PARP1 in reinforcing binge-like addictive behavior.

Trastuzumab-Resistant HER2+ Breast Cancer Cells Retain Sensitivity to Poly (ADP-Ribose) Polymerase (PARP) Inhibition.

HER2-targeted therapies, such as trastuzumab, have increased the survival rates of HER2+ breast cancer patients. However, despite these therapies, many tumors eventually develop resistance to these therapies. Our lab previously reported an unexpected sensitivity of HER2+ breast cancer cells to poly (ADP-ribose) polymerase inhibitors (PARPi), agents that target homologous recombination (HR)-deficient tumors, independent of a DNA repair deficiency. In this study, we investigated whether HER2+ trastuzumab-resistant (TR) breast cancer cells were susceptible to PARPi and the mechanism behind PARPi induced cytotoxicity. We demonstrate that the PARPi ABT-888 (veliparib) decreased cell survival in vitro and tumor growth in vivo of HER2+ TR breast cancer cells. PARP-1 siRNA confirmed that cytotoxicity was due, in part, to PARP-1 inhibition. Furthermore, PARP-1 silencing had variable effects on the expression of several NF-κB-regulated genes. In particular, silencing PARP-1 inhibited NF-κB activity and reduced p65 binding at the IL8 promoter, which resulted in a decrease in IL8 mRNA and protein expression. Our results provide insight in the potential mechanism by which PARPi induces cytotoxicity in HER2+ breast cancer cells and support the testing of PARPi in patients with HER2+ breast cancer resistant to trastuzumab. Mol Cancer Ther; 17(5); 921-30. ©2018 AACR.

TNFR2 promotes Adriamycin resistance in breast cancer cells by repairing DNA damage.

In recent years, tumor necrosis factor receptor2 (TNFR2) has attracted increasing attention for its important roles in promoting proliferation, migration and angiogenesis in several types of cancer. However, its role in drug resistance remain unclear. In the present study, TNFR2 expression levels in MDA‑MB‑231 and MCF‑7 breast cancer cells were demonstrated to be associated with Adriamycin (ADM) resistance. Silencing of TNFR2 in MCF‑7 cells significantly inhibited ADM resistance, while overexpression of TNFR2 in MDA‑MB‑231 cells significantly enhanced ADM resistance. ADM treatment induced phosphorylation of the histone family2A variant X (pH2AX), an established marker of DNA damage. Silencing of TNFR2 in MCF‑7 cells further induced pH2AX levels but inhibited the expression of the DNA damage repair protein, poly(ADP‑ribose) polymerase (PARP). By contract, overexpression of TNFR2 in MDA‑MB‑231 cells decreased pH2AX levels and enhanced PARP expression. Of note, treatment with the PARP inhibitor ABT888 significantly abrogated the effects of TNFR2 on pH2AX expression. On a molecular mechanism level, TNFR2 significantly affected the phosphorylation of AKT serine/threonine kinase 1 (AKT) in both cell lines, and treatment with the AKT inhibitor LY294002 effectively abrogated TNFR2‑induced PARP expression. Adrug resistance assay demonstrated that treatment with either LY294002 or ABT888 inhibited ADM resistance in breast cancer cells, and combination treatment with both LY294002 and ABT888 exhibited a significantly stronger inhibition effect on ADM resistance. The present results indicated that TNFR2 promoted ADM resistance in breast cancer cells by regulating the DNA damage repair protein PARP via the AKT signaling pathway.

The PARP inhibitor ABT-888 potentiates dacarbazine-induced cell death in carcinoids.

Monoagent DNA-alkylating chemotherapies like dacarbazine are among a paucity of medical treatments for advanced carcinoid tumors, but are limited by host toxicity and intrinsic chemoresistance through the base excision repair (BER) pathway via poly (ADP-ribose) polymerase (PARP). Hence, inhibitors of PARP may potentiate DNA-damaging agents by blocking BER and DNA restoration. We show that the PARP inhibitor ABT-888 (Veliparib) enhances the cytotoxic effects of dacarbazine in carcinoids. Two human carcinoid cell lines (BON and H727) treated with a combination of ABT-888 and dacarbazine resulted in synergistic growth inhibition signified by combination indices <1 on the Chou-Talalay scale. ABT-888 administered prior to varying dacarbazine doses promoted the suppression of neuroendocrine biomarkers of malignancy ASCL1 and CgA, shown by Western analysis. ATM phosphorylation and p21Waf1/Cip1 activation, indicative of DNA damage, were increased by ABT-888 when combined with dacarbazine treatment, suggesting BER pathway attenuation by ABT-888. PE Annexin V/7-AAD staining and sorting revealed a profound induction of apoptosis following combination treatment, which was further confirmed by increased PARP cleavage. These results demonstrate that ABT-888 synergizes dacarbazine treatment in carcinoids. Therefore, ABT-888 may help treat carcinoids unresponsive or refractory to mainstay therapies.

Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo.

Early studies with first-generation poly (ADP-ribose) polymerase (PARP) inhibitors have already indicated some therapeutic potential for sulfur mustard (SM) injuries. The available novel and more potential PARP inhibitors, which are undergoing clinical trials as drugs for cancer treatment, bring it back to the centre of interest. However, the role of PARP-1 in SM-induced injury is not fully understood. In this study, we selected a high potent specific PARP inhibitor ABT-888 as an example to investigate the effect of PARP inhibitor in SM injury. The results showed that in both the mouse ear vesicant model (MEVM) and HaCaT cell model, PARP inhibitor ABT-888 can reduce cell damage induced by severe SM injury. ABT-888 significantly reduced SM induced edema and epidermal necrosis in MEVM. In the HaCaT cell model, ABT-888 can reduce SM-induced NAD+/ATP depletion and apoptosis/necrosis. Then, we studied the mechanism of PARP-1 in SM injury by knockdown of PARP-1 in HaCaT cells. Knockdown of PARP-1 protected cell viability and downregulated the apoptosis checkpoints, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP and Caspase 3 following SM-induced injury. Furthermore, the activation of AKT can inhibit autophagy via the regulation of mTOR. Our results showed that SM exposure could significantly inhibit the activation of Akt/mTOR pathway. Knockdown of PARP-1 reversed the SM-induced suppression of the Akt/mTOR pathway. In summary, the results of our study indicated that the protective effects of downregulation of PARP-1 in SM injury may be due to the regulation of apoptosis, necrosis, energy crisis and autophagy. However, it should be noticed that PARP inhibitor ABT-888 further enhanced the phosphorylation of H2AX (S139) after SM exposure, which indicated that we should be very careful in the application of PARP inhibitors in SM injury treatment because of the enhancement of DNA damage.

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

Abstract 5327: Simultaneous inhibition of ATR and PARP greatly sensitizes colon cancer cell lines to irinotecan

Abstract Enhanced DNA damage repair is one mechanism behind colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these molecules at nontoxic single agent concentrations synergized with SN38 to produce a 2.2 to 3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 (Table 1). When combined, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5 to 27 fold reduction in the IC50 of SN38. Furthermore, the combination of all three agents was associated with maximal G2-M arrest and enhanced DNA-damage (γH2AX). The mechanism of this enhanced synergy was associated with maximal suppression of SN38 induced PARP activity in the presence of both inhibitors. Furthermore, VE-821 enhancement of SN38 induced DNA-PK phosphorylation was abrogated by ABT-888, resulting in more unrepaired DNA damage. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help circumvent resistance to this drug in colon cancer. Cell LineVE-821 (μM)ABT-888 (μM)SN38 (IC50) ± SE (nM)I valueP valueLoVo13.5±1.214.5±1.90.30.020.55.8±2.00.40.030.59.4±1.00.70.0610.53.0±1.20.20.010.50.54.7±1.90.40.02HCT-1168.1±0.713.5±0.50.50.010.55.2±0.60.70.030.53.9±1.10.50.0310.50.3±0.030.10.010.50.52.1±0.40.30.01HT-2920.5±1.819.3±0.60.60.010.512.2±1.30.70.020.512.9±0.70.60.0210.53.9±0.50.30.010.50.56.1±0.20.40.01 Table 1 Colon cancer cell lines LoVo, HCT-116 and HT-29 were treated with SN38 alone or in combination with various concentrations of the ATR inhibitor VE-821 and/or the PARP inhibitor ABT-888. The IC50 values of SN38 were significantly reduced in the presence of VE-821 and/or ABT-888 for all cell lines. Drug synergy (I) values were determined, according to Berenbaum (1978), by calculating the ratio: (IC50 SN38 with inhibitors/IC50 SN38 alone) + (inhibitor concentration used/IC50 inhibitor alone); I less than 1 = synergism, I equal to 1 = additive effect and I greater than 1 = antogonism. Citation Format: David Davidson, Atlal Abu-Sanad, Yunzhe Wang, Fatemeh Hasheminasab, Justin Panasci, Raquel Aloyz, Lawrence Panasci. Simultaneous inhibition of ATR and PARP greatly sensitizes colon cancer cell lines to irinotecan. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5327. doi:10.1158/1538-7445.AM2015-5327

Abstract 3632: Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound that was used for treating arthritis. Its primary mechanism of action is the inhibition of thioredoxin reductase (TRR) activity. It consequently induces oxidative/ER stress and also inhibits Heat shock protein (Hsp) 90 chaperone function. Ataxia-telangiectasia mutated and Rad3-related (ATR), Brca1, Chk1 and DNA-PK are Hsp90 client proteins that participate in DNA repair pathways. Based on our observation that AF induces DNA damage, we evaluated whether treatment with AF also impairs Hsp90-dependent DNA damage repair pathways in Mantle Cell Lymphoma (MCL) cells. It has been reported that ATM mutations are found in about 50% cases of MCL. Earlier studies have shown that ATM mutant MCL cells are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors in vitro and in vivo. We hypothesized that by inhibiting Hsp90-dependent DNA repair pathways (such as the ATR pathway), AF would sensitize ATM-proficient MCL cells to PARP inhibition by ABT-888. We demonstrate that treatment of ATM-proficient MCL cell lines, JeKo-1, MO2058 and Z138C with AF induced reactive oxygen species (ROS) levels and resulted in a concomitant decrease in TRR activity. Microarray analysis of AF-treated primary MCL cells revealed that AF significantly altered heat shock response genes, ER and oxidative stress-inducible genes and a subset of genes that were regulated by ATM or Brca1. Treatment with AF also induced Hsp90 hyperacetylation and resulted in the depletion of Hsp90 client proteins including ATR, AKT and Cyclin D1 in MCL cells. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (&amp;lt; 15% apoptosis) (p &amp;lt; 0.01). Co-treatment of MCL cells with AF and the PARP inhibitor ABT-888 resulted in synergistic increase in apoptosis of ATM-proficient MCL cells, with an increase in the induction of γH2AX foci and depletion of p-Chk1 (a downstream target of ATR signaling). This was accompanied with a concomitant increase in the levels of the ER stress-induced transcription factor CHOP (C/EBP homologous protein), cleaved Caspase 3 and PARP. Collectively, our data suggests the synthetic lethal effects observed by the simultaneous inhibition of DNA repair pathway(s) and PARP activity can be extended to ATM-proficient MCL cells. These pre-clinical studies create a strong rationale to determine the in vivo activity of the combination ABT-888 with AF in MCL. Citation Format: Siddhartha Ganguly, Trisha Home, Sumedha Gunewardena, Scott Weir, Joseph McGuirk, Rekha M. Rao. Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2015-3632

PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression.

To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosis.

Epigenetic Silencing of BRCA1 Is Linked to Homologous Recombination Repair Defects and Elevated Mir-155 Expression in Myeloid Neoplasms

Myeloid neoplasms are a heterogeneous group of clonal hematological disorders ranging from chronic myeloproliferative neoplasms (MPNs) to more aggressive mixed myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPNs) and secondary acute myeloid leukemias (sAMLs). With the exception of BCR-ABL positive chronic myelogenous leukemia (CMLs), these diseases are clinically challenging to diagnose. Due to the clinical heterogeneity and lack of new biologic insights, current therapies fail to eradicate the malignant clone and alter the natural history of the disease. However, these diagnoses share overlapping features in morphologies, cytogenetics and genetic alterations suggesting common alterations in underlying disease pathways.We have previously reported genomic loss of HR genes in MPN patients linked to defective RAD51 foci induction and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition (McDevitt et al., ASH 2011). In this study, we directly examined homologous recombination (HR) in primary human samples by measuring induction of RAD51 foci after ionizing irradiation. Freshly isolated mononuclear leukocytes from patients diagnosed with MPNs, mixed MDS/MPNs and sAMLs were collected at the Johns Hopkins Hospital from April 2012 to March 2014. 11 of the 22 samples (50%) exhibited impaired RAD51 foci induction. With little evidence for biallelic genetic inactivation of HR genes in previous work, we evaluated promoter CpG island methylation of BRCA1, which we had previously found in a pilot study of myeloid neoplasm samples (McDevitt et al., ASH 2011). Using quantitative methylation-specific PCR (qMSP) with melt curve analysis, we reported BRCA1 promoter hypermethylation in 22 of 104 samples (21.2%), and observed statistically significant down-regulation of BRCA1 transcript in samples with BRCA1 CpG methylation (p<0.05). To determine the relationship between BRCA1 epigenetic silencing and HR status, we categorized BRCA1 gene expression of the samples according to the promoter methylation and HR status. Strikingly, we noted that all BRCA1 methylated samples were defective for HR and 5/12 (41.7%) of HR defective samples had down-regulated BRCA1 levels.We next validated the role of BRCA1 repression in vitro using the AML cell line OCI-AML3 using two independent shRNAs targeting BRCA1. We found that BRCA1-silenced cells have reduced induction of RAD51 foci, recapitulating our observations in primary samples with epigenetic silencing of BRCA1. Given the selective toxicity observed with HR-deficient cancers to PARP inhibition, we treated the BRCA1 knockdown cells with the PARP inhibitor ABT-888 and observed increased drug sensitivity.Finally, we examined additional consequences of BRCA1 loss in myeloid malignancies by investigating its role in repressing miR-155. Frequent up-regulation of miR-155 in leukemia is linked to poor prognosis, and its overexpression in murine models results in myeloproliferative disorders. Our results showed a statistically significant inverse correlation between BRCA1 and miR-155 expression in patient samples (Pearson r:-0.36; p-value<0.005). We also found elevated miR-155 expression in the BRCA1-silenced OCI-AML3 cells that increased during long-term BRCA1 repression.Our results demonstrate that defective HR repair occurs in a significant subset of myeloid malignancies. This provides a rationale for PARP inhibitors in this patient subpopulation who currently lack curative therapy, a strategy currently explored in two clinical studies (J0783, PI: Keith Pratz, MD; J1051, PI: Ivana Gojo, MD). Our finding showing BRCA1 promoter methylation in samples with defective HR highlights a novel mechanism underlying HR defects in myeloid malignancies, and offers a biomarker to identify potential responders to PARP inhibition. We also provide an alternative mechanism in which BRCA1 loss contributes to disease progression, via de-repressing miR-155 linked to aberrant expansion of myeloid cells. DisclosuresNo relevant conflicts of interest to declare.

TGFβ induces "BRCAness" and sensitivity to PARP inhibition in breast cancer by regulating DNA-repair genes.

Transforming growth factor beta (TGFβ) proteins are multitasking cytokines, in which high levels at tumor sites generally correlate with poor prognosis in human patients with cancer. Previously, it was reported that TGFβ downregulates the expression of ataxia telangiectasia-mutated (ATM) and mutS homolog 2 (MSH2) in breast cancer cells through an miRNA-mediated mechanism. In this study, expression of a panel of DNA-repair genes was examined, identifying breast cancer 1, early onset (BRCA1) as a target downregulated by TGFβ through the miR181 family. Correlations between the expression levels of TGFβ1 and the miR181/BRCA1 axis were observed in primary breast tumor specimens. By downregulating BRCA1, ATM, and MSH2, TGFβ orchestrates DNA damage response in certain breast cancer cells to induce a "BRCAness" phenotype, including impaired DNA-repair efficiency and synthetic lethality to the inhibition of poly (ADP-ribose) polymerase (PARP). Xenograft tumors with active TGFβ signaling exhibited resistance to the DNA-damaging agent doxorubicin but increased sensitivity to the PARP inhibitor ABT-888. Combination of doxorubicin with ABT-888 significantly improved the treatment efficacy in TGFβ-active tumors. Thus, TGFβ can induce "BRCAness" in certain breast cancers carrying wild-type BRCA genes and enhance the responsiveness to PARP inhibition, and the molecular mechanism behind this is characterized. These findings enable better selection of patients with sporadic breast cancer for PARP interventions, which have exhibited beneficial effects in patients carrying BRCA mutations.

Abstract 5469: Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition

Abstract Background: Overexpression of HER2 in breast cancer is associated with aggressive tumor features and poor patient survival. Current therapeutic agents targeting this receptor have increased the survival rate of patients with HER2+ breast cancer; however some patients eventually develop resistance to these therapies. We have previously reported that HER2+ breast cancers are susceptible to poly (ADP-Ribose) polymerase inhibitors (PARPi) alone independent of a basal or induced DNA repair deficiency but instead via suppression of NF-κB signaling. In this study, we investigated PARPi sensitivity in HER2+ breast cancer cells that are resistant to the HER2 targeted agent trastuzumab. Materials and Methods: HER2+ breast cancer cell lines BT-474, UACC812, SKBRR3 and their trastuzumab resistant counterparts were used in this study. Cells were treated with vehicle or 10µM ABT-888 or transfected with scrambled or PARP1 siRNA. NF-κB transcriptional activity was measured using a NF-κB-driven luciferase reporter assay. Protein expression was measured by Western blot analysis. Cellular cytotoxicity and viability following PARPi was assessed with colony formation and ATPlite assays, respectively. Results: HER2+ parent and trastuzumab resistant breast cancer cells showed similar susceptibility to the PARPi ABT-888. There was a dose response reduction in survival fraction (greater than 70% at 10μM) and a greater than 40% decrease in cell viability with 10μM ABT-888. This cytotoxicity was associated with more than 50% attenuation of NF-κB transcriptional activity and reduced expression of the NF-κB activator IKKα. Suppression of NF-κB signaling was also observed utilizing PARP1 siRNA. Conclusions: HER2+ breast cancer cells resistant to trastuzumab continue to be sensitive to PARP inhibition through attenuation of the NF-κB signaling pathway. These results support the use of PARPi as part of a therapeutic strategy for patients with HER2+ breast cancer. Citation Format: Monica E. Wielgos, Tiffiny Cooper, Andres Forero, James A. Bonner, Francisco J. Esteva, C K. Osborne, Rachel Schiff, Albert F. LoBuglio, Eddy S. Yang. Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5469. doi:10.1158/1538-7445.AM2014-5469

PARP inhibition and the radiosensitizing effects of the PARP inhibitor ABT-888 in in vitro hepatocellular carcinoma models.

BackgroundHepatocellular carcinoma is the third cause of cancer related death for which new treatment strategies are needed. Targeting DNA repair pathways to sensitize tumor cells to chemo- or radiotherapy is under investigation for the treatment of several cancers with poly(ADP-ribose) polymerase (PARP) inhibitors showing great potential. The aim of this preclinical study was to evaluate the expression of PARP and PARG genes in a panel of liver cancer cell lines and primary human hepatocytes, their DNA repair capacity and assess the impact on cell survival of PARP inhibitors alone and in combination with radiotherapy.MethodsQuantitative PCR was used to measure PARP-1, -2, -3 and PARG mRNA levels and western blotting for PARP-1 protein expression and ADP-ribose polymer formation after exposure of cells to doxorubicin, a topoisomerase II poison. DNA repair capacity was assessed using an in vitro DNA lesion excision/synthesis assay and the effects on cell killing of the PARP inhibitor ABT-888 alone and in combination with ionizing radiation using clonogenic survival.ResultsAlthough a wide range in expression of the PARPs and PARG was found correlations between PARP-1 and PARP-2 mRNA levels and PARP-1 mRNA and protein levels were noted. However these expression profiles were not predictive of PARP activity in the different cell lines that also showed variability in excision/synthesis repair capacity. 4 of the 7 lines were sensitive to ABT-888 alone and the two lines tested showed enhanced radiosensitivity in the presence of ABT-888.ConclusionsPARP inhibitors combined with radiotherapy show potential as a therapeutic option for hepatocellular carcinoma.

P1-17-04: Phase I Study of PARP Inhibitor ABT-888 (Veliparib) in Combination with Cisplatin and Vinorelbine for Patients with Advanced Triple Negative Breast Cancer and/or BRCA-Mutation Associated Breast Cancer.

Abstract Background: Inhibitors of poly(ADP-ribose) polymerase (PARP) have shown preclinical and clinical activity in targeting tumors with pre-existing DNA repair defects, in particular BRCA1 and BRCA2 deficient tumors. Cisplatin (CP) has demonstrated synergy with ABT-888 (veliparib) in breast cancer xenograft models and has anti-tumor activity in triple negative (TN) and BRCA-1 deficient breast cancer. Vinorelbine (V) combined with CP has shown safety and efficacy in patients with pretreated metastatic breast cancer. Methods: We are conducting a phase I study to determine the maximum tolerated dose (MTD), pharmacokinetic (PK) and pharmacodynamic profiles (PD), and preliminary anti-tumor activity of veliparib in combination with CP and V in patients (pts) with metastatic breast cancer who are either TN or who have BRCA1 or BRCA2 mutation associated breast cancer. Cohorts receive escalating doses of ABT-888 orally BID days 1–14, CP 75 mg/m2 intravenously (IV) day 1 and V 25 mg/m2 IV days 1,8 every 21 days, in a 3+3 design. Results: As of 6/14/11, 18 eligible female pts have been enrolled. The median age at registration was 50 years (range 34–78 years). Sixteen pts received at least one prior metastatic regimen (range 0–8). Three pts had previously treated brain metastases. BRCA mutation status was as follows: BRCA1+ (3 pts); BRCA2+ (2 pts); confirmed mutation negative (8 pts); unknown (5 pts). Four pts received the 20 mg BID veliparib dose (one patient in the cohort died of progressive disease early in cycle 1 and was replaced), 3 received 30 mg BID, 6 received 40 mg BID, and 5 have been enrolled at dose level 4 (60 mg BID). Dose limiting toxicities occurred in one patient at the 40 mg BID dose (grade 4 thrombocytopenia), and one patient at the 60 mg BID dose (grade 3 neutropenic fever). Adverse events are typical for a platinum-based chemotherapy regimen and include nausea, fatigue, thrombocytopenia, and neutropenia. MTD has not been reached. Of 11 pts evaluable for radiographic response to date, 6 (55%) had a PR (3 of whom have a BRCA mutation) and 5 (45%) had stable disease. Correlative studies will use immunohistochemistry and gene expression array to evaluate the profile of TN breast cancer and predictors of response to treatment. Conclusion: Veliparib in combination with CP and V has been generally well tolerated to date. Objective anti-tumor activity was seen in BRCA mutation carriers and in pts with sporadic TN breast cancer. PK, PD, and biomarker analysis is underway. Enrollment continues in the dose escalation cohorts. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-17-04.

Cetuximab Augments Cytotoxicity with Poly (ADP-Ribose) Polymerase Inhibition in Head and Neck Cancer

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers.

The poly(ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy

Background and purposeCurrent therapies for head and neck cancer frequently are not curative, necessitating novel therapeutic strategies. Thus, we studied whether inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, could improve efficacy of radiotherapy in human head and neck cancer. Materials and methodsUM-SCC1, UM-SCC5, UM-SCC6, and FaDu human head and neck cancer cellular susceptibility to the PARP inhibitor (PARPi) ABT-888 and/or radiation (IR) was assessed using colony formation assays. DNA damage was evaluated using the alkaline comet assay and immunostaining for γ-H2AX foci. Non-homologous end-joining (NHEJ) mediated repair was measured using phospho-DNA-Pk foci. Epidermal growth factor receptor (EGFR) location was assessed by immunostaining. Poly ADP-Ribose polymerization (PAR) levels were assessed using immunoblotting. ResultsHuman head and neck cancer cells exhibited enhanced cytotoxicity with IR and ABT-888 compared to either agent alone. This increased susceptibility correlated with reduced nuclear EGFR, attenuation of NHEJ, and persistence of DNA damage following IR. Interestingly, a subset of head and neck cancer cells which had elevated basal PAR levels was susceptible to PARPi alone. ConclusionsCombining radiotherapy and PARP inhibition may improve outcomes and quality of life for head and neck cancer patients treated with radiotherapy. Furthermore, this novel strategy may also be feasible in other tumor types. Moreover, PAR levels should be investigated as a potential biomarker for tumor susceptibility to PARP inhibition.

A phase II study of the PARP inhibitor ABT-888 plus temozolomide in patients with heavily pretreated, metastatic colorectal cancer.

3502 Background: Inhibitors of poly(ADP-ribose) polymerase (PARP), such as ABT-888 are potent sensitizing agents for, and have been safely combined with DNA-damaging chemotherapeutic agents such as temozolomide (TMZ). The sensitizing effects of PARP inhibitors are magnified when cancer cells harbor underlying defects in DNA repair mechanisms. Methods: We performed a single arm, open label phase II study. Patients with metastatic, unresectable colorectal cancer (CRC) whose disease had progressed on all standard therapies were eligible. Patients received TMZ (150mg/m2 orally daily) days 1-5, and ABT-888 (40mg orally twice-a-day) days 1-7 of each 28-day cycle. Restaging studies were performed every 8 weeks. The primary endpoint was the disease control rate (DCR), defined as complete response, partial response (PR), or stable disease (after two cycles). This trial followed a Simon’s two-stage optimal design using p0=0.10 and p1=0.25 and type I and II errors of 0.10. Secondary clinical endpoints included response rate (RR) and time to progression (TTP). Archived tumor specimens were used for immunohistochemistry (IHC) to assess mismatch repair enzyme (MMR) and PTEN protein expression. Results: Between 09-2010 and 01-2011, 47 patients received treatment. The median age was 54, and most patients had an ECOG PS of 1. The average number of prior therapies was 4. The combination of ABT-888 plus TMZ was very well tolerated. There were only five grade 3 adverse events, all related to myelosuppression, which resolved with a one week treatment delay. The primary endpoint was successfully met, with a DCR of 23%. There were two confirmed PRs, for a RR of 5%. The median TTP was 11 weeks overall, but was 23 weeks in the patients who had disease control. 45 archived tumor samples were obtained, and the results of IHC for the MMR and PTEN proteins will be reported. Conclusions: In this heavily pretreated CRC patient population, the combination of ABT-888 and TMZ was very well tolerated and exhibited clinical activity. Further molecular analyses of patients’ tumor samples, and enrollment of a 20 patient MMR-deficient expansion cohort may help identify which patient subgroups are most likely to respond to therapy.

Abstract 5496: Cetuximab induces synthetic lethality with the poly (ADP-Ribose) polymerase (PARP) inhibitor ABT-888 in head and neck cancer

Abstract Purpose: Overexpression/amplification of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab, almost half of treated patients fail this therapy. PARP inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing homologous recombination (HR)-deficient tumors while maintaining minimal toxicity in normal tissues. As EGFR inhibition has been reported to alter cellular DNA repair capacity, we investigated whether cetuximab could induce a transient DNA repair deficit and subsequently induce a synthetic lethality with the PARPi ABT-888 in head and neck cancer cells. Methods: The head and neck cancer cell lines UM-SCC1, UM-SCC6, and FaDu were used in this study. Kinetics of DNA damage and repair were assessed by immunofluorescence staining of cells for γ-H2AX, Rad51, and DNA-Pk foci at various time points following irradiation (IR). Cell viability was determined via the ATPlite as well as colony formation assays. Apoptosis was evaluated by Annexin V staining as well as cleaved caspase 3 and 9 levels. Cell cycle analysis was performed via flow cytometry. Results: Cetuximab increases γ-H2AX foci, which are well established markers of DNA double strand breaks (DSBs), in head and neck tumor cells. This coincides with reduced DSB-repair as indicated by attenuation of IR-induced Rad51 and DNA-Pk foci, which are well characterized in situ markers of the homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair pathways, respectively. Importantly, cetuximab induces synthetic lethality with the PARPi ABT-888 through a mechanism involving persistent DNA damage and subsequent activation of the intrinsic pathway of apoptosis. The observed effects are not due to cell cycle redistribution. Conclusions: By generating a DSB repair deficiency, cetuximab can render head and neck tumor cells susceptible to PARP inhibition. The combination of cetuximab and the PARPi ABT-888 can thus be an innovative treatment strategy to enhance therapeutic ratio and improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5496. doi:10.1158/1538-7445.AM2011-5496