EGFR amplification predicted selective sensitivity to PARP inhibitors with high PARP-DNA trapping potential in human GBM.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2047-2047
Author(s):  
W. K. Alfred Yung ◽  
Shaofang Wu ◽  
Feng Gao ◽  
Siyuan Zheng ◽  
Jie Ding ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Parp Inhibitor ◽  
Single Agent ◽  
Parp Inhibitors ◽  
Glioma Stem Cells ◽  
Egfr Amplification ◽  
Damage Repair ◽  
Selective Sensitivity ◽  
Dna Trapping

2047 Background: Poly-ADP-ribose polymerase (PARP) is an enzyme critical for regulating a variety of DNA damage repair mechanisms such as BER/SSBR, and PARP inhibitors have been shown to have single agent activity in breast and ovarian cancer patients with BRCA ½ mutations. However, PARP inhibitor such as veliparib has limited single agent activity in GBM and identifying markers predicting sensitivity is critical to select individuals or certain groups of patients for PARP inhibitor therapy. Methods: Potency and selectivity of PARP inhibitors were analyzed in a panel of glioma stem cells (GSCs) with varying genetic background. In vivo anti-tumor activity was evaluated in xenograft models. Results: In this study, we report that PARP inhibitor, talazoparib, showed strong single-agent cytotoxicity and remarkable selective activity in glioma stem cells (GSCs). This single agent activity was strongly correlated with EGFR amplification. GSCs with EGFR amplification (which occurs in about 45% of GBMs) showed higher oxidative base damage, DNA breaks, and genomic instability than non-amplified GSCs. To sustain the elevated basal oxidative stress, EGFR-amplified GSCs had increased basal expression of DNA repair proteins. As a result of blocked DNA damage repair by talazoparib treatment, DNA damage accumulated and lead to increased PARP-DNA complexes, which was then trapped by talazoparib and resulted in high toxicity. The PARP-DNA trapping function of PARPi is essential as olaparib and veliparib, two PARP inhibitors with weak DNA-PARP trapping potential did not show sensitivity in GSCs. In contrast, Pamiparib, another PARP inhibitor with similar PARP-DNA trapping ability to that of talazoparib, showed selective sensitivity in EGFR-amplified GSC. Conclusions: Our data showed that EGFR amplified GSCs with higher basal DNA damage exhibited therapeutic vulnerability to PARP inhibitors with high PARP-DNA trapping ability, and that EGFR amplification is a potential selection or predictive biomarker for PARP inhibitor therapy in GBM.

scholarly journals EGFR Amplification Induces Increased DNA Damage Response and Renders Selective Sensitivity to Talazoparib (PARP Inhibitor) in Glioblastoma

2019 ◽  
Vol 26 (6) ◽  
pp. 1395-1407 ◽  
Author(s):  
Shaofang Wu ◽  
Feng Gao ◽  
Siyuan Zheng ◽  
Chen Zhang ◽  
Emmanuel Martinez-Ledesma ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Parp Inhibitor ◽  
Egfr Amplification ◽  
Damage Response ◽  
Selective Sensitivity

scholarly journals Loss of PTEN in Pediatric AML Confers Sensitivity to PARP Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3446-3446
Author(s):  
Jennifer Lauren Kamens ◽  
Anitria Cotton ◽  
Jeannie W Lam ◽  
Jinjun Dang ◽  
Aman Seth ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Risk ◽  
Cell Lines ◽  
Parp Inhibitor ◽  
Single Agent ◽  
Jak2 V617f ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Sensitive Cell ◽  
Pediatric Aml

Abstract Pediatric Acute Myeloid Leukemia (AML) is a rare, but deadly cancer. Outcomes over the last 20 years have remained stagnant with an overall 5-year survival rate < 70% and relapse rates around 50%. Further, few new therapies have been successfully introduced to improve these outcomes. Here we report that exploiting deficiencies in DNA damage repair (DDR) is a potential therapeutic strategy for AML. Poly-ADP Ribose Polymerase (PARP) inhibitors were initially developed to target deficient homologous recombination (HR) in BRCA1/2 mutated cancers by blocking single stranded base repair following DNA damage, leading to an accumulation of double stranded DNA breaks, thereby inducing apoptosis. To evaluate the activity of PARP inhibition in pediatric AML, talazoparib was tested as a single agent and in combination with standard chemotherapeutic agents in human AML cell lines representing low (Kasumi-1 and ME-1), intermediate (AML193), and high-risk (CTS, CMS, MOLM-13, and CHRF288-11) disease based on their genomic mutations. Talazoparib showed the highest efficacy as a single agent in all four cell lines with genomic lesions found in high-risk AML subtypes. After combination drug screens, topotecan (synergistic) and gemcitabine (additive) were chosen to move forward to in vivo testing. Our investigational combination was tested in vivo in four murine models representing pediatric AML subtypes harboring AML1-ETO9a (low risk), MLL-AF6 (high risk), CBAF2T3-GLIS2/JAK2 V617F (high risk) and NUP98-KDM5A (high risk) oncogenes. Mice received a backbone of either current standard of care chemotherapy (SOC; anthracycline plus cytarabine) or topotecan plus gemcitabine. NUP98-KDM5A and MLL-AF6 positive mice receiving single agent talazoparib were found to have prolonged survival compared to vehicle alone (p=0.019 and p<0.0001, respectively) which was further enhanced by the addition of chemotherapy irrespective of backbone (p <0.0001). Conversely, mice with AML1-ETOa positive leukemia had no response to single agent PARP inhibitor. While a few mice benefitted from the addition of talazoparib to SOC, this result was not statistically significant (p= 0.42). Early response by bioluminescent imaging confirmed that mice with MLL-AF6 and NUP98-KDM5A driven leukemias who received talazoparib in combination with chemotherapy had the lowest leukemia burdens while the AML1-ETOa cohort did not benefit from the addition of this targeted agent. Interestingly, mice harboring CBAF2T3-GLIS2/JAK2 V617F were not responsive to PARP inhibitors, which was inconsistent with the CMS cell line that has same oncogenic fusion gene but lacks the JAK2 V617F mutation. Synergy experiments with ATM inhibitor AZD0156 demonstrated tremendous synergy with talazoparib in sensitive cell lines with almost no synergy in those that were resistant, suggesting that sensitive cell lines are unable to efficiently activate the HR pathway to repair double stranded breaks induced by PARP inhibition whereas resistant cells can overcome inhibition. To determine the HR response to DNA damage in our cell lines, we exposed them to 1uM topotecan for 2 hours and then measured γH2AX response at 0, 4 and 24 hours. γH2AX is a sensor of DNA damage and therefore increases with DNA damage and decreases with repair. PARP inhibitor sensitive cell lines had persistence of gamma H2AX at 24hrs while resistant cell lines had at least partial resolution of damage, confirming that PARP inhibitor sensitive cell lines have aberrant DNA damage response through HR. RNA sequencing of our cell lines revealed a correlation between Phosphatase and tensin homolog (PTEN) transcript levels and PARP sensitivity. Western blotting confirmed that PTEN was downregulated or absent in both cell lines and murine leukemias that were sensitive to PARP inhibitors. In contrast to the CMS cell line that carries the CBFA2T3-GLIS2 fusion, murine leukemias with CBAF2T3-GLIS2/JAK2 V617F had high levels of PTEN, supporting the hypothesis that sensitivity to PARP inhibitors is due to loss of PTEN. In conclusion, we report that a subset of pediatric AML with high- risk features are sensitive to PARP inhibition due to deficient DDR through HR. Downregulation of PTEN is a candidate biomarker of response to PARP inhibitors in these patients. This data illuminates a promising therapeutic vulnerability in a patient population where new targeted treatments are vital to improve outcomes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Addition of the PARP Inhibitor, Talazoparib, to Gemtuzumab Ozogamicin Significantly Enhances Anti-Leukemic Activity in Human CD33+ Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1371-1371
Author(s):  
Scott M. Portwood ◽  
Marianna C Cantella ◽  
Tara L. Cronin ◽  
Eunice S. Wang
Keyword(s):  
Dna Damage ◽  
Parp Inhibitor ◽  
Single Agent ◽  
Parp Inhibitors ◽  
Gemtuzumab Ozogamicin ◽  
Nsg Mice ◽  
Dose Dependent ◽  
Advisory Role

Background CD33 (Siglec3) is a cell surface transmembrane receptor that is rapidly internalized and highly expressed on AML blasts but is absent on normal hematopoietic stem cells. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 antibody conjugated to a DNA strand scission inducing agent (calicheamicin) was recently FDA approved for the treatment of newly-diagnosed or relapsed/refractory CD33-positive acute myeloid leukemia (AML). GO has been shown to exert clinical activity in leukemia patients. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and by preventing the dissociation of PARP from the DNA (PARP trapping). Talazoparib is unique among clinical PARP inhibitors in displaying 10,000-fold increased PARP trapping as compared to other agents . We hypothesized that combination therapy using GO and Talazoparib would result in synergistic anti-leukemic effects on human CD33+ AML cells due to the ability of the PARP inhibitor to enhance levels of DNA damage induced by GO therapy. Materials and Methods Human AML cell lines were characterized for CD33 expression using flow cytometry after staining with antibody-linked fluorescent QuantiBrite Beads. Cells were continuously exposed to varying doses of GO (10pM - 100mM) and PARP inhibitors (1nM - 100mM) for 96h alone and in combination. Cell viability was measured immediately following treatment using a WST colorimetric assay. Treatment-induced apoptosis (annexin/PI) and DNA damage (H2AX) were quantified by flow cytometric assays. Synergy reports were generated using Compusyn software. In vivo efficacy was assessed in NSG mice systematically engrafted with luciferase labeled human CD33+ AML cells following tail vein injection. Animals were treated with varying doses of vehicle, GO (1 and 50ug/kg 1x/week for 3 weeks), or talazoparib (0.1 and 0.33mg/kg 5 days/week) either alone or in combination. Treatment effects on leukemia burden, toxicity, and survival were determined by weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity. Results Human AML cell lines (HEL, HL60) express high expression levels of CD33 molecules/cell (43,645 and 31,286 respectively) relative to negative controls. Continuous exposure to single agent GO and Talazoparib for 96h resulted in a dose dependent inhibition of human AML cell growth (HEL, HL60) . IC values for GO were 0.01 - 6.6μg/ml and for Talazoparib were 0.8-0.95μM. Combination in vitro therapy with GO (0.005 - 1μg/ml) and Talazoparib (fixed dose 100nM) resulted in synergistic anti-leukemic effects (p<0.01) significantly improving upon monotherapy. Software analyses yielded a combination Index (CI) <1 consistent with synergistic anti-leukemic effects. Combination GO and Talazoparib therapy also significantly enhanced AML cell apoptosis (p=0.0111) and levels of DNA damage (phosphorylated H2AX) (p=0.0054) over single agent activity. Evaluation of PARP trapping by western blot analysis is ongoing. In vivo administration of GO (1-50μg/kg) and Talazoparib (0.1-0.33mg/kg) in NSG mice with systemic engraftment of luciferase tagged human CD33+ AML cells was generally well tolerated with no significant weight loss or early morbidity. Single agent GO and Talazoparib therapy decreased systemic AML burden in a dose dependent manner and prolonged overall survival over vehicle treated mice (P<0.05). Concomitant GO (1μg/kg) and Talazoparib (0.33μg/kg) treatment was similarly well tolerated with no notable weight loss or toxicities. Combination GO and Talazoparib therapy significantly prolonged overall survival of leukemia xenografted mice over vehicle (p=0.0018) and single agent therapy with the same doses of GO (p=0.0018) and Talazoparib (p=0.0499), respectively). Conclusions In summary, our results demonstrate that the addition of the PARP inhibitor, Talazoparib, to the CD33 antibody drug conjugate, GO, results in potent in vitro and in vivo anti-tumor activity in human CD33+ AML preclinical models. Further studies investigating this novel combinatorial approach in AML are currently ongoing. Due to GO's FDA approval for CD33+ AML in 2018, this data strongly supports future clinical investigation using PARP inhibitors as a novel class of agents for combination therapy to significantly enhance the efficacy of ADCs. Figure 1 Disclosures Wang: Amgen: Other: Advisory role; Agios: Other: Advisory role; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau.

scholarly journals The m6A RNA Demethylase ALKBH5 Promotes Radioresistance and Invasion Capability of Glioma Stem Cells

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 40
Author(s):  
Aline Kowalski-Chauvel ◽  
Marie Géraldine Lacore ◽  
Florent Arnauduc ◽  
Caroline Delmas ◽  
Christine Toulas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
High Capacity ◽  
Glioma Stem Cells ◽  
Normal Brain ◽  
Rna Methylation ◽  
Damage Repair ◽  
Attractive Therapeutic Target ◽  
M6a Rna Methylation ◽  
The Impact

Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.

scholarly journals DDIS-03. EGFR AMPLIFICATION INDUCED INCREASED DNA DAMAGE RESPONSE AND PREDICTED SELECTIVE SENSITIVITY TO TALAZOPARIB (PARP INHIBITOR) IN GLIOBLASTOMA STEM-LIKE CELLS

2018 ◽  
Vol 20 (suppl_6) ◽  
pp. vi69-vi69
Author(s):  
Shaofang Wu ◽  
Feng Gao ◽  
Siyuan Zheng ◽  
Chen Zhang ◽  
Juan Emmanuel Martinez-Ledesma ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Parp Inhibitor ◽  
Egfr Amplification ◽  
Damage Response ◽  
Selective Sensitivity

scholarly journals PARP inhibition in UV-associated angiosarcoma preclinical models

2021 ◽  
Author(s):  
Marije E. Weidema ◽  
Ingrid M. E. Desar ◽  
Melissa H. S. Hillebrandt-Roeffen ◽  
Anke E. M. van Erp ◽  
Mikio Masuzawa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Combination Treatment ◽  
Parp Inhibitor ◽  
Treatment Options ◽  
Single Agent ◽  
Specific Treatment ◽  
Parp Inhibition ◽  
Poor Overall Survival ◽  
Damage Repair

Abstract Purpose Angiosarcoma (AS) is a rare vasoformative sarcoma, with poor overall survival and a high need for novel treatment options. Clinically, AS consists of different subtypes, including AS related to previous UV exposure (UV AS) which could indicate susceptibility to DNA damage repair inhibition. We, therefore, investigated the presence of biomarkers PARP1 (poly(ADP-ribose)polymerase-1) and Schlafen-11 (SLFN11) in UV AS. Based on experiences in other sarcomas, we examined (combination) treatment of PARP inhibitor (PARPi) olaparib and temozolomide (TMZ) in UV AS cell lines. Methods Previously collected UV AS (n = 47) and non-UV AS (n = 96) patient samples and two UV AS cell lines (MO-LAS and AS-M) were immunohistochemically assessed for PARP1 and SLFN11 expression. Both cell lines were treated with single agents PARPi olaparib and TMZ, and the combination treatment. Next, cell viability and treatment synergy were analyzed. In addition, effects on apoptosis and DNA damage were examined. Results In 46/47 UV AS samples (98%), PARP1 expression was present. SLFN11 was expressed in 80% (37/46) of cases. Olaparib and TMZ combination treatment was synergistic in both cell lines, with significantly increased apoptosis compared to single agent treatment. Furthermore, a significant increase in DNA damage marker γH2AX was present in both cell lines after combination therapy. Conclusion We showed combination treatment of olaparib with TMZ was synergistic in UV AS cell lines. Expression of PARP1 and SLFN11 was present in the majority of UV AS tumor samples. Together, these results suggest combination treatment of olaparib and TMZ is a potential novel AS subtype-specific treatment option for UV AS patients.

scholarly journals The role of PARP inhibitors in BRCA mutated pancreatic cancer

2021 ◽  
Vol 14 ◽  
pp. 175628482110148
Author(s):  
Jeffrey Chi ◽  
Su Yun Chung ◽  
Ruwan Parakrama ◽  
Fatima Fayyaz ◽  
Jyothi Jose ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp Inhibitor ◽  
Dna Damage Repair ◽  
The United States ◽  
Parp Inhibitors ◽  
Phase Iii ◽  
Tumor Cell Death ◽  
Brca Mutations ◽  
Damage Repair ◽  
The Us

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 3% of all cancers in the United States and about 7% of all cancer deaths. Despite the lower prevalence relative to other solid tumors, it is one of the leading causes of cancer-related death in the US. PDAC is highly resistant to chemotherapy as well as radiation therapy. Current standard-of-care chemotherapeutic regimens provide transient disease control but eventually tumors develop chemoresistance. Tumors that are deficient in DNA damage repair mechanisms such as BRCA mutants respond better to platinum-based chemotherapies. However, these tumor cells can utilize the poly adenosine diphosphate (ADP)-ribose polymerase (PARP) as a salvage DNA repair pathway to prolong survival. Hence, in the presence of BRCA mutations, the inhibition of the PARP pathway can lead to tumor cell death. This provides the rationale for using PARP inhibitors in patients with BRCA mutated PDAC. The phase III POLO trial showed a near doubling of progression-free survival (PFS) compared with placebo in advanced PDAC when a PARP inhibitor, olaparib, was used as maintenance therapy. As a result, the US Food and Drug Administration (FDA) approved olaparib as a maintenance treatment for germline BRCA mutated advanced PDAC that has not progressed on platinum-based chemotherapy. The success of olaparib in treating advanced PDAC opened the new field for utilizing PARP inhibitors in patients with DNA damage repair (DDR) gene defects. Currently, many clinical trials with various PARP inhibitors are ongoing either as monotherapy or in combination with other agents. In addition to germline/somatic BRCA mutations, some trials are enrolling patients with defects in other DDR genes such as ATM, PALB2, and CHEK2. With many ongoing PARP inhibitor trials, it is hopeful that the management of PDAC will continuously evolve and eventually lead to improved patient outcomes.

scholarly journals PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling

2021 ◽  
Vol 22 (10) ◽  
pp. 5112
Author(s):  
Lotte van Beek ◽  
Éilís McClay ◽  
Saleha Patel ◽  
Marianne Schimpl ◽  
Laura Spagnolo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Parp Inhibitors ◽  
Covalent Modification ◽  
Activation Mechanism ◽  
Cancer Therapies ◽  
Damage Repair ◽  
Complementary Role ◽  
Functional Understanding

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.

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