CRISPR/Cas9-Generated Models Uncover Therapeutic Vulnerabilities of Del(11q) Chronic Lymphocytic Leukemia Cells to Dual BCR and PARP Inhibition

Abstract Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies. To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition). Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P<0.01). Similar results were observed in HG3 and MEC1 BIRC3KO cells (P<0.01; P<0.05). Moreover, biallelic inactivation of BIRC3 in del(11q) cells resulted in cytoplasmic stabilization of NF-kB-inducing kinase (NIK), leading to higher nuclear NF-kB2 (p52) activation (P<0.01) as measured by ELISA. In parallel, we analyzed the DNA damage response (DDR) of these cells, and showed that del(11q) ATMKO cells displayed reduced pH2AX levels (P<0.001) and an accumulation of unrepaired double strand breaks (DSB) (P<0.001) after irradiation, as determined by comet assays. Consistently, in vivo subcutaneous xenografts showed that HG3 ATMKOBIRC3KO tumors presented proliferative advantage, higher p52 levels and greater genomic and mitotic instability than HG3WT tumors, indicating a more aggressive phenotype. We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P<0.05). Moreover, del(11q) cells were slightly more resistant to ibrutinib (IBRU) treatment compared to WT cells (mean IC50 10 uM vs. 3.7 uM; P<0.05). Interestingly, exploiting the DDR deficiencies underlying del(11q) by targeting the single strand break repair pathway with the PARP inhibitor olaparib (OLA), del(11q) ATMKO cells were not able to proliferate even 12 days after treatment (3 uM), independently of the mutational status of BIRC3 (P<0.01). In vivo intravenous HG3-derived xenografts (N=20) showed that OLA (100 mg/kg) reduced hCD45+ cells in the peripheral blood (P<0.01) and significantly improved survival of del(11q) ATMKOBIRC3KO xenografted mice (P<0.01). Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P<0.001) and HMGB1 release. Remarkably, we found that IBRU caused downregulation of the DNA repair protein RAD51, leading to impaired RAD51 foci formation in DSB lesions (P<0.01). Consistently, IBRU (1 uM) reduced the homologous recombination (HR) repair efficiency in HG3 cells (P=0.001), as determined by an HR-reporter construct. This IBRU-dependent impairment of HR repair could explain the synergistic effects with OLA by synthetic lethality. In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients. PI15/01471 SA085U16 JCyL-MQ FEHH-MH Disclosures García-Tuñón: Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

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PARP Inhibitors Are Effective in IDH1/2 Mutant MDS and AML Resistant to Targeted IDH Inhibitors

Blood ◽

10.1182/blood-2019-130814 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4222-4222 ◽

Cited By ~ 1

Author(s):

Rana Gbyli ◽

Yuanbin Song ◽

Wei Liu ◽

Yimeng Gao ◽

Namrata S Chandhok ◽

...

Keyword(s):

Research Funding ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Parp Inhibitors ◽

Parp Inhibition ◽

Resistance Mutations ◽

Single Strand Break ◽

Immunodeficient Mice ◽

Equity Ownership

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are heterogeneous clonal disorders. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations are detected in ~20% of AML and ~5% of MDS, in which they confer gain of a neomorphic function that leads to the production of (R)-2-hydroxyglutarate (2HG). Targeted inhibition of mutant IDH1/2 has resulted in significant responses in IDH1/2 mutant MDS and AML but is not curative and patients relapse (Stein et al. Blood 2016, DiNardo et al. N Engl J Med 2018). 2HG accumulation inhibits the function of histone demethylases (KDM4A and KDM4B) that are critical for the homologous recombination (HR) DNA repair pathway and consequently for the repair of DNA double strand breaks (DSBs) (Mallette et al. EMBO J 2012, Sulkowski et al. Sci Transl Med 2017). In HR deficient tumors, Poly-ADP-Ribose Polymerase (PARP) is essential for DNA single strand break (SSB) repair. In IDH mutant tumors PARP inhibitors induce synthetic lethality by suppressing the repair of SSBs, which eventually get converted into DSBs (Javle and Curtin Br J Cancer 2011). We previously demonstrated that in AML, IDH1/2 mutations impair DNA damage response by inducing a defect in HR, and that this renders leukemia cells susceptible to PARP inhibitors in vitro. We hypothesized that this vulnerability would also exist in IDH mutant MDS and more importantly, that this vulnerability would persist in MDS/AML resistant to IDH1/2 inhibitors. To determine whether PARP inhibition targets IDH mutant MDS/AML in vivo, we took advantage of 2 syngeneic mouse models of MDS and AML relying on co-mutation of SRSF2/IDH2 and FLT3/IDH2, respectively. Olaparib (PARP inhibitor) effectively targeted IDH2 mutant but not IDH2 wild type MDS/AML (Fig. 1A). We next sought to determine whether PARP inhibition mediated synthetic lethality persists in MDS/AML resistant to targeted IDH inhibition. We transduced IDH2 mutant murine cells with IDH2 WT or IDH2 MUT lentiviral vectors carrying one of two published resistance mutations. While these resistance mutations conferred resistance to the targeted IDH2 inhibitor Enasidenib, cells remained sensitive to Olaparib (Fig. 1B). Patient MDS/AML is heterogeneous and in general carries additional genetic mutations and epigenetic alterations. We therefore engrafted IDH1/2 WT and MUT MDS/AML patient samples in cytokine humanized immunodeficient mice and treated with vehicle of Olaparib. Engrafted mice were assigned to vehicle or Olaparib 8 weeks after transplantation based on equal engraftment levels determined by BM aspiration. Mice were treated with vehicle or Olaparib via IP injection for 21 days. Human engraftment levels and plasma 2-HG levels were significantly reduced in Olaparib treated animals when compared to pre-treatment and vehicle-treated mice (Fig. 1C). Of note, when equal numbers of huCD34+ cells from vehicle or Olaparib treated mice were transplanted into next generation mice, engraftment was significantly higher for recipients of human cells from vehicle than Olaparib treated mice, suggesting that Olaparib is toxic to leukemia initiating cells. In contrast, IDH WT MDS/AML was insensitive to Olaparib treatment (Fig. 1C). In conclusion, PARP inhibition is effective in vivo against IDH mutant MDS/AML and can overcome targeted IDH inhibitor resistance. Disclosures Flavell: Zai labs: Consultancy; SMOC: Equity Ownership; Troy: Equity Ownership; Artizan Biosciences: Equity Ownership; GSK: Consultancy; Rheos Biomedicines: Equity Ownership. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; novartis: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; pfizer: Honoraria. Bindra:Cybrexa: Consultancy, Equity Ownership.

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Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer

Gut ◽

10.1136/gutjnl-2019-319970 ◽

2020 ◽

pp. gutjnl-2019-319970 ◽

Cited By ~ 2

Author(s):

Johann Gout ◽

Lukas Perkhofer ◽

Mareen Morawe ◽

Frank Arnold ◽

Michaela Ihle ◽

...

Keyword(s):

Dna Damage ◽

Epithelial Mesenchymal Transition ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Copy Number Variations ◽

Ductal Adenocarcinoma ◽

Parp Inhibition ◽

Independent Manner ◽

Mesenchymal Transition ◽

Fork Stalling

ObjectiveATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC).DesignCombinational synergy screening was performed to endeavour a genotype-tailored targeted therapy.ResultsSynergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance.ConclusionAnalysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.

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CDK12 inhibition mediates DNA damage and is synergistic with sorafenib treatment in hepatocellular carcinoma

Gut ◽

10.1136/gutjnl-2019-318506 ◽

2019 ◽

Vol 69 (4) ◽

pp. 727-736 ◽

Cited By ~ 10

Author(s):

Cun Wang ◽

Hui Wang ◽

Cor Lieftink ◽

Aimee du Chatinier ◽

Dongmei Gao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Damage ◽

Cell Lines ◽

Synergistic Effects ◽

Loss Of Function ◽

Bioinformatics Analyses ◽

Sorafenib Treatment ◽

Hcc Cells

ObjectivesHepatocellular carcinoma (HCC) is one of the most frequent malignancies and a major leading cause of cancer-related deaths worldwide. Several therapeutic options like sorafenib and regorafenib provide only modest survival benefit to patients with HCC. This study aims to identify novel druggable candidate genes for patients with HCC.DesignA non-biased CRISPR (clustered regularly interspaced short palindromic repeats) loss-of-function genetic screen targeting all known human kinases was performed to identify vulnerabilities of HCC cells. Whole-transcriptome sequencing (RNA-Seq) and bioinformatics analyses were performed to explore the mechanisms of the action of a cyclin-dependent kinase 12 (CDK12) inhibitor in HCC cells. Multiple in vitro and in vivo assays were used to study the synergistic effects of the combination of CDK12 inhibition and sorafenib.ResultsWe identify CDK12 as critically required for most HCC cell lines. Suppression of CDK12 using short hairpin RNAs (shRNAs) or its inhibition by the covalent small molecule inhibitor THZ531 leads to robust proliferation inhibition. THZ531 preferentially suppresses the expression of DNA repair-related genes and induces strong DNA damage response in HCC cell lines. The combination of THZ531 and sorafenib shows striking synergy by inducing apoptosis or senescence in HCC cells. The synergy between THZ531 and sorafenib may derive from the notion that THZ531 impairs the adaptive responses of HCC cells induced by sorafenib treatment.ConclusionOur data highlight the potential of CDK12 as a drug target for patients with HCC. The striking synergy of THZ531 and sorafenib suggests a potential combination therapy for this difficult to treat cancer.

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A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis

10.1101/2021.04.22.440879 ◽

2021 ◽

Author(s):

Umar Khalid ◽

Milena Simovic ◽

Murat Iskar ◽

John KL Wong ◽

Rithu Kumar ◽

...

Keyword(s):

Synthetic Lethality ◽

Parp Inhibitor ◽

Strand Break ◽

Parp Inhibitors ◽

Synergistic Interaction ◽

Parp Inhibition ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Double Strand Break

ABSTRACTChromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

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The roles of hypoxia, PTEN, and Rad51 in mediating metastatic prostate cancer cells' responses to PARP inhibitor and topoisomerase 1 inhibitor.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13564 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13564-e13564

Author(s):

Jingsong Zhang ◽

Minghui Wu ◽

Xue Wang

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Topoisomerase I ◽

Metastatic Prostate Cancer ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Single Agent ◽

Study Results

e13564 Background: With the recent success of poly (ADP-ribose) polymerase inhibitor (PARPi) in the treatment of BRCA1 or BRCA2 mutated cancers, there is increasing interest to explore synthetic lethality in cancers with defective DNA repair pathways. Rad51 is an essential protein in the homologous recombination repair (HRR) of DNA double strand breaks. Previous studies with non metastatic prostate cancer (mCaP) cells have reported low Rad51 levels in cells with loss of PTEN or under hypoxia, which then led to their sensitivity to PARPi. Given intra tumor hypoxia and loss of PTEN is common in mCaP, we test PAPRi, ABT888 and DNA damaging topoisomerase I inhibitor, CPT11, either alone or in combination in mCaP preclinical models. Methods: mCaP cell lines with functional PTEN (DU145) and loss of PTEN (PC3) were grew under normoxia (21% O2) or hypoxia (0.2% O2). DNA damage, HRR, apoptosis were assessed with comet assay, western blot, immunofluorescence and flowcytometry. The regulation of RAD51 was studied with quantitative RT-PCR and RAD51 promoter reporter assay. PC3 xenograft was used for in vivo study. Results: Despite of its low levels of expression under hypoxia, up regulation of Rad51 was observed soon after treating hypoxic PC3 and Du145 cells with ABT888 or SN38, an active metabolite of CPT11. Such Rad51 up regulation led to less DNA damage and apoptosis under hypoxia compared to normoxia. Inhibiting RAD51 expression with siRNA overcame PC3 and Du145’s resistance to SN38. Furthermore, ABT888 enhanced the activities of SN38 as detected by clonogenic assay and flowcytometry under both normoxia and hypoxia. Consistent with the in vitro data, ABT888 by itself had limited anti-tumor activities despite the loss of PTEN in PC3 xenografts. The anti-tumor activity of single agent CPT11 was significantly improved with the ABT888 and CPT11 combination (P<0.008). Conclusions: neither loss of PTEN nor hypoxia sensitized mCaP cells to PARPi or DNA damaging drugs. Such resistance under hypoxia was at least partly due to up regulation of Rad51. Combining ABT888 with CPT11 overcame the resistance to CPT11 under hypoxia and enhanced its anti-tumor activities both in vitro and in vivo.

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EXTH-03. COMBINED TOP1 AND PARP INHIBITION ENFORCES GENOMIC INSTABILITY AND CELL DEATH IN PTEN-DEFICIENT GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.642 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi163-vi164

Author(s):

Olga Kim ◽

Madison Butler ◽

Ying Pang ◽

Guangyang Yu ◽

Mythili Merchant ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Topoisomerase I ◽

Parp Inhibitor ◽

Annexin V ◽

Parp Inhibition ◽

Pten Loss ◽

Tensin Homolog

Abstract BACKGROUND Glioblastoma is an aggressive brain tumor with high mortality. The development of new therapies is critical for improving patient outcomes. LMP400, a novel topoisomerase I (TOP1) inhibitor, traps TOP1 cleavage complexes, thereby generating DNA damage. Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair responses triggered by TOP1 inhibition. Niraparib is a potent PARP inhibitor that can cross the blood-brain barrier. Loss of phosphatase and tensin homolog (PTEN) occurs in 40% of GBM patients and is known to promote DNA damage repair deficiency. Here, we hypothesize that PTEN loss presents a vulnerability to a combined induction of DNA damage and inhibition of repair mechanisms. METHODS Human glioblastoma cells (U251, SNB-75, SF-295, LN18) and patient-derived glioblastoma stem cells (GSC923 and GSC827) were treated with LMP400 and/or Niraparib. Cell viability and apoptosis were examined using Celigo image cytometer and Annexin V/PI assay at 72h after treatment. Single clones after PTEN knockdown using shRNA were isolated after puromycin selection. For planned studies of PTEN knockout, sgRNA plasmids targeting PTEN will be transiently transfected and GFP-positive single KO clones will be isolated. PTEN will be restored in PTEN-null cells using lentiviral transduction. RESULTS CRISPR-Cas9 KO screening in GSC923 cells suggests that LMP400 is unlikely a substrate for ABC transporters. LMP400 and Niraparib synergistically induced cytotoxic effects in U251, SF-295, GSC923, GSC827 cells lacking PTEN expression. Combined LMP400/Niraparib led to increased expression of gamma-H2AX, cleaved caspase 3 and PARP, indicative of enhanced DNA damage and cell death. CONCLUSION LMP400 and Niraparib act synergistically to target PTEN-deficient glioblastoma by inducing DNA damage and cell death. These results will be further verified in isogenic cells in vitro as well as in vivo in a mouse model driven by PTEN deletion which would strongly support a novel therapeutic strategy in a subset of glioblastoma with PTEN loss.

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PARP1 Upregulation in Recurrent Oral Cancer and Treatment Resistance

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.804962 ◽

2022 ◽

Vol 9 ◽

Author(s):

Feifei Wang ◽

Odjo G. Gouttia ◽

Ling Wang ◽

Aimin Peng

Keyword(s):

Dna Damage ◽

Oral Cancer ◽

Tumor Cells ◽

Primary Tumor ◽

Parp Inhibitor ◽

Treatment Resistance ◽

Response To Treatment ◽

Parp Inhibition ◽

Recurrent Tumor

First-line treatments for oral cancer typically include surgery, radiation, and in some cases, chemotherapy. Radiation and oral cancer chemotherapeutics confer cytotoxicity largely by inducing DNA damage, underscoring the importance of the cellular DNA damage repair and response pathways in cancer therapy. However, tumor recurrence and acquired resistance, following the initial response to treatment, remains as a major clinical challenge. By analyzing oral tumor cells derived from the primary and recurrent tumors of the same patient, our study revealed upregulated PARP1 expression in the recurrent tumor cells. Cisplatin and 5-fluorouracil treatment further augmented PARP1 expression in the recurrent, but not the primary, tumor cells. Post-treatment upregulation of PARP1 was dependent on the catalytic activities of PARP and CDK7. Consistent with the established function of PARP1 in DNA repair, we showed that overexpression of PARP1 rendered the primary tumor cells highly resistant to DNA damage treatment. Conversely, PARP inhibition partially reversed the treatment resistance in the recurrent tumor cells; combinatorial treatment using a PARP inhibitor and cisplatin/5-fluorouracil significantly sensitized the tumor response in vivo. Taken together, we reported here PARP1 upregulation as a clinically relevant mechanism involved in oral cancer recurrence, and suggested the clinical benefit of PARP inhibitors, currently approved for the treatment of several other types of cancer, in oral cancer.

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ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms

Cancers ◽

10.3390/cancers12051095 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1095 ◽

Cited By ~ 4

Author(s):

Harriet E. D. Southgate ◽

Lindi Chen ◽

Deborah A. Tweddle ◽

Nicola J. Curtin

Keyword(s):

Dna Damage ◽

High Risk ◽

Cell Lines ◽

Western Blotting ◽

Ataxia Telangiectasia ◽

Parp Inhibitor ◽

Single Agent ◽

Parp Inhibition ◽

Mycn Amplification ◽

Single Strand Break

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

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CRISPR screens uncover protective effect of PSTK as a regulator of chemotherapy-induced ferroptosis in hepatocellular carcinoma

Molecular Cancer ◽

10.1186/s12943-021-01466-9 ◽

2022 ◽

Vol 21 (1) ◽

Author(s):

Yiran Chen ◽

Li Li ◽

Jie Lan ◽

Yang Cui ◽

Xiaosong Rao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Synthetic Lethality ◽

Synergistic Effects ◽

Loss Of Function ◽

Protein Coding ◽

Synthetic Lethal ◽

Chemotherapeutic Treatment ◽

Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is among the most common forms of cancer and is associated with poor patient outcomes. The emergence of therapeutic resistance has hampered the efficacy of targeted treatments employed to treat HCC patients to date. In this study, we conducted a series of CRISPR/Cas9 screens to identify genes associated with synthetic lethality capable of improving HCC patient clinical responses. Methods CRISPR-based loss-of-function genetic screens were used to target 18,053 protein-coding genes in HCC cells to identify chemotherapy-related synthetic lethal genes in these cells. Synergistic effects were analyzed through in vitro and in vivo analyses, while related mechanisms were explored through RNA-seq and metabolomics analyses. Potential inhibitors of identified genetic targets were selected through high-throughput virtual screening. Results The inhibition of phosphoseryl-tRNA kinase (PSTK) was found to increase HCC cell sensitivity to chemotherapeutic treatment. PSTK was associated with the suppression of chemotherapy-induced ferroptosis in HCC cells, and the depletion of PSTK resulted in the inactivation of glutathione peroxidative 4 (GPX4) and the disruption of glutathione (GSH) metabolism owing to the inhibition of selenocysteine and cysteine synthesis, thus enhancing the induction of ferroptosis upon targeted chemotherapeutic treatment. Punicalin, an agent used to treat hepatitis B virus (HBV), was identified as a possible PSTK inhibitor that exhibited synergistic efficacy when applied together with Sorafenib to treat HCC in vitro and in vivo. Conclusions These results highlight a key role for PSTK as a mediator of resistance to targeted therapeutic treatment in HCC cells that functions by suppressing ferroptotic induction. PSTK inhibitors may thus represent ideal candidates for overcoming drug resistance in HCC.

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Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522223113 ◽

2016 ◽

Vol 113 (30) ◽

pp. E4338-E4347 ◽

Cited By ~ 45

Author(s):

Ashish Juvekar ◽

Hai Hu ◽

Sina Yadegarynia ◽

Costas A. Lyssiotis ◽

Soumya Ullas ◽

...

Keyword(s):

Dna Damage ◽

Mouse Model ◽

Dna Synthesis ◽

Parp Inhibitor ◽

Pentose Phosphate ◽

Pi3k Inhibitor ◽

Parp Inhibition ◽

Nucleotide Synthesis ◽

Phosphoinositide 3 Kinase

We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

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