Homologous Recombination Repair Proficiency Research Articles

Abstract CT232: Phase I combination study of the CHK1 inhibitor prexasertib (LY2606368) and olaparib in patients with high-grade serous ovarian cancer and other advanced solid tumors

Abstract Background: Checkpoint kinase 1 (CHK1) plays a central role in replication fork stabilization and homologous recombination repair (HRR) through modulation of cell cycle checkpoints and phosphorylation of HRR proteins. BRCA-deficient cancers frequently upregulate CHK1 for protection of stalled replication forks. Prexasertib is an ATP-competitive inhibitor of CHK1. Addition of prexasertib to olaparib, an oral PARP inhibitor, may allow for combinatorial synergy and sensitization to PARP inhibition among tumors with de novo or acquired resistance. Methods: We conducted an open-label phase 1 study following a 3+3 design. Patients (pts) received a 7-day lead-in of olaparib alone (C0), followed by 28-day cycles with prexasertib administered on days 1 and 15 and olaparib on days 1-5 and 15-19 with starting dose of prexasertib at 80mg/m2 and olaparib at 200mg. The MTD was defined as the highest dose level at which less than 1/3rd of at least 6 pts experienced a DLT during C0+C1. Pharmacokinetic blood samples were obtained at C0, C1, and C2. Pts enrolled to the expansion phase of the study underwent tumor biopsies during C0 and on C1D16 after the combination for pharmacodynamic assessment of target effects on HRR. Patient-derived organoid cultures with functional assays for HRR proficiency and replication fork stability were conducted in parallel as an exploratory objective. Results: Twenty-one pts have been treated, 19 in the dose escalation phase. Three of 6 pts experienced DLTs at the starting dose and included Gr 3 neutropenia and Gr 3 febrile neutropenia. Lower doses for both agents were evaluated; the MTD is prexasertib at 70mg/m2 IV on days 1 and 15 in combination with olaparib at 100 mg PO BID D1-5 and 15-19. No DLTs were observed at this dose level. Drug-related adverse events occurring in ≥ 50% of pts included leukopenia, neutropenia, thrombocytopenia, anemia, and nausea. Three pts with BRCA1-mutant high-grade serous ovarian cancer (HGSOC) achieved PRs, two of whom had progressed on a prior PARP inhibitor. Tumor biopsies from pts in the expansion cohort demonstrated decreased RAD51 foci after prexasertib exposure. Pre-prexasertib patient-derived organoid cultures were successfully established and subjected to a DNA fiber assay to assess replication fork stability. The organoid derived from tumor from a non-responding patient had stable replication forks, whereas the organoid from a responding patient demonstrated baseline fork instability. Conclusion: Prexasertib in combination with an abbreviated course of olaparib has preliminary clinical activity in patients with BRCA-mutant HGSOC who have previously progressed on a PARP inhibitor. A functional assay for HRR in tumor biopsies indicates that prexasertib can compromise RAD51 focus formation, consistent with the role of CHK1 in RAD51 phosphorylation and filament formation. Preliminary data in patient-derived organoid cultures parallels that of clinical responses and confirms previously published data suggesting replication fork instability contributes to response to prexasertib. An expansion cohort in the BRCA-mutant, PARP inhibitor-resistant HGSOC population is being enrolled to further assess efficacy of the combination and to obtain additional pharmacodynamic determinants of response and resistance. Citation Format: Khanh T. Do, Sarah J. Hill, Bose Kochupurakkal, Jeffrey G. Supko, Courtney Gannon, Adrienne Anderson, Alona Muzikansky, Andrew Wolanski, Jennifer Hedglin, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Susana Campos, Ursula A. Matulonis, Alan D. D'Andrea, Geoffrey I. Shapiro. Phase I combination study of the CHK1 inhibitor prexasertib (LY2606368) and olaparib in patients with high-grade serous ovarian cancer and other advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT232.

Abstract IA17: PARP inhibitor resistance in prostate cancer

Abstract Large-scale genomic studies have demonstrated that approximately 15% of metastatic castration-resistant prostate cancer (mCRPC) harbors genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA2, BRCA1, and ATM, followed by other Fanconi anemia genes including FANCN/PALB2 and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues and PARP inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in BRCA2-deficient mCRPC where the PARPi olaparib has demonstrated improvements in progression-free survival. PARPi resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2-deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. These somatic reversions of mutant BRCA2 alleles can be monitored by sequencing of ctDNA. However, PARPi resistance may also develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism—namely, the restoration of RF stability—appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire may PARPi resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient ovarian and breast cancer cells can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. Whether or not the downregulation of the PRC2/MUS81 pathway is also a mechanism of PARPi resistance in mCRPC remains to be determined. A molecular understanding of PARPi resistance mechanisms may allow the generation of a new class of drugs, or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARPi to more tumor types. Citation Format: Alan D. D’Andrea. PARP inhibitor resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA17.

Abstract IA15: PARP inhibitor resistance and acquired vulnerability in breast cancer

Abstract Large-scale genomic studies have demonstrated that some breast cancers, especially triple-negative breast cancers (TNBCs), harbor genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA1 and BRCA2, followed by other Fanconi anemia genes including FANCN/PALB2, FANCO/RAD51, FANCJ/BRIP, and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues, topoisomerase inhibitors, and PARP inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in ovarian cancer where the PARPi olaparib is FDA approved for use in patients with germline BRCA1/2 mutations. PARP inhibitor resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2 deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. However, PARPi resistance may still develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism—namely, the restoration of RF stability—appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire PARP inhibitor resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient tumors can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. A molecular understanding of PARP inhibitor resistance mechanisms may allow the generation of a new class of drugs, or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARP inhibitors to more tumor types. Citation Format: Alan D. D’Andrea. PARP inhibitor resistance and acquired vulnerability in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA15.

Abstract CN05-02: Novel mechanisms of PARP inhibitor resistance

Abstract Large-scale genomic studies have demonstrated that approximately 50% of high-grade serous ovarian cancers (HGSOCs) harbor genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA1 and BRCA2, followed by other Fanconi anemia genes including FANCN/PALB2, FANCO/RAD51, FANCJ/BRIP, and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues, topoisomerase inhibitors, and PARP inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in ovarian cancer where multiple PARPi have been FDA approved. PARP inhibitor resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2-deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. However, PARPi resistance may still develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism–namely, the restoration of RF stability–appears to be a highly prevalent mechanism of PARPi resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire PARPi resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient tumors can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. A molecular understanding of PARPi resistance mechanisms may allow the generation of a new class of drugs or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARPi to more tumor types. Citation Format: Alan D. D'Andrea. Novel mechanisms of PARP inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr CN05-02.

Abstract B172: Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition

Abstract Cyclin-dependent kinase 12 (CDK12), a key regulator of transcriptional processing along with its binding partner cyclin K (CCNK), has been implicated in the modulation of genes involved with the DNA damage response (DDR) including, but not limited to, homologous recombination repair (HRR). However, the exact mechanism by which CDK12 is having this effect has been unclear. To identify differentially expressed transcripts between wild-type and mutant CDK12 that may indicate alternative or mis-splicing events and help understand the mechanism of CDK12 loss of function, we analyzed RNA-Seq data from TCGA ovarian cancer comparing transcripts in loss of function mutated CDK12 (~3% of ovarian) vs wild-type samples. We identified a number of transcripts that were at least twofold higher in CDK12 mutant vs wild-type samples. Deeper analysis showed an enrichment in truncated transcripts, a subset of which may arise from readthrough into an intron containing a transcriptional stop, including the DDR genes FANCI and ATM. This potential mechanism by which CDK12 may regulate DDR genes was tested in vitro using Taqman probes upstream and downstream of the introns with the stop signal for both ATM and FANCI. To validate the differential expression of FANCI and ATM transcripts observed in TCGA samples, we treated the ovarian cells lines OVCAR8 and OV90 with CDK12 siRNA for 72hrs. CDK12 knockdown resulted in truncated transcript expression and a reduction in the full-length transcripts of DDR genes FANCI and ATM. Reduction in levels of full-length transcripts also leads to decreased levels of FANCI and ATM protein. Knockdown of the closely related homolog CDK13 by siRNA did not lead to expression of truncated FANCI and ATM transcripts. Sensitization to olaparib was observed for CDK12 but not CDK13 siRNA treated ovarian cells in a 14-day clonogenic assay indicating a loss of HRR proficiency due to CDK12 depletion. A biochemical screen was developed and a synthetic chemistry plan initiated to identify CDK12 selective probe compounds to try to confirm the target biology, DDR gene modulation, and RNA processing effects seen with siRNA. Potent and selective inhibitors of CDK12/13 were identified and used to treat ovarian cancer cells. Differential expression of full-length vs truncated FANCI and ATM transcripts was seen by 4-8 hrs with CDK12/13 selective compounds. However, at higher concentrations and longer time points, effects on general transcription were observed as indicated by a decrease in full-length ATM and FANCI, potent effects on p-Ser2 of RNApolII, and growth inhibition in a 48-hr proliferation assay. The small-molecule inhibitors partially recapitulate the RNA processing effects on ATM and FANCI; however, effect on CDK13 or the retention of CCNK in an inactive complex may lead to broader transcriptional effects. To our knowledge, these are the first data linking CDK12 inhibition to a RNA processing mechanism of intronic readthrough to a transcriptional stop leading to modulation of a subset of transcripts, including the DDR genes ATM and FANCI. Citation Format: Christopher Denz, Jeffrey Johannes, Yi Yao, Meghana Kulkarni, Austin Dulak, Nin Guan, Nancy Su, Michelle Lamb, Stephen Fawell, Sylvie Guichard. Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B172.

Abstract SY29-02: Novel mechanisms of PARP inhibitor resistance

Abstract Large-scale genomic studies have demonstrated that approximately 50% of high-grade serous ovarian cancers (HGSOCs) harbor genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA1 and BRCA2, followed by other Fanconi anemia genes including FANCN/PALB2, FANCO/RAD51, FANCJ/BRIP, and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues, topoisomerase inhibitors, and PARP-inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in ovarian cancer, where the PARPi olaparib is FDA approved for use in patients with germline BRCA1/2 mutations. PARP inhibitor resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2-deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. However, PARPi resistance may still develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism—namely, the restoration of RF stability—appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire PARP inhibitor resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient tumors can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. A molecular understanding of PARP inhibitor resistance mechanisms may allow the generation of a new class of drugs or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARP inhibitors to more tumor types. Citation Format: Alan D. D'Andrea. Novel mechanisms of PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY29-02. doi:10.1158/1538-7445.AM2017-SY29-02

Abstract IA25: Novel mechanisms of PARP-inhibitor resistance in tumors with defects in the Fanconi Anemia/BRCA pathway

Abstract Large-scale genomic studies have demonstrated that approximately 50% of high-grade serous ovarian cancers (HGSOCs) harbor genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. HRR alterations have also been identified, albeit less frequently, in other human malignancies including triple negative breast, prostate, and pancreatic cancers. The most commonly altered HRR genes are BRCA1 and BRCA2 followed by other Fanconi Anemia (FA) genes (e.g. PALB2, FANCA, FANCI, FANCL, and FANCC), core RAD genes (e.g. RAD50, RAD51, RAD51C, and RAD54L) and DNA damage response genes involved in HRR, such as ATM, ATR, CHEK1, and CHEK2. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to certain types of DNA-damaging chemotherapy such as platinum analogues and topoisomerase inhibitors. HRR deficient tumors are also exquisitely sensitive to PARP-inhibitors (PARPis) which exhibit synthetic lethality to cells with defective HRR. This synthetic lethal interaction is being exploited therapeutically in diverse clinical contexts and most notably in ovarian cancer where the PARPi olaparib is FDA approved for use in patients with germline BRCA1/2 mutations who have progressed through at least 3 prior lines of therapy. The efficacy of PARPis against HRR deficient cells can be explained by various mechanisms including inhibition of base excision repair (BER), trapping of PARP-DNA complexes at the replication fork, enhancement of toxic non-homologous end joining in PARP1-deficient cells, and inhibition of PARP1/Polθ-mediated alternative end joining (alt-EJ). Underlying HRR deficiency is important for the cytotoxicity of PARPis and this is highlighted by the fact that the most prevalent mechanism of PARPi resistance is secondary genetic and epigenetic events that cancel the original HRR alteration and restore HRR proficiency. However, PARPi resistance may still develop without restoration of HR proficiency via reduced uptake and increased efflux of the drugs or via disruption of multiple proteins such as PTIP or CHD4 that leads to replication fork protection. Importantly, this latter mechanism-namely, the restoration of RF stability- appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire PARP inhibitor resistance by limiting the nucleolytic degradation of their stalled replication forks. In my presentation, I will discuss new mechanisms of RF nucleolytic degradation and novel mechanisms by which tumors can avoid this degradation and acquire PARP inhibitor resistance. A molecular understanding PARP inhibitor resistance mechanisms is important, since it may allow the generation of a new class of drugs, or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARP inhibitors to more tumor types. Citation Format: Alan D. D'Andrea. Novel mechanisms of PARP-inhibitor resistance in tumors with defects in the Fanconi Anemia/BRCA pathway [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA25.

Reciprocal positive selection for weakness - preventing olaparib resistance by inhibiting BRCA2.

Human tumor heterogeneity promotes therapeutic failure by increasing the likelihood of resistant cell subpopulations. The PARP-1 inhibitor olaparib is approved for use in BRCA-mutated ovarian cancers but BRCA2-reversion mutations lead to functional homologous recombination repair (HRR) and olaparib resistance. To overcome that resistance and expand use of PARP1 inhibition to cancers with functional HRR, we developed an antisense strategy to render the majority of tumor cells in a population BRCA2-deficient. We predicted that this strategy would render HRR-proficient tumor cells sensitive to olaparib and prevent emergence of resistance in a tumor cell population heterogeneous for HRR proficiency. We report that BRCA2 downregulation sensitized multiple human tumor cell lines (but not non-cancer human kidney cells) to olaparib and, combined with olaparib, increased aneuploidy and chromosomal translocations in human tumor cells. In a mixed HRR-proficient and HRR-deficient cell population, olaparib monotherapy allowed outgrowth of HRR-proficient cells resistant to subsequent olaparib treatment. Combined BRCA2 inhibition and olaparib treatment prevented selection of HRR-proficient cells and inhibited proliferation of the entire population. Treatment with BRCA2 siRNA and olaparib decreased ovarian xenograft growth in mice more effectively than either treatment alone. In vivo use of BRCA2 antisense oligonucleotides may be a viable option to expand clinical use of olaparib and prevent resistance.

PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia

Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor.Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting.Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity.Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Akt: A Double-Edged Sword in Cell Proliferation and Genome Stability

The Akt family of serine/threonine protein kinases are key regulators of multiple aspects of cell behaviour, including proliferation, survival, metabolism, and tumorigenesis. Growth-factor-activated Akt signalling promotes progression through normal, unperturbed cell cycles by acting on diverse downstream factors involved in controlling the G1/S and G2/M transitions. Remarkably, several recent studies have also implicated Akt in modulating DNA damage responses and genome stability. High Akt activity can suppress ATR/Chk1 signalling and homologous recombination repair (HRR) via direct phosphorylation of Chk1 or TopBP1 or, indirectly, by inhibiting recruitment of double-strand break (DSB) resection factors, such as RPA, Brca1, and Rad51, to sites of damage. Loss of checkpoint and/or HRR proficiency is therefore a potential cause of genomic instability in tumor cells with high Akt. Conversely, Akt is activated by DNA double-strand breaks (DSBs) in a DNA-PK- or ATM/ATR-dependent manner and in some circumstances can contribute to radioresistance by stimulating DNA repair by nonhomologous end joining (NHEJ). Akt therefore modifies both the response to and repair of genotoxic damage in complex ways that are likely to have important consequences for the therapy of tumors with deregulation of the PI3K-Akt-PTEN pathway.