DNA Damage-based Therapy Research Articles

CCRG-01. MYT1 BLOCKADE AS A NOVEL GLIOMA THERAPEUTICS THROUGH ABROGATIING OF THE G2/M CELL CYCLE CHECKPOINT

Abstract BACKGROUND The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. The screening hits were validated through in patient derived glioma cells and xenografts. RESULTS Our findings revealed that canonical DNA repair pathways, including the Ataxia–telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.

A DNA damage-amplifying nanoagent for cancer treatment via two-way regulation of redox dyshomeostasis and downregulation of tetrahydrofolate

DNA damage-based therapy is widely used in cancer treatment, yet its therapeutic efficacy is constrained by the redox homeostasis and DNA damage repair mechanisms of tumor cells. To address these limitations and enhance the efficacy of DNA damage-based therapy, HA-CuH@MTX, a copper-histidine metal-organic complex (CuH) loaded with methotrexate (MTX) and modified with hyaluronic acid (HA), was developed to amplify the DNA damage induced. In vitro experiments demonstrated that the presence of both Cu+ and Cu2+ in HA-CuH@MTX enables two-way regulated redox dyshomeostasis (RDH), achieved through Cu+-catalyzed generation of •OH and Cu2+-mediated consumption of glutathione, thereby facilitating efficient DNA oxidative damage. In addition, DNA damage repair is synergistically inhibited by impairing nucleotide synthesis via histidine metabolism and MTX downregulation of tetrahydrofolate, a crucial raw material in nucleotide synthesis. In vivo experiments with 4T1 tumor-bearing mice demonstrate 83.6 % inhibition of tumor growth by HA-CuH@MTX. This work provides a new strategy to amplify the DNA damage caused by DNA damage-based cancer therapies, and holds great potential for improving their therapeutic efficacy.

YY2/BUB3 Axis promotes SAC Hyperactivation and Inhibits Colorectal Cancer Progression via Regulating Chromosomal Instability.

Spindle assembly checkpoint (SAC) is a crucial safeguard mechanism of mitosis fidelity that ensures equal division of duplicated chromosomes to the two progeny cells. Impaired SAC can lead to chromosomal instability (CIN), a well-recognized hallmark of cancer that facilitates tumor progression; paradoxically, high CIN levels are associated with better therapeutic response and prognosis. However, the mechanism by which CIN determines tumor cell survival and therapeutic response remains poorly understood. Here, using a cross-omics approach, YY2 is identified as a mitotic regulator that promotes SAC activity by activating the transcription of budding uninhibited by benzimidazole 3 (BUB3), a component of SAC. While both conditions induce CIN, a defect in YY2/SAC activity enhances mitosis and tumor growth. Meanwhile, hyperactivation of SAC mediated by YY2/BUB3 triggers a delay in mitosis and suppresses growth. Furthermore, it is revealed that YY2/BUB3-mediated excessive CIN causes higher cell death rates and drug sensitivity, whereas residual tumor cells that survived DNA damage-based therapy have moderate CIN and increased drug resistance. These results provide insights into the role of SAC activity and CIN levels in influencing tumor cell survival and drug response, as well as suggest a novel anti-tumor therapeutic strategy that combines SAC activity modulators and DNA-damage agents.

Frizzled class receptor 5 contributes to ovarian cancer chemoresistance through aldehyde dehydrogenase 1A1

BackgroundChemoresistance is associated with tumor relapse and unfavorable prognosis. Multiple mechanisms underlying chemoresistance have been elucidated, including stemness and DNA damage repair. Here, the involvement of the WNT receptor, FZD5, in ovarian cancer (OC) chemoresistance was investigated.MethodsOC cells were analyzed using in vitro techniques including cell transfection, western blot, immunofluorescence and phalloidin staining, CCK8 assay, colony formation, flowcytometry, real-time PCR, and tumorisphere formation. Pearson correlation analysis of the expression levels of relevant genes was conducted using data from the CCLE database. Further, the behavior of OC cells in vivo was assessed by generation of a mouse xenograft model.ResultsFunctional studies in OC cells showed that FZD5 contributes to epithelial phenotype maintenance, growth, stemness, HR repair, and chemoresistance. Mechanistically, FZD5 modulates the expression of ALDH1A1, a functional marker for cancer stem-like cells, in a β-catenin-dependent manner. ALDH1A1 activates Akt signaling, further upregulating RAD51 and BRCA1, to promote HR repair.ConclusionsTaken together, these findings demonstrate that the FZD5-ALDH1A1-Akt pathway is responsible for OC cell survival, and targeting this pathway can sensitize OC cells to DNA damage-based therapy.

Abstract 4565: Reckless mitotic entry as a chemosensitization approach for alkylating agents

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome scale CRISPR/Cas9 chemogenomic screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Analysis on patient cohort demonstrated that PKMYT1 is not only highly presented in malignancy, but also predisposes an unfavorable disease outcome. Genetic depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James A. Cornwell, Karambir Kaur, Mirit I. Aladjem, Michael Aregger, Steven D. Cappell, Chunzhang Yang. Reckless mitotic entry as a chemosensitization approach for alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4565.

Abstract A006: Reckless mitotic entry as a novel chemosensitization approach for alkylating agents

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as a novel strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James Cornwell, Karambir Kaur, Omar Elmogazy, Wei Zhang, Meili Zhang, Hua Song, Zhonghe Sun, Xiaolin Wu, Mirit I. Aladjem, Michael Aregger, Steven Cappell, Chunzhang Yang. Reckless mitotic entry as a novel chemosensitization approach for alkylating agents [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A006.

Abrogation of the G2/M checkpoint as a chemosensitization approach for alkylating agents.

The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.

BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

Accumulating evidence indicates a correlation between circadian dysfunction and genomic instability. However, whether the circadian machinery directly regulates DNA damage repair, especially in double-strand breaks (DSBs), remains poorly understood. Here, we report that in response to DSBs, BMAL1 is activated by ATM-mediated phosphorylation at S183. Phosphorylated BMAL1 is then localized to DNA damage sites, where it facilitates acetylase CLOCK to load in the chromatin, regulating the acetylation of histone H4 (H4Ac) at DSB sites. In this way, the BMAL1-CLOCK-H4Ac axis promotes the DNA end-resection to generate single-stranded DNA (ssDNA) and the subsequent homologous recombination (HR). BMAL1 deficient cells display defective HR, accumulation of unrepaired DSBs and genome instability. Accordingly, depletion of BMAL1 significantly enhances the sensitivity of adrenocortical carcinoma (ACC) to DNA damage-based therapy in vitro and in vivo. These findings uncover non-canonical function of BMAL1 and CLOCK in HR-mediated DSB repair, which may have an implication in cancer therapeutics.

Lysine Methyltransferase Inhibitors Impair H4K20me2 and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Lethality Strategy.

BackgroundChromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs).MethodsKMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis.ResultsChaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation, and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death.ConclusionKMT inhibitors could function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.

DNA Repair Pathways in Cancer Therapy and Resistance.

DNA repair pathways are triggered to maintain genetic stability and integrity when mammalian cells are exposed to endogenous or exogenous DNA-damaging agents. The deregulation of DNA repair pathways is associated with the initiation and progression of cancer. As the primary anti-cancer therapies, ionizing radiation and chemotherapeutic agents induce cell death by directly or indirectly causing DNA damage, dysregulation of the DNA damage response may contribute to hypersensitivity or resistance of cancer cells to genotoxic agents and targeting DNA repair pathway can increase the tumor sensitivity to cancer therapies. Therefore, targeting DNA repair pathways may be a potential therapeutic approach for cancer treatment. A better understanding of the biology and the regulatory mechanisms of DNA repair pathways has the potential to facilitate the development of inhibitors of nuclear and mitochondria DNA repair pathways for enhancing anticancer effect of DNA damage-based therapy.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

Abstract A06: Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner

Abstract Post-translational modifications (PTMs) of histone tails can function as epigenomic regulators of the cellular response to DNA damage. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone PTM involved in the DNA damage response. H2Bub1 localizes to double-strand breaks where it functions to decondense chromatin, making it accessible to DNA repair factors. H2Bub1 is also known to be enriched at coding sequences of highly expressed genes, demonstrating the fundamental importance of H2Bub1 for an open chromatin structure that facilitates transcription. H2Bub1 is a dynamic process, written predominantly by the E3 ubiquitin ligase complex of the ring finger proteins RNF20 and RNF40 and erased by multiple deubiquitinases, including those from the ubiquitin-specific peptidase subfamily USP7 and USP44. RNF20 is reported to interact with p53. Platinum drugs are used as standard-of-care for the treatment of high-grade serous ovarian cancer (HGSOC) based on their capacity to damage DNA. We sought to determine the response of ubiquitinated histone tails to a platinum drug with the goal of improving the efficacy of current best practice therapy for HGSOC. Following treatment of a wild-type p53 cell line with an IC75 dose of cisplatin, we combined H2Bub1 chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to determine genomic enrichment of H2Bub1 in response to DNA damage. Further, we coupled this with RNA-seq (RNA sequencing) to enable correlation of genomic enrichment of H2Bub1 with levels of gene expression. KEGG pathway and gene ontology analyses demonstrated greatest H2Bub1 enrichment in response to DNA damage in p53 signalling pathway genes, including CDKN1A, BBC3, MDM2 and BAX. This correlated with increased expression of p53 target genes in response to DNA damage. Wild-type TP53 itself did not show H2Bub1 enrichment upon treatment with cisplatin. H2Bub1 ChIP experiments were repeated in HGSOC mutant p53 cell line models, specifically OVCAR-3 with the known gain-of-function TP53 mutation R248Q and Kuramochi with the missense TP53 mutation D281Y. H2Bub1 was not enriched in response to DNA damage at wild-type p53 gene targets in these cell lines, nor were these genes highly expressed. We therefore propose that DNA damage induced H2Bub1-enrichment in mutant p53 cell lines will have an entirely distinct genomic profile compared to wild-type p53 cell lines, with important roles in enabling the expression of mutant p53 target genes in HGSOC. Experiments are currently under way to directly address this hypothesis. Lastly, we have used clonogenic cell survival assays to demonstrate that downregulation of RNF20, either transiently using short interfering (si) RNA or stably,using short hairpin (sh) RNA, decreased the ability of HGSOC cells to survive treatment with cisplatin. Down-regulation of H2Bub1 by downregulation of its E3 ubiquitin ligase RNF20 may represent a novel treatment strategy to increase the efficacy of DNA damage-based therapy for HGSOC. Citation Format: Alexander J. Cole, Kristie-Ann Dickson, Christopher Liddle, Clare Stirzaker, Roderick Clifton-Bligh, Deborah J. Marsh. Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A06.

Selinexor reduces the expression of DNA damage repair proteins and sensitizes cancer cells to DNA damaging agents.

IntroductionThe goal of this study was to examine the effects of selinexor, an inhibitor of exportin-1 mediated nuclear export, on DNA damage repair and to evaluate the cytotoxic effects of selinexor in combination with DNA damaging agents (DDAs) in cancer cells.ResultsSelinexor reduced the expression of DNA damage repair (DDR) proteins. This did not induce significant DNA damage in tested cell lines. Inhibition of DDR protein expression resulted in enhanced cancer cell death when cells were pretreated with DDAs. In contrast, enhanced cell death was not detected in cells that were pretreated with selinexor then with DDAs. In vivo, single-agent selinexor, docetaxel, or cisplatin treatment resulted in 66.7%, 51.5%, and 26.6% tumor growth inhibition (TGI), respectively, in an MDA-MB-231 xenograft model. Consequently, combination treatment with docetaxel or cisplatin followed by selinexor in vivo resulted in 93.9% and 103.4% TGI, respectively. Immunohistochemical staining and immunoblot analysis of tumor sections confirmed reduced expression of DDR proteins.ConclusionSelinexor treatment inhibited DDR mechanisms in cancer cell lines and therefore potentiated DNA damage-based therapy. The sequential combination of DDAs followed by selinexor increased cancer cell death. This combination is superior to each individual therapy and has a mechanistic rationale as a novel anticancer strategy.MethodsCancer cells treated with selinexor ± DDAs were analyzed using reverse phase protein arrays, immunoblots, quantitative PCR and immunofluorescence. Mice bearing MDA-MB-231 tumors were treated with subtherapeutic doses of selinexor, cisplatin, docetaxel and selinexor in combination with either cisplatin or docetaxel. Tumor growth was evaluated for 25 days.

CAMP-mediated autophagy inhibits DNA damage-induced death of leukemia cells independent of p53.

Autophagy is important in regulating the balance between cell death and survival, with the tumor suppressor p53 as one of the key components in this interplay. We have previously utilized an in vitro model of the most common form of childhood cancer, B cell precursor acute lymphoblastic leukemia (BCP-ALL), to show that activation of the cAMP signaling pathway inhibits p53-mediated apoptosis in response to DNA damage in both cell lines and primary leukemic cells. The present study reveals that cAMP-mediated survival of BCP-ALL cells exposed to DNA damaging agents, involves a critical and p53-independent enhancement of autophagy. Although autophagy generally is regarded as a survival mechanism, DNA damage-induced apoptosis has been linked both to enhanced and reduced levels of autophagy. Here we show that exposure of BCP-ALL cells to irradiation or cytotoxic drugs triggers autophagy and cell death in a p53-dependent manner. Stimulation of the cAMP signaling pathway further augments autophagy and inhibits the DNA damage-induced cell death concomitant with reduced nuclear levels of p53. Knocking-down the levels of p53 reduced the irradiation-induced autophagy and cell death, but had no effect on the cAMP-mediated autophagy. Moreover, prevention of autophagy by bafilomycin A1 or by the ULK-inhibitor MRT68921, diminished the protecting effect of cAMP signaling on DNA damage-induced cell death. Having previously proposed a role of the cAMP signaling pathway in development and treatment of BCP-ALLs, we here suggest that inhibitors of autophagy may improve current DNA damage-based therapy of BCP-ALL - independent of p53.

LKB1 is a DNA damage response protein that regulates cellular sensitivity to PARP inhibitors

Liver kinase B1 (LKB1) functions as a tumor suppressor encoded by STK11, a gene that mutated in Peutz-Jeghers syndrome and in sporadic cancers. Previous studies showed that LKB1 participates in IR- and ROS-induced DNA damage response (DDR). However, the impact of LKB1 mutations on targeted cancer therapy remains unknown. Herein, we demonstrated that LKB1 formed DNA damage-induced nuclear foci and co-localized with ataxia telangiectasia mutated kinase (ATM), γ-H2AX, and breast cancer susceptibility 1 (BRCA1). ATM mediated LKB1 phosphorylation at Thr 363 following the exposure of cells to ionizing radiation (IR). LKB1 interacted with BRCA1, a downstream effector in DDR that is recruited to sites of DNA damage and functions directly in homologous recombination (HR) DNA repair. LKB1 deficient cells exhibited delayed DNA repair due to insufficient HR. Notably, LKB1 deficiency sensitized cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, we have demonstrated a novel function of LKB1 in DNA damage response. Cancer cells lacking LKB1 are more susceptible to DNA damage-based therapy and, in particular, to drugs that further impair DNA repair, such as PARP inhibitors.

Abstract C185: Targeting PARP and DNA as a novel unimolecular combination model for the enhancement of chemosensitivity

Abstract The concept of synthetic lethality arises when the alteration of a single gene A or B alone does not affect cell survival but that of both genes lead to cell death. More importantly, if A is mutated, inhibition of the functions of the gene B product should also lead to cell death. This is achievable in some tumours, predominantly in breast and ovarian cancers, where the mutation of the DNA repair proteins BRCA1/2 depletes the DNA repair capacity of the cells. The compensatory DNA repair mechanism in the cells is supported by a DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which when inhibited, leaves the cells with no compensatory DNA repair mechanisms. Hence DNA lesions accumulate in the cells and ultimately cause their death. The ability of PARP inhibitors to induce cell death in BRCA1/2 mutated tumours has been demonstrated in both preclinical models and in the clinic. While the PARP inhibitor olaparib met the requirements for recent FDA approval for the treatment of ovarian cancer, the overall survival obtained with many PARP inhibitors as single agents in patients with BRCA1,2 mutated tumours has been disappointing,. Also, acquired resistance caused by the reactivation of wild type BRCA1/2 is an emerging drawback in BRCA1/2 related therapies. Our hypothesis is that molecules termed “combi-molecules” capable of not only inhibiting PARP but also inducing DNA damage, could significantly enhance cell-killing in BRCA1/2 deficient tumours and perhaps be indicated at the earlier stages of the diseases. Here, we designed and studied the potency and mechanism of action of our first prototype EG22. The results showed that: (a) it inflicted higher levels of DNA damage than temozolomide (a clinical DNA damaging agent) and >100-fold stronger growth inhibitory potency than the latter in our cell panel, regardless of the BRCA1/2 status of the cells, (b) it showed more than 5-fold stronger growth inhibitory potency than 4-ANI, a known PARP inhibitor, in BRCA1/2 deficient cells and more than 8-fold in wild type expressing cells (c) a PARP assay showed that it induced a dose-dependent inhibition of PARP with an IC50 value almost equal to that of 4-ANI and (d) in cell-based selectivity assay involving a pair of isogenic Chinese hamster cell line (engineered to express BRCA2 wild type), it induced 20-fold selectivity for the mutant form. The results in toto suggest that EG22 is a new molecular entity with a novel and dual mechanism of action. Furthermore, reactivation of BRCA1/2 being one of the mechanisms of resistance to DNA damage-based therapy of patients with mutant BRCA1/2, the combi-molecular approach has the potential to be developed as an alternative treatment, when the tumours heterogeneously express BRCA1/2. Citation Format: Zhor Senhaji Mouhri, Elliot Goodfellow, Bertrand Jean-Claude. Targeting PARP and DNA as a novel unimolecular combination model for the enhancement of chemosensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C185.

Role of mTOR-Chk1 in enhancing DNA-damaging therapy

Mechanistic target of rapamycin (mTOR) is an atypical Ser/Thr protein kinase that acts as a cellular hub integrating cell growth signals and nutrient availability to promote cell survival.1 mTOR is the catalytic component of 2 functionally distinct complexes (mTORC1 and mTORC2), and belongs to the family of phosphatidyl-inositol(3)-like kinases (PIKKs) together with ATM, ATR and DNA-PKcs. The latter 3 kinases have well established roles in the DNA damage response (DDR) – the signaling network activated by DNA damage to promote cell survival by enhancing DNA repair and delaying cell cycle progression in order to maximize repair. However, the participation of mTOR in these pathways has only recently been recognized, aided by the generation and availability of ATP-competitive mTOR kinase inhibitors. A better understanding of the regulation of the DDR is not only vital to understand aspects of tumor development, but is also important to improve DNA damage-based cancer therapies by targeting specific aspects, such as mTOR signaling. mTOR may act to promote cell survival either by regulating DNA repair and/or cell cycle arrest. Indeed, recent work has demonstrated a role for mTOR kinase in DNA repair through the regulation of FANCD2, a key protein involved in the repair of double-strand DNA breaks.2,3 Our recent work in Oncotarget4 also implicates mTOR in control of the cell cycle arrest aspect of the DDR. We found that mTOR is required for efficient establishment of S and G2/M cell cycle arrest after DNA damage, since ablation of mTOR resulted in a reduction in the percentage of cells in S and G2/M. Interestingly, we found that mTORC2 was required for the DNA damage-induced activation of Chk1, a key regulator of cell cycle arrest. We found that mTOR inhibition significantly inhibited all 3 DNA damage-induced phosphorylations of Chk1 (S296, S317 and S345) and reduced the production of Chk1 protein. Since mTOR inhibition consistently ablated DNA damage-induced Chk1 phosphorylation under all conditions tested, whereas inhibition of protein levels was cell type specific, this suggests that there may be distinct mechanisms by which mTOR controls Chk1 and that phosphorylation of Chk1 may be of primacy. Exactly how mTOR might regulate Chk1 activity is an open question. One likely candidate could be via ATR, since its ability to phosphorylate Chk1 at Ser317 and Ser345 leading to autophosphorylation at Ser296 is well established. Alternatively, the effects on Chk1 might involve one of the substrates of mTORC2. Our results support accumulating evidence from studies in yeast implicating TORC2 in survival under conditions of DNA damage and in genome stability.5 Since approval of the rapalog, Everolimus, in combination with hormone therapies for breast cancer, the potential of mTOR inhibitors to overcome resistance to current therapies has gained increasing interest. Indeed numerous publications have demonstrated that mTOR inhibition can also enhance radio- or chemotherapy-induced cell death in various cancer types. We also investigated the translational significance of our results and found that an mTOR kinase inhibitor enhanced DNA damage-induced cell death in HEK293 cells and a panel of breast cancer cell lines.4 While this suggests that mTOR kinase inhibitors may be useful therapeutically to augment DNA damage based therapies, it is likely that these effects will be tumor-type specific since similar augmentation was not found in renal and colon cancers.6,7 We have previously shown that an mTOR kinase inhibitor prevented p53-dependent cell death in renal carcinoma cells.6 It is known that mTORC1 contributes to DNA damage-induced p53 activation via multiple mechanisms, therefore it is likely that mTOR ablation leads to p53 suppression and inhibition of p53-dependent apoptosis in these renal cells. However, HEK293 cells have non-functional p53 and the panel of breast cancer cell lines that we tested displayed a variety of either wild-type, lost or mutated p53, but all still exhibited chemosensitivity following mTOR kinase inhibition. This suggests that these latter cells may rely on mTORC2-Chk1 signaling for survival. In conclusion, it is clear that mTOR regulates the DDR response by multiple mechanisms. Whether a given tumor type is made chemosensitive or chemoresistant by mTOR kinase inhibitors will depend on which aspect of mTOR signaling is altered during tumorigenesis. In this vein, it will be important to widen our understanding of mTOR targets following DNA damage in order to gain better insight into whether specific targeting of mTORC2 will be a useful adjuvant to DNA-damaging therapies.

Vaccinia-related kinase 1 (VRK1) confers resistance to DNA-damaging agents in human breast cancer by affecting DNA damage response

Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs.