DNA Damage Response Inhibitors Research Articles

Phase II trial of olaparib in combination with ceralasertib in patients with recurrent osteosarcoma.

TPS11575 Background: Osteosarcoma is the most common primary bone tumor, occurring in children, adolescents, and young adults. In contrast to advances in treatment for most childhood cancers, there have been no significant improvements in osteosarcoma outcomes in the past 40 years. Forty percent of osteosarcoma patients will, at some point, have advanced disease which has a very poor outcome with a 5-year overall survival of approximately 20%. Genomic alterations and signatures associated with sensitivity to treatment with DNA damage response (DDR) inhibitors are observed frequently in osteosarcoma. Many osteosarcomas have a unique mutational signature (signature 3) similar to that seen in BRCA1 deficient cancer and response to PARP inhibitors has been seen in osteosarcoma cell lines. Additionally, ATRX, a protein involved in the alternative lengthening of telomeres (ALT), is often inactivated in osteosarcoma. Defects in the ALT pathway may sensitize tumor cells to ATR inhibitors and osteosarcoma cell lines have been shown to be sensitive to ATR inhibition. In vitro susceptibility of osteosarcoma cell lines to ATR and PARP inhibitors, the presence of mutations in genes involved in DDR and the presence of signature 3 in a subset of osteosarcomas serves as the basis for the development of this trial. Methods: This is an ongoing open label, multicenter, phase II clinical trial to evaluate the clinical activity of PARP inhibitor, olaparib, in combination with ATR inhibitor, ceralasertib, in 2 cohorts of patients aged 12-40 with recurrent osteosarcoma (NCT04417062). Patients with unresectable disease are enrolled into Cohort 1. Patients with resectable disease limited to the lung are enrolled into Cohort 2. Patients in both cohorts receive olaparib 300mg orally twice a day on days 1-28 and ceralasertib 160 mg orally once a day on days 1-7 of a 28-day cycle (adult maximum tolerated dose for the combination). For patients in Cohort 2, study treatment also includes surgical resection of lung metastases at protocol-specified timepoints. Patients can remain on treatment for up to 2 years if they have not progressed. For Cohort 1, the primary objective is to determine whether the combination treatment improves the 4-month event-free rate as compared to a historical benchmark from Children’s Oncology Group (COG) trials using a Simon’s two-stage design. In the first stage, ≥3 of 19 patients must be event-free at 4 months to proceed to stage 2. Enrollment into Cohort 2 continues as long as Cohort 1 enrollment is ongoing. For Cohort 2, the primary endpoint is the submission of paired pre- and post-treatment tumor samples for correlative studies. Secondary endpoints include objective response rate, event-free survival, and overall survival. Integrated correlative studies will assess tumor tissue for biomarkers of treatment response and measure circulating tumor DNA longitudinally. Enrollment began November 24, 2020 and is ongoing. Clinical trial information: NCT04417062.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality

Abstract Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

Interfaces between cellular responses to DNA damage and cancer immunotherapy

The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.

Abstract IA-008: Etiology of chromosomal rearrangements, and other DNA double-strand break repair outcomes

Abstract Defining the factors that influence chromosomal double-strand breaks (DSBs) outcomes important for developing radiosensitization strategies that target the DNA damage response (DDR). In a main project in the lab, we have used Cas9 to study mechanisms of end joining (EJ) repair that affect diverse outcomes, ranging from short insertion/deletion (indels) mutations, to large chromosomal rearrangements. In particular, we have sought to define the relative contributions of canonical non-homologous end joining (C-NHEJ), and alternative end joining (ALT-EJ), on such DSB repair outcomes. For instance, I will present evidence that C-NHEJ is absolutely required for EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (No Indel EJ). Namely, C-NHEJ does not appear to be intrinsically prone to causing indels, relative to the ALT-EJ pathway. Furthermore, using No Indel EJ as a hallmark of C-NHEJ, I will present our current directions for understanding the mechanism and regulation of C-NHEJ and ALT-EJ, and targeting these pathways by DDR inhibitors. I suggest that establishing how the DDR affects distinct EJ events will reveal the ideal contexts for using DDR inhibitors during cancer therapy and inform the development of biomarkers of response. Citation Format: Jeremy M. Stark. Etiology of chromosomal rearrangements, and other DNA double-strand break repair outcomes [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-008.

Abstract IA-011: Translation to clinical trials: Are we shooting the right arrows at the target?

Abstract Dependent on the stage of the cancer, 25-40% of patients require curative or palliative radiotherapy as part of their primary care treatment. The DNA Damage response (DDR) pathways have been a major area of interest for drug development as tumors often harbor mutations in the DDR pathways. The development of new DDR inhibitors offers wide-ranging opportunities to combine DDR inhibition with radiation therapy. Emerging evidence also supports the use of DDR inhibitors to promote immune stimulatory effects of radiation therapy by further activating the DNA sensing cGAS-STING pathway. DDR inhibitors in combination with RT have been shown to alter PDL-1 expression and have also been linked to modulating cytokine expression to alter the tumor microenvironment. This should be considered in the context of over 300 clinical trials ongoing which are combining immunotherapy with radiotherapy and important clinical studies of this combination therapy which have been published in patients with lung cancer. Significant progress is being made in understanding new drug-radiotherapy combinations and advancing them towards the clinic. Citation Format: Ricky A. Sharma. Translation to clinical trials: Are we shooting the right arrows at the target? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-011.

Abstract IA-007: Exploiting DNA repair defects in head and neck squamous cell carcinoma

Abstract DNA damage repair pathways govern genomic integrity and determine cellular sensitivities to radiation and chemotherapeutic agents. Recent genetic studies highlight the identification of an increasing number of DNA repair gene defects in many different cancer types. DNA damage response and repair inhibitors on the other hand provide opportunities to exploit such defects inherent to cancer cells. Combined with radiotherapy they are highly effective radiosensitizers that show some tumor specificity in that they cause greater radiation dose enhancement in cells with repair defects. To optimize such potent combinations, robust and sensitive biomarkers are needed to 1) assess effectivity and guide clinical trial designs and 2) to identify repair defects in individual patients. Radiotherapy is a mainstay in the treatment of advanced HPV-negative head and neck squamous cell carcinoma (HNSCC). There is a need for improved treatment options in this patient population. Indicating particular crosslink repair sensitivity and frequent cancer cell repair defects, cumulative cisplatin dose is an important factor in determining patient outcomes after chemoradiotherapy. Indeed, preclinical and genetic data highlight the frequent manifestation of DNA crosslink repair defects in HNSCC. Genetic biomarkers have therefore been generated and tested to support the identification of patients in need for novel treatment options and of crosslink repair defects in HNSCC for combination with PARP inhibitors. Citation Format: Conchita Vens. Exploiting DNA repair defects in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-007.

Abstract PR-003: Radiosensitization by targeting the NPM1/RAD51 axis

Abstract NPM1 is a chaperone involved in base excision, translesion synthesis and homologous recombination (HR). In the presence of DNA DSBs pT199 NPM1 co-localizes with H2AX foci, a consequence of binding to K63-linked ubiquitinated histones that surround DSBs via its pT199 domain comprised of an acidic tract and adjacent ubiquitin interacting motif-like domain (1-3). SUMOlyated K263 NPM1 binds to and promotes Rad51 loading onto resected 3-prime DNA at the DSB. Failure to SUMOlyate K263NPM or inhibition of NPM1 reduces Rad51 filament formation, thus inhibiting HR (4). A major effort is underway to develop DNA damage response inhibitors. The novel chemical entity YTR107 is a 5-((N-benzyl-1H-indol-3-yl) methylene) pyrimidine-2, 4,6(1H,3H,5H) trione developed using forward chemical genetics and structure/function screening (5). Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines (dose modifying factors of 1.5). Radiosensitization was NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line (3). Here we show that YTR107 inhibits radiation-induced NPM1 binding to Rad51, Rad 51 foci formation and repair of DNA DSBs.Tumor initiating cells (TICs) were radiosensitized by YTR107. YTR107 inhibited tumor growth (P = 0.027, 2-way ANOVA) and increased the survival of A549 tumor-bearing mice, determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107and 2.2 Gy(q.d. x 7) vs 20% for mice treated with 2.2 Gy alone (q.d.x 7). Cox Proportional log rank hazard ratio for irradiated vs unirradiated tumor-bearing mice was 0.24 (P = 0.0034) while the log rank hazard ratio for YTR107 plus irradiation vs unirradiated (± YTR107) tumor-bearing mice was 0.17 (P = 0.0023). Thus, these data provide support for development of YTR107 as a therapeutic radiation sensitizer. Supported in part by R44CA228756. 1.Koike A, Nishikawa H, Wu W, Okada Y, Venkitaraman AR, Ohta T.Cancer Res 2010;70:6746-56 2. Sekhar KR, Benamar M, Venkateswaran A, Sasi S, Penthala NR, Crooks PA, et al. Int J Radiat Oncol Biol Phys 2014;89:1106-14 3. Sekhar KR, Reddy YT, Reddy PN, Crooks PA, Venkateswaran A, McDonald WH, et al. Clin Cancer Res 2011;17:6490-9 4. Xu R, Yu S, Zhu D, Huang X, Xu Y, Lao Y, et al. Nat Commun 2019;10:3812 5. Reddy YT, Sekhar KR, Sasi N, Reddy PN, Freeman ML, Crooks PA. Bioorg Med Chem Lett 2010;20:600-2 Citation Format: Michael L. Freeman, Geri Traver, Konjeti R. Sekhar, Peter A. Crooks. Radiosensitization by targeting the NPM1/RAD51 axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-003.

Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors.

Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

Inhibition of the DNA damage response to activate immune responses toward tumors.

Cancer immunotherapy represents a very encouraging mode of treatment for cancer where one's immune system is utilized to eliminate tumor cells. Wayne etal. explore inhibition of DNA damage response (DDR) pathways with small molecule inhibitors as a means to prime cells with immune response. These findings suggest that a one-size-fits-all approach cannot be used when harnessing immune response via DDR inhibitors and genotoxic agents, which are required ultimately for the success of immunotherapy. Comment on: https://doi.org/10.1111/febs.15747.

Selected Articles from This Issue

Inhibition of DNA damage response and PI3K signaling has emerged as a potential strategy for treating triple-negative breast cancer (TNBC). Hong and colleagues assessed a CHK1 inhibitor, prexasertib, in combination with a PI3K/mTOR inhibitor, samotolisib, in preclinical models and a phase Ib study. This combination was synergistic or additive in 30 of 38 PDX models examined. In the clinical setting, an overall response rate (ORR) of 15.1% was observed. However, the toxicity profile of this combination limits its utility moving forward. Given the potential clinical benefit of the combined inhibition of CHK1 and PI3K/mTOR, alternative strategies to more safely target these pathways in patients are needed.Although immune checkpoint inhibition has provided clinical benefit to cancer patients, many patients do not ultimately respond to this strategy; thus, new strategies to enhance the efficacy of ICI are needed. Papadopoulos and colleagues performed a phase I study of an anti-GITR monoclonal antibody, MK-4166, as monotherap. and in combination with pembrolizumab in patients with advanced solid tumors. MK-4166 was well tolerated, and a maximum-tolerated dose was not reached. Clinical responses were observed with combination treatment, particularly in patients with ICI-naïve melanoma, with an ORR of 62%. Further study of this combination is warranted, particularly in patients not previously treated with ICI.The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed by tumor-infiltrating lymphocytes (TILs) and has emerged as an attractive target for checkpoint blockade therapies. Shaffer and colleagues developed a novel PET imaging to noninvasively quantify TIGIT expression in the tumor microenvironment. The probe was highly specific for TIGIT expressed by TILs in syngeneic mouse melanoma models as demonstrated with small animal PET imaging, ex vivo flow cytometry, and immunofluorescence microscopy. This TIGIT imaging probe shows promise as the basis of a clinically relevant tool in screening cancer patients and monitoring therapeutic efficacy.Inherited mutations in DNA repair genes commonly are associated with familial cancer. Using MSK-IMPACT, Topka and colleagues identified a subset of patients with cancer-harboring germline or somatic aberrations within the nucleotide excision repair (NER) pathway, including ERCC2 and ERCC3. Cells harboring heterozygous mutations in these genes showed increased sensitivity to irofulven. Synergy was identified between irofulven and several additional therapeutics, including cisplatin. These data suggest that further study of irofulven is warranted, especially in ERCC2/3-mutant patients.

Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer's Achilles' Heel.

Simple SummaryCancer onset and progression lead to a high rate of DNA damage, due to replicative and metabolic stress. To survive in this dangerous condition, cancer cells switch the DNA repair machinery from faithful systems to error-prone pathways, strongly increasing the mutational rate that, in turn, supports the disease progression and drug resistance. Although DNA repair de-regulation boosts genomic instability, it represents, at the same time, a critical cancer vulnerability that can be exploited for synthetic lethality-based therapeutic intervention. We here discuss the role of the error-prone DNA repair, named Alternative Non-Homologous End Joining (Alt-NHEJ), as inducer of genomic instability and as a potential therapeutic target. We portray different strategies to drug Alt-NHEJ and discuss future challenges for selecting patients who could benefit from Alt-NHEJ inhibition, with the aim of precision oncology.Error-prone DNA repair pathways promote genomic instability which leads to the onset of cancer hallmarks by progressive genetic aberrations in tumor cells. The molecular mechanisms which foster this process remain mostly undefined, and breakthrough advancements are eagerly awaited. In this context, the alternative non-homologous end joining (Alt-NHEJ) pathway is considered a leading actor. Indeed, there is experimental evidence that up-regulation of major Alt-NHEJ components, such as LIG3, PolQ, and PARP1, occurs in different tumors, where they are often associated with disease progression and drug resistance. Moreover, the Alt-NHEJ addiction of cancer cells provides a promising target to be exploited by synthetic lethality approaches for the use of DNA damage response (DDR) inhibitors and even as a sensitizer to checkpoint-inhibitors immunotherapy by increasing the mutational load. In this review, we discuss recent findings highlighting the role of Alt-NHEJ as a promoter of genomic instability and, therefore, as new cancer’s Achilles’ heel to be therapeutically exploited in precision oncology.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

SummarySARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Radiotherapy, immunotherapy, and the tumour microenvironment: Turning an immunosuppressive milieu into a therapeutic opportunity.

Immune checkpoint blockade (ICB) has revolutionised the treatment of solid tumours, yet most patients do not derive a clinical benefit. Resistance to ICB is often contingent on the tumour microenvironment (TME) and modulating aspects of this immunosuppressive milieu is a goal of combination treatment approaches. Radiation has been used for over a century in the management of cancer with more than half of all cancer patients receiving radiotherapy. Here, we outline the rationale behind combining radiotherapy with ICB, a potential synergy through mutually beneficial remodelling of the TME. We discuss the pleiotropic effects radiation has on the TME including immunogenic cell death, activation of cytosolic DNA sensors, remodelling the stroma and vasculature, and paradoxical infiltration of both anti-tumour and suppressive immune cell populations. These events depend on the radiation dose and fractionation and optimising these parameters will be key to develop safe and effective combination regimens. Finally, we highlight ongoing efforts that combine radiation, immunotherapy and inhibitors of DNA damage response, which can help achieve a favourable equilibrium between the immunogenic and tolerogenic effects of radiation on the immune microenvironment.

The promise of DNA damage response inhibitors for the treatment of glioblastoma.

Glioblastoma (GBM), the most aggressive primary brain tumor, has a dismal prognosis. Despite our growing knowledge of genomic and epigenomic alterations in GBM, standard therapies and outcomes have not changed significantly in the past two decades. There is therefore an urgent unmet need to develop novel therapies for GBM. The inter- and intratumoral heterogeneity of GBM, inadequate drug concentrations in the tumor owing to the blood–brain barrier, redundant signaling pathways contributing to resistance to conventional therapies, and an immunosuppressive tumor microenvironment, have all hindered the development of novel therapies for GBM. Given the high frequency of DNA damage pathway alterations in GBM, researchers have focused their efforts on pharmacologically targeting key enzymes, including poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, ataxia telangiectasia-mutated, and ataxia telangiectasia and Rad3-related. The mainstays of GBM treatment, ionizing radiation and alkylating chemotherapy, generate DNA damage that is repaired through the upregulation and activation of DNA damage response (DDR) enzymes. Therefore, the use of PARP and other DDR inhibitors to render GBM cells more vulnerable to conventional treatments is an area of intense investigation. In this review, we highlight the growing body of data behind DDR inhibitors in GBM, with a focus on putative predictive biomarkers of response. We also discuss the challenges involved in the successful development of DDR inhibitors for GBM, including the intracranial location and predicted overlapping toxicities of DDR agents with current standards of care, and propose promising strategies to overcome these hurdles.

SAMHD1 can suppress lung adenocarcinoma progression through the negative regulation of STING.

BackgroundThe sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) has been specifically linked to lung cancer. However, the underlying mechanisms in regulating lung adenocarcinoma (LAC) are unclear. The aim of this study was to assess the specific regulation between SAMHD1 and LAC.MethodsWe retrospectively reviewed 238 patients who underwent surgery for LAC between January 2018 and December 2019. The expression of SAMHD1 was detected by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in tumors and paired adjacent tissues. A lentivirus was used to overexpress SAMHD1 and stimulator of interferon genes (STING) in A549 cells; and RT-qPCR and western blot analysis were performed to verify their levels. Cell proliferation was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Celigo imaging cytometry. Cell apoptosis was detected by Annexin V staining. Overexpressed SAMHD1 suppressed LAC progression in a xenograft model. The DNA damage response inhibitor (DDRi) was used to assess the cell proliferation and apoptosis rate in SAMHD1-overexpressing A549 cells and the control group. A rescue experiment was carried out to evaluate the potential influence of SAMHD1 and STING.ResultsA low expression of SAMHD1 was associated with advanced disease. Overexpression of SAMHD1 decreased cell proliferation and invasion in A549 cells, and the apoptosis rate was significantly higher in the overexpressed SAMHD1 cells than those in the control group. The overexpression of SAMHD1 inhibited tumor progression in the xenograft model. The expression of STING was lower in SAMHD1-overexpressing A549 cells than those in the wild-type group. Furthermore, the inhibited cellular behaviors of LAC cells resulting from the stable SAMHD1 expression were partially reversed after STING overexpression. Treatment with DDRi could inhibit cancer cell progression.ConclusionsUpregulation of SAMHD1 could suppress the progression of LAC in vivo and in vitro through the negative regulation of STING.

SLFN11 informs on standard of care and novel treatments in a wide range of cancer models

BackgroundSchlafen 11 (SLFN11) has been linked with response to DNA-damaging agents (DDA) and PARP inhibitors. An in-depth understanding of several aspects of its role as a biomarker in cancer is missing, as is a comprehensive analysis of the clinical significance of SLFN11 as a predictive biomarker to DDA and/or DNA damage-response inhibitor (DDRi) therapies.MethodsWe used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting.ResultsSLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies.ConclusionSLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models.

MODL-02. TARGETING REPLICATION STRESS IN PEDIATRIC BRAIN TUMORS

Pediatric brain tumors harboring amplifications or high overexpression of MYC-/MYCN are often associated with poor outcome. High MYC(N) expression in these tumors leads to increased transcription, which can be in conflict with DNA replication and subsequently can cause replication stress, R-loops and DNA damage. We hypothesize that high MYC(N) expression makes them vulnerable to DNA damage response inhibitors (DDRi) and even more vulnerable to combinations of DDRi and chemotherapeutics. To test this hypothesis we performed in vitro drug experiments using Group 3 medulloblastoma (MB) and ETMR cell lines. IC50-values were evaluated of topoisomerase inhibitor Irinotecan (SN-38) and Pamiparib (BGB-290), a brain-penetrant PARP-inhibitor, in monotherapy. All cell lines were sensitive for SN-38 and showed IC50-values in the low nM-range but PARP-inhibitors were ineffective. However, a significant decrease in IC50 can be observed when SN-38 and Pamiparib are used in combination. For in vivo treatments, we injected NSG mice with luciferase labelled patient-derived xenograft- (PDX-) cells of various models (MB Group 3, MB SHH, ETMR, RELA EPN), monitored tumor growth via IVIS and randomized the mice into four groups (vehicle, BGB-290, Irinotecan and Irinotecan+Pamiparib) when a predefined threshold of tumor growth was reached. Mice were treated with Irinotecan (or vehicle) once per day i.p. and Pamiparib (or vehicle) twice per day per oral gavage. Treatment with Pamiparib did not show any survival benefit, but mice treated with Irinotecan or the combination showed a clear survival benefit. Treatments are ongoing and more results will be presented at the conference.

Cytotoxic and genotoxic effects of epigenetic inhibitors on bladder cancer cells

Urothelial carcinoma (UC) are characterized by frequent mutations and incorrect expression of epigenetic regulator proteins. Thus epigenetic inhibitors (Epi-I) may be suitable as new therapeutic approaches or supplements to standard therapy. Previous work demonstrated additional cytotoxic effects of Epi-I which may result from disturbed DNA synthesis and DNA damage response (DDR). These were systematically investigated to identify options for novel combination therapies. UC cell lines VMCUB-1 and UM-UC-3 were treated with GSK126 (EZH2), GSK-J4 (inhibitor of histone demethylases such as UTX and JMJD3), PLX51107 (bromodomain protein (BET) inhibitor) and the class I HDAC inhibitor romidepsin. In addition to the effects on short and long-term proliferation, effects on cell cycle and apoptosis induction were investigated using flow cytometry and Western blot analyses. Induction of DNA damage and DDR were analysed using immunocytochemistry and Western blot. Synergy of combination treatment was evaluated by the Chou-Talalay method. RNA of UCCs treated with romidepsin and PLX51107 was subjected to RNA sequencing. Normal HBLAK control cells were used as a control. All Epi-I reduced cell viability and colony formation in both cell lines. Cells responded most weakly to GSK126, while PLX51107 had most significant effects on apoptosis induction and regulation. The apparently aneugenic mode of action of GSKJ4 induced mitotic disorders recognizable by accumulation of micronuclei, "lagging chromosomes" and chromosome bridges and altered expression of DDR markers. Although the clastogenic mode of action of romidepsin and PLX51107 resulted in accumulation of DNA double-strand breaks, no activation of checkpoint kinases was found. On the contrary, expression of CHK1 and factors of homologous recombination was downregulated. GSK126 was cytotoxic without induction of DNA damage. Combination treatments of PLX51107 and inhibitors of DDR or chemotherapeutic compounds were highly synergistic with tolerable toxicity in benign control cells. The genotoxic mode of action of Epi-I, particularly of HDAC and BET proteins, enables new approaches for a combination therapy with inhibitors of DNA repair mechanisms or standard chemotherapy.

Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.