DNA Damage Response Inhibitors Research Articles

Radiation in Combination with Immune Checkpoint Blockade and DNA Damage Response Inhibitors in Mice: Dosage Optimization in MC38 Syngeneic Tumors via Modelling and Simulation.

Clinical trials assessing the impact of radiotherapy (RT) in combination with DNA damage response pathway inhibitors (DDRis) and/or immune checkpoint blockade are currently ongoing. However, current methods for optimizing dosage and schedule are limited. A mathematical model was developed to capture the impacts of RT in combination with DDRi and/or anti-PD-L1 [immune checkpoint inhibitor (ICI)] on tumor immune interactions in the MC38 syngeneic tumor model. The model was fitted to datasets that assessed the impact of RT in combination with the DNA protein kinase inhibitor (DNAPKi) AZD7648. The model was further fitted to datasets from studies that were used to assess both RT/ICI combinations as well as RT/ICI combinations followed by concurrent administration of the poly ADP ribose polymerase inhibitor (PARPi) olaparib. Nonlinear mixed-effects modeling was performed followed by internal validation with visual predictive checks (VPC). Simulations of alternative dosage regimens and scheduling were performed to identify optimal candidate dosage regimens of RT/DNAPKi and RT/PARPi/ICI. Model fits and VPCs confirmed a successful internal validation for both datasets and demonstrated very small differences in the median, lower, and upper percentile values of tumor diameters between RT/ICI and RT/PARPi/ICI, which indicated that the triple combination of RT/PARPi/ICI at the given dosage and schedule does not provide additional benefit compared with ICI in combination with RT. Simulation of alternative dosage regimens indicated that lowering the dosage of ICI to between 2 and 4 mg/kg could induce similar benefits to the full dosage regimen, which could be of translational benefit. SIGNIFICANCE STATEMENT: This work provides a mixed-effects model framework to quantify the effects of combination radiotherapy/DNA damage response pathway inhibitors/immune checkpoint inhibitors in preclinical tumor models and identify optimal dosage regimens, which could be of translational benefit.

Suppression of TREX1 deficiency-induced cellular senescence and interferonopathies by inhibition of DNA damage response

TREX1 encodes a major DNA exonuclease and mutations of this gene are associated with type I interferonopathies in human. Mice with Trex1 deletion or mutation have shortened life spans accompanied by a senescence-associated secretory phenotype. However, the contribution of cellular senescence in TREX1 deficiency-induced type I interferonopathies remains unknown. We found that features of cellular senescence present in Trex1-/- mice are induced by multiple factors, particularly DNA damage. The cGAS-STING and DNA damage response pathways are required for maintaining TREX1 deletion-induced cellular senescence. Inhibition of the DNA damage response, such as with Checkpoint kinase 2 (CHK2) inhibitor, partially alleviated progression of type I interferonopathies and lupus-like features in the mice. These data provide insights into the initiation and development of type I interferonopathies and lupus-like diseases, and may help inform the development of targeted therapeutics.

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

Phase I/II study of stereotactic radiosurgery with concurrent olaparib followed by adjuvant durvalumab and physician’s choice systemic therapy in patients with breast cancer brain metastases.

TPS1133 Background: Despite progress in the treatment of brain metastasis for HER2+ breast cancer, outcomes for patients with HER2-negative breast cancer brain metastases remain poor. Current standard of care consists of local therapies, including surgery and radiotherapy, followed by systemic therapy. Preclinical studies show inhibitors of poly(ADP-ribose) polymerase (PARP) are effective in combination with radiation therapy as a DNA damage response inhibitor. Triple-negative breast cancer (TNBC) has higher rates of homologous recombination deficiency compared to other breast cancer subtypes, and together with HER2-negative, BRCA-mutated breast cancer would be particularly sensitive to PARP inhibition. PARP inhibition has also demonstrated promising efficacy combined with immunotherapy in patients with germline BRCA-mutant and metastatic TNBC in clinical trials (MEDIOLA, TOPACIO). In addition, immunotherapy with stereotactic radiosurgery (SRS) is associated with favorable intracranial control and survival in patients with brain metastases. We hypothesize that this biologically-driven combination will enhance local control of SRS-treated brain metastases through synergy with PARP inhibition, while controlling micrometastatic disease in the brain and extracranial sites via potentiation of the immune response. Methods: We are conducting a multi-institution, Phase I/II trial of SRS plus olaparib, followed by durvalumab (with physician’s choice systemic therapy), for patients with TNBC (any BRCA status) or HER2-negative with BRCA-mutated (germline or somatic) breast cancer brain metastases [NCT04711824]. A total of 41 patients are planned for enrollment at 8 sites. The primary objectives are to evaluate safety and tolerability (Phase I) and determine intracranial disease control at 6 months (Phase II) of this treatment combination. Secondary objectives include determining clinical activity via intracranial and global progression-free survival, overall survival, and intracranial and extracranial response rate. Exploratory objectives will assess potential biomarkers of treatment response, including changes in circulating tumor cells and DNA in blood and cerebrospinal fluid, germline and tumor mutations in DNA repair pathway genes, and PD-L1 expression, as well as quality of life and patient-reported outcomes. A surgical sub-study (n=5) will evaluate olaparib concentration/distribution in resected brain metastases. As of January 2023, cohort 1 of phase I has been completed without dose-limiting toxicity. Clinical trial information: NCT04711824 .

Aligning Germline Cancer Predisposition With Tumor-Based Next-Generation Sequencing for Modern Oncology Diagnosis, Interception, and Therapeutic Development.

In the era of precision medicine, genomic interrogation for identification of both germline and somatic genetic alterations has become increasingly important. While such germline testing was usually undertaken via a phenotype-driven single-gene approach, with the advent of next-generation sequencing (NGS) technologies, the widespread utilization of multigene panels, often agnostic of cancer phenotype, has become a commonplace in many different cancer types. At the same time, somatic tumor testing in oncology performed for the purpose of guiding therapeutic decisions for targeted therapies has also rapidly expanded, recently starting to incorporate not just patients with recurrent or metastatic cancer but even patients with early-stage disease. An integrated approach may be the best approach for the optimal management of patients with different cancers. The lack of complete congruence between germline and somatic NGS tests does not minimize the power or importance of either, but highlights the need to understand their limitations so as not to overlook an important finding or omission. NGS tests built to more uniformly and comprehensively evaluate both the germline and tumor simultaneously are urgently required and are in development. In this article, we discuss approaches to somatic and germline analyses in patients with cancer and the knowledge gained from integration of tumor-normal sequencing. We also detail strategies for the incorporation of genomic analysis into oncology care delivery models and the important emergence of poly(ADP-ribose) polymerase and other DNA Damage Response inhibitors in the clinic for patients with cancer with germline and somatic BRCA1 and BRCA2 mutations.

A Very Long-acting Exatecan and Its Synergism with DNA Damage Response Inhibitors.

A circulating conjugate that slowly releases Exa is described. It is efficacious after a single dose and synergistic with ATR and PARP inhibitors.

Targeting Replication Stress and Chemotherapy Resistance with a Combination of Sacituzumab Govitecan and Berzosertib: A Phase I Clinical Trial.

Despite promising preclinical studies, toxicities have precluded combinations of chemotherapy and DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted chemotherapy delivery might enable clinical translation of such combinations. In a phase I trial, we combined sacituzumab govitecan, antibody-drug conjugate (ADC) that delivers topoisomerase-1 inhibitor SN-38 to tumors expressing Trop-2, with ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib. Twelve patients were enrolled across three dose levels. Treatment was well tolerated, with improved safety over conventional chemotherapy-based combinations, allowing escalation to the highest dose. No dose-limiting toxicities or clinically relevant ≥grade 4 adverse events occurred. Tumor regressions were observed in 2 patients with neuroendocrine prostate cancer, and a patient with small cell lung cancer transformed from EGFR-mutant non-small cell lung cancer. ADC-based delivery of cytotoxic payloads represents a new paradigm to increase efficacy of DDR inhibitors. See related commentary by Berg and Choudhury, p. 3557.

SP-0190 Improving radiotherapy-induced immune stimulation by DNA damage response inhibition

SP-0190 Improving radiotherapy-induced immune stimulation by DNA damage response inhibition

PD-0826 Radiosensitization by DNA Damage response inhibitors correlates with Replication stress & HR scores

PD-0826 Radiosensitization by DNA Damage response inhibitors correlates with Replication stress & HR scores

Olaparib and durvalumab in patients with relapsed small cell lung cancer (MEDIOLA): An open-label, multicenter, phase 1/2, basket study

IntroductionPreclinical studies have demonstrated increased efficacy with combined DNA damage response inhibition and immune checkpoint blockade compared with either alone. We assessed olaparib in combination with durvalumab in patients with relapsed small cell lung cancer (SCLC). MethodsPatients with previously treated limited or extensive-stage SCLC received oral olaparib 300 mg twice daily, as run-in for 4 weeks, then with durvalumab (1500 mg intravenously every 4 weeks) until disease progression. Primary endpoints were safety, tolerability, and 12-week disease control rate (DCR). Secondary endpoints included 28-week DCR, objective response rate (ORR), duration of response, progression-free survival, overall survival, change in tumor size, and programmed death-ligand 1 (PD-L1) expression subgroup analyses. ResultsForty patients were enrolled and analyzed for safety; 38 were analyzed for efficacy. Eleven patients (28.9% [90% confidence interval (CI), 17.2–43.3]) had disease control at 12 weeks. ORR was 10.5% (95% CI, 2.9–24.8). Median progression-free and overall survival were 2.4 (95% CI, 0.9–3.0)months and 7.6(95% CI, 5.6–8.8)months, respectively. The most common adverse events (≥40.0%) were anemia, nausea, and fatigue. Grade ≥ 3 adverse events occurred in 32 patients (80.0%). PD-L1 levels, tumor mutational burden, and other genetic mutations were evaluated, but no significant correlations with clinical outcomes wereobserved. ConclusionsTolerability of olaparib with durvalumab was consistent with the safety profile of each agent alone. Although the 12-week DCR did not meet the prespecified target (60%), four patients responded, and median overall survival was promising for a pretreated SCLC population. Further analyses are required to identify patients most likely to benefit from this treatment approach.

Abstract CT268: Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial

Abstract Despite provocative preclinical results, dose-limiting toxicities have precluded rational combinations of cytotoxic chemotherapies that increase DNA damage with DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted delivery of cytotoxic chemotherapy might enable tolerable and active combinations with DDR inhibitors. We conducted a phase I clinical trial combining ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib with sacituzumab govitecan, a trophoblast cell surface antigen 2 (Trop-2) directed antibody drug conjugate (ADC) that delivers high tumoral concentrations of topoisomerase 1 (TOP1) inhibitor SN-38. Depletion of ATR, the main transducer of replication stress is synthetically lethal with double-strand breaks (DSB) generated by TOP1 inhibitors. Patients with DDR gene-mutated or high replication stress solid tumors were enrolled since such tumors are particularly susceptible to ATR inhibition. Primary end point was identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Using 3 + 3 dose escalation, sacituzumab govitecan (8-10 mg/m2, days 1, 8) and berzosertib (140-210 mg/m2, days 2, 9) were administered to 12 patients across three dose levels in 21-day cycles. The combination was well tolerated, with improved safety profile over conventional chemotherapy-based combinations, which allowed dose escalation to the highest planned dose level. There were no dose limiting toxicities. Common treatment-related adverse events (TRAE) were neutropenia (41.7%), diarrhea (50%), and fatigue (50%). Grade 3 TRAEs occurred in 58.3% of patients and included neutropenia (25%) and diarrhea (8.3%). There were no instances of febrile neutropenia or clinically significant grade 4 TRAEs. Pharmacodynamic studies showed evidence of ATR inhibition and enhanced DNA DSB in response to the combination. While no tumor responses were seen in three patients with DDR defects including BRCA1 and ATM mutations, two patients with neuroendocrine prostate cancer, a highly aggressive subtype of prostate cancer, showed partial or metabolic responses. A patient with EGFR-transformed small cell lung cancer (SCLC) also experienced partial response, together yielding objective responses in 3 of 12 evaluable patients (25%). Ongoing phase II expansion cohorts are evaluating efficacy of sacituzumab govitecan 10mg/m2 and berzosertib 210mg/m2 in patients with SCLC, extra-pulmonary small cell cancers, and DDR-mutated solid tumors. ADC-based delivery of cytotoxic payload represents a new therapeutic paradigm to extend the benefit of DDR inhibitors to target replication stress and chemotherapy resistance, with minimal added toxicities. Clinical trial information: NCT04826341 Citation Format: Melissa L. Abel, Nobukyuki Takahashi, Parth Desai, Cody Peer, Christophe Redon, Samantha Nichols, Rasa Vilimas, Min-Jung Lee, Sunmin Lee, Linda Sciuto, Danielle Pinkiert, Meenakshi Shelat, Chante Graham, Seth Steinberg, William D. Figg, Mirit Aladjem, Jane Trepel, Yves Pommier, Anish Thomas. Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT268.

Abstract CT198: Immunomodulatory effects of the ATR inhibitor ceralasertib in a window of opportunity biomarker trial in patients with head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is frequently locally advanced, but has a high risk of recurrence after initial treatment. Immune checkpoint blockade with PD-1/PD-L1 inhibitors has revolutionized treatment in the recurrent setting, but resistance to these therapies frequently occurs. Combination of checkpoint inhibitors with the ATR inhibitor ceralasertib could provide an exciting opportunity, with encouraging clinical activity reported in melanoma and non-small cell lung cancer (Kwon et al. 2022; Kim et al. 2022; Besse et al. OA15.05 IASLC 2022 WCLC). This phase 1b trial provided a unique opportunity to understand the immunomodulatory impact of DNA Damage Response (DDR) inhibitors in a clinical setting. NCT03022409 is an open-label, randomized window of opportunity trial where patients with HNSCC were treated with either the PARP inhibitor olaparib (300mg BID) or ceralasertib (160mg BID) for 9-21 days, prior to definitive surgery (or on-treatment biopsy). 21 patients were randomized; n=12 to ceralasertib and n=9 to olaparib, and ceralasertib data will be presented here. Translational endpoints on frozen and fixed tumor biopsies included spatial pharmacokinetics, pharmacodynamic biomarkers, multiplexed fluorescence of tumor infiltrating immune cells and gene expression panels. Blood assessments included cytokine detection, immune cell phenotyping and gene expression. Primary endpoint analysis utilized a bespoke prognostic immune-focused gene signature and secondary endpoint an IHC immunoscore, both aimed to measure if tumors would shift from a ‘cold’ to ‘hot/active’ immune state. We observed an ‘on-drug’ selective suppression of proliferating Ki67+ T-cells (but not total T-cells) in the tumor microenvironment and periphery, followed by a repopulation and median rebound of 63.8% and 187.5% above baseline levels for peripheral helper (CD4+Ki67+; n=8) and cytotoxic (CD8+Ki67+; n=7) T-cells respectively, when ceralasertib dose stopped. IL-12 plasma cytokine levels dropped on ceralasertib treatment and returned to baseline levels ‘off-drug’ in 8/10 patients. Type-I interferon (IFN1) gene expression associated signatures were also upregulated in PBMCs, suggestive of an immune priming effect. 0/2 and 2/4 patients on ceralasertib met their primary and secondary endpoints respectively, however, interpretation is limited due to small numbers of evaluable patients. 1 patient had a grade 3 serious adverse event of chest pain in the ceralasertib arm. There were no unexpected safety findings for either drug and adverse events were generally low grade. The translational data has generated new insights into the immunomodulatory effect of ceralasertib. Further evaluation of the combination of ceralasertib with immune checkpoint blockade is warranted to explore this novel immune mechanism of action. Citation Format: Gemma N. Jones, Sonia Iyer, Marta Milo, Pei-Jen Lou, Melonie A. Nance, Carlos A. Gomez-Roca, Nathan Standifer, Paola Marco-Casanova, Shaan Gill, Michael Surace, Richard Bystry, Maria Alexandrova, Sophie Willis, Jaime Rodriguez-Canales, Andreas Dannhorn, Bienvenu Loembé, Alan Lau, Natalia Lukashchuk, Elizabeth A. Harrington, Elhan Sanai, Emma Dean, Umamaheswar Duvvuri. Immunomodulatory effects of the ATR inhibitor ceralasertib in a window of opportunity biomarker trial in patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT198.

Abstract 6189: Targeting DNA damage responsive pathways in cancer therapy

Abstract DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results provide a better understanding of DDR pathways, which may facilitate the use of these DDR-targeting agents in cancer therapy. Moreover, To provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting-based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage-signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6189.

Abstract 482: Discovery of a novel and oral CHK1 inhibitor for the treatment of solid tumors

Abstract Genomic instability is a key hallmark of cancer that arises owing to defects in the DNA damage response (DDR), a complex network of genes responsible for sensing and responding to specific types of DNA damage, in which numerous interdependent signaling pathways and machineries are involved. Although deficiencies in these responses promote cancer initiation and progression via accumulated mutations, they also provide targetable vulnerabilities relatively specific to cancer cells that can be exploited with the use of DDR inhibitors. The cell cycle checkpoint kinases CHK1 and CHK2 are prime targets of DDR inhibitors, working with the key upstream regulators of the DDR signaling cascade, ATR and ATM, respectively, to halt cell cycle progression and initiate DNA repair via distinct but overlapping pathways. CHK1 and/or CHK2 inhibitors have been developed in the past. However, many of them were discontinued before making to phase III clinical trials, in most cases owing to severe side effects induced by their non-specific nature. Others are related to their limited clinical efficacy, such as SRA737 which selectively inhibits CHK1 but not CHK2. Prexasertib (LY2606368), one of the few agents to undergo clinical trials, specifically inhibits both CHK1 and CHK2, displaying obvious therapeutic effects but also raising inevitable target-associated drug toxicities and a potential compliance issue due to intravenous administration. Given the fact that CHK2 aids in the coordination with CHK1 pathway, adjustment in the intensity of inhibition to both kinases could provide a suitable balance between efficacy and toxicity. Here, we describe the characterization of XS-02, a novel and orally bioavailable CHK1 inhibitor with moderate potency against CHK2, which shows manageable side effects and broad antitumor activity in various solid tumors, including tumors resistant to PARP inhibitors. XS-02 specifically inhibited both CHK1 and CHK2 kinases in different degree with IC50 values of 2 and 282 nM, respectively. In vitro western blotting assays demonstrated that XS-02 decreased phosphorylation of CHK1 completely but partially for CHK2 in OVCAR3 cancer cells. Its potency against proliferation of cells with low nM IC50 was exhibited in multiple cancer cell lines instead of normal cells. Oral dosing of XS-02 as a single agent induced dose-dependent tumor growth inhibition of OVCAR3 and MD-MBA-436 xenografts. Moreover, the combination of XS-02 and olaparib, a PARP inhibitor, accelerated tumor regression, which synergy was confirmed in a patient-derived tumor xenograft animal model of acquired resistance to olaparib, without significant body weight changes. In vivo assays showed that XS-02 was generally well tolerated with an acceptable oral bioavailability across multiple non-clinical species. These results support the agent as a clinical candidate for the treatment of solid tumors. An Investigational New Drug application is planned for 2023. Citation Format: Xin Gao, Yonggang Liao, Qi Li, Ling Yang, Chunyan Zhao, Yanfen Teng, Lei Liu, Wenyuan Qian, Xiaohong Yu. Discovery of a novel and oral CHK1 inhibitor for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 482.

Abstract 6217: Inhibiting ataxia-telangiectasia mutated and RAD3-related (ATR) by BAY 1895344 overcomes chemoresistance to oxaliplatin and promotes synergistic anti-tumor effect in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis and a limited response to most of the treatments. Despite a platinum-based drug such as oxaliplatin or cisplatin is one of the most effective chemotherapy drugs for PDAC, resistance to it is a major limiting factor in PDAC treatment, indicating an urgent need for new approaches. Recently, targeting major DNA damage response (DDR) regulators such as ATM (Ataxia-telangiectasia mutated) or ATR (Ataxia telangiectasia mutated and Rad3-related) kinase has shown therapeutic potential in cancer treatment. This shows that it may be possible to enhance the responsiveness of platinum medicines via a DDR inhibition strategy. The most recently developed ATR inhibitor with the greatest potency, BAY 1895344, showed an anti-proliferative effect in clinical trials. Here, we aimed to evaluate the effect of ATR inhibition using BAY 1895344 on responsiveness to oxaliplatin in pancreatic cancer, for the first time. CFPAC-1 and Capan-2 are selected among six kinds of pancreatic cancer cell lines as oxaliplatin-sensitive and -resistant cells, respectively. According to BRAID analysis, combining the BAY 1895344 and oxaliplatin resulted in strong synergistic effects in both cell lines, particularly in Capan-2. The synergism is also confirmed in all four organoids derived from PDAC patients. We found that p-Chk1, coordinating DDR and cell cycle checkpoint, was considerably suppressed by the combined treatments, which was associated with elevated γ-H2AX intensity, cell cycle arrest and apoptosis. Moreover, we investigated the in vivo synergistic anti-tumor efficacy of combination therapy using a tumor-bearing nude mice model with CFPAC-1 and Capan-2 cells, demonstrating a substantial reduction of tumor growth in combination therapy when compared to single treatment. In conclusion, ATR inhibition enhanced the anticancer effect of oxaliplatin, and this combined therapeutic strategy may be effective in overcoming chemo-resistance in PDAC. (This study is supported by National Cancer Center, Republic of Korea (No. 2212470, 2010330)) Citation Format: Hye Won Shon, Jung Won Chun, Jeong Eun Gong, Mi Rim Lee, Yu-Sun Lee, Sumin Kang, Sunshin Kim, Sang Myung Woo, In Rae Cho, Woo Hyun Paik, Woo Jin Lee, Ji Kon Ryu, Yong-Tae Kim, Sang Hyub Lee, Yun-Hee Kim. Inhibiting ataxia-telangiectasia mutated and RAD3-related (ATR) by BAY 1895344 overcomes chemoresistance to oxaliplatin and promotes synergistic anti-tumor effect in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6217.

ATM inhibition enhances the efficacy of radiation across distinct molecular subgroups of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.

ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias

Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1−/− murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1−/− cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1−/− and NUP98-PMX1;Abl1−/− cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors.

Changes in the Proteome Profile of A549 Cells Following Helichrysetin-Induced Apoptosis Suggest the Involvement of DNA Damage Response and Cell Cycle Arrest-Associated Proteins.

Our previous findings demonstrated that Helichrysetin possessed promising anti-cancer activity. It was able to induce apoptosis in the A549 cell line. However, its mechanism of action is unknown. The present study aimed to unravel possible underlying molecular mechanisms of helichrysetin-induced apoptosis in A549 (human lung carcinoma) cells using comparative quantitative proteomics (iTRAQ labeled), followed by an exhaustive bioinformatics analysis. Our results suggested that DNA damage response (DDR) and cell cycle arrest were responsible for lung cancer cell death with helichrysetin treatment. Among proteins that changed in abundance were Nrf2 and HMOX1. They are oxidative stress-related proteins and were increased in abundance. BRAT1 was also increased in abundance, suggesting an increase in DNA damage repair, indicating the occurrence of DNA damage due to oxidative stress. However, several essential DDR downstream proteins such as p-ATM, BRCA1, FANCD2, and Rb1 that would further increase DNA damage were found to be dramatically decreased in relative abundance. Cell cycle-related proteins, p53, p21, and cyclin D1, were increased while cyclin A, cyclin E, and cdk2 were decreased. This is predicted to facilitate S-phase arrest. Furthermore, excessive DNA damage and prolonged arrest would in turn result in the induction of mitochondrial-mediated apoptosis. Based on these observations, we postulate that the effects of helichrysetin were in part via the suppression of DNA damage response which led to DNA damage and prolonged cell cycle arrest. Subsequently, this event initiated mitochondrial-mediated apoptosis in A549 lung cancer cells.

Morusinol extracted from Morus alba induces cell cycle arrest and apoptosis via inhibition of DNA damage response in melanoma by CHK1 degradation through the ubiquitin-proteasome pathway

BackgroudFlavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear. PurposeIn present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma. MethodsThe anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The ɣ-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity. ResultsWe demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly, CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo. ConclusionMorusinol induces CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.

DNA protein kinase promotes cellular senescence in dental follicle cells

ObjectiveShort telomeres and genomic DNA damage are causes of cellular senescence in dental follicle cells (DFCs). DesignThis study examined the role of the DNA damage response (DDR) during cellular senescence of DFCs by β-galactosidase activity and DNA damage by comet assay. Expression of genes/proteins was determined by Western Blots and reverse transcription-quantitative polymerase chain reaction, while glycolysis was enzymatically estimated. Cell cycle stages and reactive oxygen species (ROS) were investigated by flow cytometry. ResultsDuring the induction of cellular senescence gene expression of DDR genes were down-regulated, while DNA double-strand breaks occurred at the same time. Furthermore, inhibition of DNA protein kinase (DNA-PK) reduced senescence and ROS, both of which are associated with cellular senescence. In contrast, while these data suggest that inhibition of DDR is associated with the induction of cellular senescence, inhibition of DNA-PK did not result in renewal of DFCs, as inhibition resulted in typical features of depleted cells such as increased cell size and reduced cell proliferation rate. DNA-PK repression inhibited both osteogenic differentiation potential and glycolysis, which are typical features of cellular exhaustion. Moreover, DNA-PK affects cellular senescence via activation of AKT1 (protein kinase B). ConclusionOur results suggest that DNA-PK promotes cellular senescence, but DFCs may control the induction of cellular senescence via down-regulation of DDR genes. However, we also showed that inhibition of DNA-PK cannot renew senescent DFCs