Loss Of Ataxia Telangiectasia Mutated Research Articles

Dual inhibition of ATR and DNA-PKcs radiosensitizes ATM-mutant prostate cancer.

In advanced castration resistant prostate cancer (CRPC), mutations in the DNA damage response (DDR) gene ataxia telangiectasia mutated ( ATM ) are common. While poly(ADP-ribose) polymerase inhibitors are approved in this context, their clinical efficacy remains limited. Thus, there is a compelling need to identify alternative therapeutic avenues for ATM mutant prostate cancer patients. Here, we generated matched ATM-proficient and ATM-deficient CRPC lines to elucidate the impact of ATM loss on DDR in response to DNA damage via irradiation. Through unbiased phosphoproteomic screening, we unveiled that ATM-deficient CRPC lines maintain dependence on downstream ATM targets through activation of ATR and DNA-PKcs kinases. Dual inhibition of ATR and DNA-PKcs effectively inhibited downstream γH2AX foci formation in response to irradiation and radiosensitized ATM-deficient lines to a greater extent than either ATM-proficient controls or single drug treatment. Further, dual inhibition abrogated residual downstream ATM pathway signaling and impaired replication fork dynamics. To circumvent potential toxicity, we leveraged the RUVBL1/2 ATPase inhibitor Compound B, which leads to the degradation of both ATR and DNA-PKcs kinases. Compound B effectively radiosensitized ATM-deficient CRPC in vitro and in vivo , and impacted replication fork dynamics. Overall, dual targeting of both ATR and DNA-PKcs is necessary to block DDR in ATM-deficient CRPC, and Compound B could be utilized as a novel therapy in combination with irradiation in these patients.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

ATM deficiency confers specific therapeutic vulnerabilities in bladder cancer.

Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair-targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer.

Investigating the Influence of Germline ATM Variants on Susceptibility to Chronic Lymphocytic Leukemia and Other Malignancies

Introduction: Chronic lymphocytic leukemia (CLL) patients (pts) face an increased susceptibility to secondary cancers. Furthermore, the hereditary predisposition to CLL is influenced by factors that have yet to be fully elucidated by GWAS-identified common variants. Among the hereditary cancer risk mutations observed, germline Ataxia-telangiectasia mutated ( ATM) aberrations are among the most prevalent. Rare germline ATM variants are detected in 24% of CLL pts, surpassing the frequency observed in other hematologic neoplasms and the general population. In CLL, loss of ATM via 11q23 deletion is linked to an unfavorable prognosis and a shortened progression-free survival. The extent to which germline ATM variants predispose CLL pts to other malignancies remains unclear; to examine this potential risk we conducted a retrospective study to assess the influence of germline ATM variants on the predisposition to secondary neoplasms in CLL pts and their relatives. Methods: Five hundred and eighty-five pts seen at Dana-Farber Cancer Institute and who had NGS performed to evaluate germline ATM status, either through direct germline sequencing of saliva or by inference according to the hierarchical algorithm we have previously published (Lampson, 2022), were mailed a questionnaire that investigated: demographics; personal and family history of any cancer; non-medical radiation and Agent Orange exposure; and Ataxia-Telangiectasia syndrome (AT). Of these, 333 replied (57%). Information collected from our CLL database included FISH, IGHV status and treatment status. European ancestry was categorized based on the United Nations geoscheme (UNSD, 2019). Pt characteristics are in Table 1. Pts were stratified into 4 groups, based on ATM mutational status and del(11q): Group-1, ATM germline variants alone (N=63); Group-2, ATM germline with somatic ATM mutation and/or del(11q)(N=22); Group-3, ATM somatic aberration alone (including del(11q))(N=41); and Group-4, no ATM aberration (N=207). Results: Of the 333 pts in our cohort, baseline demographic and clinical characteristics among the 4 groups were balanced, except for unmutated IGHV status. Nineteen (86%) and 36 (86%) pts of groups 2 and 3, carrying somatic ATM aberrations, had unmutated IGHV, compared to 21 (33%) and 76 (37%) of groups 1 and 4, without somatic ATM aberrations(p<0.01). Regarding treatment, 60% of our cohort had received at least one line of therapy (median: 1 (1-7)). Among the 164 pts (49%) with history of an additional non-CLL neoplasm, 221 cancers were reported. Excluding pts with only non-melanoma skin cancers, the incidence is reduced to 31% (104 pts). The median number of second cancers per pt is 1 (1-4). Of the 134 pts with available age of onset data for the secondary malignancy, 80 (60%) were diagnosed with the secondary cancer after CLL. Comparing pts with germline ATM variants (N=85) to the ones without germline ATM variants (N=248), no significant difference in the incidence of secondary cancer was found (p=0.73). The median age of secondary cancer onset was also similar between the groups (median: 63 and 64 years in germline ATM present vs absent, p=0.28). Of the whole cohort, 283 pts (85%) reported at least one relative with a cancer history (median 2 relatives affected (1-17)). The incidence of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in pts with germline ATM variants (32%) compared to those without germline ATM variants (21%), including familial CLL (25% vs18%) (p=0.04). Familial CLL was also strongly associated with Ashkenazi Jewish ethnicity (39% incidence in Ashkenazi pts vs 16% in non-Ashkenazi, p=0.004). No difference in the incidence of other hematologic or solid malignancies was found between the relatives in the two groups. No pts reported a familial or personal history of AT. Compared to pts without any ATM aberration, time to first treatment (TTFT) was shorter in pts harboring somatic ATM events, while no difference was observed between pts with germline ATM variants (median TTFT: 82, 59, 52, 90 months for group 1, 2, 3 and 4 respectively, p=0.01) (Figure 1). Conclusions: Our results suggest a higher incidence of B-cell lymphoproliferative disorders, including familial CLL, in the relatives of CLL pts carrying germline ATM variants. The presence of these germline variants did not impact TTFT, compared to pts harboring somatic ATM mutations.

ATM Mutations Associate with Distinct Co-Mutational Patterns and Therapeutic Vulnerabilities in NSCLC.

Ataxia-telangiectasia mutated (ATM) is the most frequently mutated DNA damage repair gene in non-small cell lung cancer (NSCLC). However, the molecular correlates of ATM mutations and their clinical implications have not been fully elucidated. Clinicopathologic and genomic data from 26,587 patients with NSCLC from MD Anderson, public databases, and a de-identified nationwide (US-based) NSCLC clinicogenomic database (CGDB) were used to assess the co-mutation landscape, protein expression, and mutational processes in ATM-mutant tumors. We used the CGDB to evaluate ATM-associated outcomes in patients treated with immune checkpoint inhibitors (ICI) with or without chemotherapy, and assessed the effect of ATM loss on STING signaling and chemotherapy sensitivity in preclinical models. Nonsynonymous mutations in ATM were observed in 11.2% of samples (2,980/26,587) and were significantly associated with mutations in KRAS, but mutually exclusive with EGFR (q < 0.1). KRAS mutational status constrained the ATM co-mutation landscape, with strong mutual exclusivity with TP53 and KEAP1 within KRAS-mutated samples. Those ATM mutations that co-occurred with TP53 were more likely to be missense mutations and associate with high mutational burden, suggestive of non-functional passenger mutations. In the CGDB cohort, dysfunctional ATM mutations associated with improved OS only in patients treated with ICI-chemotherapy, and not ICI alone. In vitro analyses demonstrated enhanced upregulation of STING signaling in ATM knockout cells with the addition of chemotherapy. ATM mutations define a distinct subset of NSCLC associated with KRAS mutations, increased TMB, decreased TP53 and EGFR co-occurrence, and potential increased sensitivity to ICIs in the context of DNA-damaging chemotherapy.

Germline ATM Mutations Detected by Somatic DNA Sequencing in Lethal Prostate Cancer

BackgroundGermline mutations in the ataxia telangiectasia mutated (ATM) gene occur in 0.5–1% of the overall population and are associated with tumour predisposition. The clinical and pathological features of ATM-mutated prostate cancer (PC) are poorly defined but have been associated with lethal PC. ObjectiveTo report on the clinical characteristics including family history and clinical outcomes of a cohort of patients with advanced metastatic castration-resistant PC (CRPC) who were found to have germline ATM mutations after mutation detection by initial tumour DNA sequencing. Design, setting, and participantsWe acquired germline ATM mutation data by saliva next-generation sequencing from patients with ATM mutations in PC biopsies sequenced between January 2014 and January 2022. Demographics, family history, and clinical data were collected retrospectively. Outcome measurements and statistical analysisOutcome endpoints were based on overall survival (OS) and time from diagnosis to CRPC. Data were analysed using R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria). Results and limitationsOverall, seven patients (n = 7/1217; 0.6%) had germline ATM mutations detected, with five of them having a family history of malignancies, including breast, prostate, pancreas, and gastric cancer; leukaemia; and lymphoma. Two patients had concomitant somatic mutations in tumour biopsies in genes other than ATM, while two patients were found to carry more than one ATM pathogenic mutation. Five tumours in germline ATM variant carriers had loss of ATM by immunohistochemistry. The median OS from diagnosis was 7.1 yr (range 2.9–14 yr) and the median OS from CRPC was 5.3 yr (range 2.2–7.3 yr). When comparing these data with PC patients sequenced by The Cancer Genome Atlas, we found that the spatial localisation of mutations was similar, with distribution of alterations occurring on similar positions in the ATM gene. Interestingly, these include a mutation within the FRAP-ATM-TRRAP (FAT) domain, suggesting that this represents a mutational hotspot for ATM. ConclusionsGermline ATM mutations are rare in patients with lethal PC but occur at mutational hotspots; further research is warranted to better characterise the family histories of these men and PC clinical course. Patient summaryIn this report, we studied the clinical and pathological features of advanced prostate cancers associated with germline mutations in the ATM gene. We found that most patients had a strong family history of cancer and that this mutation might predict the course of these prostate cancers, as well as response to specific treatments.

Abstract 6180: Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations

Abstract Introduction: The ataxia telangiectasia and RAD3-related (ATR) kinase is a key component of the DNA damage response (DDR) and functions in conjunction with ataxia telangiectasia mutated (ATM). ATM loss or functional deficiency may enhance reliance on ATR signaling. Preclinical and clinical studies have pointed to a potential synthetic lethality of ATR inhibition and ATM loss. In this study, we sought to further elaborate potential candidates for ATR inhibition by testing the anti-tumor effect of elimusertib (BAY 1895344) in patient-derived tumor xenograft (PDX) models with DDR alterations including ATM loss. Methods: Patient derived xenograft (PDX) models were implanted into athymic nu/nu mice. Once tumors reached an appropriate size of approximately 200-400mm3 treatments were started. Mice were randomized (N= 3-8) to different treatment arms for assessment of anti-tumor activity of elimusertib as monotherapy and in combination therapy with copanlisib. Monotherapy treatment was performed with 20 mg/kg and 40 mg/kg doses, applied twice daily for 3 days with 4 days treatment break (BID 3 days on/4 days off). Efficacy read-out was either Event-Free Survival (EFS-2), defined as time for doubling of tumor volume relative to baseline, or response based on RECIST criteria:(partial response [PR]= &amp;gt;30% decrease, stable disease [SD]= between 20% increase and 30% decrease, and progressive disease [PD]= &amp;gt;20% increase). For assessment of pharmacodynamic effects, tumors were harvested 10 days after treatment start and analyzed by immunohistochemistry (IHC) and RPPA (Reverse Phase Proteomics Array). Results: Elimusertib was tested in 21 PDX models with various DDR alterations. Of those 11 showed a statistically significant prolongation of event free survival compared to control. In 4 PDX models elimusertib reached a partial response (PR), and another 4 models showed stable disease (SD). Amongst the 5 models with ATM protein Ioss (IHC), one was PR and another one SD. In 3 of 5 PARP inhibitor resistant there was statistically significant prolongation of EFS upon elimusertib treatment vs control. We found an increase in DNA damage in tumors, as indicated by increased level of ƳH2AX (on IHC and RPPA). We also detected an increase in PI3K/mTOR pathway signaling as determined by p-MTOR and pS6 (RPPA) in 2 of 4 models (PR and PD model) with different sensitivity to ATRi and PARPi alone. We tested the combination of elimusertib with the PI3K inhibitor copanlisib achieved a statistically significant increase of EFS compared to monotherapy in 3 of 11 models tested. Conclusion ATR inhibition shows potent monotherapy activity in selected PDX models that harbor DDR defects, including models with intrinsic and acquired PARPi resistance. Further work is needed to identify markers predicting sensitivity or resistance that providing rationale for synergistic combination therapies. Citation Format: Kaushik Varadarajan, Christian X. Pico, Kurt Evans, Maria G. Raso, Yasmeen Rizvi, Xiaofeng Zheng, Timothy P. Diperi, Bailiang Wang, Stephen Scott, Ming Zhao, Argun Akcakanat, Antje M. Wengner, Timothy A. Yap, Funda Meric-Bernstam. Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations. [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 6180.

Abstract 2405: Delineating molecular vulnerabilities of ATM mutant prostate cancers

Abstract Background: Mutations in DNA Damage Response (DDR) genes, including Ataxia, Telangiectasia, Mutated (ATM), are common in advanced castration-resistant prostate cancers (CRPCs). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in DDR mutant CRPCs, but demonstrate limited clinical efficacy in CRPCs with ATM mutations. In this project, we sought to specifically define the impact of ATM loss on DDR pathways in CRPC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO CRPC cell lines were generated via CRISPR-Cas9 mediated knockout. ATM loss and abolishment of downstream ATM kinase activity was confirmed via western blot. We performed an unbiased phospho-proteomic evaluation of DDR pathways in parental and ATM-KO cells after ionizing radiation (IR). Clonogenic survival assays were performed with either an ATR inhibitor (VX970), the selective DNA-PKcs inhibitor (M3814), or combination therapy. Kinetics of DDR protein recruitment and resolution were interrogated with immunofluorescence (IF) staining for γH2ax, 53BP1, MDC1, and Rad51 foci. Results: ATM-KO cells were able to effectively repair DNA damage following IR, as measured by recruitment and resolution of γH2ax, 53BP1, MDC1, and Rad51 foci. Phospho-proteomic studies demonstrated that ATM-KO cells maintain canonical DDR pathways through ATR and DNA-PKcs kinase activation. Treatment of ATM-KO cells with either VX-970 or M3814 only incrementally affected DDR in ATM-KO cells compared to parental controls, as evidenced by clonogenic survival assays and maintenance of DDR foci. Importantly, combination treatment with VX-970 and M3814 prevented downstream DDR foci recruitment and radio-sensitized ATM-KO CRPC to a greater extent than parental controls. This suggested that activity of any of the trinity of kinases is sufficient to mediate DDR, and that blockade of both ATR and DNA-PKcs is required to effectively prevent DDR in ATM-KO CRPC. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which significantly attenuates levels of these kinases in lung cancer cells. We confirmed that Compound B treatment attenuated ATR and DNA-PKcs protein expression and kinase activity in ATM-KO CRPC cells, and demonstrated sensitivity of ATM-KO cells to Compound B. Conclusions: Our data demonstrates that dual targeting of ATR and DNA-PKcs is necessary in ATM-KO CRPC, as either kinase is independently capable of mediating DDR following IR. Our initial studies indicate that the RUVBL1 ATPase inhibitor Compound B may effectively block DDR in ATM-mutant CRPC, and could be utilized as a novel therapeutic strategy in this molecular subtype. Citation Format: Mia Hofstad, Lan Yu, Andrea Woods, Zoi Sychev, Collin Gilbreath, Xiaofang Huo, Ralf Kittler, Justin M. Drake, Ganesh V. Raj. Delineating molecular vulnerabilities of ATM mutant prostate cancers [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 2405.

Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC

Abstract Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for &amp;gt;2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [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 6192.

ATM deficiency aggravates the progression of liver fibrosis induced by carbon tetrachloride in mice

Ataxia telangiectasia mutated (ATM) is a pivotal sensor during the DNA damage response that slows cell passage through the cell cycle checkpoints to facilitate DNA repair, and liver fibrosis is an irreversible pathological consequence of the sustained wound-healing process, However, the effects of ATM on the development of liver fibrosis are still not fully understood. Therefore, the aim of the study was to investigate the effects and potential mechanisms of ATM on the progression of liver fibrosis. Wild-type and ATM-deficient were administered with carbon tetrachloride (CCl4, 5 ml/kg, i.p.) for 8 weeks to induce liver fibrosis, and the liver tissues and serum were collected for analysis. KU-55933 (10 μM) was used to investigate the effects of ATM blockage on CCl4-induced hepatocyte injury in vitro. The results showed that ATM deficiency aggravated the increased serum transaminase levels and liver MDA, HYP, and 8-OHdG contents compared with the model group (p < 0.05). Sirius red staining showed that ATM deficiency exacerbated liver collagen deposition in vivo, which was associated with the activation of TGF-β1/Smad2 signaling. Furthermore, blocking ATM with KU-55933 exacerbated the production of ROS and DNA damage caused by CCl4 exposure in HepG2 cells, and KU-55933 treatment also reversed the downregulated expression of CDK1 and CDK2 after CCl4 exposure in vitro. Moreover, the loss of ATM perturbed the regulation of the hepatic cell ChK2-CDC25A/C-CDK1/2 cascade and apoptosis in vivo, which was accompanied by increased Ki67-positive and TUNEL-positive cells after chronic CCl4 treatment. In conclusion, our results indicated that ATM might be a critical regulator of liver fibrosis progression, and the underlying mechanisms of exacerbated liver fibrosis development in ATM-deficient mice might be associated with the dysregulation of hepatic cell proliferation and apoptosis.

ATM inhibition drives metabolic adaptation via induction of macropinocytosis.

Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.

Abstract 9727: Reduced Bmpr2 Causes a Decrease in Foxf1 That Links Unrepaired DNA Damage to Persistent Pulmonary Hypertension

Introduction: B MPR2 mutation is the most common genetic cause of pulmonary arterial hypertension (PAH). Loss of BMPR2 causes pulmonary arterial (PA) endothelial cell (EC) dysfunction and impairs EC-mediated suppression of smooth muscle cells proliferation. These are characteristic features of PAH vascular cells as is the presence of DNA damage that can be induced by oxidative stress. We previously showed that EC- Bmpr2 -/- mice have persistent pulmonary hypertension (PH) with reoxygenation after hypoxia. However, the role of DNA damage in causing persistent PH related to loss of BMPR2 and oxidative stress is unknown. Hypothesis: We hypothesize that loss of BMPR2 causes unrepaired DNA damage and persistent PH by altering the expression of genes that maintain EC homeostasis. Methods and Results: DNA damage assessed by γH2AX foci was observed in PAEC of EC- Bmpr2 -/- mice in room air, that was more extensive after reoxygenation (10% hypoxia for 3 weeks followed by recovery in room-air for 4 weeks). Deletion of the DNA damage sensor ataxia-telangiectasia mutated (ATM) in EC of mice (EC- Atm -/- ) also resulted in persistent DNA damage and PH. RNA sequencing of PAEC after reoxygenation in EC- Bmpr2 -/- and EC- Atm -/- mice vs. controls showed downregulated EC differentiation and development genes enriched in biding motifs for Foxf1, a transcription factor associated with DNA repair and angiogenesis. Foxf1 expression was reduced in PAEC with loss of BMPR2 or ATM. Loss of Foxf1 decreased angiogenic genes Cldn5 and Vegfr2 , and DNA damage response genes Atm and p53 , and impaired PAEC migration and tube formation. Foxf1 overexpression increased these genes in PAEC. Immunohistochemistry showed decreased FOXF1 in PAEC of human pulmonary vascular lesions. Delivery of Foxf1 to EC- Bmpr2 -/- mice with an AAV2-peptide targeting the PA endothelium attenuated DNA damage in PAEC, restored angiogenesis genes and completely reversed persistent PH returning right ventricular systolic pressure to normal levels. Conclusions: We link loss of BMPR2 and DNA damage sensing in PAEC to persistent DNA damage and PAH and show that both are mediated by reduced FOXF1 . In EC- Bmpr2 -/- mice, Foxf1 gene therapy attenuated DNA damage restored angiogenesis genes, and prevented persistent PH.

Abstract CT006: Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects

Abstract Introduction: ATR kinase is a critical component of DDR machinery and is activated by DNA damage or replication stress. Elimusertib is a selective ATR inhibitor with promising antitumor activity in pts with advanced solid tumors with ataxia telangiectasia mutated (ATM) protein loss and/or ATM putative deleterious alterations (Yap et al. Cancer Discov 2021). We report here the safety and efficacy of elimusertib in expansion cohorts of pts with a range of cancer types and DDR deficiencies and/or ATM loss. We also explored an alternative dose schedule in pts with ATM aberrations. Methods: Pts with advanced solid tumors resistant or refractory to standard treatment were screened for pathogenic DDR defects by next-generation sequencing or ATM protein loss by immunohistochemistry (IHC) analysis of baseline tumor tissue. Pts were assigned to cohorts: colorectal cancer (CRC); castration-resistant prostate cancer (CRPC); HER2− breast cancer (BC); gynecologic (GYN, mainly ovarian and endometrial); and advanced cancers with ATM IHC loss. Pts were treated with elimusertib 40 mg twice daily (BID) 3 days on/4 days off (3 on/4 off). A schedule of 3 on/11 off was also explored by dose escalation in pts with ATM loss or inactivating mutation. Results: 143 pts received ≥1 dose of elimusertib 40 mg BID 3 on/4 off, including 24 CRC, 19 CRPC, 19 BC, 45 GYN, and 36 ATM loss. 56% of pts had ≥4 prior therapy lines. Drug-related grade 3 and 4 treatment-emergent adverse events (TEAEs) were observed in 69% and 15% of pts, respectively, mainly anemia, leukopenia/neutropenia, and thrombocytopenia leading to dose modification but not withdrawal. 32 pts were treated 3 on/11 off with doses from 60 to 120 mg BID. Compared with 3 on/4 off, drug-related grade ≥3 hematologic TEAEs and dose modifications improved at lower doses, with 80 mg 3 on/11 off determined as the recommended dose. In pts receiving elimusertib 3 on/4 off, 5 RECIST partial responses were observed: 1 in ovarian (BRCA1 heterozygous mutation), 1 in BC (BRCA2 mutation), and 3 in ATM IHC loss: 2 CRPC pts both with ATM mutations (1 heterozygous, 1 homozygous), including 1 with FANCA mutation; 1 esophageal with ATM mutation. In pts with ATM IHC loss, objective response rate (ORR) was 9% and disease control rate (DCR) was 65%. A similar ORR was seen on 3 on/11 off in pts with ATM aberrations. In GYN pts, ORR was 2.3% and DCR was 73%. Conclusions: Elimusertib monotherapy demonstrated clinical activity in pts with DDR defects. Overall safety was manageable and hematologic toxicity improved on the 3 on/11 off schedule. Further biomarker analysis is underway to identify potential gene signatures associated with response. Clinical development of elimusertib in combination with checkpoint inhibitors and chemotherapy is ongoing (NCT04095273, NCT04514497). Citation Format: Timothy A. Yap, David S. Tan, Anastasios Stathis, Geoffrey I. Shapiro, Satoru Iwasa, Markus Joerger, Jingsong Zhang, Ruth Plummer, Michael Sawyer, Aaron C. Tan, Vincent Castonguay, Nashat Gabrail, Nobuaki Matsubara, Gary Wilkinson, Matthias Ludwig, Yinghui Zhou, Claudia Merz, Joseph Hreiki, Neelesh Sharma, Johan deBono. Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT006.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins’ expression. Additionally, ATM expression suppressed cystathionine β-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Abstract P054: RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key mediator of cellular DNA damage repair (DDR) and is activated in response to DNA replication stress. ATR is attractive as a drug target in tumors with loss-of-function alterations in complimentary DDR pathways, including ataxia-telangiectasia mutated (ATM) and BRCA. RESULTS: RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor. RP-3500 is highly potent with ATR kinase IC50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. It is highly selective with &amp;gt;30-fold selectivity over mTOR and &amp;gt;2000-fold selectivity over ATM, DNA-PK and PI3Kα kinases. Preclinical tumor xenograft models harboring synthetic lethal (SNIPRx) gene mutations whose loss of function sensitizes to RP-3500 were selected for in vivo studies. RP-3500 treatment resulted in potent single-agent in vivo efficacy and/or tumor regression in multiple models at minimum effective doses (MED) of 5–7 mg/kg once daily. Pharmacodynamic assessments validated target engagement, with a proportional relationship between tumor pCHK1(Ser345) inhibition and circulating RP-3500 plasma levels (IC80 = 18.6 nM). Circulating free plasma levels of RP-3500 at the MED indicate that exposure above the in vivo tumor pCHK1(Ser345) IC80 for 10–12 hours is sufficient for efficacy and dose proportional phosphorylation of DNA damage markers γ-H2AX, pDNA-PKcs and pKAP1. In vitro, tumor cells with ATM loss exhibited increased susceptibility to RP-3500 and a 3-day compound exposure was sufficient to generate a sustained DNA damage response compared to cells with functional ATM expression. In ATM-deficient mouse models, short-duration intermittent (weekly 3 days on/4 days off or 5 days on/2 days off) dosing schedules maximized tumor growth inhibition while minimizing the impact on hematology parameters, including red blood cell depletion. These results emphasize the reversible nature of erythroid toxicity with RP-3500 and the advantage of intermittent dosing schedules to alleviate anemia. The 3 days on/4 days off intermittent treatment schedule also substantially improved the efficacy and tolerability of RP-3500 and PARP inhibitor combinations compared to continuous treatment schedules. Intermittent treatments of RP-3500 given concomitantly with reduced doses of olaparib or niraparib demonstrated synergistic efficacy in Granta-519 (ATM mutn) and SUM149PT (BRCA1mutn) models with minimal hematological adverse effects and superior efficacy to sequential treatment. CONCLUSIONS: These results provide a strong preclinical rationale to support clinical investigation of the novel ATR inhibitor RP-3500 on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a means of maximizing clinical benefit. RP-3500 is currently evaluated in the ongoing phase 1 TRESR (Treatment Enabled by SNIPRx) study (NCT04497116). Citation Format: Anne Roulston, Michal Zimmerman, Robert Papp, Alex Skeldon, Charles Pellerin, Émilie Dumas-Bérube, Valerie Dumais, Stephane Dorich, Sara Fournier, Li Li, Marie-Ève Leclaire, Shou Yun Yin, Amandine Chefson, Hunain Alam, William Yang, Chloe Fugère-Desjardins, Sabrina Hammond, Kathryn Skorey, Amina Mulani, Victoria Rimkunas, Artur Veloso, Martine Hamel, Rino Stocco, Yael Mamane, Zuomei Li, Jordan Young, Mike Zinda, Cameron Black. RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P054.

Cellular functions of the protein kinase ATM and their relevance to human disease

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.

HGG-16. DISSECTING THE EFFECT OF ATM DELETION ON RADIOSENSITIVITY IN DIFFUSE MIDLINE GLIOMAS WITH H3K27M MUTATION

Abstract Diffuse midline gliomas (DMGs) are responsible for a large proportion of childhood brain tumor deaths. Currently, radiation therapy is thought to be one of the most effective treatment options, but more than 90% of children still die within 2 years of diagnosis. DMGs are defined by somatic histone 3 K27M (H3K27M) mutations that have been shown to promote the G0/G1 to S cell cycle transition. The majority of DMGs also contain loss-of-function mutations in TP53. Prior research demonstrated that orthotopic xenograft and primary mouse models of non-H3K27M-mutated gliomas with inactivation of p53 are preferentially radiosensitized by inactivation of Ataxia Telangiectasia Mutated (ATM), a kinase that mediates DNA repair in response to DNA damage caused by radiation. The high frequency of mutations that deregulate p53 in DMGs raises the possibility that H3K27M-mutant DMGs may also be radiosensitized by ATM inhibition, representing a unique therapeutic opportunity. Here, we hypothesize that H3K27M-mutant DMGs that have loss of function of p53 will be radiosensitized by loss of ATM. To test this hypothesis, we used the RCAS-TVA viral gene delivery system to generate genetically-faithful primary mouse models of H3K27M-mutant DMG with p53 deletion, and we used Cre recombinase to delete Atm in the tumor cells of these mice and generated littermate controls that retained Atm. Mice were imaged weekly via luciferase-based bioluminescence to track tumor development and irradiated with three daily fractions of 10 Gy after tumor detection. We subsequently quantified the survival of mice without neurological decline following irradiation. In separate cohorts, we collected primary tumors after irradiation to verify H3K27M expression and to assess cell cycle arrest and mechanisms of cell death. These studies will elucidate mechanisms by which ATM inactivation can radiosensitize H3K27M-mutant DMGs with nonfunctioning p53, which will guide the design of clinical trials testing ATM inhibitors in DMG patients.

ATM Expression as a Prognostic Marker in Patients With Advanced Biliary Tract Cancer Treated With First-line Gemcitabine and Platinum Chemotherapy

Biliary tract cancer (BTC) has a poor prognosis due to its highly invasive and metastatic potential. Ataxia-telangiectasia mutated (ATM) is a key regulator of DNA damage response and an emerging therapeutic target; however, the association between the expression of ATM and the prognosis in advanced BTC is unknown. We aimed to identify the relationship between ATM expression, clinicopathological characteristics, and survival outcomes in patients with advanced BTC. We analyzed 113 patients with advanced BTC who received first-line gemcitabine and platinum. The tumor location was intrahepatic cholangiocarcinoma (IH-CCC) in 43 patients, extrahepatic cholangiocarcinoma (EH-CCC) in 49, and gallbladder (GB) cancer in 21 patients. Fifty-four patients (47.8%) exhibited loss of ATM protein expression. The overall response rate (ORR) of ATM loss and intact ATM was 13.3% and 19.6%, respectively. In a subgroup analysis, EH-CCC patients with ATM loss tended to have improved PFS after platinum-based chemotherapy compared to those with intact ATM (7.9 vs. 6.2 months, respectively; p=0.050). We demonstrated that ATM loss could be a prognostic marker after platinum-based chemotherapy in patients with advanced EH-CCC.

First-in-Human Trial of the Oral Ataxia Telangiectasia and RAD3-Related (ATR) Inhibitor BAY 1895344 in Patients with Advanced Solid Tumors.

Targeting the ataxia telangiectasia and RAD3-related (ATR) enzyme represents a promising anticancer strategy for tumors with DNA damage response (DDR) defects and replication stress, including inactivation of ataxia telangiectasia mutated (ATM) signaling. We report the dose-escalation portion of the phase I first-in-human trial of oral ATR inhibitor BAY 1895344 intermittently dosed 5 to 80 mg twice daily in 21 patients with advanced solid tumors. The MTD was 40 mg twice daily 3 days on/4 days off. Most common adverse events were manageable and reversible hematologic toxicities. Partial responses were achieved in 4 patients and stable disease in 8 patients. Median duration of response was 315.5 days. Responders had ATM protein loss and/or deleterious ATM mutations and received doses ≥40 mg twice daily. Overall, BAY 1895344 is well tolerated, with antitumor activity against cancers with certain DDR defects, including ATM loss. An expansion phase continues in patients with DDR deficiency. SIGNIFICANCE: Oral BAY 1895344 was tolerable, with antitumor activity in heavily pretreated patients with various advanced solid tumors, particularly those with ATM deleterious mutations and/or loss of ATM protein; pharmacodynamic results supported a mechanism of action of increased DNA damage. Further study is warranted in this patient population.See related commentary by Italiano, p. 14.This article is highlighted in the In This Issue feature, p. 1.