ATM Inhibitor Research Articles

ATAXIA TELANGIECTASIA MUTATED PROTECTS AGAINST LIPOPOLYSACCARIDE-INDUCED BLOOD-BRAIN BARRIER DISRUPTION BY REGULATING ATK/DRP1-MEDIATED MITOCHONDRIAL HOMEOSTASIS.

Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of ATM in lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) disruption remains unknown. This study aimed to investigate the role and underlying mechanism of ATM in the regulation of the BBB function in sepsis. Methods: We used LPS to induce BBB disruption in vivo and to establish an in vitro model of cerebrovascular endothelial cells. Blood-brain barrier disruption was assessed by measuring Evans blue leakage and expression of vascular permeability regulators. To investigate the role of ATM, its inhibitor AZD1390 and clinically approved doxorubicin, an anthracycline that can activate ATM, were administered as scheduled. To explore the underlying mechanism, protein kinase B (AKT) inhibitor MK-2206 was administered to block the AKT/dynamin-related protein 1 (DRP1) pathway. Results: Lipopolysaccharide challenge induced significant BBB disruption, ATM activation, and mitochondrial translocation. Inhibiting ATM with AZD1390 aggravated BBB permeability as well as the following neuroinflammation and neuronal injury, while activation of ATM by doxorubicin abrogated these defects. Further results obtained in brain microvascular endothelial cells showed that ATM inhibition reduced the phosphorylation of DRP1 at serine (S) 637, promoted excessive mitochondrial fission, and resulted in mitochondrial malfunction. By activating ATM, doxorubicin increased the protein binding between ATM and AKT and promoted the phosphorylated activation of AKT at S473, which could directly phosphorylate DRP1 at S637 to repress excessive mitochondrial fission. Consistently, the protective role of ATM was abolished by the AKT inhibitor MK-2206. Conclusions: Ataxia telangiectasia mutated protects against LPS-induced BBB disruption by regulating mitochondrial homeostasis, at least in part, through the AKT/DRP1 pathway.

A Chemical Proteomics Approach to Discover Regulators of Innate Immune Signaling.

Innate immune pathways are tightly regulated to balance an appropriate response to infectious agents and tolerable levels of inflammation. Dysregulation of innate immune pathways can lead to severe autoinflammatory disorders or susceptibility to infections. Here, we aimed to identify kinases in common cellular pathways that regulate innate immune pathways by combining small-scale kinase inhibitor screening with quantitative proteomics. We found that inhibitors of kinases ATM, ATR, AMPK, and PLK1 reduced the induction of interferon-stimulated gene expression in response to innate immune pathway activation by poly(I:C) transfection. However, siRNA depletion of these kinases did not validate findings with kinase inhibitors, suggesting that off-target effects may explain their activities. We mapped the effects of kinase inhibitors to various stages in innate immune pathways. Determining the mechanisms by which kinase inhibitors antagonize these pathways may illuminate novel mechanisms of innate immune pathway control.

ATM Inhibition-Induced ISG15/IFI27/OASL Is Correlated with Immunotherapy Response and Inflamed Immunophenotype.

Immune checkpoint blockade (ICB) therapy can improve the survival of cancer patients with a high tumor mutation burden (TMB-H) or deficiency in DNA mismatch repair (dMMR) in their tumors. However, most cancer patients without TMB-H and dMMR do not benefit from ICB therapy. The inhibition of ATM can increase DNA damage and activate the interferon response, thus modulating the tumor immune microenvironment (TIME) and the efficacy of ICB therapy. In this study, we showed that ATM inhibition activated interferon signaling and induced interferon-stimulated genes (ISGs) in cisplatin-resistant and parent cancer cells. The ISGs induced by ATM inhibition were correlated with survival in cancer patients who received ICB therapy. In oral cancer, high expressions of ISG15, IFI27, and OASL were associated with low expressions of ATM, the activation of inflamed immune pathways, and increased tumor-infiltrating scores of CD8+ T, natural killer, and dendritic cells. The high expressions of ISG15, IFI27, and OASL were also correlated with complete remission in patients with cervical cancer treated with cisplatin. These results suggest that ATM inhibition can induce the interferon response and inflamed TIME, which may benefit ICB therapy.

Selective inhibition of ATM-dependent double-strand break repair and checkpoint control synergistically enhances the efficacy of ATR inhibitors.

Ataxia Telangiectasia and Rad3-Related Protein (ATR) kinase regulates a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell cycle checkpoints controlled by Ataxia Telangiectasia Mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1 phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell cycle checkpoints and DSB repair, lowered the p53 protective barrier and extended the life of ATR inhibitor induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Further, a combination study in 26 patient-derived xenograft models of triple negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared to single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.

Abstract CT171: A first-in-human phase I study of the ATM inhibitor M4076 in patients with advanced solid tumors (DDRiver Solid Tumors 410): Part 1A results

Abstract M4076 is a potent and selective oral inhibitor of ataxia-telangiectasia mutated (ATM), a key kinase of the DNA damage response (DDR) involved in double-strand break repair. Preclinically, M4076 when combined with DNA damage-inducing therapy or other DDR inhibitors caused unrestricted cell cycle progression and DNA damage accumulation, resulting in tumor cell death. Part 1A of this ongoing open-label study (NCT04882917) evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and maximum tolerated dose (MTD) of M4076 monotherapy in patients (pts) with advanced solid tumors. Dose escalation was guided by safety, along with Bayesian analysis, PK, and PD. In total, 22 pts received M4076 at 4 different dose levels (100-400 mg once daily [QD]); 21 pts (95.5%) discontinued treatment (progressive disease: 11 [50%], adverse events [AEs]: 5 [22.7%]). Dose-limiting toxicities (DLTs) were reported in 4 pts. Overall, 6 pts had ≥1 treatment-related AE (TRAE) of Grade ≥3 (including one Grade 4 hypersensitivity). The most common TRAEs were rash and anemia (Table 1). MTD was determined at 300 mg QD. As per preliminary PK across cohorts, M4076 was rapidly absorbed with median Tmax ~0.5-2 h and mean elimination half-life (t1/2) ~2-10 h; exposure increased in a dose-related manner, with minimum accumulation after multiple QD doses. At ≥200 mg QD, unbound steady state plasma concentration exceeded in vivo pCHK2 IC90 throughout the dosing interval. PD evaluation was based on the modulation of γ-H2AX (a direct ATM target) in ex vivo bleomycin stimulated CD45+ lymphocytes, by flow cytometry. Preliminary analyses showed a trend of reduction of γ-H2AX levels from day 1 to day 2, reaching 80-100% of target inhibition on day 2 with the dose range 100-400 mg QD. The MTD of M4076 was determined, and target exposure and engagement were achieved without significant hematological toxicity. Future investigations on M4076 as combination therapy are planned. Table 1. Safety overview Dose (Safety set) Patients with TRAEs (Grade ≥3) DLT analysis set (Patients with ≥80% of the planned dose or DLT per investigator) DLT DLT AE terms 100 mg(n = 2) No n = 2 No NA 200 mg(n = 7) n = 1 (decreased lymphocyte count, maculo-papular rash) n = 5 Yes(n = 1) Grade 3 maculo-papular rash (TRAE, required drug withdrawal; resolved) 300 mg(n = 9) n = 3 (anemia, spontaneous bacterial peritonitis) n = 7 Yes(n = 1) Grade 1 maculo-papular rash; Grade 2 fever (TRAEs, required treatment discontinuation; resolved) 400 mg(n = 4) n = 2 (nausea, maculo-papular rash, hypersensitivity) n = 4 Yes(n = 2) Grade 3 maculo-papular rash (TRAEs, required dose reduction/interruption and/or concomitant medication; resolved) MedDRA version 23.0, NCI-CTCAE version 5.0. Data cutoff: 2 Nov 2022 AE, adverse event; DLT, dose-limiting toxicity; NA, not applicable; TRAE, treatment-related AE Citation Format: Lillian L. Siu, Timothy A. Yap, Sofia Genta, Gregory Pennock, Christine Hicking, Xiaoli You, Jatinder K. Mukker, Giuseppe Locatelli, Anthony W. Tolcher. A first-in-human phase I study of the ATM inhibitor M4076 in patients with advanced solid tumors (DDRiver Solid Tumors 410): Part 1A results [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 CT171.

Design, synthesis, and biological evaluation of pyrido[3,2-d]pyrimidine derivatives as novel ATR inhibitors

Targeting ataxia telangiectasia mutated and Rad3-related (ATR) kinase is being pursued as a new therapeutic strategy for the treatment of advanced solid tumor with specific DNA damage response deficiency. Herein, we report a series of pyrido[3,2-d]pyrimidine derivatives with potent ATR inhibitory activity through structure-based drug design. Among them, the representative compound 10q exhibited excellent potency against ATR in both biochemical and cellular assays. More importantly, 10q exhibited good liver microsomes stability in different species and also showed moderate inhibitory activity against HT-29 cells in combination treatment with the ATM inhibitor AZD1390. Thus, this work provides a promising lead compound against ATR for further study.

Liposomal ATM siRNA delivery for enhancing triple-negaitive breast cancer immune checkpoint blockade therapy.

Triple-negative breast cancer (TNBC), which accounts for 10%-20% of breast cancer cases, is characterized by a higher metastasis rate, higher recurrence risk, and worse prognosis. Traditional treatments such as chemotherapy, surgery, and radiotherapy have limited therapeutic effects. Although immune checkpoint blockade (ICB) therapy represented by anti-programmed death 1 (aPD-1) antibody has made further progress in treating TNBC, its therapeutic effect is still not optimistic. Ataxia telangiectasia mutated (ATM) is a critical factor in the DNA damage response (DDR) pathway, which is associated with the development of tumors. Recent studies have found that it can regulate the tumor immune microenvironment, affecting ICB responsiveness. Inhibition of ATM could enhance ICB therapy by promoting mitochondrial DNA cytoplasmic leakage and activating the innate immune signaling pathway. To explore the effect of ATM siRNA(siATM) on the ICB responsiveness of TNBC, we designed and synthesized nanoparticles using 1,2-dioleoyl-glycero-3-phosphatidylcholine (DOPC) liposomes to deliver siATM. In vitro and in vivo experiments demonstrated that DOPC/siATM could enhance the ability of siRNA to enter tumor cells and effectively inhibit the expression of ATM protein. Our study found that nanoparticles carrying siATM could activate cytotoxic T lymphocytes and regulate the immunosuppressive tumor microenvironment (ITM) by activating the cGAS-STING pathway. Its combination with aPD-1 may be a potential way to improve the efficacy of TNBC.

Abstract 6212: Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors

Abstract Ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated (ATM) protein kinases play key roles in the DNA damage response (DDR) by responding to replication stress and double-strand DNA breaks respectively to pause the cell cycle and promote DNA repair. ATR kinase inhibition in cancer cells disrupts cell cycle control and causes unrepaired DNA lesions and cytotoxicity. Accordingly, several ATR inhibitors (ATRi) are in clinical development as monotherapies and in combination with DNA damaging chemotherapies and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that while treatment of cancer cells with an ATRi inhibits the ATR pathway, it simultaneously activates the ATM signaling pathway as shown by increased levels of p-ATM, p-CHK2, p-KAP1 and p-p53. In p53 wild-type cancer cells, ATR inhibition causes an ATM-mediated G1 cell cycle arrest which diminishes the DNA lesions and cytotoxic effects of ATR inhibition. Combination of an ATRi with a selective ATM inhibitor (ATMi) synergistically potentiated efficacy in cancer cells in vitro and increased efficacy in vivo at doses that did not show overt toxicities. In a panel of patient-derived xenograft (PDX) models of triple-negative breast cancer treated with an ATRi/ATMi combination, substantial improvement in efficacy was observed. Thus, activation of the ATM pathway by an ATRi acts as a compensatory resistance pathway for ATR inhibition. These results suggest a novel and efficacious combination approach for cancer therapy by dual inhibition of these two key DDR kinases. Citation Format: Audrey Turchick, Astrid Zimmermann, Li-Ya Chiu, Heike Dahmen, Brian Elenbaas, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors. [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 6212.

Abstract 6208: Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells

Abstract Introduction: Rapid cell proliferation requires intact and faithful DNA damage repair mechanisms. DNA polymerase theta (POLQ) plays a key role in repairing DNA double-strand breaks through the microhomology-mediated end-joining (MMEJ), which is one of the three main pathways involved in repairing replication-induced double-strand breaks. Limited data have suggested that concurrent depletion of POLQ and ataxia-telangiectasia mutated (ATM) could be embryonic lethal. Hence, this phenomenon has the potential to be exploited for therapeutic benefit in cancers where ATM mutations are commonly seen. Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma marked by (11;14) translocation with ATM alterations seen in 40-50% of patients. There is an unmet need to find novel therapeutic strategies, especially in relapsed and/or refractory (R/R) MCL. Here, we investigated whether targeting POLQ and ATM could be a potential therapeutic strategy in MCL. Methods: In vitro studies were conducted by using MCL cell lines. CRISPR-Cas9 system was used to genetically deplete POLQ and ATM genes and sgRNAs co-expressing fluorescence markers were used to track the cell population with respective genotypes over time. Cell viability was assessed by CellTiter-Glo assay and flow cytometry. All cell lines were profiled for ATM expression and activity. A p-value of < 0.05 was considered statistically significant. The combination index of <1 was defined as synergistic. Results: CRISPR-Cas9-mediated depletion of POLQ significantly decreased cell proliferation in multiple MCL cell lines. In particular, Granta-519, which possesses a single copy of kinase-dead ATM that is reduced in expression, was most sensitive to POLQ depletion. Concurrent genetic depletion of ATM and POLQ resulted in a synergistic antiproliferative effect in ATM-proficient MCL cell lines. Subsequently, this cellular phenotype caused by the genetic intervention was recapitulated by using two POLQ inhibitors (novobiocin and ART558) and an ATM inhibitor (AZD0156). In vitro, single-agent treatment with novobiocin or ART558 caused a significant cytotoxic effect at physiologically relevant concentrations in ATM-deficient cells and co-treatment of novobiocin or ART558 with AZD0156 was synergistic in killing ATM-proficient MCL cells. Importantly, POLQ inhibitors significantly decreased the cell viability of MCIR1, which is an ibrutinib-resistant MCL cell line. Mechanistically, novobiocin or ART558 treatment induced gH2AX and cleaved PARP upregulation, which was further enhanced by ATM depletion, suggesting that co-inhibition of POLQ and ATM caused apoptosis due to the accumulation of unrepaired DNA damage. Conclusion: POLQ is a promising target in MCL, especially in ATM-deficient setting. In ATM-proficient MCL, targeting ATM and POLQ is synergistic. Our data has the potential to uncover novel biomarker-driven drug therapy of POLQ inhibitors in R/R MCL. Citation Format: Jithma Prasad Abeykoon, Shuhei Asada, Kalindi Parmar, Xiaosheng Wu, Thomas Witzig, Geoffrey Shapiro, Alan D. D'Andrea. Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells. [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 6208.

Abstract 3804: Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment

Abstract The human Plasmacytoma Variant Translocation 1 (PVT1) gene encodes for 176 linear (PVT1, lncipedia.org) and 29 circular isoforms (circPVT1, www.circbase.org). The most common isoform of circPVT1 is 410bp and is a product of back-splicing containing the whole exon 2 of PVT1 in a closed loop-like structure (hsa_circ_0001821). PVT1 and circPVT1 have oncogenic roles in several tumor types and can contribute to the suppression of anti-tumor immune responses. Our study aims to investigate whether and how PVT1 isoforms and circPVT1 promote an aggressive phenotype in acute myeloid leukemia (AML) and are involved in the crosstalk between leukemic and immune cells. Six out of the 14 PVT1 isoforms expressed by white blood cells and lymphnodes (noncode.org) were detected in a panel of AML cell lines along with circPVT1. Downregulation (KD) of groups of linear isoforms (based on sequence homology) or of circPVT1 by antisense-oligonucleotides (ASOs) in the t(8;21) KASUMI-1 and the NPM1-mut OCI-AML3 cells induced an impairment of leukemia cell growth especially when all the 6 linear isoforms were silenced by ASO combination, in both cell lines, under normoxia and hypoxia mimicking the bone-marrow microenvironment. Interestingly circPVT1-KD induced apoptosis in OCI-AML3 cells. Moreover, MYC protein expression decreased by ASO combination or circPVT1_KD in OCI-AML3 under normoxia and in KASUMI-1 under hypoxia. To further investigate the biological consequences of PVT1/circPVT1-KD, we performed RNAseq. Analysis on AML cell lines revealed that downregulation of PVT1 and, especially circPVT1, altered the expression of genes involved in RNA transport and degradation, as expected for a long non-coding genes, but also in DNA damage response (DDR) and immunological pathways, as also confirmed by transcriptomic data of primary AML cases and proteomic analysis of both cell lines. Based on these novel findings, we tested the downregulation of PVT1/circPVT1 in combination with inhibition of ATM or ATR, key DDR transducers. Interestingly, circPVT1-KD sensitized OCI-AML3 to ATM (71% vs 95% live cells) or ATR (75% vs 90% live cells) inhibitors. PVT1 and circPVT1 expression was also detected at the level of cell-free RNA in primary AML samples, the latter showing higher levels. We then analyzed extracellular vesicles (EVs) from AML peripheral blood and healthy subjects. EVs from patients were larger, increased in number and expressed higher levels of myeloid lineage markers. Finally AML EVs had more circPVT1 cargo. In conclusion, we uncovered novel potential roles of PVT1 isoforms and circPVT1 in the DDR and in the tumor microenvironment that pave the way for novel therapeutic combinations. Citation Format: Martina Ghetti, Lorenzo Ledda, Ivan Vannini, Eugenio Fonzi, Maria Teresa Bochicchio, Tristan Cardon, Andrea Ghelli Luserna di Rorà, Matteo Paganelli, Chiara Servili, Francesco Fabbri, Sabina Marianini, Giovanni Marconi, Michela Rondoni, Roberta Chicchi, Rino Biguzzi, Francesco Lanza, Michel Salzet, Giovanni Martinelli, Giorgia Simonetti. Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment. [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 3804.

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.

Phosphorylation stabilized TET1 acts as an oncoprotein and therapeutic target in B cell acute lymphoblastic leukemia.

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

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.

Plumbagin Exhibits Genotoxicity and Induces G2/M Cell Cycle Arrest via ROS-Mediated Oxidative Stress and Activation of ATM-p53 Signaling Pathway in Hepatocellular Cells

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), a naturally occurring naphthoquinone mainly isolated from the plant Plumbago zeylanica L., has been proven to possess anticancer activities towards multiple types of cancer. Although there has been an increasing amount of research regarding its anticancer effects, the association between oxidative stress, genotoxicity and the cell cycle arrest induced by PLB still remains unclear. Therefore, it is important to investigate their potential connections and the involvement of DNA damage and the ataxia telangiectasia mutated protein (ATM)-p53 signaling pathway in PLB's anticancer mechanism. The present study showed that PLB exposure significantly reduced HCC cell viability and colony formation. In addition, PLB-induced G2/M cell cycle arrest, oxidative stress, and DNA damage was detected, which could be almost blocked by NAC pretreatment. PLB could trigger a DNA damage response by activating cell cycle checkpoints such as ATM, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) and p53. Meanwhile, the key modulator of the G2/M transition factor, Cell Division Cycle 25C (cdc25C), was significantly downregulated in an ROS-dependent manner. Furthermore, pretreatment with ATM and p53 inhibitors (KU55933 and Pifithrin-α) could reduce the occurrence of G2/M cell cycle arrest by inhibiting the activation of the ATM-p53 pathway. Taken together, these results indicate that ROS-mediated oxidative stress plays a key role in PLB-induced G2/M cell cycle arrest mediated by the ATM-p53 pathway.

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.

Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation.

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions. Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3‐like kinases including Ataxia‐Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination‐mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long‐term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation‐induced apoptosis. This acute, ATM‐dependent apoptosis is a key determinant of long‐term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

ATM-SPARK: A GFP phase separation-based activity reporter of ATM.

The kinase ataxia telangiectasia mutated (ATM) plays a key role in the DNA damage response (DDR). It is thus essential to visualize spatiotemporal dynamics of ATM activity during DDR. Here, we designed a robust ATM activity reporter based on phosphorylation-inducible green fluorescent protein phase separation, dubbed ATM-SPARK (separation of phases-based activity reporter of kinase). Upon ATM activation, it undergoes phase separation via multivalent interactions, forming intensely bright droplets. The reporter visualizes spatiotemporal dynamics of endogenous ATM activity in living cells, and its signal is proportional to the amount of DNA damage. ATM-SPARK also enables high-throughput screening of biological and small-molecule regulators. We identified the protein phosphatase 4 that blocks ATM activity. We also identified BGT226 as a potent ATM inhibitor with a median inhibitory concentration of~3.8 nanomolars. Furthermore, BGT226 sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting that BGT226 might be combined with radiotherapeutic treatment. ATM-SPARK achieves large dynamic range, bright fluorescence, and simple signal pattern.

Abstract P2-17-03: Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models

Abstract Background: Triple negative breast cancer (TNBC), defined by negative hormone receptor (HR) status and the lack of HER2 gene amplification, is a significant clinical challenge because of its aggressive clinical course and association with a poor prognosis. Recent studies demonstrated that approximately a third of TNBC has low expression of HER2, so called HER2-low TNBC, which could be targeted by the new generation HER2-directed antibody-drug conjugate (ADC). Trastuzumab deruxtecan (also known as T-Dxd, DS-8201a) is a novel HER2-directed ADC with a topoisomerase 1 inhibitor payload and a tetrapeptide-based cleavable linker, which has been FDA approved for patients with metastatic HER2-positive, or amplified breast cancer. T-Dxd has also shown to improve survival outcomes when compared to physicians’ choice chemotherapy in patients with metastatic HR-negative/HER2-low breast cancer in the Phase 3 DESTINY-Breast04 trial. However, resistance is invariable. We hypothesize that small molecule inhibitors against DNA damage response (DDR) pathways could potentiate the DNA damaging effect of T-Dxd and improve its anti-tumor activity in HER2-low TNBC. Method: We conducted preclinical experiments to assess the anti-tumor activity of T-Dxd and each of the DDR pathway inhibitors, including the ATR inhibitor AZD6738, the ATM inhibitor AZD1390, and the PARP inhibitor olaparib, as well as the Wee 1 inhibitor AZD1775, either alone or in combination, compared to the vehicle treatment, in a panel of HER2-low TNBC PDX models in vivo, as well as mechanistic studies using HER2-low TNBC cell lines in vitro. HER2-low was defined as 1+ or 2+ on HER2 immunohistochemistry. Results: Four HER2-low TNBC PDX models, including WHIM2 (TP53 mutated, basal-like), WHIM6 (TP53 WT, basal-like), WHIM12 (TP53, PIK3CA, and PTEN mutated, claudin-low), and WHIM30 (BRCA1 and TP53 mutated, basal-like), as well as a panel of 4 TNBC cell lines with variable HER2 expression were included in the study. We demonstrated that the addition of DDR pathway inhibitors to T-Dxd led to further tumor shrinkage and delayed tumor progression in HER2-low TNBC PDX models in vivo and induced synergistic anti-tumor effects in vitro. Enhanced DNA damaging effect and apoptosis induction were observed with combination therapies in vitro. Biomarker analysis on xenograft tumors harvested 3 days following treatment with either Vehicle, T-Dxd, olaparib, or T-Dxd plus olaparib in WHIM6, was also performed, which demonstrated that the addition of olaparib enhanced DNA damage (increased pH2AX), and promoted apoptosis (increased cleaved PARP). Further mechanistic studies in HER2-low TNBC cell lines demonstrated the formation of DNA topoisomerase 1 covalent complexes (TopoIcc) following treatment with T-Dxd. Interestingly, while the formation of Topolcc was reduced with the addition of either ATR or ATM inhibitor, no reduction was observed with the addition of olaparib. Additionally, we observed that combination therapies reduced cell migration in vitro. The combination of olaparib with T-Dxd, in particular, reduced mammosphere formation and Epithelial Mesenchymal Transition (EMT) modulation markers, including SNAIL, SLUG and/or TWIST. Conclusion: Our study demonstrated that the combination of T-Dxd and DDR pathway inhibitors induced additive or synergistic anti-tumor effect in HER2-low TNBC. Our data from the studies of cell lines and PDX models provided preclinical rationale for the combination of T-Dxd and DDR pathway inhibitors in patients with HER2-low TNBC. The olaparib combination showed unique mechanistic characteristics compared with other DDR inhibitors, which are being further examined in additional HER2-low TNBC models. Citation Format: Adrian Gonzalez-Gonzalez, Zhanfang Guo, Alice Meroni, Emily Cybulla, Jeremy Hoog, Alessandro Vindigni, Cynthia Ma. Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-03.

Abstract P6-10-05: Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition

Abstract Background: Heterozygous hotspot mutations in the RNA splicing factor SF3B1, occur in 3% of unselected breast cancers and are associated with oestrogen receptor (ER+) breast cancer (BC) where they are enriched in metastatic disease and are associated with a poor clinical outcome. SF3B1 mutations drive distinct signatures of alternative splicing through cryptic 3’ splice site selection leading to global transcriptomic and proteomic changes. The functional consequences of the mis-splicing events and resultant genetic vulnerabilities are poorly understood and precision medicine approaches that exploit these characteristics are not clinically available (Table 1). Methods: To understand the role of SF3B1 mutations in ER+ BC, we generated a series of SF3B1 mutant (SF3B1MUT) isogenic cell lines which were characterised using RNA-sequencing and high content mass-spectrometry proteomic profiling. SF3B1 interactome analysis was also performed using immunoprecipitation of SF3B1 followed by mass-spectrometry. The molecular consequences of aberrant splicing were investigated using a targeted screening approach of 280 genes predicted to be alternatively spliced in SF3B1MUT BC, while high-throughput drug screens were used to identify novel therapeutic options for patients with SF3B1MUT breast cancer using isogenic cells. Hits were validated in vitro and in vivo using cell line and patient derived xenografts. Results: Transcriptomic and proteomic profiling of SF3B1MUT cells identified global alternative 3’ splice site selection and subsequent proteomic changes induced by the mutations. Investigation of the SF3B1K700E interactome identified an enrichment of SF3B1K700E binding with ER, aberrant splicing of ER target genes, global rewiring of ER chromatin binding and resistance to endocrine therapy. Silencing of the aberrantly spliced candidate genes PPIH, TRIM37, HIGD1A, BRD9, and PHKG2 significantly enhanced the growth of the SF3B1 mutant cells, suggestive of a dose dependent tumour suppressive effect. Through synthetic-lethal drug screens we found that SF3B1MUT cells are selectively sensitive to PARP inhibitors. SF3B1MUT cells display a defective response to PARPi induced replication stress. Mechanistically, this occurs via defective ATR signalling in SF3B1MUT cells, which upon PARPi exposure leads to increased replication origin firing and loss of pChk1 (S317) induction. The resultant replication stress leads to failure to resolve DNA replication intermediates via the endonuclease MUS81 and cell cycle stalling at the G2/M checkpoint. These defects can be further targeted by ATM, CDK7 or FACT inhibition, when used in combination with PARPi treatment. This SF3B1MUT selective PARPi sensitivity is preserved across multiple cell lines and patient derived tumour models. In vivo, PARPi produce profound anti-tumour effects in multiple SF3B1MUT cancer models and eliminate distant metastases. Conclusions: Our integrative analysis reveals mechanistic insight into the role of SF3B1 mutations in endocrine therapy response in ER+ breast cancers, where altered SF3B1 induces ER-transcriptional re-programming. We further identified a robust synthetic-lethal relationship of mutant SF3B1 with PARP inhibition that is caused by a defective response to PARPi induced replication stress. Furthermore, we identified several potential selective combination strategies together with PARPi that are selective for SF3B1MUT cells. Together, these data provide the pre-clinical and mechanistic rationale for assessing already-approved PARPi in a biomarker-defined subset of advanced ER+ BC. Table 1. Identified potential therapies for SF3B1 mutant cancers from this study and the literature Citation Format: Phil Bland, Harry Saville, Abigail Read, Patty Wai, Gareth Muirhead, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Marie John, Somaieh Hedayat, Holly Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Mark Allen, Patrycja Gazinska, Helen Pemberton, Aditi Gulati, Sarah Nash, Farzana Noor, Naomi Guppy, Ioannis Roxanis, Samantha Barlow, Helen Kalirai, Sarah Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher Lord, Rachael Natrajan. Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-05.