Although several ATR inhibitors are in development, there are unresolved questions regarding their differential potency, molecular signatures of patients with cancer for predicting activity, and most effective therapeutic combinations. Here, we elucidate how to improve ATR-based chemotherapy with the newly developed ATR inhibitor, M4344 using in vitro and in vivo models. The potency of M4344 was compared with the clinically developed ATR inhibitors BAY1895344, berzosertib, and ceralasertib. The anticancer activity of M4344 was investigated as monotherapy and combination with clinical DNA damaging agents in multiple cancer cell lines, patient-derived tumor organoids, and mouse xenograft models. We also elucidated the anticancer mechanisms and potential biomarkers for M4344. We demonstrate that M4344 is highly potent among the clinically developed ATR inhibitors. Replication stress (RepStress) and neuroendocrine (NE) gene expression signatures are significantly associated with a response to M4344 treatment. M4344 kills cancer cells by inducing cellular catastrophe and DNA damage. M4344 is highly synergistic with a broad range of DNA-targeting anticancer agents. It significantly synergizes with topotecan and irinotecan in patient-derived tumor organoids and xenograft models. Taken together, M4344 is a promising and highly potent ATR inhibitor. It enhances the activity of clinical DNA damaging agents commonly used in cancer treatment including topoisomerase inhibitors, gemcitabine, cisplatin, and talazoparib. RepStress and NE gene expression signatures can be exploited as predictive markers for M4344.

Significance: Schlafen-11 (SLFN11) inactivation leads to chemoresistance of a broad range of DNA-damaging agents. We uncover an expanded Ataxia Telangiectasia- and Rad3-related (ATR)-mediated signaling network that overcomes chemoresistance by an unbiased genome-wide RNAi screen in SLFN11 -knockout cells and is validated with clinically developing ATR/CHK1 inhibitors. ATR inhibition induces CDT1 phosphorylation, leading to mitotic catastrophe cell death in SLFN11-deficient cells. We identify a key role of SLFN11 for CDT1 degradation by binding to DDB1–CUL4 ^CDT2 ubiquitin ligase during DNA damage, thereby blocking replication reactivation by which SLFN11-deficient cells cause chemoresistance. Our findings provide a rationale for combination therapy with ATR inhibitor and DNA-targeted drugs, and reveal how SLFN11 irreversibly arrests replication during DNA damage acting as a cofactor of CUL4 ^CDT2 ubiquitin ligase.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody–drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide–exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1,978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2α, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than in WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors. Significance: This study uncovers that SLFN11 deficiency induces proteotoxic stress and sensitizes cancer cells to TAK-243, suggesting that profiling SLFN11 status can serve as a therapeutic biomarker for cancer therapy.

Precision medicine aims to implement strategies based on the molecular features of tumors and optimized drug delivery to improve cancer diagnosis and treatment. DNA replication is a logical approach because it can be targeted by a broad range of anticancer drugs that are both clinically approved and in development. These drugs increase deleterious replication stress (RepStress); however, how to selectively target and identify the tumors with specific molecular characteristics are unmet clinical needs. Here, we provide background information on the molecular processes of DNA replication and its checkpoints, and discuss how to target replication, checkpoint, and repair pathways with ATR inhibitors and exploit Schlafen 11 (SLFN11) as a predictive biomarker.

Small‐cell lung cancer (SCLC) is the most lethal type of lung cancer. Specifically, MYC‐driven non‐neuroendocrine SCLC is particularly resistant to standard therapies. Lurbinectedin was recently approved for the treatment of relapsed SCLC, but combinatorial approaches are needed to increase the depth and duration of responses to lurbinectedin. Using high‐throughput screens, we found inhibitors of ataxia telangiectasia mutated and rad3 related (ATR) as the most effective agents for augmenting lurbinectedin efficacy. First‐in‐class ATR inhibitor berzosertib synergized with lurbinectedin in multiple SCLC cell lines, organoid, and in vivo models. Mechanistically, ATR inhibition abrogated S‐phase arrest induced by lurbinectedin and forced cell cycle progression causing mitotic catastrophe and cell death. High CDKN1A /p21 expression was associated with decreased synergy due to G1 arrest, while increased levels of ERCC5 /XPG were predictive of increased combination efficacy. Importantly, MYC‐driven non‐neuroendocrine tumors which are resistant to first‐line therapies show reduced CDKN1A /p21 expression and increased ERCC5 /XPG indicating they are primed for response to lurbinectedin–berzosertib combination. The combination is being assessed in a clinical trial NCT04802174.