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Kinase-Dead ATM Protein Is Highly Oncogenic and Can Be Preferentially Targeted by Topo

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Kinase-Dead ATM Protein Is Highly Oncogenic and Can Be Preferentially Targeted by Topo 1 Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo- 2 isomerase I inhibitors 3 4 Kenta Yamamoto1,2, Jiguang Wang3, Lisa Sprinzen1,2, Jun Xu5, Christopher J. Haddock6, Chen 5 Li1, Brian J. Lee1, Denis G. Loredan1, Wenxia Jiang1, Alessandro Vindigni6, Dong Wang5, Raul 6 Rabadan3 and Shan Zha1,4 7 8 1 Institute for Cancer Genetics, Department of Pathology and Cell Biology, College of Physicians 9 and Surgeons, Columbia University, New York City, NY 10032 10 2 Pathobiology and Molecular Medicine Graduate Program, Department of Pathology and Cell 11 Biology, Columbia University, New York City, NY 10032 12 3 Department of Biomedical Informatics and Department of Systems Biology, College of 13 Physicians & Surgeons, Columbia University, New York City, NY 10032 14 4 Division of Pediatric Oncology, Hematology and Stem Cell Transplantation, Department of 15 Pediatrics, College of Physicians & Surgeons, Columbia University, New York City, NY 10032 16 5 Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California San Diego, 17 La Jolla, CA 92093 18 6 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University 19 School of Medicine, St. Louis, MO 63104 20 21 Short Title: Topo1 inhibitors target ATM mutated cancers 22 Key Words: ATM, missense mutations, Topo I inhibitors 23 24 Address Correspondence to: Shan Zha at [email protected] 25 26 1 27 ABSTRACT 28 Missense mutations in ATM kinase, a master regulator of DNA damage responses, are 29 found in many cancers, but their impact on ATM function and implications for cancer therapy are 30 largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense 31 mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) 32 is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more 33 frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of 34 ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at 35 the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo- 36 isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and 37 preferentially eliminate AtmKD/-, but not Atm-proficient or Atm-/- leukemia in animal models. These 38 findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a 39 biomarker for Topo-isomerase I inhibitor based therapy. 40 2 41 INTRODUCTION 42 ATM kinase is a tumor suppressor that has a central role in the DNA damage responses. 43 Germline inactivation of ATM causes ataxia-telangiectasia (A-T), which is associated with 44 greatly increased risk of lymphoma and leukemia1. Somatic mutations of ATM occur frequently 45 in mantle cell lymphomas (MCL), chronic lymphoblastic leukemia (CLL) and T-cell 46 prolymphocytic leukemia (TPLL)2-4. In lymphomas, ATM mutations often occur with concurrent 47 heterozygous deletion of 11q23 including ATM2-4, potentially leading to the expression of 48 mutated ATM in the absence of the wild-type (WT) protein. Recent sequencing studies also 49 identified recurrent ATM mutations in 2-8% of breast, pancreas or gastric cancers5,6. While the 50 majority of A-T patients (~90%) have truncating ATM mutations that result in little or no ATM 51 protein expression7, missense ATM mutations are more common in cancers and with the 52 exception of the few that cause A-T, their biological functions are unknown. 53 As a serine/threonine protein kinase, ATM is recruited and activated by DNA double 54 strand breaks (DSBs) through direct interactions with the MRE11, RAD50 and NBS1 (MRN) 55 complex8-11. Activated ATM phosphorylates >800 substrates implicated in cell cycle checkpoints, 56 DNA repair, and apoptosis to suppress genomic instability and tumorigenesis. ATM activation is 57 also associated with inter-molecular autophosphorylation12,13. Studies in human cells suggest 58 that auto-phosphorylation is required for ATM activation12,13. However, alanine substitutions at 59 one or several auto-phosphorylation sites do not measurably affect ATM kinase activity in 60 transgenic mouse models14,15, leaving the biological function of ATM auto-phosphorylation 61 unclear. In this context, we and others generated mouse models expressing kinase dead (KD) 62 ATM protein (Atm-KD)16,17. In contrast to the normal development of Atm-/- mice, AtmKD/- and 63 AtmKD/KD mice die during early embryonic development with severe genomic instability16-18, 64 implying that the expression of Atm-KD in the absence of WT ATM further inhibits DNA repair 65 beyond the loss of ATM. Among the major DNA DSB repair pathways, non-homologous end- 66 joining (NHEJ) is uniquely required for the repair phases of lymphocyte specific V(D)J 3 67 recombination and class switch recombination (CSR). ATM promotes NHEJ19,20, and Atm-/- mice 68 and A-T patients suffer from primary immunodeficiency21,22. The end-joining defects in V(D)J 69 recombination and CSR are similar in AtmKD/- and Atm-/- lymphocytes, suggesting that Atm-KD 70 protein is not dominant-inhibitory for NHEJ. Meanwhile, AtmKD/- cells show moderate yet 71 significant hypersensitivity to PARP inhibitors in comparison to both Atm-/- and Atm+/+ cells17, and 72 ATM kinase inhibitor, but not loss-of-ATM, reduced homologous recombination (HR) as 73 measured by sister chromatid exchange (SCE)23 and the DR-GFP HR reporter24,25, suggesting a 74 role of ATM auto-phosphorylation in replication associated homology dependent repair. Yet, it is 75 unknown how ATM auto-phosphorylation contributes to DNA replication and HR beyond its 76 previously identified signaling roles. Finally, consistent with the “inter”-molecular 77 autophosphorylation model, Atm+/KD mice are largely normal16, suggesting that ATMKD mutation 78 carriers could be asymptomatic and somatic loss of the WT allele in the carriers might create 79 the Mut/Del status reported in human cancers and lymphomas. 80 Here we report that 72% of human cancer-associated ATM mutations are missense 81 mutations that are highly enriched in the kinase domain. We further show that conditional 82 expression of Atm-KD protein alone (somatic inactivation of the conditional allele (AtmC) in 83 AtmKD/C mice to generate the AtmKD/- cells) in murine hematopoietic stem cells (HSCs) is more 84 oncogenic than the complete loss of ATM (Atm-/-). AtmKD/- cells are selectively hypersensitive to 85 Topo Isomerase I (Topo1) inhibitors, in part because the Atm-KD protein physically blocks 86 replication-dependent strand cleavage upon Topo1 inhibition. Correspondingly, Topo1 inhibitor 87 selectively eradicates Notch1-driven AtmKD/- leukemia, but not the isogenic parental Atm 88 proficient leukemia, identifying Topo1 inhibitors as a targeted therapy for human cancers 89 carrying missense ATM kinase domain mutations. 90 RESULTS 91 Cancer-associated ATM mutations are enriched for kinase domain missense mutations. 4 92 Among the 5402 cases in The Cancer Genome Atlas (TCGA), we identified 286 unique 93 non-synonymous mutations of ATM. While truncating (nonsense/frameshift) mutations compose 94 83% (373/447) of A-T associated point mutations (>1000 patients), 72% (206/286) of non- 95 synonymous point mutations of ATM in TCGA are missense mutations [Figure 1A, Supplement 96 file 1A and 1B]. Permutation analyses show that ATM gene is not hyper-mutated, but the 97 kinase-domain is mutated 2.5 fold more frequently than otherwise expected in TCGA [Figure 1- 98 figure supplement 1A, p<0.01]. The mutation density calculated using the Gaussian Kernel 99 model revealed that cancer associated missense ATM mutations in TCGA cluster around the C- 100 terminal kinase domain, while truncating mutations (in A-T or TCGA) span the entire ATM 101 protein [Figure 1B and Figure 1-figure supplement 1B]. Given the severe phenotype of AtmKD/-, 102 but not AtmKD/+ cells, we further analyzed the subset (~105/286) of ATM missense mutations in 103 TCGA that are concurrent with heterozygous loss of ATM (shallow deletion) or truncating 104 mutations in the same case, and found that, again, ATM missense mutations cluster around the 105 C-terminal kinase domain even in this smaller subset [Figure 1B]. The kinase and FATC 106 domains of ATM share 31% sequence identity with mTOR, a related phosphatidylinositol 3- 107 kinase-related protein kinase (PIKK) for which the high resolution crystal structure is available26. 108 Homology modeling using mTOR (PDB 4JSP)26 revealed that 64% (27/42) (at 18 unique amino 109 acids) of ATM kinase domain missense mutations from TCGA, affect highly conserved residues 110 and 50% (21/42) of the mutations (red on the ribbon structure) likely to abolishes kinase activity 111 based on structural analyses [Figure 1C, Figure 1-figure supplement 1C]. Specifically, residues 112 K2717, D2720, H2872, D2870, N2875 and D2889 of human ATM are predicted to bind ATP or 113 the essential Mg+ ion [Figure 1-figure supplement 1D]. Notably, N2875 is mutated in two TCGA 114 cases at the time of initially analyses. One of the two cases have concurrent shallow deletion in 115 this region [Supplementary file 1B]. Since then, one additional N2875 mutation were reported in 116 a prostate cancer case (TCGA-YL-A8S9) with an allele frequency of 0.92, consistent with 117 homozygosity. Mutations corresponding to N2875K of human ATM were previously engineered 5 118 into the AtmKD/+ mice together with the corresponding D2870A mutation of human ATM. This 119 combination was found to abolish ATM kinase activity without significantly affecting ATM protein 120 levels16,27. Finally, immunoblotting confirmed the expression of catalytically inactive ATM protein 121 in several human cancer cell lines with missense mutations around the kinase domain (CCLE, 122 Broad Institute) [Figure 1-figure supplement 1E-1F]. 123 Expression of Atm-KD is more oncogenic than loss of Atm.
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