A Comprehensive Investigation of MTH1 as a Novel Therapeutic Target in RAS-Driven Cancers.

The nucleotide pool is significantly more susceptible to oxidative damage induced by reactive oxygen species (ROS) compared to genomic DNA, due to DNA being more protected by the double helix and nucleosome packing. The NUDIX pyrophosphatase, MutT Homolog 1 (MTH1), maintains nucleotide pool integrity by hydrolyzing oxidized purines thus preventing their genomic incorporation. If incorporated into DNA the accumulation of these oxidized purines can lead to DNA strand breaks, genomic instability, and ultimately cell death. Our prior work has shown that MTH1 is critical in facilitating multiple pro-tumorigenic phenotypes in oncogenic RAS-mutated cancer cells, with its depletion leading to decreased tumor formation in lung cancer xenograft models. As MTH1 loss is well tolerated in normal tissues, it was predicted to be an excellent anti-cancer therapeutic target with a wide therapeutic index; yet the first wave of MTH1 chemical inhibitors have yielded inconsistent results in their tumoricidal effects. Critical contributing factors to these discrepancies are 1) lack of an assay to measure endogenous cell/tissue-specific MTH1 8-oxo-dGTPase enzymatic activity, and 2) till-date unexplored biological redundancy in cellular 8-oxo-dGTPase activity that can compensate in the absence of functional MTH1. In order to address these critical gaps in the field, in collaboration with a synthetic chemistry group at Stanford, I utilized a novel ATP-releasing guanine-oxidized (ARGO) probe-based assay to measure endogenous 8-oxo-dGTPase activity in cancer cell lines and tissue specimens. I did an extensive comparative study on the effects of five independently developed small molecule MTH1 inhibitors on 8-oxo-dGTPase activity, oxidative DNA damage and cell viability in multiple cancer cell lines. My research helped establish that cancer cell lines and patient tissues possess variable levels of 8-oxo-dGTPase activity that cannot be decreased either by MTH1 shRNA-mediated depletion or the first-in-class MTH1 inhibitors, indicating previously unidentified redundancy in this function. My work additionally identified that the reported cytotoxicity from the first-in-class MTH1 inhibitors occurs through off-target mechanisms, implicating polypharmacology as their mechanism of action. Given this complication in MTH1 therapeutic targeting, in order to comprehensively investigate the biology of MTH1 in cancer initiation and progression, I generated the first mouse models of spontaneous KRAS-driven tumorigenesis in the background of MTH1 loss. I identified that the MTH1 null mouse also exhibits robust redundancy in the 8-oxodGTPase pathway. I next assessed how germline MTH1-knockout (KO) affected spontaneous in vivo oncogenic KRAS-driven pancreatic ductal adenocarcinoma and have found evidence that the functional 8-oxo-dGTPase activity redundancy existing in the MTH1-KO animals impacts the development of aggressive pancreatic cancer relative to the MTH1-wildtype animals. Our identification of MTH1-independent 8-oxo-dGTPase activity unveils previously undiscovered complexity in 8-oxo-dGTPase biology and its role in cancer. Identifying the source of this MTH-independent 8-oxo-dGTPase activity will be critical in understanding potential resistance mechanisms to the on-target effects of MTH1 inhibition. As the cytotoxic MTH1 inhibitors appear to exert their effects independent of MTH1 inhibition, through polypharmacology, our data imply that the utility of the on-target MTH1 inhibitors in the clinic may lie in combinatorial treatment regimens rather than as a monotherapy. Our findings have strong clinical implications, as the first-in-man clinical trial with Karonudib, an MTH1 inhibitor based on the same drug scaffold as the first-in-class inhibitors, is currently underway in patients with advanced solid tumors