KRAS was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRAS between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRAS. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRAS-specific inhibitors with promising therapeutic potential.

The gene coding for bovine prolactin was shown to exist as a single copy per haploid genome. Three restriction fragment polymorphisms were detected in the prolactin gene by Southern blot analysis of DNA obtained from the semen of pedigreed bulls representing eight breeds. The organization of the bovine prolactin gene was determined by restriction mapping of a clone isolated from a genomic library and by genomic blots. The 5'-flanking region and two exons were sequenced and the transcription start site mapped by primer extension. Comparison of the bovine prolactin sequence reported here with the published sequence of the rat prolactin gene revealed extensive homology (79%), extending 360 nucleotides upstream from the cap site, after which the sequences diverge. The homology exceeds that of the coding regions. A possible alternate intron-exon splice site was noted within the sequence coding for the signal peptide.

The RAS/RAF/MEK pathway is a major driver of malignant progression, particularly in cancers arising from mutations in RAS, BRAF and NF1. Although both BRAF and MEK inhibitors have been approved for treatment of melanoma, their clinical activity is commonly limited by acquired resistance due to reactivation of the pathway downstream of their targets. Since the ERK1/2 kinases are the final node in the MAPK signaling pathway, they are not subject to the feedback reactivation mechanisms that can undermine RAF or MEK blockade, offering the possibility of clinical benefit in settings where earlier drugs are ineffective. Here we describe the characterization of KO-947, a potent and selective inhibitor of ERK1/2 kinases, in biochemical, cellular and in vivo antitumor activity assays. KO-947 was profiled in vitro in biochemical activity assays, competition binding assays, and a probe-based competition binding assay in cell lysates, and subsequently screened in a large panel of human tumor cell lines focused on MAPK-dysregulated tumor types. In vivo studies were carried out in well-characterized xenograft models of BRAF-, KRAS- and NRAS-mutant disease and subsequently extended to a large panel of early-passage patient-derived xenograft (PDX) models representative of a wide range of tumor types and molecular profiles. Biochemical assays reveal that KO-947 is a 10nM inhibitor of ERK with at least 50-fold selectivity against a panel of 450 kinases. KO-947 blocks ERK signaling and proliferation of tumor cells exhibiting dysregulation of MAPK pathway signaling, including mutations in BRAF, NRAS or KRAS, at low nanomolar concentrations. KO-947 is differentiated from other published ERK inhibitors by an extended residence time and high potency in cell engagement that translate into prolonged pathway inhibition in vitro and in vivo. In cell-line derived xenograft studies, the drug profoundly suppresses ERK signaling for up to five days after a single dose and induces regressions in RAS- and RAF-mutant melanoma, NSCLC and pancreatic cancer models on administration schedules ranging from daily to weekly. Intermittent dosing enables comparable antitumor activity at reduced dose-intensity. PDX screening confirms and extends these findings to include RAS and BRAF mutant colorectal, gastric and cervical carcinoma models, and robust activity is also seen on both weekly and Q2D schedules in tumor models lacking BRAF/RAS mutations but with other dysregulation of the MAPK pathway. Thus, the favorable ADME properties of KO-947 enable the achievement of optimal antitumor activity with intermittent dosing, which may provide an opportunity to maximize the therapeutic window with flexible administration routes and schedules. These results demonstrate the potential clinical utility of KO-947 in MAPK pathway dysregulated tumors. Citation Format: Francis Burrows, Linda Kessler, Jeffrey Chen, Xin Gao, Rasmus Hansen, Shuangwei Li, Carol Thach, Levan Darjania, Yvonne Yao, Yi Wang, Ata Zarieh, Ke Yu, Tao Wu, Jingchuan Zhang, Dana Hu-Lowe, Liansheng Li, Pingda Ren, Yi Liu. KO-947, a potent ERK inhibitor with robust preclinical single agent activity in MAPK pathway dysregulated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2017-5168

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