SBIR-STTR Award

Somatic kRAS mutations, termed oncogenic kRas, are found at a high rate in many cancers. For example, in colon cancer, the rate is 40%. Mutated KRAS allows the EGF system to bypass its natural controls and operate continuously, locked in a pro-growth mode (wild-type kRas, but not oncogenic KRAS, is self- inactivating). Consistent with this mechanism, the presence of mutated KRas predicts lack of response to anti- EGF monoclonal antibody therapeutics such as panitumumab (Vectibix) and cetuximab (Erbitux) in colorectal cancer. The goal of this grant is to discover compounds that specifically inhibit oncogenic kRas without affecting normal kRAS. Such molecules would be expected to restore the anti-cancer efficacy of anti-EGF therapeutics such as panitumumab and cetuximab when administered to patients with oncogenic kRas. Unfortunately, many previous efforts to target oncogenic KRas have failed, and kRAS has been declared an 'intractable' target by many cancer researchers. However, we recently developed a unique screening model using a genetically modified D. melanogaster that expresses mutant, oncogenic kRAS (G12V) in their wings while maintaining normal kRAS in the rest of their bodies. We plan to use this fly as a screening platform to search for drugs that inhibit mutant kRAS while not affecting wild-type kRAS. The phenotype for the mutated fly is a 'crimped' wing, and a 'hit' in the assay would be a compound that prevents formation of the crimped wing defect. The use of the fly permits screening in a whole animal, which is expected to produce more reliable 'hits' than screening in cell culture or in biochemical assays. In addition, the whole animal model permits a preliminary evaluation of safety, because only compounds that permit development of normal, healthy flies while blocking the mutant kRAS in their wings will be selected as 'hits'. Compounds toxic in flies will not pass the screen. Activities to be funded by the grant include further characterizing the transgenic fly and the assay, establishing an assay to differentiate inhibitors of oncogenic and wild-type kRAS, screening for potential inhibitors, structural optimization of 'hits', and characterization of optimized 'hits' in PK, safety and kRAS-relevant xenograft studies.

Thesaurus Terms:
Absorption;Affect;Animal Model;Animals;Base;Biochemical;Biological Assay;Bypass;Cancer Patient;Cancer Therapy;Cell Culture System;Cell Culture Techniques;Cell Proliferation;Cetuximab;Chemicals;Colon Carcinoma;Colorectal Cancer;Data;Defect;Development;Drosophila Melanogaster;Drug Candidate;Drug Formulations;Egf Gene;Epidermal Growth Factor Receptor;Erbitux;Evaluation;Excretory Function;Fly;Funding;Future;Genes;Goals;Grant;Growth;Guanosine Triphosphate Phosphohydrolases;Human;Inhibitor/Antagonist;Kras2 Gene;Label;Libraries;Location;Malignant Neoplasms;Map Kinase Gene;Metabolism;Modeling;Monkeys;Mus;Mutant;Mutate;Mutation;Oncogenic;Operating System;Panitumumab;Patients;Performance;Pharmaceutical Preparations;Phase;Phenotype;Poisons;Pre-Clinical;Prevent;Property;Proto-Oncogene Proteins C-Akt;Public Health Relevance;Reproducibility;Research Personnel;Response;Rest;Safety;Safety Study;Scaffold;Scale Up;Screening;Signal Transduction;Small Business Innovation Research Grant;Small Molecule;Small Molecule Libraries;Success;System;Testing;Therapeutic;Therapeutic Monoclonal Antibodies;Toxic Effect;Transgenic Organisms;Tumor;Update;Vectibix;Wing;Writing;Xenograft Model;Xenograft Procedure;

kRAS is a GTPase which is the main mediator (on/off) of downstream signaling from the EGF receptor (EGF-R) on the surface of cells. kRAS controls major signaling systems such as the RAF/MEK1/2/ERK1/2 cell proliferation pathway and the PI3K/AKT/mTOR cell survival pathway. Somatic kRAS mutations, termed oncogenic kRAS, are common in many cancers: 90% incidence in pancreatic, 45% in colorectal, and 35% in lung cancer. Mutated kRAS allows the EGF-R system to bypass its natural controls and operate nearly continuously in a pro-growth mode. Wild-type kRAS is self-inactivating, while oncogenic kRAS is not. Consistent with this mechanism, the presence of G12V mutated kRAS blocks the efficacy of monoclonal antibody drugs against EGF-R, such as panitumumab (Vectibix®) and cetuximab (Erbitux®), which are used to treat colorectal cancer, head and neck, and other cancers. Tosk’s Phase I SBIR research used a proprietary, genetically modified Drosophila melanogaster strain that expresses G12V kRAS in its wings to identify two chemical scaffolds (“hits”) that suppress kRAS- related activity. In addition to phenotype reversal of anti-G12V activity in the mutant fly, the hits were tested in relevant cell culture, protein kinase, computational active site docking, RAF, and SOS pull down assays. Assessment of ERK activity, xenograft model studies, and short-term safety and PK studies were also performed. Information about the presence, location, and activity of the mutant kRAS gene was also obtained. The repeatability and reproducibility of the G12V fly screens were evaluated and confirmed. Although not conclusive, MOA studies, including knockout studies in the G12V-expressing fly, strongly suggest that the hits inhibit kRAS activity in the RAS/RAF/MEK/ERK pathway. Computational docking studies further suggest a direct interaction of the hits with kRAS, possibly leading to interference with the kRAS-RAF protein-protein interaction. The primary goals of this Phase II SBIR application are to: (1) perform additional screening using an improved version of the Phase I fly assay to discover new kRAS inhibitors, (2) optimize and further characterize the two inhibitors discovered in the SBIR Phase I and any newly discovered inhibitors from Phase II, and (3) use mechanism, efficacy, safety, and pharmacokinetic studies to select a candidate and one back-up ready for IND-enabling tests needed to file an IND for an inhibitor of mutant kRAS.

Public Health Relevance Statement:
Principal Investigator: Garland, WA NARRATIVE The presence of G12V mutated kRAS blocks the response to epidermal growth factor receptor (EGFR) antibody drugs such as panitumumab (Vectibix®) and cetuximab (Erbitux®), which otherwise are effective cancer therapies. Tosk’s Phase I SBIR research used a proprietary, genetically modified Drosophila melanogaster strain that expresses G12V kRAS in its wings to identify two chemical scaffolds (“hits”) that suppress kRAS-related activity. The goal of this Phase II SBIR application is to build on the Phase I SBIR findings to identify a candidate suitable for Tosk-funded IND-enabling studies for a small molecule kRAS suppressor drug. A successful Tosk anti-mutant kRAS drug should be an effective companion treatment for the approximately 40% of cancer patients currently unable to benefit from EGFR drugs. Such a kRAS inhibiting drug should also prove effective in treating patients with mutant kRAS positive tumors, which are common in pancreatic, colorectal, and lung cancers.

Project Terms:
Active Sites; ADME Study; Back; base; Biological Assay; Bypass; Cancer Patient; cancer therapy; Cell Culture Techniques; Cell Proliferation; Cell surface; Cell Survival; Cetuximab; Chemicals; Clinical; Colorectal; Colorectal Cancer; Companions; design; Development; Docking; Drosophila genus; Drosophila melanogaster; Drug Kinetics; Drug Receptors; EGF gene; Epidermal Growth Factor Receptor; Erbitux; Extramural Activities; Fc Receptor; fly; FRAP1 gene; Funding; Genes; Goals; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Head and neck structure; improved; in vivo; Incidence; inhibitor/antagonist; insight; Knock-out; Letters; Location; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Malignant Neoplasms; MAP2K1 gene; MAPK3 gene; Mediator of activation protein; meetings; MEKs; Monoclonal Antibodies; Mus; mutant; Mutate; Mutation; Oncogenic; Pancreas; panitumumab; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; PI3K/AKT; Principal Investigator; programs; Protein Kinase; protein protein interaction; Protocols documentation; Ras/Raf; Reproducibility; Research; Research Personnel; Resources; response; S Phase; Safety; safety study; scaffold; screening; Signal Transduction; Small Business Innovation Research Grant; small molecule; small molecule libraries; Son of Sevenless Proteins; Study models; System; Testing; Therapeutic Uses; tumor; Vectibix; Vendor; Wing; Writing; Xenograft Model