Activation Induced Cytidine Deaminase (AID) is a DNA directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. Unlike site-specific recombinases (e.g. RAG1/2), AID lacks target site specificity and can generate DNA damage at widespread locations throughout the genome. In cancer cells, AID expression promotes high levels of DNA replication stress, and results in dependency upon the homologous recombination factor RAD51. We have shown previously that a novel RAD51 inhibitor, CYT01B, functions by disrupting RAD51 focus formation which reduces the nuclear concentration of RAD51 and promotes RAD51 protein degradation. Here we present the in vitro and in vivo pharmacological characterization of CYT01B. We first analyzed the permeability and stability of the compound using Caco-2 and liver microsome assays. CYT01B shows low efflux and is stable with low intrinsic clearance rates (< 30 ml/min/mg protein) in mouse, rat, dog, and human, but not in cynomolgus monkey liver microsomes. In vivo, CYT01B showed oral bioavailability that correlated well with Caco-2 permeability (36.9% in monkeys up to 86.5% in rats). Additionally, CYT01B exhibited a minimum half-life of 4 hours in all species tested (mouse, rat, dog, and monkey). We then examined off target liabilities by performing kinome inhibition and Panlabs safety panel screens. CYT01B showed negligible (<50%) inhibition across 371 kinases tested, while only two targets showed greater than 50% inhibition in the Panlabs panel. CYT01B displayed activity in several human-to-mouse cell line xenograft models. In a systemic model of chronic lymphoblastic leukemia (CLL), the tumor burden in the bone marrow of CYT01B-treated mice was reduced by approximately 30%. In two different subcutaneous engraftment models of either a Burkitt's lymphoma cell line (Daudi) or an acute lymphoblastic leukemia cell line (CCRF-SB), mice treated with CYT01B showed tumor growth inhibition of 49% and 88%, respectively. Lastly, CYT01B was well tolerated in rat 7-day dose range finding studies. Rats were treated with 20, 80, and 240 mg/kg once per day by oral gavage. There were no observed changes in hematology or clinical chemistry, and no observed histopathological toxicities. Taken together, these data demonstrate that CYT01B is a cell permeable, metabolically stable, and orally bioavailable small molecule, with anti-cancer activity in multiple preclinical lymphoid cancer models. Overall, this provides the basis for continued preclinical development of an AID/RAD51 synthetic lethal therapeutic paradigm that may be applicable to various hematologic malignancies.
Patrick:Cyteir Therapeutics: Consultancy. Bedard:Cyteir Therapeutics: Consultancy. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.