Resistance to Danusertib (formerly PHA-739358) in BCR-ABL-Positive Cells Is Mediated by Upregulation of the Drug Transporter Abcg2 and Can Be Suppressed in Vitro by Combination Treatment with Imatinib.
Authors: Brummendorf Tim H , Gontarewicz Artur , Keller Gunhild , Moll Jürgen , Braig Melanie , Rohe Imke , Jost Edgar , Galm Oliver , Bokemeyer Carsten , Holyoake Tessa , Balabanov Stefan 
Published: October 2019

Abstract
Abstract 1724
Poster Board I-750
The success of imatinib (IM, formerly STI571, Gleevec®) in the treatment of chronic myeloid leukemia (CML) is compromised by the development of primary or acquired IM-resistance, particularly in advanced phase disease as well as by a limited IM-effect on immature hematopoietic stem cells, emphasizing the need for novel therapeutic strategies. The small molecule inhibitor Danusertib (formerly PHA-739358) potently inhibits Aurora and ABL kinases. Here, the individual contribution of each pathway to the effect of Danusertib was investigated. Starting at very low concentration, a dose-dependent reduction of BCR-ABL activity was observed, whereas inhibition of Aurora kinase activity, assessed by phosphorylation of histone H3-Ser10, required substantially higher concentrations. In primary CD34+ CML cells, including initially quiescent leukemic stem cells, combination therapy with IM and Danusertib revealed a synergistic anti-proliferative activity, which also affected immature CD34+38- cells. Neither mono- nor combination therapy led to substantial induction of apoptosis in quiescent stem cells. Interestingly, under treatment with Danusertib, the emergence of resistant clones in a well-established murine Ba/F3-p210 cell model was considerably less frequent than with IM. Surprisingly, Danusertib-resistant cells did not have mutations in BCR-ABL or Aurora kinase domains and remained IM-sensitive. Analysis of resistance mechanisms using DNA-microarray suggests an overexpression of Abcg2 efflux transporter to be causative for the resistance arising under Danusertib treatment. In support of this finding, stable retroviral overexpression of Abcg2 in parental Ba/F3-p210 cells induced a resistant phenotype against Danusertib. Furthermore, the Abcg2 inhibitor Fumitremorgin C (FTC) could restore the sensitivity of resistant cells to Danusertib. Finally, significant re-expression of Abcg2 in parental Ba/F3-p210 cells upon treatment with the demethylating agent 5-Azacytidine suggests that an epigenetic mechanism might play a role in the regulation of Abcg2 gene expression in resistant clones. Detailed analyses of the methylation patterns of the Abcg2 promoter region are currently being performed.
In conclusion, simultaneous in vitro exposure of Ba/F3-p210 cells to Danusertib and IM significantly reduced the emergence of drug resistance, raising hope that both epigenetic modulation of drug transporters involved in development of resistance as well as hypothesis-driven combinations of kinase inhibitors may eventually achieve durable disease control even in 2nd and 3rd line treatment of CML.

Disclosures
Brummendorf: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Moll:Nerviano MS: Employment. Jost:MSD: Research Funding. Bokemeyer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holyoake:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Balabanov:Novartis : Research Funding; Bristol Myers Squibb: Research Funding.