Establishment and Characterization of Mouse Models with Primary and Acquired BTK Inhibitor Resistance

Background: The second-generation Bruton's tyrosine kinase (BTK) inhibitor acalabrutinib is effective for treatment of patients with chronic lymphocytic leukemia (CLL), including high-risk and relapsed patients. Resistance to BTK inhibitors can develop not only due to on-target mutations, but also by bypassing the BTK signaling pathway. Mouse models represent useful tools in studying mechanisms of resistance as well as testing the efficacy of alternative therapy options to overcome resistance. Eµ-TCL1-transgenic (TCL1-tg) mice have the advantage of immunocompetence and resemble the human CD49d-high CLL cohort (Szenes et al., 2020). Transplantation of tumors from these mice to wild-type recipients allows acceleration of pathophysiology, selection of specific tumor clones, and more efficient testing of drug combinations. Here, we developed acalabrutinib resistance models by serial transplantation and long-term administration of the drug in drinking water, characterized these models, and compared molecular resistance signatures with those of human patient samples.

Methods: Models were developed starting from four primary TCL1-tg mice at overt disease with tumor burden in spleen ranging up to 95% of lymphocytes. Serial retransplantation to wild-type C57BL/6J recipients for two to four rounds and acalabrutinib therapy pressure resulted in several drug resistant pedigrees. Mice were sacrificed at end-stage disease and tumor burden in spleen, bone marrow and blood was analyzed by flow cytometry using anti-CD5/CD19 antibodies. Purified leukemic cells were subjected to RNA-seq, real-time quantitative PCR and mutational analyses.

Results: Three response patterns were identified in recipient mice: a) sustained sensitivity (models were stopped after 23 weeks of treatment), b) acquired resistance (with initial response followed by development of resistance under continuous therapy pressure and c) retransplantation-induced resistance in a case of an aggressive tumor clone that was responding to acalabrutinib in the parental mouse. The sensitive models displayed a 63% reduction in spleen weight upon treatment and a concomitant 65% reduction in blood tumor burden. In acquired or primary resistant mice, spleen weight and tumor burden was not significantly different from that of untreated mice, with blood tumor burden in the magnitude of 100,000 leukemic cells/µl and spleen tumor burden of 500-1000 million leukemic cells. Resistance could not be attributed to the mutational landscape of BTK and PLCγ2. Mutations in p53, Myc, Notch1, Atm, and Sf3b1 were detected but found to be intrinsic in individual ancestors and further present in the follow-up transplantations. RNA-seq analysis revealed 863 differentially expressed genes in mice with acquired resistance to acalabrutinib (compared to untreated, Figure 1) and 114 shared differentially expressed genes in mice with acquired or transplantation-based resistance. Gene set enrichment analysis demonstrated the presence of MAPK signaling and NF-kB signaling gene signatures within the resistant tumors. In particular, acquired resistance was accompanied by changes in the B cell receptor downstream components. We also compared the murine 863 gene signature to human samples from acalabrutinib resistant patients (progression on drug versus baseline) and found 13 genes are differently expressed in both, human and mouse. Validation for this gene expression data is ongoing and updated results will be presented.

Conclusion: We have developed novel TCL1-tg based murine models of acalabrutinib resistance. Our models provide well-characterized tools to study primary as well as acquired BTK inhibitor resistance and allow the current testing of potential new therapeutic approaches to overcome resistance.

Polcik:AstraZeneca: Research Funding. Dannewitz Prosseda:AstraZeneca: Research Funding. Parsa:AstraZeneca: Current Employment. Bibikova:Acerta Pharma: Current equity holder in publicly-traded company; AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wiestner:Acerta Pharma: Research Funding; Merck: Research Funding; Abbvie company: Research Funding; Pharmacyclics: Research Funding; Nurix: Research Funding; GenMab: Research Funding; Verastem: Research Funding. Hartmann:AstraZeneca: Research Funding.