Single Cell Signaling Pharmacodynamics in a Phase 1b Trial of the Axl Inhibitor BGB324 in Acute Myeloid Leukemia
Authors: Hellesøy Monica , Gullaksen Stein-Erik , Tislevoll Benedicte Sjo , Fagerholt Oda Helen Eck , Reikvam Hakon , Loges Sonja , Cortes Jorge , Heuser Michael , Chromik Jörg , Skavland Jørn , Sulen André , Bruserud Øystein , Lorens Jim , Gausdal Gro , Micklem David , Gjertsen Bjorn T. 
Published: October 2019

Abstract
Axl is a receptor tyrosine kinase that has been shown to have a strong oncogenic potential in many cancer types. Overexpression and activation of Axl is found in many cancers, and is linked to increased proliferation, migration/invasion and resistance to apoptosis. Axl overexpression has been shown to be a poor prognostic marker, and recently overexpression of Axl has also been linked to the acquired resistance to chemotherapy and other anticancer therapies in many malignancies, including AML.
BGB324 (BerGenBio AS) is a first-in-class highly specific small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in a clinical safety trial in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in a phase 1b clinical trial in patients with refractory/relapsed AML and MDS (BGBC003, ClinicalTrials.gov Identifier:NCT02488408; Loges S et al. J Clin Oncol 34, 2016 suppl; abstr 2561). 20 AML and 4 MDS patients have been treated at the following dose levels (loading dose/continuation dose): 400/100mg, 600/200mg and 900/300mg. Objective responses were observed in 2/4 MDS patients and 2/20 AML patients including one CR (AML). Enrollment continues to define MTD.
The effect of BGB324 on intracellular signaling and the immune profile of leukemic blasts in patients treated in the clinical study was investigated using phospho-flow cytometry. Blasts were identified using surface markers (CD45low, CD66b-, CD38-, and CD117+ and/or CD34+), and the following direct and indirect downstream targets of Axl were explored: phosphorylated (p)-Akt(S473 and T308), pErk(T202/Y204), pp38(T180/Y182), pPLCγ1(Y783), pNFκB(S529), pCREB(S113) and pSTAT1(Y701), 3(Y705), 5(Y694)and 6(Y641). Preliminary analyses of blood samples from six patients show very rapid responses in signaling pathways downstream of Axl (including Akt, Erk, NFκB and PLCγ1) within hours or days of ingestion of the first dose, although the response patterns varies from patient to patient (Figure 1A). Two distinct blast populations were identified: one CD117+/CD34- and one CD117+/CD34+. In most patients the CD117+/CD34- population displayed the most extensive signaling changes during treatment, and this population also decreased during treatment with BGB324. In contrast, the CD117+/CD34+ population expanded during the course of the treatment (Figure 1B).
White cell differential counts of peripheral blood from two patients treated with BGB324 for a prolonged period of time (15 weeks or more) showed a decrease in peripheral blast count, and a corresponding increase in granulocyte and monocyte counts, suggesting that Axl inhibition may push the blasts towards differentiation.
The clinical trial is ongoing, and the signaling profile of leukemic blasts in blood and bone marrow of treated patients will be further examined by conventional phosphoflow cytometry and mass cytometry searching for signaling profiles with prognostic information.
In conclusion, BGB324 has unique pharmacodynamic properties and molecular responses to exposure can be observed in peripheral blood leukemic blasts by phospho-flow cytometry within hours of ingestion of the first treatment dose. Further studies may establish whether single cell signal profiling can discriminate responders from non-responders and provide information about dose-response in a clinically meaningful way.

Disclosures
Cortes: Astellas: Research Funding; Arog: Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Ambit: Research Funding. Heuser:Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Lorens:BerGenBio AS: Employment, Equity Ownership, Research Funding. Gausdal:BerGenBio AS: Employment. Micklem:BerGenBio AS: Employment, Equity Ownership. Gjertsen:BerGenBio AS: Consultancy, Research Funding.