BGB324 Inhibits BCR-ABL TKI-Resistant Chronic Myeloid Leukemia
Authors: Ben Batalla Isabel , Erdmann Robert , Jørgensen Heather , Mitchell Rebecca , Ernst Thomas , von Amsberg Gunhild , Schafhausen Philippe , Rankin Stephen , Clark Richard E. , Koschmieder Steffen , Schultze Alexander , Mitra Subir , Vandenberghe Peter , Brümmendorf Tim H. , Carmeliet Peter , Helgason Gudmundur , Hochhaus Andreas , Pantel Klaus , Bokemeyer Carsten , Holyoake Tessa L , Loges Sonja 
Published: October 2019

Abstract
Resistance and CML stem cell persistence preclude cure for the majority of patients treated with tyrosine kinase inhibitor (TKI) therapies.
We demonstrated that the receptor tyrosine kinase (RTK) Axl of the Tyro-3, Axl, Mer (TAM) family is expressed by TKI-sensitive and -resistant CML cells (Erdmann R. et al. ASH 2013 and 2014). We have shown that blockade of the Gas6-Axl axis by the small molecule Axl inhibitor BGB324 (BerGenBio) represents a therapeutic target in AML. We are currently investigating BGB324 in a Phase 1b trial in refractory AML patients and in those non-eligible for intensive chemotherapy (BGBC003, NCT02488408). We hypothesised that Axl represents a tractable therapeutic target even in the most resistant forms of CML.
Upon treatment with imatinib KCL-22 and K562 cells showed upregulation of Axl at the protein level indicating that Axl might be involved in resistance towards TKIs in CML. Consistently, Axl levels were higher in MNCs of TKI-resistant patients compared to -sensitive patients after 6 months of treatment (n=17/20, 1±0.4 vs. 0.16±0.03; *p<0.05). Upon combined treatment of KCL-22 and K562 cells with BGB324 and imatinib we detected an additive effect of growth inhibition (KCL-22 cells; n=3, viability 66.0±0.5% BGB324, 52.0±1.1% imatinib, 42.3±1.5% combo; combo vs. IM *p<0.01 and combo vs. BGB324 *(p<0.0001), and not shown). Analysis of intracellular signal transduction in these cell lines indicated that Axl induces phosphorylation of Stat5 by BCR-ABL independent pathways because we detected an additive effect of inhibition of Stat5 phosphorylation when combining imatinib and BGB324. We could not detect an additive inhibitory effect on phosphorylation of Erk and Akt. Consistently, combined BCR-ABL and Axl blockade by means of imatinib and shRNA respectively, demonstrated an additive effect in reducing cell viability in KCL-22 and K562 cells (KCL-22 cells; n=3, viability 84.5±0.8% shControl+imatinib, 74.5±2.6% shAxl, 50.4±0.9% shAxl+imatinib; shAxl+IM vs. IM *(p<0.0001) and vs. shAxl *(p<0.001) and not shown).
We next investigated Axl activation in TKI insensitive BCR-ABL+ cell lines. In addition we tested a novel Ponatinib-resistant cell line KCL-22 PonR generated by subcloning parental KCL-22 in increasing concentrations of ponatinib. BCR-ABL is unmutated in these cells; oncoprotein kinase activity is switched off but cell death is not induced with 2mM ponatinib. We found that Axl phosphorylation was higher in the TKI-resistant cell lines BaF3/T315I, KCL-22 T315I and KCL-22 PonR when compared to the parental cell lines (n=3, 139±3.8% KCL-22 T315I, 214±1.3% KCL-22 PonR with respect to KCL-22 WT, *p<0.001 for both comparisons; 169±8.7% with respect to BaF3/p210, *p<0.005). Treatment with BGB324 inhibited cell proliferation with an IC50of 726, 3178 and 2720nM for BaF3/T315I, KCL-22 T315I and KCL-22 PonR, respectively. BGB324 could induce apoptosis and reduce proliferation in these cell lines. Furthermore, BGB324 blocked growth of colonies and induced apoptosis of T315I-mutated and pan-TKI-resistant (including ponatinib) primary CML MNCs.
The finding that BGB324 inhibits TKI-resistant CML was further corroborated with KCL-22 T315I mutated and KCL-22 PonR xenograft models. In both models we observed a significant tumor growth reduction upon treatment with 25 mg/kg BGB324 twice daily compared to placebo leading to a 34% and 58% reduction in tumor volume, p=0.0044, p=0.0021 for KCL-22 T315I and KCL-22 PonR, respectively). Cell proliferation was quantified by pHH3 analysis indicating a significant reduction in KCL-22 T315I and KCL-22 PonR tumors (n=8, 242.8±10.9 vs. 182.2±8.1, *p<0.0001; n=9, 259.5±9.3 vs. 213.2±6.8; *p<0.0001, respectively). Furthermore, we observed a significant decrease of Axl, Erk and Stat5 phosphorylation after treatment with BGB324 for 8 days.
We also investigated the therapeutic effect of BGB324 in a systemic model, by transplantation of KCL-22 PonR into sublethally irradiated NSG mice. In this model, treatment with 25 mg/kg BGB324 twice daily resulted in significant prolongation of overall survival (median OS 36 days (control) vs 43 days (BGB324), n=5 *p<0.05).
In summary, our data highlight the advantage to be gained from inhibition of Axl even in the most resistant CML cells, and support the need for human clinical trials of the novel inhibitor BGB324 alone and in combination with TKIs.

Disclosures
Schafhausen: Novartis: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hochhaus:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Holyoake:Novartis: Research Funding; BMS: Research Funding. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding.