ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

Abstract

Background: Chronic myelogenous leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) are caused by the t(9;22)(q34;q11.2) chromosome translocation, resulting in fusion of the BCR and ABL1 genes on the Philadelphia chromosome to encode constitutively active ABL1 kinase. Despite the dramatic progress made over the past decade with tyrosine kinase inhibitors (TKIs) in the treatment of CML, allogeneic stem cell transplant is considered the only proven curative therapy. To achieve cure or benefit from treatment-free remissions with pharmacologically-based therapies, it is estimated that patients will likely need to achieve a sustained reduction in tumor burden of 4 logs (MR4) or deeper (MR4.5). Currently, only 39% and 18% of patients achieve MR4 by 24 months of treatment with single agent nilotinib or imatinib, respectively. Furthermore, for a subset of CML patients and the majority of Ph+ ALL patients, resistance develops to current TKI’s as a result of emergence of point mutations in the ATP site of the kinase domain. ABL001 is a potent, selective BCR-ABL inhibitor that maintains activity across most mutations, including T315I, with a distinct, allosteric mechanism of action which recently entered Phase I development for the treatment of patients with CML and Ph+ ALL. ABL001 was developed to be dosed in combination with nilotinib to provide greater pharmacological coverage of BCR-ABL disease and prevent the emergence of resistance. Methods: Based on X-ray crystallography, NMR and molecular modeling, ABL001 is the result of a structure-guided medicinal chemistry program targeting the myristoyl pocket of the ABL1 kinase. In vitro cell based assays were performed using the Ba/F3 isogenic cell system and a panel of over 300 cell lines. KCL-22 cells were used to develop an in vivo xenograft model to assess the efficacy of ABL001 and the PD marker, pSTAT5, was used to monitor the inhibition of BCR-ABL signaling. Results: In contrast to TKIs that bind to the ATP-site of the ABL1 kinase domain, NMR and X-Ray crystallography studies confirmed that ABL001 binds to a pocket on the BCR-ABL kinase domain that is normally occupied by the myristoylated N-terminus of ABL1. Upon fusion with BCR, this myristoylated N-terminus that serves to autoregulate ABL1 activity is lost. ABL001 functionally mimics the role of the myristoylated N-terminus by occupying its vacant binding site and restores the negative regulation of the kinase activity. Cell proliferation studies demonstrate that ABL001 selectively inhibited the growth of CML and Ph+ ALL cells with potencies ranging from 1-10nM range. In contrast, BCR-ABL-negative cell lines remained unaffected at concentrations 1000-fold higher. With resistance emerging in the clinic to current TKI’s as a result of point mutations in the ATP-site, ABL001 was tested for activity against clinically observed mutations and found to be active in the low nM range. In the KCL-22 mouse xenograft model, ABL001 displayed potent anti-tumor activity with complete tumor regression observed and a clear dose-dependent correlation with pSTAT5 inhibition. The KCL-22 xenograft model was also used to compare the dosing of ABL001 and nilotinib as single agents to dosing a combination of ABL001 and nilotinib. Single agent dosing regimens led to tumor regressions; however, despite continuous dosing, all tumors relapsed within 30-60 days with evidence of point mutations in the resistant tumors. In contrast, animals treated with the combination of ABL001 and nilotinib achieved sustained tumor regression with no evidence of disease relapse either during the 70 days of treatment or for > 150 days after treatment stopped. Conclusion: ABL001 selectively inhibited the proliferation of cells expressing the BCR-ABL fusion gene and was active against clinically important mutations that arise with current TKI therapy in CML. In an in vivo model of CML, the combination of ABL001 and nilotinib resulted in complete and sustained tumor regression with no evidence of disease relapse. These results provide proof-of-principle that simultaneous targeting of the myristoyl pocket and ATP-pocket by ABL001 and nilotinib, respectively, promotes a more sustained overall efficacy and prevents the emergence of resistance via acquisition of point mutations in the respective binding sites. ABL001 is currently being evaluated in a Phase 1 study in patients with CML and Ph+ ALL. Disclosures Wylie: Novartis Institutes for Biomedical Research, Inc: Employment. Schoepfer:Novartis Institutes for Biomedical Research: Employment. Berellini:Novartis Institutes for Biomedical Research: Employment. Cai:Novartis Institutes for Biomedical Research: Employment. Caravatti:Novartis Institutes for Biomedical Research: Employment. Cotesta:Novartis Institues for Biomedical Research: Employment. Dodd:Novartis Institutes for Biomedical Research: Employment. Donovan:Novartis Institutes for Biomedical Research: Employment. Erb:Novartis Institutes for Biomedical Research: Employment. Furet:Novartis Institutes for Biomedical Research: Employment. Gangal:Novartis Institutes for Biomedical Research: Employment. Grotzfeld:Novartis Institutes for Biomedical Research: Employment. Hassan:Novartis Institutes for Biomedical Research: Employment. Hood:Novartis Institutes for Biomedical Research: Employment. Iyer:Novartis Institutes for Biomedical Research: Employment. Jacob:Novartis Institutes for Biomedical Research: Employment. Jahnke:Novartis Institutes for Biomedical Research: Employment. Lombardo:Novartis Institutes for Biomedical Research: Employment. Loo:Novartis Institutes for Biomedical Research: Employment. Manley:Novartis Institutes for Biomedical Research: Employment. Marzinzik:Novartis Institutes for Biomedical Research: Employment. Palmer:Novartis Institutes for Biomedical Research: Employment. Pelle:Novartis Institutes for Biomedical Research: Employment. Salem:Novartis Institutes for Biomedical Research: Employment. Sharma:Novartis Institutes for Biomedical Research: Employment. Thohan:Novartis Institutes for Biomedical Research: Employment. Zhu:Novartis Institutes for Biomedical Research: Employment. Keen:Novartis Institutes for Biomedical Research: Employment. Petruzzelli:Novartis Institutes for Biomedical Research: Employment. Vanasse:Novartis: Employment, Equity Ownership. Sellers:Novartis: Employment.

The new allosteric inhibitor asciminib is susceptible to resistance mediated by ABCB1 and ABCG2 overexpression in vitro

“…Preclinical modeling in Ba/F3 cells [ 23 , 29 ] indicates that asciminib is transported by ABCB1, however, we now show that asciminib is also transported by ABCG2. Asciminib-mediated cell death was evaluated in K562-Dox (ABCB1 overexpressing) and K562-ABCG2 overexpressing cells compared with parental K562 cells (negligible ABCB1 and ABCG2 expression).…”

“…Native c-Abl1 contains a myristate moiety that functions as an auto regulator; however, the myristate group is lost upon fusion to Bcr causing the constitutive activation associated with Bcr-Abl [ 20 , 21 ]. Asciminib and other allosteric inhibitors mimic the myristate group locking Bcr-Abl in an inactive conformation and inhibiting kinase activity [ 22 , 23 ]. Following preclinical modelling, which demonstrated both sustained elimination of tumors in a mouse model of leukemia (when used in combination with nilotinib) and activity against clinically relevant kinase domain mutations in vitro [ 23 ], asciminib entered open label phase I clinical trial for patients with refractory CML or Ph+ ALL ( http://clinicaltrials.gov/show/NCT02081378 ) alone and in combination with imatinib or nilotinb or dasatinib.…”

Relative Bioavailability and Food Effect Evaluation for 2 Tablet Formulations of Asciminib in a 2‐Arm, Crossover, Randomized, Open‐Label Study in Healthy Volunteers

“…5 The KCL-22 cell line is a blast crisis (CML-BC) Ph+ cell line with no BCR-ABL mutations. 5 In a KCL-22 murine subcutaneous xenograft model, when asciminib was administered as a single agent, tumor regression was observed at doses of 7.5 mg/kg twice daily and above. Efficacy in the KCL-22 xenograft model correlated with the complete inhibition of the downstream pharmacodynamic (PD) marker STAT5.…”

“…6 Asciminib displays potent antitumor activity in vivo, with a clear pharmacokinetic (PK)/PD/efficacy relationship. 5 Recent phase 1 clinical trials on asciminib in patients with CML and Ph+ ALL have shown that in patients intolerant or resistant to at least 2 TKIs, a majority of the patients achieved major molecular response after 12 months of treatment. 7 Based on the results from a first-in-human study, the 40-mg twice-daily dose of asciminib was declared the recommended dose in patients with chronic myeloid leukemia in chronic phase (CML-CP) without T315I mutations.…”

Combating mutations in genetic disease and drug resistance: understanding molecular mechanisms to guide drug design

“…ABL001 binds to the myristoyl pocket of ABL1 kinase leading to a formation of an inactive kinase conformation 84 . Recent studies have shown that treatment with ABL001 combined with ATP-competitive inhibitors can help prevent resistance in chronic myeloid leukemia 85,86 .…”