Combinatorial treatment options for highly resistant compound mutations in the kinase domain of the <scp>BCR</scp>::<scp>ABL1</scp> fusion gene in Ph‐positive leukemias
Authors: Isabella Sponseiler, Anna‐Maria Bandian, Petra Pusic, Thomas Lion
Journal: American Journal of Hematology (2023)

The introduction of tyrosine kinase inhibitor (TKI)-based therapies has dramatically changed the outcome in patients with chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph + ALL).1 However, the occurrence of point mutations in the BCR::ABL1 kinase domain (KD) can impair or completely inhibit the efficacy of individual TKIs, and the 3rd generation compound ponatinib is currently the only agent capable of controlling leukemic cells carrying any individual point mutation in the BCR::ABL1 KD.2 The most recently approved 3rd generation compound asciminib, an allosteric inhibitor of the BCR::ABL1 kinase, is effective against cells carrying most mutations causing resistance to other TKIs, including the important gatekeeper mutation T315I, but different mutations at the residue F359 and a number of newly identified single mutations around the myristoylation site can cause resistance to this agent.3 In Ph + ALL and in advanced phases of CML, patients frequently develop compound mutations (CMs), defined as the presence of more than one mutation on the same BCR::ABL1 molecule in a leukemic cell, which are often highly or even completely resistant to any individual TKI.4, 5 Some CMs were shown to respond to high doses of the currently most potent available TKI, ponatinib, but dose-related toxicity of this agent requires judicious selection of adequate dosing regimens.4, 6 Moreover, the most frequently occurring CMs, which include the gatekeeper mutation T315I, are often completely resistant to any individual TKI available, including ponatinib and asciminib.3, 4 We and others have recently demonstrated in vitro that treatment approaches combining ponatinib with asciminib, hydroxyurea and other agents can suppress the growth of cells carrying CMs resistant to any single agent tested, and anecdotal observations in patients indicate that these effects can also be seen in the clinical setting.3, 7, 8 Our earlier findings indicated that certain CM constellations including the T315I mediate the upregulation of the cyclin-dependent kinase 6 (CDK6), thereby rendering the cells vulnerable to specific and non-specific inhibitors of this kinase, such as palbociclib and hydroxyurea, respectively, and the inclusion of these agents in combinatorial treatment approaches was indeed effective in vitro against certain compound mutant cells.7, 8 We set out to determine whether combinations of ponatinib with other drugs might also display efficacy against challenging CMs, with the aim to broaden the spectrum of treatment options in these settings. Moreover, we were interested in assessing the lowest drug concentrations required for growth suppression of various CM constellations, to account for dose-dependent toxicities of individual drugs, with a particular focus on ponatinib. To address these issues, we have performed in vitro analyses using a commonly employed cell line model based on BaF3 cells (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) expressing 12 judiciously selected CMs in BCR::ABL1 constructs, which had been introduced into the cells by a transposon-mediated approach we described earlier. In short, the pITR-ETP vector containing the full-length wildtype coding sequence of the human p210 BCR::ABL1 gene as well as the genes for puromycin resistance and the fluorescence protein dTomato was used as a template for site-directed mutagenesis by exploiting the QuikChange II XL Kit (Agilent Technologies, Santa Clara, CA, USA), to generate a panel of compound mutant constructs. The vector constructs were nucleofected (Cell Line Nucleofector® Kit V, Lonza, Basel, Switzerland) into BaF3 cells and subjected to sequential IL-3 and puromycin selection. For the enrichment of cells carrying a single mutant construct, monitoring by flow cytometry (BD LSRFortessa, BD Bioscience, Franklin Lakes, NJ, USA) and cell sorting according to fluorescence signal intensity was performed using a BD FACSAriaFusion instrument (BD Bioscience, Franklin Lakes, NJ, USA). The presence of single insertion copies of the mutant constructs is essential for precluding skewed test results potentially caused by multiple copies of the mutant construct in a relevant proportion of cells. The cells expressing various BCR::ABL1 CMs pre-tested in this way were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were exposed for 72 h to three different concentrations of ponatinib (10, 23, 28 nM) as a single agent and in combination with three different concentrations of other candidate inhibitors including asciminib (62.5, 125, 250 nM), hydroxyurea (25, 50, 100 μM), ibrutinib (625, 1250, 2500 nM), venetoclax (200, 400, 800 nM), vodobatinib (500, 1000, 2000 nM), and crizotinib (300, 600, 1200 nM) (all from MedChemExpress EU, Sollentuna, Sweden), as well as Palbociclib (50, 100, 200 nM), and ruxolitinib (250, 500, 1000 nM) (both from SelleckChemicals, Houston, TX, USA). All indicated compounds were also tested as single agents using the same concentrations, which corresponded to the spectrum of respective dosing regimens investigated or used routinely in the clinical setting. For the lead compound ponatinib, the concentrations tested reflected the average effective plasma concentrations achieved in patients with commonly used dosing regimens of 15, 30, and 45 mg daily, as indicated in Table 1.2 The responses to drug exposure were determined by a luminescent cell viability assay (CellTiter-Glo®, Promega GmbH, Walldorf, Deutschland), and the data were processed using the PRISM (GraphPad, Boston, USA) and Excel software (Microsoft Corporation, Redmond, USA). The responses were evaluated by determining the drug concentrations required for growth inhibition at defined levels, which were based on our earlier experience with these assays.4 The compound mutant cells were regarded as sensitive to individual drugs or drug combinations if the IC50 or even better responses were achievable at the respective drug concentrations tested. Conversely, the cells were deemed resistant if the highest drug concentrations used revealed residual cell viability above 60% (i.e., responses worse than IC40) after the standardized exposure time. Partial resistance was defined by the best achievable response values between IC50 and IC40, as specified in Table 1 showing a heatmap established by compiling the readouts of multiple independent measurements. The cell viability assays performed indicated that CMs responding only to elevated concentrations of ponatinib could be suppressed by combinatorial treatment with low concentrations of ponatinib and different other agents tested. Moreover, CMs displaying high levels of resistance to all available TKIs applied as single agents, mostly revealed adequate in vitro responses to individual drug combinations with ponatinib, most commonly including asciminib, hydroxyurea, or crizotinib. Only the most resistant CM constellation tested, T315I/F359V, failed to respond to the compounds investigated and only showed a limited response to the combination of ponatinib and hydroxyurea at high concentrations of both drugs. The failure to respond to combinations of ponatinib with asciminib at any concentration tested might be attributable to the fact that asciminib is apparently ineffective against mutations at the amino acid residue F359.3 A good response of this CM in vitro was only achieved by a combination of ponatinib with a very high concentration of crizotinib (Table 1), which might be too toxic in the clinical setting. This CM therefore requires further testing by additional candidate drugs. Overall, combinatorial treatment of BCR::ABL1 CMs indicated that relatively low doses of the drugs used are capable of effectively inhibiting in vitro survival of mutant cells in most instances. Thus far, there are only anecdotal preliminary observations on the efficacy of some indicated combinatorial treatment options against BCR::ABL1 CMs in the clinical setting. However, since our earlier data indicated a good correlation between in vitro test results and clinical responses,4, 7, 8 the present observations may serve as a basis for effective treatment options in patients with Ph + leukemias displaying BCR::ABL1 KD-mutations with challenging CM constellations. The study was performed with financial support from Incyte. Thomas Lion has received research funding and honoraria from Incyte and Novartis as well as honoraria from Angelini, Pfizer, and Bristol Myers Squibb. The other authors have no relevant disclosures. The data underlying the results presented are available as part of the article and no additional source data are required.