AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance

Abstract

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Figure 1. Chemical Structure of AP24534 and Co-Crystal Structure with ABL^T315I

(A) Chemical structure of AP24534. (B) Crystal structure of AP24534 in complex with the ABL^T315I mutant kinase. AP24534 is shown in green with translucent molecular surface. The side chain of the mutated gatekeeper residue Ile315 is shown in red. The side chains of Y253 and E255, locations of point mutations appearing in the resistant outgrowth screen of AP24534 are shown in grey. The C-helix is labeled (αC). (C) Key interactions of AP24534 with ABL^T315I at the ATP binding site. Hydrogen bonds are highlighted with pink dashed lines. Residues making critical contact with the imidazo[1,2b]pyridazine core and the ethynyl linker group of AP24534 are also labeled. (D) and (E) Superposition of imatinib and AP24534 highlighting the effect of the Thr to Ile mutation. The superposition was based on the C positions of ABL residues 312–321 in the T315I mutant and in native ABL kinase complexed with imatinib (shown in brown; PDB code 1IEP).

Figure 2. AP24534 Inhibits the Autophosphorylation of ABL and ABL^T315I

In vitro [γ-³²P]-ATP autophosphorylation of full-length (A) ABL or (B) ABL^T315I kinase treated with AP24534, imatinib, nilotinib, or dasatinib. After incubation of tyrosine-dephosphorylated enzyme with the indicated inhibitor in the presence of [γ-³²P]-ATP and separation by SDS-PAGE, signal intensity was measured by autoradiography.

Figure 3. AP24534 Inhibits BCR-ABL Signaling in CML Cell Lines Expressing Native BCR-ABL or BCR-ABL^T315I

(A) Immunoblot analysis of CrkL phosphorylation in Ba/F3 cells expressing native BCR-ABL treated with imatinib, nilotinib, dasatinib, or AP24534. Cells were cultured for 4 hr with inhibitors, harvested, lysed, and analyzed by immunoblot using an antibody for CrkL, a substrate of BCR-ABL whose phosphorylation is an established clinical marker of BCR-ABL kinase activity. Both the phosphorylated and non-phosphorylated forms are resolved by electrophoretic mobility, and bands are quantitated by densitometry and expressed as a % phosphorylated CrkL. (B) Immunoblot analysis of CrkL phosphorylation in Ba/F3 BCR-ABL^T315I cells treated with imatinib, nilotinib, dasatinib, or AP24534. Assays and analysis were carried out as described above in panel (A). Abbreviations: NT, no treatment.

Figure 4. Ex Vivo Treatment of CML Primary Cells with AP24534 Inhibits Cellular Proliferation and BCR-ABL-Mediated Signaling

(A) Cellular proliferation assays for ex vivo AP24534-treated mononuclear cells from CML myeloid blast crisis (M-BC) patients harboring native BCR-ABL (N=3) or BCR-ABL^T315I (N=1) and from healthy individuals (N=3). For reference, the dashed line indicates 50% cell viability relative to untreated cells. (B) Immunoblot analysis of CrkL phosphorylation in mononuclear cells from a CML lymphoid blast crisis (L-BC) patient harboring BCR-ABL^T315I following ex vivo exposure to AP24534, imatinib, nilotinib, or dasatinib. Cells were cultured overnight with inhibitors, harvested, lysed, and analyzed by CrkL immunoblot. Both the phosphorylated and non-phosphorylated forms are resolved by electrophoretic mobility, and bands are quantitated by densitometry and expressed as a % phosphorylated CrkL. (C) FACS analysis of global tyrosine phosphorylation in mononuclear cells from the CML L-BC BCR-ABL^T315I patient in panel (B). After overnight culture with inhibitors, cells were fixed and permeabilized, incubated with a FITC-labeled antibody for phosphorylated tyrosine, and analyzed by FACS. Values reported are as fold increase in mean fluorescence intensity relative to unstained controls. Abbreviations: NT, no treatment.

Figure 5. AP24534 is Effective in Mouse Xenograft Models of BCR-ABL-Driven and BCR-ABL^T315I-Driven Tumor Growth

(A) Effect of AP24534 on survival of SCID mice after intravenous injection of Ba/F3 cells expressing native BCR-ABL (left) or BCR-ABL^T315I (right). Ba/F3 cells expressing native BCR-ABL or BCR-ABL^T315I were injected into the tail vein of SCID mice, and animals were treated once daily by oral gavage with vehicle, AP24534, or dasatinib for the indicated dosing period (days 3–21). (B) In vivo efficacy and BCR-ABL signaling suppression by AP24534 in a subcutaneous xenograft model using Ba/F3 BCR-ABL^T315I cells. Tumor-bearing animals were treated once daily by oral gavage with vehicle or the indicated doses of AP24534 for 19 consecutive days (dosing period indicated) with mean tumor volume plotted (error bars represent S.E.M.). Each AP24354 treatment group was compared to the vehicle group using Dunnett’s test, with statistical significance (p<0.05) indicated by an asterisk. BCR-ABL and CrkL phosphorylation were evaluated by immunoblot in animals treated with a single oral dose of vehicle or 30 mg/kg AP24534 (N=3 per group).

Figure 6. Single-Agent AP24534 Completely Suppresses Resistant Outgrowth in Cell-Based Mutagenesis Screens

(A) Resistant clones recovered from ENU-treated Ba/F3 cells starting from native BCR-ABL cultured with graded concentrations of AP24534 (10, 20, 40 nM). Each bar represents the relative percentage of the indicated BCR-ABL kinase domain mutant among recovered subclones. Since the percentage of surviving resistant subclones and the concentration of AP24534 are inversely related, a different number of sequenced subclones are represented in the graph for each concentration of AP24534 (Table S2). The percent of wells surveyed that contained outgrowth is indicated to the right of each graph. (B) ENU-treated Ba/F3 BCR-ABL^T315I cells were cultured with graded concentrations of AP24534 (40, 80, 160, 320, 640 nM). Experiments and data analysis were performed as described in panel (A). All recovered subclones contain T315I in addition to the secondary mutation indicated on each graph.