Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency

Abstract

Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.

Figure 1. Sequential use of tyrosine kinase inhibitors can select for cells harboring compound kinase domain mutations.

Schematic of sequential kinase inhibitor therapy and predicted BCR-ABL kinase domain (KD) genotypes of dasatinib-resistant cases treated after imatinib failure. Blue box represents imatinib-resistant, dasatinib-sensitive mutation.

Figure 2. The dasatinib-resistant V299L mutation is clinically sensitive to imatinib and nilotinib.

(A) wbc count (blue) and peripheral blood myeloblast percentage (red) in an accelerated-phase CML patient (AP-1) who developed dasatinib resistance associated with the V299L mutation in BCR-ABL and responded to imatinib. (B) CRKL immunoblot performed after exposure of leukocytes isolated from patient AP-1 (harboring BCR-ABL/V299L) to varying concentrations of imatinib. (C) wbc count in a second accelerated-phase CML patient (AP-2) who developed resistance to dasatinib due to the V299L mutation and was sequentially treated with imatinib and nilotinib. D, dasatinib; I, imatinib; NI, nilotinib.

Figure 3. Additive BCR-ABL inhibitory properties of 2 drugs can lead to clinical responses in cases harboring compound mutations resistant to both agents.

(A) wbc count (blue) and peripheral blood myeloblast percentage (red) in MBC-1, who developed resistance associated with the compound mutation M244V/T315A/L364I in BCR-ABL in 22 of 27 clones sequenced, then responded to a combination of dasatinib and imatinib. This patient initially relapsed on imatinib with 2 subclones, 1 with the mutant L364I and a second clone bearing the M244V/L364I compound mutant (both M244V and L364I are known imatinib-resistant mutants). (B) Dasatinib and imatinib cooperatively inhibit the growth of Ba/F3 cells harboring BCR-ABL/M244V/T315A/L364I in vitro. Combination therapy had no effect on parental IL-3–dependent Ba/F3 cells (data not shown). (C) CRKL Western immunoblot of Ba/F3–BCR-ABL/V299L exposed to clinically relevant concentrations of imatinib and dasatinib.

Figure 4. Sequential kinase inhibitor therapy can result in compound mutations that increase transformation potency and growth rate in vitro.

(A) Bone marrow transformation assays with select drug-resistant BCR-ABL kinase domain mutants are shown. After infection with mutant or wild-type p210 BCR-ABL retrovirus, primary mouse bone marrow cells were plated at the serial dilutions indicated then monitored for oncogenic transformation. The time to outgrowth in assays performed in triplicate at 5 separate dilutions is shown on the left, and the right panel shows the percentage growth rate of various BCR-ABL isoforms relative to wild-type BCR-ABL. (B) Schematic of cell growth competition experiment. (C) Representative sequencing results following cell growth competition assay reveal BCR-ABL/M244V/T315A/L364I to be capable of conferring a growth advantage over BCR-ABL/M244V/L364I and BCR-ABL/T315A.

Figure 5. The most common dasatinib-resistant mutations are sensitive to the Aurora kinase inhibitor VX-680 in vitro.

(A) CrkL immunoblot of Ba/F3 cells transformed with dasatinib-resistant BCR-ABL isoforms depicted after exposure to varying concentrations of the kinase inhibitors indicated. (B) Primary CML cells harboring the dasatinib-resistant BCR-ABL/V299L mutation retain biochemical sensitivity to VX-680.