A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Abstract

Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach.

Figure 1

Imatinib and dual-resistant K562 cells are resistant to all three ABL-TKI therapies, whereas the KCL-22 cells are highly sensitive to ponatinib. (a) Sensitivity of the K562 parental, imatinib- and dual-resistant cell lines to 0–10 μm imatinib, dasatinib or ponatinib after treatment for 72 h as measured by CellTiter-Glo assays. (b) Sensitivity of the KCL-22 parental and resistant cells to 0–10 μm imatinib, dasatinib or ponatinib after treatment for 72 h as measured by CellTiter-Glo assays. The cell viability was measured and corrected relative to time zero (time of treatment). The data shows % control 0.001–10 μm ABL-TKI-treated cells relative to vehicle (DMSO)-treated cells, which is set to 100% growth (upper dotted line). The data represent the average of three independent experiments. The error bars represent standard deviation.

Figure 2

Reverse-phase protein arrays reveal elevated levels of p-ERK, BCL-2 and FOXO1 in TKI-resistant versus parental K562 cells. (a) The baseline proteomic signature of the parental K562 cells (Pa) is compared with the dual-resistant cells (DR) and resistant clones (C4–C20). The baseline proteomic signature of the parental KCL-22 cells (KP) compared with the resistant KCL-22 cells (KR) is also shown. The data are log2-transformed and normalized to the respective parental cell line. Only the end points that show a >1.6-fold difference between the resistant cells and their respective parental cells are shown. (b) Immunoblot analyses confirmed elevated levels of FOXO1, p-ERK T202/Y204 and BCL-2 expression, as well as decreased p-ABL Y245, in the dual-resistant cells compared with the parental cell line. FOXO3a, FOXO4 and beta-actin were analyzed as controls.

Figure 3

Dual-resistant cells and clones are sensitive to GDC-0941-mediated growth inhibition (a) Sensitivity of the dual-resistant K562 cells and resistant KCL-22 cells to 0–10 μm GDC-0941 or 0–10 μm GDC-0973 after 72 h treatment. The cells were treated for 72 h with the indicated drug and cell viability was measured and corrected relative to time zero (time of treatment). The data shows % control 0.001–10 μm TKI-treated cells relative to vehicle (DMSO)-treated cells, which is set to 100% (upper dotted line). The data represent the average of three independent experiments. The error bars represent standard deviation. (b) The percentage of cells undergoing early apoptosis induced by 50 nm dasatinib or 300 nm GDC-0941 in the dual-resistant and parental K562 cells 48 h after treatment using AnnexinV/PI FACS analysis. The error bars represent standard deviation. *P<0.05.

Figure 4

GDC-0941 inhibits phosphorylation of PI3K substrates in the dual-resistant cells. Responsiveness of phospho-c-ABL Y245, phospho-Akt T308, phospho-PRAS40 T246, phospho-p70 S6 kinase T389, FOXO1 and BCL-2 to vehicle (DMSO, V), 100 nm dasatinib (D), 300 nm GDC-0941 (0941) or 300 nm GDC-0973 (0973) treatment for 1 h in parental K562 (Pa) or dual-resistant K562 cells (DR) by reverse-phase protein array. The data were normalized to total protein and log2-transformed. The error bars represent standard deviation. *P<0.05.

Figure 5

The BCL-2 inhibitor, ABT-737, in combination with GDC-0941, inhibits growth of the dual-resistant but not parental K562 cells. Parental and dual-resistant K562 cells were treated with 0–10 μm of the BCL-2 inhibitor, ABT-737, alone or in combination with 0–10 μm dasatinib or GDC-0941. The cell viability after 72 h was measured using a CellTiter-Glo assay and mean percentage growth inhibition derived from quadruplicates at each dose or combination is shown.

Figure 6

Elevated FOXO1 accumulates in the cytoplasm of the dual-resistant K562 cells and translocates into the nucleus upon GDC-0941 treatment. Immunofluorescence cytochemistry revealed that FOXO1 was localized primarily within the cytoplasm of dual-resistant K562 cells treated with DMSO or 100 nm dasatinib, whereas it was low to undetectable in the parental TKI-sensitive controls. The treatment of dual-resistant K562 cells with 1 μm GDC-0941 resulted in a translocation of FOXO1 to the nucleus. Representative images of the treatment groups are shown under × 40 magnification using confocal microscopy. For clarity, single-cell images are shown for the dual-resistant cells.

Figure 7

FOXO1 levels are elevated in a subset of relapsed patient samples and are sensitive to GDC-0941-mediated survival inhibition. (a) The baseline proteomic signature of TKI-naive samples (samples 180, 266 and 300) compared with samples from TKI-relapsed patients (resistant) lacking BCR–ABL1 mutations (samples 109-197) or harboring a BCR–ABL1 kinase domain mutation (samples 077 and 252). Samples with elevated FOXO1 expression are denoted by a red asterisk. (b) TKI-naive samples (180, 266 and 300) and patient samples with high FOXO1 (077, 109, 142, 185, 197 and 252) or low FOXO1 (123 and 186) were treated with dasatinib (50 nm) and/or GDC-0941 (300 nm) in colony-formation assays. The data represents percent of vehicle-treated controls. The error bars represent standard deviation. *P<0.05, ***P<0.001.