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.