BCR-ABL1 kinase domain mutations are detected in 30-60% of patients who develop resistance to tyrosine kinase inhibitors (TKIs) such as imatinib. However, the underlying mechanism(s) of resistance in the remaining patients are not known. To identify BCR-ABL1-independent mechanisms of resistance to TKIs, we used K562 cells that were adapted for long-term growth in 1 µM imatinib (K562-R). These cells lack BCR-ABL1 kinase domain mutations and survive despite continued suppression of BCR-ABL1 kinase activity. To screen for novel genes associated with BCR-ABL1-independent resistance, parental K562 and K562-R cells were lentivirally infected with a pooled shRNA library containing 27,000 shRNAs targeting ∼5,000 genes with known roles in cell signaling (Cellecta, Human Module 1). Using an unbiased, customized algorithm, we identified 18 genes with multiple shRNA hits that were depleted more than three-fold in K562-R cells compared to parental controls. Among the top five genes were RAN and XPO1 (CRM1), both of which encode components of a nucleocytoplasmic transport complex. Parental K562 cells also showed depletion for RAN and XPO1 shRNAs, suggesting a potential role in pathogenesis of CML and magnified activity in TKI resistance. Recently, enhanced expression of XPO1 was shown in Philadelphia chromosome positive leukemia and investigated as a potential therapeutic target in blast crisis CML (Walker et al. ASH 2012).
To validate the data from our shRNA library screen, we cloned a RAN shRNA into a tet-inducible lentiviral vector (Cellecta). Transduced parental and K562-R cells were treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline. Parental and K562-R cells were treated with various concentrations of the oral XPO1 inhibitor Selinexor (KPT-330), which is now being evaluated in Phase 1 clinical trials in patients with solid and hematological malignancies. Following 72 hours of treatment, proliferation was measured by MTS assay (Promega). Additionally, CD34+ cells from newly diagnosed CML patients were lentivirally transduced with RAN shRNA and treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline (n=3) or KPT-330 (n=1). Survival was assessed by colony formation assays. Normal cord blood CD34+cells were used as controls.
Transduction of parental or K562-R cells with the inducible RAN shRNA and treatment with doxycycline reduced RAN mRNA levels by 57 and 76% respectively compared to the untreated control. This level of RAN suppression was sufficient to reduce the proliferation of parental K562 by 54%. It also reduced the IC50 of imatinib by > 3-fold in K562-R cells. In CD34+ cells from newly diagnosed CML patients, RAN shRNA alone had no significant effect on colony formation, whereas RAN shRNA in combination with imatinib (2.5 µM) reduced colony formation by 46% compared to imatinib alone. The IC50 of KPT-330 was 30-35% less in K562-R cells compared to parental controls suggesting a higher sensitivity of K562-R cells to XPO1 inhibition. We observed that 25 nM KPT-330 reduced colony formation by 52% in primary CML cells without reducing cord blood CD34+colonies. Combination of 2.5 µM imatinib and 25 nM KPT-330 reduced the colony formation significantly compared to imatinib alone in primary CML cells with no change in the control.
An unbiased, shRNA library screen and subsequent validations with primary CML cells suggested that the activity of nucleocytoplasmic transport is crucial for the survival of CML cells. Genetic and pharmacologic disruption revealed that nucleocytoplasmic transport is a target in both TKI sensitive and resistant CML cells. A deeper understanding of this pathway in CML will be important for understanding BCR-ABL1 independent resistance to TKI treatment
Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Schacham:Karyopharm: Membership on an entity’s Board of Directors or advisory committees. Chenchik:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Bonneau:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.
Asterisk with author names denotes non-ASH members.