Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl

Abstract

Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to eradicate Bcr-Abl(+) leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl(+) leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib mesylate in CML cells. This screen identified numerous components of a Wnt/Ca(2+)/NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production, and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl(+) acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl(+) leukemias.

Figure 1. Knockdown of FZD-8 sensitizes CML cells to imatinib and impairs NFAT activity

A) Diagrammatic representation of the Wnt/Ca²⁺/NFAT pathway. Pathway details are described in the text. B) K562 CML cells were stably transduced with shRNAs (shA or shB) targeting FZD-8 or a negative control shRNA. Real-time PCR (qPCR) was performed on resultant cell lines to examine FZD-8 expression levels normalized to 18S ribosomal RNA. C) K562 cell lines were left untreated or treated with imatinib at 0.1 μM for 72 hr, after which the cells were reseeded in the absence of drug and cultured for an additional 4 days. The number of viable cells (based on PI-exclusion) was counted by flow cytometry at the indicated time points. D) K562 cell lines were infected with an adenoviral NFAT-luciferase reporter. After 32 hr, the cells were left untreated or treated with ionomycin at 1 μg/ml for 16 hr after which the cells were harvested and luciferase activity was assayed. Error bars +/− SD. See also Figure S2.

Figure 2. Inhibition of CaMKII sensitizes CML cells to Bcr-Abl inhibition and impairs NFAT activity

A) K562 cells were treated with imatinib and KN93 alone or in combination as indicated (increasing KN93 concentrations of 0, 2, 5, and 10 μM are indicated by the triangles) for 48 hr and viable cells (based on PI-exclusion) were counted by flow cytometry. Values were normalized to untreated (control) cells and graphed. Representative flow profiles (side scatter vs. forward scatter) from these experiments are shown in B. The “cell” gate was defined based on the scatter profile of untreated viable K562 cells (upper left panel). PI⁺ cells are colored in red and PI⁻ in violet. C) K562 cells were treated with dasatinib and KN93 as in A for 48 hr and viable cells were counted. D) K562 cells were infected with an NFAT-luciferase reporter. After 32 hr, the cells were treated with the indicated concentrations of KN93 alone or together with ionomycin at 1 μg/ml for 16 hr after which cells were harvested and assayed for luciferase activity. Error bars +/− SD. See also Figure S3.

Figure 3. Inhibition of calcineurin-NFAT by CsA sensitizes Ph⁺ leukemia cells to Bcr-Abl inhibition

A) K562 cells were treated with imatinib and CsA alone or in combination as indicated (increasing CsA concentrations of 0, 1, 2.5, and 5 μM are indicated by the triangles) for 72 hr and viable cells were counted by flow cytometry. B) K562 cells were treated with dasatinib and CsA as in A for 48 hr and viable cells were counted. C) K562 cells were infected with an NFAT-luciferase reporter. After 32 hr, the cells were treated with the indicated concentrations of CsA alone or together with ionomycin at 1 μg/ml for 16 hr after which cells were harvested and assayed for luciferase activity. D) K562 cells were treated with imatinib and CsA as indicated. After 24 hr, the cells were harvested and lysates subjected to western blot analysis for phosphorylated (p) Bcr-Abl, CrkL, STAT5 and total phosphotyrosine or EIF-4E (loading control). E) KBM7 CML cells were treated with imatinib and CsA as in A for 48 hr and viable cells were counted. F) SUP-B15 Ph⁺ ALL cells were treated with imatinib and CsA as in A for 72 hr and viable cells were counted. Error bars +/− SD. See also Figure S4.

Figure 4. CsA sensitizes primary CML cells and cells with imatinib-resistant forms of Bcr-Abl to imatinib

A) Colony formation assays were performed on CD34⁺ cells purified from bone marrow of two patients with chronic phase CML in the presence of imatinib and/or cyclosporine at the indicated concentrations. B) Ba/F3 cells transduced with vector, wild-type Bcr-Abl, or imatinib-resistant mutant forms of Bcr-Abl (M351T or E255K) were treated with imatinib and CsA alone or in combination as indicated (increasing CsA concentrations of 0, 2.5, and 5 μM are indicated by the triangles) for 72 hr and viable cells were counted. Error bars +/− SD. See also Figure S5.

Figure 5. NFAT protects CML cells from imatinib-induced cell death

A) K562 cells were treated with ionomycin (1 μg/ml) alone or in combination with PMA (10 ng/ml). After 16 hr, the cells were treated −/+ 1 μM imatinib for 48 hr as indicated and the number of viable cells was counted. B) K562 cells were treated with PMA and/or ionomycin, or CsA for 16 hr and harvested. Whole cells lysates and nuclear extracts were prepared and subjected to western blotting for NFATc1. The nuclear extract blot was stripped and reprobed for lamin C as a loading control. C) K562 cells were infected with retrovirus expressing GFP alone or constitutively active NFATc1-GFP. Forty-eight hours after infection, the cells were treated with imatinib at the indicated concentrations and/or CsA (5 μM) for 72 hr and the percentages of viable GFP⁺ (relative to all viable cells) were determined using flow cytometry. Error bars +/− SD.

Figure 6. IL-4 production is NFAT and FZD-8 dependent and protects Ph⁺ cells from imatinib

A) Conditioned media from K562 cells treated −/+ CsA at 5μM was used for cytokine array analysis. Cytokines that were consistently detectable are labeled. B) Conditioned media from K562 cells expressing FZD-8 shA (no knockdown) or shB (FZD-8 knockdown) was prepared and used for cytokine array analysis. C) Ba/F3 cells expressing Bcr-Abl (described in Figure 4) were treated with imatinib and neutralizing IL-4 antibody (11B11) alone or in combination (increasing 11B11 concentrations of 0, 25, 50, and 100 μg/ml are indicated by the triangles) for 72 hr and viable cells were counted. D. Ba/F3 cells expressing Bcr-Abl were treated with recombinant murine IL-4 together with imatinib and CsA at the indicated concentrations for 72 hr and viable cells were counted. Error bars +/− SD. See also Figure S6.

Figure 7. CsA enhances elimination of Ph⁺ ALL cells by dasatinib in vivo

C57BL/6 mice were inoculated intravenously with 5 × 10⁵ ARF-/- p185 Bcr-Abl/GFP B-ALL cells. After 3 days, groups of mice were treated once daily by oral gavage with vehicle (80 mM citric acid, pH 3.1; n=5), CsA (25 mg/kg; n=5), dasatinib (20 mg/kg; n=10), or dasatinib and CsA combined (n=10) up to day 52 post-inoculation. A) After 3 days of therapy, peripheral blood from all mice was immunostained for B220 and Mac-1 and analyzed by flow cytometry. The percentage of GFP⁺ cells in the B-lineage (B220⁺, Mac-1⁻) population was determined and plotted. B) Peripheral blood was taken on the indicated days and analyzed as in A and plotted over time. The limit of detection of GFP in peripheral blood, based on analyses of blood from control C57Bl/6 mice is ~0.1%. C) Kaplan-Meier curve showing survival of mice receiving the indicated therapy. Mice were sacrificed when moribund, and all showed clear evidence of leukemia in blood, bone marrow and spleen. See also Figure S7.