Abstract
Introduction
While childhood acute lymphoblastic leukemia (ALL) is highly curable, up to 20% of children will relapse, with dismal prognosis, warranting the need for novel therapies. Previously, using an integrated genomic approach on matched diagnosis-relapse samples, we identified overactivation of the Wnt pathway as a mechanism of disease recurrence at relapse (Hogan et al, Blood 2011). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin and more recently hematologic malignancies. To validate our findings and determine if Wnt inhibition could restore chemosensitivity in relapsed ALL, we sought to examine directly whether Wnt is activated at relapse in paired samples (examining expression of activated b-catenin and its downstream target Survivin (BIRC5) using multiparameter phosphoflow cytometry) and tested the efficacy of a recently developed small molecule Wnt inhibitor, iCRT14, that specifically interferes with the b-catenin-TCF interaction (Gonzalves et al, PNAS 2011), in ALL cell lines and patient samples.
Methods
B and T-ALL cell lines were treated with iCRT14 and the expression of target genes were determined by quantitative RT-PCR.10 paired diagnosis-relapse patient samples obtained from the Children’s Oncology Group were washed, fixed and stained simultaneously with caspase 3, CD10, activated b-catenin and survivin and the change in expression of activated b-catenin and survivin from diagnosis to relapse was measured by multiparameter phosphoflow cytometry in each patient by gating on the caspase 3 negative, CD10 positive leukemic blasts. To test the effect of Wnt inhibition on chemosensitivity, B-ALL cell lines were pretreated with iCRT14 for 48 hours prior to incubation with traditional chemotherapy for an additional 24 hours. The response to increasing doses of iCRT14 and chemo, alone and in combination, was assessed by cell viability (Cell Titer-Glo Luminescent Assay (Promega)) and apoptosis (FACS analysis with AnnexinV-PE/7AAD staining (BD Bioscience)). Protein levels of apoptotic markers were assessed. Also, 4 newly diagnosed and 4 relapsed patient samples were treated ex vivo with iCRT14 (20 and 30 uM) and prednisolone, alone and in combination. Drug combination results were analyzed using the Calcusyn program which calculates a Combination Index (CI): CI>1.1=antagonism, 0.9-1.1=additive and <0.9=synergy.
Results
Previously, we reported that treatment of ALL cell lines with iCRT14, downregulated the mRNA expression of the Wnt target genes BIRC5, axin 2, and c-myc (Romanos et al, ASPHO 2012 # 414). Comparison of Mean Fluorescent Intensity of activated b-catenin and survivin in the 10 pairs showed upregulation of activated b-catenin at relapse in 6 of 10 patients. Survivin expression was increased in 6 patients and in 4 patients the upregulation of activated b-catenin and survivin was concordant. iCRT14 pretreatment of cell lines followed by chemotherapy (prednisolone, etoposide, doxorubicin, cytarabine and 6TG) demonstrated additive to synergistic effects on viability. UOCB1 cells showed synergism with all 5 chemotherapy agents (CI=0.1-0.88). Nalm6 cells were very sensitive to iCRT, hence the combination with chemotherapy showed additive to synergistic effects (CI=0.05-1.I). In Reh cells, all agents besides cytarabine showed robust synergism (CI=0.03-0.55). FACS analysis revealed that iCRT14 alone contributed significantly to apoptosis and combination with chemotherapy further increased cell death with >80% apoptosis by hour 72 with the maximal chemotherapy dose in all cell lines. Change in the protein levels of cleaved PARP and cleaved caspase 3 was seen. The 4 diagnosis patients were very sensitive to prednisolone as expected, precluding synergism with iCRT14. The relapsed patient samples were much less sensitive to prednisolone alone (40% decrease in viability in relapsed patients vs 80% in new diagnoses). Interestingly, all the relapsed patients showed enhanced chemosensitivity with Wnt inhibition. 3 out of 4 relapsed patients showed strong synergism (CI=0.03-0.6) with both doses of iCRT14 and 1 patient showed additive to synergistic effects (CI=0.7 and 1).
Conclusion
Overactivation of the Wnt pathway may lead to chemoresistance in relapsed ALL. Wnt Inhibition restores chemosensitivity and induces apoptosis in ALL cell lines and primary patient samples making it a potential therapeutic approach.
Disclosures:
No relevant conflicts of interest to declare.
Methylation status of Wnt signaling pathway genes affects the clinical outcome of Philadelphia-positive acute lymphoblastic leukemia
