ALBERT

Description: Background: Survival rates for acute lymphoblastic leukemia (ALL) have risen dramatically but up to 20% of patients relapse and their prognoses are dismal. Resistance to glucocorticoid (GC) agonists is a hallmark of relapsed ALL and a strong predictor of outcome at diagnosis (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). However, the underlying biological pathways that mediate sensitivity to GCs remain to be determined. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cells. Methods: Genome-wide screening was performed using pooled shRNA libraries coupled with next generation sequencing to identify genes that when depleted promote GC sensitivity. Statistical analysis using Bioinformatics for Next Generation Sequencing (BiNGS) and Redundancy and Fold Change (RFC) were employed to identify candidate genes that mediate prednisolone sensitivity (Porter et. al, Leukemia 2012). Validation of hits from the primary screen were performed in Reh and RS4;11 cells. Knockdown of candidate genes MEK2 and MEK4 was determined by western blot. Changes in chemosensitivity upon MEK2 and MEK4 knockdown were determined by Cell Titer-Glo assay (Promega). The levels of apoptotic cells upon chemotherapy treatment in control and knockdown cell lines was determined by Annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA), followed by flow cytometry using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The levels of downstream GC target genes including NR3C1, GILZ, and BIM were determined by RT-PCR. The levels of target proteins including GR, pERK, ERK, MEK1, and p53 were determined by western blot. pERK levels in primary matched pairs were determined by multiparameter phosphoflow cytometry. Results: In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with integrated analysis of relapse-specific genetic and epigenetic changes (Hogan et. al, Blood 2012) allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. Interestingly, depletion of MAPK pathway members, MEK2 and MEK4, increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to GCs by increasing the mRNA and protein levels of the glucocorticoid receptor (GR). This resulted in greater induction of the GR target genes GILZ and BIM upon prednisolone exposure over time. Importantly, depletion of MEK4 did not affect sensitivity of ALL cells to other chemotherapy agents (doxorubicin, etoposide, and 6-thioguanine). By contrast, MEK2 knockdown increased the sensitivity of cells to each of the chemotherapy agents tested including prednisolone, doxorubicin, etoposide, and 6-thioguanine. Depleting MEK2 decreased activated pERK and increased levels of p53. Over expression of a dominant negative p53 in MEK2 deficient cells reversed sensitivity to doxorubicin and prednisolone, indicating that MEK2 expression mediates chemosenstivity in a p53 dependent manner. Furthermore, inhibition of MEK1/2 pharmacologically with trametinib increased sensitivity of ALL cells to chemotherapy. Trametinib treatment also resulted in increased levels of p53. To determine if activation of the MAPK pathway in patients is associated with recurrent disease we examined seven matched diagnosis and relapse primary samples for MAPK activation as determined by pERK staining, and observed increased pERK levels at relapse in all samples tested. Conclusion: Our data indicate that activation of the MAPK pathway promotes chemoresistance and may drive the development of recurrent disease in pediatric ALL.Asdisrupting MEK2 and MEK4 sensitizes cells to chemotherapy, this makes the MAPK pathway an attractive target for therapeutic intervention in relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: 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 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.