ALBERT

Description: Background Burkitt Lymphoma (BL) is an aggressive non-Hodgkin B cell neoplasm primarily affecting children. While overall cure rates are high, prognosis for the 20% of patients with relapsed or refractory is dismal with a 16% survival rate at four years, indicating the need for novel therapeutic approaches. While epigenetic modulators have shown potential therapeutic benefit in other hematologic malignancies, their use in BL has been limited. Here, we evaluate whether histone deacetylase (HDAC) inhibitors could enhance cell death in chemotherapy resistant BL cells. Methods The BL cell lines Ramos, Raji, or previously generated/characterized immunochemotherapy resistant Raji BL cell lines (Raji 2R and Raji 4RH) (Barth et al., Br J Haematol, 2012) were treated for 48 hours with 4-hydroperoxycyclophosphamide (4-HC, the active metabolite of cyclophosphamide), cytarabine, prednisolone, HDAC inhibitors (suberoyanilide hydroxamic acid (SAHA, vorinostat), and romidepsin), or vehicle control. Cell viability was measured on a Via Cell, by Alamar Blue staining (Invitrogen), or by measuring Caspase-3 activation by flow cytometry. Levels of pro- (survivin, XIAP, MCL-1, livin, and BCL-XL) and anti- (Bax, Bak) apoptotic proteins were evaluated using standard western blot techniques. To evaluate the wiring of signaling networks in the basal state or in response to drug treatment, samples were fixed, permeabilized, and simultaneously stained for Caspase-3, phospho GSK-3 (pGSK3, the inactive form of this protein), active β-catenin, and survivin. Cells were processed on a BD FACSVerse and analyzed by gating on Caspase-3 negative (chemotherapy resistant) cells and monitoring the activity of the pGSK3, active β-catenin, and survivin using FlowJO (V9.6) software. Results Relative to the BL cell line Ramos, Raji BL cells were resistant to all doses of conventional chemotherapy (cyclophosphamide, ara-c, and prednisolone) tested. Because hyper-activation of the WNT/b-catenin network due to epigenetic modulation has been implicated in chemotherapy resistance in other B-lineage pediatric malignancies (Hogan et al., Blood 2011), we treated cells with chemotherapy or vehicle and used phosphoflow cytometry to evaluate the expression of pGSK-3, active β-catenin, and its downstream target survivin. Relative to vehicle control, we found that exposure to cytotoxic chemotherapy resulted in rewiring of the cellular networks with increased levels of pGSK3, β-catenin, and survivin. Western blot analyses of the immunochemotherapy resistant Raji 2R and Raji 4RH cells revealed similar upregulation of anti-apoptotic proteins including survivin, downregulation of pro-apoptotic proteins Bak and Bax, and increased expression of pGSK3 compared to Raji cells. To test whether alterations in these signaling axes might enhance the survival of chemorefractory BL cells, we treated Raji cells with the HDAC inhibitor SAHA and found induction of apoptosis at nanomolar doses. Sensitivity to SAHA correlated with down-regulation of active β-catenin and survivin in a dose-dependent manner. To extend these results, we also tested whether immunochemotherapy resistant Raji 2R and Raji 4RH cells were sensitive to HDAC inhibition and found similar robust induction of cell death upon treatment with a panel of HDAC inhibitors. Conclusions Active β-catenin and survivin are upregulated in chemotherapy resistant BL cells. HDAC inhibition results in their rapid down regulation and enhanced apoptotic cell death. Further evaluation of inhibitors of the WNT/ β-catenin pathway and HDAC inhibitors is warranted in chemotherapy resistant BL and could have a role in treating refractory or relapsed BL patients. Disclosures: No relevant conflicts of interest to declare.

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.

Description: Key Points Genetic or pharmacologic inhibition of MEK4 and MEK2 enhances prednisolone-induced cell death in ALL models. MAPK signaling cascades are activated at relapse compared to diagnosis in ALL samples and have enhanced response to MEK inhibition.

Description: Background Burkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma in children. Although curable in most cases, those patients who relapse have a dismal prognosis with a 4-year survival of 16% (Cairo, Blood 2007). Identification of biomarkers to predict relapse/refractory disease is needed, and an understanding of the underlying mechanisms is vital to develop novel targeted therapies. We previously identified a recurrent gain of chromosome 13q31 that was associated with relapse and increased expression of miR-17-92 in pediatric BL (Schiffman/Miles, Br J Haematol, 2011). In the present study, we sought to determine the role of miR-17-92 expression in vitro using TALENs mediated deletion of the miR-17-92 locus (MIR17HG) in Raji BL cells that express high levels of miR-17-92 (Shiffman/Miles, 2011). Methods The modified restriction enzyme and ligation assembly method (Lee/Cairo, ASH 2012) was used to construct MIR17HG TALENs. Expression of miR-17 was assayed by TaqMan assay, and expression of the miR-17-92 target PTEN mRNA and protein were assayed by RT-PCR and western blot, respectively. Proliferation and caspase activity were measured at baseline and after treatment with cyclophosphamide. AKT and phospho (p)AKT were assayed by western blotting. To evaluate mTOR and MAPK pathway activation, pS6 and pERK were assayed by phosphoflow cytometry. In addition, caspase 3 activity was assayed by phosphoflow cytometry to assess induction of apoptosis. Cells were treated with chemotherapy (cyclophosphamide or 4-hydroperoxycyclophosphamide, 4HC), rapamycin, the PI3 kinase inhibitor PIK90, and the dual PI3K/mTOR inhibitor BEZ235 (Axon Medchem). To further validate the role of miR-17 in chemoresistance, miR-17 expression levels were measured by RT-PCR TaqMan assay in the previously developed rituximab-chemotherapy resistant Raji cell lines (RCRCL) Raji 2R and Raji 4RH (Barth et al. Br J Haematol, 2012). Results MIR17HG TALENs were used to generate two Raji cell lines with a hemizygous deletion of MIR17HG, #31(+/-) and #48(+/-). Relative to vector control Raji cells, cell lines with hemizygous MIR17HG deletion showed lower expression of miR-17: Raji = 1.0, #31= 0.80-fold, and #48= 0.71-fold. PTEN mRNA was higher in #31 and #48 cells than in the control (Raji = 1.0, #31 = 1.38-fold, and #48 = 2.24-fold), but there was no apparent difference in PTEN protein by western blot. Western blotting showed decreased pAKT with no difference in total AKT. Cyclophosphamide (10mM) induced a significant decrease in proliferation at 48 hours (90.5 +/- 2.9%, p=0.007) in #31 cells vs. control cells that was associated with a trend toward increased caspase 3/7 activity (148.3+/-8.9%, p=0.08). By phosphoflow cytometry, Raji #48 cells showed decreased pS6 compared to control cells and a modestly greater induction of caspase 3 in response to 4HC (3.4 uM) or PIK90 after 48 hours. The combination of 4HC and PIK90, however, significantly induced caspase 3 in Raji #48 cells relative to control Raji cells. Raji cells showed high basal levels of pERK and pS6. BEZ235 or PIK90 treatment decreased p-ERK. Rapamycin did not affect p-ERK but did decrease pS6. While BEZ235 and 4HC each modestly induced caspase alone in Raji cells, the combination caused a much greater increase in caspase 3. In RCRCLs Raji 2R and Raji 4RH, which show high pAKT levels by western blot and phosphoflow cytometry, miR-17 levels were increased (1.73- and 1.69-fold, respectively) relative to chemosensitive Raji cells (1.0) that exhibit lower pAKT levels. Conclusions Increased miR-17-92 expression in BL may contribute to therapy resistance, which is further supported by finding increased miR-17 expression in chemotherapy resistant Raji cell lines. Hemizygous deletion of the miR-17-92 locus in BL cells led to increased PTEN mRNA, decreased mTOR pathway activation (decreased pAKT and pS6), and increased sensitivity to chemotherapy. Resistance to PIK90 and 4HC in parental Raji cells that overexpresses miR-17-92 may be due in part to a compensatory upregulation of the MAPK and mTOR pathways, as demonstrated by increased pERK and pS6. Inhibiting this survival mechanism with the PI3K/mTOR inhibitor BEZ235 restored sensitivity to cytotoxic chemotherapy. These findings suggest potential mechanisms underlying BL therapy resistance as well as targeted therapies to overcome resistance. Disclosures: Cairo: Roche/Genentech: advisory board Other.

Description: Abstract 3174 Introduction Anemia is prevalent in Indian children 1.5) and those having non iron deficient anemia of inflammation (Non ID-AI) (sTfR-F index value