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. 2014 Aug 1;4(8):e232.
doi: 10.1038/bcj.2014.52.

A Pre-Clinical Model of Resistance to Induction Therapy in Pediatric Acute Lymphoblastic Leukemia

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Free PMC article

A Pre-Clinical Model of Resistance to Induction Therapy in Pediatric Acute Lymphoblastic Leukemia

A L Samuels et al. Blood Cancer J. .
Free PMC article

Abstract

Relapse and acquired drug resistance in T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. This study was designed to establish a preclinical model of resistance to induction therapy in childhood T-ALL to examine the emergence of drug resistance and identify novel therapies. Patient-derived T-ALL xenografts in immune-deficient (non-obese diabetic/severe combined immunodeficient) mice were exposed to a four-drug combination of vincristine, dexamethasone (DEX), L-asparaginase and daunorubicin (VXLD). 'Relapse' xenografts were characterized by responses to drugs, changes in gene expression profiles and Connectivity Map (CMap) prediction of strategies to reverse drug resistance. Two of four xenografts developed ex vivo and in vivo drug resistance. Both resistant lines showed altered lipid and cholesterol metabolism, yet they had a distinct drug resistance pattern. CMap analyses reinforced these features, identifying the cholesterol pathway inhibitor simvastatin (SVT) as a potential therapy to overcome resistance. Combined ex vivo with DEX, SVT was significantly synergistic, yet when administered in vivo with VXLD it did not delay leukemia progression. Synergy of SVT with established chemotherapy may depend on higher drug doses than are tolerable in this model. Taken together, we have developed a clinically relevant in vivo model of T-ALL suitable to examine the emergence of drug resistance and to identify novel therapies.

Figures

Figure 1
Figure 1
In vivo drug treatment of T-ALL xenografts. (a) ALL-31, (b) ALL-27, (c) ALL-29 and (d) ALL-33 were treated with either single (VXLD) or multiple (VXLD2) rounds of treatment, or saline (control), to generate lines resistant to multidrug chemotherapy. Acquired drug resistance to single agent ASP (e, f), single agent DNR and VCR (i, j) and single agent DEX, or VXLD combination therapy (g, h) was assessed in ALL-31R (derived from VXLD2-treated ALL-31) and compared with passage-matched ALL-31C, with time course of huCD45+ cells (e, g, i) and survival plots (f, h, j) for the groups of mice in each drug treatment. Baseline engraftment ALL-31C or ALL-31R treated with saline only is indicated in e.
Figure 2
Figure 2
Gene expression analysis of acquired drug resistance in T-ALL xenografts. Volcano plots of differential gene expression (VXLD treated vs passage matched control) for (a) ALL-27R and (b) ALL-31R. (c) Correlation between differences in gene expression and drug resistance phenotype for VXLD-treated xenografts. (d) Gene expression comparison of control or VXLD-treated ALL-31R with ALL-31b (derived from the day103 relapse specimen of same patient). (e) Venn diagram showing overlap of resistance-associated gene expression signatures between ALL-27R and ALL-31R (unadjusted P<0.05 with direction of fold-change taken into account, that is, overlapping genes move in the same direction). (f) Ingenuity Pathway Analysis of resistance-associated gene expression changes affecting ALL-27R and ALL-31R in common (that is, the 399 transcript clusters from Venn diagram intersect) or separately (top 1000 most-significant transcript clusters from either line). Heatmap shows top biological functions, canonical pathways and upstream-network regulators (for example, transcription factors or chemical perturbations) associated with resistance signatures (VXLD-treated vs control). Highlighted pathways are those with a predicted directionality of effect (green, pathway activated in resistant cells; red, pathway downregulated in resistant cells) or networks directly associated with agents used in this study (DEX and SVT, blue highlight). (g) MYC-regulation network associated with acquired resistance in ALL-27R (Ingenuity Pathway Analysis). Colors represent log-fold change (green, expression increased in VXLD-treated xenografts; red, decreased expression in VXLD-treated xenografts; a larger version of this figure can be found in Supplementary Figure 7).
Figure 3
Figure 3
Ex vivo drug synergy assessment in T-ALL xenograft lines. The cytotoxicity of the indicated drugs in cells from resistant (VXLD2-treated) xenografts was assessed by MTT assay both as single agents and in combination, using fixed IC50 ratios. A comparison of observed (mean±s.e.m.) and predicted cytotoxic drug responses is shown for (a) ALL-27R (combined DEX+SVT), (b) ALL-31R (combined DEX+SVT), (c) ALL-27R (combined DEX+SAHA) and (d) ALL-31R (combined DEX+SAHA), with deviations from the predicted curve corresponding to antagonistic (less than additive cytotoxic effects) or synergistic (greater than additive cytotoxicity) effects as indicated. (e) The size of the deviation from Bliss additivity across all IC50 ratios was averaged across experiments (n=3 to 5 independent experiments per drug combination) to obtain the net synergy effect over all drug concentrations for that combination (±s.e.m.). Positive scores represent a mean cytotoxic effect greater than that predicted by additive effects alone and thus indicate a net synergistic relationship between the two drugs over all drug concentrations tested (vice versa for antagonism). Significant positive or negative deviation of the mean from zero was assessed by one-tailed t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4
Efficacy of SVT (20 mg/kg) for treatment of drug-resistant T-ALL. (ac) Engraftment of ALL-31R (% Hu CD45+ cells) over time in (a) spleen, (b) bone marrow or (c) peripheral blood in mice treated with vehicle (black solid line), SVT (gray dotted line), VXLD (gray solid line) or VXLD+SVT (black dotted line). (d, e) Effect of vehicle (black solid line), SVT (gray dotted line), VXLD (gray solid line) and SVT+VXLD (black dotted line) on survival (d) and the time course for the appearance of human CD45+ cells in peripheral blood (e).

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