Comprehensive profiling of DNA methylation in colorectal cancer reveals subgroups with distinct clinicopathological and molecular features

<p>Abstract</p> <p>Background</p> <p>Most previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups.</p> <p>Methods</p> <p>DNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate^®methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in <it>BRAF </it>and <it>KRAS</it>.</p> <p>Results</p> <p>A total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of <it>KRAS </it>and <it>BRAF </it>(<it>P </it>< 0.001).</p> <p>Conclusions</p> <p>Comprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both <it>KRAS </it>and <it>BRAF </it>mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.</p

Pharmacokinetic Modeling of an Induction Regimen for In Vivo Combined Testing of Novel Drugs against Pediatric Acute Lymphoblastic Leukemia Xenografts

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and l-asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo, VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL

Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia: an in vivo study

Intrinsic or acquired resistance to vincristine (VCR), an antimicrotubule agent used in the treatment of childhood acute lymphoblastic leukemia (ALL), is a major clinical problem. Using a clinically relevant NOD/SCID mouse xenograft model of ALL, we established that alterations in the actin and tubulin cytoskeleton are involved in in vivo VCR resistance. Altered protein expression between VCR-sensitive ALL xenografts, and xenografts with intrinsic or acquired VCR resistance, was identified using 2-D DIGE coupled with MS. Of the 19 proteins displaying altered expression, 11 are associated with the actin cytoskeleton. Altered expression of the actin- and/or tubulin-binding proteins gelsolin, moesin, ezrin, tropomyosin, CAP-G, HSP27, HSP70, TCP-1, and stathmin were associated with in vivo VCR resistance. The actin-regulating protein gelsolin was increased in both acquired and resistant leukemia as confirmed by immunoblotting and gene expression. The major cytoskeletal protein, γ-actin, was down-regulated in the VCR-resistant leukemia xenografts; in contrast, there was no significant change in β-actin expression. This study provides the first evidence for a role of the actin cytoskeleton in intrinsic and acquired in vivo antimicrotubule drug resistance in childhood leukemia and highlights the power of 2-D DIGE for the discovery of resistance markers, pharmacoproteomics, and signaling pathways in cancer

<i>In vivo</i> responses of ALL xenografts to ABT-737, VXL or VXL/ABT-737 combination treatments.

<p>Significant values are shown in bold.</p

Combination Indices of <i>in vitro</i> cytotoxicity assays.

<p>VCR, vincristine; DEX, dexamethasone; ASP, l-asparaginase.</p

Synergy between VCR, DEX and ASP against ALL cell lines <i>in vitro</i>.

<p>Cell lines were exposed to VCR (open circles), DEX (open triangles), ASP (open squares), or the triple combination VXL (closed circles), at fixed ratios, and dose-responses were assessed using the DIMSCAN assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033894#s4" target="_blank">Materials and methods</a>. Fractional survival of treated vs. untreated control cells is shown. Each condition included 12 replicates and error bars represent standard deviation. The data shown are representative of two independent experiments.</p

<i>In vivo</i> sensitivity of ALL xenografts to VXL and VXL/ABT-737 combination treatments.

<p>Female mice were engrafted with: ALL-2 (<b>A</b>); ALL-8 (<b>B</b>); ALL-10 (<b>C</b>); or ALL-17 (<b>D</b>) and treated with a diluent vehicle (controls, dashed black lines), ABT-737 (25 mg/kg, solid grey lines), VXL combination (solid black lines), or VXL+ABT-737 quadruple combination (dashed grey lines). Engraftment kinetics indicated by %huCD45⁺ cells in PB of individual mice (left panel) and Kaplan-Meier survival curves (EFS) (right panel) are shown. Shaded boxes represent the treatment period. All events were leukemia-related except for 1 and 4 in the VXL/ABT-737-treated group of the ALL-8, and ALL-10, respectively. In the ALL-17 quadruple drug combination cohort all mice were culled due to leukemia or toxicity unrelated morbidity.</p

<i>In vivo</i> sensitivity of ALL xenografts to VXL and VXL/ATO combination treatments.

<p>Female mice were engrafted with: ALL-4 (<b>A</b>); ALL-7 (<b>B</b>); ALL-8 (<b>C</b>); or ALL-19 (<b>D</b>) and treated with a diluent vehicle (controls, dashed black lines), ATO (2.5 mg/kg, solid grey lines), VXL combination (solid black lines), or VXL+ATO quadruple combination (dashed grey lines). Engraftment kinetics indicated by %huCD45⁺ cells in PB of individual mice (left panel) and Kaplan-Meier survival curves (EFS) (right panel) are shown. Shaded boxes represent the treatment period.</p

LGD in response to VXL treatment in xenografts stratifies according to patient outcome.

<p>Median LGD obtained by VXL treatment for a panel of ALL xenografts derived from long term survivors (Alive) and from patients who died of their disease (DOD). There is evidence that the two groups are different (<i>p</i> = 0.0159) by Mann-Whitney two tailed test.</p

<i>In vivo</i> sensitivity of ALL-19 to low dose VCR, DEX and ASP.

<p>Female mice were engrafted with ALL-19 cells and treated with vehicle (<b>A</b>); DEX (5 mg/kg) (<b>B</b>); VCR (0.15 mg/kg) (<b>C</b>); and ASP (1000 U/kg) (<b>D</b>); as single agents or the combination of the three drugs at the same doses (VXL) (<b>E</b>). The %huCD45⁺ cells in PB of individual mice (<b>A–E</b>); control vehicle-treated mice (dashed lines); drug-treated mice (solid lines). Kaplan-Meier analysis of EFS (<b>F</b>) control (grey solid line), VCR (grey dashed line), DEX (black dashed line), ASP (black dotted line), VXL (solid black line). All events were leukaemia-related. Shaded boxes represent the treatment period.</p