PTEN contributes to, but does not define IGF dependence.

<p>Cell growth as measured by resazurin reduction assay. (<b>A</b>) PTEN-negative cell lines (P12 Ichikawa and PF-382) or (<b>B</b>) PTEN-positive cell lines (HPB-ALL and ALL-SIL) were transduced with lentiviral PTEN expression or knock-down constructs, respectively, FACS sorted, and then cultured <i>in vitro</i> with IGF1R blocking antibody (1 μg/ml CP-751,871) or dual IGF1R/InsR tyrosine kinase inhibitor (0.5 μM BMS-754807) for 2–3 days. Mean resorufin fluorescence values +/- SD after normalization to respective mock-treated controls are plotted for assays performed in triplicate. <i>*</i>, <i>p<0</i>.<i>05; **</i>, <i>p<0</i>.<i>01; ns</i>, <i>not significant (2-way ANOVA with Sidak’s multiple comparisons test</i>).</p

Constitutive activation of AKT, but not RAS rescues T-ALL cells from IGF1R inhibition.

<p>(<b>A,B</b>) Cell growth as measured by resazurin reduction. T-ALL cells were transduced with lentiviral vectors as indicated, FACS sorted, and then cultured <i>in vitro</i> with IGF1R blocking antibody (1 μg/ml CP-751,871) for 3 days. Mean resorufin fluorescence values +/- SD after normalization to respective mock-treated controls are plotted for assays performed in triplicate. <i>ns</i>, <i>not significant (2-way ANOVA with Sidak’s multiple comparisons test)</i>. (<b>C,D</b>) Cell size as measured by forward light scatter. (<b>C</b>) T-ALL cells were treated with CP-751,871 (1 μg/ml) for 3 days. Data are depicted for gated live events. (<b>D</b>) T-ALL cells were transduced with lentiviral vectors as indicated. Data are depicted for gated live GFP+ events. Mean forward light scatter values +/- 95% confidence interval are plotted after normalization to mock-treated control cells (<b>C</b>) or to untransduced cells in the same culture, then scaled to the empty virus control (<b>D</b>). <i>*</i>, <i>p<0</i>.<i>05; **</i>, <i>p<0</i>.<i>01 (Student’s t-test)</i>.</p

IGF1R Derived PI3K/AKT Signaling Maintains Growth in a Subset of Human T-Cell Acute Lymphoblastic Leukemias

<div><p>Insulin-like growth factor 1 receptor (IGF1R) is a prevalent signaling pathway in human cancer that supports cell growth/survival and thus contributes to aggressive biological behavior. Much work has gone into development of IGF1R inhibitors; however, candidate agents including small molecule tyrosine kinase inhibitors and blocking antibodies have yet to fulfill their promise clinically. Understanding cellular features that define sensitivity versus resistance are important for effective patient selection and anticipation of outgrowth of a resistant clone. We previously identified an important role for IGF signaling in T-cell acute lymphoblastic leukemia (T-ALL) relying primarily upon genetically defined mouse models. We present here an assessment of IGF1R dependence in human T-ALL using a broad panel of 27 established cell lines that capture a spectrum of the genetic variation that might be encountered in clinical practice. We observed that a subset of cell lines are sensitive to IGF1R inhibition and are characterized by high levels of surface IGF1R expression and PTEN positivity. Interestingly, lentiviral expression or knock-down of PTEN in PTEN-negative/positive cell lines, respectively, had limited effects on their response to IGF1R inhibition, suggesting that PTEN contributes to, but does not define IGF dependence. Additionally, we characterize downstream PI3K/AKT signaling as dominant over RAS/RAF/MEK/ERK in mediating growth and/or survival in this context. Finally, we demonstrate that IGF and interleukin-7 (IL-7) fulfill non-overlapping roles in supporting T-ALL growth. These findings are significant in that they reveal cellular features and downstream mechanisms that may determine the response of an individual patient’s tumor to IGF1R inhibitor therapy.</p></div

Combined inhibition of IGF1R and PI3Kγ does not block growth of PTEN negative CCRF-CEM cells.

<p>Cell growth as measured by resazurin reduction assay. CCRF-CEM cells were cultured <i>in vitro</i> with IGF1R blocking antibody (1 μg/ml CP-751,871) with or without a selective PI3Kγ inhibitor (50 μM AS-604850) for 3 days. Mean resorufin fluorescence values +/- SD after normalization to the mock/DMSO control are plotted for assays performed in triplicate. <i>*</i>, <i>p<0</i>.<i>05; ns</i>, <i>not significant (2-way ANOVA with Sidak’s multiple comparisons test</i>).</p

Sensitivity to IGF1R inhibition correlates with surface IGF1R expression level.

<p>Plots of cell growth (normalized resorufin fluorescence data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161158#pone.0161158.g001" target="_blank">Fig 1</a>) vs. surface IGF1R expression level (mean fluorescence intensity by flow cytometry from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161158#pone.0161158.s003" target="_blank">S3 Fig</a>). Linear regression lines are depicted with the 95% confidence interval indicated by flanking dotted lines. Pearson correlation r values and associated significance p-values are as indicated.</p

Correlation between IGF1R inhibitor efficacy and surface IGF1R expression level holds up in PTEN-positive cell lines, but less so in PTEN-negative cell lines.

<p>Linear regression lines are shown separately for PTEN-positive and -negative subsets, with 95% confidence intervals indicated by flanking dotted lines. Pearson correlation r values and associated significance p-values are indicated for each subset. <i>Plotted data are identical to those presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161158#pone.0161158.g002" target="_blank">Fig 2</a>, but now segregated into PTEN-positive and -negative subsets</i>.</p

Constitutive activation of IL7R does not confer resistance to IGF1R inhibition.

<p>HPB-ALL cells were transduced with a constitutively active IL-7R lentivirus bearing the p.L242_L243 insLSRC mutation, FACS sorted, and cultured <i>in vitro</i> with IGF1R blocking antibody (1 μg/ml CP-751,871) for 3 days. (<b>A</b>) Cell growth as measured by resazurin reduction assay. Mean resorufin fluorescence values +/- SD after normalization to respective mock-treated controls are plotted for assays performed in triplicate. <i>**</i>, <i>p<0</i>.<i>01; ***</i>, <i>p<0</i>.<i>001; ns</i>, <i>not significant (2-way ANOVA with Sidak’s multiple comparisons test</i>). (<b>B</b>) Flow cytometric analysis for intracellular phospho-AKT (S473) level. Mean fluorescence intensity after normalization to mock-treated, empty virus control is plotted for a representative example of assays performed in duplicate.</p

Pharmacological inhibition of IGF1R restricts growth of a subset of human T-ALL cell lines.

<p>Cell growth as measured by resazurin reduction assay. Twenty-seven human T-ALL cell lines were cultured <i>in vitro</i> for 3 days with either (<b>A</b>) IGF1R blocking antibody (CP-751,871; 1 μg/ml) versus PBS vehicle control (mock) or (<b>B</b>) dual IGF1R/InsR tyrosine kinase inhibitor (BMS-754807; 0.5 μM) versus DMSO vehicle control (mock). Mean resorufin (reduced resazurin) fluorescence values +/- SD after normalization to mock-treated controls are plotted for assays performed in triplicate. Cell lines are rank ordered left-to-right by decreasing effect of the CP-751,871 blocking antibody. The horizontal dotted line indicates the 20% growth inhibition level.</p

Treatment with Insulin Analog X10 and IGF-1 Increases Growth of Colon Cancer Allografts

<div><p>Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured <i>in vitro</i> correlate with tumor growth-promoting effects <i>in vivo</i>. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls <i>in vitro</i>, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) <i>in vitro</i> and <i>in vivo</i>. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic <i>in vitro</i> and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.</p></div

Tumor growth <i>in vivo</i>.

<p>95% CI = 95% confidence interval.</p>†<p>fold change, i.e., mean value for a given treatment expressed relative to the mean value of the vehicle-treated group.</p>*<p>, ** and *** indicates <i>P</i><0.05, <0.001 and <0.0001 respectively.</p