Epigenetic Restoration of Fetal-like IGF1 Signaling Inhibits Leukemia Stem Cell Activity

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High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

Notch-driven expression of IGF1R promotes the growth, viability, and transplantability of T-ALL cells

Characterization of select downstream effectors of oncogenic NOTCH1 in T-cell acute lymphoblastic leukemia

Oncogenic NOTCH1 signalling is a major driver of T cell acute lymphoblastic leukemia (T-ALL) transformation and growth. Although some downstream effectors of this function are known, they cannot explain all observed pro-growth and leukemogenic phenotypes and there are undoubtedly other effectors yet to be described and investigated. This study identifies microRNAs (miRNAs) regulated by NOTCH1 in T-ALL and further characterizes the actions of insulin-like growth factor 1 receptor (IGF1R) and protein kinase C theta (PKCθ), two signalling molecules I was previously involved in identifying as being regulated by NOTCH1 in a T-ALL context. I found that NOTCH1 can negatively regulate miR-223 expression, contributing to its ability to enhance IGF1R expression. In turn, IGF1R signalling is important to maintain growth in a subset of T-ALL cell lines and is a major positive effector of the PI3K/AKT signalling pathway. IGF1R downstream signalling pathways may be negatively affected by PKCθ. As expression of PKCθ is negatively regulated by NOTCH1 in T-ALL, here, I have attempted to identify its direct phosphorylation targets in this context. I have done this through the combined use of an analog sensitive (AS) kinase screen and an ascorbate peroxidase (APEX) based chemical labelling proximity screen. Candidate direct PKCθ phosphorylation targets identified include potential IGF1R downstream signalling components such as IRS4, mTOR, RICTOR, RAF1 and ARAF. Some of these targets were also found to be proximal to PKCθ in a T-ALL cellular context. This suggests that, in addition to regulating IGF1R signalling at the transcript or protein (via miR-223 repression) level, NOTCH1 also has the potential to positively regulate this pathway through repressing PKCθ phosphorylation of downstream components. Further studies are required to validate this hypothesis. Other candidate direct PKCθ phosphorylation targets identified here may also be worth further investigation and may suggest the involvement of PKCθ in additional cellular processes in T-ALL. Further development of my novel combined approach for the identification of direct phosphorylation targets may prove to be useful for the investigation of other kinases in a broad range of cell types.Medicine, Faculty ofGraduat

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

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

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

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