High 4E-BP1 expression associates with chromosome 8 gain and CDK4/6 sensitivity in Ewing sarcoma
Cornelius M. Funk, Anna C. Ehlers, Martin F. Orth, Karim Aljakouch, Jing Li, Tilman L.B. Hölting, Rainer Will, Florian H. Geyer, A. Katharina Ceranski, Franziska Willis, Endrit Vinca, Shunya Ohmura, Roland Imle, Jana Siebenlist, Angelina Yershova, Maximilian M.L. Knott, Felina Zahnow, Ana Sastre, Javier Alonso, Felix Sahm, Heike Peterziel, Anna Loboda, Martin Schneider, Ana Banito, Gabriel Leprivier, Wolfgang Hartmann, Uta Dirksen, Olaf Witt, Ina Oehme, Stefan M. Pfister, Laura Romero-Pérez, Jeroen Krijgsveld, Florencia Cidre-Aranaz, Thomas G.P. Grünewald, Julian Musa
Cornelius M. Funk, Anna C. Ehlers, Martin F. Orth, Karim Aljakouch, Jing Li, Tilman L.B. Hölting, Rainer Will, Florian H. Geyer, A. Katharina Ceranski, Franziska Willis, Endrit Vinca, Shunya Ohmura, Roland Imle, Jana Siebenlist, Angelina Yershova, Maximilian M.L. Knott, Felina Zahnow, Ana Sastre, Javier Alonso, Felix Sahm, Heike Peterziel, Anna Loboda, Martin Schneider, Ana Banito, Gabriel Leprivier, Wolfgang Hartmann, Uta Dirksen, Olaf Witt, Ina Oehme, Stefan M. Pfister, Laura Romero-Pérez, Jeroen Krijgsveld, Florencia Cidre-Aranaz, Thomas G.P. Grünewald, Julian Musa
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Research Article Genetics Oncology

High 4E-BP1 expression associates with chromosome 8 gain and CDK4/6 sensitivity in Ewing sarcoma

Abstract

Chromosome 8 (chr8) gains are common in cancer, but their contribution to tumor heterogeneity is largely unexplored. Ewing sarcoma (EwS) is defined by FET::ETS fusions with few other recurrent mutations to explain clinical diversity. In EwS, chr8 gains are the second most frequent alteration, making it an ideal model to study the relevance of chr8 gains in an otherwise silent genomic context. We report that chr8 gain–driven expression patterns correlate with poor overall survival of patients with EwS. This effect is mainly mediated by increased expression of the translation initiation factor binding protein 4E-BP1, encoded by EIF4EBP1 on chr8. Among all chr8-encoded genes, EIF4EBP1 expression showed the strongest association with poor survival and correlated with chr8 gains in EwS tumors. Similar findings emerged across multiple cancer entities in The Cancer Genome Atlas. Multiomics profiling revealed that 4E-BP1 orchestrates a pro-proliferative proteomic network. Silencing 4E-BP1 reduced proliferation, clonogenicity, spheroidal growth in vitro, and tumor growth in vivo. Drug screens demonstrated that high 4E-BP1 expression sensitizes EwS to pharmacological CDK4/6-inhibition. Chr8 gains and elevated 4E-BP1 emerge as prognostic biomarkers in EwS, with poor outcomes driven by 4E-BP1–mediated pro-proliferative networks that sensitize tumors to CDK4/6 inhibitors. Testing for chr8 gains may enhance risk stratification and therapy in EwS and other cancers.

Authors

Cornelius M. Funk, Anna C. Ehlers, Martin F. Orth, Karim Aljakouch, Jing Li, Tilman L.B. Hölting, Rainer Will, Florian H. Geyer, A. Katharina Ceranski, Franziska Willis, Endrit Vinca, Shunya Ohmura, Roland Imle, Jana Siebenlist, Angelina Yershova, Maximilian M.L. Knott, Felina Zahnow, Ana Sastre, Javier Alonso, Felix Sahm, Heike Peterziel, Anna Loboda, Martin Schneider, Ana Banito, Gabriel Leprivier, Wolfgang Hartmann, Uta Dirksen, Olaf Witt, Ina Oehme, Stefan M. Pfister, Laura Romero-Pérez, Jeroen Krijgsveld, Florencia Cidre-Aranaz, Thomas G.P. Grünewald, Julian Musa

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Figure 4

High 4E-BP1 expression sensitizes to targeted CDK4/6 inhibitor treatment with palbociclib and ribociclib.

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High 4E-BP1 expression sensitizes to targeted CDK4/6 inhibitor treatment...
(A) IC50 analysis of CDK4/6 inhibitors palbociclib and ribociclib in A-673 cells containing indicated Dox-inducible shRNAs, as measured by resazurin colorimetry. Cells were treated with or without Dox as well as with serial dilutions of respective inhibitors. Horizontal bars represent means; whiskers represent the SEM; n ≥ 5 biologically independent experiments. P values were determined via 1-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. (B) IC50 analysis of CDK4/6 inhibitors palbociclib and ribociclib in EwS cells with high and low endogenous 4E-BP1 expression. Horizontal bars represent means; whiskers represent the SEM; n ≥ 3 biologically independent experiments. P values were determined via 1-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. (C) NSG mice xenografted with A-673 EwS cells containing a Dox-inducible sh4E-BP1 construct, treated with or without Dox and either vehicle or palbociclib. Mice were randomized to the treatment groups when tumors were palpable. For each condition, the mean tumor volume and SEM of 4–6 mice over the time of treatment are shown. P values were determined via 2-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. (D) Representative H&E-stained micrographs of A673/sh4E-BP1 xenografts [Dox (–)] treated with either vehicle or palbociclib, as described in (C) (shown as an overview with ×12.5 magnification and as a high-power field [HPF] at ×400 magnification). Scale bar: 2.5 mm (×12.5) and 100 μm (×400). (E) Quantification of mitoses in micrographs of xenografts described in (C). Horizontal bars represent means; whiskers represent the SEM; n ≥ 2 samples per condition. P values were determined via 2-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. Rel, relative. (F) EIF4EBP1 gene expression data from EwS tumors of 14 patients treated within the INFORM registry and stratified according to matched palbociclib or ribociclib drug-sensitivity data from 3D tumor cell cultures into a CDK4/6 inhibitor nonresponsive and responsive groups. ***P < 0.001, **P < 0.01, *P < 0.05. P values were determined via 2-tailed Mann-Whitney test if not otherwise specified.