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 3

RNAi-mediated knockdown of 4E-BP1 inhibits EwS growth.

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RNAi-mediated knockdown of 4E-BP1 inhibits EwS growth. (A) Relative viab...
(A) Relative viable cell count of A-673, SK-N-MC, and TC-71 cells containing either Dox-inducible specific shRNA constructs directed against EIF4EBP1 (sh4E-BP1_1 or sh4E-BP1_2) or an shCtr, as measured by trypan blue exclusion. Cells were grown either with or without Dox for 120 hours. Horizontal bars represent means; whiskers represent the SEM; n ≥ 4 biologically independent experiments. P values were determined via 1-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. (B) Relative colony numbers of EwS cells containing indicated Dox-inducible shRNA constructs. Cells were grown either with or without Dox for 8–14 days. Horizontal bars represent means; whiskers the SEM; n ≥ 4 biologically independent experiments. P values were determined via 2-tailed Mann-Whitney test and adjusted for multiple comparisons with the Benjamini-Hochberg method. Representative images of colony formation are shown on the right. (C) Sphere formation in EwS cells containing indicated Dox-inducible shRNA constructs, treated with or without Dox for 8–14 days. Horizontal bars represent means; whiskers represent the SEM; n ≥ 3 biologically independent experiments. P values were determined by 2-tailed unpaired t test with Welch′s correction and adjusted for multiple comparisons with the Benjamini-Hochberg method. Representative images of spheres are shown on the right. (D) Kaplan-Meier analysis of event-free survival of NSG mice xenografted with A-673 cells containing indicated Dox-inducible shRNA constructs. Once tumors were palpable, mice were randomly assigned to treatment with either vehicle (–) or Dox (+); n ≥ 5 animals per condition. An “event” was recorded when tumors reached a size maximum of 15 mm in 1 dimension. P values were determined via Mantel-Haenszel test. (E) Quantification of mitoses in H&E-stained slides of xenografts described in (D). Five high-power fields (HPFs) were counted per sample. Horizontal bars represent means; whiskers represent the SEM; n ≥ 4 samples per condition. Rel, relative. (F) Kaplan-Meier analysis of event-free survival of NSG mice orthotopically xenografted into the proximal tibia with TC-71 cells containing a Dox-inducible shRNA construct directed against EIF4EBP1. n = 5 animals per condition. P values were determined via Mantel-Haenszel test. ***P < 0.001, **P < 0.01, *P < 0.05, determined via 2-tailed Mann-Whitney test if not otherwise specified.