Chromosome 8 gain drives cancer progression by hijacking the translation factor 4E-BP1 sensitizing for targeted CDK4/6 inhibition

Abstract

To refine patient care in the context of precision oncology, it is increasingly important to understand mechanisms underlying inter-individual tumor heterogeneity, especially in oligomutated cancers. Ewing sarcoma (EwS) is genetically characterized by pathognomonic FET::ETS gene fusions (in most cases EWSR1::FLI1) while featuring a general paucity of other recurrent somatic alterations that might account for observed diversity in clinical patient outcome. Following FET::ETS fusions, chromosome (chr) 8 gain is the second most common recurrent somatic alteration in EwS. However, its pathophysiological role and potential clinical implications remain unclear. Here, we report that gene expression signatures indicative for chr8 gain are significantly associated with poor overall survival of EwS patients, and that this effect is predominantly mediated by expression of the translation initiation factor binding protein EIF4EBP1 (4E-BP1). High EIF4EBP1 expression shows the strongest association with poor overall survival of EwS patients among all genes located on chr8, which as well holds true in a subgroup analysis of patients with localized disease, emphasizing the prognostic role of 4E-BP1 in EwS. Consistently, chr8 gain was associated with higher intra-tumoral EIF4EBP1 expression in EwS patient samples. RNA interference-mediated knockdown of 4E-BP1 significantly decreased proliferation, clonogenicity, and spheroidal growth of EwS cells in vitro as well as tumor growth of EwS xenografts in vivo. To identify potential mechanisms via which 4E-BP1 mediates its phenotypic effect, we performed integrated proteomic and transcriptomic profiling of EwS cell lines with and without silencing of 4E-BP1 revealing that 4E-BP1 guides a multifunctional proteomic network including hubs associated with RNA processing, translational regulation, and chromatin modification. Collectively, we establish a significant association between chr8 gain and unfavorable prognosis in EwS. We show that this association primarily depends on the expression of the translation initiation factor binding protein 4E-BP1 regulating a multifunctional proteomic network. Our data suggest that cytogenetic testing for chr8 gain in EwS tumors may help to improve risk-stratification of patients and adaptation of the therapeutic management.

TheEIF4EBP1gene encoding 4EBP1 is transcriptionally upregulated by MYC and linked to shorter survival in medulloblastoma

“…Given the clinical relevance we uncovered for EIF4EBP1 mRNA and 4EBP1 protein expression in MB patients, we wondered whether 4EBP1 exerts a pro-tumorigenic function in this tumor entity, as reported in gliomas [36] and Ewing sarcomas [21]. Furthermore, we previously reported that 4EBP1 promotes the survival of MB cells under glucose starvation [36], a feature that has been linked to tumorigenic promotion [27].…”

“…As glucose levels are particularly low in MB [3], as compared to other pediatric brain cancers, high 4EBP1 expression may confer resistance to MB cells against such metabolic stress conditions. Since molecular mechanisms of tumor adaptation to glucose starvation are similar to the ones promoting tumorigenesis [27, 36], and since we and others reported that 4EBP1 promotes tumorigenesis of glioblastoma and Ewing’s sarcoma cells in vivo [21, 36], we explored 4EBP1 function in MB cell growth in vitro . Our findings suggest that 4EBP1 contributes to the tumorigenic capacity, i.e.…”

“…EIF4EBP1 mRNA expression is upregulated in numerous tumor entities [36, 67] and high EIF4EBP1 mRNA levels correlate with poor survival in several cancer types [67, 36, 21, 60, 64, 28, 58, 8, 9]. In MB, the amount of phosphorylated 4EBP1, i.e., inactive 4EBP1, was reported to be lower in non-SHH/non-WNT MBs when compared to SHH and WNT MBs [66].…”

“…While 4EBP1 appears to exert tumor suppressor activity, since it blocks the oncoprotein eIF4E [33], inhibits cellular proliferation [19] and restricts tumor growth in genetically engineered mouse models of prostate [18] as well as head and neck squamous cell carcinoma (HNSCC) [65], pro-tumorigenic functions also have been reported for 4EBP1. Indeed, it was reported that 4EBP1 promotes angiogenesis in ovarian and breast cancer models, thereby facilitating tumor growth under hypoxia [6, 34], supports oncogenic transformation [36, 53], and promotes glioma and Ewing sarcoma tumorigenicity [36, 21]. However, the role of 4EBP1 in MB is currently unknown.…”

Glutaredoxin 3 (GLRX3) confers a fusion oncogene-dependent vulnerability to Ewing sarcoma

“…Of the 87 drugs, 9 had a Δ DSS ≥ 5 and 6 had a Δ DSS ≤ –5. Among the drugs with higher efficacy in GLRX3-high conditions, we focused in independent validation experiments – using a colorimetric resazurin assay 50 – on the CDK4/6 inhibitor palbociclib because it is currently in clinical trials that included EwS patients 51 ( NCT04129151 , NCT03155620 , NCT03526250 , NCT03709680 ) and as we and others have shown previously that CDK4/6 inhibition is effective in preclinical EwS models 52–54 . Furthermore, we also paid particular attention to CDK4/6 inhibitors because the mechanism is based on a disruption of the cell cycle machinery, and since our data pointed to a strong connection of GLRX3 and cell cycle signatures in vitro and in situ ( Fig.…”