A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly “undruggable” therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor I-BET151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in four models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of I-BET151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that I-BET151 and alisertib are effective in prolonging survival in four xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly "undruggable" therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor IBET-151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in three models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of IBET-151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that IBET-151 and alisertib are effective in prolonging survival in three xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.

The EWS-FLI1 fusion protein drives oncogenesis in the Ewing sarcoma family of tumors (ESFT) in humans, but its toxicity in normal cells requires additional cellular events for oncogenesis. We show that the lncRNA HOTAIR maintains cell viability in the presence of EWS-FLI1 and redirects epigenetic regulation in ESFT. HOTAIR is consistently overexpressed in ESFTs and is not driven by EWS-FLI1. Repression of HOTAIR in ESFT cell lines significantly reduces anchorage-independent colony formation in vitro and impairs tumor xenograft growth in vivo. Overexpression of HOTAIR in human mesenchymal stem cells (hMSCs), a putative cell of origin of ESFT, and IMR90 cells induces colony formation. Critically, HOTAIR-expressing hMSCs and IMR90 cells remain viable with subsequent EWS-FLI1 expression. HOTAIR induces histone modifications and gene repression through interaction with the epigenetic modifier LSD1 in ESFT cell lines and hTERT-hMSCs. Our findings suggest that HOTAIR maintains ESFT viability through epigenetic dysregulation.SignificanceWhile the EWS-FLI1 fusion gene was determined to be the oncogenic driver in the overwhelming majority of ESFT, it is toxic to cell physiology and requires one or more additional molecular events to maintain cell viability. As these tumors have surprisingly few genetic mutations at diagnosis, epigenetic changes have been considered to be such an event, but the mechanism by which these changes are driven remains unclear. Our work shows that HOTAIR is consistently expressed among ESFT and induces epigenetic and gene expression changes that cooperate in tumorigenesis. Furthermore, expression of HOTAIR allows for cell viability in the setting of subsequent EWS-FLI1 expression. Our findings elucidate new steps of malignant transformation in this cancer and identify novel therapeutic targets.

Background: Ewing sarcoma (ES) is an undifferentiated cancer of the bone and soft tissue canonically defined by expression of EWS-FLI1 or similar fusion proteins. The cell of origin remains undefined but is likely either a human mesenchymal stem cell (hMSC) or neural crest stem cell. The ES cells are depending on EWS-FLI1 for viability, but paradoxically exogenous EWS-FLI1 expression causes cell death in virtually all other cells. Expression of EWS-FLI1 in hMSCs has been variably successful and unreliable, and the two prior reports of stable expression did not elucidate why those efforts were successful, preventing reproducibility. Our work has shown that expression of the lncRNA HOTAIR allows for subsequent stable expression of EWS-FLI1 in hTERT-immortalized hMSCs and primary hMSCs but without induction of tumorigenesis in vivo. Methods: We used established ES cell lines, hTERT-hMSCs and primary hMSCS, to evaluate mRNA and protein expression at baseline and in the context of modulation of expression of HOTAIR, EWS-FLI1, MYC, and MYCN. Results: We demonstrated by RT-qPCR that HOTAIR is highly expressed across all ES cell lines as compared to hTERT-hMSCs and primary hMSCS. We also found that MYC is highly expressed in most ES cells lines and hMSCs. In contrast, MYCN is overexpressed in ES cell lines as compared to hMSCs. Repression of MYC in ES cell lines had no significant effect on proliferation of ES cells in vitro; repression of MYCN by shRNA in ES cell lines decreased proliferation in vitro and slowed tumor growth in vivo. Exogenous expression of HOTAIR in hMSCs allowed for viable subsequent expression of EWS-FLI1 in those cells, but without tumorigenesis in vivo (0/10 for hTERT- or primary hMSCs). Overexpression of MYC (0/6) or MYCN (1/6) in hMSCs expression HOTAIR did not reliably induce tumorigenesis. Overexpression of MYC or MYCN in hMSCs did not allow for viable EWS-FLI1 expression. Overexpression of HOTAIR, then EWS-FLI1 and MYCN, to levels comparable with ES cell lines, induced tumorigenesis from hTERT-hMSCS and primary hMSCs (6/6 for each), when cells were implanted subcutaneously into SCID mice with Matrigel. The resultant tumors were positive by immunohistochemistry for CD99 and NKX2.2, clinical markers of ES. RNA-Seq demonstrated the HOTAIR-EWS-FLI1-MYCN expressing hMSCs had gene expression profiles comparable to ES tumors. Conclusions: We have generated the first genetically defined tumorigenic models of ES from non-neoplastic human cells. This model can be used to elucidate the biology of EWS-FLI1 in malignant transformation and tumor formation. This work supports our prior data demonstrating a necessary role for HOTAIR in ES biology and suggests that MYCN may also be a driver of tumorigenesis in ES. Additional work is needed to define the underlying factors that induce HOTAIR and MYCN expression in ES (i.e., are they basally or aberrantly expressed in the cell of origin?) and if they are therapeutically targetable. This abstract is also being presented as Poster B77. Citation Format: Joshua Felgenhauer, Laura Tomino, Hasan Siddiqui, Emily Bopp, Julia Selich-Anderson, Jessica Mormol, Cenny Taslim, Nilay Shah. Generation of the first genetically defined tumorigenic model of Ewing sarcoma expressing EWS-FLI1 [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR02.

Background: Ewing sarcoma (ES), a bone cancer affecting children, adolescents, and young adults, is driven by the EWS-FLI1 fusion protein in the majority of cases, but the mechanisms of tumorigenesis are still being elucidated. Consequentially, therapeutic advances have been limited in the last 25 years. Two therapeutic targets have been recently examined. Aurora kinase A (AURKA) promotes cell cycling and posttranslational stabilization of the oncoprotein MYC, while bromodomain-containing protein 4 (BRD4) promotes gene expression epigenetically. Studies of AURKA and BRD4 inhibitors showed some impairment of tumorigenesis in ES preclinical models, but this efficacy was limited in single-agent use. AURKA and BRD4 activate common oncogenic pathways through different mechanisms, so we hypothesized dual inhibition would be synergistic against tumorigenesis. Aims: (a) Define the synergistic antineoplastic effects of BRD4 inhibitor I-BET151 and AURKA inhibitor alisertib against ES cell lines in vitro. (b) Confirm the mechanism of activity of each agent in these cell line models. (c) Define the efficacy of I-BET 151 and alisertib alone and in combinations with each other, and with the chemotherapeutic drug vincristine against ES tumor xenografts in vivo. Methods and results: I-BET151 and alisertib synergistically inhibit viability in ES cell lines SK-ES, TC71, and ES2 in vitro. Alisertib alone upregulates transcriptional expression of its targets, but combined use of I-BET151 mitigates that upregulation, likely contributing to the drug synergy and downregulating RNA and protein expression of key oncogenic pathways as shown by RT-qPCR and western blot. Alisertib and I-BET151 significantly prolong survival in three ES xenograft models in vivo, and this efficacy is augmented by the addition of vincristine. Conclusion: Dual targeting of oncogenic drivers in ES epigenetically and posttranslationally, specifically by use of BRD4 and AURKA inhibitors, may have significant clinical efficacy in ES alone and/or in combination with current chemotherapy regimens.