EWS/ETS-Driven Ewing Sarcoma Requires BET Bromodomain Proteins

Gollavilli, Paradesi Naidu; Pawar, Aishwarya; Wilder-Romans, Kari; Natesan, Ramakrishnan; Engelke, Carl G.; Dommeti, Vijaya L.; Krishnamurthy, Pranathi M.; Nallasivam, Archana; Apel, Ingrid J.; Xu, Tianlei; Qin, Zhaohui S.; Feng, Felix Y.; Asangani, Irfan A.

doi:10.1158/0008-5472.can-18-0484

Cited by 56 publications

(83 citation statements)

References 47 publications

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“…We also confirmed AURKA kinase inhibition by alisertib and concomitant MYC repression, consistent with prior studies . Our work does not preclude other biological impacts of alisertib and I‐BET151 on ES biology; in fact, we do confirm that BRD4 inhibition does impair EWS‐FLI1‐mediated transcriptional activation as shown by I‐BET151's repression of NR0B1, consistent with recent data . Nonetheless, our data does demonstrate a mechanism of synergy between the two drugs, further augmented by use of chemotherapy such as vincristine.…”

Section: Discussionsupporting

confidence: 92%

“…BRD4 is active and oncogenic in cancers by promoting expression of multiple targets, including CDK4/6, MCL1, BCL2, MYC,, and AURKA . BRD4 has been more recently shown to be critical for EWS‐FLI1‐directed transcriptional reprogramming . BRD4 inhibitors showed some preclinical efficacy against ES; while BRD4 inhibition alone could not induce ES tumor regression in these studies, the data did show BRD4 inhibition could inhibit tumor formation .…”

Section: Introductionmentioning

confidence: 83%

“…BRD4 has been more recently shown to be critical for EWS‐FLI1‐directed transcriptional reprogramming . BRD4 inhibitors showed some preclinical efficacy against ES; while BRD4 inhibition alone could not induce ES tumor regression in these studies, the data did show BRD4 inhibition could inhibit tumor formation . These data suggest that BRD4 inhibition may have cytostatic effects that limit its use as a solo agent against ES but may augment other molecular therapeutic approaches.…”

Section: Introductionmentioning

confidence: 84%

“…Those oncogenic drivers could serve as novel therapeutic targets to augment current treatment strategies. Prior studies demonstrated that AURKA and BRD4 both promote oncogenic phenotypes in ES, making them attractive therapeutic targets. However, preclinical and clinical studies using agents targeting these proteins showed limited efficacy against ES when used alone .…”

Section: Discussionmentioning

confidence: 99%

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Combinatorial BRD4 and AURKA inhibition is synergistic against preclinical models of Ewing sarcoma

et al. 2019

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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.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

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Combinatorial BRD4 and AURKA inhibition is synergistic against preclinical models of Ewing sarcoma

et al. 2019

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“…Prior work indicates that BET bromodomain inhibitors and degraders may have applications in Ewing Sarcoma. 24 To investigate potential synergy between direct and indirect repression of the EWS/FLI transcriptional program, we evaluated degradation of EWS/FLI in combination with BET bromodomain degradation. We observed that dBET6, a CRBN-recruiting, pan-BET bromodomain degrader, 25 synergized strongly with VHL-mediated EWS/FLI degradation (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Nabet

Ferguson

Seong

et al. 2020

Preprint

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35Chemical biology strategies for directly perturbing protein homeostasis including the 36 degradation tag (dTAG) system provide temporal advantages over genetic approaches and 37 improved selectivity over small molecule inhibitors. We describe dTAG V -1, an exclusively 38 selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12 F36V -tagged proteins. 39 dTAG V -1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to 40 degrade recalcitrant oncogenes, supports combination degrader studies and facilitates 41 investigations of protein function in cells and mice. 42 43 We next evaluated the utility of dTAG V -1 for combinatorial degrader studies. dTAG V -1 94 was effectively combined with THAL-SNS-032, a previously reported CRBN-recruiting CDK9 95 degrader 21 (Fig. 1e). Pronounced degradation of LACZ-FKBP12 F36V and CDK9 was observed to 96 levels comparable to those with treatment of each degrader alone, avoiding potential substrate 97 competition effects. 22 To confirm the utility of dTAG V -1 for target validation, we evaluated 98 KRAS G12V degradation, an oncogenic driver of PDAC, in PATU-8902 FKBP12 F36V -KRAS G12V ;; 99 KRAS -/cells. 17 dTAG V -1 treatment led to rapid KRAS G12V degradation, which was rescued by 100 use of dTAG V -1-NEG, pre-treatment with proteasome-inhibitor (carfilzomib) or Nedd8 activating 101 enzyme inhibitor (MLN4924), and VHL knockout, consistent with the mechanism of action of a 102 VHL-recruiting degrader ( Fig. 1f-g and Supplementary Fig. 2a-b). We also observed the 103 expected collapse in cellular signaling and diminished cell proliferation in 2D-monolayer and 3D-104 spheroid cultures upon FKBP12 F36V -KRAS G12V degradation with dTAG V -1 treatment, to levels 105 comparable to CRBN-recruiting dTAG molecules ( Fig. 1h and Supplementary Fig. 2c-e). 106To confirm the in vivo applicability of dTAG V -1, we characterized the pharmacokinetic 107 (PK) and pharmacodynamic (PD) profile of dTAG V -1 in mice. dTAG V -1 demonstrated improved 108properties compared to dTAG-13, with a longer half-life (T1/2 = 4.43, 2.41 h respectively) and 109 greater exposure (AUCinf = 18517, 6140 hr*ng mL -1 respectively) by intraperitoneal 110 administration at 10 mg/kg ( Supplementary Table 1). To report on the PD profile of dTAG 111 molecules, we employed MV4;;11 luciferase-FKBP12 F36V (luc-FKBP12 F36V ) cells 6 that allow non-112 invasive monitoring of bioluminescent signal upon dTAG molecule administration in mice. 113Following tail vein injection of MV4;;11 luc-FKBP12 F36V cells and establishment of leukemic 114 burden, we performed daily bioluminescent measurements 4 h after vehicle, 35 mg/kg dTAG-13 115 or 35 mg/kg dTAG V -1 administration. Striking loss of bioluminescent signal was achieved 4 h 116 after the first administration of dTAG V -1 ( Fig. 1i and Supplementary Fig. 3). Consistent loss of 117 bioluminescent signal was observed 4 h after each of the three dTAG-13 or dTAG V -1 118 administrations. Compared to dTAG-13, improved duration of degradation w...

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FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

et al. 2022

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FET fusion oncoproteins containing one of the FET (FUS, EWSR1, TAF15) family proteins juxtaposed to alternative transcription-factor partners are characteristic of more than 20 types of sarcoma and leukaemia. FET oncoproteins bind to the SWI/SNF chromatin remodelling complex, which exists in three subtypes: cBAF, PBAF and GBAF/ncBAF. We used comprehensive biochemical analysis to characterize the interactions between FET oncoproteins, SWI/SNF complexes and the transcriptional coactivator BRD4. Here, we report that FET oncoproteins bind all three main SWI/SNF subtypes cBAF, PBAF and GBAF, and that FET oncoproteins interact indirectly with BRD4 via their shared interaction partner SWI/SNF. Furthermore, chromatin immunoprecipitation sequencing and proteomic analysis showed that FET oncoproteins, SWI/SNF components and BRD4 co-localize on chromatin and interact with mediator and RNA Polymerase II. Our results provide a possible molecular mechanism for the FET-fusion-induced oncogenic transcriptional profiles and may lead to novel therapies targeting aberrant SWI/SNF complexes and/or BRD4 in FET-fusion-caused malignancies.

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EWS/ETS-Driven Ewing Sarcoma Requires BET Bromodomain Proteins

Cited by 56 publications

References 47 publications

Combinatorial BRD4 and AURKA inhibition is synergistic against preclinical models of Ewing sarcoma

Combinatorial BRD4 and AURKA inhibition is synergistic against preclinical models of Ewing sarcoma

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

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