FDA-approved drug screen identifies proteasome as a synthetic lethal target in MYC-driven neuroblastoma.

MYCN amplification in neuroblastoma predicts poor prognosis and resistance to therapy. Yet pharmacological strategies of direct MYC inhibition remain unsuccessful due to its "undruggable" protein structure. We herein developed a synthetic lethal screen against MYCN-amplified neuroblastomas using clinically approved therapeutic reagents. We performed a high-throughput screen, from a library of 938 FDA-approved drugs, for candidates that elicit synthetic lethal effects in MYC-driven neuroblastoma cells. The proteasome inhibitors, which are FDA approved for the first-line treatment of multiple myeloma, emerge as top hits to elicit MYC-mediated synthetic lethality. Proteasome inhibition activates the PERK-eIF2α-ATF4 axis in MYC-transformed cells and induces BAX-mediated apoptosis through ATF4-dependent NOXA and TRIB3 induction. A combination screen reveals the proteasome inhibitor bortezomib (BTZ) and the histone deacetylase (HDAC) inhibitor vorinostat (SAHA) concertedly induce dramatic cell death in part through synergistic activation of BAX. This combination causes marked tumor suppression in vivo, supporting dual proteasome/HDAC inhibition as a potential therapeutic approach for MYC-driven cancers. This FDA-approved drug screen with in vivo validation thus provides a rationale for clinical evaluation of bortezomib, alone or in combination with vorinostat, in MYC-driven neuroblastoma patients.

Cell cycle-dependent degradation of the methyltransferase SETD3 attenuates cell proliferation and liver tumorigenesis.

Histone modifications, including lysine methylation, are epigenetic marks that influence many biological pathways. Accordingly, many methyltransferases have critical roles in various biological processes, and their dysregulation is often associated with cancer. However, the biological functions and regulation of many methyltransferases are unclear. Here, we report that a human homolog of the methyltransferase SET (SU(var), enhancer of zeste, and trithorax) domain containing 3 (SETD3) is cell cycle-regulated; SETD3 protein levels peaked in S phase and were lowest in M phase. We found that the ß-isoform of the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7ß) specifically mediates SETD3 degradation. Aligning the SETD3 sequence with those of well known FBXW7 substrates, we identified six potential non-canonical Cdc4 phosphodegrons (CPDs), and one of them, CPD1, is primarily phosphorylated by the kinase glycogen synthase kinase 3 (GSK3ß), which is required for FBXW7ß-mediated recognition and degradation. Moreover, depletion or inhibition of GSK3ß or FBXW7ß resulted in elevated SETD3 levels. Mutations of the phosphorylated residues in CPD1 of SETD3 abolished the interaction between FBXW7ß and SETD3 and prevented SETD3 degradation. Our data further indicated that SETD3 levels positively correlated with cell proliferation of liver cancer cells and liver tumorigenesis in a xenograft mouse model, and that overexpression of FBXW7ß counteracts the SETD3's tumorigenic role. We also show that SETD3 levels correlate with cancer malignancy, indicated by SETD3 levels that the 54 liver tumors are 2-fold higher than those in the relevant adjacent tissues. Collectively, these data elucidated that a GSK3ß-FBXW7ß-dependent mechanism controls SETD3 protein levels during the cell cycle and attenuates its oncogenic role in liver tumorigenesis.

Stabilization of Notch1 by the Hsp90 Chaperone Is Crucial for T-Cell Leukemogenesis.

Purpose: Notch1 deregulation is assuming a focal role in T-cell acute lymphoblastic leukemia (T-ALL). Despite tremendous advances in our understanding of Notch1 transcriptional programs, the mechanisms by which Notch1 stability and turnover are regulated remain obscure. The goal of the current study is to identify intracellular Notch1 (ICN1, the activated form of Notch1) binding partner(s) regulating its stability and activity. Experimental Design: We employed immunoaffinity purification to identify ICN1-associating partner(s) and used coimmunoprecipitation to verify the endogenous protein interaction. Pharmacologic or short hairpin RNA-mediated inhibition was applied in loss-of-function assays to assess the role of tentative binding partner(s) in modulating ICN1 protein stability as well as affecting T-ALL cell expansion in vitro and in vivo Mechanistic analysis involved protein degradation and polyubiquitination assays. Results: We identify the Hsp90 chaperone as a direct ICN1-binding partner essential for its stabilization and transcriptional activity. T-ALL cells exhibit constitutive endogenous ICN1-Hsp90 interaction and Hsp90 depletion markedly decreases ICN1 levels. The Hsp90-associated E3 ubiquitin ligase Stub1 mediates the ensuring proteasome-dependent ICN1 degradation. Administration of 17-AAG or PU-H71, two distinct Hsp90 inhibitors, depletes ICN1, inhibits T-ALL cell proliferation, and triggers dramatic apoptotic cell death. Systemic treatment with PU-H71 reduces ICN1 expression and profoundly inhibits murine T-ALL allografts as well as human T-ALL xenografts. Conclusions: Our findings demonstrate Hsp90 blockade leads to ICN1 destabilization, providing an alternative strategy to antagonize oncogenic Notch1 signaling with Hsp90-selective inhibitors. Clin Cancer Res; 23(14); 3834-46. ©2017 AACR.

Polo-like Kinase-1 Regulates Myc Stabilization and Activates a Feedforward Circuit Promoting Tumor Cell Survival.

MYCN amplification in human cancers predicts poor prognosis and resistance to therapy. However, pharmacological strategies that directly target N-Myc, the protein encoded by MYCN, remain elusive. Here, we identify a molecular mechanism responsible for reciprocal activation between Polo-like kinase-1 (PLK1) and N-Myc. PLK1 specifically binds to the SCFFbw7 ubiquitin ligase, phosphorylates it, and promotes its autopolyubiquitination and proteasomal degradation, counteracting Fbw7-mediated degradation of N-Myc and additional substrates, including cyclin E and Mcl1. Stabilized N-Myc in turn directly activates PLK1 transcription, constituting a positive feedforward regulatory loop that reinforces Myc-regulated oncogenic programs. Inhibitors of PLK1 preferentially induce potent apoptosis of MYCN-amplified tumor cells from neuroblastoma and small cell lung cancer and synergistically potentiate the therapeutic efficacies of Bcl2 antagonists. These findings reveal a PLK1-Fbw7-Myc signaling circuit that underlies tumorigenesis and validate PLK1 inhibitors, alone or with Bcl2 antagonists, as potential effective therapeutics for MYC-overexpressing cancers.

Myc promotes glutaminolysis in human neuroblastoma through direct activation of glutaminase 2.

Deamidation of glutamine to glutamate by glutaminase 1 (GLS1, also called GLS) and GLS2 is an essential step in both glutaminolysis and glutathione (GSH) biosynthesis. However, mechanisms whereby cancer cells regulate glutamine catabolism remains largely unknown. We report here that N-Myc, an essential Myc family member, promotes conversion of glutamine to glutamate in MYCN-amplified neuroblastoma cells by directly activating GLS2, but not GLS1, transcription. Abrogation of GLS2 function profoundly inhibited glutaminolysis, which resulted in feedback inhibition of aerobic glycolysis likely due to thioredoxin-interacting protein (TXNIP) activation, dramatically decreasing cell proliferation and survival in vitro and in vivo. Moreover, elevated GLS2 expression is significantly elevated in MYCN-amplified neuroblastomas in comparison with non-amplified ones, correlating with unfavorable patient survival. In aggregate, these results reveal a novel mechanism deciphering context-dependent regulation of metabolic heterogeneities, uncovering a previously unsuspected link between Myc, GLS2 and tumor metabolism.

ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 activation.

Amplification of the MYCN gene in human neuroblastoma predicts poor prognosis and resistance to therapy. We previously showed that MYCN-amplified neuroblastoma cells constantly require large amounts of glutamine to support their unabated growth. However, the identity and regulation of the transporter(s) that capture glutamine in MYCN-amplified neuroblastoma cells and the clinical significance of the transporter(s) in neuroblastoma diagnosis remain largely unknown. Here, we performed a systemic glutamine influx analysis and identified that MYCN-amplified neuroblastoma cells predominantly rely on activation of ASCT2 (solute carrier family 1 member 5, SLC1A5) to maintain sufficient levels of glutamine essential for the TCA cycle anaplerosis. Consequently, ASCT2 depletion profoundly inhibited glutaminolysis, concomitant with a substantial decrease in cell proliferation and viability in vitro and inhibition of tumourigenesis in vivo. Mechanistically, we identified ATF4 as a novel regulator which coordinates with N-Myc to directly activate ASCT2 expression. Of note, ASCT2 expression, which correlates with that of N-Myc and ATF4, is markedly elevated in high-stage neuroblastoma tumour samples compared with low-stage ones. More importantly, high ASCT2 expression is significantly associated with poor prognosis and survival of neuroblastoma patients. In aggregate, these findings elucidate a novel mechanism depicting how cell autonomous insults (MYCN amplification) and microenvironmental stresses (ATF4 induction) in concert coordinate ASCT2 activation to promote aggressive neuroblastoma progression, and establish ASCT2 as a novel biomarker in patient prognosis and stratification.