Annette Vu

ATF4 Regulates MYC-Mediated Neuroblastoma Cell Death upon Glutamine Deprivation

Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA, and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis, and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.

Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma

Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single-agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with noncurative chemotherapy even for highest risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria.

Polyamine antagonist therapies inhibit neuroblastoma initiation and progression

Purpose: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC. Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. Experimental Design: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. Results: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. Conclusions: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391–404. ©2016 AACR.

Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma

Cancer treatments induce cell stress to trigger apoptosis in tumor cells. Many cancers repress these apoptotic signals through alterations in the Bcl2 proteins that regulate this process. Therapeutics that target these specific survival biases are in development, and drugs that inhibit Bcl2 activities have shown clinical activity for some cancers. Mcl1 is a survival factor for which no effective antagonists have been developed, so it remains a principal mediator of therapy resistance, including to Bcl2 inhibitors. We used a synthetic-lethal screening strategy to identify genes that regulate Mcl1 survival activity using the pediatric tumor neuroblastoma (NB) as a model, as a large subset are functionally verified to be Mcl1 dependent and Bcl2 inhibitor resistant. A targeted siRNA screen identified genes whose knockdown restores sensitivity of Mcl1-dependent NBs to ABT-737, a small molecule inhibitor of Bcl2, BclXL and BclW. Three target genes that shifted the ABT-737 IC50 >1 log were identified and validated: PSMD14, UBL5 and PRPF8. The latter two are members of a recently characterized subcomplex of the spliceosome that along with SART1 is responsible for non-canonical 5′-splice sequence recognition in yeast. We showed that SART1 knockdown similarly sensitized Mcl1-dependent NB to ABT-737 and that triple knockdown of UBL5/PRPF8/SART1 phenocopied direct MCL1 knockdown, whereas having no effect on Bcl2-dependent NBs. Both genetic spliceosome knockdown or treatment with SF3b-interacting spliceosome inhibitors like spliceostatin A led to preferential pro-apoptotic Mcl1-S splicing and reduced translation and abundance of Mcl1 protein. In contrast, BN82865, which inhibits the second transesterification step in terminal spliceosome processing, did not have this effect. These findings demonstrate a prominent role for the spliceosome in mediating Mcl1 activity and suggest that drugs that target either the specific UBL5/PRPF8/SART1 subcomplex or SF3b functions may have a role as cancer therapeutics by attenuating the Mcl1 survival bias present in numerous cancers.

Abstract 2459: Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering

Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies. Citation Format: Jorida Coku, Elizabeth O. Scadden, Kangning Liu, Annette Vu, David M. Booth, Michelle Chen, Sharon Kim, C. Patrick Reynolds, Gyorgy Hajnoczky, Michael D. Hogarty. Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2459.

Abstract B203: Polyamine blockade modulates the neuroblastoma microenvironment

Neuroblastoma (NB) accounts for a considerable portion of childhood cancer-related mortalities. Despite improvements in therapy over the last few decades, 5-year survival rates in patients with high-risk disease remain poor (40-50%). High-risk disease results from amplification of MYCN and dysregulation in downstream Myc-related pathways. Polyamine (PA) synthesis is one such pathway, as MYCN-amplified high-risk NBs have elevated polyamine levels, and the gate-keeper enzyme in this pathway, ornithine decarboxylase (ODC1), is a direct target of Myc. In a mouse model of MYCN-driven NB (TH-MYCN+/+ transgenic mice), inhibition of PA synthesis with an irreversible inhibitor of ODC1, difluoromethylornithine (DFMO), led to reductions in NB burden that seemed out of proportion to DFMO9s ability to limit tumor growth in vitro, suggesting that PA blockade may result in additional tumor cell-extrinsic effects. Consistent with this possibility, PAs have been shown to favor the differentiation and function of suppressive, tumor-supportive cell types, while PA depletion results in reversal of immune suppression via increased number and function of tumor-infiltrating leukocytes (TILs) and concomitant inhibition of infiltration and differentiation of suppressive cell types. However, these previous studies focused on investigating the effects of PA depletion in vitro or in vivo in tumor-induction models or immune-deficient mice. We therefore sought to characterize the tumor microenvironment of NB in TH-MYCN+/+ mice in the presence or absence of DFMO-mediated PA blockade. Tumors at terminal disease were dissected, mechanically and enzymatically dissociated, and the number and frequencies of various TIL subsets were assessed using an optimized flow cytometry-based protocol. Results indicate that DFMO therapy reduces tumor growth and results in distinct and reproducible alterations in the cellular composition of the NB tumor microenvironment at terminal disease. Specifically, we found an increased frequency of dendritic cells, natural killer cells, and a late accumulation of CD4-negative invariant natural killer T cells and granulocytic tumor-associated myeloid cells. Increases in these specific TIL subsets are consistent with the hypothesis that polyamine blockade induces distinct tumor cell-extrinsic changes in the microenvironment that allow for more efficient immune control of NB. We hope that these studies will complement the data being accrued from phase I/II clinical studies using DFMO in various therapeutic strategies for NB and will allow for an increased understanding of how to employ PA blockade in the immunotherapy of this disease. Citation Format: Adriana D. Benavides, Annette T. Vu, Jennie B. Altman, Gabrielle M. Ferry, Michael D. Hogarty, Hamid Bassiri. Polyamine blockade modulates the neuroblastoma microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B203.

Abstract 4848: A synthetic-lethal screen identifies novel Mcl1 regulators in neuroblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Neuroblastoma (NB) is a lethal pediatric tumor. Despite multimodal therapy survival remains poor due to emergent chemoresistance governed by Bcl2-homology (BH) proteins. Agents that antagonize BH proteins, such as ABT-737, are in clinical development. ABT-737 does not antagonize Mcl1, a Bcl2-homologue correlated with ABT-737 resistance in many tumors. We defined the survival bias present in NBs using functional mitochondrial profiling in response to diverse BH3 death-domain peptides. Those with a functional Bcl2-dependence were exquisitely sensitive to ABT-737 in vitro and in vivo. Those with an Mcl1-dependence were relatively ABT-737 resistant (3-5 uM IC50s), yet Mcl1 knock-down restored ABT-737 activity in this subset (nM IC50s). We therefore undertook a synthetic-lethal screen for Mcl1 regulators using siRNA against deubiquitinating enzymes (DUBs), as Mcl1 is highly regulated with a short half-life due to ubiquitin-mediated degradation and this genomic space has many plausible druggable targets. We treated Mcl1-dependent NB cell lines (IMR5 and NLF) with siRNA targeting DUBs and ABT-737 at a sublethal concentration (200 nM). This screen identified 5 targets (of 98) demonstrating synthetic lethality (Z 10-fold decrease in IC50 for 4 of 5 targets. Three further demonstrated a >10-fold decrease in ABT-737 IC50 with multiple independent siRNA supporting these as on-target effects (4/4 for PSMD14, 3/4 for PRPF8, and 2/4 for UBL5). We show that we can rescue the PSMD14 and UBL5 knock-down phenotypes by expression of an siRNA-resistant expression plasmid. While knock-down of all three targets decreases Mcl1 function, only knock-down of UBL5 (downregulates) and PSMD14 (upregulates) alter Mcl1 protein expression. We hypothesize that knock-down of PSMD14 and PRPF8 decrease Mcl1 activity through inhibitory post-translational modifications despite paradoxical or absent effects on Mcl1 protein expression, while UBL5 stabilizes Mcl1 as a ubiquitin-like PTM. Thus, we have demonstrated the robustness of a novel synthetic lethal screen to identify modulators of Mcl1 activity in vitro and identified three novel therapeutic targets regulating Mcl1 activity in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4848. doi:1538-7445.AM2012-4848