Targeting Glutaminolysis Shows Efficacy in Both Prednisolone-Sensitive and in Metabolically Rewired Prednisolone-Resistant B-Cell Childhood Acute Lymphoblastic Leukaemia Cells.

The prognosis for patients with relapsed childhood acute lymphoblastic leukaemia (cALL) remains poor. The main reason for treatment failure is drug resistance, most commonly to glucocorticoids (GCs). The molecular differences between prednisolone-sensitive and -resistant lymphoblasts are not well-studied, thereby precluding the development of novel and targeted therapies. Therefore, the aim of this work was to elucidate at least some aspects of the molecular differences between matched pairs of GC-sensitive and -resistant cell lines. To address this, we carried out an integrated transcriptomic and metabolomic analysis, which revealed that lack of response to prednisolone may be underpinned by alterations in oxidative phosphorylation, glycolysis, amino acid, pyruvate and nucleotide biosynthesis, as well as activation of mTORC1 and MYC signalling, which are also known to control cell metabolism. In an attempt to explore the potential therapeutic effect of inhibiting one of the hits from our analysis, we targeted the glutamine-glutamate-α-ketoglutarate axis by three different strategies, all of which impaired mitochondrial respiration and ATP production and induced apoptosis. Thereby, we report that prednisolone resistance may be accompanied by considerable rewiring of transcriptional and biosynthesis programs. Among other druggable targets that were identified in this study, inhibition of glutamine metabolism presents a potential therapeutic approach in GC-sensitive, but more importantly, in GC-resistant cALL cells. Lastly, these findings may be clinically relevant in the context of relapse-in publicly available datasets, we found gene expression patterns suggesting that in vivo drug resistance is characterised by similar metabolic dysregulation to what we found in our in vitro model.

Suppression of MYC by PI3K/AKT/mTOR pathway inhibition in combination with all-trans retinoic acid treatment for therapeutic gain in acute myeloid leukaemia.

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes.

The RARγ Oncogene: An Achilles Heel for Some Cancers.

Cancer "stem cells" (CSCs) sustain the hierarchies of dividing cells that characterize cancer. The main causes of cancer-related mortality are metastatic disease and relapse, both of which originate primarily from CSCs, so their eradication may provide a bona fide curative strategy, though there maybe also the need to kill the bulk cancer cells. While classic anti-cancer chemotherapy is effective against the dividing progeny of CSCs, non-dividing or quiescent CSCs are often spared. Improved anti-cancer therapies therefore require approaches that target non-dividing CSCs, which must be underpinned by a better understanding of factors that permit these cells to maintain a stem cell-like state. During hematopoiesis, retinoic acid receptor (RAR) γ is selectively expressed by stem cells and their immediate progeny. It is overexpressed in, and is an oncogene for, many cancers including colorectal, renal and hepatocellular carcinoma, cholangiocarcinomas and some cases of acute myeloid leukemia that harbor RARγ fusion proteins. In vitro studies suggest that RARγ-selective and pan-RAR antagonists provoke the death of CSCs by necroptosis and point to antagonism of RARγ as a potential strategy to treat metastatic disease and relapse, and perhaps provide a cure for some cancers.

Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.

A Case of AML Characterized by a Novel t(4;15)(q31;q22) Translocation That Confers a Growth-Stimulatory Response to Retinoid-Based Therapy.

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.

Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer.

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Interference with Sin3 function induces epigenetic reprogramming and differentiation in breast cancer cells.

Sin3A/B is a master transcriptional scaffold and corepressor that plays an essential role in the regulation of gene transcription and maintenance of chromatin structure, and its inappropriate recruitment has been associated with aberrant gene silencing in cancer. Sin3A/B are highly related, large, multidomian proteins that interact with a wide variety of transcription factors and corepressor components, and we examined whether disruption of the function of a specific domain could lead to epigenetic reprogramming and derepression of specific subsets of genes. To this end, we selected the Sin3A/B-paired amphipathic alpha-helices (PAH2) domain based on its established role in mediating the effects of a relatively small number of transcription factors containing a PAH2-binding motif known as the Sin3 interaction domain (SID). Here, we show that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction of a SID decoy that interferes with PAH2 binding to SID-containing partner proteins reverted the silencing of genes involved in cell growth and differentiation. In particular, the SID decoy led to epigenetic reprogramming and reexpression of the important breast cancer-associated silenced genes encoding E-cadherin, estrogen receptor alpha, and retinoic acid receptor beta and impaired tumor growth in vivo. Interestingly, the SID decoy was effective in the triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cell line, restoring sensitivity to 17beta-estradiol, tamoxifen, and retinoids. Therefore, the development of small molecules that can block interactions between PAH2 and SID-containing proteins offers a targeted epigenetic approach for treating this type of breast cancer that may also have wider therapeutic implications.

The biguanide polyamine analog verlindamycin promotes differentiation in neuroblastoma via induction of antizyme.

Deregulated polyamine biosynthesis is emerging as a common feature of neuroblastoma and drugs targeting this metabolic pathway such as DFMO are in clinical and preclinical development. The polyamine analog verlindamycin inhibits the polyamine biosynthesis pathway enzymes SMOX and PAOX, as well as the histone demethylase LSD1. Based on our previous research in acute myeloid leukemia (AML), we reasoned verlindamycin may also unblock neuroblastoma differentiation when combined with all-trans-retinoic acid (ATRA). Indeed, co-treatment with verlindamycin and ATRA strongly induced differentiation regardless of MYCN status, but in MYCN-expressing cells, protein levels were strongly diminished. This process was not transcriptionally regulated but was due to increased degradation of MYCN protein, at least in part via ubiquitin-independent, proteasome-dependent destruction. Here we report that verlindamycin effectively induces the expression of functional tumor suppressor-antizyme via ribosomal frameshifting. Consistent with previous results describing the function of antizyme, we found that verlindamycin treatment led to the selective targeting of ornithine decarboxylase (the rate-limiting enzyme for polyamine biosynthesis) as well as key oncoproteins, such as cyclin D and Aurora A kinase. Retinoid-based multimodal differentiation therapy is one of the few interventions that extends relapse-free survival in MYCN-associated high-risk neuroblastoma and these results point toward the potential use of verlindamycin in this regimen.

The Protozoan Inhibitor Atovaquone Affects Mitochondrial Respiration and Shows In Vitro Efficacy Against Glucocorticoid-Resistant Cells in Childhood B-Cell Acute Lymphoblastic Leukaemia.

Childhood acute lymphoblastic leukaemia (cALL) accounts for about one third of all paediatric malignancies making it the most common cancer in children. Alterations in tumour cell metabolism were first described nearly a century ago and have been acknowledged as one of the key characteristics of cancers including cALL. Two of the backbone chemotherapeutic agents in the treatment of this disease, Glucocorticoids and L-asparaginase, are exerting their anti-leukaemic effects through targeting cell metabolism. Even though risk stratification and treatment regimens have improved cure rates to nearly 90%, prognosis for relapsed children remains poor. Therefore, new therapeutic approaches are urgently required. Atovaquone is a well-tolerated drug used in the clinic mainly against malaria. Being a ubiquinone analogue, this drug inhibits co-enzyme Q10 of the electron transport chain (ETC) affecting oxidative phosphorylation and cell metabolism. In this study we tested the effect of Atovaquone on cALL cells in vitro. Pharmacologically relevant concentrations of the inhibitor could effectively target mitochondrial respiration in both cALL cell lines (REH and Sup-B15) and primary patient samples. We found that Atovaquone leads to a marked decrease in basal respiration and ATP levels, as well as reduced proliferation, cell cycle arrest, and induction of apoptosis. Importantly, we observed an enhanced anti-leukaemic effect when Atovaquone was combined with the standard chemotherapeutic Idarubicin, or with Prednisolone in an in vitro model of Glucocorticoid resistance. Repurposing of this clinically approved inhibitor renders further investigations, but also presents opportunities for fast-track trials as a single agent or in combination with standard chemotherapeutics.

Differentiation therapy of acute myeloid leukemia: past, present and future.

PURPOSE OF REVIEW: Since the 1970s, the concept of differentiation therapy has been viewed as a promising and revolutionary approach for the treatment of acute myeloid leukemia (AML) and other cancers. However, the successful clinical application of differentiation therapy has only been realized since the late 1980s and only in one subtype of AML, acute promyelocytic leukemia (APL). The use of all-trans-retinoic acid (ATRA) and arsenic trioxide, both of which induce degradation of the progressive multifocal leukoencephalopathy/retinoic acid receptor alpha oncoprotein, in combination with chemotherapy is currently the accepted treatment of APL, presenting a potential paradigm for differentiation therapy in clinical oncology. RECENT FINDINGS: We have begun to understand why ATRA fails to induce differentiation in AML. The underlying reasons identified thus far are associated with an inability to target the removal of leukemogenic fusion proteins, aberrant epigenetic regulation of genes involved in the ATRA signaling pathway and the presence of factors that interfere with proper retinoic acid receptor alpha function. SUMMARY: Here, we examine the reasons why the exquisite sensitivity of APL to ATRA-based differentiation therapy has not been extended to other of AML subtypes. Current differentiation-based combinatorial approaches to target AML will also be analyzed. Finally, we will evaluate the potential of novel strategies, high-throughput screening, and functional genomics to uncover new differentiation-based therapies for AML.

Retinoic acid receptor γ is a therapeutically targetable driver of growth and survival in prostate cancer.

BACKGROUND: Prostate cancer (PC) tissue contains all-trans retinoic acid (ATRA) at a very low level (10-9 M), at least an order of magnitude lower than in adjacent normal healthy prostate cells or benign prostate hyperplasia. When this is coupled with deregulated expression of the intracellular lipid-binding proteins FABP5 and CRABP2 that is frequently found in PC, this is likely to result in the preferential delivery of ATRA to oncogenic PPARß/δ rather than retinoic acid receptors (RARs). There are three isotypes of RARs (RARα, RARß, and RARγ) and recent studies have revealed discrete physiological roles. For example, RARα and RARγ promote differentiation and self-renewal, respectively, which are critical for proper hematopoiesis. AIMS: We have previously shown that ATRA stimulates transactivation of RARγ at sub-nanomolar concentrations (EC50 0.24 nM), whereas an 80-fold higher concentration was required for RARα-mediated transactivation (EC50 19.3 nM). Additionally, we have shown that RAR pan-antagonists inhibit the growth of PC cells (at 16-34 nM). These findings, together with the low level of ATRA in PC, led us to hypothesize that RARγ plays a role in PC pathogenesis and that RARγ-selective antagonism may be an effective treatment. METHODS AND RESULTS: We found that concentrations of 10-9 M and below of ATRA promoted survival/proliferation and opposed adipogenic differentiation of human PC cell lines by a mechanism that involves RARγ. We also found that a RARγ-selective antagonist (AGN205728) potently induced mitochondria-dependent, but caspase-independent, cell death in PC cell lines. Furthermore, AGN205728 demonstrated synergism in killing PC cells in combination with cytotoxic chemotherapeutic agents. CONCLUSION: We suggest that the use of RARγ-selective antagonists may be effective in PC (and potentially other cancers), either as a single agent or in combination with cytotoxic chemotherapy.

Correction: The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML.

To date, only one subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) can be effectively treated by differentiation therapy utilizing all-trans retinoic acid (ATRA). Non-APL AMLs are resistant to ATRA. Here we demonstrate that the acetyltransferase GCN5 contributes to ATRA resistance in non-APL AML via aberrant acetylation of histone 3 lysine 9 (H3K9ac) residues maintaining the expression of stemness and leukemia associated genes. We show that inhibition of GCN5 unlocks an ATRA-driven therapeutic response. This response is potentiated by coinhibition of the lysine demethylase LSD1, leading to differentiation in most non-APL AML. Induction of differentiation was not correlated to a specific AML subtype, cytogenetic, or mutational status. Our study shows a previously uncharacterized role of GCN5 in maintaining the immature state of leukemic blasts and identifies GCN5 as a therapeutic target in AML. The high efficacy of the combined epigenetic treatment with GCN5 and LSD1 inhibitors may enable the use of ATRA for differentiation therapy of non-APL AML. Furthermore, it supports a strategy of combined targeting of epigenetic factors to improve treatment, a concept potentially applicable for a broad range of malignancies.

Molecular target-based treatment of human cancer: summary of the 10th international conference on differentiation therapy.

The 10th International Conference on Differentiation Therapy was held between April 29 and May 3, 2004, in Shanghai, China. In the tradition of previous conferences from this series, which have been held biannually since the first meeting organized 20 years ago by Samuel Waxman and Giovanni Rossi in Sardinia, the organizers of the 10th International Conference on Differentiation Therapy aimed to gather basic and clinical cancer investigators in a setting of plenary sessions, workshops, and poster presentations to maximize the effective exchange of information and foster the establishment of collaborative interactions. Approximately 300 scientists attended the meeting with a mission to discuss targeted approaches to cancer treatment, which stem from our understanding of basic biological processes and the mechanisms of their deregulation during tumorigenesis.

Benzodithiophenes potentiate differentiation of acute promyelocytic leukemia cells by lowering the threshold for ligand-mediated corepressor/coactivator exchange with retinoic acid receptor alpha and enhancing changes in all-trans-retinoic acid-regulated gene expression.

Differentiation induction is an effective therapy for acute promyelocytic leukemia (APL), which dramatically responds to all-trans-retinoic acid (ATRA). Recent studies have indicated that combinatorial use of retinoid and nonretinoid compounds, such as histone deacetylase inhibitors, arsenics, and PKA agonists, has higher therapeutic value in this disease and potentially in other malignancies. In a screen of 370 compounds, we identified benzodithiophene analogues as potent enhancers of ATRA-induced APL cell differentiation. These effects were not associated with changes in global histone acetylation and, for the most potent compounds, were exerted at very low nanomolar concentrations, and were paralleled by enhancement of some, but not all, ATRA-modulated gene expressions. Investigating the mechanism underlying the effects of these drugs on ATRA-induced APL cell differentiation, we have shown that benzodithiophenes enhance ATRA-mediated dissociation and association of corepressor N-CoR and coactivator p300 acetyltransferase, respectively, with retinoic acid receptor (RAR) alpha proteins. These data suggest that benzodithiophenes act at the level of receptor activation, possibly by affecting posttranslational modification of the receptor (and/or coregulators), thus leading to an enhancement in ATRA-mediated effects on gene expression and APL cell differentiation. Given the specificities of these low benzodithiophene concentrations for PML-RARalpha and RARalpha, these drugs may be useful for combinatorial differentiation therapy of APL and possibly other acute myelogenous leukemia subtypes in which the overall ATRA signaling is suppressed.

Clinical translation of epigenetics in cancer: eN-CORe--a report on the second workshop.

Recent advances in understanding the role that epigenetics plays in cancer pathogenesis and understanding the mechanisms through which these processes regulate gene expression have stimulated considerable interest in developing clinically viable antineoplastic agents that target enzymatic components of transcriptional regulatory complexes responsible for the establishment of pathologic epigenetic modifications that lead to deregulated gene expression in cancer. In January 2003, a workshop was held in Baltimore to discuss the therapeutic potential of several agents that can modify chromatin structure. A follow-up meeting on "Clinical Translation of Epigenetics in Cancer" was held in Charleston, SC, in January 2005. The aim of this workshop was to assess the progress that has been made over the past 2 years in bringing effective therapeutic protocols that use agents capable of reverting pathologic epigenetic changes into the clinic. The meeting was attended by approximately 70 investigators and included formal presentations, panel group discussions, and two break-out sessions that addressed targeted therapies in hematologic and solid tumors. The aim of this article is to summarize topics discussed at this workshop and highlight conclusions as to the immediate and long-term future of epigenetic therapy in cancer.

Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice.

Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eµ). Here, we report that the Eµ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eµ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.

p53 Loss in MYC-Driven Neuroblastoma Leads to Metabolic Adaptations Supporting Radioresistance.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.

Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

Neuroblastoma Arginase Activity Creates an Immunosuppressive Microenvironment That Impairs Autologous and Engineered Immunity.

Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.