Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

O-linked ß-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibits decreased PRPS1 O-GlcNAcylation and activity. Together, our findings establish a direct connection among O-GlcNAc signals, de novo nucleotide synthesis and human diseases, including cancer and Arts syndrome.

Medulloblastoma rendered susceptible to NK-cell attack by TGFß neutralization.

BACKGROUND: Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor ß (TGF-ß). Here, we address this challenge in in vitro models of MB. METHODS: CB-derived NK cells were modified to express a dominant negative TGF-ß receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. RESULTS: We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-ß-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-ß (decreased TGF-ß levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). CONCLUSIONS: CB NK cells expressing a TGF-ß DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-ß-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Subtypes of medulloblastoma have distinct developmental origins.

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1(+) precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.

A Proteogenomic Approach to Understanding MYC Function in Metastatic Medulloblastoma Tumors.

Brain tumors are the leading cause of cancer-related deaths in children, and medulloblastoma is the most prevalent malignant childhood/pediatric brain tumor. Providing effective treatment for these cancers, with minimal damage to the still-developing brain, remains one of the greatest challenges faced by clinicians. Understanding the diverse events driving tumor formation, maintenance, progression, and recurrence is necessary for identifying novel targeted therapeutics and improving survival of patients with this disease. Genomic copy number alteration data, together with clinical studies, identifies c-MYC amplification as an important risk factor associated with the most aggressive forms of medulloblastoma with marked metastatic potential. Yet despite this, very little is known regarding the impact of such genomic abnormalities upon the functional biology of the tumor cell. We discuss here how recent advances in quantitative proteomic techniques are now providing new insights into the functional biology of these aggressive tumors, as illustrated by the use of proteomics to bridge the gap between the genotype and phenotype in the case of c-MYC-amplified/associated medulloblastoma. These integrated proteogenomic approaches now provide a new platform for understanding cancer biology by providing a functional context to frame genomic abnormalities.

WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum.

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated ß-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although ß-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation.

OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma.

PURPOSE: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. EXPERIMENTAL DESIGN: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. RESULTS: We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. CONCLUSIONS: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

MYC Drives Group 3 Medulloblastoma through Transformation of Sox2+ Astrocyte Progenitor Cells.

A subset of group 3 medulloblastoma frequently harbors amplification or overexpression of MYC lacking additional focal aberrations, yet it remains unclear whether MYC overexpression alone can induce tumorigenesis and which cells give rise to these tumors. Here, we showed that astrocyte progenitors in the early postnatal cerebellum were susceptible to transformation by MYC. The resulting tumors specifically resembled human group 3 medulloblastoma based on histology and gene-expression profiling. Gene-expression analysis of MYC-driven medulloblastoma cells revealed altered glucose metabolic pathways with marked overexpression of lactate dehydrogenase A (LDHA). LDHA abundance correlated positively with MYC expression and was associated with poor prognosis in human group 3 medulloblastoma. Inhibition of LDHA significantly reduced growth of both mouse and human MYC-driven tumors but had little effect on normal cerebellar cells or SHH-associated medulloblastoma. By generating a new mouse model, we demonstrated for the first time that astrocyte progenitors can be transformed by MYC and serve as the cells of origin for group 3 medulloblastoma. Moreover, we identified LDHA as a novel, specific therapeutic target for this devastating disease. SIGNIFICANCE: Insights from a new model identified LDHA as a novel target for group 3 medulloblastoma, paving the way for the development of effective therapies against this disease.

Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key ß-Cell-Specific Disease Genes.

Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct ß cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both ß-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.