Inhibitors of the Oncogenic PA2G4-MYCN Protein-Protein Interface.

MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R1 position. Replacing the trifluoromethyl group on the phenyl ring at the R2 position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.

Identification of Novel CSF-Derived miRNAs in Treated Paediatric Onset Spinal Muscular Atrophy: An Exploratory Study.

The availability of disease modifying therapies for spinal muscular atrophy (SMA) have created an urgent need to identify clinically meaningful biomarkers that provide insight into disease progression and therapeutic response. microRNAs (miRNA) have been shown to be involved in the pathogenesis of SMA and have the potential to provide insight within the field of SMA. miRNA-sequencing was utilized to identify differential miRNA expression in the cerebrospinal fluid (CSF) in six children with SMA treated with nusinersen in this exploratory study. Fourteen differentially expressed miRNAs were significantly altered in CSF from baseline to follow-up during treatment with nusinersen. The greatest magnitude of change was noted in miR-7-5p, miR-15a-5p, miR-15b-3p/5p, miR-126-5p, miR-128-2-5p and miR-130a-3p which encompassed a spectrum of functions predominantly in neurogenesis, neuronal differentiation and growth. The dominant signaling pathways identified in this study were the mammalian target of rapamycin and the mitogen-activated protein kinase signaling pathways. This study identified multiple miRNAs that were involved in the complex interplay between neurodevelopment and neurodegeneration.

Combined thioredoxin reductase and glutaminase inhibition exerts synergistic anti-tumor activity in MYC-high high-grade serous ovarian carcinoma.

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

Optimized flow cytometric detection of transient receptor potential vanilloid-1 (TRPV1) in human hematological malignancies.

The ectopic overexpression of transient receptor potential vanilloid-1 (TRPV1) has been detected in numerous solid cancers, including breast, prostate, pancreatic, and tongue epithelium cancer. However, the expression of TRPV1 in hematological malignancies remains unknown. Here we show through in silico analysis that elevated TRPV1 mRNA expression occurs in a range of hematological malignancies and presents an optimized flow cytometry method to rapidly assess TRPV1 protein expression for both cell lines and primary patient samples. Three anti-TRPV1 antibodies were evaluated for intracellular TRPV1 detection using flow cytometry resulting in an optimized protocol for the evaluation of TRPV1 in hematological malignant cell lines and patients' peripheral blood mononuclear cells (PBMC). Overexpression of TRPV1 was observed in THP-1 (acute monocytic leukemia) and U266B1 (multiple myeloma, MM), but not U937 (histiocytic lymphoma) compared to healthy PBMC. TRPV1 was also detected in all 49 patients including B-cell non-Hodgkin's lymphoma (B-NHL), MM, and others and 20 healthy controls. TRPV1 expression was increased in 8% of patients (MM = 2, B-NHL = 2). In conclusion, we provide an optimized flow cytometry method for routine expression analysis of clinical samples and show that TRPV1 is increased in a subset of patients with hematological malignancies.

Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma.

BACKGROUND: Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA. METHODS: We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each). RESULTS: Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10-4 (0.01%)-10-5 (0.001%) mononuclear cells. In ALL, WGS-MRD and Ig/TCR-MRD were highly concordant. WGS-MRD assays also showed good concordance between quantitative PCR and droplet digital PCR formats. In serial clinical samples, WGS-MRD correlated with disease course. In solid tumours, WGS-MRD assays were more sensitive than RNA-MRD assays. CONCLUSIONS: WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours.

Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

An ALYREF-MYCN coactivator complex drives neuroblastoma tumorigenesis through effects on USP3 and MYCN stability.

To achieve the very high oncoprotein levels required to drive the malignant state cancer cells utilise the ubiquitin proteasome system to upregulate transcription factor levels. Here our analyses identify ALYREF, expressed from the most common genetic copy number variation in neuroblastoma, chromosome 17q21-ter gain as a key regulator of MYCN protein turnover. We show strong co-operativity between ALYREF and MYCN from transgenic models of neuroblastoma in vitro and in vivo. The two proteins form a nuclear coactivator complex which stimulates transcription of the ubiquitin specific peptidase 3, USP3. We show that increased USP3 levels reduce K-48- and K-63-linked ubiquitination of MYCN, thus driving up MYCN protein stability. In the MYCN-ALYREF-USP3 signal, ALYREF is required for MYCN effects on the malignant phenotype and that of USP3 on MYCN stability. This data defines a MYCN oncoprotein dependency state which provides a rationale for future pharmacological studies.

Suppression of ABCE1-Mediated mRNA Translation Limits N-MYC-Driven Cancer Progression.

The ability of the N-MYC transcription factor to drive cancer progression is well demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN amplification heralds a poor prognosis, with only 11% of high-risk patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is "undruggable." Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here, we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of nonmalignant cells or MYCN-nonamplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together, ABCE1-mediated translation constitutes a critical process in the progression of N-MYC-driven and c-MYC-driven cancers that warrants investigations into methods of its therapeutic inhibition. SIGNIFICANCE: These findings demonstrate that N-MYC-driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.

Comparative analysis of milk microRNA in the therian lineage highlights the evolution of lactation.

Milk is a complex secretion that has an important role in mammalian reproduction. It is only recently that sequencing technologies have allowed the identification and quantification of microRNA (miRNA) in milk of a growing number of mammalian species. This provides a novel window on the study of the evolution and functionality of milk through the comparative analysis of milk miRNA content. Here, milk miRNA sequencing data from five species (one marsupial (tammar wallaby) and four eutherians (human, mouse, cow and pig)) have been retrieved from public depositories and integrated in order to perform a comparison of milk miRNA profiles. The study shows that milk miRNA composition varies widely between species, except for a few miRNAs that are ubiquitously expressed in the milk of all mammals and indicates that milk miRNA secretion has broadly evolved during mammalian evolution. The putative functions of the most abundant milk miRNAs are also discussed.