Targeting NRAS via miR-1304-5p or farnesyltransferase inhibition confers sensitivity to ALK inhibitors in ALK-mutant neuroblastoma.

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

Patient-derived xenograft models of ALK+ ALCL reveal preclinical promise for therapy with brigatinib.

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by the oncogenic anaplastic lymphoma kinase (ALK), accounting for approximately 15% of all paediatric non-Hodgkin lymphoma. Patients with central nervous system (CNS) relapse are particularly difficult to treat with a 3-year overall survival of 49% and a median survival of 23.5 months. The second-generation ALK inhibitor brigatinib shows superior penetration of the blood-brain barrier unlike the first-generation drug crizotinib and has shown promising results in ALK+ non-small-cell lung cancer. However, the benefits of brigatinib in treating aggressive paediatric ALK+ ALCL are largely unknown. We established a patient-derived xenograft (PDX) resource from ALK+ ALCL patients at or before CNS relapse serving as models to facilitate the development of future therapies. We show in vivo that brigatinib is effective in inducing the remission of PDX models of crizotinib-resistant (ALK C1156Y, TP53 loss) ALCL and furthermore that it is superior to crizotinib as a second-line approach to the treatment of a standard chemotherapy relapsed/refractory ALCL PDX pointing to brigatinib as a future therapeutic option.

The Contribution of Autophagy and LncRNAs to MYC-Driven Gene Regulatory Networks in Cancers.

MYC is a target of the Wnt signalling pathway and governs numerous cellular and developmental programmes hijacked in cancers. The amplification of MYC is a frequently occurring genetic alteration in cancer genomes, and this transcription factor is implicated in metabolic reprogramming, cell death, and angiogenesis in cancers. In this review, we analyse MYC gene networks in solid cancers. We investigate the interaction of MYC with long non-coding RNAs (lncRNAs). Furthermore, we investigate the role of MYC regulatory networks in inducing changes to cellular processes, including autophagy and mitophagy. Finally, we review the interaction and mutual regulation between MYC and lncRNAs, and autophagic processes and analyse these networks as unexplored areas of targeting and manipulation for therapeutic gain in MYC-driven malignancies.

The Role of Autophagy and lncRNAs in the Maintenance of Cancer Stem Cells.

Cancer stem cells (CSCs) possess properties such as self-renewal, resistance to apoptotic cues, quiescence, and DNA-damage repair capacity. Moreover, CSCs strongly influence the tumour microenvironment (TME) and may account for cancer progression, recurrence, and relapse. CSCs represent a distinct subpopulation in tumours and the detection, characterisation, and understanding of the regulatory landscape and cellular processes that govern their maintenance may pave the way to improving prognosis, selective targeted therapy, and therapy outcomes. In this review, we have discussed the characteristics of CSCs identified in various cancer types and the role of autophagy and long noncoding RNAs (lncRNAs) in maintaining the homeostasis of CSCs. Further, we have discussed methods to detect CSCs and strategies for treatment and relapse, taking into account the requirement to inhibit CSC growth and survival within the complex backdrop of cellular processes, microenvironmental interactions, and regulatory networks associated with cancer. Finally, we critique the computationally reinforced triangle of factors inclusive of CSC properties, the process of autophagy, and lncRNA and their associated networks with respect to hypoxia, epithelial-to-mesenchymal transition (EMT), and signalling pathways.

Core regulatory circuitries in defining cancer cell identity across the malignant spectrum.

Gene expression programmes driving cell identity are established by tightly regulated transcription factors that auto- and cross-regulate in a feed-forward manner, forming core regulatory circuitries (CRCs). CRC transcription factors create and engage super-enhancers by recruiting acetylation writers depositing permissive H3K27ac chromatin marks. These super-enhancers are largely associated with BET proteins, including BRD4, that influence higher-order chromatin structure. The orchestration of these events triggers accessibility of RNA polymerase machinery and the imposition of lineage-specific gene expression. In cancers, CRCs drive cell identity by superimposing developmental programmes on a background of genetic alterations. Further, the establishment and maintenance of oncogenic states are reliant on CRCs that drive factors involved in tumour development. Hence, the molecular dissection of CRC components driving cell identity and cancer state can contribute to elucidating mechanisms of diversion from pre-determined developmental programmes and highlight cancer dependencies. These insights can provide valuable opportunities for identifying and re-purposing drug targets. In this article, we review the current understanding of CRCs across solid and liquid malignancies and avenues of investigation for drug development efforts. We also review techniques used to understand CRCs and elaborate the indication of discussed CRC transcription factors in the wider context of cancer CRC models.

Diagnostic accuracy of circulating-free DNA for the determination of MYCN amplification status in advanced-stage neuroblastoma: a systematic review and meta-analysis.

BACKGROUND: MYCN amplification (MNA) is the strongest indicator of poor prognosis in neuroblastoma (NB). This meta-analysis aims to determine the diagnostic accuracy of MNA analysis in circulating-free DNA (cfDNA) from advanced-stage NB patients. METHODS: A systematic review of electronic databases was conducted to identify studies exploring the detection of MNA in plasma/serum cfDNA from NB patients at diagnosis using PCR methodology. Pooled estimates for sensitivity, specificity and diagnostic odds ratio (DOR) were calculated by conducting a bivariate/HSROC random-effects meta-analysis. RESULTS: Seven studies, with a total of 529 advanced-stage patients, were eligible. The pooled sensitivity of cfDNA-based MNA analysis was 0.908 (95% CI, 0.818-0.956), the pooled specificity was 0.976 (0.940-0.991) and the DOR was 410.0 (-103.6 to 923.7). Sub-grouped by INSS stage, the sensitivity for stage 3 and 4 patients was 0.832 (0.677-0.921) and 0.930 (0.834-0.972), respectively. The specificity was 0.999 (0.109-1.000) and 0.974 (0.937-0.990), respectively, and the DOR was 7855.2 (-66267.0 to 81977.4) and 508.7 (-85.8 to 1103.2), respectively. CONCLUSIONS: MNA analysis in cfDNA using PCR methodology represents a non-invasive approach to rapidly and accurately determine MNA status in patients with advanced-stage NB. Standardised methodology must be developed before this diagnostic test can enter the clinic.

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

Opportunities and challenges of circulating biomarkers in neuroblastoma.

Molecular analysis of nucleic acid and protein biomarkers is becoming increasingly common in paediatric oncology for diagnosis, risk stratification and molecularly targeted therapeutics. However, many current and emerging biomarkers are based on analysis of tumour tissue, which is obtained through invasive surgical procedures and in some cases may not be accessible. Over the past decade, there has been growing interest in the utility of circulating biomarkers such as cell-free nucleic acids, circulating tumour cells and extracellular vesicles as a so-called liquid biopsy of cancer. Here, we review the potential of emerging circulating biomarkers in the management of neuroblastoma and highlight challenges to their implementation in the clinic.

Early detection of pre-malignant lesions in a KRASG12D-driven mouse lung cancer model by monitoring circulating free DNA.

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a 'liquid biopsy' for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by KrasG12D (the KrasLSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the KrasLox-G12D allele from the KrasLSL-G12D and KrasWT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm3 Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling.A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3UThis article has an associated First Person interview with the joint first authors of the paper.

ALK in Neuroblastoma: Biological and Therapeutic Implications.

Neuroblastoma (NB) is the most common and deadly solid tumour in children. Despite the development of new treatment options for high-risk NB, over half of patients relapse and five-year survival remains at 40-50%. Therefore, novel treatment strategies aimed at providing long-term disease remission are urgently sought. ALK, encoding the anaplastic lymphoma kinase receptor, is altered by gain-of-function point mutations in around 14% of high-risk NB and represents an ideal therapeutic target given its low or absent expression in healthy tissue postnatally. Small-molecule inhibitors of Anaplastic Lymphoma Kinase (ALK) approved in ALK fusion-positive lung cancer are currently undergoing clinical assessment in patients with ALK-mutant NB. Parallel pre-clinical studies are demonstrating the efficacy of ALK inhibitors against common ALK variants in NB; however, a complex picture of therapeutic resistance is emerging. It is anticipated that long-term use of these compounds will require combinatorial targeting of pathways downstream of ALK, functionally-related 'bypass' mechanisms and concomitant oncogenic pathways.