The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ...ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
For children with cancer, the clinical integration of precision medicine to enable predictive biomarker–based therapeutic stratification is urgently needed.
We have developed a hybrid-capture ...next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)–specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings.
A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma.
We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel–based approach can identify actionable genetic alterations in a high proportion of patients.
•The hybrid-capture panel gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded tissue.•A tailored sequencing approach identifies ≥1 genetic alteration in 70% of samples and potentially actionable genetic alterations in 51% of patients.•In paired samples, from different treatment time points, there were differences in the genetic alterations detected.
Neuroblastomas harbor ALK aberrations clinically resistant to crizotinib yet sensitive pre-clinically to the third-generation ALK inhibitor lorlatinib. We conducted a first-in-child study evaluating ...lorlatinib with and without chemotherapy in children and adults with relapsed or refractory ALK-driven neuroblastoma. The trial is ongoing, and we report here on three cohorts that have met pre-specified primary endpoints: lorlatinib as a single agent in children (12 months to <18 years); lorlatinib as a single agent in adults (≥18 years); and lorlatinib in combination with topotecan/cyclophosphamide in children (<18 years). Primary endpoints were safety, pharmacokinetics and recommended phase 2 dose (RP2D). Secondary endpoints were response rate and
I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45-115 mg/m
/dose in children and 100-150 mg in adults. Common adverse events (AEs) were hypertriglyceridemia (90%), hypercholesterolemia (79%) and weight gain (87%). Neurobehavioral AEs occurred mainly in adults and resolved with dose hold/reduction. The RP2D of lorlatinib with and without chemotherapy in children was 115 mg/m
. The single-agent adult RP2D was 150 mg. The single-agent response rate (complete/partial/minor) for <18 years was 30%; for ≥18 years, 67%; and for chemotherapy combination in <18 years, 63%; and 13 of 27 (48%) responders achieved MIBG complete responses, supporting lorlatinib's rapid translation into active phase 3 trials for patients with newly diagnosed high-risk, ALK-driven neuroblastoma. ClinicalTrials.gov registration: NCT03107988 .
Abstract INTRODUCTION MYC-amplified Group 3 medulloblastomas (MBGroup3-MYC) do not respond to conventional up-front therapies and are almost universally fatal (<5% survival). MYC is not directly ...targetable. Thus, the systematic development of therapies which target complementary MYC-dependencies offers clear potential to rationally re-design effective therapeutic strategies. METHODS Isogenic cell-based models with regulable shRNA MYC-knockdown were developed in three independent MYC-amplified backgrounds (D283Med; D425Med; HD-MB03). These were utilised to i) define the MYC-dependent transcriptome/proteome in MBGroup3, through analysis in conjunction with our primary tumour cohort, and ii) as the basis of an automated MYC-dependent high-throughput drug screen (HTS; >500 clinically-actionable compounds). Bespoke integrative AI tools and bioinformatic analysis, and subsequent siRNA/pharmacodynamic validation, identified and validated candidate MYC-dependent therapeutic vulnerabilities. Prioritised drugs were advanced to pre-clinical trials across two in vivo MBGroup3 models (GTML/Trp53KI/KI (GEMM; MYCN-driven) and GMYC (GEMM-derived allografts; MYC-driven)) to evaluate candidate efficacy and toxicity. RESULTS shRNA-mediated MYC knockdown could be maintained for 28 days without escape in all models and, as anticipated, was associated with cell cycle abrogation and elevated caspase-3/-7 activity. HTS across all models revealed that MYC expression confers differential dependencies to small molecule inhibitors, with 82 compounds spanning 11 major drug classes demonstrating MYC-dependent activity. AI-based integration of HTS data with model and primary patient MYC-dependent transcriptome data shortlisted multiple druggable molecular targets which, in turn, were independently validated. To date, we have progressed alisertib (AURKAi), volasertib (PLK1i) and prexasertib (CHK1i) to in vivo pre-clinical trials. Promisingly, both alisertib and prexasertib demonstrated efficacy within MBGroup3-MYC models. CONCLUSIONS Integrated human tumour and model-based screening approaches have enabled identification and validation of critical MYC co-dependencies, targetable using established small-molecules, to inhibit MBGroup3 tumour growth. Their further evaluation, including integration into established and/or rationally-designed combinations, will be essential next steps in their (pre-)clinical development.
Abstract BACKGROUND Medulloblastoma is the most frequent malignant brain tumour of childhood. Whilst advances in risk-stratification and upfront multimodal therapy have led to five-year survival ...rates around 70%, a subset of tumours remain refractory to current therapies. Group 3 medulloblastomas (MBGRP3) are enriched for amplification or overexpression of the proto-oncogene MYC which conveys a dismal prognosis (<10% survival), highlighting the urgent need for novel therapeutic strategies. Recent advances have revealed a role for metabolic reprogramming in MYC-amplified MBGRP3 tumorigenesis, providing novel opportunities for selective therapeutic targeting. METHODS & RESULTS To investigate MYC-dependent metabolic alterations in MBGRP3, we generated three independent isogenic MBGRP3 cell-based models with regulable MYC expression (iMBGRP3-MYC) using a doxycycline-inducible shRNA system. Using 1H high-resolution magic angle spectroscopy and stable isotope-resolved metabolomics, we identified upregulation of the de novo serine/glycine biosynthesis pathway (SGP), attributable to elevated expression of the rate-limiting enzyme PHGDH, as a novel therapeutic target. Genetic and pharmacological (NCT-503) inhibition of PHGDH induced greater cell death in MYC-expressing cells compared to MYC knockdown cells and increased survival across two independent MYC-amplified MBGRP3 mouse models. The decrease in tumour growth observed was moderate; we therefore hypothesised that a compensatory reliance on exogenous serine/glycine may play a role in sustaining tumour progression under therapy. To explore this, we investigated the impact of exogenous serine/glycine starvation on MBGRP3-MYC tumour development. Starvation resulted in a marked MYC-dependent reduction in cell viability in vitro across independent iMBGRP3-MYC models. Notably, dietary serine/glycine starvation was well-tolerated, delayed tumour progression, and increased survival (by 50%) in an MBGRP3-MYC mouse model (p=0.005), highlighting the metabolic plasticity of MYC-amplified tumours to utilise both de novo and exogenous serine/glycine sources to survive and proliferate. CONCLUSIONS These findings identify serine/glycine metabolism as a targetable therapeutic vulnerability within MYC-amplified MBGroup3 and a novel therapeutic strategy to treat this poor-prognosis disease group.
Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic ...approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.
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•Palbociclib inhibits neuroblastoma cell proliferation and drives differentiation•The transcriptome and epigenome are reprogrammed favoring neuronal differentiation•Palbociclib inhibits tumor growth in in vivo mouse neuroblastoma models•PB and RA together drive transcriptional changes associated with improved prognosis
Ferguson and Gillen et al. show that the CDK4/6 inhibitor, palbociclib, reactivates a latent ability of neuroblastoma cells to undergo differentiation and it improves survival in mouse neuroblastoma models. Moreover, combined treatment of cells with retinoic acid further enhances differentiation, suggesting that adding palbociclib may improve outcomes to the current treatment for neuroblastoma.
Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill ...this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.
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•Single-cell RNAseq identifies 17 distinct immune subsets infiltrating neuroblastoma•Dysfunctionality of T and NK cells alternates throughout the course of chemotherapy•Interaction analysis identifies the NECTIN2-TIGIT axis as crucial immune checkpoint•TIGIT (+PD-L1) blockade enhances immune responses against neuroblastoma in vivo
Wienke et al. analyze the immune landscape of neuroblastoma pre- and post-chemotherapy and identify the NECTIN2-TIGIT axis as a crucial immune checkpoint, which correlates with dysfunction of T/NK cells. TIGIT (+PD-L1) blockade induces numerous complete responses (CR) in vivo, even against chemotherapy-resistant neuroblastoma, highlighting TIGIT blockade as promising immunotherapy for neuroblastoma.
Increased stiffness in the extracellular matrix (ECM) contributes to tumor progression and metastasis. Therefore, stromal modulating therapies and accompanying biomarkers are being developed to ...target ECM stiffness. Magnetic resonance (MR) elastography can noninvasively and quantitatively map the viscoelastic properties of tumors
and thus has clear clinical applications. Herein, we used MR elastography, coupled with computational histopathology, to interrogate the contribution of collagen to the tumor biomechanical phenotype and to evaluate its sensitivity to collagenase-induced stromal modulation. Elasticity (
) and viscosity (
) were significantly greater for orthotopic BT-474 (
= 5.9 ± 0.2 kPa,
= 4.7 ± 0.2 kPa,
= 7) and luc-MDA-MB-231-LM2-4 (
= 7.9 ± 0.4 kPa,
= 6.0 ± 0.2 kPa,
= 6) breast cancer xenografts, and luc-PANC1 (
= 6.9 ± 0.3 kPa,
= 6.2 ± 0.2 kPa,
= 7) pancreatic cancer xenografts, compared with tumors associated with the nervous system, including GTML/
medulloblastoma (
= 3.5 ± 0.2 kPa,
= 2.3 ± 0.2 kPa,
= 7), orthotopic luc-D-212-MG (
= 3.5 ± 0.2 kPa,
= 2.3 ± 0.2 kPa,
= 7), luc-RG2 (
= 3.5 ± 0.2 kPa,
= 2.3 ± 0.2 kPa,
= 5), and luc-U-87-MG (
= 3.5 ± 0.2 kPa,
= 2.3 ± 0.2 kPa,
= 8) glioblastoma xenografts, intracranially propagated luc-MDA-MB-231-LM2-4 (
= 3.7 ± 0.2 kPa,
= 2.2 ± 0.1 kPa,
= 7) breast cancer xenografts, and Th-
neuroblastomas (
= 3.5 ± 0.2 kPa,
= 2.3 ± 0.2 kPa,
= 5). Positive correlations between both elasticity (
= 0.72,
< 0.0001) and viscosity (
= 0.78,
< 0.0001) were determined with collagen fraction, but not with cellular or vascular density. Treatment with collagenase significantly reduced
(
= 0.002) and
(
= 0.0006) in orthotopic breast tumors. Texture analysis of extracted images of picrosirius red staining revealed significant negative correlations of entropy with
(
= -0.69,
< 0.0001) and
(
= -0.76,
< 0.0001), and positive correlations of fractal dimension with
(
= 0.75,
< 0.0001) and
(
= 0.78,
< 0.0001). MR elastography can thus provide sensitive imaging biomarkers of tumor collagen deposition and its therapeutic modulation. SIGNIFICANCE: MR elastography enables noninvasive detection of tumor stiffness and will aid in the development of ECM-targeting therapies.
Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) ...initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain.
This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials.
Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial.
Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration.
In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy.
•The Neuroblastoma New Drug Development Strategy initiative aims to accelerate new drugs development.•Twenty two of 40 targets were identified as high priority for neuroblastoma.•Targeting telomere maintenance, ATRX, BRIP1, RRM2 are high priority for drug discovery.•Drugs targeting CDK2/9, CDK7, ATR should enter paediatric clinical development rapidly.•Combining targeted therapies with immunological agents is crucial.
The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable ...outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.
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