Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set-point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile, and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with non-curative chemotherapy even for highest-risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria.

Pediatric patients with high-risk neuroblastoma often relapse with chemotherapy-resistant, incurable disease. Relapsed neuroblastomas harbor chemo-resistant mesenchymal tumor cells and increased expression/activity of the transcriptional co-regulator, the Yes-Associated Protein (YAP). Patients with relapsed neuroblastoma are often treated with immunotherapy such as the anti-GD2 antibody, dinutuximab, in combination with chemotherapy. We have previously shown that YAP mediates both chemotherapy and MEK inhibitor resistance in relapsed RAS mutated neuroblastoma and so posited that YAP might also be involved in anti-GD2 antibody resistance. We now show that YAP genetic inhibition significantly enhances sensitivity of mesenchymal neuroblastomas to dinutuximab and gamma delta (γδ) T cells both in vitro and in vivo. Mechanistically, YAP inhibition induces increased GD2 cell surface expression through upregulation of ST8SIA1, the gene encoding GD3 synthase and the rate-limiting enzyme in GD2 biosynthesis. The mechanism of ST8SIA1 suppression by YAP is independent of PRRX1 expression, a mesenchymal master transcription factor, suggesting YAP may be the downstream effector of mesenchymal GD2 resistance. These results therefore identify YAP as a therapeutic target to augment GD2 immunotherapy responses in patients with neuroblastoma.

GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.

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 123I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45–115 mg/m2/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/m2. 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.

Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.

Locally advanced cholangiocarcinoma has a poor prognosis, with long-term survival only for patients where complete surgical resection is achieved. Median overall survival with chemotherapy alone is less than 1 year. Novel strategies combining conventional chemotherapy and radiotherapy followed by targeted agents can lead to durable treatment responses and are applicable to cholangiocarcinoma management. Pediatric cholangiocarcinoma is exceedingly rare, with an estimate of 15-22 cases reported in the last 40 years. As such, no standard therapeutic regimen exists. We present a case of a 16-year-old previously healthy patient with unresectable cholangiocarcinoma whose tumor genetic sequencing revealed a novel, actionable neuregulin-1 (NRG1) gene translocation. The patient underwent standard systemic chemotherapy with gemcitabine and cisplatin followed by hypofractionated proton radiation therapy for local control. The patient then started an oral pan-ERBB (erythroblastic B receptor tyrosine kinases including ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4) family inhibitor as a maintenance medication, remaining with stable disease and excellent quality of life for over 2 years. This case highlights a novel NRG1 fusion in a rare clinical entity that provided an opportunity to utilize a multimodal therapeutic strategy in the pediatric setting.

An effective therapy regimen for relapsed/refractory high-risk neuroblastoma (NB) includes the anti-GD2 monoclonal antibody, dinutuximab, in combination with temozolomide and irinotecan, supporting a role for chemo-immunotherapy in NB. γδ T cells are an attractive anti-tumor immunotherapy because of their direct cytotoxic activity mediated through cell surface receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands whereas CD16 recognizes antibody bound to tumors and activates mechanisms of antibody-dependent cellular cytotoxicity (ADCC). This study demonstrates an efficient method for expanding and storing γδ T cells from NB patient-derived apheresis products at clinically relevant amounts. The expanded patient-derived γδ T cells were cytotoxic against the K562 cell line and multiple NB cell lines. Combining γδ T cells with dinutuximab led to a 30% increase in tumor cell lysis compared to γδ T cells alone. Furthermore, low-dose temozolomide in combination with expanded γδ T cells and dinutuximab resulted in increased IFNγ secretion and increased γδ T-cell surface expression of FasL and CD107a. IMR5 NB cell line xenografts established subcutaneously in NSG mice were treated with a regimen of dinutuximab, temozolomide, and γδ T cells. This combination caused targeted killing of NB xenografts in vivo, reducing tumor burden and prolonging survival. These data support the continued preclinical testing of dinutuximab and temozolomide in conjunction with γδ T-cell immunotherapy for patients with recurrent/refractory NB.

Neuroblastoma (NB) is the most common extracranial tumor in children resulting in substantial morbidity and mortality. A deeper understanding of the NB tumor microenvironment (TME) remains an area of active research but there is a lack of reliable and biomimetic experimental models. This study utilizes a 3D bioprinting approach, in combination with NB spheroids, to create an in vitro vascular model of NB for exploring the tumor function within an endothelialized microenvironment. A gelatin methacryloyl (gelMA) bioink is used to create multi-channel cubic tumor analogues with high printing fidelity and mechanical tunability. Human-derived NB spheroids and human umbilical vein endothelial cells (HUVECs) are incorporated into the biomanufactured gelMA and cocultured under static versus dynamic conditions, demonstrating high levels of survival and growth. Quantification of NB-EC integration and tumor cell migration suggested an increased aggressive behavior of NB when cultured in bioprinted endothelialized models, when cocultured with HUVECs, and also as a result of dynamic culture. This model also allowed for the assessment of metabolic, cytokine, and gene expression profiles of NB spheroids under varying TME conditions. These results establish a high throughput research enabling platform to study the TME-mediated cellular-molecular mechanisms of tumor growth, aggression, and response to therapy.

Neuroblastoma is the most common extra-cranial pediatric solid tumor that accounts for more than 15% of childhood cancer-related deaths. High risk neuroblastomas that recur during or after intense multimodal therapy have a <5% chance at a second sustained remission or cure. The solid tumor microenvironment (TME) has been increasingly recognized to play a critical role in cancer progression and resistance to therapy, including in neuroblastoma. The Yes-Associated Protein (YAP) in the Hippo pathway can regulate cancer proliferation, tumor initiation, and therapy response in many cancer types and as such, its role in the TME has gained interest. In this review, we focus on YAP and its role in neuroblastoma and further describe its demonstrated and potential effects on the neuroblastoma TME. We also discuss the therapeutic strategies for inhibiting YAP in neuroblastoma.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Nonresistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.