Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Development of X-ray micro-focus computed tomography to image and quantify biofilms in central venous catheter models in vitro.

Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems.Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated matlab script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single- and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimized method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture. X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.

Invasive bacterial and fungal infections in paediatric patients with cancer: incidence, risk factors, aetiology and outcomes in a UK regional cohort 2009-2011.

BACKGROUND: Cancer is the second most common cause of childhood deaths in the United Kingdom and infection contributes to a quarter of all cancer-related deaths. This study aimed to estimate the risk, aetiology and outcome of bloodstream bacterial and fungal infections in children with cancer within a geographically defined region in South-West London over a 3-year period. METHODS: Web-based questionnaires were completed using case records of children with positive blood cultures admitted to five London hospitals during 2009-2011. RESULTS: A total of 112 children with a median age of 5.4 (IQR 3.6-11.2) years had 266 significant blood cultures during 149 infection episodes. Haematological malignancy affected 68 patients (60.7%) and solid tumours 44 (39.3%). The overall bloodstream infection rate was 1.5 episodes per 1,000 days-at-risk (95% CI, 1.2-1.8) and was similar for those with haematological malignancies and solid tumours. Most episodes were attributed to central venous catheter infection (120/149, 80.5%). Coagulase-negative staphylococci were isolated in almost half the bloodstream infections (127/266; 47.7%), while Gram-negative organisms accounted for a further quarter (64/266; 24.1%). Fungal isolates from blood were uncommon (8/112 children, 7.1%) but significantly associated with neutropenia (18/149 [12.1%] vs. 1/114 [0.9%], P = 0.0004). Six children (5.4%) died, including three (2.7%; 95% CI, 0.6-7.6%) whose deaths were infection-related. CONCLUSIONS: This study provides an updated risk estimate for bloodstream infections in children with cancer and adds to the framework for developing evidence-based guidance for management of suspected infections in this highly vulnerable group.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

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.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

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.

In Vivo Modeling of Chemoresistant Neuroblastoma Provides New Insights into Chemorefractory Disease and Metastasis.

Neuroblastoma is a pediatric cancer that is frequently metastatic and resistant to conventional treatment. In part, a lack of natively metastatic, chemoresistant in vivo models has limited our insight into the development of aggressive disease. The Th-MYCN genetically engineered mouse model develops rapidly progressive chemosensitive neuroblastoma and lacks clinically relevant metastases. To study tumor progression in a context more reflective of clinical therapy, we delivered multicycle treatment with cyclophosphamide to Th-MYCN mice, individualizing therapy using MRI, to generate the Th-MYCN CPM32 model. These mice developed chemoresistance and spontaneous bone marrow metastases. Tumors exhibited an altered immune microenvironment with increased stroma and tumor-associated fibroblasts. Analysis of copy number aberrations revealed genomic changes characteristic of human MYCN-amplified neuroblastoma, specifically copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. RNA sequencing revealed enriched expression of genes associated with 17q gain and upregulation of genes associated with high-risk neuroblastoma, such as the cell-cycle regulator cyclin B1-interacting protein 1 (Ccnb1ip1) and thymidine kinase (TK1). The antiapoptotic, prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, and treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival. Our results highlight that under treatment conditions that mimic chemotherapy in human patients, Th-MYCN mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease. The Th-MYCN CPM32 model therefore is a useful tool to dissect in detail mechanisms that drive metastasis and chemoresistance, and highlights dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease. SIGNIFICANCE: An in vivo mouse model of high-risk treatment-resistant neuroblastoma exhibits changes in the tumor microenvironment, widespread metastases, and sensitivity to JAK1/2 inhibition.

Very low rates of culture-confirmed invasive bacterial infections in a prospective 3-year population-based surveillance in Southwest London.

OBJECTIVES: To estimate the incidence, clinical characteristics and risk factors for culture-confirmed invasive bacterial infections in England. DESIGN: Prospective, observational, study of all children with positive blood and/or cerebrospinal fluid (CSF) culture over a 3-year period (2009-2011). SETTING: All five hospitals within a geographically defined region in southwest London providing care for around 600 000 paediatric residents. PATIENTS: Children aged 1 month to 15 years MAIN OUTCOME MEASURES: Rates of community-acquired and hospital-acquired invasive bacterial infections in healthy children and those with co-morbidities; pathogens by age group, risk group and clinical presentation. RESULTS: During 2009-2011, 44 118 children had 46 039 admissions, equivalent to 26 admissions per 1000 children. Blood/CSF cultures were obtained during 44.7% of admissions, 7.4% were positive but only 504 were clinically significant, equivalent to 32.9% of positive blood/CSF cultures, 2.4% of all blood/CSF cultures and 1.1% of hospital admissions. The population incidence of culture-confirmed invasive bacterial infection was 28/100 000. One-third of infections were hospital acquired and, of the community-acquired infections, two-thirds occurred in children with pre-existing co-morbidities. In previously healthy children, therefore, the incidence of community-acquired invasive bacterial infection was only 6.4/100 000. CONCLUSIONS: Although infection was suspected in almost half the children admitted to hospital, a significant pathogen was cultured from blood or CSF in only 2.4%, mainly among children with pre-existing co-morbidities, who may require a more broad-spectrum empiric antibiotic regime compared to previously healthy children. Invasive bacterial infection in previously healthy children is now very rare. Improved strategies to manage low-risk febrile children are required.

Hepatoblastoma in a child with a paternally-inherited ABCC8 mutation and mosaic paternal uniparental disomy 11p causing focal congenital hyperinsulinism.

Hepatoblastoma is a tumour of early childhood occurring in association with genetic syndromes including Beckwith-Wiedemann Syndrome (BWS) which results from dominance of paternally-inherited genes on chromosome 11p15. We report a child without clinical BWS, neonatally diagnosed with focal congenital hyperinsulinism resulting from a paternally-inherited recessively-acting mutation of ABCC8 and pancreatic paternal uniparental disomy (UPD) for chromosome 11p15, who subsequently developed hepatoblastoma. Genetic testing showed UPD 11p15 in the pancreas and liver but not systemically, allowing the expression of mutated ABCC8 in both tissues. Infants with large or multifocal forms of focal congenital hyperinsulinism may be at risk of BWS-like tumours due to mosaic UPD despite negative whole-blood and buccal DNA testing and tumour surveillance should be considered for this minority.