Andrew J. Gifford

P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.

We have previously described a series of methotrexate (MTX)‐selected CCRF‐CEM sublines (CEM/MTX R1–3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P‐glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P‐glycoprotein‐mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1–3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross‐resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P‐glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P‐glycoprotein. Our data have implications for the P‐glycoprotein‐mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited. Int. J. Cancer 78:176–181, 1998.© 1998 Wiley‐Liss, Inc.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma

Histone chaperone FACT acts in a positive feedback loop with MYCN and is a therapeutic target in neuroblastoma. Uncovering the FACTs in neuroblastoma Neuroblastoma is a common pediatric cancer of the nervous system. It is often difficult to treat, and tumors with amplifications of the MYC oncogene are particularly aggressive. Carter et al. have identified a histone chaperone called FACT as a mediator of MYC signaling in neuroblastoma and demonstrated its role in a feedback loop that allows tumor cells to maintain a high expression of both MYC and FACT. The authors then used curaxins, which are drugs that inhibit FACT, to break the vicious cycle. They demonstrated that curaxins work in synergy with standard genotoxic chemotherapy to kill cancer cells and treat neuroblastoma in mouse models. Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models

Background Diffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG. Methods A collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo. Results In vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice. Conclusion Our collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, Kis for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

Reduced folate carrier and methylenetetrahydrofolate reductase gene polymorphisms: associations with clinical outcome in childhood acute lymphoblastic leukemia

Reduced folate carrier and methylenetetrahydrofolate reductase gene polymorphisms: associations with clinical outcome in childhood acute lymphoblastic leukemia

Rare MYC-amplified Neuroblastoma With Large Cell Histology:

Background Although MYCN (aka N-myc) amplification is reported in ∼20% of neuroblastomas, MYC (aka C-myc) amplification appears to be a rare event in this disease. As of today, only 2 MYC-amplified neuroblastomas have been briefly mentioned in the literature. Methods We studied here the clinicopathological features of 3 MYC-amplified neuroblastomas. Results All 3 patients (2 females and 1 male) had stage 4 disease. One female is currently alive and well 52 months after the diagnosis, while the other female and male patients died of disease 24 and 20 months after the diagnosis, respectively. Further analysis on 2 tumors revealed unfavorable histology with MYC protein overexpression but with neither MYCN amplification nor MYCN protein overexpression. Both of these tumors exhibited “large cell neuroblastoma” histology with enlarged, uniquely open nuclei and nucleolar hypertrophy, along with “aberrant” desmin expression. Conclusions MYC-amplified neuroblastomas are extremely rare and seem to present with distinct clinicopathological features.

Antenatal gastrointestinal anomalies in neonates subsequently found to have alveolar capillary dysplasia

Alveolar capillary dysplasia (ACD) is a rare condition with variable presentation and clinical course. Clinicians should consider this diagnosis in neonates presenting with nonlethal congenital gastrointestinal malformation, a period of well‐being after birth then unremitting hypoxemia and refractory pulmonary hypertension. Lung biopsy and FOXF1 gene testing may help in diagnosis.

Unresectable VIP-secreting neuroblastoma: Efficacy of debulking and steroids for symptom control

To the Editor: Neuroblastoma is a malignant tumour arising from embryonal cells of the autonomic nervous system.1 A small percentage of patients have paraneoplastic syndromes2–4 including vasoactive intestinal peptide (VIP) associated secretory diarrhoea.5,6 VIP induces smooth muscle relaxation, stimulating the secretion of water, electrolytes and pancreatic bicarbonate leading to severe watery diarrhoea, hypokalaemia, achlorhydria, hyperglycaemia and hypercalcemia.6–8 VIP secretion at presentation is usually associated with well-differentiated and localised tumours with excellent longtermoutcomes following complete resection.8 However, the syndrome of VIP secretion can lead to intractable secretory diarrhoea5,6 and severe systemic symptoms. The management of unresectable VIP secreting tumours has not beenwell defined. Here, we describe a child with a VIP-secreting neuroblastoma that was unresectable, non-responsive to standard chemotherapy, and caused severe systemic symptoms. The control of symptoms was achieved after partial debulking surgery and steroid therapy. An 11-month-old female presented with failure to thrive and profound diarrhoea for 10 weeks. On presentation, she weighed less than the 3rd centile, was hypotensive, severely dehydrated and had an intra-abdominal mass. Computed tomography showed an extensive retroperitoneal mass located anterior to the lumbar and upper sacral spine that displaced the lower portion of the abdominal aorta away from the vertebral bodies and encased the origins and proximal portions of both common iliac arteries, and was therefore not amenable to surgical resection. The tumour was avid on metaiodobenzyguanidine (MIBG) scan and urine catecholamine was raised. Electrolytes were deranged with hyponatraemia and severe hypokalaemia. Open biopsy demonstrated an NMYC non-amplified, well-differentiated neuroblastoma,with no bonemarrow involvement, consistent with a Stage III, intermediate risk, VIP-secreting neuroblastoma. Chemotherapy was commenced as per A3961 protocol for intermediate risk neuroblastoma. Two cycles consisting of carboplatin, etoposide, cyclophosphamide and doxorubicin were administered without improvement.9 A single cycle of high-risk therapy was trialled using cyclophosphamide and topotecanwithout response. Loperamide and octreotide were tried to manage the diarrhoea without clinical benefit. The patient experienced clinical deterioration with persistent diarrhoea greater than 30 mL/kg/day, electrolyte loss and metabolic acidosis, features that appeared to be related to the underlying VIP secretion. Tumour debulking surgery was the next step where 85% of the tumour was resected, with a significant residual bulk of tumour remaining (Figure 1). The residual tumour measured 6.1 × 2.0 × 3.6 cm compared with 7.7 × 4.4 × 9.3 cm at diagnosis. The histopathology was consistent with the diagnostic biopsy. The patients clinical status improved immediately following the operation with a dramatic reduction in the volume of diarrhoea and resolution of systemic symptoms. However, due to persistence of reduced volume of diarrhoea, and significant residual tumour on post-operative imaging, six courses of retinoic differentiation therapy and a course of oral non-steroidal were trialled. No response was observed, and the patient remained well but with persistent loose, watery stools. During an intercurrent respiratory viral infection corticosteroids were commenced, with an immediate and complete cessation of her diarrhoea, which lasted for 2 months. Her diarrhoea then recurred but continued to slowly improve over the subsequent years. She is now 7 years after diagnosis and her diarrhoea has completely resolved.Her residual tumour has not changed in size since the debulking operation, but has become less MIBG avid, suggesting ongoing differentiation. The metabolic consequences of VIP-associated diarrhoea may complicate induction chemotherapy and be difficult to manage, as in this case. Somatostatin is an effective treatment to control diarrhoea associated with VIP secreting pancreatic tumours in adults. However, limited case reports suggest that it may not be effective in neuroblastoma.10 Resection of both VIP secreting neuroblastoma and ganglioneuroma demonstrate a resolution of diarrhoea within hours. However, there is no clear treatment pathway for unresectable tumours. In the series from Bourdeaut et al., four children with unresectable tumours received neoadjuvant chemotherapy, but no improvement in diarrhoea was seen.5 Our case supports the suggestion that tumour debulking should be considered early in the management of unresectable tumours. Corticosteroids have been reported to be of benefit in adults with VIP secreting pancreatic tumours. Cooperman et al. described control of diarrhoea in a 65-year-old man with a benign VIP secreting pancreatic islet cell tumour on 10 mg of oral prednisone a day.11 Our patient received corticosteroids and experienced a sudden and prolonged improvement in VIP-related diarrhoea, suggesting a possible role in this disease. The further gradual resolution of diarrhoea over subsequent years, alongwith reducedMIBGuptake, suggests thatVIPsecreting neuroblastomas, which are already well differentiated, may reduce their secretion as differentiation continues. This case indicates that debulking should be considered early in themanagement of unresectable VIP-secreting neuroblastoma to control VIP-associated diarrhoea and metabolic complications. Steroid

Targeted Doxorubicin-loaded Bacterially Derived Nano-cells for the Treatment of Neuroblastoma

Advanced stage neuroblastoma is an aggressive disease with limited treatment options for patients with drug-resistant tumors. Targeted delivery of chemotherapy for pediatric cancers offers promise to improve treatment efficacy and reduce toxicity associated with systemic chemotherapy. The EnGeneIC Dream Vector (EDVTM) is a nanocell, which can package chemotherapeutic drugs and target tumors via attachment of bispecific proteins to the surface of the nanocell. Phase I trials in adults with refractory tumors have shown an acceptable safety profile. Herein we investigated the activity of EGFR-targeted and doxorubicin-loaded EDVTM (EGFREDVTMDox) for the treatment of neuroblastoma. Two independent neuroblastoma cell lines with variable expression of EGFR protein [SK-N-BE(2), high; SH-SY-5Y, low] were used. EGFREDVTMDox induced apoptosis in these cells compared to control, doxorubicin, or non-doxorubicin loaded EGFREDVTM. In three-dimensional tumor spheroids, imaging and fluorescence life-time microscopy revealed that EGFREDVTMDox had a marked enhancement of doxorubicin penetration compared to doxorubicin alone, and improved penetration compared to non-EGFR-targeted EDVTMDox, with enhanced spheroid penetration leading to increased apoptosis. In two independent orthotopic human neuroblastoma xenograft models, short-term studies (28 days) of tumor-bearing mice led to a significant decrease in tumor size in EGFREDVTMDox-treated animals compared to control, doxorubicin, or non-EGFR EDVTMDox. There was increased TUNEL staining of tumors at day 28 compared to control, doxorubicin, or non-EGFR EDVTMDox. Moreover, overall survival was increased in neuroblastoma mice treated with EGFREDVTMDox (P < 0007) compared to control. Drug-loaded bispecific-antibody targeted EDVsTM offer a highly promising approach for the treatment of aggressive pediatric malignancies such as neuroblastoma. Mol Cancer Ther; 17(5); 1012–23. ©2018 AACR.

Integration of genomics, high throughput drug screening, and personalized xenograft models as a novel precision medicine paradigm for high risk pediatric cancer

ABSTRACT Pediatric high grade gliomas (HGG) are primary brain malignancies that result in significant morbidity and mortality. One of the challenges in their treatment is inter- and intra-tumoral heterogeneity. Precision medicine approaches have the potential to enhance diagnostic, prognostic and/or therapeutic information. In this case study we describe the molecular characterization of a pediatric HGG and the use of an integrated approach based on genomic, in vitro and in vivo testing to identify actionable targets and treatment options. Molecular analysis based on WGS performed on initial and recurrent tumor biopsies revealed mutations in TP53, TSC1 and CIC genes, focal amplification of MYCN, and copy number gains in SMO and c-MET. Transcriptomic analysis identified increased expression of MYCN, and genes involved in sonic hedgehog signaling proteins (SHH, SMO, GLI1, GLI2) and receptor tyrosine kinase pathways (PLK, AURKA, c-MET). HTS revealed no cytotoxic efficacy of SHH pathway inhibitors while sensitivity was observed to the mTOR inhibitor temsirolimus, the ALK inhibitor ceritinib, and the PLK1 inhibitor BI2536. Based on the integrated approach, temsirolimus, ceritinib, BI2536 and standard therapy temozolomide were selected for further in vivo evaluation. Using the PDX animal model (median survival 28 days) we showed significant in vivo activity for mTOR inhibition by temsirolimus and BI2536 (median survival 109 and 115.5 days respectively) while ceritinib and temozolomide had only a moderate effect (43 and 75.5 days median survival respectively). This case study demonstrates that an integrated approach based on genomic, in vitro and in vivo drug efficacy testing in a PDX model may be useful to guide the management of high risk pediatric brain tumor in a clinically meaningful timeframe.