Andrew Hallahan

The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas

To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or γ secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a γ secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

The Smo/Smo Model: Hedgehog-Induced Medulloblastoma with 90% Incidence and Leptomeningeal Spread

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies.

Sonic Hedgehog regulates Hes1 through a novel mechanism that is independent of canonical Notch pathway signalling

Aberrant regulation of signalling mechanisms that normally orchestrate embryonic development, such as the Hedgehog, Wnt and Notch pathways, is a common feature of tumorigenesis. In order to better understand the neoplastic events mediated by Hedgehog signalling, we identified over 200 genes regulated by Sonic Hedgehog in multipotent mesodermal cells. Widespread crosstalk with other developmental signalling pathways is evident, suggesting a complex network of interactions that challenges the often over-simplistic representation of these pathways as simple linear entities. Hes1, a principal effector of the Notch pathway, was found to be a target of Sonic Hedgehog in both C3H/10T1/2 mesodermal and MNS70 neural cells. Desert Hedgehog also elicited a strong Hes1 response. While Smoothened function was found necessary for upregulation of Hes1 in response to Sonic Hedgehog, the mechanism does not require γ-secretase-mediated cleavage of Notch receptors, and appears to involve transcription factors other than RBP-Jκ. Thus, we have defined a novel mechanism for Hes1 regulation in stem-like cells that is independent of canonical Notch signalling.

Improved outcomes of children with malignancy admitted to a pediatric intensive care unit.

ObjectiveTo assess the acute and long-term outcomes of children admitted to the intensive care unit with cancer or complications after bone marrow transplantation. DesignRetrospective analysis of databases from a prospective pediatric intensive care unit (PICU) database supplemented by case notes review. SettingA PICU in a tertiary pediatric hospital. PatientsAll children with malignancy admitted to the PICU between May 1, 1987, and April 30, 1996. InterventionsNone. Measurements and Main ResultsThere were 206 admissions to the PICU during a 9-yr study period of 150 children with malignancies or complications after bone marrow transplantation. Forty patients died in the PICU (27% mortality rate). The most frequent indications for PICU admission were shock and respiratory disease. Of 56 children admitted with shock, there were 16 deaths (29% mortality rate). In 24 episodes of sepsis, inotropic and ventilatory support were required and 13 patients (54%) survived. Analysis of long-term survival gave estimates of 50% survival for all oncology patients admitted to the PICU and 42% for those admitted for shock. ConclusionsA high proportion of oncology patients admitted to the PICU requiring intensive intervention survive and go on to be cured of their malignancy. Our study suggests the PICU outcome for these patients has improved.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Investigating the cost-effectiveness of videotelephone based support for newly diagnosed paediatric oncology patients and their families: design of a randomised controlled trial

BackgroundProviding ongoing family centred support is an integral part of childhood cancer care. For families living in regional and remote areas, opportunities to receive specialist support are limited by the availability of health care professionals and accessibility, which is often reduced due to distance, time, cost and transport. The primary aim of this work is to investigate the cost-effectiveness of videotelephony to support regional and remote families returning home for the first time with a child newly diagnosed with cancerMethods/designWe will recruit 162 paediatric oncology patients and their families to a single centre randomised controlled trial. Patients from regional and remote areas, classified by Accessibility/Remoteness Index of Australia (ARIA+) greater than 0.2, will be randomised to a videotelephone support intervention or a usual support control group. Metropolitan families (ARIA+ ≤ 0.2) will be recruited as an additional usual support control group. Families allocated to the videotelephone support intervention will have access to usual support plus education, communication, counselling and monitoring with specialist multidisciplinary team members via a videotelephone service for a 12-week period following first discharge home. Families in the usual support control group will receive standard care i.e., specialist multidisciplinary team members provide support either face-to-face during inpatient stays, outpatient clinic visits or home visits, or via telephone for families who live far away from the hospital. The primary outcome measure is parental health related quality of life as measured using the Medical Outcome Survey (MOS) Short Form SF-12 measured at baseline, 4 weeks, 8 weeks and 12 weeks. The secondary outcome measures are: parental informational and emotional support; parental perceived stress, parent reported patient quality of life and parent reported sibling quality of life, parental satisfaction with care, cost of providing improved support, health care utilisation and financial burden for families.DiscussionThis investigation will establish the feasibility, acceptability and cost-effectiveness of using videotelephony to improve the clinical and psychosocial support provided to regional and remote paediatric oncology patients and their families.

Using videotelephony to support paediatric oncology-related palliative care in the home: from abandoned RCT to acceptability study

Videotelephony (real-time audio-visual communication) has been used successfully in adult palliative home care. This paper describes two attempts to complete an RCT (both of which were abandoned following difficulties with family recruitment), designed to investigate the use of videotelephony with families receiving palliative care from a tertiary paediatric oncology service in Brisbane, Australia. To investigate whether providing videotelephone-based support was acceptable to these families, a 12-month non-randomised acceptability trial was completed. Seventeen palliative care families were offered access to a videotelephone support service in addition to the 24 hours ‘on-call’ service already offered. A 92% participation rate in this study provided some reassurance that the use of videotelephones themselves was not a factor in poor RCT participation rates. The next phase of research is to investigate the integration of videotelephone-based support from the time of diagnosis, through outpatient care and support, and for palliative care rather than for palliative care in isolation. Trial registration ACTRN 12606000311550

Canonical Notch signaling is not required for the growth of Hedgehog pathway-induced medulloblastoma.

Current treatment for medulloblastoma is successful in more than half of all cases but results in substantial disability in survivors. Accordingly, there is considerable interest in drugs that may target specific signaling pathways activated in the tumors, with inhibitors of both the Hedgehog and Notch pathways currently proposed as possible therapeutics. Here, we tested the hypothesis that Notch pathway inhibition in vivo may block the formation of Hedgehog-dependent medulloblastoma. We took the general approach of using a cre recombinase under the control of the GFAP promoter to generate medulloblastoma in mice carrying a conditional Ptc1 allele and introduced a conditional RBP-J allele to ablate canonical Notch signaling. Loss of RBP-J from the developing cerebellum led to a modest loss of stem cells and an overall developmental delay. These phenotypes could be partially compensated by activation of the Hedgehog pathway. Hedgehog-dependent medulloblastoma were not blocked by loss of RBP-J, indicating that canonical Notch signaling is not required for tumor initiation and growth in this model.

ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma.

BackgroundResistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death.MethodsUsing the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place.ResultsIsolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome.ConclusionsABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma.