Holmen Lab Research - Malignant Glioma

There has been a 35% increase in the incidence of pediatric brain tumors over the last thirty five years and brain tumors have become the single most common cause of cancer-related mortality in children. The primary treatment is surgical excision, but this is challenging in cases where the tumor is located within the optic pathways, the brain stem, or diencephalon (located near the midline of the brain). Prior to the recent discovery of BRAF mutations in these tumors, very little was known about the genetic alterations in pediatric malignant gliomas. These gliomas comprise World Health Organization (WHO) grades II-IV and account for 50% of cerebral tumors. Despite the addition of radiotherapy and chemotherapy, the prognosis for pediatric high-grade gliomas remains poor and 5-year survival rates are less than 20%. Recently, BRAFV600E mutations were identified in 18% of WHO grade II, 33% of WHO grade III, and 18% of WHO grade IV pediatric gliomas (a combined 23% for grades II-IV). While these findings represent a significant breakthrough, the precise role of this alteration in the formation and maintenance of this disease remains to be defined. Whether BRAF or relevant downstream signaling mediators can be productively targeted for therapeutic intervention in glioma patients has yet to be determined.

BRAF mutations have also been observed in adult grade III and IV gliomas. Glioblastoma multiforme (GBM) (WHO grade IV astrocytoma) is the most common and aggressive primary brain tumor in adults. It is also the most fatal. Despite major improvements in treatment, the prognosis for patients with this disease has not changed in the last twenty years. The current standard of care for this disease includes surgical resection followed by adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. However, with standard treatment mean survival is only 15 months. Novel treatment strategies are badly needed to improve the outcome for these patients. We are working to better understand the biology of these gliomas to guide our development of new therapies, which we hope will improve survival and reduce morbidity in these patients.

High-throughput efforts such as The Cancer Genome Atlas (TCGA) pilot project and an extensive sequencing project have yielded novel information about GBM. In 2008, a multi-group collaboration sequenced >20,000 genes in 22 GBMs and identified a common point mutation in the metabolic gene isocitrate dehydrogenase 1 (IDH1) in secondary GBM. Numerous publications have since followed demonstrating that the majority of low-grade gliomas and secondary GBMs possess mutations in IDH1. The few exceptions that do not contain a mutation in IDH1 have an equivalent mutation in the related gene IDH2. This mutation had never before been linked to cancer and the function remains unclear. There have been reports suggesting the gene functions as a tumor suppressor and others proposing the mutation renders it oncogenic. At the heart of the debate are the findings that IDH1 mutations are associated with better prognosis. While these findings represent a significant breakthrough, they remain to be validated to demonstrate a clear role for IDH1 and IDH2 in the etiology of this disease. Whether mutant IDH or products of its activity can be productively targeted for therapeutic intervention in glioma patients has yet to be determined. A better understanding of the biology of these gliomas will guide the development of new therapies to improve survival and reduce morbidity in these patients.

Our laboratory utilizes a robust somatic cell gene delivery glioma mouse model based on the RCAS/TVA system originally pioneered by Dr. Harold Varmus and Dr. Eric Holland. In this model, the retroviral receptor TVA is expressed under the control of the Nestin promoter, which is active in neural and glial progenitors. This mouse model allows efficient and cost-effective modeling of gliomas because testing of a new gene simply requires generating a new retroviral vector not a new transgenic mouse. Because a new virus can be made and evaluated quickly (<4 weeks) in comparison to the time required to make a new transgenic mouse, we were able to quickly demonstrate a role for BRAFV600E in the etiology of this disease. In addition, multiple genetic alterations can be introduced into the same animal without the cost associated with mating multiple strains of transgenic or knockout mice allowing us to assess cooperating events. Our group also extended the utility of this mouse model system by engineering the viruses to be responsive to doxycycline in the presence of Tet-off or Tet-on proteins. This allows us to regulate the expression of the delivered genes post-infection in vivo and define the role of specific genes in tumor maintenance. This genetic approach is extremely useful for target identification because pharmacological inhibition of a target can introduce additional variables in the experiment. If tumor growth is not affected following drug treatment it may be unclear if this is due to the target or the drug. We have successfully used this approach to assess the role of mutant KRAS in the context of active AKT and Ink4a/Arf deficiency. We plan to apply this approach to validate the genes identified by TCGA.