Investigating cell division mode in the normal and diseased brain
Cell division modes decide about cell fate, and changes in cell division mode may contribute to diseases, such as gliomas and demyelinating diseases. Vice versa, disease-related signals may alter the cell division modes and thereby change cell fates. Proliferative oligodendrocyte precursor cells (OPCs) in the uninjured adult brain are capable of three cell division modes: symmetric self-renewing divisions, symmetric differentiating divisions, and asymmetric divisions, generating one OPC and one differentiating daughter cell. Studies ongoing in the lab into the poorly understood regulation of cell division modes and their impact on cell fate in the postnatal and adult brain are expected to yield novel points of disruption, to which therapies can be targeted.

Asymmetry-defective adult oligodendrocyte precursor cells as glioma origin

The purpose of this work is to determine whether restoring normal asymmetric cell division in oligodendrocyte precursor cells (OPCs) can prevent tumor initiation. We are in particular focused on oligodendrogliomas, which are progressive brain tumors. The proposed research is a paradigm for understanding the role of asymmetric cell divisions in all cancers and might have a far-reaching therapeutic impact.

Gliomas are the most common and deadly brain tumors in adults and can arise from mutant OPCs by a largely unknown mechanism. Specific treatments to eliminate these malignant OPCs have not been developed, primarily because the underlying mechanisms associated with their malignant transformation are not well understood. Finding the underlying causes for malignant transformation of OPCs by investigating defects in asymmetric cell division is part of our lab’s effort.  It should lead us to identify and test specific pharmacological agents targeted to re-store these defects for their ability to impact tumor growth. In 2011, we published molecular evidence that OPCs are the cellular origin of experimental oligodendroglioma.  We further showed that OPCs divide asymmetrically to self-renew and generate mature oligodendrocytes.  In this work, we showed that the transmembrane proteoglycan NG2 distributes asymmetrically during mitosis and thereby promotes activation of epidermal receptor growth factor and self-renewal and prevents differentiation of the NG2+ progeny. OPC with defective NG2 asymmetry aberrantly self-renew, fail to differentiate and turn into glioma –initiating cells. The work raised the question whether losing cellular asymmetry transforms OPC and changes their cell fate. As a consequence – we hypothesize – OPC rather self-renew and proliferate uncontrollably, rather than self-renew and differentiate at a one-to-one rate. Ongoing work in the lab addresses whether OPC-specific loss of cell polarity regulator Lethal giant larvae-1 (LGL1) disrupts asymmetric cell division and cell fate and thereby causes NG2-dependent tumor initiation. We pursue this question by generating transgenic mice with OPC deficient for LGL1. The phenotypic analyses of OPCs in this model enables us to determine a causal link between loss between LGL1, disrupted NG2 asymmetry and tumor initiation (Daynac et al, In submission, Nature Neuroscience).

Collaborators: Dr. Valerie Vasioukhin, Fred Hutchinson Cancer Research Center, Seattle

Investigating ASPM regulation of asymmetric cell division for therapeutic opportunities

Novel therapeutic approaches are desperately needed to improve the prognosis for glioma patients. Developing such approaches will require the delineation of key regulatory networks specific to glioma cells and leading to neoplastic transformation and enabling tumor growth. Past studies in mouse models identified oligodendrocyte precursor cells (OPCs) as the putative cellular origin of astrocytoma and oligodendroglioma. Data from our lab showed that the switch from asymmetric, self-sustaining to symmetric, self-renewing divisions is a critical step in the neoplastic transformation of OPC. Therapies specifically interfering with aberrant symmetric cell divisions are expected to eliminate malignant OPCs and disrupt tumor growth. It is our long-term goal to develop such therapies, by defining the effect of glioma-associated genetic alterations on the asymmetric-to-symmetric cell division mode switch. The objective of this project is to test the hypothesis that abnormal spindle microcephaly associated Aspm mRNA expression is upregulated in glioma cells by constitutive-active receptor tyrosine kinase signaling via PI3K-AKT. We further propose that Aspm protein positively regulates symmetric divisions and thereby promotes neoplastic transformation and malignant growth. Functionally, Aspm positively regulates mitotic spindle integrity and positioning. We expect that by pursuing our work, we will identify the molecular switch from asymmetric to symmetric division in OPCs, and demonstrate that this switch is regulated by an extrinsic signal through modulation of Aspm levels.  We will validate Aspm as a proto-oncogene and Aspm functions as therapeutic opportunities and anticipate that Aspm spindle regulatory functions provide novel therapeutic vulnerabilities in glioma cells. These studies can delineate a paradigm for enforcing symmetric cell division and tumorigenesis in all progenitor-driven tumors. By unraveling Aspm upregulation as a specific step in the neoplastic transformation of progenitor cells we identify a point of susceptibility for cancer therapies and provide a treatment paradigm for other progenitor-derived cancers.

Collaborators: Dr. Noemi Andor, Stanford University, Drs. Annette Molinaro, Anders Persson and Joe Costello, UCSF

The development of therapeutic approaches based on the unique properties of lower-grade glioma

Lower-grade glioma is driven by a variety of factors that contribute to tumor formation and persistence. A key factor in lower grade glioma development is mutant IDH. The IDH gene encodes isocitrate dehydrogenase, and enzyme that converts isocitrate to alpha ketoglutarate (αKG).  Nearly 80% of lower grade glioma, but not higher-grade glioma, contain mutations in the IDH gene.  The mutant IDH produced exhibits a novel enzymatic activity that converts αKG to 2-hydroxyglutatate (2HG) and leads to accumulation of 2HG in cells.  High levels of 2HG in turn inhibit the activity of αKG-dependent enzymes, including those responsible for histone methylation, leading to changes in patterns of gene expression that contribute to inhibition of cellular differentiation, cellular transformation and tumor formation.  The basis for the contribution of mutant IDH1 to transformation is incompletely understood, and if better defined could contribute to the development of therapies targeting the lesions that drive tumor formation. We determine if IDH mutation and/or metabolic changes disrupt asymmetric cell division of OPC and thereby contribute to increased self-renewal and changes in cell fate. These studies will help define how mutant IDH expression drives the tumorigenic process and in the process may lead to the identification of pathways that can be targeted for therapeutic intervention.

Collaborators: The Loglio Initiative, including Dr. Russ Pieper, Department of Neurosurgery, Dr. Sabrina Ronen, Department of Radiology and Biomedical Imaging and Dr. Anders Persson, Department of Neurology, UCSF. 

Cellular origin and immune therapy of BRAFV600E mutant malignant astrocytoma

Our work is aimed to generate new information regarding the cellular origin of BRAFVE induced malignant astrocytoma (MA) and to provide insight into the molecular mechanisms of neoplastic transformation resulting in brain tumor development. 

A mutant, activated form of BRAF, BRAFV600E, and concomitant homozygous deletion of CDKN2A, encoding p16, have been found in a significant fraction of pediatric MA and in a small number of adult glioblastoma.  BRAFVE is known to promote proliferation while suppressing normal cellular differentiation. Whether increased production of self-renewing cells through increasing symmetric cell divisions (i.e., decreased asymmetric divisions) is manifested in association with BRAFV600E induced cell transformation is currently unknown. Many pediatric brain tumors, including malignant astrocytomas (MA), are thought to originate from neural stem cells (NSC) which, due to the occurrence and accumulation of growth-promoting gene alterations, may give rise to various cell subpopulations, including tumor-propagating cells (TPCs). TPCs are also referred to as cancer stem cells when expressing neural stem cell markers such as CD133.  TPCs are considered to have increased resistance to conventional therapy for MA, and consequently are important contributors to recurrence. We have recently shown that TPCs undergo asymmetric division by a mechanism that involves elevated polo-like-kinase 1 (Plk1) activity. Pharmacologic inhibition of Plk1 inhibits the growth of glioma xenografts and more effectively eliminating TPCs than anti- BRAFV600E-targeted therapy.  A Plk1-controlled polarity checkpoint was uncovered in TPCs that renders them especially sensitive to Plk1 inhibition (Lerner et al, Cancer Research, 2015). We continue to bridge the gap in our understanding of BRAFVE-induced transformation, and its relationship with ACD, we examine effects of BRAFVE in p16 deficient NSCs as well as in corresponding p16 deficient astrocytes of mouse and human origin. In addition, we investigate relationships between BRAFVE–p16 deficient tumors and their adaptation to BRAFVE targeted therapy, with our primary focus directed to tumor-propagating, therapy-resistant cell subpopulation and changes in cell division mode resulting from treatment. We investigate BRAFVE tumor cells and tumor tissues, ex vivo and in vivo, using genetically engineered mouse models (GEMMs), respectively, for molecular changes, changes in cellular composition, and asymmetric division in association with response to BRAFVE targeted therapy. We have recently generated a mouse allograft model in immune competent mice (Grossauer et al, Oncotarget, 2016) and use it to test combinations of BRAFVE targeted therapy and immune therapies.

Collaborators: Dr. C. David James and Dr. Derek Wainwright, Department of Neurosurgery, Northwestern University, Chicago, Dr. Theodore Nicolaides, Department of Pediatrics, UCSF, Dr. Joanna Phillips, Department of Pathology, UCSF, San Francisco; Dr. Stefan Grossauer and Dr. Katharina Koeck, Department of Neurological Surgery, University Hospital Bochum, Germany.