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Analysis of MELK Function In Vivo in CNS Neural Progenitors And in Brain Tumors

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$2 732 400
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
In most tissues, including the central nervous system, differentiation from a somatic multipotent stem cell proceeds through transit-amplifying progenitors (TAPs). These highly mitotic progenitors are the first committed cells, which may remain multipotent, but have limited self-renewal capacity. Despite the recent progress in understanding the pathways operating in stem cells, the molecular mechanisms that function selectively in TAPs remain poorly studied. TAPs are likely critical cells that determine the extent of adult neural cell growth. TAPs may also be the key cells responsible for the transition from lifetime self-renewing stem cells to highly proliferative but short-lived progenitors. This is a very important point in neural cell development and thus, TAPs may be potential targets for neoplastic transformations. We have identified a novel protein (MELK) as a functional marker of proliferating cells in the adult brain. MELK is likely to selectively modulate stem-to-progenitor cell transition and proliferation of TAPs. On the other hand MELK has emerged as a promising target for many types of cancer, in particular glioblastomas. An important difference between normal neural progenitors and CD133+ cancer stem cells is that MELK will inhibit growth in normal neural progenitors but will kill cancer stem cells. However, nothing is known about the role of MELK in vivo.. The goal of this proposal is to address this critical issue. Our hypothesis is that MELK is upregulated in neural progenitors in vivo but its function is not needed for survival of normal TAPs. We also suggest that MELK activity in cancer stem cells is required for brain tumor survival and growth. Our Aims are to determine the exact areas of MELK expression and its activity in the central nervous system using mouse models. We will also determine whether MELK acitivity is required for tumor formation in the mouse. . Finally, we will test whether MELK inhibitors kill primary human glioblastomas. My laboratory generated a diverse set of reagents such as genetically modified mice and efficient tools to manipulate the MELK gene. In collaboration with Dr. Abagyan (Scripps) we have produced molecules that inhibit MELK expression and we established a partnership with the neurosurgeon Dr. Jandial (UCSD) to obtain primary glioblastoimas. Thus, my laboratory is uniquely positioned to address the role of MELK in neural progenitors under normal conditions and evaluate the its potential as target for anti-tumor drug development for primary human brain tumors. Understanding the exact role of MELK and its mechanism of action will allow manipulation of neural progenitor compartment for therapeutic purposes. On the other hand the different sensitivity to MELK inhibition seen in normal TAPs and glioblastomas makes it a perfect target for treatment of brain tumors.
Statement of Benefit to California: 
Cell therapies proposed for traumatic CNS injuries, ischemia and several neurodegeneration conditions such as Parkinson’s, Alzheimer’s and ALS rely on our ability to manipulate neural stem and progenitor cells. Their therapeutic use depends on our understanding of the genes and pathways that govern proliferation of multipotent stem cells and progenitors as well as their differentiation under the appropriate stimuli. At the same time, it became clear that many tumors arise from the stem/progenitor cell compartments and the notion of cancer stem cells has been proposed to better characterize the cellular and molecular mechanism of tumor initiation and progression. It is now increasingly evident that the same genes and pathways are operating in both normal and cancer stem cells. However, the lack of fundamental knowledge in this area impedes technological advancements. The PI’s laboratory has recently identified Maternal Leucine Zipper Kinase (MELK) as a key candidate gene that modulates proliferation of both normal neural progenitors and malignant cancer stem cells in human glioblastomas and we have developed several lead small molecule inhibitors of MELK function. Our analysis will unequivocally determine the in vitro role of MELK in both normal neurogenesis and growth of primary glioblastomas. Here we will use a mouse model of malignant glioma that mimics common genetic lesions underlying primary glioblastoma in humans. Moreover, we will test whether small molecule inhibitors of MELK kill primary glioblastomas. Our hypothesis is that MELK regulates neural progenitors within the adult germinal zones in vivo. If this is correct the agonist of MELK function will increase adult neurogenesis and, likely, will result in benefits currently associated with treatments such as Prozac. If MELK is also indispensable for brain tumor growth, small molecule inhibitors of MELK will lead to a major clinical breakthrough in treatment of malignant brain tumors. An effective, straightforward, and understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The novel therapies that will be developed as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will go to the state and its citizens from stem cell research programs financed by the CIRM. The second source will be several types of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.
Review Summary: 
SYNOPSIS: This proposal is focused on Maternal Embryonic Leucine Zipper Kinase (MELK) gene product as a functional marker of transit-amplifying progenitors (TAPs) in the adult brain and as a key factor with respect to brain tumors. In most tissues, including the CNS, differentiation from a somatic multipotent stem cell proceeds through transit-amplifying progenitors (TAPs). Despite recent progress in understanding the pathways operating in stem cells, genes that function selectively in TAPs remain poorly studied. TAPS may be especially important in determining the extent of adult neurogenesis. TAPs transition from lifetime self-renewing stem cells to highly proliferative but short-lived progenitors, making them a potential target for neoplastic transformations. The applicant identified the MELK gene product as a functional marker of TAPs in the adult brain. MELK is upregulated in the neural progenitors in the SVZ and DG where it regulates stem-to-progenitor cell transition and proliferation of TAPS; MELK is not found in GFAP+ stem cells in germinal zones or in terminally differentiated cells. MELK modulates stem-to-progenitor cell transition and proliferation of TAPs in vitro. In addition, MELK appears to be involved in brain tumor formation. Knocking down MELK activity induces apoptosis in primary glioblastomas. The hypotheses of this proposal are that MELK is upregulated in neural progenitors, modulating the proliferation of TAPs, and that MELK activity in cancer stem cells is required for glioblastoma survival and growth, making it a unique cancer stem cell-specific target in CNS tumors. The present proposal is focused on the role of MELK in vivo. In specific aim 1, the PI will determine whether MELK expression and function in the adult CNS is restricted to neural progenitors in the SVZ and DG of the hippocampus. In the first part of this specific aim, the PI will transplant into the lateral ventricle of neonatal mice neural progenitors tagged with mCherry from a MELK/GFP+ mouse that he generated and examine proliferation of these cells in vivo after BrdU labeling and Ara-C treatment. In these studies, the PI will also make use of a transgenic mouse (which is presently being generated by the PI) expressing nuclear GFP driven by the nestin promoter. In the second part of this aim, the PI will determine the role of MELK in CNS neural progenitors using MELK null mice (and conditional MELK floxed mice) that he generated. Morphology and cell markers (GFAP, etc.) will be assessed as well as the proliferation of neural progenitors (testing BrdU incorporation and markers of the stem/progenitor compartment) under normal conditions, hypoxia-ischemia (in collaboration with Dr. Stuart Lipton), and exposure to an “enriched” environment. The expression level and phosphorylation of putative MELK targets will be assessed. Lastly, the MELK null mouse will be crossed with a transgenic mouse expressing nuclear GFP driven by the nestin promoter. The PI has preliminary data that include evidence that INK4alpha/Arf null mice X MELK-GFP demonstrate MELK activation in the brain tumor and that MELK inhibits proliferation and induces apoptosis of primary glioblastomas. In specific aim 2, the PI will determine whether MELK function is required for tumorigenesis using a mouse model glioblastoma and test the activity of MELK inhibitors in primary human glioblastomas. In the first part of this specific aim, the PI will determine whether knocking out the MELK gene decreases the frequency and type of brain tumors in a mouse model of glioblastoma (the INK4alpha/Arf null mouse). Because the PI found that brain tumors in the INK4alpha/Arf null are rare, he will take cultured neurospheres derived from E14.5 brains of INK4alpha/Arf null mice with or without being crossed with the MELK null mice and transduce them with a retrovirus expressing a constitutively active mutation of epidermal growth factor receptor (EGFRvIII) - and then transplant them into the striatum of C57 BL/6 mice. In the second part of the specific aim 2, the PI will test whether small molecule inhibitors of MELK catalytic activity inhibit growth of primary human glioblastomas. The PI demonstrated that MELK knockdown in CD133+ cancer stem cells from primary human glioblastoma inhibts CD133+ cell proliferation. In collaboration with Dr. Abagyan at Scripps, small molecule inhibitors of MELK were produced. The PI has established a collaboration with the neurosurgeon Dr. Jandial (UCSD) to obtain primary glioblastomas from patients. CD133+ cells will be fractionated from the tumors by FACS using CD133 antibody. These cells will be tested along with control fibroblasts to determine the expression of MELK protein and the effect of treatment with MELK shRNA and selected small molecules. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The involvement of MELK in progenitor cells and its implication in brain tumors are exciting findings that could have an important impact and high payoff in the field of stem cells and brain tumors. However, although MELK could be a key regulator of TAP cells in the adult SVZ and DG, as well as some cancer stem cells, the studies described in this proposal are diffuse, not particularly innovative, and lacking in depth and analysis. The relevance to regenerative medicine appears limited as half of the proposal is on the role of MELK in tumor progression. The only relevance to stem cell research is that the PI is testing whether MELK is required for adult neural stem cell proliferation or differentiation. There also could be a role for MELK in embryonic neural progenitors, but this is not addressed, which is a weakness. There are strengths to the proposal. The focus of this grant on glioblastoma and on the in vivo properties of MELK are important albeit difficult ones. Although unfocused, the experiments are logical and straightforward and are each likely to provide meaningful results. Experiments to determine whether small molecule inhibitors of MELK catalytic function block the growth or kill primary human brain tumors are potentially exciting, especially when one considers the remarkable success of Gleevec, a Bcr-Abl kinase inhibitor. The PI is well-trained and in a productive interactive milieu. He has valuable reagents, including MELK null mice that he developed as well as small molecules that inhibit MELK activity. Although the PI is new to the field, he has established collaborations with productive investigators. The proposal’s weaknesses outweigh its strengths. The experiments are feasible but the applicant does not convey significant focus and rigor in his experimental design, defining his scientific goals too broadly with little attention to detail. For example, in the first aim he describes a logical set of experiments intended to determine whether MELK-expressing cells are TAP cells, and whether MELK null animals display decreased neurogenesis. The phenotype in the null mutant, however, could arise from an embryonic defect, which is not discussed, although the key experiment is noted as a possible follow-up to address embryonic compensation for MELK by other genes (GFAP-Cre CKO). He states that the experiments will test “self-renewal” of the MELK+ cells by transplanting them into the neonatal brain, yet he does not define “self-renewal” in the context of this cell population, which consists presumably of transient amplifying cells. In fact the experimental design, including BrdU labeling, does not allow lineage tracing, nor will the experimenters be able to ascertain that glial or neuronal progeny in vivo are derived clonally. Is self-renewal then defined as homing in on the SVZ? Or is it defined as simple proliferation? Or is it differentiation into multiple fates as some of his readout plans suggest (eg looking for granule neurons in the OB)? A question that is not addressed is when and where is MELK expressed in embryo? In the pitfalls section, the applicant discusses the difficulties of in utero transplantation, yet the transplantations he describes are in the neonate. Reviewers agree that transplantation in E14 would have been a better option, but he does not propose it in his research plan. Other troubleshooting suggestions are overarching, eg using ischemic models, enrichment, etc. in the setting of the MELK transgenics. In these studies, the PI will also make use of a transgenic mouse (which is presently being generated by the PI) expressing nuclear GFP driven by the nestin promoter. Use of the latter mouse is a clever and powerful approach (and used in a recent publication by another group) to assess the relationship of MELK to neural progenitors in the adult brain, and provides a much cleaner read-out. However, these mice are not currently available. In aim 2 the PI will test whether MELK is required for progression of a model glioma. The inactivation of the MELK/INK4/Arf locus results in multiple spontaneous tumors, including brain tumors, and his preliminary data demonstrate that his MELK-GFP reporter seems to reflect endogenous expression in tumors. However, since these brain tumors are rare, he has decided to transduce neural stem/progenitor cells from INK4/Arf locus null embryos with a retrovirus containing a constitutively-active mutation of epidermal growth factor receptor to increase frequency of tumors. The experiments in this section are fairly superficial and require selection in neurospheres, meaning that they lack immediate relevance to patient tumors and could be complicated by selecting cells in which MELK may be required independent of tumor progression. The tumor experiments do not take advantage of any biological finding or definition the applicant could have harnessed from his MELK experiments in the first aim and are even less hypothesis driven, beyond the obvious and the empirical. Reviewers were unconvinced that the present plan would yield a satisfactory and robust model for glioblastoma and that differences in tumor formation with and without MELK expression might not be adequately addressed. In the second part of the specific aim 2, the PI will test whether small molecule inhibitors of MELK catalytic activity inhibit growth of primary human glioblastomas. The PI notes that it is critically important to also test the effect of treatment of animals with brain tumors with MELK inhibitors, but writes that “these efforts, however, are clearly outside of the scope of the current proposal due to funding and time limitations,” and that “we have established a dialogue with Perry Scientific (San Diego), who specializes in these kinds of in vivo studies.” QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: This is a good applicant with solid training and a moderate publication record. The PI was a Ph.D. student at the University of Lausanne (1991-1997) and a post-doctoral fellow with Irv Weissman (1997-2001). From 2002—2006 he was an Assistant Professor at the Brain and Mind Institute, EPFL, Lausanne Switzerland, and since 2002 an Assistant Professor at the Burman Institute for Medical Research, La Jolla, CA, USA. For the past 10 years, the applicant’s research has been focused on the biology of somatic and embryonic stem (ES) cells. The applicant obtained his PhD 10 years ago, following which he was a postdoc in Irv Weissman's lab for 3 years. Those were his most productive years in terms of first paper authorship and focus. He has only been a collaborative middle level author since then. The applicant has two first author papers with Dr. Weissman and 3 first/senior authorship publications since 2002 in mid-level journals. The PI has published 15 papers (including chapters and reviews) on stem cells. The first authored or senior authored refereed publications were 1 in 2001, 1 in 2003, 2 in 2005, and 1 in press. This productivity seems somewhat modest for an individual in his 5th year of Assistant Professorship. His CV lists important papers in 2005 and in press concerning the role of MELK in regulating neural progenitor proliferation and MELK as a key regulator of the proliferation of malignant brain tumors. However, the applicant is neither the first nor last author on either of these papers, but appears as a middle author. This suggests that his collaborators from UCLA may have been responsible for some of the important ideas and data with respect to the involvement of MELK in TAPs and brain cancer - and one has some concern that they will continue to be the prime leaders in this field. There are letters from Dr. Kornblum (who notes that they have obtained RO1 and R21 joint funding and "are actively pursuing a number of projects together") and Dr. Geschwind at UCLA that fail to completely dispel this perception. It would have been valuable for the applicant to have addressed this issue more directly. The applicant will have a mentoring committee of 4 faculty that he will interact with individually on a weekly basis, quarterly as a group and annually will submit a written report on progress and plans. The applicant has funding from multiple sources covering a variety of subjects (neural crest derivation, mammary tumors, brain tumors, protein acetylation in ES cells, neural differentiation, aging) suggesting perhaps that the applicant's efforts are being diluted over a diffuse spectrum of activities. The PI has funding from CIRM that ends in 2007 for a project entitled “Analysis of Candidate Neural Crest Cells derived from Human ES Cells”. There is funding from NICHHD until 2008 for a project entitled “Protein Acetylation Signature of ES Cell Differentiation”. He is also co-PI on an NIH proposal entitled “Neural Progenitor Genes and Brain Tumors” and PI on a Keck Foundation grant. The largest portion of his effort in the previous year (35%) was dedicated to a CIRM grant addressing neural crest derivation from hES cells. Will this project play a role in his career development? A comment by the applicant about this would have helped alleviate an overwhelming impression of diminished focus and absence of scientific rigor conveyed by his research plan and perhaps reflected in his CV and career development plan. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant has adequate letters of support and institutional background. The laboratory is located in 1800 square feet of laboratory space at The Burnham Institute, where the applicant has been Assistant Professor since 2002. There are a number of shared facilities, including animal/transgenic, microscopy and imaging, proteomics, gene analysis, structural biology cores. There has been extensive recruitment in the stem cell field including Evan Snyder as Program Director of the Stem Cells and Regenerative Medicine program; three new faculty were recruited this year. The Burnham Institute is the recipient of a number of CIRM grants DISCUSSION: This application comes from a PI with a moderate publication record in hematopoetic stem cells who is proposing a number of straighforward experiments in neurons. The PI and the institution were good and the proposal was potentially interesting but poorly developed. The project studies an interesting kinase (MELK) and raises a provocative potential relationship to brain tumors, but the PI asks the wrong questions. Overall, the experimental design is weak and experiments are unfocused and not relevant to the stated questions. Panelists were not sure why PI looks at MELK in adult brain since embryonic progenitors also express MELK; they weren’t clear why the focus on brain tumors, since there doesn’t seem to be any indication that MELK is linked to brain tumors in humans. Reviewers commented that PI is studying a null phenotype in a mouse model but does not discuss the possibility that phenotypes could be due to embryonic defects. A further weakness is the reliance on neurospheres as a model, since MELK may be required independent of tumor progression in these cells. Reviewers were only moderately enthusiastic about the proposal and the applicant. They felt that the diffuse focus of the proposal is reflective of the CV and career path of the investigator, which is reflected in his average publication record.

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