Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

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Targeting mitochondria-lysosome crosstalk as a therapeutic strategy for glioblastoma multiforme
Translational
Brain Tumors (General)
Lay Summary

Glioblastoma is the most common brain tumor. It is aggressive, and the therapeutic strategies focused on surgery, radiotherapy and chemotherapy are unable to destroy the tumor, which typically reappears post-treatment. The regrowth happens because the treatments are not able to destroy the cells that originate the tumor, called glioblastoma stem cells. Patients have an average lifespan of 12-15 months after diagnosis. Recently, it was shown that glioblastoma stem cells have a particular metabolic signature: they rely on a signaling pathway that normally is used to respond to acute lack of cellular energy (ATP). This pathway is known as AMPK (AMP-activated protein kinase) signaling. Genetic ablation of AMPK in glioblastoma stem cells results in their death.

We propose to explore this vulnerability of the glioblastoma stem cells. Because the available AMPK inhibitors are non-specific, we will focus on a key protein that is regulated by AMPK. This strategy will be tested in glioblastoma stem cells, and is expected to result in their death. By targeting the stem cells, this approach is expected to yield the first therapeutic window to kill glioblastoma stem cells, and therefore solve the problem of tumor regrowth, and significantly increase the lifespan of patients.

We have shown that AMPK is an essential regulator of lysosomes, and we also found that the expression of lysosomal genes is increased in glioblastoma stem cells. The lysosomes, a cellular organelle that coordinates recycling of damaged components to regenerate building blocks for synthesis of new molecules. They also coordinate the cellular response to the amount of building blocks available. AMPK is required not only for the expression of lysosomal genes but also for lysosomal function. We have identified the protein that is directly activated by AMPK on the lysosomes, and how its product regulates a key channel for lysosomal function. In contrast to the AMPK inhibitors, the inhibitors for these two lysosomal proteins are highly selective, and therefore testable as a therapeutic strategy.

AMPK occurs in pools associated with different cellular compartments, including lysosomes, mitochondria (the cellular “power plants”), nucleus (where the DNA is located, and where the decision of which genes are made into proteins) and others. Most studies address AMPK as a single entity, which dilutes the contributions of the different pools. We found that inhibition of the lysosomal AMPK pool is sufficient to perturb lysosomal function and promote cell death. We will now test the approach of blocking lysosomal function in glioblastoma stem cells by shutting down the lysosomal AMPK pool, which is expected to result in death of glioblastoma stem cells.

Another function of AMPK in glioblastoma stem cells is the division (fission) of mitochondria. Mitochondria can present as long filaments or fragmented into smaller structures. The longer filaments are more efficient in cellular energy production, while the fragmented mitochondria are less efficient. The fragmentation is promoted by AMPK. Recent evidence shows that the lysosomes have a key role in activating the mitochondrial AMPK pool to promote mitochondrial fragmentation. Glioblastoma stem cells present fragmented low-efficiency mitochondria, and use other faster energy-producing pathways to fuel their growth. We will test how blocking the process of mitochondrial fragmentation impairs the survival of glioblastoma stem cells.

This project is entirely focused on glioblastoma stem cells, because their destruction is essential to avoid tumor regrowth and successfully treat glioblastoma. Here, we propose three strategies, focused on one pathway, AMPK signaling, that glioblastoma stem cells have excessively activated. By tackling different branches of AMPK signaling, we will test three different strategies to kill glioblastoma stem cells, using highly selective compounds that can rapidly be translated for clinical practice.

Executive Summary

This proposal tackles a fundamental problem in glioblastoma treatment: how to kill the glioblastoma stem cells (GSC), to avoid tumor regrowth after chemotherapy. We propose to explore a metabolic vulnerability of these cells.

Both glioblastomas and GSC are dependent on a key signaling mediator, AMP-activated protein kinase (AMPK). We have shown that AMPK is essential for basal and induced lysosomal function (Fernández-Mosquera et al., 2017; Fernandez-Mosquera et al., 2019), and we have observed that GBM and GSC have increased expression of lysosomal genes (unpublished data). Specifically, we have shown that AMPK is necessary for the activity of the enzyme PIKfyve, which is the only source for the signaling lipid PI(3,5)P2 in the lysosomal membrane (Fernandez-Mosquera et al., 2019). PI(3,5)P2 regulates the activity of many lysosomal membrane proteins, and in particular of the lysosomal cation export channel MCOLN1 (Fernandez-Mosquera et al., 2019). By specifically targeting the lysosomal AMPK branch in GSC, via specific inhibitors of PI(3,5)P2 and MCOLN1, we aim to ablate the competitive advantage that excessive lysosomal activity confers to GSC, and reduce their ability to proliferate and survive. Furthermore, using tools and methodology developed in my lab (Fernández-Mosquera et al., 2017; Fernandez-Mosquera et al., 2019; Fonseca et al., 2019; Yambire et al., 2019a; Yambire et al., 2019b), we will assess which other downstream effectors of AMPK signaling are required, alone or in combination with the AMPK-lysosome branch, to trigger GSC death. Our approach proposes a novel therapeutic strategy to kills the GSC, an essential step to effectively ablate the tumor, avoid recurrence and increase patient lifespan. Genetic ablation of AMPK signaling resulted in death of primary glioblastoma stem cells and inability to grow tumors in nude mice. By tackling three different aspects of AMPK signaling with highly selective pharmacologic agents, we will unveil the best strategies to kill glioblastoma stem cells:

1) Blocking PIKfyve will decrease the amount of PI(3,5)P2 in the lysosome, and therefore decrease the function of many lysosomal membrane proteins whose activity requires PI(3,5)P2. One of those proteins is the lysosomal cation export channel MCOLN1. We propose to inhibit PIKfyve and/or MCOLN1 using inhibitors with high specificity. We expect that ablation of PIKfyve signaling has a detrimental effect on the GSCs, resulting in their death, and that inhibition of MCOLN1 phenocopies these effects. This strategy will establish how to kill glioblastoma stem cells using two molecular targets (PIKfyve, MCOLN1) with highly-selective, readily translatable inhibitors.

2) There are several intracellular AMPK pools, and the lysosomal AMPK pool is pivotal in the regulation of the lysosomal activity. We expect that by perturbing the activity of lysosomal AMPK, without interfering with the others (mitochondrial, cytoplasmic, nuclear), we will specifically target the dependency of the lysosome on AMPK activity. This approach will allow the definition of therapies to kill glioblastoma stem cells specifically by targeting the lysosomal AMPK pool.

3) AMPK regulates many aspects of cellular signaling, including lysosomal function and biogenesis, as discussed, but also mitochondrial biogenesis and mitochondrial fission. Brain tumor-initiating cells have fragmented mitochondria, and inhibition of mitochondrial fission triggers these cells to die. Both lysosomal activity and AMPK signaling promote mitochondrial fission in glioblastoma stem cells. We will tackle this pathway by blocking mitochondrial fission with highly selective inhibitors of the process, and determine how it impacts the death of glioblastoma stem cells. This strategy will allow us to define how the mitochondrial AMPK pool can be harnessed to kill glioblastoma stem cells.

All reagents and cells are available in the lab. I have worked in AMPK for over 10 years (Fernández-Mosquera et al., 2017; Fernandez-Mosquera et al., 2019; McKay et al., 2015; Raimundo et al., 2012; Zhao et al., 2020), and additional unpublished data, both in basic mechanistic aspects as well as its involvement in pathology. My doctoral studies were focused background on tumor metabolism (Raimundo et al., 2008; Raimundo et al., 2011; Raimundo et al., 2009; Tyynismaa et al., 2010). My lab was created in 2014, in Germany, with elite funding from the European Research Council, the most prestigious and competitive cross-european funding agency. The focus of my lab has been the communication between mitochondria and lysosomes, and its role in physiology and pathology. AMPK is a key component of mitochondria-lysosomal communication, as well as of mitochondrial and lysosomal function and homeostasis (Deus et al., 2020). Therefore, my lab has the expertise and tools to tackle this very feasible project here proposed. The experimental part will be carried out by a senior postdoctoral scientist that I recently recruited, and whose doctoral thesis was focused on the metabolism of astrocytes and oligodendrocytes. Thus, his expertise and training is very appropriate for the project here described. All the methods required for this project are established in my lab, as documented by several publications in the last five years. Importantly, I have been working with stem cells for several years, with the help of a close collaborator (as documented in (Simão et al., 2018)). The primary glioblastoma stem cells for this project were a kind gift of our collaborator Dr. Dr. Biplab Dasgupta (Cincinatti Children’s Hospital).

My lab is currently relocating to Penn State University College of Medicine (PSU-CM), where I will take the position of Associate Professor at the Department of Cellular and Molecular Physiology, staring September 01, 2020. This move is supported by a generous start-up package that allowed me to equip the laboratory with top-line features for cell culture, including stem cells, cellular biology (including confocal microscopy in live and fixed cells, electron microscopy), biochemistry, molecular biology (including cloning), cell viability and assessment of mitochondrial and lysosomal functions (including physiological assays), morphology and dynamics, as well as assessment of signaling pathways using western blotting, immunohistochemistry or mass spectrometry, and quantitative real-time PCR. Furthermore, there is a rich environment in cancer research at PSU-CM, and through our local collaborator Dr. Brad Zacharia, a specialist in brain tumors, we have access to patient samples for further validation of results.

Description of Research Proposal

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Budget

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.

Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.

Morbi orci urna, ornare non pretium eget, pulvinar eget magna. Ut consectetur efficitur varius. Fusce ac aliquet mauris, at mattis ligula. Quisque est libero, interdum id orci et, ornare luctus diam. Proin commodo lectus id accumsan blandit. Nulla eu turpis interdum, luctus ante ac, imperdiet tellus. In semper enim eu tristique aliquam.

Collaborations and Conflicts of Interest

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Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.