Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

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Targeting hypoxia and mitochondrial metabolism with repurposing drugs as an approach of radiosensitization for diffuse intrinsic pontine gliomas
Translational
DIPG, Childhood (Brain Cancer)
Lay Summary

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable brain tumor that arises in the brainstem of children predominantly between the ages of 6 and 9.  Unlike many brain tumors, DIPGs cannot be removed through surgery due to its sensitive location. The standard of care today remains radiation therapy (RT) alone. Unfortunately, almost all DIPGs recur locally within 12 months secondary to radioresistance. Therefore, it is important to understand the mechanisms of radioresistance, as this may be used to improve the radiosensitivity and offer a pathway to the development of novel therapies for this deadly brain tumor. Hypoxia, a condition in which the body is deprived of adequate oxygen supply at the tissue level, is a common microenvironmental feature of all solid tumors, playing a vital role in radioresistance. Recent reports showed that DIPGs are exposed to a hypoxic microenvironment, suggesting targeting of hypoxia may be effective to improve their radiosensitivity. My preliminary data have shown that the radiosensitivity of DIPG cells was significantly improved when treated with biguanide, a class of diabetes drug that can reduce hypoxia, and this radiosensitising effect was further improved when a second drug was combined to further modulate sugar metabolism. These findings could potentially form the basis for pharmaceutically targeting hypoxia and tumor metabolism as a new radiosensitising treatment for incurable DIPG.

Lay summary

Children currently diagnosed with DIPG have no hope of cure and are offered palliative treatment only.  RT is the only effective treatment for DIPG to date although it only provides relief of tumor-related symptoms in roughly 70% patients. However, all DIPGs recur locally secondary to radioresistance, thus improving the effect of RT remains the most promising avenue to better outcomes in DIPG patients.  Cells under hypoxia, a condition where the tissues do not have enough oxygen supply, are 2-3 times more resistant to RT than cells that are well oxygenated at the time of irradiation [1]. A recent study reported that DIPG cells are exposed to a hypoxic microenvironment, suggesting these cells are intrinsically resistant to RT [2]. These findings also suggest that targeting hypoxia may be an effective strategy to overcome the radioresistance of DIPG cells. Biguanides (metformin/phenformin) are a class of drugs that are currently used in clinic for the treatment of Type II diabetes. Apart from lowering blood sugar level, biguanides can also reduce the oxygen consumption of cells by targeting mitochondria, the rod-shaped organelles that can be considered the power generators of cells. By lowering tumor demand for oxygen, the hypoxic condition can thus be lessened.  In our pilot studies, we have observed that metformin largely improved the efficacy of RT in a mouse model carrying DIPG cells in their brains. This exciting result led us to further optimize this strategy such that a better efficacy can be achieved. Given metformin is reliant on certain transporters to enter tumor cells [3], we proposed to use a similar drug phenformin as it does not rely on those transporters to enter cells [4], thereby allowing higher drug concentration in tumor cells. Strikingly, phenformin is ~30 times stronger than metformin to slow the growth of DIPG cells. Phenformin reduced oxygen consumption rate which in turn increased lactic acid production. Given high level of lactic acid is the primary adverse effect of phenformin and strongly correlates with poor clinical outcome, a strategy is thus needed to counteract this unfavorable side effect. Dichloroacetate (DCA), a compound that lowers blood lactic acid levels, was selected to offset this side effect, not only because it is an orphan drug to treat high level of lactic acid, it is also well tolerated by children [5]. When DCA was combined with phenformin, the high level of lactic acid production was largely blocked. Surprisingly, the combination also led to a much stronger cell killing effect by depriving tumor cells of energy supply and damaging their DNA. Moreover, the combination also reduced the level of two master regulators that collaboratively enhance the cancer cell growth/survival and metabolic needs through increased uptake of sugar and contribute significantly to radioresistance. When RT was combined with phenformin and DCA, the most effective activity was observed, with the triple combination leading to the lowest number of surviving DIPG cells. More importantly, both phenformin and DCA can readily cross blood-brain-barrier (BBB), have long been used in clinics, and are very well tolerated by young children. Therefore, they are the drugs with the most amount of testing and closest to being tested in clinical trials. Given the results generated from this project will be rapidly translated to clinic and benefit children with DIPG, the anticipated impact of this research project is of great significance: it may lead to a change in treatment regimen resulting in longer survival rates for the pediatric patients with newly diagnosed DIPG.

Executive Summary

Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating, aggressive childhood brain tumor arising in the ventral pons, comprising approximately 10-15% of pediatric brain tumors [6]. Half of all malignant pediatric gliomas occur in the brainstem, with DIPG being the most common tumor subtype in this anatomical region, constituting 80% of brainstem gliomas overall [7]. With an estimated 200-300 children affected by DIPG annually in the United States and 20-30 children in Australia, it is the second most common malignant brain tumor of childhood. In the absence of effective therapies, the prognosis of DIPG is bleak. The median age at diagnosis is 6-9 years, with median survival of 9 months; 90% of children will die from the disease within 2 years of initial diagnosis, with less than 1% surviving after 5 years [8]. To date, RT is the only form of treatment that offers a transient benefit in DIPG. Unfortunately, almost all DIPGs recur locally within 12 months secondary to radioresistance. Therefore, improving the effect of RT remains the most promising avenue to better outcomes in children with DIPGs.

Hypoxia is a common microenvironmental feature of solid tumors [9] that exists because the supply of oxygen is insufficient to meet the metabolic demand of the tumor [10]. Recent reports showed that DIPGs are hypoperfused compared to surrounding brain tissue, suggesting that the tumor cells are exposed to a hypoxic microenvironment [2]. This stimulus may induce widespread transcriptional changes through activation of hypoxia inducible factor (HIF), which have been associated with invasion, metastasis, angiogenesis, and resistance to therapeutics. Hypoxia has been recognized as a barrier to effective RT long time ago [11], because molecular oxygen (O2) is an electrophile that enhances the efficacy of RT by fixing radiation-induced DNA damage. It has also been reported that drugs that target metabolic diseases are clinically important in the treatment of cancer. Biguanide (metformin/phenformin) is a class of hypoglycemic agent currently used in clinic that can also modify tumor mitochondrial metabolism to reduce oxygen consumption rate. By targeting mitochondrial complex I, biguanide reduces oxygen consumption rate of tumor cells, which in turn alleviates the hypoxic condition. Our preliminary data have shown that metformin significantly improve the radiosensitivity of DIPGs to extend the median survival of an orthotopic model bearing patient-derived DIPG cells. Strikingly, phenformin, another biguanide derivative, demonstrated a much more potent anti-DIPG activity which reduced the half maximal inhibitory concentration (IC50) up to 30 times compared to metformin. To minimize lactic acidosis induced by phenformin, a drug combination was developed by combining phenformin with DCA. Not only did this unique combination significantly alleviate phenformin’s glycolytic effect, it also augmented cell-killing effect via a mechanism of inducing energy crisis, apoptosis and DNA double-strand breaks. Moreover, molecular signaling analysis revealed that this combination effectively inhibited HIF-1 and c-Myc, two master regulators that collaboratively enhance the cancer cell growth/survival and metabolic needs through increased uptake of sugar and contribute significantly to radioresistance. Additionally, a significant upregulation of TKT, a gene encoding the key enzyme transketolase that controls pentose phosphate pathway (PPP), was also observed in treatment resistant clones, suggesting the inhibition of PPP may also be needed when acquired resistance is developed. These findings indicate that targeting hypoxia and mitochondrial metabolism with repurposing drugs represents a novel radiosensitising approach for incurable DIPG.

Overall, we have strong preliminary data suggesting that targeting hypoxia and mitochondrial metabolism potently improve the radiosensitivity of DIPG cells, thus providing an innovative therapeutic opportunity for the treatment of DIPG. Children currently diagnosed with DIPG have no hope of cure and are offered palliative treatment only. Both phenformin and DCA are generic drugs that are clinically available and have been well characterised as treatments for Type II diabetes and congenital mitochondrial deficiency, respectively. Compared to other novel targeted anti-neoplastic agents, these repurposed drugs will significantly reduce the cost of treatment and shorten the time of translating positive results generated from our proposed project to clinical testing. Importantly, as a potential treatment for brain cancer, both phenformin and DCA have been shown to readily cross the BBB via oral administration [12, 13]. We will perform the first comprehensive analysis to assess efficacy of this novel treatment, identify potential metabolic signatures, optimize the timing of sequential treatment, and explore resistant mechanism for developing salvage therapy using in vitro and in vivo DIPG models. In doing so, we aim to develop the quantum of preclinical data required for personalized treatment and rapidly translate this novel therapy to the clinic.

Our team has all the necessary expertise that will ensure the success of the proposed project and ultimately the implementation of the discoveries into the clinic. In collaboration with Prof Michelle Monje (Stanford University, USA) and Dr Ángel Montero Carcaboso (Hospital Sant Joan de Déuin, Spain), we have 10 patient-derived DIPG cells cultured in our lab, consisting of tumors that were established from biopsies at diagnosis and those from early post-mortem autopsies. Using this panel of DIPG isolates allows us to identify the capability of our proposed treatment to overcome the intrinsic (biopsies at diagnosis) and acquired (early post-mortem autopsies) radioresistance of DIPGs both in vitro and in vivo. Dr Han Shen (Westmead Institute for Medical Research) is an early career post-doctoral researcher with particular expertise in DIPG cell culture and xenograft models, cancer cell biology, drug discovery and radiation oncology. Dr Shen will perform most of the experiments proposed in this application. Dr Raluca Maltesen (Aalborg University Hospital, Denmark) is a metabolomics and data mining scientist specialized with 7 years’ experience in systematic biology and multivariate data analysis. She will provide the guidance and analyze the data from metabolomics studies. Dr Eric Hau (Westmead Institute for Medical Research, Crown Princess Mary Cancer Centre Westmead Hospital) is a clinician-scientist and leader in Radiation Oncology with 10 years’ experience in treating patients with high-grade brain tumors. Dr Hau will supervise the progression of whole project and facilitate the translation of our laboratory findings from this project into clinical practice with the already established collaborations at Westmead Hospital and the adjacent Westmead Children’s Hospital. We will also be collaborating with Sydney Informatics Hub based at University of Sydney for analysis of single cell transcriptomics data.  The support from Dr Raluca Maltesen, Dr Eric Hau, Sydney Informatics Hub, Sydney West Radiation Oncology Network, and Westmead Institute for Medical Research provides an invaluable environment to ensure the success of this novel research project.

The total grant amount of USD $100,000 is requested for this proposed project.

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.