Grants & Publications

Funded Research by Brains Together and Our Community

2018

Development of combinatorial EGFRviii/lL13Ra2 directed Chimeric Antigen Receptor T-cell therapy in Glioblastoma

Abstract

A new form of therapy to treat cancer has emerged called Chimeric Antigen Receptor T-cell therapy, or CAR-T. CAR-T uses the patient’s own white blood cells which are then genetically modified to attach an antibody to the T-cell which is specific to a tumor marker. The antibody is coupled to a signaling cascade within the T-cell to activate it and engage the immune response. This creates, in essence, a living drug that can survive in a patient to prevent disease recurrence. The first FDA approval of this therapy was in 2017, approved for the treatment of lymphoma and a type of leukemia. Additionally, CAR-T technology has been demonstrated to have a dramatic effect against glioblastoma targeting a tumor specific marker, IL-13Ra2. Another recent case series of patients with glioblastoma treated with a CAR-T targeting a different tumor cell marker (EGFRviii) was recently published and seems to reinforce that there is efficacy against glioblastoma with this method of treatment. A major barrier with previous targeted treatment in glioblastoma has been the diversity of the tumor cell population. For example, not all tumor cells in a patient’s tumor will express EGFRviii or IL-13Ra2 and therefore CAR-T targeting one of the two targets leads to escape of those cells that do not express the CAR-T target. Our group’s novel approach is to combine these two previously published (and safe in human) targets for CAR-T (IL 13Ra2 and EGFRviii) to create a dual antigen CAR so as to increase the targeting efficiency of this mode of therapy. We are ready to begin evaluation of this dual construct against tumor cells that have either target on them. We are preparing to demonstrate that our construct can correctly recognize and kill glioblastoma cells with either marker; specifically, we will look to see if the use of our construct prolongs the survival of mice implanted with glioblastoma cell lines, prior to initiating a phase 1 clinical trial in human beings. We hypothesize that a dual antigen CAR such as ours will be more efficacious than prior CAR-T cells in glioblastoma and have minimal systemic toxicity.

2018

Disrupting glioblastoma-mediated immunosuppression by blocking extracellular vesicles

Abstract

Glioblastoma (GBM) is the most common primary malignant brain tumor. Average survival remains 14-15 months despite surgery, radiation, and chemotherapy. lmmunotherapy (stimulating the immune system to attack the tumor) has been promising but is limited by GBM’s ability to shut down the immune system. Like many cancers, GBM’s release small cell particles called extracellular vesicles (EV’s). Preliminary data suggests that GBM EV’s are an important mechanism these tumors use to suppress immune responses. In this project, we will test this in detail using a series of GBM models that have been genetically engineered to increase or decrease expression of a protein (PD-L 1) that inhibits the immune system. In addition, we will test whether the commonly administered blood thinner heparin can block the uptake of GBM EV’s by white blood cells and reverse some of the immune suppression caused by these tumors. Heparin administration could easily be combined with existing GBM immunotherapies including those targeting PD-L 1, improving their success rate. Ultimately, we expect that this research will lead to new therapeutic strategies that prolong survival for patients with this devastating disease.

2017

Glioblastoma-secreted extra cellular vesicles induce novel immunosuppressive non-classical monocyte in vitro, and contain microRNA signatures that correlate with glioblastoma subtype

Abstract

Glioblastoma is the most common malignant tumor derived from brain tissue, and remains nearly universally fatal within two years despite aggressive treatment. A receptor on glioblastoma-secreted extra-cellular vesicles called programmed death ligand-1 (PDLl) prevents white blood cells from destroying them, and converts white blood cells throughout the body into immunosuppressive cell types.

In this grant, we propose to monitor both the vesicles and the immunosuppressive monocytes, as our data suggests this may help improve current diagnostic tools and judge the effectiveness of treatments. Ultimately, this should open up the path to screen people for glioblastoma based on a blood sample.

Finally, in an ongoing effort to describe and interfere with the way GBM uses these vesicles to communicate and control surrounding cells, we aim to create new cell lines where we take out key genes and study the effect on EV production and adherence. Undermining GBMs at this fundamental level may lead to novel drugs that can block GBM EVs in patients.

2017

Elucidating Innate Immune contributors to radiation-induced cognitive impairment

Abstract

Brain radiation is essential for the treatment of brain tumors but interferes with cognitive functions such as learning, memory, attention and mood. Certain new cells must be generated in the brain throughout life for it to function normally: new neurons in the hippocampus enhance learning and memory, while other continually generated cells help maintain the insulation around neurons, which is important for attention, multitasking, and complex processing. Additionally, neurons must have appropriate numbers of the right kind of synapses in order to transmit and receive information. All of these processes are damaged by brain radiation, and there is evidence that inflammatory cells regulate this injury. Certain inflammatory cells normally live in the healthy brain and are called microglia, while others, “bone marrow-derived monocytes (BMMs)” only enter the brain after injury. How these different cell types may help or harm the brain after radiation is controversial. In this proposal, we will evaluate the relative impacts of microglia and BMMs, alone and in combination, on the radiated brain. To do this, we will use an experimental system that allows mouse brain cells to be grown in a dish, whilst maintaining their normal connections and functions. By adding and subtracting microglia and/or BMMs, we will determine which cause or prevent injury after radiation. Since human patients who undergo brain surgery have sometimes previously received brain radiation, we will also be able to validate our findings, learning how cells from the human brain function and interact after radiation injury. Knowledge from these experiments will allow new treatments to be developed that help patients think more clearly and enjoy life more fully after treatment for brain tumors.

2016

Investigating Immune-independent growth effects of PDL1 on Glioblastoma multiforme, using RNA sequencing and mouse in-vivo glioma models

Abstract

Glioblastoma is the most common malignant tumor derived from brain tissue, and remains nearly universally fatal within two years despite aggressive treatment. A receptor on glioblastoma cells called programmed death ligand-1 (POL 1) prevents white blood cells from destroying them, and converts white blood cells throughout the body into immunosuppressive cell types.

Antibodies against POL 1 hold therapeutic promise, but may not be able to get into the brain due to the blood-brain barrier. We will implant tumors in either mice flanks or brains and periodically inject POL 1 antibodies / POL 1 proteins to see if either can cross the blood-brain barrier. This will help us understand how important POL 1 is in terms of survival. We will also compare the effects the immune system has on survival by comparing mice with functional and dysfunctional immune systems.

We discovered that POL 1 also stimulates tumor growth independently of the immune system. In this grant, we propose to analyze the genome of glioblastomas in which we have artificially increased and decreased POL 1 expression. This will help us understand what genes and pathways are activated or deactivated in order for POL 1 to stimulate GBM growth. Understanding how POL 1 achieves this effect will likely give us new and exiting treatment options.

2016

Targeting Glutamine Metabolism in Pediatric High Grade Gliomas with the H3K27M Mutation

Abstract

Glioblastoma, the most common type of primary brain tumor, is unfortunately the most severe type and affects~ 15,000 people each year. They are highly aggressive and infiltrative and the median survival after diagnosis is only 14 months despite maximal therapy which includes surgical resection, radiation and chemotherapy. Despite the recent advances in medicine that include a better understanding of cancer mechanisms, the overall life expectancy has not changed much for several decades. It is for these reasons researchers are now turning their attention toward pathways that are implicated in malignant glioma formation and progression.

There is mounting evidence that STAT3, a key protein that promotes tumor activation, growth and survival is playing an active role in malignant glioma tumor formation. We hypothesize that a small molecule can be developed through a rational drug discovery process, that block STAT3’s ability to activate malignant gliomas-thus stopping tumor activation and growth. This project can be broken down into three distinct phases. First, using computer aided modeling, we will design drug structures that block the key step in STAT3 activation. Next, these compounds will be synthesized using standard organic chemistry techniques and characterized fully. We will test these compounds ability to inhibit STAT3s activity in test tubes. Finally, we will determine how effective these compounds are in malignant glioma cells and animal models of glioblastoma. The ultimate long-term goal is translational, including the development of a novel anticancer drug and a pilot clinical trial to determine how well patients respond.

2015

Combined Therapy for Glioblastoma Multiforme

Continued funding 2nd year

Abstract

In a pilot project supported by BTFC, we determined that our vaccination approach used in conjunction with anti-angiogenic therapy had a synergistic treatment effect against glioma in our model system. Coinciding with this effect was an increase in tumor specific CD8 T cells. In this new project, we will determine if the combined therapy is unique to picornavirus vaccination, or if this approach is translatable to immunotherapies at large. In addition, we will determine the extent anti-angiogenic therapy contributes to enhance T cell responses against tumor. The results of this study will help guide immunotherapy approaches and determine if Bevacizumab can be used in conjunction with vaccines in the clinic.

2015

Reversing the Tides of Glioma Induced lmmunosuppression

Abstract

Glioblastoma (GBM) is a devastating disease with median survivals ranging from 12 to 15 months with optimal current therapies. lmmunotherapies have emerged as a potentially effective strategy for treatment of GBM with minimal morbidity. Pre_-clinical animal models have shown impressive results, while human clinical trials have only shown modest increases in survival. Patients with GBM have reductions in their total T-cells and an increase in the number of circulating myeloid-derived suppressor cells (MDSC), leaving them with an immunosuppressive phenotype similar to patients with human immune deficiency virus (HIV). This effectively results in patients’ inability to fight off the tumor with their own immune system. Our lab has focused on the mechanisms responsible for MDSC development using in vivo mouse models of GBM, as well as by using in vitro co-culture experiments with normal human monocytes and primary human GBM cell lines derived from surgical samples obtained at Mayo Clinic. In the work funded by Brains Together for a Cure, we have demonstrated that two factors expressed by tumor cells, STAT-3 and B7-H1 (PDL1), appear to be critical for the formation of MDSCs from normal white blood cells exposed to human GBM cells in culture. Furthermore, we have shown in culture that curcumin, a natural compound derived from the spice turmeric that has anti-STAT-3 properties, inhibits the human GBM-mediated development of MDSCs and reverses their ability to suppress T cells. Our laboratory is now proceeding with studies in mouse glioma models to further characterize the role of STAT-3 and B7-H1 in MDSC development and determine the effectiveness of curcumin in reversing GBM-mediated immunosuppression.

2014

Modeling the effects of STAT3 and B7-Hl on Myeloid derived suppressor cells {MDSC} mediated immunosuppression in a human in-vitro and mouse in-vivo glioma model

Abstract

Modeling the effects of STAT3 and B7-H1 on Myeloid Derived Suppressor Cell (MDSC) mediated immunosuppression in a human in-vitro and mouse in-vivo glioma model.

Glioblastoma (GBM) is a devastating disease with median survivals ranging from 12 to 15 months with optimal current therapies. lmmunotherapies have emerged as a potentially effective strategy for treatment of GBM with minimal morbidity. Pre-clinical animal models have shown impressive results, while human clinical trials have only shown modest increases in survival. A Patients with GBM have reductions in their total T-cells and an increase in the number of circulating myeloid-derived suppressor cells (MDSC), leaving them with an immunosuppressive phenotype similar to patients with human immune deficiency virus (HIV). This effectively results in the patients inability to fight off the tumor with their own immune system. Our lab has focused on the mechanisms responsible for MDSC development using in vivo mouse models of GBM, as well as by using in vitro co-culture experiments with normal human monocytes and primary human GBM cell lines derived from surgical samples obtained at Mayo Clinic. We have developed an animal model that mimics the MDSC mediated immunosuppression seen in human GBM patients. This allows us to trial strategies that may result in more effective immunotherapies in humans. STAT3 and B7-H1 proteins have received attention as two potentially import factors in glioma mediated immunosuppression. STAT3 is a cytoplasmic signaling molecule that results in the release of immunosuppressive cytokines. B7-H1 is a cell surface ligand present on both glioma cells and MDSCs that influences MDSC formation and Teel! anergy. We plan to knockout STAT3 and B7-H1 in mouse and human glioma cell lines and evaluate their effect on glioma immunosuppression using an in-vitro model and our mouse MDSC model.

2014

Combined therapy for glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is among the most lethal of cancers, with an average survival of 12-15 months post diagnosis. Complete surgical resection of the tumor is not feasible in the vast majority of cases. Current treatment entails radiation plus temozolamide and anti-angiogenesis treatment with a drug called Bevacizumab. Vaccines to promote an immune response against GBM have shown some promise in clinical trials. Therefore, a continued effort to develop vaccines for GBM will continue to be a high priority. In work performed in my laboratory, we have determined that mouse GBM behaves similarly to human GBM with antiangiogenesis treatment. We have also developed a new vaccine approach to mouse GBM. In this pilot experiment, we are going to determine if both anti-angiogenic and vaccination can be used together in a combined therapy. This will be important to provide a rational approach to clinical trials with GBM patients who will likely benefit from both forms of therapy. Also, through the experiments in this pilot project, we will determine the extent anti-angiogenic therapy alters the immune response to mouse GBM.

2013

An evaluation of the blood brain barrier penetration of PARP inhibitors with a dual MDRl/BCRP expressing in vitro model

Abstract

The blood brain barrier is an interface between the brain and the bloodstream that is well known for its ability to limit the entry of chemotherapy drugs into both the normal brain and even into portions of brain tumors. Drugs that are unable to cross this barrier cannot be used to treat brain tumors. So, the ability to find out if a drug can cross this barrier would be very useful. One of the most important ways that this barrier prevents drug entry is through the activity of certain proteins that actively remove chemotherapy drugs from brain tumors by pumping them back into the bloodstream. The focus of this work is to develop and test a model that can predict which drugs will be excluded from the brain by these pumps. This will allow an easy prediction of which chemotherapy drugs penetrate into brain tumors and are more likely to be effective in people. In this way, this model will speed up the process of finding effective drugs against brain tumors.

2013

Impact of Apolipoprotein E Genotype on baseline neurocognition and radiation-induced neurocognitive dysfunction in patients with brain tumors

Abstract

Radiation is the cornerstone of treatment for patients with brain tumors, yet there are concerns regarding toxicity. An especially significant fear of radiation to the brain is the potential decline in cognitive abilities. However the negative impact of radiation on cognitive function is unpredictable with some patients showing no decline in function while other patients who received the same dose of radiation deteriorate into dementia. The unpredictability of radiation toxicity suggests different genetic sensitivities with some patients more resilient to the effects of radiation while others are much more sensitive. One possible predictive genetic marker is Apolipoprotein E (ApoE). Certain ApoE genetic types are predictive of Alzheimer’s dementia, and this is important since Alzheimer’s dementia closely resembles radiation-induced dementia. Therefore we plan to perform analyses of ApoE on blood samples of patients with brain tumors treated with radiation in two clinical trials. Identiying genes that predict radiation induced neurocognitive decline would have a significant impact on patient care. Knowledge of these genes would assist considerably in treatment decisions (e.g. whether to treat with radiation). Additionally this knowledge could lead to the development of new treatments that could prevent the development of radiation cognitive toxicity.

2012

Autophagy inhibition for treatment of childhood medulloblastoma

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor of children. Although current therapies have improved the overall outlook for patients, there are still a significant number of refractory and metastatic cases of MB, which not infrequently are fatal. In addition, survivors have increased rates of longterm toxicities from radiation, surgery, or chemotherapy that impact their quality of life. Clearly, improved treatments are urgently needed for this tumor type. A new type of therapy termed “metronomic” therapy is designed to attack the blood supply to the tumor cells. This could improve the response of the tumor, because the blood vessels do not typically develop resistance to chemotherapy drugs. However, although recent clinical tests show some promising results, it is also being discovered that cancer cells can partially avoid being killed by metronomic therapy by managing to get nutrients in other ways than directly from the blood supply. One way is to begin to digest their own cellular components, for recycling and re-use. This process (called “autophagy”) may allow the tumor cells to survive long enough to resist metronomic therapy. We want to test whether inhibiting autophagy with chloroquine (a well known drug that is used to treat or prevent malaria)’ could improve the response of MB to metronomic therapy. We propose to test the combination of chloroquine with metronomic chemotherapy in mice to see if it is safe to give, and to determine whether it would improve the treatment of MB tumors. If this work is successful, it could ultimately lead to a new phase I trial in children with MB, combining chloroquine with currently used metronomic drugs, in order to improve rates of tumor response in humans.

2012

The Rational Design, Synthesis and Evaluation of Novel STAT3 Inhibitors for Treatment of Malignant Gliomas

Continuous Funding 2nd Year

Abstract

Our results show we have developed novel small-molecule STAT3 inhibitors using a rational structure-based design. We have evaluated the effects of our compounds in four separate tumor cell lines including two glioblastoma, medulloblastoma and human colorectal carcinoma tumor cells. We found our compounds are potent inhibitors of STAT3 phosphorylation in a dose-dependent manner. Our inhibitors produce both an antiproliferative and proapoptotic effect by downregulating downstream targets of ST AT3 signaling including Cyclin D1 and Bcl-2. More importantly, we found potent inhibition of tumor cell proliferation in glioblastoma, medulloblastoma and human colorectal carcinoma tumor cells with our compounds. We also showed that cell death occurs in an apoptotic manner.

Based on these findings, our pyrazole-based compounds should be suitable lead structures for targeting cancer cells that use STAT3 signaling for tumorigenesis owing to their ability to inhibit STAT3 phosphorylation and potently block tumor cell proliferation. Furthermore, our compounds therapeutic potential are greatly increased over available STAT3 inhibitors because the core pyrazole structure is based on a FDA approved drug and our drugs will likely retain similar pharmacokinetic and toxicity properties. Additionally, many other pathological processes may use STAT signaling, thus our compounds may have extended application outside of oncology, including use in drug eluting stents, and in treating polycystic kidney disease.

This work has led to a patent application surrounding these novel structures and a manuscript is also in the works. However, much more work needs to be done. We need to further characterize the properties of our current compounds. A critical step is to prove that our compounds are direct STAT3 inhibitors and not just upstream or downstream inhibitors. This will be done by using a cell-free fluorescence binding assay. We will also start preliminary in vivo work and address pharmacokinetic aspects of our drugs. Based on these results, we feel we _can further optimize our current compounds and select a suitable drug candidate for pre-clinical studies. We believe we can develop a potent STAT3 inhibitor that ultimately will show promise as a novel chemotherapeutic agent and will help alleviate some of the suffering of patients with malignant brain tumors.

2011

Utility of F-FDOPA-PET for neurosurgical planning and radiotherapy target delineation in glioma patients: Biopsy validation of F-FDOPA-PET uptake and biodistribution in brain tumors

Continuous Funding 2nd Year

Abstract

Seeking additional funding: A primary goal of our research is to identify and incorporate imaging techniques which will allow us to more accurately determine where the most aggressive disease is located. This means we can personalize patient care by targeting these specific regions and possibly spare more healthy tissue if we do not have,to use the large margins required with conventional imaging where we know there is diseased tissue beyond what we’re able to see.

Our initial research has focused on first validating that 18F-FDOPA-PET imaging correlates with pathology, and our preliminary results have clearly demonstrated this imaging technique is able to show regions of aggressive disease not seen with conventional imaging. Based on these results we believe radiation therapy target volumes defined per conventional imaging are under-treating regions of highly aggressive disease. While we continue to accrue patients for the initial biopsy study funded by the BTFC grant, we are seeking additional funding to pilot the next phase of our research. This next phase will involve incorporating 18F-FDOPA-PET imaging into target volume delineation for radiation therapy, and studying if the change in our target volumes will improve individual patient outcomes. Patterns of failure will be correlated with tumor extent treated with the addition of 18F-FDOPA-PET imaging and previously treated volumes based solely on conventional MRI volumes. Specifically, patients will be followed to determine if the addition of 18F-FDOPA-PET imaging for treatment planning increases the time to progression and/or improves patient survival. In order to use 18F-FDOPA-PET in a diagnostic fashion, i.e. to include that information when defining the radiation target volume, the FDA requires that we submit an lnvestigational New Drug (IND) application (please see the budget and budget justification, attachments C and D). The continued support of BTFC would allow us to apply for the IND, and to run a second pilot study in which patients will undergo an 18F-FDOPA-PET scan just prior to radiation treatment planning. Patients in the initial biopsy-correlation study will be also eligible for the proposed treatment planning study, but this second pilot study will also be open to patients who did not or could not participate in the biopsycorrelation study. We also plan to acquire advanced MR imaging for these glioma patients. Advanced imaging techniques such as perfusion and diffusion MRI will allow us to further study the underlying structure of brain tum,ors and can provide additional information about tumor grade and extent.

In addition to bringing us closer to individualizing radiation target volumes, the continued support of BTFC would help secure extramural funding for future research with a larger patient population. We are actively seeking extramural funding and have recently been selected by the Mayo Cancer Center to apply for the National Brain Tumor Society Innovation Grant Award. In addition we are currently writing a National Institutes of Health (NIH) Research Project Grant (R0l). The R0l will incorporate ?8F-FDOPA-PET imaging data with additional advanced MR imaging of gliomas including on a large scale both a neurosurgery component as funded initially by BTFC as well as a radiation treatment planning and outcomes component as proposed in pilot form here.

Once again we thank you for the generosity you have shown us, and we hope to continue this productive collaboration in the future.

2011

The Rational Design, Synthesis and Evaluation of Novel STAT3 Inhibitors for Treatment of Malignant Gliomas

Abstract

There is mounting evidence that STAT3, a key protein that promotes tumor activation, growth and survival is playing an active role in malignant glioma tumor formation. We hypothesize that a small molecule can be developed through a rational drug discovery process, that block STAT3 ‘s ability to activate malignant gliomas-thus stopping tumor activation and growth. This project can be broken down into three distinct phases. First, using computer aided modeling, we will design drug structures that block the key step in STAT3 activation. Next, these compounds will be synthesized using standard organic chemistry techniques and characterized fully. We will test these compounds ability to inhibit STAT3s activity in test tubes. Finally, we will determine how effective these compounds are in malignant glioma cells and animal models of glioblastoma. The ultimate long-term goal is translational, including the development of a novel anticancer drug and a pilot clinical trial to determine how well patients respond.

2010

Utility of F-FDOPA-PET for neurosurgical planning and radiotherapy target delineation in glioma patients: Biopsy validation of F-FDOPA-PET uptake and biodistribution in brain tumors

Abstract

Surgery and radiation are the cornerstones of treatment for malignant brain tumors. Accurate diagnosis is important for tailoring therapy. The extent of surgical resection is increasingly being associated with improved survival in brain tumors such that patients who are able to have a more extensive resection are more likely to live longer. Currently, contrast-enhanced MRI is used for surgical planning of most brain tumors. Previous biopsy studies done at Mayo have shown that brain tumor cells are present in what appears to be swelling on the MRI, thus MRI is inadequate for determining tumor extent. Metabolic imaging techniques such as PET scanning show promise in being able to better differentiate tumor from normal tissue in the brain. Metabolic techniques rely on the different uptake of certain nutrients in tumors as compared to normal brain. Recent reports suggest ¹⁸F-FDOPA is a nutrient that is taken up preferentially by tumor cells and very minimally by normal brain. Additionally, areas that have very high uptake of ¹⁸F-FDOPA could be higher grade or more aggressive. The purpose of this study is to describe how ¹⁸F-FDOPA-PET images correlate with brain tumor extent by performing biopsies of suspicious areas before the tumor is removed surgically and comparing the 1 F-FDOPA -PET scans to the MRI. A better understanding of ¹⁸F-FDOPA-PET imaging could allow more accurate diagnosis as the surgeon could focus on the more aggressive looking areas for biopsy, and guide surgical resection to allow the surgeon to remove tumor areas that were not visible on the MRI. In the many patients, 18 complete tumor removal is not possible because of the risks of injury to the brain. F-FDOPA-PET could also eventually guide radiotherapy such that areas that are currently not seen on MRI could be targeted with radiation.