Lay Summary

This project will develop novel combination therapies for the most aggressive childhood cancer, diffuse intrinsic pontine glioma (DIPG). Our team performed a comprehensive high-throughput drug screen, testing 3,570 drugs for their ability to prevent DIPG cell growth and found ACT001 to be one of the most effective drugs tested. ACT001 is known to have both antioxidant and anti-inflammatory properties and readily crosses the blood brain barrier. We have initiated a world first Phase 1 pediatric clinical trial of ACT001 for children with relapsed/refractory solid or CNS tumors, including patients with DIPG/DMG. Currently, fifteen patients, seven of which are DIPG/DMG patients, are enrolled and no dose-limiting toxicities have been observed. Excitingly, clinical activity has been demonstrated in three DIPG/DMG patients including improvement in the appearance of the tumor on MRI imaging, as well as improvement in patient symptoms. Using our preclinical models, we now seek to determine precisely how ACT001 works in DIPG and then use this information to evaluate potential ACT001 combination therapies, to enhance the effectiveness of ACT001. As such, this project will provide valuable information for developing new combination treatments that may lead to ACT001 as the first active treatment for children suffering from DIPG.

Executive Summary

Background

DIPG is the most aggressive of all childhood cancers accounting for up to 80% of pediatric brainstem tumors^1,2. There are no effective treatments and current therapeutic strategies are palliative only³. Due to its location within the brainstem, the tumor cannot be removed surgically, it does not respond to chemotherapy, and radiotherapy only slows their growth temporarily. Novel and innovative treatment approaches are therefore urgently needed to counter this tumor and its ongoing poor prognosis⁴.

ACT001 (also known as dimethylaminomicheliolide (DMAMCL)), is a guaianolide sesquiterpene lactone and synthetic analogue of parthenolide, a natural component of the medicinal plant “feverfew”.  ACT001 (Accendatech Co.) has been certified as an orphan drug by the United States Food and Drug Administration (FDA) and the European Union. ACT001 is currently undergoing phase I and phase II clinical trials.^5,6 ACT001 has shown activity in adult glioblastoma (GBM) phase I clinical trials⁷.  We have initiated a world first Phase 1 pediatric clinical trial of ACT001 for children with relapsed/refractory solid or CNS tumors, with an expansion cohort planned for patients with DIPG/DMG. So far, 15 patients have been enrolled, of which 7 are DIPG/DMG patients. Dose escalation from dose level 1 at 188mg/m2 bd to dose level 4 at 700mg/m2 bd has been completed with no dose limiting or Grade 3/4 toxicities observed. At the highest dose level reached so far, clinical activity has been demonstrated in 3 patients with DIPG/DMG including objective radiological responses. Two DIPG patients had a minor reduction in disease burden on MRI imaging, and another DMG patient with H3K27M mutation has had an objective radiographic and clinical response.

In GBM it has been shown that ACT001 targets both adipocyte enhancer binding protein 1 (AEBP1) and plasminogen activator inhibitor I (PAI-1), inhibiting glioma cell proliferation, invasion and metastasis through the Phosphoinositide 3-kinase (PI3K)/ Protein kinase B (AKT) pathway^8,9. Additionally, ACT001 inhibits the Nuclear factor-κB (NF-κB) pathway by direct IKKβ binding and phosphorylation inhibition, thereby inducing reactive oxygen species (ROS)-mediated G2/M phase arrest and apoptosis in glioblastoma cells¹⁰. ACT001 has also been found to inhibit Signal transducer and activator of transcription 3 (STAT3), Programmed death-ligand 1 (PD-L1) transcription and modulates the anti-tumor immune response in glioma bearing mice¹¹. A summary of these proposed mechanisms of action in GBM are graphically outlined below in Figure 1.

Figure 1. Proposed mechanism of action of ACT001 in GBM

Supporting data

Our group performed a high-throughput drug screen (HTS) with 3,570 biologically active, clinically approved compounds against a panel of DIPG cultures. Despite having a library enriched with anti-cancer compounds, only 30 brain penetrant drugs showed significant activity against DIPG. Following validation across an expanded panel of DIPG neurosphere cultures, parthenolide was identified as one of the most effective drugs tested, demonstrating significant anti-tumor activity across the entire panel. Unfortunately, however, parthenolide showed limited in vivo efficacy against DIPG PDX models. We have now shown that a synthetic analogue of parthenolide, ACT001, inhibited DIPG colony formation and decreased DIPG cell viability in a dose dependent manner when examined across a panel of DIPG neurosphere cultures, without any adverse effect on normal healthy cells. Even more exciting, using an orthotopic DIPG mouse model we found that treatment with ACT001 resulted in a significant decrease in proliferating DIPG tumor cells within the brain, concomitantly significantly increasing mouse survival. We have also shown that ACT001 treatment in vitro does not significantly alter cell cycle progression but does induce apoptosis in DIPG cells. We also have preliminary data that suggests that ACT001 may induce ROS expression, with subsequent activation of the NRF2 antioxidant pathway. Furthermore, in our world first Phase 1 pediatric clinical trial of ACT001, clinical activity has been demonstrated in three DIPG/DMG patients, resulting in either stable disease or a marked reduction in disease burden observed. One patient with a DMG with H3K27M mutation has had a clear reduction in tumor burden as seen in Figure 2, and two further DIPG patients have had minor reductions in disease on MRI imaging.

Figure 2. MRI of clinical response observed in a patient with DMG with H3K27M mutation treated with ACT001. (A) shows T2 weighted MRI pre study enrolment, (B) shows T2 weighted MRI after 6 months of treatment with ACT001.

Hypothesis

We have both preclinical and clinical evidence that ACT001 is active as a single-agent against DIPG/DMG and hypothesize that the development of rational combination therapies will significantly increase its clinical efficacy.

Design, methods and goals

We aim to build on these exciting preclinical and clinical data by determining the precise mechanism(s) of action of ACT001 within the setting of DIPG. Elucidating this mechanism is of primary importance for the identification of biomarkers, and potential synergistic drug combinations.  To achieve this, we will assess the molecular changes in DIPG in response to ACT001 treatment at both the gene and protein level, by qPCR and western blotting, respectively. We will examine key drivers of DIPG including epigenetic machinery such as H3K27 acetylation and H3K27 trimethylation as well as other pathways including the aforementioned mechanisms of action of ACT001 in GBM. These acquired data will be further strengthened through flow cytometric analysis of the ability of ACT001 to cause increased ROS levels and subsequent apoptosis.

Next, we will identify and evaluate potential ACT001 combinations. Candidate drugs for combination with ACT001 will be selected initially through HTS and their ability to cause DIPG cytotoxicity and colony formation inhibition further evaluated in vitro. We have already identified a number of promising drug candidates for potential use in combination with ACT001. These include the dopamine receptor antagonist compounds ONC201/206/213, the thioredoxin reductase agonist auranofin, the HDAC inhibitor CBL0137, the epigenetic modifier SAHA, the chemotherapeutic carboplatin, and the serotonin receptor agonist GR127935. Importantly, all these drug candidates are either currently in clinical trial or clinically approved pharmaceutical compounds. We aim to assess the mechanism of synergistic interaction between ACT001 and the candidate drugs. To achieve this, we will assess the molecular changes in DIPG in response to ACT001 in combination with our candidate drugs, through qPCR and western blotting, as above. Furthermore, to assess if the drug combinations employ novel mechanisms at the transcriptional level, we will perform global gene expression analysis in DIPG cells treated with either drug alone or in combination using the Affymetrix Human Genome-U133 Plus 2.0. These experiments will provide information for the selection of potential biomarkers for an assessment of response in both animal studies and, ultimately, DIPG patients.

From these data we will identify the two most favorable lead compounds to be further investigated and will evaluate the therapeutic efficacy of these combination treatments in a DIPG orthotopic animal model. We will use a patient derived xenograft (PDX) DIPG model contain the K27M mutation of histone H3.3 that is characteristic of DIPG and is genetically modified to express luciferase which allows real time monitoring of tumor engraftment and progression. Our PDX models are developed using a specialized stereotactic device to introduce patient derived DIPG cells at the exact anatomical region the tumor develops in children. 100% of animals harboring these PDXs develop tumors, with the onset of neurological signs at 6-10 weeks that precisely mimic the clinical condition. Histologically the tumors recapitulate the human disease with diffuse infiltration of the pons, and the capacity to progress further to regions such as the cerebellum and midbrain.

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. Dr Dannielle Upton is an early career post-doctoral scientist with expertise in DIPG cell cultures and xenograft models, drug combinations and mechanistic studies. Her research focuses on understanding the cytotoxic effects of drugs identified from high throughput screening and their effectiveness in PDX animal models of diffuse intrinsic pontine gliomas (DIPG). She is also developing novel combination therapeutic strategies with a focus on the translation to clinical trials. Dr Upton has over 10 years’ experience in drug efficacy studies and animal experimentation. Dr Upton will undertake in vitro, and in vivo studies involved in this project, performing cytotoxicity assays, colony forming assays, molecular analysis, mechanistic studies, intracranial injections, drug treatments, and survival studies in animals. She will be responsible for managing all aspects of the in vitro and in vivo research project, the production of data and manuscripts resulting from this work. With the support and guidance of A/Prof Ziegler and Dr Rayner, she has generated all the background pre-clinical data described in this project. Dr Rayner has extensive expertise in cell and molecular biology and will be assisting with molecular analysis and mechanistic pathway analysis involved in the project. A/Prof Ziegler has preclinical expertise in pediatric malignant brain tumors and established the first DIPG research program in Australia. He has overseen the preclinical development of several novel therapies for DIPG that are each being translated to clinical trial. His clinical focus on early phase clinical trials will facilitate translation of positive results to the bedside. The support of A/Prof Ziegler and Prof Haber at Children’s Cancer Institute provides an invaluable environment to ensure the success of this novel research program, and with the ultimate goal of improving outcomes for children diagnosed with DIPG.

Outcomes and Clinical significance

This project focuses on the development of therapies that are rapidly translatable from 'bench to bedside' using state-of-the-art pre-clinical models that precisely recapitulate DIPG. At the conclusion of this study, we would have positioned a combination drug therapy regime for direct translation into the clinic. As DIPG shares many biological characteristics with other childhood cancers, novel treatment strategies discovered through this grant will be readily applicable to other forms of cancer, benefiting a wider patient base.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest