Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
Funding Progress: $§ / $§§§§§

Signup to see this grant's details

Chromatin defects and therapeutic targets in pediatric brain cancers
Informational
DIPG, Childhood (Brain Cancer)
Lay Summary

DNA is wrapped around an octamer of histone proteins, known as a nucleosome, to form the basic repeating unit of chromatin. Genes encoding two chromatin regulators, ATRX and the histone H3.3, are frequently mutated in pediatric glioblastoma (pGBM) which is a lethal form of brain cancer. To translate this new knowledge into novel therapeutic strategies for treating pGBM, we have established pGBM cell models to uncover genomic aberrations linked to ATRX and H3.3 mutations. We have new data that mutant H3.3 affects PML nuclear body formation, and this in turn affects proper chromatin assembly and block neural differentiation in pGBMs. We hypothesise that 1) Mutant H3.3 drives the formation of defective PML bodies that block the pGBM from undergoing proper neural differentiation, trapping the pGBM in an undifferentiated, stem-like tumour state that causes the cancers to multiply; 2) Targeting factors regulating the undifferentiated glioma phenotype will drive pGBM differentiation and represents a therapeutic strategy for pGBM. We aim to 1) Investigate the ability of H3.3-mutant PML bodies to drive cancer growth; 2) Identify factors which can restore normal differentiation programs in pGBM. Outcomes from these studies will identify novel targets for therapeutic intervention in pGBM.

Histones are proteins that protect and organize our DNA. They are also described as suitcases that sort our DNA to regulate gene activities. Recent studies have identified that a high mutation rate of the histone H3.3 genes in pediatric brain cancer. This exciting discovery brings renewed hope for developing effective therapies for pediatric brain cancer and has profound implications for understanding molecular mechanisms underlying cancer in general. To translate this new knowledge into clinical patient management and patient outcomes, we propose to investigate how histone H3.3 mutations alter DNA organization and gene activity. 

PML (promyelocytic leukemia) body is a structural compartment found within the nucleus of normal cells and required for regulating genome organization, DNA stability and gene activity. We have findings that the mutant histones are located within the PML bodies where they are incorporated into DNA. We hypothesize that mutant histones may interfere with the function of PML bodies, which may then affect the global genome organization and gene activity. PML bodies were named for their prominent role in Acute Promyelocytic Leukemia (APL), which arises due to a mutation in the PML gene. This leukemia can be cured by removing the abnormal PML bodies with a combination of drug treatments. We propose that the histone mutations in pediatric brain cancer are analogous to the PML mutations in APL. Supporting this, we have evidence that the presence of mutant H3.3 within PML bodies changes the protein composition and function of these compartments. To further investigate this, we will use cutting edge genomics approaches to investigate how H3.3 mutations affect PML body formation and global genome organization. In addition, we hypothesise that targeting genetic determinants maintaining the undifferentiated state in pGBM will drive differentiation of these tumours, thereby providing a therapeutic strategy for pGBM. We will employ the CRISPR/Cas9 screening system to identify these genetic regulators.

Research Outcomes: The pathway to new drug treatments from basic research is still at least 10-15 years, however, without basic research in this area, the long-term benefits will not be achieved. There are currently no effective treatments or promising leads for treatment of pGBM. While this proposal has an element of risk, we are pursuing if a target (PML body) which is proven to be clinically druggable and highly effective in treating leukemia. This study will uncover if PML bodies (and the proteins that control cell differentiation) a good therapeutic target for pGBM. Positive outcomes from this study would drive the development of new ways to target pGBMs. 
 

Executive Summary

I) Scientific merit

Hypothesis, goals and methods:
Treatment for paediatric glioblastoma (pGBM) remains an unfulfilled need in clinical neuro-oncology. The standard treatment involves surgical removal followed by radiation and chemotherapy. While there is evidence for the prognostic impact of surgical removal of the tumor mass, the concerns of irradiation on the developing brain and contradictory results of various chemotherapy regimens in pGBM make the treatment decisions rather complicated and difficult in children. The high mortality rate in pGBM demonstrates a clear need for therapies, and progress in this area requires an understanding of the fundamental biology of pGBM. DNA sequencing studies have identified frequent mutations of the histone H3.3 and ATRX genes in pGBM. Histone H3.3 is deposited into DNA by ATRX within the PML (promyelocytic leukemia) bodies, which are nuclear compartments controlling processes including gene expression, DNA repair, and cell growth. The discovery of ATRX and H3.3 mutations in pGBM was a breakthrough in the cancer field. However, this key knowledge has not been translated into therapeutic strategies because of a poor understanding of i) the molecular changes underlying the pathway to immortalization and malignancy, ii) chromatin abnormalities linked to ATRX and H3.3 mutations.

We aim to uncover new therapeutic strategies for pGBM. To achieve this goal, we have created mutant cell models carrying mutations in ATRX, H3.3 and other co-driver gene mutations in pGBM  to investigate key features of ATRX/H3.3-mutated pGBM:
a.    pGBM cells show large-scale genome organisation disruption and altered gene activity;
b.    pGBM cells are trapped in an undifferentiated, stem-like tumour state and divide indefinitely. 
Using our cell models, we have made significant discoveries that form the basis of this study. We show that mutant H3.3 inhibits KDM4 histone demethylase [Voon Nature Comm 2018, Udugama Proc Natl Acad Sci (PNAS) 2018] and KDM4B inactivation is the missing factor, acting together with ATRX mutations, to drive immortality in pGBM (Udugama Nature Comm 2021). We now have new evidence that H3.3 mutations interfere with PML body function and impair genome organisation. 

Based on these new findings, we have formulated the following hypotheses and aims:
1)    Mutant H3.3 nucleates the formation of defective PML bodies blocking pGBM from undergoing proper neural differentiation and trapping them in an undifferentiated stem-like tumour state. Thus, we will investigate the ability of H3.3-mutant PML bodies to drive cancer growth.
2)    Targeting factors regulating the undifferentiated stem-like tumour phenotype will drive pGBM differentiation, and represents a therapeutic strategy for pGBM. Thus, we will identify factors which can restore normal differentiation programs in pGBM.

Clinical significance and research outcomes: 
The long-term impact of this research will be to identify new treatments for childrens suffering from GBM. The pathway to new drug treatments from basic research is still at least 10-12 years, however, without basic research in this area, the long-term benefits will not be achieved. There are currently no effective treatments or promising leads for treatment of pGBM. While this proposal has an element of risk, this study will provide the first discovery and knowledge framework for understanding how ATRX and H3.3 mutations promote the ALT pathway and drive cancer. It will identify novel targets for therapeutic intervention in pGBM. Furthermore, histone mutations are common in other rare cancers (chondroblastoma, giant cell tumor of bone) where these findings may also be relevant. If we are able to provide good biological evidence for targeting novel factors associated with PML bodies in pGBM, this lays the foundation for long-term investment into technical optimizations (e.g. delivery and uptake, drug development) for treatment of pGBM.


II) Feasibility and Expertise 
Team and resources:  Chief Investigator Wong’s lab has made influential discoveries on genome biology (published in Nature Comm, PNAS, Genome Res, Nucl Acids Res), placing them at the forefront of the field. CIA has established advanced “omics” and cell biology to study chromatin biology and will apply this expertise here to define for the first time how ATRX and H3.3 mutations impair pGBM’s capacity for differentiation by promoting activation of the ALT pathway and formation of defective PML bodies. This study is carried out in collaboration with Prof Philippe Collas (University of Oslo) and Prof Nada Jabado (McGill University). By combining their resources and skills, the CI Wong and her collaborators are in a unique position to build a comprehensive understanding of genomic aberrations in pGBM to inform on future treatments. They will also synergize with other collaborators at Monash University, who have expertise in proteomics (Monash Proteomics and Metabolomics) and functional genomics (Monash Functional Genomics Platform). 

Chief Investigator Wong: CI Wong leads a research team focused on chromatin pathways governing genome stability at Monash Uni. She has authored 53 articles (>5900 cites, h index 35, 33 as first/senior author). In the last 10 years, she has a number of project grants which led to 26 papers (16 first/senior author) e.g. Nat Comm (3), PNAS (2), Genome Res (5), NAR (6), PLoS Genet (4) and Nat Immunol (1). These studies demonstrate her ability to lead high-quality and high-impact research. She has identified H3.3 and ATRX as key factors preserving telomere and genome integrity. These studies preceded the break-through discovery of ATRX mutations in cancers and their link to ALT telomere maintenance pathway, which is essential for continual cell growth in cancer. In recent studies, she has created ATRX/H3.3 mutated cell models and uncovered novel phenotypes associated with ALT pathway (Udugama 2018 PNAS, Voon 2018 Nat Comm, Udugama Nat Comm 2021). She has the expertise, knowledge and tools to lead this study. She has assembled a team with strong skills in chromatin biology, as shown by our recent papers in PNAS, NAR, Genome Res and Nat Comm.

Collaborators:
Prof Collas is a leading genome biologist at the University of Oslo (published in Nature Genetics, Genome Biol, Genome Res, Nature Protocols, Nucl Acids Res). He has pioneered the development of wet-lab and computational tools for genomics studies, including small cell number Chromatin immunoprecipitation assays, a genomic domain calling algorithm and a widely used 3D genome computational modelling platform for analyses and predictions of 3D genome structure. CI Wong and Prof Collas have collaborated for several years and published 8 papers together on ATRX, H3.3 and telomere biology. 
Prof Jabado (>28,000 cites, h index 88) is an oncologist and geneticist specialising in pGBM who had published >200 papers including those in Nature, Cell, Cancer Cell, Science, Nature Genetics, Nature Comm, Cancer Discovery. She was the first to identify H3.3 mutations in pGBM and had set up human pGBM cell models to be used to investigate pGBM. 

Risks and feasibility:
The risks are low. CI Wong and her team are experienced in molecular and cellular biology, and in chromatin and genomics studies. Also, they are supported by a team of collaborators with strong expertise in proteomics and functional genomic screening. They have also established all the essential cell models (ATRX/H3.3 ALT cell models and human pGBM cell lines) and techniques, e.g. protein isolation protocol, ChIP-sequencing, in vitro differentiation assay and DNA based functional genomics screen. Therefore, we are unlikely to encounter issues which cannot be resolved.
 

 

Description of Research Proposal

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.

Integer fermentum vestibulum lacinia. Duis id aliquam nibh, ut mattis leo. Nulla ac dui at sapien posuere facilisis ut quis ex. Aliquam vestibulum blandit tristique. Integer pretium dui ac nulla accumsan, et finibus velit euismod. Proin placerat, nunc eu sodales facilisis, tellus justo efficitur risus, non blandit diam nulla ac ligula. Aliquam ullamcorper quam leo, porttitor dictum ex tempor ac. Ut efficitur, justo et auctor volutpat, ex ex pulvinar est, sed consequat turpis leo nec ipsum. Nunc tempor, turpis ut ullamcorper tempor, dolor dui varius dui, et congue quam nisi vel nunc.

Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec faucibus, turpis sit amet maximus dapibus, sapien nisl bibendum turpis, pharetra commodo tellus libero vel nulla. Sed nec velit viverra, congue sapien et, gravida libero. Proin eget ante eget turpis egestas accumsan. Aliquam arcu nibh, aliquam rhoncus vulputate in, pellentesque at sem. Maecenas cursus tempus nibh id tempus. Mauris dolor sapien, lacinia sit amet condimentum at, dapibus eget lectus. Phasellus vel pellentesque ex. Nunc aliquam in ligula at tristique. In mollis suscipit felis eu finibus. Nullam non dignissim nibh, nec suscipit ex. Suspendisse tincidunt et mauris id finibus. Aliquam vehicula a sem quis venenatis.

Suspendisse leo odio, rutrum et viverra ut, consequat finibus enim. Vivamus dolor nisl, viverra eu egestas vel, blandit in nulla. Curabitur auctor purus non est volutpat bibendum. Proin fringilla magna sed metus maximus, in dictum neque suscipit. Sed ornare ut mi ut sodales. Nulla efficitur urna nunc, non molestie nunc egestas ut. Nunc arcu lorem, semper ut tincidunt ac, eleifend quis elit.

Sed at tortor et tortor tincidunt feugiat id in dui. Vivamus eget justo nisl. Aenean congue laoreet nisl a elementum. Nunc consectetur velit non ligula sollicitudin, quis eleifend urna sollicitudin. Sed tincidunt, nisl quis varius venenatis, dui massa condimentum tellus, sed sollicitudin diam magna mollis augue. Sed venenatis commodo purus id malesuada. Aenean volutpat elit vel gravida consectetur. Vestibulum diam quam, lacinia ac tortor eget, tincidunt dapibus dui. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Proin nisl leo, pretium sed arcu imperdiet, hendrerit sollicitudin sem. Duis non magna at nunc sagittis ullamcorper a id est. Maecenas cursus nisl in faucibus hendrerit.

Sed hendrerit vitae purus et tempor. Aenean vitae varius velit. Nullam aliquet ipsum elit. Mauris vestibulum purus et metus imperdiet, quis gravida eros pretium. Curabitur rutrum nunc vitae tincidunt condimentum. Aenean sit amet augue velit. Nunc tristique quis lorem id pharetra. Pellentesque sollicitudin, eros sed egestas rutrum, nulla nisl sodales elit, ut imperdiet nunc lectus sit amet nulla. Ut malesuada finibus libero. Curabitur mi dolor, sollicitudin quis bibendum quis, dictum sed enim.

Pellentesque nibh erat, egestas sit amet sagittis malesuada, rhoncus at neque. Mauris maximus commodo tortor, non egestas magna finibus vitae. Sed hendrerit nulla vel venenatis tempor. Suspendisse tristique tincidunt libero et placerat. Nam tincidunt condimentum lorem, vel pharetra est iaculis sed. Nullam ac tincidunt orci. Quisque pharetra ut sem sit amet aliquam. Phasellus risus libero, varius in condimentum vel, commodo id ipsum. Aliquam in metus cursus, mattis diam ut, aliquam magna. Suspendisse facilisis dui et orci varius, suscipit facilisis augue dapibus. In eget nibh ipsum. Suspendisse eget pharetra est, quis condimentum felis. Fusce scelerisque congue libero, sed aliquam mi elementum a. Etiam scelerisque ante non auctor porta. Nam eu nunc id ex finibus dictum. Praesent dui ex, dictum ac massa eget, rutrum gravida nisi.

Sed egestas arcu in dui euismod, eget faucibus massa iaculis. Etiam efficitur lectus et purus lobortis, ac blandit eros rutrum. Proin bibendum consectetur leo vel gravida. Etiam et ultricies sapien. Nam lacinia tellus erat, id facilisis est consequat et. Morbi quis risus in neque iaculis pharetra ut consectetur libero. Aenean feugiat tempor mi eu posuere.

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

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.