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123 abc lane, Townsville, ZZ 00000, USA
Funding Progress: $§ / $§§§§§
Protein degradation as a strategy to target mutant WIP1 in Diffuse Intrinsic Pontine Glioma
TranslationalDIPG, Childhood (Brain Cancer)
Lay Summary
Close to 20% of Diffuse Intrinsic Pontine Gliomas (DIPGs) harbor a mutation in a gene called PPM1D that leads to expression of a truncated form of a protein called WIP1. We have found PPM1D-mutant DIPG cells to require expression of WIP1, rendering it an attractive therapeutic target for these tumors. However it has been difficult to develop new drugs to directly inhibit the function of WIP1. An alternate approach to targeting proteins is to hijack normal regulatory processes to induce their degradation. In this project, we will explore strategies to degrade WIP1, leveraging new drugs developed by our group. These experiments will lay the groundwork to introduce an entirely novel therapeutic strategy for children with DIPG.
Executive Summary
Hypothesis proposed and anticipated results
Diffuse Intrinsic Pontine Gliomas (DIPGs) are one of the most lethal cancers and afflict young children. We have found up to 20% of all DIPGs to harbor PPM1D mutations at diagnosis. The mutations afflict exon 6, the site of the negative regulatory domain, resulting in over-expression of truncated WIP1 (WIP1tr). Our preliminary experiments reveal expression of WIPtr to be preferentially essential in patient-derived PPM1D mutant DIPG cell lines (compared to those that are PPM1D wild-type), highlighting it as a possible therapeutic candidate. Protein phosphatases have been traditionally difficult to directly inhibit. Protein degradation has rapidly emerged as an alternate strategy to target proteins that are otherwise difficult to inhibit.
Our preliminary analysis of genome-scale CRISPR-cas9 screens across more than 500 cell lines reveals a co-dependency of PPM1D and the ubiquitin specific peptidase USP7 in TP53 wild-type cell lines, suggesting a potential role for USP7 in the protein regulation of WIP1. USP7 also regulates other key members of the TP53, including MDM2, in addition to proteins that have oncogenicity in other contexts. Indeed, the Burhlage lab currently has extensive efforts to generate clinically relevant USP7 inhibitors.
These findings form the basis of our central hypothesis that WIP1 protein degradation represents a potential therapeutic target for children with PPM1D-mutant DIPGs. We will address our hypothesis through the following specific aims:
Aim 1 will test the prediction that USP7 expression is necessary and represents a therapeutic target for PPM1D-mutant DIPGs. Our analysis of genetic dependencies across more than 500 cancer cell lines reveal a co-dependency of TP53 wild-type lines on PPM1D and USP7. We will determine whether PPM1D-mutant DIPG cell lines (which we have confirmed to be dependent on WIP1 expression) exhibit a similar dependency on expression of USP7 through a series of assays in which we first apply CRISPR-cas9 technology to genetically ablate expression of USP7 followed by assays in which we assess the efficacy of small-molecule inhibitors of USP7 developed by the Burhlage lab. We will assess the effect on cellular proliferation, induction of apoptosis and effects on cell-cycle of PPM1D-mutant lines (relative to wild-type lines). While USP7 is known to be a key regulator of MDM2 stability, its effect on WIP1 protein stability is uncharacterized. We will therefore assess whether USP7 inhibition results in degradation of PPM1D and/or MDM2 using fluorescent reporter constructs. We will also assess whether WIP1 or MDM2 are the key downstream targets of USP7 inhibition through a series of rescue experiments in which WIP1 and MDM2 are exogenously expressed following genetic ablation of USP7. Finally, our preliminary data show potential synergy between MDM2 and WIP1 inhibition. We will determine whether similar synergies exist between USP7 inhibition and MDM2 inhibition in our panel of patient-derived DIPG cell lines.
Aim 2 will comprehensively catalogue the deubiquitinases that regulate WIP1 protein stability, identifying those that can be inhibited in therapeutic approaches. Wild-type WIP1 is subjected to ubiquitin-mediated proteosomal degradation1. Thus, strategies that increase levels of WIP1-ubiquitination, for example through the use of deubiquitinase inhibitors (DUBi)2, may facilitate protein degradation of WIP1. We will leverage CRISPR-cas9 technology to genetically ablate all 100 deubiquitinases, coupling with a WIP1-reporter construct (to overexpress both full length WIP1 and WIP1tr), to identify specific DUBs that regulate WIP1’s degradation. In parallel, we complete a systematic small molecule screen leveraging the panel of DUB inhibitors synthesized by the Burhlage lab to identify those that facilitate degradation of truncated WIP1, representing therapeutic promise for PPM1D-mutant DIPGs. We will validate candidate WIP1-regulatory DUBs in low-throughput assays. DUB inhibitors that score in our screens will also be validated in low-throughput assays in vitro using patient derived PPM1D-mutant and wild-type DIPG lines.
Study Team: Drs. Bandopadhayay and Buhrlage will serve as Co-Principle Investigators for this application and will work in close collaboration with Dr. Cory Johannessen and Dr Keith Ligon. Dr. Bandopadhayay, a pediatric neuro-oncologist, leads a research group focused on applying genomic approaches to find novel treatments for children with brain tumors. Dr. Bandopadhayay serves as the DFCI Principal Investigator for the DIPG Registry. Dr. Burhlage, an Assistant Professor of chemistry has tremendous experience in leveraging the ubiquitin system to therapeutically degrade proteins and her lab have developed a number of deubiquitinases (DUB) inhibitors that are being developed as potential cancer therapies. Both investigators are Associate Members of the Broad Institute. Dr. Johannessen is a senior group leader of the Cancer Program at the Broad Institute with expertise in leveraging genome-scale modifier screens to identify new therapeutic targets for cancers and characterizing resistance mechanisms. Dr. Ligon, a neuropathologist, completes this study team. His team have developed a panel of patient-derived DIPG models (including those that harbor PPM1D mutations) that will provide an invaluable resource to validate the candidate DUB inhibitors.
In summary, PPM1D has emerged as a potential therapeutic target for up to 20% of DIPGs however inhibiting the protein phosphatase remains a challenge in drug development. This proposal explores protein degradation as an alternate therapeutic approach directed towards truncated WIP1 (encoded by mutant PPM1D) and will leverage the collaborative scientific community of Dana-Farber Cancer Institute and the Broad Institute.
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
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.