Diffuse Midline Gliomas H3K27-altered (DMGs) represent a subtype of pediatric high-grade brain tumors that are almost universally fatal with an overall survival of 8 – 12 months post-diagnosis. Because surgery is often not an option and radiation remains the only therapy available to patients, there is an enormous unmet need for targeted therapeutics that is based on an understanding of the unique biology of these tumors and the pathways that interact to drive tumorigenesis. Here we investigate the cooperation between the phosphatase PPM1D and the PI3K pathway in driving DMGs. Mutations in PPM1D are present in 8 -12% of DMGs – mutually exclusive with TP53 mutations - that mainly lead to a C-terminally truncated protein that is thought to drive tumorigenesis at least in part due to its enhanced phosphatase activity on targets such at p53, ATM and CHK1/2. However, because p53 is still intact in PPM1Dtr tumors, we find that that its activation is suppressed but not completely ablated. Leveraging a CRISPR screen performed in isogenic mouse neural stem cell (mNSC) system, we find that activation of the PI3K/mTOR pathway provides a selective fitness advantage to PPM1Dtr-expressing cells. Consistent with that, we show that PPM1D mutations significantly cooccur with alterations in the PI3K/mTOR pathway in an analysis of ∼180 pediatric high-grade brain tumors. Using an in utero electroporation (IUE) murine DMG model, we also demonstrate that PI3K pathway activation indeed accelerates tumor formation and reduces survival of neonatal mice. To explore the mechanism behind the PPM1Dtr – PI3K pathway cooperation, we investigated the response to irradiation-induced DNA damage and replication stress. Interestingly, PI3K pathway activation further suppressed p53 activation in addition to PPM1Dtr. Moreover, in patient-derived PPM1D-mutant cell lines, CRISPR activation of PI3K/MAPK genes rescued cells from small molecule inhibition of PPM1D and allowed cells to progress through the cell cycle despite wildtype p53 activation. Taken together, our results suggest that PPM1D mutant DMGs evolve differently from TP53-mutant ones and acquire PI3K pathway alterations to enhance gliomagenesis. Future work will aim to unravel genetic dependencies of PPM1D-PI3K co-altered tumors and identify therapeutic strategies to target them. Adam Fiseha Kebede, Eric Morin, Ipsita Kundu, Apichaya Sethaudom, Nicolas Poux, Daren Zhang, Leslie Lupien, Veronica Rendo, Timothy N. Phoenix, Pratiti Bandopadhayay. Truncated PPM1D cooperates with the PI3K pathway to drive Diffuse Midline Gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A050.

Every year, up to 300 children are diagnosed with H3K27-altered Diffuse Midline Gliomas (DMGs), a type of high-grade glioma (HGG) that originates in the midline structures of the brain. Its location renders surgical removal challenging, and current treatment options, namely chemotherapy and radiation, are ineffective. This highlights the urgency for a better understanding of the disease and more effective therapies. While DMGs are aggressive tumors, a subset retains wild-type TP53, presenting p53 reactivation therapy as a potential therapeutic strategy. However, the p53 pathway may be functionally suppressed by alteration of negative regulators such as PPM1D and MDM2/4. PPM1D, a phosphatase with several targets in the DNA Damage Response pathway including p53, is frequently altered in DMGs, largely mutually exclusively with TP53 mutations. Whole Genome Sequencing of approximately 180 pediatric HGGs found that 10% of DMGs harbor nonsense mutations in PPM1D, leading to a truncated and more stable phosphatase (PPM1Dtr). PPM1Dtr DMGs therefore present a relevant model for exploring the therapeutic potential of p53 reactivation therapy in DMGs. To investigate potential mechanisms of resistance to p53 reactivation in DMGs, a CRISPR activation (CRISPRa) screen was performed in a p53 wild-type, PPM1Dtr patient derived cell line (PDCL) under treatment with inhibitors of PPM1D and MDM2. The screen identified MAPK pathway genes as significantly enriched, suggesting the pathway’s involvement in resisting p53 reactivation. To validate that the hits are responsible for the phenotype, the PDCL constitutively expressing dCas9 fused to transcriptional activator VP64 was transduced with sgRNAs targeting those hits to activate their endogenous transcription. CRISPRa-mediated upregulation was confirmed in the transduced cells via western blotting. To assess their sensitivity to p53 reactivation, dose-response curves were performed using CellTiter-Glo assay to quantify cell viability. Cells transduced with CRISPRa sgRNAs targeting candidate genes displayed a rightward shift in their curves and higher IC50 values compared to cells transduced with non-targeting control sgRNAs. As a follow-up experiment, the cells were monitored for proliferation in drug treatment using a live-cell imaging system. Cells transduced with CRISPRa sgRNAs targeting candidate genes increased proliferation compared to the controls, further supporting the role of the MAPK pathway in conferring resistance to p53 reactivation therapy. Adam F Kebede, Eric Morin, Ipsita Kundu, Apichaya Sethaudom, Nicolas Poux, Daren Zhang, Leslie Lupien, Veronica Rendo, Timothy N Phoenix, Pratiti Bandopadhayay. MAPK pathway mediates resistance to p53 reactivation therapies in Diffuse Midline Glioma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A046.

Oncogenic alterations in fibroblast growth factor receptor (FGFR)-family proteins occur across cancers, including pediatric gliomas. Our genomic analysis of 11,635 gliomas across ages finds that 5.3% of all gliomas harbor FGFR alterations, with an incidence of almost 9% in pediatric gliomas. Alterations in FGFR proteins are differentially enriched by age, tumor grade, and histology, with FGFR1 alterations associated with glioneuronal histologies. Leveraging isogenic systems, we confirm FGFR1 alterations to induce downstream Mitogen Activated Protein Kinase (MAPK) and mTOR signaling pathways, drive gliomagenesis, activate neuronal transcriptional programs and exhibit sensitivity to MAPK pathway and pan-FGFR inhibitors. Finally, we perform a retrospective analysis of clinical responses in children diagnosed with FGFR-altered gliomas and find that treatment with currently available inhibitors is largely associated with stability of disease. This study provides key insights into the biology of FGFR1-altered gliomas, therapeutic strategies to target them and associated challenges that still need to be overcome.

Diffuse Midline Gliomas (DMGs) are universally fatal pediatric brain tumors. The current standard of care is radiation therapy, which provides only a transient benefit. The genetic landscape of DMG has revealed mutual exclusivity between TP53-mutant and PPM1D-mutant tumors, likely due to functional redundancy. While both TP53 and PPM1D mutations participate in DMG formation, different mechanisms of p53 pathway suppression may present unique therapeutic opportunities. We hypothesize that PPM1D-mutant tumors retain a suppressed yet functional p53 which can be reactivated under therapy stress. Targeting other complementary pathways and PPM1D-specific targets could help attain better overall outcomes. Moreover, PPM1D-mutant tumors frequently co-occur with sub-clonal PI3K pathway activating mutations leading to our hypothesis that they help accelerate tumorigenesis and confer therapy resistance. To investigate this, we have generated isogenic DMG IUE mouse models of Trp53-mutant, PPM1D-mutant, and PIK3CA-mutant (in the context of both Trp53- and PPM1D-mutant) to uncover shared and unique functions of these mutations. Ongoing studies are aimed to elucidate the differential response to radiation therapy using a combination of in vitro assays and transcriptomic profiling in each condition. We aim to test whether Trp53- and PPM1D-mutant DMG models display differences in response to CHK1 inhibition, which is a direct target of PPM1D phosphatase activity and regulator of DNA damage response and cell cycle progression. Similarly, experiments are focused on the use of PI3K/AKT/mTOR inhibitors, examining their in vitro and in vivo ability to improve response to DNA damaging agents in PPM1D-mutant DMGs. We expect to reveal PPM1D and PIK3CA specific targets that can be pursued to improve the outcomes for PPM1D-mutant DMG patients. This will help strategize specific targeted therapeutic approaches in combination with radiation to better treat PPM1D-mutant DMGs.

Pediatric brain cancers are the leading cause of cancer-related deaths in children. We (and others) have reported that close to 10% of all pediatric gliomas, encompassing low-grade and high-grade gliomas, harbor recurrent drive alterations in Fibroblast Growth Factor Receptor proteins (FGFR). In pediatric low-grade gliomas FGFR1 alterations are the most common and occur as structural variants, including kinase duplications and fusion proteins, or kinase-activating single nucleotide variants. Recurrent FGFR1 alterations and the existence of multiple FDA-approved (for adult) pan-FGFR inhibitors represent an attractive therapeutic target for precision medicine approaches, however a challenge with FGFR inhibitors has been the toxicities associated with them and the lack of clinical trials in children. A major aim of this project is to identify the best preclinical FGFR inhibitor candidates for these patients. To address this, we have generated isogenic mouse and human neural stem cells models driven by FGFR1 and BRAF alterations found in pLGGS. We have found that these models grow independent of growth factor and form tumors in mice. The FGFR1-altered models exhibit sensitivity to MEK inhibitors. They also exhibit efficacy to panFGFR inhibitors in vitro and in vivo. We are currently testing the PK characteristics and blood brain barrier efficacy of all available FGFR inhibitors to inform the best candidate for early phase clinical trials. Overall, this project aims to perform preclinical testing using our novel isogenic models to determine a candidate FGFR inhibitor for patients with pLGGs. April A Apfelbaum, Sangita Pal, Sarah W Lamson, Eric Morin, Georges Ayoub, Sher Bahadur, Jeromy DiGiacomo, Margaret M Cusick, Prem Prabhakar, Connor C Bossi, Sehee Oh, Hyesung Jeon, Jinhua Wang, Hong Yue, Yuan Xiong, Amy Cameron, Patrick Rechter, Quang-De Nguyen, Sara J. Buhrlage, Eric S Fischer, Michael J Eck, Keith L Ligon, Pratiti Bandopadhayay. Preclinical investigation of FGFR inhibitors in pediatric low-grade gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B020.

Abstract Diffuse Intrinsic Pontine Glioma (DIPGs) are universally fatal pediatric tumors with a median survival of only 9-12 months. PPM1D-truncating mutations (PPM1Dtr), which promote its phosphatase activity and suppress TP53 function, are found in approximately 15% of DIPG tumors. PPM1Dtr are mutually exclusive to TP53 mutations, and present unique therapeutic opportunities to reactivate wild-type TP53. Another distinct feature of PPM1Dtr DIPGs is the co-occurrence of PI3K/AKT/mTOR pathway alterations, such as PIK3CA. There is an unmet need to elucidate the underlying molecular mechanisms of DIPG and identify novel therapeutic vulnerabilities that will improve outcomes of this devastating tumor. We hypothesize that PPM1D-mutant DIPGs can be therapeutically targeted by reactivating the p53 signaling pathway and other complementary pathways which cooperate with PPM1Dtr. To investigate this, we have generated Trp53- and Ppm1d-mutant isogenic DIPG murine models to determine how specific oncogenic alterations function in DIPG formation, growth and treatment sensitivity. Current studies aim to examine differences in sensitivity to standard of care treatment modalities and leverage next generation sequencing techniques to uncover additional vulnerabilities. We found notable differences upon addition of PIK3CA mutations in the context of PPM1Dtr DIPG tumor formation, growth, survival, and lineage identity. Ongoing studies using whole genome transcriptomics to compare tumor models will provide insight into the molecular signals and cell state changes driven by individual genetic alterations. Furthermore, by comparing isogenic TP53 and PPM1Dtr mutant DIPG models, we aim to further delineate the molecular mechanisms that drive differential sensitivity of TP53 wild-type tumors. The immune-competent nature of our murine models also provides us with a unique opportunity to delineate tumor immune microenvironment changes. Leveraging this information and our novel murine models, our long-term goal is to develop combinatorial therapeutic strategies to improve outcomes for this deadly disease.

Abstract BACKGROUND Pediatric brain cancers are the leading cause of cancer-related deaths in children. We (and others) have recently found that close to 10% of all pediatric gliomas, encompassing low-grade gliomas (LGGs) and high-grade gliomas (HGGs), harbor recurrent driver alterations in FGFR proteins, most frequently FGFR1, the gene encoding Fibroblast Growth Factor Receptor 1. In pediatric gliomas, FGFR1 alterations present as either structural variants (SVs), including kinase duplications and fusion proteins, or kinase-activating single nucleotide variants (SNVs). Recurrent FGFR1 alterations represent a promising therapeutic target for precision medicine approaches, however a challenge has been the lack of clinical trials for FGFR inhibitors. A major goal of this project is to identify preclinical FGFR inhibitor candidates for FGFR1-altered pediatric gliomas. METHODS We have generated isogenic mouse and human neural stem cell (NSC) models driven by FGFR1 and BRAF alterations commonly seen in pediatric gliomas to test the oncogenic properties and therapeutic capacities of these alterations. RESULTS We found that NSC lines grow independent of growth factors and form tumors in mice. RNA-sequencing of these lines revealed the FGFR1-altered lines are enriched in neuronal gene programs. The FGFR1-altered models are exquisitely sensitive to the four FDA-approved panFGFR inhibitors Infigratinib, Erdafitinib, Pemigatinib, and Futibatinib. However, the FGFR1 SNV + PTPN11 co-occurring SNV line is the least sensitive to FGFR inhibition, which suggests combination therapies may be needed. Combination drug studies with FGFR inhibition in our FGFR1-altered lines reveal the best synergy with mTOR inhibition. Finally, we present examples of case studies of patients with FGFR1-altered tumors treated with targeted inhibitors. CONCLUSIONS Taken together, our studies have established models that have generated key insights into the biology of FGFR1-altered gliomas and how to best target them therapeutically.

Abstract Mutations in PPM1D – coding for the phosphatase WIP1 - are present in 8 -12% of all Diffuse Midline Glioma (DMG), H3 K27-altered – a universally fatal subtype of pediatric high-grade tumors. PPM1D mutations are mutually exclusive with TP53 mutations and mainly lead to a truncation of its C-terminal regulatory domain. Truncated PPM1D (PPM1Dtr) has increased protein stability and impairs the p53-dependent DNA Damage Response (DDR) by dephosphorylating its numerous targets (e.g p53, H2A.X, CHK1) which contributes to its oncogenicity. However, because wildtype p53 is present in PPM1Dtr tumors, its activation is suppressed but not completely ablated suggesting that PPM1D mutant tumors may evolve differently from p53 mutant ones. Our analysis of whole genome sequencing datasets indeed found that PPM1D mutations uniquely co-occur with alterations in the PI3K/mTOR pathway suggesting potential cooperativity in enhancing tumorigenesis. Using mouse neural stem cells (mNSCs) as an isogenic model system, we have engineered specific combinations of oncogenic alterations to study their impact on cell proliferation, PI3K/mTOR pathway activation and response to DNA damage and replication stress. We find that PPM1Dtr cooperates with PIK3CA alterations to increase proliferation and signaling through the PI3K/mTOR pathway. Notably, in an in utero electroporation model, mutant PIK3CA accelerates the development of high-grade tumors that infiltrate deep into the brainstem. Our study also indicates a vulnerability to inhibition of the PI3K/mTOR pathway in PPM1Dtr-PI3K co-altered tumors. Future work will aim at 1) exploring the mechanism of cooperation and impact on replicative stress response and 2) developing and testing a rational therapeutic strategy for PPM1D-mutant tumors.

Abstract p53 pathway reactivation through pharmacologic inhibition of negative regulators was identified as a promising therapeutic strategy for TP53 wild-type (WT) high-grade gliomas, including Diffuse Midline Gliomas (DMGs). Despite widespread theoretical promise, the therapeutic efficacy of MDM2 inhibitors and PPM1D inhibitors as single agents have each been limited by issues, including sub-lethal effects on cancer cells and innate or emerging resistance leading to tumor progression. We carried out a genome-scale CRISPR activating (CRISPRa) screen to identify genes that, when overexpressed, confer resistance to p53 reactivation therapies. NAD(P)H quinone dehydrogenase 1 (NQO1) emerged as a top hit and was confirmed through secondary CRISPRa screens and low-throughput validations as driving a resistance phenotype in TP53 WT DMGs. Our subsequent work has aimed to elucidate the mechanisms through which NQO1 mediates the effects of p53 reactivation therapies, and assess the feasibility and impact of exploiting NQO1 in combination therapies towards a cure.

Alterations in Fibroblast growth factor receptor (FGFR)- family proteins frequently occur as oncogenes in many cancers, including a subset of pediatric gliomas. Here, we performed a genomic analysis of 11,635 gliomas across ages and found that 4.5% of all gliomas harbor FGFR alterations including structural variants (SV) and single nucleotide variants (SNV), with an incidence of almost 10% in pediatric gliomas. FGFR family members are differentially enriched by age, tumor grade, and histological subtype, with FGFR1-alterations associated with glioneuronal histologies and pediatric low-grade gliomas. Across development, we find FGFR1 expression in both neuronal and glial precursors, while FGFR3 expression is largely restricted to astrocytic lineages. Leveraging novel isogenic model systems, we confirm FGFR1 alterations to be sufficient to activate MAPK and mTOR signaling, drive gliomagenesis, activate neuronal transcriptional programs and exhibit sensitivity to MAPK pathway inhibitors, including pan-FGFR inhibitors. Models driven by FGFR1 SVs exhibited different patterns of sensitivity compared to those driven by SNVs. Finally, we performed a retrospective analysis of clinical responses in children diagnosed with FGFR-driven gliomas and found that targeted MAPK or FGFR-inhibition with currently available inhibitors is largely associated with stability of disease. This study provides key insights into the biology of FGFR1-altered gliomas, therapeutic strategies to target them and associated challenges that still need to be overcome. ### Competing Interest Statement PB serves on paid advisory boards for DayOne Biopharmaceuticals and has served on a paid advisory board for QED Therapeutics. Her lab has received grant funding from Novartis Institute of Biomedical Research. SHR has employment at Labcorp Oncology. CMVT is on advisory boards for Alexion, Bayer, and Novartis. OW is advisory board member or has received research grants from Janssen, Day One Biopharmaceuticals, and Novartis. K.L.L. disclosures: Equity: Travera Inc.; Consulting- Travera Inc., BMS, Servier, LEK, Integragen, Blaze Bioscience; Research.

Pediatric low-grade gliomas (pLGGs) are the most common solid tumors in children and are associated with devastating lifelong morbidities and mortality. Recent genomic profiling efforts have revealed that these tumors are largely driven by single-driver events that activate MAPK signaling. These insights have led to early clinical trials evaluating the role of MAPK inhibitors for these children with promising initial results. However, pLGGs are not cured by MAPK inhibitors, and tumors often rapidly rebound upon cessation of treatment. Therefore, continuous dosing of these inhibitors is required throughout a child’s development–often with significant and potentially permanent toxicities. In addition, 30-40% of tumors do not respond to MAPK inhibition, indicating primary resistance. We hypothesized that pediatrlc low-grade gliomas may also harbor additional dependencies beyond the MAPK pathway that may represent potential therapeutic targets. To address this, we generated isogenic neural stem cell models that expressed pLGG relevant oncogenes (BRAF family members, FGFR family members and MYB transcription factors, and subjected them to genome-scale CRISPR-cas9 screens. Compared to vector controls, pLGG models exhibited genetic dependencies on pathways that included regulators of cell-cycle and differentiation, in addition to genes involved in regulating the mTOR and MAPK pathways. Cell lines expressing MYB family transcription factors also harbored dependencies on genes involved in the DREAM complex. Together, these genes and pathways represent potential targets for combination treatments with MAPK pathway inhibitors for the treatment of pediatric low-grade gliomas.

Abstract Pediatric low-grade gliomas (pLGGs) have excellent survival, however, with current standard of care, most patients suffer lifelong severe sequalae. pLGGs are almost exclusively driven by single activating mutations in the MAPK pathway. Clinical trials with small molecule inhibitors in BRAF-altered pLGGs are showing promising results in early clinical trials, and similar efforts are now underway for FGFR1-altered tumors, however the underlying biology and treatment response has not been thoroughly explored in a pre-clinical setting. To explore the genetic landscape of FGFR altered gliomas we assembled a cohort of 87 patients with FGFR1-4 altered gliomas across Dana-Farber Cancer Institute, Boston Children’s Hospital and Brigham and Women’s Hospital. Within this cohort we observed that pLGGs harboring FGFR1 kinase hotspot mutations (FGFR1-N546K or -K656E) frequently harbored a second alteration associated with activation of the MAPK or mTOR pathways, most commonly in the phosphatase PTPN11, NF1 or within the FGFR1 gene itself. Additionally, we observed two previously described structural variants of FGFR1, an FGFR1 internal kinase tandem duplication (FGFR-ITD) and a fusion with TACC1 (FGFR1:TACC1). The relative impact of the different FGFR1 alterations on oncogenicity, therapeutic response and resistance has not been previously explored. To address this, we have established mouse neural stem cell models overexpressing the structural variants and hot spot mutant FGFR1 alone or in combination with a second alteration. Immunoblotting revealed that the addition of a second alteration attenuated phosphorylation of ERK, AKT and S6 and influenced cell proliferation both in normal growth conditions and in absence of growth factor. Treatment with inhibitors of FGFR (Infigratinib) and MEK (Trametinib) revealed variable sensitivity both targeted therapies, suggesting that treatment of FGFR1 driven pLGG might require tailoring to the specific FGFR1 alteration.