Mutations In ACVR1 Research Articles

PATH-13. MOLECULAR AND BIOLOGY CORRELATIVE RESULTS FROM THE PHASE 1B TRIAL OF UNESBULIN (PTC596) WITH RADIOTHERAPY IN CHILDREN NEWLY-DIAGNOSED WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE NETWORK FOR NEURO-ONCOLOGY CLINICAL TRIALS (CONNECT)

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (>23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.

DIPG-22. A NOVEL ROLE FOR ACVR1/ALK2 IN REGULATING CHOLESTEROL BIOSYNTHESIS PROVIDES A NEW COMBINATORIAL THERAPEUTIC APPROACH FOR PATIENTS WITH DMG

Abstract BACKGROUND The discovery of somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, in 20-25% of diffuse midline glioma H3K27-altered (DMG-H3K27) patients has led to the development of numerous chemotypes of ALK2 inhibitor (ALK2i). However, in models of ACVR1-mutant DMG-H3K27 the efficacy of single-agent ALK2i’s has been modest and new combinatorial treatment approaches are desperately needed. METHODS We performed unbiased combinatorial genetic and drug screens in patient-derived ACVR1-mutant DMG-H3K27 cells in vitro, using multiple chemotypes of ALK2i. We also generated in vitro transcriptomic, proteomic and metabolomic profiles of three ACVR1-mutant DMG-H3K27 models following ALK2i treatment. RESULTS The top ALK2i sensitising CRISPR hits (vehicle vs. ALK2i) were analysed using gene set enrichment analysis (GSEA) which revealed specific genetic dependencies in cholesterol synthesis/regulation following both M4K2009 and TP-0184 treatment. The top CRISPR hits included multiple key nodes of cholesterol biosynthesis such as HMGCR (HMG-CoA>mevalonate), LSS (squalene-2-2-epoxide>lanosterol) and DHCR24 (lanosterol>cholesterol). These hits were pharmacologically mirrored in the top hits identified from the drug screen, including drugs that either directly or indirectly target cholesterol synthesis. Integration of the multi-omic data revealed a raft of changes associated with cholesterol biosynthesis/efflux. The expression of HMGCR/HMGCS1 (cholesterol synthesis) and LXR/ABCA1 (cholesterol export) were down and upregulated, respectively, with metabolomic data showing a reduction in cholesterol and it’s derivative 4-cholesten-3-one and an increase in desmosterol, an immediate precursor to cholesterol synthesis. Highly synergistic interactions were observed between multiple ALK2i and a panel of pharmacological agents targeting the cholesterol synthesis/efflux pathway, including routinely used drugs such as simvastatin and lovastatin, clinically well-tolerated and with good CNS penetrance in immuno-compromised and immune-competent mouse models, in whom in vivo combination studies are ongoing. CONCLUSIONS These data provide evidence for a previously unappreciated link between ALK2 signalling and metabolism, and identify readily translatable combination partners for ALK2i in the clinic.

DIPG-31. IS RE-IRRADIATION A POTENTIAL SURVIVAL BENEFIT FOR PATIENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMA?

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is an aggressive infiltrative glioma with dismal prognosis. Radiation therapy (RT) offers a transient benefit and re-irradiation (reRT) may be an option at disease progression. OBJECTIVE To evaluate the outcome of patients with DIPG at progression in a single institution. METHODS Between 2010-2023, 55 patients were evaluated, retrospectively, in respect of their treatment-one RT course versus re-RT) and concomitant medications. Overall survival (OS) was measured from diagnosis to date of death and from reRT to death using Kaplan–Meier and log-rank test. RESULTS Median age was 7.1 years (range 2.9-16.2y). Twenty-two patients underwent biopsy with 11 performing immunohistochemistry for H3K27M (9 mutated). In nine cases molecular analyses was evaluated: 6 H3.3K27M-mutant (2 simultaneously TP53 + 1PTPRZ1-MET fusion), 1 H3.1K27M-mutant, 1 H3C2-mutant + ACVR1 mutation and 1 PDGFRA + KIT amplifications. Thirty-five patients completed the initial course of RT (54Gy- 13-33 daily fraction), 30/35 also received valproic acid (VA), 5/35 bevacizumab, 3/35 nimotuzumab and 1/35 nivolumab+ipilimumab. Twenty patients underwent re-RT at first progression, four with 54 Gy (30x 180Gy), nine with 50Gy (25x 200Gy) and seven with median dose of 30Gy (26-39Gy), depending on their clinical status. These group also received valproic acid(8/20), bevacizumab(8/20), nivolumab(1/20) and ONC 201(1/20). Patients who received reRT showed prolonged OS compared to no reRT (median 524 vs 351 days; p=0.013). ReRT was well tolerated with 15(75%) patients experiencing stable to improved neurologic function. Patients with better clinical status and 50-54Gy reRT doses, showed prolonged OS (median 238 vs 135 days; p=0.1). Median OS from re-RT to death was 174 days (range, 14-218 days) with eight patients alive to date. CONCLUSIONS Re-irradiation with 50-54Gy seems safe and feasible for DIPG patients after disease progression from the first treatment, with mostly patients experiencing neurological improvement and prolonged life period.

IMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva

BackgroundFibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP.MethodsIn the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively.ResultsWe found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs.ConclusionsThese results offer a new perspective for treating FOP through stem cell therapy.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood–brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

A fibrodysplasia ossificans progressiva patient with a rare missense mutation in ACVR1 detected on 18F-FDG PET/CT

Fibrodysplasia ossificans progressiva (FOP) is an exceedingly rare human genetic disorder characterized by the progressive and incapacitating formation of ectopic bone outside the skeleton. We report a case of FOP patient with mutations within the ACVR1 gene (c.982G>A; p.G328R). 18F-FDG positron emission tomography/computed tomography (PET/CT) was carried out for disease assessment. Previous studies have shown increased FDG uptake in regions of heterotopic ossification (HO) in FOP. However, in our study, the PET/CT features demonstrate that active ossificans exhibit increased 18F-FDG uptake, whereas end-stage ossifications do not. Collectively, 18F-FDG PET/CT emerges as a prospective approach to evaluate medication efficacy in the early stages, directing early intervention and pharmacological management of FOP before ossifications formation.

TMIC-11. SPATIAL PROFILING OF TUMOR VASCULATURE AT AUTOPSY IN BRAINSTEM GLIOMAS

Abstract BACKGROUND Diffuse midline glioma (DMG) is a lethal pediatric tumor, and new treatment strategies are urgently needed. To design therapies that can cross the blood-tumor barrier, we designed a pilot study to investigate protein expression on DMG tumor vasculature using spatial profiling, with attention to targetable surface proteins. METHODS We identified 5 patients and 4 controls consented to institutional tissue banking protocols with adequate autopsy tissue from multiple anatomical regions within the brainstem. Portions of formalin-fixed, paraffin-embedded tissue were arranged into tissue microarrays profiled using the nanoString GeoMX platform. Briefly, a multiplexed cocktail of 51 primary antibodies tagged with UV-photocleavable oligonucleotide barcodes and fluorescent morphology markers were applied to tissues and 10-14 regions of interest (ROIs) were segmented by vessel area for each patient. ROIs were processed using UV light and barcodes were quantified using ex situ digital counting. RESULTS Five patients were included in the pilot study with average age 9.8 years (range 8-13), and overall survival was 9.5 months (range 3-18 months). Three tumors were classified as DMG, H3K27-mutant, WHO grade 4; one as high grade glioma without identified histone alteration but with recurrent DMG drivers (ACVR1 and PIK3CA mutations) and one as infiltrating atypical glioma with high proliferative rate and no DMG driver mutations. Controls ranged from 4-15 years and did not have pathological changes in the brain at death. Protein abundance was compared between cases and control and within cases where tumor-adjacent normal tissue was identified. Several proteins were significantly more abundant on tumor vasculature compared to non-tumor adjacent or control brainstem vessels. Validation studies in primary tissue are ongoing. CONCLUSIONS Digital spatial profiling is a promising technology for investigating tumor vasculature and is feasible in autopsy specimens. Work is ongoing to validate candidate biomarkers and expand the cohort to develop a vascular atlas for DMG.

OR29-06 Flare-Ups In Patients With Fibrodysplasia Ossificans Progressiva Reduced By Garetosmab Treatment: LUMINA-1 Data

Abstract Disclosure: K.M. Dahir: Consulting Fee; Self; Alexion Pharmaceuticals, Inc., Ultragenyx, Inozyme, AM Pharma. Grant Recipient; Self; Regeneron Pharmaceuticals, Alexion Pharmaceuticals, Inc., AstraZeneca, Ultragenyx. J. McGinniss: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. E. Forleo-Neto: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. S. Mellis: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. R.J. Sanchez: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. M. Di Rocco: Research Investigator; Self; Regeneron Pharmaceuticals, Ipsen. R. Keen: Advisory Board Member; Self; International Clinical Council on FOP and IFOPA Registry. Research Investigator; Self; Ipsen, Regeneron Pharmaceuticals, Clementia. P. Orcel: Research Investigator; Self; Regeneron Pharmaceuticals. C. Roux: Consulting Fee; Self; Alexion Pharmaceuticals, Inc., Amgen Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Kyowa, Kirin Brewery, Alexion Pharmaceuticals, Inc. J. Tabarkiewicz: Speaker; Self; Merck, Novartis Pharmaceuticals. Other; Self; SoftSystem. J. Bachiller-Corral: Research Investigator; Self; Regeneron Pharmaceuticals. A.M. Cheung: Consulting Fee; Self; Ipsen. Grant Recipient; Self; Ipsen, Incyte, Regeneron Pharmaceuticals. M. Al Mukaddam: Grant Recipient; Self; Ipsen, Clementia, Incyte, Regeneron Pharmaceuticals. K. Mohammadi: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. D. Srinivasan: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. A. Rankin: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. A.N. Economides: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. D. Gonzalez Trotter: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. F.S. Kaplan: Research Investigator; Self; Regeneron Pharmaceuticals, Clementia, Ipsen. M.W. Eekhoff: Grant Recipient; Self; Regeneron Pharmaceuticals, Ipsen, AstraZeneca, IMI, IFOPA. R.J. Pignolo: Grant Recipient; Self; Regeneron Pharmaceuticals, Clementia, Ipsen, Incyte. Background: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare autosomal dominant disorder driven by missense mutations in ACVR1. This results in inappropriate activation by activin-A. FOP is characterized by progressive heterotopic ossification and painful soft tissue inflammatory events known as flare-ups. Garetosmab, an investigational, fully human monoclonal antibody against activin-A, prevents formation of new heterotopic ossification lesions in FOP. Here we describe the impact of garetosmab on flare-up events in the LUMINA-1 study (NCT03188666). Methods: This was a post hoc analysis of phase 2 LUMINA-1, a randomized double-blind placebo-controlled study that evaluated the safety and efficacy of garetosmab 10 mg/kg/every 4 weeks intravenous vs placebo in adult patients with FOP over 28 weeks (Period 1), followed by a 28-week open-label treatment period (Period 2) and subsequent open-label extension (Period 3). Patient-reported flare-ups were collected via a patient diary and severity of symptoms was reported as mild, moderate, or severe. Clinician-reported flare-ups were collected as adverse events. Results: In Period 1, there was a significant reduction in the proportion of patients reporting one or more flare-ups (35% vs 71%, p=0.032) and clinician-reported flare-ups (10% vs 42%, p=0.039) with garetosmab vs placebo, respectively. The overall number of patient-reported flare-ups (13 vs 34) and mean days experiencing new flare-ups (42.6 days vs 65.4 days) were also reduced with garetosmab vs placebo, respectively. Most flare-ups occurred in the back in garetosmab-treated patients, and most flare-ups occurred in the lower extremities and back for those on placebo. Pain was the most frequent symptom among both cohorts. Flare-up associated joint stiffness and severity of flare-up associated symptoms were nominally reduced at both patient and flare-up level of assessment in those treated with garetosmab. No patients reported severe swelling or severe decrease in movement. One patient reported severe pain and joint stiffness in the garetosmab cohort. In Period 2, there were significant reductions in the proportion of patients experiencing flare-ups (68% vs 14%, p=0.0002) and number of patient-reported flares (31 vs 11) among those who crossed over from placebo in Period 1 to garetosmab. Patients who continued on garetosmab through Period 2 maintained a sustained reduction in number of flare-ups (12 vs 6) and proportion of patients experiencing flare-ups (33.3% vs 22.2%). Reductions were maintained through the open-label extension. Conclusions: Patients treated with garetosmab experienced significant and sustained reductions in the frequency, duration, and severity of flare-ups. The ability of garetosmab to reduce flare-up events may provide a clinically meaningful benefit for patients with FOP. Presentation: Sunday, June 18, 2023

Novel Therapeutic Targets for Fibrodysplasia Ossificans Progressiva: Emerging Strategies and Future Directions.

Fibrodysplasia ossificans progressiva (FOP), also known as Stoneman syndrome, is a rare genetic disorder characterized by abnormal bone development caused by activating mutations of the ACVR1 gene. FOP affects both the developmental and postnatal stages, resulting in musculoskeletal abnormalities and heterotopic ossification. Current treatment options for FOP are limited, emphasizing the need for innovative therapeutic approaches. Challenges in the development of management criteria for FOP include difficulties in recruitment due to the rarity of FOP, disease variability, the absence of reliable biomarkers, and ethical considerations regarding placebo-controlled trials. This narrative review provides an overview of the disease and explores emerging strategies for FOP treatment. Gene therapy, particularly the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system, holds promise in treating FOP by specifically targeting the ACVR1 gene mutation. Another gene therapy approach being investigated is RNA interference, which aims to silence the mutant ACVR1 gene. Small molecule inhibitors targeting glycogen synthase kinase-3β and modulation of the bone morphogenetic protein signaling pathway are also being explored as potential therapies for FOP. Stem cell-based approaches, such as mesenchymal stem cells and induced pluripotent stem cells, show potential in tissue regeneration and inhibiting abnormal bone formation in FOP. Immunotherapy and nanoparticle delivery systems provide alternative avenues for FOP treatment.

DIPG-06. PRECLINICAL EVALUATION OF A NOVEL ACVR1 INHIBITOR IN DIFFUSE INTRINSIC PONTINE GLIOMAS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable brain disease in children. DIPG is refractory to current available therapies. Hence, new therapeutic approaches are urgently needed. About 80% of DIPGs harbor histone 3 lysine to methionine (H3K27M) mutation, in H3-3A (encoding H3.3 histone) and H3C2 (encoding H3.1 histone) genes. One quarter of DIPG tumors harbor somatic mutations in ACVR1 gene, encoding the serine/threonine kinase receptor ALK2. ACVR1 mutations co-segregate with H3.1K27M mutation, representing a specific tumour subtype with distinct clinicopathological and molecular features. These mutations are associated with an aberrant activation of SMAD pathway believed to promote cell proliferation in DIPGs. In the present studies, we evaluated the effectiveness of an orally bioavailable ALK inhibitor, TP-0184 (Tolero Pharmaceuticals) in ACVR1 mutated (ACVR1-mut) and wildtype (ACVR1- WT) DIPG models. Our results indicate that TP-0184 effectively inhibited cell proliferation in primary-patient derived DIPG cell lines. However, we did not observe any selectivity toward ACVR1 mutations. TP-0184 does not induce cell death, but rather cell cycle inhibition, arresting cells in G2/M phase. Interestingly, when treated with ionizing radiation (IR), ACVR1-mut cells preferentially activated SMAD signaling compared to ACVR1-WT. We are currently evaluating the combination efficacy of TP-0184 with IR in our in vitro and in vivo DIPG models. Data on tumor growth kinetics, survival and the effect on SMAD signaling pathway will be presented and discussed.

DIPG-29. A CONNECT CONSORTIUM PRECLINICAL STUDY IDENTIFIES MEK INHIBITION AND RADIATION AS POTENTIAL COMBINATION PARTNERS FOR THE ALK2 INHIBITOR TP-0184 IN ACVR1-MUTANT DMG

Abstract Somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, are found in 20-25% of DMG-H3K27 patients. Treatment of ACVR1-mutant orthotopic xenografts with multiple chemotypes of ALK2 inhibitors (ALK2i) results in extended survival in vivo but, as single agents, these inhibitors are unable to achieve a complete anti-tumour response. To identify novel combination strategies alongside ALK2 inhibition, five international laboratories pooled resources to evaluate the ALK2i TP-0184 across 26 patient-derived DMG-H3K27 models in vitro, demonstrating GI50 values of ~0.5-10uM, although with no significant difference between ACVR1-mutant and wild-type. Genome-scale CRISPR screens of ALK2i-treated ACVR1-mutant models revealed specific genetic dependencies in critical nodes of several signalling pathways including MTOR, as well as PPP2R1A, known to play a role in MAPK pathway activation. Biomarker analysis showed inhibition of phospho-SMAD1/5/9 by western blot, with RNAseq and proteomic analysis indicating downstream signalling consequences in BMP/SMAD signalling following TP-0184 treatment. We observed synergistic pharmacological interactions between TP-0184 and both everolimus (mTORi) and trametinib (MEKi) in 7 to 8 ACVR1-mutant DMG models, respectively, with the latter validated across laboratories using orthogonal assays. Importantly, combining TP-0184 with radiation in vitro showed a profound radiosensitisation effect of the ALK2i in three ACVR1-mutant models using both clonogenic and cell viability assays in multiple laboratories, in part due to an observed activation of phospho-SMAD1/5/9 by radiation. Finally, in vivo tolerability and pharmacokinetic assays in multiple immuno-compromised and immune-competent mouse strains suggested a dose of 150mg/kg TP-0184 and 0.4mg/kg trametinib for use in in vivo efficacy studies, with good CNS penetrance of both drugs in excess of in vitro GI50 values. Such studies have been initiated in six DMG-H3K27 models across our laboratories to robustly assess the combination of TP-0184 and trametinib/radiation to support rapid potential translation to clinical trial.

DIPG-50. INSIGHTS INTO TUMORIGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMA-ONCOHISTONE AND SIGNALING

Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable pediatric brain cancer with survival of less than one year. Understanding the tumorigenesis mechanisms of DIPG and identifying potential therapeutic strategy are major research foci in the DIPG field. The most lethal subtype of diffuse intrinsic pontine glioma (DIPG) is the H3K27M subtype. Although ACVR1 mutations have been implicated in the pathogenesis of this presently incurable disease, the impacts of BMP signaling on more than 60% of H3K27M DIPG carrying ACVR1 wild-type remain unknown. Here, we show that BMP ligands exert potent tumor suppressive effects against H3.3 K27M and ACVR1 WT DIPG in a SMAD-dependent manner. Specifically, clinical data revealed that many DIPG tumors have exploited CHRDL1’s capacity to hijack BMP ligands. We discovered activation of BMP signaling promotes the exit of DIPG tumor cells from “prolonged-stem-cell-like” state to differentiation by epigenetically regulating CXXC5, which acts as a tumor suppressor and positive regulator of BMP signaling. Beyond showing how BMP signaling impacts DIPG, our study also identified potent anti-tumor efficacy of Dacinostat for DIPG. Thus, our study delineates context-dependent features of BMP signaling pathway in DIPG subtype.

Palovarotene (Sohonos)


 CADTH recommends that Sohonos be reimbursed by public drug plans to reduce the formation of heterotopic ossification (HO) in adults and children aged 8 years and above for females and 10 years and above for males with fibrodysplasia ossificans progressiva (FOP), if certain conditions are met.
 Sohonos should only be covered to treat patients who have a clinical diagnosis of FOP and the R206H ACVR1 mutation as confirmed by genetic testing, and who do not have completely fused joints over the whole body.
 Sohonos should only be reimbursed if prescribed by an expert in the diagnosis and management of FOP and if the cost of Sohonos is reduced. While receiving Sohonos, patients (and their caregivers, for pediatric patients) and their physicians should have ongoing and regular discussions to assess the benefits and risks of treatment with Sohonos.

Abstract 4990: Dual inhibitor of ACVR1 and MEK 1/2 E6201 and PI3K/mTOR inhibitor paxalisib synergistically inhibit cell growth in DMG

Abstract Diffuse midline glioma (DMG) is an aggressive pediatric glioma with no curative treatment options. Mutation in MAPK pathway is common in many DMG tumors, however, has not been largely explored before. E6201 is a dual inhibitor of ACVR1 and MEK 1/2. It is currently in a phase 1 clinical trial for BRAF/MEK mutant melanoma. E6201 achieves higher levels in brain compared to other MEK inhibitors, suggesting it might be a promising therapeutic option for DMG. Previously, E6201 was effective in DMG cell lines with ACVR1 mutation, such as HSJD007. We have evaluated the effect of E6201 in two MAP kinase pathway activated, novel DMG cell lines, DIPG2JA and JHH-DIPG17. DIPG2JA has a KRAS mutation, leading to upregulation of MAPK pathway, and JHH-DIPG17 has two NF1 alterations predicted to disrupt this tumor suppressor, resulting in activation of MAPK pathway. Treatment of DIPG2JA with E6201 identified an IC50 of 44 nM, which is comparable to an IC50 of 71nM in the ACVR1 mutant DMG HSJD007. E6201 suppressed phospho—ERK and induced apoptosis as measured by cPARP western blot in DMG cells. Evaluation of cell viability by CellTiterBlue viability assay demonstrated that E6201 suppressed DMG growth between 0.05uM to 1uM concentrations. (DIPG2JA p=0.0004, JHH-DIPG17 p<0.0001 by t-test compared to control). Compensatory upregulation of mTOR pathway is often associated with downregulation of MAPK pathway, and we hypothesized that E6201 will synergize with a PI3K/mTOR inhibitor to induce DMG cell death. We selected the brain-penetrant PI3K/mTOR inhibitor paxalisib, because this drug is currently being tested in early phase clinical trials for DMG. Through CellTiterBlue cell viability assay, we showed that E6201 synergizes with paxalisib to inhibit cell growth. The ZIP synergy scores were 13.1 for HSJD007 and 12.7 for DIPG2JA according to the SynergyFinder program, suggesting synergistic interaction between E6201 and paxalisib. In vitro experiments for further evaluation of the combination treatment of E6201 and paxalisib are underway and in vivo studies are pending. From our data, E6201 as a single agent is a promising therapeutic option for DMG. With further evaluation of combination of E6201 and paxalisib, we believe that combination therapy will be an even more effective treatment option for DMG. Citation Format: Hyuk Jean Kwon, Abigail Tindall, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Dual inhibitor of ACVR1 and MEK 1/2 E6201 and PI3K/mTOR inhibitor paxalisib synergistically inhibit cell growth in DMG. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4990.

Histone H3-wild type diffuse midline gliomas with H3K27me3 loss are a distinct entity with exclusive EGFR or ACVR1 mutation and differential methylation of homeobox genes

Diffuse midline gliomas (DMG) harbouring H3K27M mutation are paediatric tumours with a dismal outcome. Recently, a new subtype of midline gliomas has been described with similar features to DMG, including loss of H3K27 trimethylation, but lacking the canonical H3K27M mutation (H3-WT). Here, we report a cohort of five H3-WT tumours profiled by whole-genome sequencing, RNA sequencing and DNA methylation profiling and combine their analysis with previously published cases. We show that these tumours have recurrent and mutually exclusive mutations in either ACVR1 or EGFR and are characterised by high expression of EZHIP associated to its promoter hypomethylation. Affected patients share a similar poor prognosis as patients with H3K27M DMG. Global molecular analysis of H3-WT and H3K27M DMG reveal distinct transcriptome and methylome profiles including differential methylation of homeobox genes involved in development and cellular differentiation. Patients have distinct clinical features, with a trend demonstrating ACVR1 mutations occurring in H3-WT tumours at an older age. This in-depth exploration of H3-WT tumours further characterises this novel DMG, H3K27-altered sub-group, characterised by a specific immunohistochemistry profile with H3K27me3 loss, wild-type H3K27M and positive EZHIP. It also gives new insights into the possible mechanism and pathway regulation in these tumours, potentially opening new therapeutic avenues for these tumours which have no known effective treatment.This study has been retrospectively registered on clinicaltrial.gov on 8 November 2017 under the registration number NCT03336931 (https://clinicaltrials.gov/ct2/show/NCT03336931).

Multimodal MRI radiomic models to predict genomic mutations in diffuse intrinsic pontine glioma with missing imaging modalities.

Predicting H3.1, TP53, and ACVR1 mutations in DIPG could aid in the selection of therapeutic options. The contribution of clinical data and multi-modal MRI were studied for these three predictive tasks. To keep the maximum number of subjects, which is essential for a rare disease, missing data were considered. A multi-modal model was proposed, collecting all available data for each patient, without performing any imputation. A retrospective cohort of 80 patients with confirmed DIPG and at least one of the four MR modalities (T1w, T1c, T2w, and FLAIR), acquired with two different MR scanners was built. A pipeline including standardization of MR data and extraction of radiomic features within the tumor was applied. The values of radiomic features between the two MR scanners were realigned using the ComBat method. For each prediction task, the most robust features were selected based on a recursive feature elimination with cross-validation. Five different models, one based on clinical data and one per MR modality, were developed using logistic regression classifiers. The prediction of the multi-modal model was defined as the average of all possible prediction results among five for each patient. The performances of the models were compared using a leave-one-out approach. The percentage of missing modalities ranged from 6 to 11% across modalities and tasks. The performance of each individual model was dependent on each specific task, with an AUC of the ROC curve ranging from 0.63 to 0.80. The multi-modal model outperformed the clinical model for each prediction tasks, thus demonstrating the added value of MRI. Furthermore, regardless of performance criteria, the multi-modal model came in the first place or second place (very close to first). In the leave-one-out approach, the prediction of H3.1 (resp. ACVR1 and TP53) mutations achieved a balanced accuracy of 87.8% (resp. 82.1 and 78.3%). Compared with a single modality approach, the multi-modal model combining multiple MRI modalities and clinical features was the most powerful to predict H3.1, ACVR1, and TP53 mutations and provided prediction, even in the case of missing modality. It could be proposed in the absence of a conclusive biopsy.

TGF-Beta Induces Activin A Production in Dermal Fibroblasts Derived from Patients with Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic, ultra-rare disease of heterotopic ossification caused by genetic defects in the ACVR1 gene. The mutant ACVR1 receptor, when triggered by an inflammatory process, leads to heterotopic ossification of the muscles and ligaments. Activin A has been discovered as the main osteogenic ligand of the FOP ACVR1 receptor. However, the source of Activin A itself and the trigger of its production in FOP individuals have remained elusive. We used primary dermal fibroblasts from five FOP patients to investigate Activin A production and how this is influenced by inflammatory cytokines in FOP. FOP fibroblasts showed elevated Activin A production compared to healthy controls, both in standard culture and osteogenic transdifferentiation conditions. We discovered TGFβ1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFβ1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFβ1 in both cell groups. We present for the first time TGFβ1 as a triggering factor of Activin A production in FOP. As TGFβ1 can promote the induction of the main driver of FOP, TGFβ1 could also be considered a possible therapeutic target in FOP treatment.

Bone morphogenetic protein signaling is a possible therapeutic target in gynecologic cancer.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily. BMPs play crucial roles in embryogenesis and bone remodeling. Recently, BMP signaling has been found to have diverse effects on different types of tumors. In this review, we summarized the effects of BMP signaling on gynecologic cancer. BMP signaling has tumor-promoting effects on ovarian cancer (OC) and endometrial cancer (EC), whereas it has tumor-suppressing effects on uterine cervical cancer (UCC). Interestingly, EC has frequent gain-of-function mutations in ACVR1, encoding one of the type I BMP receptors, which are also observed in fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. Little is known about the relationship between BMP signaling and other gynecologic cancers. Tumor-promoting effects of BMP signaling in OC and EC are dependent on the promotion of cancer stemness and epithelial-mesenchymal transition (EMT). In accordance, BMP receptor kinase inhibitors suppress the cell growth and migration of OC and EC. Since both cancer stemness and EMT are associated with chemoresistance, BMP signaling activation might also be an important mechanism by which OC and EC patients acquire chemoresistance. Therefore, BMP inhibitors are promising for OC and EC patients even if they become resistant to standard chemotherapy. In contrast, BMP signaling inhibits UCC growth in vitro. However, the in vivo effects of BMP signaling have not been elucidated in UCC. In conclusion, BMP signaling has a variety of functions, depending on the types of gynecologic cancer. Therefore, targeting BMP signaling should improve the treatment of patients with gynecologic cancer.

K27M in canonical and noncanonical H3 variants occurs in distinct oligodendroglial cell lineages in brain midline gliomas.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate.