A road map for the treatment of pediatric diffuse midline glioma.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways.

Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293.

Receptor tyrosine kinase (RTK) targeting in pediatric high-grade glioma and diffuse midline glioma: Pre-clinical models and precision medicine.

Pediatric high-grade glioma (pHGG), including both diffuse midline glioma (DMG) and non-midline tumors, continues to be one of the deadliest oncologic diagnoses (both henceforth referred to as "pHGG"). Targeted therapy options aimed at key oncogenic receptor tyrosine kinase (RTK) drivers using small-molecule RTK inhibitors has been extensively studied, but the absence of proper in vivo modeling that recapitulate pHGG biology has historically been a research challenge. Thankfully, there have been many recent advances in animal modeling, including Cre-inducible transgenic models, as well as intra-uterine electroporation (IUE) models, which closely recapitulate the salient features of human pHGG tumors. Over 20% of pHGG have been found in sequencing studies to have alterations in platelet derived growth factor-alpha (PDGFRA), making growth factor modeling and inhibition via targeted tyrosine kinases a rich vein of interest. With commonly found alterations in other growth factors, including FGFR, EGFR, VEGFR as well as RET, MET, and ALK, it is necessary to model those receptors, as well. Here we review the recent advances in murine modeling and precision targeting of the most important RTKs in their clinical context. We additionally provide a review of current work in the field with several small molecule RTK inhibitors used in pre-clinical or clinical settings for treatment of pHGG.

Serial H3K27M cell-free tumor DNA (cf-tDNA) tracking predicts ONC201 treatment response and progression in diffuse midline glioma.

BACKGROUND: Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. METHODS: We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n = 24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). RESULTS: Change in H3.3K27M VAF over time ("VAF delta") correlated with prolonged PFS in both CSF and plasma samples. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .085). VAF "spikes" (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression. CONCLUSION: Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.

Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma.

Pediatric and adult high-grade gliomas (HGGs) frequently harbor PDGFRA alterations. We hypothesized that cotreatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib. We performed dose-response, synergism, P-glycoprotein inhibition, and pharmacokinetic studies in in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus. We found that dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with a reduction in mTOR signaling that persisted after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended the survival of PPK tumor-bearing mice (mutant TP53, mutant PDGFRA, H3K27M). Six pediatric patients with glioma tolerated this combination without significant adverse events, and 4 patients with recurrent disease (n = 4) had a median overall survival of 8.5 months. Our results show that the efficacy of dasatinib treatment of PDGFRα-driven HGG was enhanced with everolimus and suggest a promising route for improving targeted therapy for this patient population.

Papel da Stress Inducible Phosphoprotein One (STI1) e da proteína prion celular na angiogênese tumoral / The role of Stress Inducible Phosphoprotein One (STI1) and cellular prion protein in tumor angiogenesis

Proteínas multifuncionais podem ser consideradas uma vantagem evolutiva celular, ao passo que uma única proteína divide-se, simultaneamente, entre suas funções intracelulares e a sinalização extracelular para complementar mecanismos de responsividade tecidual. No papel de co-chaperona, a STI1 se complexa com HSP70/90, auxiliando no dobramento de proteínas. Reciprocamente, a secreção não convencional da STI1, em geral associada a vesículas extracelulares, permite a sua integração dinâmica com a demanda fisiológica pelas suas funções neurotróficas, ou até vasculogênicas, pelo recrutamento de precursores endoteliais para a regeneração do tecido cerebral durante a isquemia, sendo ambas mediadas via receptor PrPC (proteína príon). Em glioblastomas, a sinalização via STI1-PrPC também se mostrou determinante para a progressão tumoral. Dessa maneira, a STI1 apresenta um grande potencial de estudo como agente regulador dos processos angiogênicos tumorais em glioblastomas. As funções da STI1 foram avaliadas no modelo de glioma murino GL261 in vitro, ex vivo e in vivo em relação à invasão perivascular e à angiogênese peritumoral. A secreção de STI1 pelas células GL261 ocorre de maneira abundante, em sua maior parte na forma solúvel, não associada a vesículas extracelulares, por mecanismos ainda a serem desvendados, mas não controlados por hipóxia. O papel da STI1 no estímulo à giogênese sobre células endoteliais foi verificado em ensaios de formação de tubos (2D) e microvasos (3D) e em anéis aórticos. Porém, a contaminação por LPS nas preparações bacterianas de STI1 recombinante tornou inválidos nossos esforços e os ensaios foram interrompidos. Os ensaios in vivo foram realizados por meio da injeção intracerebral (ortotópica) de células GL261 em animais tipo-selvagem, haploinsuficientes (STI1+/-) e superexpressores de STI1 (TgA). A sobrevida dos animais STI1+/- e TgA foi estatisticamente superior a dos animais tipo-selvagem. Imunofluorescências com o marcador de células endoteliais CD31 indicaram uma maior vascularização intratumoral nos animais STI1+/-, enquanto animais TgA apresentaram marcação menor que animais tipo-selvagem. Tratamentos com anticorpos neutralizantes para STI1, da mesma forma que implantes ortotópicos de GL261 em animais deficientes para PrPC (PrP0/0), não produziram alterações no crescimento tumoral, que apresentou o mesmo padrão de invasão perivascular e de angiogênese peritumoral. A ausência de resposta também se reproduziu nos ensaios ex vivo dos tumores de GL261, a partir de co-culturas organotípicas com fatias de cérebros de animais STI1+/- e PrP0/0, em relação aos animais tipo-selvagem. Como abordagem final, as células GL261, previamente silenciadas para a expressão de STI1 ou HIF-1α, foram implantadas ortotopicamente no cérebro de animais tipo-selvagem. Apesar de terem sido selecionados os clones com menor nível de expressão de cada proteína, novamente não se verificaram alterações do crescimento tumoral e da angiogênese. Em conjunto, os resultados indicam que a proteína STI1 não possui efeito sobre o crescimento dos tumores de GL261, especialmente no que diz respeito à invasão perivascular e a vascularização intra/peritumoral. Linhagens celulares, porém, como a GL261, podem perder sua assinatura genética pela pressão seletiva a que são submetidas durante o seu período em cultura, o que compromete a sua representatividade in vivo em relação ao fenótipo tumoral infiltrativo próprio dos gliomas. A função extracelular de STI1 como modulador da angiogênese sobre células endoteliais ainda pode ser determinada com a utilização de proteína recombinante livre de LPS, ou sendo esta produzida por sistema heterólogo não-bacteriano

Multifunctional proteins might provide cells with an evolutionary advantage since a sole protein engages in concerted mechanisms of tissue responsiveness by simultaneously acting as a mediator of complementary intracellular and extracellular signaling pathways. Intracellularly, the cochaperone STI1 is an adaptor protein that aids in the folding of client proteins through the HSP90/70 complex. Conversely, the availability of cellular stocks of STI1 enables its dynamic mobilization by unconventional secretion, mostly associated with extracellular vesicles, in order to meet physiological demands extracellularly. Originally described as a neurotrophic growth factor, STI1 also holds vasculogenic functions, given the recruitment of endothelial progenitor cells positive for PrPC (prion protein) receptors from bone marrow to ischemic regions of mice brain, where it prompts vascular and functional recovery. In glioblastomas, signaling via STI1-PrPC axis was also showed to govern tumor progression. Thus, STI1 represents a potential target for modulation of tumor angiogenesis in glioblastomas (GBMs). We used the GL261 murine GBM cell line as an in vitro, ex vivo and in vivo model to evaluate the role of STI1 in certain aspects of perivascular invasion and peritumoral angiogenesis. GL261 cells secrete large amounts of freely soluble STI1, which is not influenced by hypoxia, and a small proportion associated to extracellular vesicles, in agreement with previously studied human GBM cell lines, although the mechanism for this protein segregation is still unknown. Recombinant STI1 apparently stimulated the endothelial sprouting as verified in 2D and 3D in vitro assays (matrigel tube formation and microcarrier-based microvessels formation), besides 3D ex vivo aortic ring assays. However, during these experiments, we found our recombinant protein with a high level of LPS contamination, due to failures in the established purification techniques used to process our bacterial preparations, which prevented the confirmation of STI1 as a pro-angiogenic factor. Orthotopic tumors were obtained by intracerebral injection of GL261 cells in wild-type, superexpressing STI1 (TgA) and haploinsufficient for STI1 (STI1+/-) C57BL/6J mice. Survival of STI1+/-and TgA mice was statistically higher compared to wild-type ones. Immunofluorescence staining of brain tissue sections with the endothelial cell marker CD31 revealed enhanced intratumoral vascularization, related to the wild-type controls, in STI1+/- mice, whereas TgAs presented as the least vascularized. Despite variations in intratumoral vascularization, tumor growth remained encapsulated within the brain parenchyma, without the typical difuse infiltration of GBMs associated with peritumoral angiogenesis. Even when tumors in wild-type mice were treated with neutralizing antibody for STI1, or GL261 cells were orthotopically implanted in PrP0/0 mice brains, tumor histology remained the same, reproducing results from organotypic co-cultures of wild-type, STI1+/-and PrP0/0 mice brain slices with GL261 tumors ex vivo. Unexpectedely, the highest knockdown of STI1 or HIF-1α obtained by lentiviral transduction in selected GL261 cell clones equally produced no effect over orthotopic tumor growth, perivascular invasion, and both intratumoral and peritumoral angiogenesis in wild-type mice brains. Taken together, our results show that STI1 might not affect GL261 tumor growth, and also raises conceptual caveats to the experimental utility of cancer cell lines as an in vivo representative of tumor phenotype. Future experiments might reveal the true angiogenic potential of LPS-free recombinant STI1 produced by a nonbacterial heterologous system

Crosstalk between kinases, phosphatases and miRNAs in cancer.

Reversible phosphorylation of proteins, performed by kinases and phosphatases, is the major post translational protein modification in eukaryotic cells. This intracellular event represents a critical regulatory mechanism of several signaling pathways and can be related to a vast array of diseases, including cancer. Cancer research has produced increasing evidence that kinase and phosphatase activity can be compromised by mutations and also by miRNA silencing, performed by small non-coding and endogenously produced RNA molecules that lead to translational repression. miRNAs are believed to target about one-third of human mRNAs while a single miRNA may target about 200 transcripts simultaneously. Regulation of the phosphorylation balance by miRNAs has been a topic of intense research over the last years, spanning topics going as far as cancer aggressiveness and chemotherapy resistance. By addressing recent studies that have shown miRNA expression patterns as phenotypic signatures of cancers and how miRNA influence cellular processes such as apoptosis, cell cycle control, angiogenesis, inflammation and DNA repair, we discuss how kinases, phosphatases and miRNAs cooperatively act in cancer biology.