Pediatric high-grade glioma: aberrant epigenetics and kinase signaling define emerging therapeutic opportunities.

INTRODUCTION: Supratentorial pediatric high-grade gliomas (pHGGs) are aggressive malignancies that lack effective treatment options. Deep genomic sequencing by multiple groups has revealed that the primary alterations unique to pHGGs occur in epigenetic and kinase genes. These mutations, fusions, and deletions present a therapeutic opportunity by use of small molecules targeting epigenetic modifiers and kinases that contribute to pHGG growth. METHODS: Using a targeted search of the pre-clinical literature and clinicaltrials.gov for kinase and epigenetic pathways in pHGG, we collectively describe how these mechanisms are being targeted in pre-clinical animal models and in current clinical trials, as well as propose unexplored therapeutic possibilities for future investigations. RESULTS: Relevant pHGG kinases are targetable by several FDA-approved or clinical-stage kinase inhibitors, including altered BRAF/MET/NTRK/ALK and wild-type PI3K/EGFR/PDGFR/VEGF/AXL. Epigenetic proteins implicated in pHGG are also clinically targetable and include histone erasers, writers and readers such as HDACs, demethylases LSD1/JMJD3, methyltransferase EZH2, chromatin reader bromodomains, and chromatin remodeler subunit BMI-1. Crosstalk between these pathways can occur involving kinases such as EGFR and AMPK interacting with epigenetic modifiers such as HDACs or EZH2. Single agent trial results of kinase inhibitors or epigenetic targets alone are underwhelming and hampered by poor pharmacokinetics, adaptive resistance, and broad inclusion criteria. CONCLUSIONS: The genetic and phenotypic diversity of pHGGs is now well characterized after large-scale sequencing studies on patient tissue. However, clinical treatment paradigms have not yet shifted in response to this information. Combination therapies targeting multiple kinases or epigenetic targets may hold more promise, especially if attempted in selected patient populations with hemispheric pHGG tumors and relevant targeted therapeutic biomarkers.

Double haploidentical hematopoietic stem cell transplantation results in successful engraftment of bone marrow from both donors without graft-versus-host or graft-versus-graft effects.

We established double-haploidentical (DH) hematopoietic stem cell transplantation (HSCT) murine models to explore competitive engraftment, graft-versus-graft effect and graft-versus-host disease (GVHD). T cell-depleted (TCD) bone marrow (BM) cells from B6SJF1 (donor 1 [D1]) and B6D2F1 (donor 2 [D2]) mice achieved >90% donor engraftment when transplanted into B6CBAF1 mice. B6CBAF1 recipients survived without evidence of GVHD when undergoing HSCT with TCD-BM from 2 haploidentical donors, D1 and D2. DH-HSCT recipients had significantly higher leukocyte and neutrophil counts than single-haploidentical HSCT recipients from either D1 or D2. DH recipients consistently showed successful mixed chimerism in both BM and spleen. Two other DH-HSCT models, B6D2F1 + C3D2F1→B6C3F1 and B6CBAF1 + B6SJLF1→B6D2F1, showed similar engraftment patterns. Low-dose T cell infusion from both D1 and D2 increased the degree of early engraftment of the respective donors in BM and spleen; however, this early engraftment pattern did not determine long-term engraftment dominance. In the long term, minimally engrafted D1 BM recovered and comprised >50% of all donor- derived B, T, and natural killer cells. We conclude that early BM engraftment is determined by donor T cell immunodominance, but long-term engraftment is related to the engraftment potential of stem cells after DH-HSCT.

Computational immune infiltration analysis of pediatric high-grade gliomas (pHGGs) reveals differences in immunosuppression and prognosis by tumor location.

Immunotherapy for cancer has moved from pre-clinical hypothesis to successful clinical application in the past 15 years. However, not all cancers have shown response rates in clinical trials for these new agents, high-grade gliomas in particular have proved exceedingly refractory to immunotherapy. In adult patients, there has been much investigation into these failures, and researchers have concluded that an immunosuppressive microenvironment combined with low mutational burden render adult glioblastomas "immune cold". Pediatric cancer patients develop gliomas at a higher rate per malignancy than adults, and their brain tumors bear even fewer mutations. These tumors can also develop in more diverse locations in the brain, beyond the cerebral hemispheres seen in adults, including in the brainstem where critical motor functions are controlled. While adult brain tumor immune infiltration has been extensively profiled from surgical resections, this is not possible for brainstem tumors which can only be sampled at autopsy. Given these limitations, there is a dearth of information on immune cells and their therapeutic and prognostic impact in pediatric high-grade gliomas (pHGGs), including hemispheric tumors in addition to brainstem. In this report we use computational methods to examine immune infiltrate in pHGGs and discover distinct immune patterns between hemispheric and brainstem tumors. In hemispheric tumors, we find positive prognostic associations for regulatory T-cells, memory B-cells, eosinophils, and dendritic cells, but not in brainstem tumors. These differences suggest that immunotherapeutic approaches must be cognizant of pHGG tumor location and tailored for optimum efficacy.

Scaffolding LSD1 Inhibitors Impair NK Cell Metabolism and Cytotoxic Function Through Depletion of Glutathione.

Cell therapies such as chimeric-antigen receptor (CAR) T-cells and NK cells are cutting-edge methods for treating cancer and other diseases. There is high interest in optimizing drug treatment regimens to best work together with emerging cell therapies, such as targeting epigenetic enzymes to stimulate recognition of tumor cells by immune cells. Herein, we uncover new mechanisms of the histone demethylase LSD1, and various inhibitors targeting unique domains of LSD1, in the function of NK cells grown for cell therapy. Catalytic inhibitors (tranylcypromine and the structural derivatives GSK LSD1 and RN-1) can irreversibly block the demethylase activity of LSD1, while scaffolding inhibitors (SP-2509 and clinical successor SP-2577, also known as seclidemstat) disrupt epigenetic complexes that include LSD1. Relevant combinations of LSD1 inhibitors with cell therapy infusions and immune checkpoint blockade have shown efficacy in pre-clinical solid tumor models, reinforcing a need to understand how these drugs would impact T- and NK cells. We find that scaffolding LSD1 inhibitors potently reduce oxidative phosphorylation and glycolysis of NK cells, and higher doses induce mitochondrial reactive oxygen species and depletion of the antioxidant glutathione. These effects are unique to scaffolding inhibitors compared to catalytic, to NK cells compared to T-cells, and importantly, can fully ablate the lytic capacity of NK cells. Supplementation with biologically achievable levels of glutathione rescues NK cell cytolytic function but not NK cell metabolism. Our results suggest glutathione supplementation may reverse NK cell activity suppression in patients treated with seclidemstat.

Pharmacologic inhibition of lysine-specific demethylase 1 as a therapeutic and immune-sensitization strategy in pediatric high-grade glioma.

BACKGROUND: Diffuse midline gliomas (DMG), including brainstem diffuse intrinsic pontine glioma (DIPG), are incurable pediatric high-grade gliomas (pHGG). Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, rendering them therapeutically sensitive to small-molecule inhibition of chromatin modifiers. Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) is clinically relevant but has not been carefully investigated in pHGG or DIPG. METHODS: Patient-derived DIPG cell lines, orthotopic mouse models, and pHGG datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Immune cell cytotoxicity was assessed in DIPG cells pretreated with LSD1 inhibitors, and informatics platforms were used to determine immune infiltration of pHGG. RESULTS: Selective cytotoxicity and an immunogenic gene signature were established in DIPG cell lines using clinically relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit of this LSD1-dependent gene signature. Pretreatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. Catalytic LSD1 inhibitors induced tumor regression and augmented NK cell infusion in vivo to reduce tumor burden. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival, while CD8 T cells are negatively prognostic. Catalytic LSD1 inhibitors are nonperturbing to NK cells, while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene signature in DIPG cells. CONCLUSIONS: LSD1 inhibition using catalytic inhibitors is selectively cytotoxic and promotes an immune gene signature that increases NK cell killing in vitro and in vivo, representing a therapeutic opportunity for pHGG. KEY POINTS: 1. LSD1 inhibition using several clinically relevant compounds is selectively cytotoxic in DIPG and shows in vivo efficacy as a single agent.2. An LSD1-controlled gene signature predicts survival in pHGG patients and is seen in neural tissue from LSD1 inhibitor-treated mice.3. LSD1 inhibition enhances NK cell cytotoxicity against DIPG in vivo and in vitro with correlative genetic biomarkers.

Cutting Edge Therapeutic Insights Derived from Molecular Biology of Pediatric High-Grade Glioma and Diffuse Intrinsic Pontine Glioma (DIPG).

Pediatric high-grade glioma (pHGG) and brainstem gliomas are some of the most challenging cancers to treat in children, with no effective therapies and 5-year survival at ~2% for diffuse intrinsic pontine glioma (DIPG) patients. The standard of care for pHGG as a whole remains surgery and radiation combined with chemotherapy, while radiation alone is standard treatment for DIPG. Unfortunately, these therapies lack specificity for malignant glioma cells and have few to no reliable biomarkers of efficacy. Recent discoveries have revealed that epigenetic disruption by highly conserved mutations in DNA-packaging histone proteins in pHGG, especially DIPG, contribute to the aggressive nature of these cancers. In this review we pose unanswered questions and address unexplored mechanisms in pre-clinical models and clinical trial data from pHGG patients. Particular focus will be paid towards therapeutics targeting chromatin modifiers and other epigenetic vulnerabilities that can be exploited for pHGG therapy. Further delineation of rational therapeutic combinations has strong potential to drive development of safe and efficacious treatments for pHGG patients.

New interleukin-15 superagonist (IL-15SA) significantly enhances graft-versus-tumor activity.

Interleukin-15 (IL-15) is a potent cytokine that increases CD8+ T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8+ T cells (specifically CD44+ memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8+ T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8+ T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells.We then evaluated IL-15SA's effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.

Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo.

New therapies for glioblastoma (GBM) are needed, as five-year survival is <10%. The proteasome inhibitor marizomib (MRZ) has inhibitory and death-inducing properties unique from previous inhibitors such as bortezomib (BTZ), and has not been well examined in GBM. We evaluated the mechanism of death and in vivo properties of MRZ in GBM. The activation kinetics of initiator caspases 2, 8, and 9 were assessed using chemical and knockdown strategies to determine their contribution to cell death. Blood brain barrier permeance and proteasome inhibition by MRZ and BTZ were examined in an orthotopic GBM model. Blockade of caspase 9, relative to other caspases, was most protective against both MRZ and BTZ. Only MRZ increased the proteasome substrate p27 in orthotopic brain tumors after a single injection, while both MRZ and BTZ increased p21 levels after multiple treatments. Cleavage of caspase substrate lamin A was increased in orthotopic brain tumors from mice treated with MRZ or BTZ and the histone deacetylase inhibitor vorinostat. Our data indicate that MRZ induces caspase 9-dependent death in GBM, suggesting drug efficacy biomarkers and possible resistance mechanisms. MRZ reaches orthotopic brain tumors where it inhibits proteasome function and increases death in combination with vorinostat.