Diffuse intrinsic pontine glioma (DIPG) remains one of the most lethal brain tumors in all of childhood with no effective treatments besides radiation, which only extends survival a few months. Against this backdrop, our lab recently executed a focused CRISPR negative selection screen in DIPG cell lines after treatment with the histone deacetylase (HDAC) inhibitor panobinostat and discovered a strong co-dependence with the histone demethylase LSD1. To further explore the therapeutic potential of this synergistic interaction, we tested a drug library of HDAC- and LSD1- targeting drugs with the goal of identifying a combination with optimal synergy and blood brain barrier (BBB) penetration suitable for clinical translation. We were surprised to find that traditional catalytic LSD1 inhibitors had minimal effect in isolation and did not seem to synergize with HDAC inhibitors, while a recently described CoREST/LSD1 degrader named UM171 phenocopied the effects seen in our CRISPR screen. Degraders are a class of compounds that recruit an E3 ubiquitin ligase to a protein-of-interest and cause target ubiquitination and proteasomal degradation. Given our unexpected finding, we hypothesized that UM171 induces synergy with HDAC inhibitors through elimination of a scaffolding function of LSD1. To prove this, we knocked out LSD1 using CRISPR/Cas9 and subsequently treated with a panel of HDAC inhibitors, which showed a signification sensitization of DIPG cells to HDAC inhibitors compared to standard controls. We also confirmed that UM171 interacts with the CoREST complex (members include LSD1, RCOR1, HDAC1/2) by performing streptavidin bead pull down with a newly synthesized biotin-conjugated UM171 probe. In summary, our results show that targeting LSD1 for degradation in combination with HDAC inhibition is a synergistic strategy in DIPG worthy of further translational study.
Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are uniformly lethal malignancies with currently no targeted therapies available. They exclusively occur in the cerebral hemispheres of adolescents and young adults, and have been linked to a distinct interneuronal lineage of origin. The developmental spectrum and functional role of this interneuronal lineage in DHG-H3G34 remain incompletely understood. Here, through integrating bulk and single-cell multi-omics with genome-wide CRISPR-Cas9 screens, we resolve a putative cellular hierarchy that follows a continuum of interneuronal lineage development, ranging from a self-renewing progenitor-like cell to a more differentiated cell resembling early immature GABAergic interneurons, along with quiescent astrocyte-like and mesenchymal-like cells. We validate these single-cell states in patient DHG-H3G34 tissue sections by multiplexed immunofluorescence, and describe spatial structures that resemble nests of early migratory interneurons surrounded by progenitor cells, characteristic of human embryonal interneuron development. Intriguingly, we reveal the majority of CRISPR-Cas9 screen-derived gene dependencies are upregulated in interneuronal lineage tumor cells, specifically in less differentiated progenitor-like cells, highlighting these as a driver of DHG-H3G34. We validate the essentiality of these interneuronal lineage associated targets in patient-derived in vitro and in vivo models, and highlight CDK6 as a druggable target selectively upregulated in DHG-H3G34. Inhibition of CDK6 leads to a decrease of undifferentiated progenitor-like signatures, reduced tumor growth, and prolonged survival of patient-derived xenograft models. Encouraged by these findings, we treated a patient upon a second relapse of a DHG-H3G34 with ribociclib on a compassionate use basis, who, as of the time of submission, has shown stable disease within four cycles of ribociclib treatment after progression on PCV chemotherapy. In sum, we reveal CDK6 inhibition as a rationally informed and clinically actionable therapeutic avenue that selectively perturbs the unique interneuronal lineage in DHG-H3G34, paving the way for rapid clinical translation.
Histone 3 lysine27-to-methionine mutations (H3-K27M) frequently occur in childhood diffuse midline gliomas (DMGs) of the pons, thalamus, and spinal cord, presumed to be driven by the specific spatiotemporal context of these midline locations during postnatal development. While most common in the pons and at mid-childhood ages, the same oncohistone mutation is recurrently detected in adult DMGs and throughout different midline regions. The potential heterogeneity of tumors at different ages and in different anatomical locations of the midline are vastly understudied. Through dissecting the transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs - spanning the age range from 2-68 years and locations from spinal cord to thalamus - at single cell resolution, we delineate how age- and location-dependent contexts shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We identify that oligodendrocyte precursor (OPC)-like cells constitute the stem-like compartment in H3-K27M DMGs across all clinico-anatomical groups, however, depending on location, display varying levels of maturity resembling less differentiated pre-OPCs or more mature OPCs further differentiated along the oligodendroglial lineage. We further demonstrate increased mesenchymal cell states in adult tumors, which we link to age-related differences in glioma-associated immune cell compartments, in particular an increase of macrophages in adult compared to pediatric tumors. Furthermore, we resolve the spatial organization of H3-K27M DMG cell types and states in intact patient tissues, identifying a local niche of the oligodendroglial lineage. Our study provides a powerful resource for rational modeling and therapeutic frameworks taking into account determinants of age and location in this lethal glioma group.
High-grade gliomas harboring H3 G34R/V mutations exclusively occur in the cerebral hemispheres of adolescents and young adults, suggesting a distinct neurodevelopmental origin. Combining multimodal bulk and single-cell genomics with unbiased genome-scale CRISPR/Cas9 approaches, we here describe a GABAergic interneuron progenitor lineage as the most likely context from which these H3 G34R/V mutations drive gliomagenesis, conferring unique and tumor-selective gene targets essential for glioma cell survival, as validated genetically and pharmacologically. Phenotypically, we demonstrate that while H3 G34R/V glioma cells harbor the neurotransmitter GABA, they are developmentally stalled, and do not induce the neuronal hyperexcitability described in other glioma subtypes. These findings offer a striking counter-example to the prevailing view of glioma origins in glial precursor cells, resulting in distinct cellular, microenvironmental, and therapeutic consequences.
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