Single nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of combined CDK4/6 and mTOR inhibition in a phase 0/1 trial of recurrent high-grade glioma.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A / B deletion or CDK4 / 6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740 .

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Quantitative protein expression of blood-brain barrier transporters in the vasculature of brain metastases of patients with lung and breast cancer.

This study determined absolute transporter protein abundances in isolated microvessels of human noncancerous cerebral cortex as well as brain metastases of patients with lung and breast cancer, using a validated targeted proteomics approach. As compared with those in microvessels of noncancerous cerebral cortex, the median protein abundances of glucose transporter 1 (a brain endothelial marker) and sodium-potassium pump (Na/K ATPase, a plasma membrane marker) were decreased by ~ 80% in brain metastasis microvessels. The major efflux transporters (ABCB1 and ABCG2) fell to undetectable in microvessels of more than 80% of brain metastasis specimens. Monocarboxylate transporter 1 was undetectable in microvessels of more than 80% of brain metastases, whereas large neutral amino acid transporter 1 levels remained unchanged. In conclusion, the integrity of the physical and biochemical barrier with respect to transporter protein expression is largely disrupted in brain metastasis tumor vasculatures. Differential transporter protein abundances at the blood-brain barrier and blood-brain tumor barrier provided mechanistic and quantitative basis for prediction of heterogeneous drug penetration into human brain and brain tumors, which is critical not only to the understanding of the success or failure of systemic chemotherapy in the treatment of brain tumors but also to the development of more effective therapeutic strategies.

A Phase 0 Trial of Ribociclib in Recurrent Glioblastoma Patients Incorporating a Tumor Pharmacodynamic- and Pharmacokinetic-Guided Expansion Cohort.

PURPOSE: CDK4/6-dependent cell-cycle regulation is disrupted in most glioblastomas. This study assesses the central nervous system (CNS) pharmacokinetics and tumor pharmacodynamics of ribociclib, a highly selective CDK4/6 inhibitor, in patients with recurrent glioblastoma. PATIENTS AND METHODS: Patients with recurrent glioblastoma with intact retinoblastoma protein (RB) expression and CDKN2A deletion or CDK4/6 amplification were treated with ribociclib daily (900 mg) for 5 days before tumor resection. Blood, tumor, and cerebrospinal fluid (CSF) samples were collected, and total and unbound ribociclib concentrations were determined. Pharmacodynamic effects, assessed by RB and FOXM1 phosphorylation, were compared with matched archival tissue. Patients with positive pharmacokinetic and pharmacodynamic effects were enrolled into the expansion cohort for preliminary assessment of progression-free survival (PFS). RESULTS: Twelve patients were enrolled. The mean unbound ribociclib concentrations in CSF, nonenhancing, and enhancing tumor regions were 0.374 µmol/L, 0.560, and 2.152 µmol/kg, respectively, which were more than 5-fold the in vitro IC50 for inhibition of CDK4/6 (0.04 µmol/L). G1-to-S phase suppression was inferred by decreases in phosphorylation of RB (P < 0.01) and cellular proliferation (P < 0.05). Six of 12 patients were enrolled into the pharmacokinetic/pharmacodynamic-guided expansion cohort and demonstrated a median PFS of 9.7 weeks. Examination of recurrent tumors following monotherapy indicated upregulation of the PI3K/mTOR pathway. CONCLUSIONS: Ribociclib exhibited good CNS penetration, and target modulation was indicated by inhibition of RB phosphorylation and tumor proliferation. Ribociclib monotherapy showed limited clinical efficacy in patients with recurrent glioblastoma. Combination therapy with CDK4/6 and PI3K/mTOR inhibitors may be explored for treating recurrent glioblastoma.

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

A Sequentially Priming Phosphorylation Cascade Activates the Gliomagenic Transcription Factor Olig2.

During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/ß [GSK3α/ß], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged "acid blob" in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics.

Lineage-Restricted OLIG2-RTK Signaling Governs the Molecular Subtype of Glioma Stem-like Cells.

The basic helix-loop-helix (bHLH) transcription factor OLIG2 is a master regulator of oligodendroglial fate decisions and tumorigenic competence of glioma stem-like cells (GSCs). However, the molecular mechanisms underlying dysregulation of OLIG2 function during gliomagenesis remains poorly understood. Here, we show that OLIG2 modulates growth factor signaling in two distinct populations of GSCs, characterized by expression of either the epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor alpha (PDGFRα). Biochemical analyses of OLIG2 function in normal and malignant neural progenitors reveal a positive feedforward loop between OLIG2 and EGFR to sustain co-expression. Furthermore, loss of OLIG2 function results in mesenchymal transformation in PDGFRα(HIGH) GSCs, a phenomenon that appears to be circumscribed in EGFR(HIGH) GSCs. Exploitation of OLIG2's dual and antithetical, pro-mitotic (EGFR-driven), and lineage-specifying (PDGFRα-driven) functions by glioma cells appears to be critical for sustaining growth factor signaling and GSC molecular subtype.

Oligodendrocyte precursors migrate along vasculature in the developing nervous system.

Oligodendrocytes myelinate axons in the central nervous system and develop from oligodendrocyte precursor cells (OPCs) that must first migrate extensively during brain and spinal cord development. We show that OPCs require the vasculature as a physical substrate for migration. We observed that OPCs of the embryonic mouse brain and spinal cord, as well as the human cortex, emerge from progenitor domains and associate with the abluminal endothelial surface of nearby blood vessels. Migrating OPCs crawl along and jump between vessels. OPC migration in vivo was disrupted in mice with defective vascular architecture but was normal in mice lacking pericytes. Thus, physical interactions with the vascular endothelium are required for OPC migration. We identify Wnt-Cxcr4 (chemokine receptor 4) signaling in regulation of OPC-endothelial interactions and propose that this signaling coordinates OPC migration with differentiation.

Rapamycin ameliorates age-dependent obesity associated with increased mTOR signaling in hypothalamic POMC neurons.

VIDEO ABSTRACT: The prevalence of obesity in older people is the leading cause of metabolic syndromes. Central neurons serving as homeostatic sensors for body-weight control include hypothalamic neurons that express pro-opiomelanocortin (POMC) or neuropeptide-Y (NPY) and agouti-related protein (AgRP). Here, we report an age-dependent increase of mammalian target of rapamycin (mTOR) signaling in POMC neurons that elevates the ATP-sensitive potassium (K(ATP)) channel activity cell-autonomously to silence POMC neurons. Systemic or intracerebral administration of the mTOR inhibitor rapamycin causes weight loss in old mice. Intracerebral rapamycin infusion into old mice enhances the excitability and neurite projection of POMC neurons, thereby causing a reduction of food intake and body weight. Conversely, young mice lacking the mTOR-negative regulator TSC1 in POMC neurons, but not those lacking TSC1 in NPY/AgRP neurons, were obese. Our study reveals that an increase in mTOR signaling in hypothalamic POMC neurons contributes to age-dependent obesity.

Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord.

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.

Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity.

Rapamycin, a specific inhibitor for mTOR complex 1, is an FDA-approved immunosuppressant for organ transplant. Recent developments have raised the prospect of using rapamycin to treat cancer or diabetes and to delay aging. It is therefore important to assess how rapamycin treatment affects glucose homeostasis. Here, we show that the same rapamycin treatment reported to extend mouse life span significantly impaired glucose homeostasis of aged mice. Moreover, rapamycin treatment of lean C57B/L6 mice reduced glucose-stimulated insulin secretion in vivo and ex vivo as well as the insulin content and beta cell mass of pancreatic islets. Confounding the diminished capacity for insulin release, rapamycin decreased insulin sensitivity. The multitude of rapamycin effects thus all lead to glucose intolerance. As our findings reveal that chronic rapamycin treatment could be diabetogenic, monitoring glucose homeostasis is crucial when using rapamycin as a therapeutic as well as experimental reagent.

Rich regulates target specificity of photoreceptor cells and N-cadherin trafficking in the Drosophila visual system via Rab6.

Neurons establish specific synaptic connections with their targets, a process that is highly regulated. Numerous cell adhesion molecules have been implicated in target recognition, but how these proteins are precisely trafficked and targeted is poorly understood. To identify components that affect synaptic specificity, we carried out a forward genetic screen in the Drosophila eye. We identified a gene, named ric1 homologue (rich), whose loss leads to synaptic specificity defects. Loss of rich leads to reduction of N-Cadherin in the photoreceptor cell synapses but not of other proteins implicated in target recognition, including Sec15, DLAR, Jelly belly, and PTP69D. The Rich protein binds to Rab6, and Rab6 mutants display very similar phenotypes as the rich mutants. The active form of Rab6 strongly suppresses the rich synaptic specificity defect, indicating that Rab6 is regulated by Rich. We propose that Rich activates Rab6 to regulate N-Cadherin trafficking and affects synaptic specificity.

Phosphorylation state of Olig2 regulates proliferation of neural progenitors.

The bHLH transcription factors that regulate early development of the central nervous system can generally be classified as either antineural or proneural. Initial expression of antineural factors prevents cell cycle exit and thereby expands the pool of neural progenitors. Subsequent (and typically transient) expression of proneural factors promotes cell cycle exit, subtype specification, and differentiation. Against this backdrop, the bHLH transcription factor Olig2 in the oligodendrocyte lineage is unorthodox, showing antineural functions in multipotent CNS progenitor cells but also sustained expression and proneural functions in the formation of oligodendrocytes. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino-terminal domain. In the phosphorylated state, Olig2 maintains antineural (i.e., promitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma.

The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors.

Cell fate decisions mediated by the Notch signalling pathway require direct cell-cell contact between adjacent cells. In Drosophila melanogaster, an external sensory organ (ESO) develops from a single sensory organ precursor (SOP) and its fate specification is governed by differential Notch activation. Here we show that mutations in actin-related protein-3 (Arp3) compromise Notch signalling, leading to a fate transformation of the ESO. Our data reveal that during ESO fate specification, most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Using immunohistochemistry and transmission electron microscopy (TEM) analyses, we show that the ARS contains numerous microvilli on the apical surface of SOP progeny. In Arp2/3 and WASp mutants, the surface area of the ARS is substantially reduced and there are significantly fewer microvilli. More importantly, trafficking of Delta-positive vesicles from the basal area to the apical portion of the ARS is severely compromised. Our data indicate that WASp-dependent Arp2/3 actin polymerization is crucial for apical presentation of Delta, providing a mechanistic link between actin polymerization and Notch signalling.

A Notch updated.

Cell-cell signaling mediated by the Notch receptor is iteratively involved in numerous developmental contexts, and its dysregulation has been associated with inherited genetic disorders and cancers. The core components of the signaling pathway have been identified for some time, but the study of the modulation of the pathway in different cellular contexts has revealed many layers of regulation. These include complex sugar modifications in the extracellular domain as well as transit of Notch through defined cellular compartments, including specific endosomes.

Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster.

Notch-mediated cell-cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

Negative-feedback regulation of proneural proteins controls the timing of neural precursor division.

Neurogenesis requires precise control of cell specification and division. In Drosophila, the timing of cell division of the sensory organ precursor (SOP) is under strict temporal control. But how the timing of mitotic entry is determined remains poorly understood. Here, we present evidence that the timing of the G2-M transition is determined by when proneural proteins are degraded from SOPs. This process requires the E3 ubiquitin ligase complex, including the RING protein Sina and the adaptor Phyl. In phyl mutants, proneural proteins accumulate, causing delay or arrest in the G2-M transition. The G2-M defect in phyl mutants is rescued by reducing the ac and sc gene doses. Misexpression of phyl downregulates proneural protein levels in a sina-dependent manner. Phyl directly associates with proneural proteins to act as a bridge between proneural proteins and Sina. As phyl is a direct transcriptional target of Ac and Sc, our data suggest that, in addition to mediating cell cycle arrest, proneural protein initiates a negative-feedback regulation to time the mitotic entry of neural precursors.

Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles.

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.

Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin.

The basic helix-loop-helix (bHLH) proneural proteins Achaete and Scute cooperate with the class I bHLH protein Daughterless to specify the precursors of most sensory bristles in Drosophila. However, the mechanosensory bristles at the Drosophila wing margin have been reported to be unaffected by mutations that remove Achaete and Scute function. Indeed, the proneural gene(s) for these organs is not known. Here, we show that the zinc-finger transcription factor Senseless, together with Daughterless, plays the proneural role for the wing margin mechanosensory precursors, whereas Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages. We provide evidence that Senseless and Daughterless physically interact and synergize in vivo and in transcription assays. Gain-of-function studies indicate that Senseless and Daughterless are sufficient to generate thoracic sensory organs (SOs) in the absence of achaete-scute gene complex function. However, analysis of senseless loss-of-function clones in the thorax implicates Senseless not in the primary SO precursor (pI) selection, but in the specification of pI progeny. Therefore, although Senseless and bHLH proneural proteins are employed during the development of all Drosophila bristles, they play fundamentally different roles in different subtypes of these organs. Our data indicate that transcription factors other than bHLH proteins can also perform the proneural function in the Drosophila peripheral nervous system.

POINT: a database for the prediction of protein-protein interactions based on the orthologous interactome.

One possible path towards understanding the biological function of a target protein is through the discovery of how it interfaces within protein-protein interaction networks. The goal of this study was to create a virtual protein-protein interaction model using the concepts of orthologous conservation (or interologs) to elucidate the interacting networks of a particular target protein. POINT (the prediction of interactome database) is a functional database for the prediction of the human protein-protein interactome based on available orthologous interactome datasets. POINT integrates several publicly accessible databases, with emphasis placed on the extraction of a large quantity of mouse, fruit fly, worm and yeast protein-protein interactions datasets from the Database of Interacting Proteins (DIP), followed by conversion of them into a predicted human interactome. In addition, protein-protein interactions require both temporal synchronicity and precise spatial proximity. POINT therefore also incorporates correlated mRNA expression clusters obtained from cell cycle microarray databases and subcellular localization from Gene Ontology to further pinpoint the likelihood of biological relevance of each predicted interacting sets of protein partners.