Hedgehog target genes regulate lipid metabolism to drive basal cell carcinoma and medulloblastoma.

Hedgehog (Hh) signaling is essential for development, homeostasis, and regeneration1. Misactivation of the Hh pathway underlies medulloblastoma, the most common malignant brain tumor in children, and basal cell carcinoma (BCC), the most common cancer in the United States2. Primary cilia regulate Hh signal transduction3, but target genes that drive cell fate decisions in response to ciliary ligands or oncogenic Hh signaling are incompletely understood. Here we define the Hh gene expression program using RNA sequencing of cultured cells treated with ciliary ligands, BCCs from humans, and Hh-associated medulloblastomas from humans and mice (Fig. 1a). To validate our results, we integrate lipidomic mass spectrometry and bacterial metabolite labeling of free sterols with genetic and pharmacologic approaches in cells and mice. Our results reveal novel Hh target genes such as the oxysterol synthase Hsd11ß1 and the adipokine Retnla that regulate lipid metabolism to drive cell fate decisions in response to Hh pathway activation. These data provide insights into cellular mechanisms underlying ciliary and oncogenic Hh signaling and elucidate targets to treat Hh-associated cancers.

Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling.

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.

Sterol regulation of developmental and oncogenic Hedgehog signaling.

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic tissue patterning and postembryonic tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that endogenous cholesterol and oxidized cholesterol derivatives (oxysterols) bind and modulate SMO activity. Here we discuss the myriad sterols that activate or inhibit the Hh pathway, with emphasis on endogenous 24(S),25-epoxycholesterol and 3ß,5α-dihydroxycholest-7-en-6-one, and propose models of sterol regulation of SMO. Synthetic inhibitors of SMO have long been the focus of drug development efforts. Here, we discuss the possible utility of steroidal SMO ligands or inhibitors of enzymes involved in sterol metabolism as cancer therapeutics.

Meningioma surgical outcomes and complications in patients aged 75 years and older.

OBJECTIVE: Meningioma incidence increases with age, yet limited data exist on how comorbidities impact complication rates in elderly patients undergoing meningioma resection. The objective of this study was to report surgical outcomes and identify risk factors for perioperative complications. METHODS: We performed a retrospective study of patients 75 years and older undergoing meningioma resection. Outcomes included survival and complications. Major complications were those requiring surgical intervention or causing permanent neurological deficit. Recursive partitioning, Kaplan-Meier survival, univariate and multi-variate (MVA) analyses were performed. RESULTS: From 1996 to 2014, 103 patients with a median age of 79 years (IQR 77-83 years) underwent cranial meningioma resection. Median follow-up was 5.8 years (IQR 1.7-8.7 years). Median actuarial survival was 10.5 years. Complications occurred in 32 patients (31.1%), and 13 patients (12.6%) had multiple complications. Major complications occurred in 16 patients (15.5%). Increasing age was not a significant predictor of any (p = 0.6408) or major complication (p = 0.8081). On univariate analysis, male sex, Charlson Comorbidity Index greater than 8, and cardiovascular comorbidities were significantly associated with major complications. On MVA only cardiovascular comorbidities (OR 3.94, 95% CI 1.05-14.76, p = 0.0238) were significantly associated with any complication. All patients with major complications had cardiovascular comorbidities, and on MVA male gender (OR 3.78, 95%CI 1.20-11.93, p = 0.0212) was associated with major complications. CONCLUSIONS: Cardiovascular comorbidities and male gender are significant risk factors for complications after meningioma resection in patients aged 75 years and older. While there is morbidity associated with meningioma resection in this cohort, there is also excellent long-term survival.

Smoothened-activating lipids drive resistance to CDK4/6 inhibition in Hedgehog-associated medulloblastoma cells and preclinical models.

Medulloblastoma is an aggressive pediatric brain tumor that can be driven by misactivation of the Hedgehog (HH) pathway. CDK6 is a critical effector of oncogenic HH signaling, but attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to single-agent molecular therapy. We identified mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma by performing orthogonal CRISPR and CRISPR interference screens in medulloblastoma cells treated with a CDK4/6 inhibitor and RNA-Seq of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6. Our concordant in vitro and in vivo data revealed that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to ER stress and activation of the unfolded protein response (UPR). These pathways increased the activity of enzymes producing Smoothened-activating (SMO-activating) sterol lipids that sustained oncogenic HH signaling in medulloblastoma despite cell-cycle attenuation. We consistently demonstrated that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing SMO-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. Our data reveal what we believe to be a novel pathway of resistance to CDK4/6 inhibition as well as a novel combination therapy to treat the most common malignant brain tumor in children.

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway.

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.

Meningioma cells express primary cilia but do not transduce ciliary Hedgehog signals.

Meningiomas are the most common primary intracranial tumors, but treatment options for meningioma patients are limited due to incomplete understanding of tumor biology. A small percentage of meningiomas harbor somatic variants in the Hedgehog pathway, a conserved gene expression program that is essential for development and adult stem cell homeostasis. Hedgehog signals are transduced through primary cilia, and misactivation of the Hedgehog pathway is known to underlie cancer. Nevertheless, the mechanisms of Hedgehog signaling in meningioma are unknown. Here, we investigate mechanisms of ciliary Hedgehog signaling in meningioma using tissue microarrays containing 154 human meningioma samples, NanoString transcriptional profiling, primary meningioma cells, pharmacology, and CRISPR interference. Our results reveal that meningiomas of all grades can express primary cilia, but that cilia are less prevalent among anaplastic tumors. Moreover, we find that expression of Smoothened alleles that are oncogenic in other contexts fail to activate the Hedgehog transcriptional program or promote proliferation in primary meningioma cells. These data reveal that meningiomas can express the subcellular structure necessary for canonical Hedgehog signaling, but suggest that they do not transduce ciliary Hedgehog signals.

Multiplatform Molecular Profiling Reveals Epigenomic Intratumor Heterogeneity in Ependymoma.

Ependymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma.

Clinical Applications of Quantitative 3-Dimensional MRI Analysis for Pediatric Embryonal Brain Tumors.

PURPOSE: To investigate the prognostic utility of quantitative 3-dimensional magnetic resonance imaging radiomic analysis for primary pediatric embryonal brain tumors. METHODS AND MATERIALS: Thirty-four pediatric patients with embryonal brain tumor with concurrent preoperative T1-weighted postcontrast (T1PG) and T2-weighted fluid-attenuated inversion recovery (FLAIR) magnetic resonance images were identified from an institutional database. The median follow-up period was 5.2 years. Radiomic features were extracted from axial T1PG and FLAIR contours using MATLAB, and 15 features were selected for analysis based on qualitative radiographic features with prognostic significance for pediatric embryonal brain tumors. Logistic regression, linear regression, receiver operating characteristic curves, the Harrell C index, and the Somer D index were used to test the relationships between radiomic features and demographic variables, as well as clinical outcomes. RESULTS: Pediatric embryonal brain tumors in older patients had an increased normalized mean tumor intensity (P = .05, T1PG), decreased tumor volume (P = .02, T1PG), and increased markers of heterogeneity (P ≤ .01, T1PG and FLAIR) relative to those in younger patients. We identified 10 quantitative radiomic features that delineated medulloblastoma, pineoblastoma, and supratentorial primitive neuroectodermal tumor, including size and heterogeneity (P ≤ .05, T1PG and FLAIR). Decreased markers of tumor heterogeneity were predictive of neuraxis metastases and trended toward significance (P = .1, FLAIR). Tumors with an increased size (area under the curve = 0.7, FLAIR) and decreased heterogeneity (area under the curve = 0.7, FLAIR) at diagnosis were more likely to recur. CONCLUSIONS: Quantitative radiomic features are associated with pediatric embryonal brain tumor patient age, histology, neuraxis metastases, and recurrence. These data suggest that quantitative 3-dimensional magnetic resonance imaging radiomic analysis has the potential to identify radiomic risk features for pediatric patients with embryonal brain tumors.

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors.

p53 protects against genome instability following centriole duplication failure.

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.

Binding of STIL to Plk4 activates kinase activity to promote centriole assembly.

Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle-regulated accumulation of STIL.