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

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

The role of Hedgehog signaling in lipid metabolism and cancer

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic development and tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched (PTCH) 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 oxysterol lipids bind and modulate SMO activity. This thesis explores the central theme that lipids regulate the Hh pathway which in turn regulates lipid metabolism. First, I review the myriad of sterols that activate or inhibit the Hedgehog pathway. I discuss the possibility utility of Smo ligands or inhibitors in sterol metabolism as cancer therapeutics. Next, I define the Hh gene expression program using RNA sequencing of cultured cells treated with ciliary ligands, human BCCs, and Hh-associated medulloblastomas from humans and mice. These results reveal Hh target genes such as the oxysterol synthase Hsd111 and the adipokine Retnla regulate lipid metabolism to drive cell fate decisions in response to Hh pathway activation. Lastly, I demonstrate that Hedgehog signaling can be co-opted to drive resistance to pharmacological blockade of CDK6, an important single-agent molecular therapy in cancer therapeutics. Through CRISPR screens and RNA-sequencing of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6, I demonstrate that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). I then demonstrate concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. In sum, this research program demonstrates that lipid signaling is an avenue to target Hh-dependent cancers and I demonstrate a proof-of-principle therapeutic to target lipid signaling in Hh signaling

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 3β,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 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

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

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

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

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