Mechanistic Studies of Vertebrate Hedgehog Signaling

Metazoans use Hedgehog signaling to direct many stages of embryonic development, and deregulation of this pathway is implicated in many types of cancer. I investigated several steps of Hedgehog pathway transduction that were poorly understood in mechanistic terms. The mature Hedgehog ligand is produced by a self-proteolysis reaction that covalently attaches a cholesterol molecule to the signaling half of the protein. I showed that the catalytic cysteine forms a disulfide bridge that is essential for the folding and function of the C-terminal tail of Hedgehog, and identified two protein disulfide isomerases that remodel this bridge to free the catalytic thiol group after folding is complete. Using pulse chase assays to follow Hedgehog processing, I demonstrated that the self-proteolysis reaction takes place in the endoplasmic reticulum, that the cleaved C-terminal tail of Hedgehog is degraded before moving to the Golgi, and that Hedgehog mutants defective in processing get degraded in their entirety by the same route. Lipidated Hedgehog ligand requires the transmembrane protein Dispatched for secretion. I devised a system to test Dispatched function in cultured cells, and showed that some inactive Dispatched mutants fail to bind Hedgehog, while others bind more tightly than the wild type protein. Scube2 was implicated as a Hedgehog pathway component in zebrafish genetic studies. I showed that Scube2 is a secreted protein that binds Hedgehog via its cholesterol adduct and solubilizes it in aqueous media. Dispatched and Scube2 bind Hedgehog on opposing faces, and they function synergistically to release it from the membrane. Vertebrate Hedgehog signaling relies on intraflagellar transport through an antenna-like organelle called the primary cilium. The Hedgehog receptor Patched and transducer protein Smoothened localize to primary cilia in a mutually exclusive pattern, depending on Hedgehog ligand presence. I showed that cytoplasmic components of the pathway Suppressor of Fused (SuFu, a pathway inhibitor) and Glioma-associated oncogene transcription factors (the Gli family, the effectors of the pathway) localize to primary cilia and accumulate there when Smoothened is activated. SuFu and Gli form a complex that dissociates when the pathway is turned on, and this dissociation depends on trafficking through the cilium

A Mechanism for Vertebrate Hedgehog Signaling: Recruitment to Cilia and Dissociation of SuFu-Gli Protein Complexes

In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu–Gli complexes to cilia, suggesting a model in which Smo activates Gli by relieving inhibition by SuFu. In support of this model, we find that Hh causes rapid dissociation of the SuFu–Gli complex, thus allowing Gli to enter the nucleus and activate transcription. Activation of protein kinase A (PKA), an inhibitor of Hh signaling, blocks ciliary localization of SuFu–Gli complexes, which in turn prevents their dissociation by signaling. Our results support a simple mechanism in which Hh signals at vertebrate cilia cause dissociation of inactive SuFu–Gli complexes, a process inhibited by PKA

Pan-cancer interrogation of MUTYH variants reveals biallelic inactivation and defective base excision repair across a spectrum of solid tumors

Purpose Biallelic germline pathogenic variants of the base excision repair (BER) pathway gene MUTYH predispose to colorectal cancer (CRC) and other cancers. The possible association of heterozygous variants with broader cancer susceptibility remains uncertain. This study investigated the prevalence and consequences of pathogenic MUTYH variants and MUTYH loss of heterozygosity (LOH) in a large pan-cancer analysis. Materials and Methods Data from 354,366 solid tumor biopsies that were sequenced as part of routine clinical care were analyzed using a validated algorithm to distinguish germline from somatic MUTYH variants. Results Biallelic germline pathogenic MUTYH variants were identified in 119 tissue biopsies. Most were CRCs and showed increased tumor mutational burden (TMB) and a mutational signature consistent with defective BER (COSMIC Signature SBS18). Germline heterozygous pathogenic variants were identified in 5,991 biopsies and their prevalence was modestly elevated in some cancer types. About 12% of these cancers (738 samples: including adrenal gland cancers, pancreatic islet cell tumors, nonglioma CNS tumors, GI stromal tumors, and thyroid cancers) showed somatic LOH for MUTYH, higher rates of chromosome 1p loss (where MUTYH is located), elevated genomic LOH, and higher COSMIC SBS18 signature scores, consistent with BER deficiency. Conclusion This analysis of MUTYH alterations in a large set of solid cancers suggests that in addition to the established role of biallelic pathogenic MUTYH variants in cancer predisposition, a broader range of cancers may possibly arise in MUTYH heterozygotes via a mechanism involving somatic LOH at the MUTYH locus and defective BER. However, the effect is modest and requires confirmation in additional studies before being clinically actionable

Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

Autocatalytic processing of the Hedgehog ligand from its precursor protein relies on protein disulfide isomerase and ER-associated degradation

Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

In higher eukaryotes, the endoplasmic reticulum (ER) contains a network of membrane tubules, which transitions into sheets during mitosis. Network formation involves curvature-stabilizing proteins, including the reticulons (Rtns), as well as the membrane-fusing GTPase atlastin (ATL) and the lunapark protein (Lnp). Here, we have analyzed how these proteins cooperate. ATL is needed to not only form, but also maintain, the ER network. Maintenance requires a balance between ATL and Rtn, as too little ATL activity or too high Rtn4a concentrations cause ER fragmentation. Lnp only affects the abundance of three-way junctions and tubules. We suggest a model in which ATL-mediated fusion counteracts the instability of free tubule ends. ATL tethers and fuses tubules stabilized by the Rtns, and transiently sits in newly formed three-way junctions. Lnp subsequently moves into the junctional sheets and forms oligomers. Lnp is inactivated by mitotic phosphorylation, which contributes to the tubule-to-sheet conversion of the ER. DOI: http://dx.doi.org/10.7554/eLife.18605.00

Dispatched and Scube Mediate the Efficient Secretion of the Cholesterol-Modified Hedgehog Ligand

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand

A High Percentage of NSCLC With Germline CHEK2 Mutation Harbors Actionable Driver Alterations: Survey of a Cancer Genomic Database and Review of Literature.

INTRODUCTION: Germline CHEK2 mutations are rare and have not been associated with increased risk of NSCLC. METHODS: We identified two sequential primary NSCLCs harboring distinct actionable driver alterations (EGFR E746 _S752 delinsV and CD74-ROS1) in a patient with NSCLC with a novel germline CHEK2 mutation S5fs∗54 (c.14_20delCGGATGT). We queried a genomic database of NSCLC samples profiled by plasma next-generation sequencing (Foundation Medicine Inc.) and performed a literature search of germline CHEK2 mutations in NSCLC. RESULTS: Of 6101 patients with unique NSCLC profiled by plasma next-generation sequencing, 53 cases (0.87%) of germline CHEK2 mutation were identified (male-to-female ratio, 49%:51%; median age = 75 y). The median allele frequency of CHEK2 was 49% (interquartile range: 49%-51%). Ten unique CHEK2 germline mutations were identified. Literature review identified 15 additional cases of germline CHEK2 mutations in NSCLC. Overall, a total of 70 CHEK2 germline mutations (21 unique CHEK2 alterations) were identified. Among these 70 CHEK2 germline mutations, 54.3% were amino acid substitutions (point mutation), 40.0% were frameshift mutations, and 5.7% were splice site mutations. Of these 70 total cases assessed, 29 (41.4%) potentially actionable driver alterations were identified with KRAS G12C mutation (27.6%) being the most common and KRAS G12A/C/D/R/S/V mutations together constituting 51.7% of these driver mutations. CONCLUSIONS: Germline CHEK2 mutations are rare in NSCLC. A large proportion of these cases harbor actionable driver alterations. The relationship between germline CHEK2 mutations and actionable driver alterations in NSCLC may be worth further investigation

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres

Molecular, immunologic, and clinicodemographic landscape of MYC-amplified (MYCamp) advanced prostate cancer (PCa)

5041 Background: The MYC oncogene is one of the most commonly amplified genes in PCa, contributes to androgen independent growth, and is potentially targetable. We sought to define the molecular, immunologic, and clinicodemographic landscape of MYCamp in advanced PCa to better understand progression and establish rationale for personalized treatments and combinations. Methods: Hybrid capture-based comprehensive genomic profiling (CGP) was performed on tumor samples from predominantly advanced PCa samples. MYCamp was defined as copy number (CN) ≥6. PD-L1 IHC was performed using Dako 22C3. A subset of patients (pts) with advanced PCa were selected from the Flatiron Health- Foundation Medicine (FM) clinicogenomic database (CGDB), a nationwide de-identified EHR-derived clinical DB linked to FM CGP data for pts treated from 01/2011-12/2020. The de-identified data originated from approximately 280 US cancer clinics (̃800 sites of care). Results: The genomic profiles of 12,528 tissue samples from unique PCa pts (including hormone sensitive and castrate resistant) were evaluated. MYCamp was detected in 10.6%, with a median MYC CN of 8. Median age was 67 years (67 for MYCwt versus 68 for MYCamp). MYCamp occurred at a higher frequency in men with African (N = 190/1,473, 12.9%) versus European (N = 996/9,796, 10.2%) ancestry (P = 0.002), was more frequent in metastatic biopsy sites vs primary (15.7% vs 6.2%, P 15 was enriched for PD-L1 positivity (26.1%) compared with MYCwt (9.8%) or MYCamp CN 6-15 (11.5%) (CN > 15 vs wt P = 0.025). In pts with MYCamp vs MYCwt PCa AR, RAD21, PTEN, CCND1, ZNF703, FGF19, FGFR1, and FGF3 each had significantly higher rates of CN changes (all p 0) from PCa pts MYCamp was detected in 2.0% (28/1,402), and in 4.5% (20/445) with cTF > 20%. Among evaluable PCa pts in the CGDB, (67 MYCamp and 658 MYCwt) MYCamp did not significantly impact treatment decisions, with the majority receiving novel hormone therapies (35.8% MYCamp vs. 31.5% MYCwt) or chemotherapy containing regimens (37.3% MYCamp vs. 27.7% MYCwt) as first therapy after CGP report. Conclusions: Herein, we report the largest analysis to date of molecular, immunologic, and clinicodemographic features of MYCamp advanced PCa. These findings suggest that MYCamp defines a biologically distinct subset of PCa pts for whom personalized combination treatments utilizing targeted and/or immunotherapies may be effective. Independent cohorts are needed to validate these findings