Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors.

Autophagy, particularly with BECN1, has paradoxically been highlighted as tumor promoting in Ras-driven cancers, but potentially tumor suppressing in breast and ovarian cancers. However, studying the specific role of BECN1 at the genetic level is complicated due to its genomic proximity to BRCA1 on both human (chromosome 17) and murine (chromosome 11) genomes. In human breast and ovarian cancers, the monoallelic deletion of these genes is often co-occurring. To investigate the potential tumor suppressor roles of two of the most commonly deleted autophagy genes in ovarian cancer, BECN1 and MAP1LC3B were knocked-down in atypical (BECN1+/+ and MAP1LC3B+/+) ovarian cancer cells. Ultra-performance liquid chromatography mass-spectrometry metabolomics revealed reduced levels of acetyl-CoA which corresponded with elevated levels of glycerophospholipids and sphingolipids. Migration rates of ovarian cancer cells were increased upon autophagy gene knockdown. Genomic instability was increased, resulting in copy-number alteration patterns which mimicked high grade serous ovarian cancer. We further investigated the causal role of Becn1 haploinsufficiency for oncogenesis in a MISIIR SV40 large T antigen driven spontaneous ovarian cancer mouse model. Tumors were evident earlier among the Becn1+/- mice, and this correlated with an increase in copy-number alterations per chromosome in the Becn1+/- tumors. The results support monoallelic loss of BECN1 as permissive for tumor initiation and potentiating for genomic instability in ovarian cancer

Altered H3 histone acetylation impairs high-fidelity DNA repair to promote cerebellar degeneration in spinocerebellar ataxia type 7.

A common mechanism in inherited ataxia is a vulnerability of DNA damage. Spinocerebellar ataxia type 7 (SCA7) is a CAG-polyglutamine-repeat disorder characterized by cerebellar and retinal degeneration. Polyglutamine-expanded ataxin-7 protein incorporates into STAGA co-activator complex and interferes with transcription by altering histone acetylation. We performed chromatic immunoprecipitation sequencing ChIP-seq on cerebellum from SCA7 mice and observed increased H3K9-promoter acetylation in DNA repair genes, resulting in increased expression. After detecting increased DNA damage in SCA7 cells, mouse primary cerebellar neurons, and patient stem-cell-derived neurons, we documented reduced homology-directed repair (HDR) and single-strand annealing (SSA). To evaluate repair at endogenous DNA in native chromosome context, we modified linear amplification-mediated high-throughput genome-wide translocation sequencing and found that DNA translocations are less frequent in SCA7 models, consistent with decreased HDR and SSA. Altered DNA repair function in SCA7 may predispose the subject to excessive DNA damage, leading to neuron demise and highlights DNA repair as a therapy target

ATF6-mediated signaling contributes to PARP Inhibitor Resistance in Ovarian Cancer.

High grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy resistant disease. Poly(ADP)-ribose polymerase inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-seq and an shRNA screen, we identified Activating Transcription Factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. Implications: This study highlights that a novel p38-ATF6 mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors

Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer.

Identification of specific oncogenic gene changes has enabled the modern generation of targeted cancer therapeutics. In high-grade serous ovarian cancer (OV), the bulk of genetic changes is not somatic point mutations, but rather somatic copy-number alterations (SCNAs). The impact of SCNAs on tumour biology remains poorly understood. Here we build haploinsufficiency network analyses to identify which SCNA patterns are most disruptive in OV. Of all KEGG pathways (N=187), autophagy is the most significantly disrupted by coincident gene deletions. Compared with 20 other cancer types, OV is most severely disrupted in autophagy and in compensatory proteostasis pathways. Network analysis prioritizes MAP1LC3B (LC3) and BECN1 as most impactful. Knockdown of LC3 and BECN1 expression confers sensitivity to cells undergoing autophagic stress independent of platinum resistance status. The results support the use of pathway network tools to evaluate how the copy-number landscape of a tumour may guide therapy

Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors.

Autophagy, particularly with BECN1, has paradoxically been highlighted as tumor promoting in Ras-driven cancers, but potentially tumor suppressing in breast and ovarian cancers. However, studying the specific role of BECN1 at the genetic level is complicated due to its genomic proximity to BRCA1 on both human (chromosome 17) and murine (chromosome 11) genomes. In human breast and ovarian cancers, the monoallelic deletion of these genes is often co-occurring. To investigate the potential tumor suppressor roles of two of the most commonly deleted autophagy genes in ovarian cancer, BECN1 and MAP1LC3B were knocked-down in atypical (BECN1+/+ and MAP1LC3B+/+) ovarian cancer cells. Ultra-performance liquid chromatography mass-spectrometry metabolomics revealed reduced levels of acetyl-CoA which corresponded with elevated levels of glycerophospholipids and sphingolipids. Migration rates of ovarian cancer cells were increased upon autophagy gene knockdown. Genomic instability was increased, resulting in copy-number alteration patterns which mimicked high grade serous ovarian cancer. We further investigated the causal role of Becn1 haploinsufficiency for oncogenesis in a MISIIR SV40 large T antigen driven spontaneous ovarian cancer mouse model. Tumors were evident earlier among the Becn1+/- mice, and this correlated with an increase in copy-number alterations per chromosome in the Becn1+/- tumors. The results support monoallelic loss of BECN1 as permissive for tumor initiation and potentiating for genomic instability in ovarian cancer

Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance

Disaccharide composition of heparan sulfate molecules in <i>Ndst1</i>^<i>f/f</i><i>tga20</i>^<i>+/+</i><i>GFAPCre</i>- and <i>GFAPCre</i>+ brain.

Disaccharide composition of heparan sulfate molecules in Ndst1f/ftga20+/+GFAPCre- and GFAPCre+ brain.</p

<i>Ndst1</i>^<i>f/f</i><i>SynCre-</i> and <i>SynCre+</i> mice infected with subfibrillar prions: survival times, brain lesions, and biochemical properties.

(A) Survival curves of SynCre- and SynCre+ mice intracerebrally inoculated with RML or ME7 prions (RML: n = 7 SynCre- and 8 SynCre+ mice; ME7: n = 16 SynCre- and 17 SynCre+ mice). (B) Brain sections (hippocampus) immunolabelled for PrPSc or GFAP or stained with hematoxylin and eosin (HE) show prion aggregate distribution and morphology, spongiform degeneration, and astrogliosis in SynCre- and SynCre+ brains. Scale bar = 50 μm. (C) Lesion profiles of RML- and ME7-infected SynCre- and SynCre+ mice (1-dorsal medulla, 2-cerebellum, 3-hypothalamus, 4-medial thalamus, 5-hippocampus, 6-septum, 7-cerebral cortex, 8- cerebral peduncle and 9- cerebellar peduncle) (RML: n = 5 mice per genotype; ME7: n = 6 SynCre- and 7 SynCre+ mice). (D) PrPSc immunoblots (post-PK digest) show electrophoretic mobility and glycoprofile of PK-digested PrPSc. N = 6 per genotype. (E) Immunolabelling for microglia (Iba1) (representative images of hippocampus) and (F) quantification of the Iba1-stained area in CC, CT, HP, TH. Scale bar = 50 μm. (G) Representative immunoblots for PrPSc (post PK digest) from 30 μg brain, and (H) quantification of PrPSc levels in the whole brain lysate at terminal disease. *P (TIF)</p

Reducing neuronal HS sulfation increases mCWD solubility and decreases fibril length.

(A) Representative western blot of mCWD aggregates after PK digestion and centrifugation over an Optiprep layer (P = pellet, S = supernatant). (B) Quantification of PrPSc in pellet and supernatant fractions. N = 6 samples per genotype. (C) Representative electron microscopy images of purified PrPSc fibrils from mCWD-infected mice. Scale bars represent 100 nm. Fibril measurements (bottom panel) are 193 nm (SynCre-) and 144 nm (SynCre+). (D) Quantification of the fibril lengths. N = 49 and 42 fibrils measured from SynCre- and SynCre+ brains, respectively (n = 6 mice per genotype). *P< 0.05, unpaired two-tailed t-test with Bonferroni’s post test (panels B and D).</p