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Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation 1 – 5 . Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass 1 . Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer 6 , 7 . Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth 1 , 8 . Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7 -deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly regulated circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7 . Dietary supplementation of Atg7 -deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer. Mice with whole-body or liver-specific deletion of Atg7 release circulating arginase I and have reduced levels of serum arginine, which impairs the growth of allografted arginine-auxotrophic tumours.

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

Macroautophagy (hereafter autophagy) degrades and recycles intracellular components to sustain metabolism and survival during starvation. Host autophagy promotes tumor growth by providing essential tumor nutrients. Autophagy also regulates immune cell homeostasis and function and suppresses inflammation. Although host autophagy does not promote a T-cell anti-tumor immune response in tumors with low tumor mutational burden (TMB), whether this was the case in tumors with high TMB was not known. Here we show that autophagy, especially in the liver, promotes tumor immune tolerance by enabling regulatory T-cell function and limiting stimulator of interferon genes, T-cell response and interferon-γ, which enables growth of high-TMB tumors. We have designated this as hepatic autophagy immune tolerance. Autophagy thereby promotes tumor growth through both metabolic and immune mechanisms depending on mutational load and autophagy inhibition is an effective means to promote an antitumor T-cell response in high-TMB tumors.

In this Letter, ‘released’ should have been ‘regulated’ in the sentence starting: ‘Deletion of Atg5 in the host similarly regulated circulating arginine and suppressed tumorigenesis...’ This has been corrected online.

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

Genome-wide analysis of Cas9-DNA interactions in mammalian cells shows widespread binding but low levels of cleavage. Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

Most women with breast cancer in sub-Saharan Africa (SSA) are diagnosed with late-staged disease. The current study assesses patient-related barriers among women from a general SSA population to better understand how patient-related barriers contribute to diagnostic delays. Using convenience-based sampling, 401 Ugandan women without breast cancer were surveyed to determine how prior participation in cancer detection practices correlate with patient-related barriers to prompt diagnosis. In a predominantly poor (76%) and rural population (75%), the median age of the participants was 38. Of the women surveyed, 155 (46%) had prior exposure to breast cancer education, 92 (27%) performed breast self-examination (BSE) and 68 (20%) had undergone a recent clinical breast examination (CBE), breast ultrasound or breast biopsy. The most commonly identified barriers to prompt diagnosis were knowledge deficits regarding early diagnosis (79%), economic barriers to accessing care (68%), fear (37%) and poor social support (24%). However, only women who reported knowledge deficits-a modifiable barrier-were less likely to participate in cancer detection practices (p<0.05). Women in urban and rural areas were similarly likely to report economic barriers, knowledge deficits and/or poor social support, but rural women were less likely than urban women to have received breast cancer education and/or perform BSE (p<0.001). Women who have had prior breast cancer education (p<0.001) and/or who perform BSE (p = 0.02) were more likely to know where she can go to receive a diagnostic breast evaluation. These findings suggest that SSA countries developing early breast cancer detection programs should specifically address modifiable knowledge deficits among women less likely to achieve a diagnostic work-up to reduce diagnostic delays and improve breast cancer outcomes.

BH3 mimetic drugs that inhibit BCL-2, BCL-XL, or MCL-1 have limited activity in solid tumors. Through assessment of xenograft-derived 3D prostate cancer models and cell lines we find that tumors with RB1 loss are sensitive to BCL-XL inhibition. In parallel, drug screening demonstrates that disruption of nucleotide pools by agents including thymidylate synthase inhibitors sensitizes to BCL-XL inhibition, together indicating that replication stress increases dependence on BCL-XL. Mechanistically we establish that replication stress sensitizes to BCL-XL inhibition through TP53/CDKN1A -dependent suppression of BIRC5 expression. Therapy with a BCL-2/BCL-XL inhibitor (navitoclax) in combination with thymidylate synthase inhibitors (raltitrexed or capecitabine) causes marked and prolonged tumor regression in prostate and breast cancer xenograft models. These findings indicate that BCL-XL inhibitors may be effective as single agents in a subset of solid tumors with RB1 loss, and that pharmacological induction of replication stress may be a broadly applicable approach for sensitizing to BCL-XL inhibitors. While BH3-mimetics can be effective for treatment of haematological malignancies, their efficacy in solid tumours is limited. Here, using a range of patient-derived prostate cancer models, the authors demonstrate that increased replication stress induced by RB1 loss confers sensitivity to BH3 mimetics targeting BCL-XL.

Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis- regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis -linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning. In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes. In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants.