While the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a nontraditional role for Cu as a modulator of kinase signaling is emerging. We discovered that Cu is required for the activity of the autophagic kinases ULK1/2 through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt Cu-binding reduced ULK1/2-dependent signaling and autophagosome complex formation. Elevated intracellular Cu levels are associated with starvation induced autophagy and sufficient to enhance ULK1 kinase activity and in turn autophagic flux. The growth and survival of lung tumors driven by KRAS G12D is diminished in the absence of Ctr1 , depends on ULK1 Cu-binding, and is associated with reduced autophagy levels and signaling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to forestall autophagy signaling to limit proliferation and survival in cancer.

Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1+/−;MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).

Chromatin modifying genes are frequently mutated in human lung adenocarcinoma, but the functional impact of these mutations on disease initiation and progression is not well understood. Using a CRISPR-based approach, we systematically inactivated three of the most commonly mutated chromatin regulatory genes in two KrasG12D-driven mouse models of lung adenocarcinoma to characterize the impact of their loss. Targeted inactivation of SWI/SNF nucleosome remodeling complex members Smarca4 (Brg1) or Arid1a had complex effects on lung adenocarcinoma initiation and progression. Loss of either Brg1 or Arid1a were selected against in early stage tumors, but Brg1 loss continued to limit disease progression over time, whereas loss of Arid1a eventually promoted development of higher grade lesions. In contrast to these stage-specific effects, loss of the histone methyltransferase Setd2 had robust tumor promoting consequences. Despite disparate impacts of Setd2 and Arid1a loss on tumor development, each resulted in a gene expression profile with significant overlap. Setd2 inactivation and subsequent loss of H3K36me3 led to the swift expansion and accelerated progression of both early and late stage tumors. However, Setd2 loss per se was insufficient to overcome a p53-regulated barrier to malignant progression, nor establish the pro-metastatic cellular states that stochastically evolve during lung adenocarcinoma progression. Our study uncovers differential and context-dependent effects of SWI/SNF complex member loss, identifies Setd2 as a potent tumor suppressor in lung adenocarcinoma, and establishes model systems to facilitate further study of chromatin deregulation in lung cancer.

Autophagy is an intracellular self-digestion mechanism, by which cellular components are sorted into double-membrane autophagosomes and delivered to lysosomes for degradation. Cells utilize autophagy to dispose of wastes and eliminate hazards, while recycling nutrients and tuning metabolism in the process. Through these functions, autophagy promotes cell fitness, genome integrity, tissue homeostasis, and cell survival and growth under stress. Autophagy up- and down-regulation have both been found in human cancers, suggesting a complex role in tumor development. Accumulating results from autophagy-deficient mice and mouse models of human cancers have demonstrated that autophagy generally suppresses tumor initiation, but promotes tumor progression, in a manner that is dependent on timing and context and modified by specific tumorigenic events. Given the role of autophagy in facilitating tumor growth, autophagy inhibition has gained wide attention as a potential anticancer therapy. Here, we summarize relevant genetic, preclinical and clinical studies and discuss the multi-faceted role of autophagy in cancer, as well as the prospects of autophagy inhibition for cancer therapy.

Mutations in the Retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma 1 , 2 , 3 . Despite being the first tumour suppressor to be identified, the molecular and cellular basis underlying selection for persistent RB loss in cancer remains unclear 4 – 6 . Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and currently under evaluation in lung adenocarcinoma 7 – 9 . Whether RB pathway reactivation will have therapeutic effects and if targeting CDK4/6 is sufficient to reactivate RB pathway activity in lung cancer is unknown. Here, we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in the mouse. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for MAPK signal amplification during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to CDK4/6 inhibition. Second, RB inactivation deregulates expression of cell state-determining factors, facilitates lineage infidelity, and accelerates the acquisition of metastatic competency. In contrast, reactivation of RB reprograms advanced tumours toward a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs they control, and critical determinants of successful therapy.

Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here, we report the development and characterization of a synthetic gene switch that when targeted in the mouse germline enables conditional inactivation, reports gene expression, and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap-reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR to denote each of the allelic states and the associated expression patterns of the targeted gene: expressed (XTR), Trapped (TR), and Restored (R).

Targeting aberrant kinase activity in cancer relies on unmasking cellular inputs such as growth factors, nutrients, and metabolites that contribute to cancer initiation and progression 1 . While the transition metal copper (Cu) is an essential nutrient that is traditionally viewed as a static cofactor within enzyme active sites 2 , a newfound role for Cu as a modulator of kinase signaling is emerging 3,4 . We discovered that Cu is required for the activity of the autophagic kinases ULK1/2 through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt Cu-binding reduced ULK1/2-dependent signaling and autophagosome complex formation. Elevated intracellular Cu levels are associated with starvation induced autophagy and sufficient to enhance ULK1 kinase activity and in turn autophagic flux. Targeting autophagy machinery is a promising therapeutic strategy in cancers 5 , but is limited by the absence of potent inhibitors and the emergence of resistance. The growth and survival of lung tumors driven by KRAS G12D is diminished in the absence of Ctr1, depends on ULK1 Cu-binding, and is associated with reduced autophagy levels and signaling. These findings suggest a new molecular basis for exploiting Cu-chelation therapy to forestall autophagy signaling to limit proliferation and survival in cancer.By default, the dynamic and adaptive nature of signaling networks allows them to respond and, in some cases, sense extracellular and intracellular inputs 6 . While growth factors, nutrients, and metabolites are well-appreciated regulators of cell proliferation, the contribution of transition metals to cellular processes that support proliferation and contribute to malignant transformation are understudied. The transition metal copper (Cu) is essential for a diverse array of biological processes from cellular proliferation, neuropeptide processing, free radical detoxification, and

SUMMARY Expression of oncogenic KrasG12D initiates lung adenomas in a MAPK signal-dependent manner from only a subset of cell types in the adult mouse lung. Amplification of MAPK signaling is associated with progression to malignant adenocarcinomas, but whether this is a cause or consequence of disease progression is not known. To better understand the effects of MAPK signaling downstream of KrasG12D expression, we capitalized on the ability of Braf inhibition to selectively amplify MAPK pathway signaling in KrasG12D-expressing epithelial cells. MAPK signal amplification indeed promoted the rapid progression of established adenomas to malignant adenocarcinomas. However, we surprisingly observed a greater number of overall tumor-initiating events after MAPK signal amplification, due to induced proliferation of cell types that are normally refractory to KrasG12D-induced transformation. Thus, MAPK signaling in the lung is thresholded not only during malignant progression, but also at the moment of tumor initiation.