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Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu–ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.Tsang et al. show that copper modulates the activity of autophagic kinases ULK1/2 to control autophagy, and is required for KRASG12D-driven tumour growth and cancer survival in response to starvation.

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53. [PUBLICATION ABSTRACT]

Metastasis is the leading cause of cancer-related deaths and enables cancer cells to compromise organ function by expanding in secondary sites. Since primary tumours and metastases often share the same constellation of driver mutations, the mechanisms that drive their distinct phenotypes are unclear. Here we show that inactivation of the frequently mutated tumour suppressor gene LKB1 (encoding liver kinase B1) has evolving effects throughout the progression of lung cancer, which leads to the differential epigenetic re-programming of early-stage primary tumours compared with late-stage metastases. By integrating genome-scale CRISPR–Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumours. Using an in vivo model of metastatic progression, we further show that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumours. The expression of SOX17 is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution. Pierce et al perform genome-wide CRISPR screening and identify LKB1 as a regulator of chromatin accessibility and metastatic progression in lung cancer through a mechanism of SOX17-mediated epigenetic changes.

Targeting KRAS cancers Mutations in genes of the RAS family are preset on about 20% of human cancers, making RAS proteins prime potential targets for cancer therapy. Direct targeting of RAS proteins has not so far been productive, but two papers published in this issue offer the prospect of alternative targets in a signalling pathway downstream of RAS. Using a synthetic lethality RNAi screen, Barbie et al . identify TBK1 as a kinase in the NF-κB signalling pathway that is essential for the survival of KRAS -transformed cells. TBK1 induces anti-apoptotic signals and may be a therapeutic cancer target. And in an elegant mouse model for lung cancer driven by Kras mutation and loss of p53, Meylan et al . show that NF-κB signalling is activated by the concerted actions of these two alterations and required for tumour initiation and tumour maintenance. NF-κB transcription factors have been implicated in cellular transformation and tumorigenesis, but despite extensive biochemical characterization of NF-κB signalling, its requirement in tumour development is not completely understood. Here, the NF-κB pathway is shown to be required for the development of tumours in a mouse model of lung adenocarcinoma in a p53-status-dependent manner, providing support for the development of NF-κB inhibitory drugs as targeted therapies. NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival 1 . They have also been implicated in cellular transformation and tumorigenesis 2 , 3 , 4 , 5 , 6 . However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo , particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo , from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Tumor suppressor genes play critical roles orchestrating anti-cancer programs that are both context dependent and mechanistically diverse. Beyond canonical tumor suppressive programs that control cell division, cell death, and genome stability, unexpected tumor suppressor gene activities that regulate metabolism, immune surveillance, the epigenetic landscape, and others have recently emerged. This diversity underscores the important roles these genes play in maintaining cellular homeostasis to suppress cancer initiation and progression, but also highlights a tremendous challenge in discerning precise context-specific programs of tumor suppression controlled by a given tumor suppressor. Fortunately, the rapid sophistication of genetically engineered mouse models of cancer has begun to shed light on these context-dependent tumor suppressor activities. By using techniques that not only toggle “off” tumor suppressor genes in nascent tumors, but also facilitate the timely restoration of gene function “back-on again” in disease specific contexts, precise mechanisms of tumor suppression can be revealed in an unbiased manner. This review discusses the development and implementation of genetic systems designed to toggle tumor suppressor genes off and back-on again and their potential to uncover the tumor suppressor’s tale.

The p53 pathway is a universal tumor suppressor mechanism that limits tumor progression by triggering apoptosis or permanent cell cycle arrest, called senescence. In recent years, efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained traction with the development of a range of small molecules targeting mutant p53. However, knowledge of the downstream mediators of p53 reactivation in different oncogenic contexts has been limited. Here, we utilized a panel of murine cancer cell lines from three distinct tumor types susceptible to alternative outcomes following p53 restoration to define unique and shared p53 transcriptional signatures. While we found that the majority of p53-bound sites and p53-responsive transcripts are tumor-type specific, analysis of shared targets identified a core signature of genes activated by p53 across all contexts. Furthermore, we identified repression of E2F and Myc target genes as a key feature of senescence. Characterization of p53-induced transcripts revealed core and senescence-specific long noncoding RNAs (lncRNAs) that are predominantly chromatin associated and whose production is coupled to cis-regulatory activities. Functional investigation of the contributions of p53-induced lncRNAs to p53-dependent outcomes highlighted Pvt1b, the p53-dependent isoform of Pvt1, as a mediator of p53-dependent senescence via Myc repression. Inhibition of Pvt1b led to decreased activation of senescence markers and increased levels of markers of proliferation. These findings shed light on the core and outcome-specific p53 restoration signatures across different oncogenic contexts and underscore the key role of the p53-Pvt1b-Myc regulatory axis in mediating proliferative arrest.

Limits to antitumour effect of p53 restoration Inactivation of the p53 tumour-suppressor pathway is a common feature of human cancers, prompting suggestions that restoring p53 function in established tumours might be an effective therapy. However, two papers in this week's Nature highlight a practical limitation of p53-directed cancer therapeutics. They show in a K-Ras-driven lung-cancer model that p53-mediated tumour suppression is engaged only at a late stage of tumour progression, when the K-Ras oncogenic signal reaches a threshold that is sufficient to activate the ARF-p53 pathway. This means that p53 re-expression fails to restrict the early stages of tumorigenesis, although it does induce regression of more aggressive tumours. p53 is an important tumour suppressor gene. Two papers now show in a Kras-driven lung cancer model that p53-mediated tumour suppression is only engaged late during tumour progression, when the Kras oncogenic signal reaches a threshold sufficient to activate the ARF–p53 pathway. Therefore, p53 re-expression in p53-deficient lung tumours does not restrict early stages of tumorigenesis, but induces tumour regression of more aggressive tumours. Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway 1 , 2 , 3 . Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma 1 – 3 . Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies 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 are currently under evaluation for the treatment of lung adenocarcinoma 7 – 9 . Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards 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 that they control and critical determinants of successful cancer therapy. Loss of RB promotes both malignant progression and the development of metastatic disease; however, whereas reactivation of the RB pathway can revert metastatic tumour cell states to non-metastatic cell states, malignant cell proliferation is supported by MAPK–CDK2-dependent suppression of RB.

LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPα, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation. LKB1 tumour suppressor gene is frequently mutated in lung adenocarcinoma. Here the authors show that in genetically engineered mouse models of lung cancer Lkb1 restoration induces growth arrest and drives neoplastic cells toward a more differentiated and less proliferative alveolar type II cell-like state via C/EBP-mediated reprogramming.

The p53 tumor suppressor regulates multiple context-dependent tumor suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here, using a mouse model of SCLC in which endogenous p53 expression can be conditionally and temporally regulated, we show that SCLC tumors maintain a requirement for p53 inactivation. However, we identify tumor subtype heterogeneity between SCLC tumors such that p53 reactivation induces senescence in a subset of tumors, while in others, p53 induces necrosis. We pinpoint cyclophilins as critical determinants of a p53-induced transcriptional program that is specific to SCLC tumors and cell lines poised to undergo p53-mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53 transcriptional output without impacting p53 chromatin binding. Our study demonstrates that intertumoral heterogeneity in SCLC influences the biological response to p53 restoration, describes a cyclophilin-dependent mechanism of p53-regulated cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant tumor type. p53 inactivation is nearly universal in small-cell lung cancer (SCLC), but its tumor suppressive role in this cancer type is poorly understood. Here the authors show that intertumoral heterogeneity in SCLC influences the biological mechanisms of p53-mediated tumor suppression and identify a role for cyclophilins in p53-dependent necrotic cell death.