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.

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.

Non–small cell lung cancer (NSCLC) is often characterized by mutually exclusive mutations in the epidermal growth factor receptor (EGFR) or the guanosine triphosphatase KRAS. We hypothesized that blocking EGFR palmitoylation, previously shown to inhibit EGFR activity, might alter downstream signaling in the KRAS-mutant setting. Here, we found that blocking EGFR palmitoylation, by either knocking down the palmitoyltransferase DHHC20 or expressing a palmitoylation-resistant EGFR mutant, reduced activation of the kinase PI3K, the abundance of the transcription factor MYC, and the proliferation of cells in culture, as well as reduced tumor growth in a mouse model of KRAS-mutant lung adenocarcinoma. Knocking down DHHC20 reduced the growth of existing tumors derived from human KRAS-mutant lung cancer cells and increased the sensitivity of these cells to a PI3K inhibitor. Palmitoylated EGFR interacted with the PI3K regulatory subunit PIK3R1 (p85) and increased the recruitment of the PI3K heterodimer to the plasma membrane. Alternatively, blocking palmitoylation increased the association of EGFR with the MAPK adaptor Grb2 and decreased that with p85. This binary switching between MAPK and PI3K signaling, modulated by EGFR palmitoylation, was only observed in the presence of oncogenic KRAS. These findings suggest a mechanism whereby oncogenic KRAS saturates signaling through unpalmitoylated EGFR, reducing formation of the PI3K signaling complex. Future development of DHHC20 inhibitors to reduce EGFR-PI3K signaling could be beneficial to patients with KRAS-mutant tumors.

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.

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.

SETD2 is a tumor suppressor that is frequently inactivated in several cancer types. The mechanisms through which SETD2 inactivation promotes cancer are unclear, and whether targetable vulnerabilities exist in these tumors is unknown. Here we identify heightened mTORC1-associated gene expression programs and functionally higher levels of oxidative metabolism and protein synthesis as prominent consequences of Setd2 inactivation in KRAS-driven mouse models of lung adenocarcinoma. Blocking oxidative respiration and mTORC1 signaling abrogates the high rates of tumor cell proliferation and tumor growth specifically in SETD2-deficient tumors. Our data nominate SETD2 deficiency as a functional marker of sensitivity to clinically actionable therapeutics targeting oxidative respiration and mTORC1 signaling.

<p>Supplementary Figure 1: Locations and types of chromatin regulator mutations found in human lung adenocarcinomas. Supplementary Figure 2: Identification of high efficiency Brg1, Arid1a and Setd2 sgRNAs using a fluorescent reporter-based sensor assay. Supplementary Figure 3: Examples of tumors with mixed expression of Brg1, Arid1a and H3K36me3. Supplementary Figure 4: Proliferative and apoptotic rates of KrasG12D/+ tumors targeted with sgRNAs against GFP, Brg1, Arid1a, or Setd2. Supplementary Figure 5: Identification of chromatin regulator expression status and measurement of tumor cell purity in RNA-Seq samples. Supplementary Figure 6: Ingenuity Pathway Analysis of top 10 enriched canonical pathways in Arid1a and Setd2 deficient tumors. Supplementary Figure 7: Proliferative and apoptotic rates of KrasG12D/+;p53-/- tumors targeted with sgRNAs against GFP, Brg1, Arid1a, or Setd2. Supplementary Figure 8: Analysis of markers of tumor cell identity in early and late KrasG12D/+;p53-/- tumors. Supplementary Figure 9: Representative images of tumors from KrasG12D/+;p53flox/flox mice, and the distinct morphology of Setd2 deficient tumors. Supplementary Figure 10: Analysis of multinucleate giant cells, aberrant mitoses and DNA damage in Setd2 deficient tumors.</p>