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Supplementary Table 3: Gene ontology analysis for genes deregulated by both Arid1a and Setd2 loss.

Figure legends for supplementary figures.

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation.Significance:This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160

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 prometastatic 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. Cancer Res; 77(7); 1719–29. ©2017 AACR.

Supplementary Table 2: Ingenuity Pathway Analysis results for Setd2 vs. GFP tumors.

Supplementary Table 1: Ingenuity Pathway Analysis results for Arid1a vs. GFP tumors.

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.

Supplemental Figures and Tables

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.

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. Significance: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms. See related commentary by Spasevska and Myklebust, p. 3160

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1–3. 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 unclear4–6. 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 adenocarcinoma7–9. 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.

Type 1 conventional dendritic cells (cDC1s) are typically thought to be dysregulated secondarily to invasive cancer. Here, we report that cDC1 dysfunction instead develops in the earliest stages of preinvasive pancreatic intraepithelial neoplasia (PanIN) in the KrasLSL-G12D/+ Trp53LSL-R172H/+ Pdx1-Cre–driven (KPC) mouse model of pancreatic cancer. cDC1 dysfunction is systemic and progressive, driven by increased apoptosis, and results in suboptimal up-regulation of T cell–polarizing cytokines during cDC1 maturation. The underlying mechanism is linked to elevated IL-6 concomitant with neoplasia. Neutralization of IL-6 in vivo ameliorates cDC1 apoptosis, rescuing cDC1 abundance in tumor-bearing mice. CD8+ T cell response to vaccination is impaired as a result of cDC1 dysregulation. Yet, combination therapy with CD40 agonist and Flt3 ligand restores cDC1 abundance to normal levels, decreases cDC1 apoptosis, and repairs cDC1 maturation to drive superior control of tumor outgrowth. Our study therefore reveals the unexpectedly early and systemic onset of cDC1 dysregulation during pancreatic carcinogenesis and suggests therapeutically tractable strategies toward cDC1 repair.

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.

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 prometastatic 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. Cancer Res; 77(7); 1719–29. ©2017 AACR.

Cancer is a heterogeneous disease that arises from the accumulation of genetic mutations that lead to a loss of tumor suppressor genes and activation of oncogenes. Mutational inactivation of genes that regulate epigenetic mechanisms, such as post translational modifications to DNA and histones, also contribute to cancer progression but the mechanisms are poorly understood. Trimethylation on histone 3 lysine 36 (H3K36me3) is encoded by the histone lysine methyltransferase (SETD2). SETD2 is recurrently inactivated by missense and frameshift mutations in multiple tumor types. Previously, we identified that Setd2 loss has potent tumor promoting activities in a mouse model of oncogenic Kras-induced lung adenocarcinoma. However, whether Setd2 loss has cooperating effects with other tumor suppressor gene mutations remains unstudied. To simultaneously inactivate distinct tumor suppressor genes, we use a lentiviral-based CRISPR/Cas gene editing approach to express a combination of specific gRNA-pairs along with Cas9 and Cre recombinase. Inhalation of these lentiviral particles leads activation of an oncogenic KrasLSL-G12D allele and simultaneously inactivation two gRNA-targeted genes. We hypothesize that SETD2 will collaborate with other tumor suppressor gene mutations in lung adenocarcinoma. To clone lentivirus vectors that have two different gRNA we use oligonucleotides encoding gRNAs targeting Setd2 and either Lkb1, Apc, or Rb1 to PCR amplify the 3' region of the murine U6 (mU6) promoter and the 5' region of the gRNA scaffold. Overlapping sequences in the 148-bp PCR amplicon, facilitate recombination with a 415-bp donor fragment derived from BbsI-digested pDonor plasmid using the Gibson cloning method. The newly circularized fragment is then re-linearized via BbsI digestion and cloned into LentiCRISPRv2Cre vector through the Golden Gate method using BsmBI type IIs restriction endonuclease and T4 DNA ligase. Our data demonstrate the development and presence of dual gRNA nucleotides through NotI/EcoRI restriction enzymes digestion and DNA Sequencing. Moreover, to study the cooperative effects of these mutations in mice, we created viruses and corroborated through Flow cytometry and Fluorescence microscopy. In conclusion, we generate lentiviral particles expressing pairs of gRNAs targeting distinct tumor suppressor genes, to identify cooperative effects of their mutation in lung adenocarcinoma models. Citation Format: Mabel G. Perez-Oquendo, David M. Feldser, David Walter. Identifying cooperative effects of tumor suppressor gene mutations in lung adenocarcinoma using paired CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-195.

We examined the role of IL-18 in preventing the development of and in reversing established allergen-induced airway inflammation and airway hyperreactivity (AHR), the cardinal features of asthma. IL-18, which potently induces IFN-γ, was administered into the respiratory tract as cDNA in a replication-deficient adenovirus (Adv). Treatment of OVA-sensitized mice with the IL-18-expressing Adv reduced allergen-specific IL-4 production, airway eosinophilia, and mucus production, increased IFN-γ production, and prevented the development of AHR. The effects of the IL-18 Adv treatment were dependent on the presence of IFN-γ and IL-12. Moreover, administration of the IL-18 Adv to mice with established AHR greatly reduced AHR and IL-4 production and increased IFN-γ production. These results demonstrate that IL-18, when administered by Adv into the respiratory tract, effectively reduces AHR and replaces an established Th2-biased immune response with a Th1-biased response.

Intracellular protein traffic involves a tightly regulated series of events in which a membrane-bounded vesicles bud from one compartment and are specifically targeted to the next compartment, where they dock and fuse. A cell-free system that reconstitutes vesicle trafficking between the cis and medial Golgi cisternae has been used previously to identify several proteins involved in vesicular transport (N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins, p115, and p16); however, these factors are insufficient to drive the transport reaction. We have used a modified version of this in vitro intra-Golgi transport assay to guide purification of a new transport-stimulating activity. The active component is a 13 S hetero-oligomeric complex consisting of at least five polypeptides (approximately 110, 109, 90, 82, and 71 kDa), which we term Golgi transport complex (GTC). Hydrodynamic properties suggest that GTC is approximately 800 kDa and nonglobular. We obtained peptide sequence information from the 90-kDa subunit (GTC-90) that allowed us to identify a number of GTC-90 cDNAs. Comparison of these cDNAs with one another and with the genomic sequence suggests that the GTC-90 mRNA is alternatively spliced. Anti-GTC-90 antibodies inhibit the in vitro Golgi transport assay, confirming the functionality of the purified complex. Subcellular fractionation indicates that GTC-90 exists in both membrane and cytosolic pools, with the cytosolic pool associated exclusively with the GTC complex. The membrane-associated pool of GTC-90 is localized to the Golgi apparatus.

Vaccination with naked DNA encoding a specific allergen has been shown previously to prevent, but not reverse, the development of allergen-induced airway hyperresponsiveness (AHR). To enhance the effectiveness of DNA vaccine therapies and make possible the treatment of established AHR, we developed a DNA vaccination plasmid containing OVA cDNA fused to IL-18 cDNA. Vaccination of naive mice either with this fusion DNA construct or with an OVA cDNA-containing plasmid protected the mice from the subsequent induction of AHR. Protection from AHR correlated with increased IFN-γ production and reduced OVA-specific IgE production. The protection appeared to be mediated by IFN-γ and CD8+ cells because treatment of mice with neutralizing anti-IFN-γ mAb or with depleting anti-CD8 mAb abolished the protective effect. Moreover, vaccination of mice with preexisting AHR with the OVA-IL-18 fusion DNA, but not with the OVA cDNA plasmid, reversed established AHR, reduced allergen-specific IL-4, and increased allergen-specific IFN-γ production. Thus, combining IL-18 cDNA with OVA cDNA resulted in a vaccine construct that protected against the development of AHR, and that was unique among cDNA constructs in its capacity to reverse established AHR.