Setd2 inactivation sensitizes lung adenocarcinoma to inhibitors of oxidative respiration and mTORC1 signaling.

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.

RB depletion is required for the continuous growth of tumors initiated by loss of RB.

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

NPM-ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation.

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.

Type 1 conventional dendritic cells are systemically dysregulated early in pancreatic carcinogenesis.

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.

Blocking EGFR palmitoylation suppresses PI3K signaling and mutant KRAS lung tumorigenesis.

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.

RB constrains lineage fidelity and multiple stages of tumour progression and metastasis.

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.

Systematic In Vivo Inactivation of Chromatin-Regulating Enzymes Identifies Setd2 as a Potent Tumor Suppressor in Lung Adenocarcinoma.

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.