Cellular Origins of EGFR-Driven Lung Cancer Cells Determine Sensitivity to Therapy

Targeting the epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKIs) is one of the major precision medicine treatment options for lung adenocarcinoma. Due to common development of drug resistance to first- and second-generation TKIs, third-generation inhibitors, including osimertinib and rociletinib, have been developed. A model of EGFR-driven lung cancer and a method to develop tumors of distinct epigenetic states through 3D organotypic cultures are described here. It is discovered that activation of the EGFR T790M/L858R mutation in lung epithelial cells can drive lung cancers with alveolar or bronchiolar features, which can originate from alveolar type 2 (AT2) cells or bronchioalveolar stem cells, but not basal cells or club cells of the trachea. It is also demonstrated that these clones are able to retain their epigenetic differences through passaging orthotopically in mice and crucially that they have distinct drug vulnerabilities. This work serves as a blueprint for exploring how epigenetics can be used to stratify patients for precision medicine decisions

H3K9 Methyltransferases and Demethylases Control Lung Tumor-Propagating Cells and Lung Cancer Progression

Epigenetic regulators are attractive anticancer targets, but the promise of therapeutic strategies inhibiting some of these factors has not been proven in vivo or taken into account tumor cell heterogeneity. Here we show that the histone methyltransferase G9a, reported to be a therapeutic target in many cancers, is a suppressor of aggressive lung tumor-propagating cells (TPCs). Inhibition of G9a drives lung adenocarcinoma cells towards the TPC phenotype by de-repressing genes which regulate the extracellular matrix. Depletion of G9a during tumorigenesis enriches tumors in TPCs and accelerates disease progression metastasis. Depleting histone demethylases represses G9a-regulated genes and TPC phenotypes. Demethylase inhibition impairs lung adenocarcinoma progression in vivo. Therefore, inhibition of G9a is dangerous in certain cancer contexts, and targeting the histone demethylases is a more suitable approach for lung cancer treatment. Understanding cellular context and specific tumor populations is critical when targeting epigenetic regulators in cancer for future therapeutic development

Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer

As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically-engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways leading to epigenetic transformation independent from canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a novel and potent EZH2 inhibitor JQEZ5 that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer

\u3cem\u3eLkb1\u3c/em\u3e Inactivation Drives Lung Cancer Lineage Switching Governed by Polycomb Repressive Complex 2

Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours

The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations

Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of <0.2% in general population databases. Epithelial sodium channel (ENaC) mutants were generated via site-directed mutagenesis and expressed for Xenopus oocyte assays. Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of the ENaC. Separately, an independently enriched rare variant in SCNN1D was identified in the Exome Variant Server database associated with a milder pulmonary disease phenotype. Functional analysis using Xenopus oocytes revealed that two of the three variants in δ-ENaC encoded by SCNN1D exhibited hypomorphic channel activity. Our data suggest a potential role for δ-ENaC in controlling sodium reabsorption in the airways, and advance the plausibility of ENaC as a therapeutic target in CF

FIGURE 5 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

EZH2 inhibition alone and combined with immunotherapy is effective at controlling tumor burden in mouse models of LSCC. A, Representative MRI scans of autochthonous mice from each treatment arm at baseline and after treatment. B, Waterfall plot showing change in tumor volume for each mouse on all treatment arms, ****, P P P P post hoc test on log2-transformed values. C, H&E and HALO nuclear phenotyper images showing the cells within an autochthonous Lkb1/Pten tumor and a syngeneic graft seeded from Lkb1/Pten tumoroids. D, Percentage tumor growth from the syngeneic mouse model during 14 days of indicated treatments. ***, P = 0.0004; ****, P post hoc test, *, P = 0.024 by two-tailed t test on log2-transformed values, Mice/tumors n are placebo = 4/8, EPZ6438 = 5/9, anti-PD1 = 6/8, combo = 5/9, mean ± SEM. is plotted. E, Flow cytometry analysis of dissociated tumors from the syngeneic grafts from the indicated treatment arms at day 14. Percentage of EpCAM+ cells expressing IA/IE or PD-L1 are graphed, mean ± SEM is plotted, placebo n = 7, EZH2 inhibitor n = 7, anti-PD1 n = 8, combo n = 7 with two experimental replicates each, *, P = 0.035; ***, P = 0.0008 by one-way ANOVA with multiple comparisons and Holm-Šídák post hoc test. F, From the same tumor grafts, MFI for HLA-A in the EpCAM+ cells was graphed, mean ± SEM is plotted, placebo n = 7, EZH2 inhibitor n = 7, anti-PD1 n = 8, combo n = 7 with 2 experimental replicates each, **, P = 0.0015 by one-way ANOVA with multiple comparisons and Holm-Šídák post hoc test. G, From tumor grafts, PD1+/CD3+/CD4+ cells and PD1+/CD3+/CD8+ were gated and percentage of cells bound to Rat-IgG2A antibody are graphed, mean ± SEM is plotted, placebo n = 6, EZH2 inhibitor n = 6, anti-PD1 n = 7, combo n = 7; **, P P = 0.0001; ****, P post hoc test. H, From the grafts, percentage of CD3+/SSC-low cells within the CD45+ fraction and percentage of CD8+ cells within the CD3+ fraction were graphed, please see Supplementary Fig. S5C for representative gates, placebo n = 8, EZH2 inhibitor n = 8, anti-PD1 n = 9, combo n = 7 with two experimental replicates each, *, P = 0.0197; ****, P post hoc test. See also Supplementary Fig. S5.</p

FIGURE 1 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

EZH2 inhibition allows upregulation of MHCI and MHCII in 2D human LSCC cell lines. A, Schematic for proposed mechanism: Inhibition of EZH2 methyltransferase activity by the drugs GSK126 or EPZ6438 will lead to derepression of antigen presentation genes that can then be more effectively activated by IFNγ. B, qRT-PCR in the indicated four human lung cancer cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the genes B2M, HLA-A, CIITA, and HLA-DRA, mean ± SEM is graphed, n = 4 individual cultures, *, P P P P post hoc test. C, Flow cytometry analysis of indicated four human lung cancer cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the cell surface proteins HLA-A,B,C and HLA-DR; mean ± SEM is graphed; n = 4 individual cultures; *, P P P P post hoc test. Representative histograms from HCC15 cell lines are shown, G = GSK126, E = EPZ6438, I = IFNγ, I+G = IFNγ+GSK126, and I+E = IFNγ+EPZ6438. D, Western blotting of A549 and HCC15 cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the proteins B2M, HLA-DR, DQ, DP, EZH2, H3K27me3 and total histone H3. Data are representative of two individual cultures. See also Supplementary Fig. S1.</p

Supplementary Figure 5 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 5 shows additional analysis of tumors from EZH2 inhibition and anti-PD1 treated mice.</p

FIGURE 3 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Murine LSCC organoids share derepression of MHC and pro-T cell cytokines with human models. A, Schematic: Generation of murine tumoroids in air-liquid interface from tumor induced in Lkb1/Pten mice by adenoCre administration, showing H&E stain of tumoroids, scale bar = 100 µm, and brightfield microscopy, scale bar = 200 µm. B, Flow cytometry analysis of two separate murine tumoroid models treated for 11 days with 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 9 stained for cell surface expression of NGFR, PD-L1, H2Kd,Dd, and I-A/I-E, n = 5 individual experiments except mouse 2 I-A/I-E and PD-L1; n = 4 individual experiments; *, P P P = 0.0002; ****, P post hoc test. C, Heat maps of log2 fold change in expression level from patient-derived and murine tumoroids treated for 11 days with 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added in on day 9, G = GSK126, E = EPZ6438, I = IFNγ, I+G = IFNγ+GSK126, and I+E = IFNγ+EPZ6438. For each map, the first columns are sample 1, the second columns are sample 2. Expression relative to vehicle control (left) and relative to IFNγ only (right) are depicted. See also Supplementary Fig. S3.</p

Supplementary Figure 2 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 2 shows changes in NGFR and CD274 (PD-L1) gene and protein expression in human lung cancer tumoroids in response to EZH2 inhibitor and interferon-gamma treatment.</p