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Supplementary Tables S1-S2 from An Embryonic Stem Cell–Like Signature Identifies Poorly Differentiated Lung Adenocarcinoma but not Squamous Cell Carcinoma

APOBEC and AICDA expression after exposure to gefitinb.

PDF file - 90K, Notch pathway genes categorized based on function.

Purpose: An embryonic stem cell (ESC) profile correlates with poorly differentiated breast, bladder, and glioma cancers. In this article, we assess the correlation between the ESC profile and clinical variables in lung cancer.Experimental Design: Microarray gene expression analysis was done using Affymetrix Human Genome U133A on 443 samples of human lung adenocarcinoma and 130 samples of squamous cell carcinoma (SCC). To identify gene set enrichment patterns, we used the Genomica software.Results: Our analysis showed that an increased expression of the ESC gene set and a decreased expression of the Polycomb target gene set identified poorly differentiated lung adenocarcinoma. In addition, this gene expression signature was associated with markers of poor prognosis and worse overall survival in lung adenocarcinoma. However, there was no correlation between this ESC gene signature and any histologic or clinical variable assessed in lung SCC.Conclusions: This work suggests that not all poorly differentiated non–small cell lung cancers exhibit a gene expression profile similar to that of ESC, and that other characteristics may play a more important role in the determination of differentiation and survival in SCC of the lung. (Clin Cancer Res 2009;15(20):6386–90)

Uracil-DNA glycosylase (UDG) hydrolysis assay.

PDF file - 467K, Representative immunohistochemical staining for Hes-1 protein, in primary NSCLC tissue micro array and NCI-H1299 xenograft.

PDF file - 357K, NSCLC cell lines transduced with Notch reporter.

PDF file - 184K, Supporting data for Notch reporter specificity, Notch activity and MRK003 specific targeting of Notch pathway.

Supplementary Table S3 from An Embryonic Stem Cell–Like Signature Identifies Poorly Differentiated Lung Adenocarcinoma but not Squamous Cell Carcinoma

AICDA CRISPR1 and CRISPR2 mutations.

XLSX file - 63K, Microarray Notch pathway components gene expression data on 443 human lung adenocarcinomas.

Patients' clinical data.

Sensitivity of T790M ddPCR assay.

AICDA and RELB expression after TKI exposure was inhibited upon pretreatment with BAY11-7802.

PDF file - 196K, Stable transduction of NCI-H1299 with Notch reporter, where GFP-bright cells regenerate GFP-dim cells, while GFP-dim cells are unable to generate GFP-bright cells.

PDF file - 84K, Immunohistochemistry and PCR assays methodology.

Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it.Significance:These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.

Purpose: The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation, and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells.Experimental Design: We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a γ-secretase inhibitor (GSI), which inhibits Notch pathway activity.Results: Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival.Conclusions: Notch activity can identify lung cancer stem cell–like population and its inhibition may be an appropriate target for treating lung adenocarcinoma. Clin Cancer Res; 19(8); 1972–80. ©2013 AACR.

PDF file - 82K, Relative expression of Notch receptors and ligands in multiple NSCLC cell lines transduced with Notch reporter.

PDF file - 89K, Relative gene expression of cancer stem cell related genes based on Notch activity.

Supplementary Figures S1-S2 from An Embryonic Stem Cell–Like Signature Identifies Poorly Differentiated Lung Adenocarcinoma but not Squamous Cell Carcinoma

Deamination of Cytosine or 5-methylcytosine to Uracil or Thymine respectively.

PDF file - 59K, Notch1 receptor mutation analysis in multiple NSCLC cell lines.

PDF file - 102K, Xenograft tumor growth curves of NCI-H1299 cell lines, in mice treated with MRK003, docetaxel, and combination with evidence of blocking Notch activity with MRK003 treatment.

PDF file - 76K, Clinical variables of the lung adenocarcinoma patients.

Improving the clinical outcomes of patients withIn this review, we survey the epidemiology ofWhile sotorasib's development has been unique and exciting, questions persist regarding its intracranial penetrance, optimal dose, and efficacy relative to standard-of-care therapy. Improvements in the clinical activity of KRAS inhibition will hinge on better understanding of resistance mechanisms, the development of broad-spectrum inhibitors with activity beyond G12C mutations, and combination therapy targeting multiple mediators of KRAS signaling and alternative pathways. From a regulatory perspective, sotorasib's development may, in time, prove to be an instructive example for early-phase clinical trialists and regulators focused on dose optimization.