Paul Yenerall

Essential role of aldehyde dehydrogenase 1A3 for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway

Purpose: Lung cancer stem cells (CSC) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic, and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. Experimental Design: A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ versus ALDH− cells. RT-PCR, Western blot analysis, and Aldefluor assay were used to validate identified genes. To explore the function in CSCs, we manipulated their expression followed by colony and tumor formation assays. Results: We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ versus ALDH− cells. shRNA-mediated knockdown of ALDH1A3 in non–small cell lung cancer (NSCLC) resulted in a dramatic reduction in ALDH activity, clonogenicity, and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. In contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated STAT3 than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well-differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival. Conclusions: Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs. Clin Cancer Res; 20(15); 4154–66. ©2014 AACR.

GLI1 Blockade Potentiates the Antitumor Activity of PI3K Antagonists in Lung Squamous Cell Carcinoma

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.

Harnessing the nuclear receptor PPARγ to inhibit the growth of lung adenocarcinoma by rewiring metabolic circuitries.

Altered metabolism and nuclear receptor activity have been reported in various cancer types. Here, we discuss our recent finding that the metabolic state of lung adenocarcinoma cells expressing the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) can be modulated by thiazolidinediones, culminating in accumulation of reactive oxygen species and decreased proliferation.

Minireview: Familiar Faces in Unfamiliar Places: The Emerging Role of Nuclear Receptors in Lung Cancer

Nuclear hormone receptors (NRs) are a superfamily of 48 transcription factors that are frequently modulated by ligands and control various cancer-relevant cellular pathways, such as differentiation, proliferation, migration, and metabolism. These properties make them excellent therapeutic targets in cancers dependent upon their activity, and as such, 3 NRs, estrogen receptor-α, androgen receptor, and retinoic acid receptor-α (more specifically, the promyelocytic leukemia-retinoic acid receptor-α translocation), have been targeted clinically in breast cancer, prostate cancer, and acute promyelocytic leukemia, respectively. Recently, a number of studies have highlighted a putative role for NRs in nonsmall cell lung cancer (NSCLC), a highly lethal type of lung cancer with relatively few targeted agents. Here, we review the potential roles of selected NRs in NSCLC and offer insights on how NRs may be leveraged in NSCLC to improve patient outcomes.

Abstract IA12: Developing precision medicine-based new lung cancer therapeutics

We have used a “chemistry first” approach to discover druggable acquired vulnerabilities that have been acquired in the pathogenesis of non-small cell lung cancer (NSCLC). We screened chemical libraries (~200,000 compounds) for chemical toxins that killed subsets of NSCLC but not normal human lung epithelial cells (HBECs). We first screened a panel of 12 NSCLC lines that represented a variety of known oncogenotypes and identified chemicals with large Z scores and appropriate properties, including re-supply, chemistry, and reproducible drug response phenotypes, and from this narrowed down a list of 202 chemicals and 18 drugs with known targeting (we called our “Precision Oncology Probe” set, POPS). These and a panel of 30 clinically available drugs, targeted therapies, and drug combinations, already in use or in trials for NSCLC treatment, were then tested on a panel of 96 NSCLC lines for their drug response phenotypes in 12-point dose response curves. This information was analyzed using scanning ranked KS (Kolmogorov–Smirnov) and elastic net biostatistics approaches to identify molecular biomarkers (mutations, mRNA expression, copy number variation, protein expression, and metabolomics) that could predict for sensitivity or resistance to a particular chemical toxin or treatment regimen. From this we have discovered that our approach identifies already known molecular biomarker drug sensitivities (e.g., EGFR mutations and EGFR TK inhibitors); many clinically available chemotherapy agents have molecular biomarkers predicting preclinical model drug responses; the POP set of chemical toxins provides novel drug-response phenotype patterns in the large NSCLC panel different from those found with clinically available agents including a therapeutic window; many of the POP toxins only hit a small % (~5%) of the NSCLC panel but the POP set as a whole provides “coverage” of the entire NSCLC panel; there are simple, one- or two-component molecular biomarkers (mutations, mRNA expression) that predict responses to the different chemical toxins in the NSCLC panel; and that the molecular biomarkers provide some information on the targets and pathways involved in response to the chemical toxins. Thus, we have identified a group of chemical toxins with selectivity for subsets of NSCLC and associated tumor molecular biomarkers to facilitate their development for precision medicine and also, in some cases, information on the targets and pathways interdicted by these chemical compounds. In addition, we have discovered NSCLC predictive biomarkers for clinically available agents. University of Texas SPORE in Lung Cancer (P50CA70907), NCI CTD2N (U01 CA176284), and CPRIT Grants. Citation Format: John D. Minna, Elizabeth McMillan, Luc Girard, Michael Peyton, Kenneth Huffman, Dhruba Deb, Paul Yenerall, Amit Das, Longshan Li, Maithili Dalvi, Boning Gao, Yang Xie, Yonghao Yu, Suzie Hight, Rachel Vaden, Caroline Diep, Michael Roth, Bruce Posner, John MacMillan, Ralph Deberardinis, David Wheeler, John V. Heymach, Ignacio I. Wistuba, Adi Gazdar, Michael White. Developing precision medicine-based new lung cancer therapeutics [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA12.

Abstract 1111: The chromatin remodelers RUVBL1 and RUVBL2 are prognostic factors and therapeutic targets in non-small cell lung cancer due to their roles in DNA replication, repair, and radiosensitization

Despite advances in targeted agents and immunotherapy, non-small cell lung cancer (NSCLC) remains the number one cause of cancer-related death. To identify new therapeutic targets in NSCLC, we performed an siRNA screen directed against genes involved in chromatin remodeling. This screen showed that RUVBL1 and RUVBL2 (herein collectively referred to as RUVBL1/2) were universally but differentially required for the viability of 24 NSCLC cell lines, which was an on-target effect. Various independent gene expression datasets/platforms show that NSCLC patient tumors have increased levels of RUVBL1 and RUVBL2 mRNAs, in comparison to normal lung, and that patients with high levels of RUVBL1 or RUVBL2 have a poorer prognosis, suggesting that RUVBL1/2 may play an important role in NSCLC tumors. To confirm this at the protein level, we validated an antibody against RUVBL1 for immunohistochemistry, stained clinically annotated NSCLC tissue microarrays for RUVBL1, and found that patients with higher levels of RUVBL1 protein also have a poorer prognosis. To better understand the role of RUVBL1/2 in NSCLC at a molecular level, we measured the distribution of cells in the cell cycle following RUVBL1/2 KD, determined RUVBL1/2 interacting proteins by immunoprecipitation followed by tandem mass-spec (IP-MS/MS), and measured gene expression changes following RUVBL1/2 KD by RNA-seq. Depletion of RUVBL1/2 arrested cells in S-phase and promoted pan-γH2AX positivity, IP-MS/MS showed an over-representation of proteins involved in DNA repair and replication, and gene set enrichment analysis of the RNA-seq data displayed a downregulation of transcripts involved in DNA replication and repair, strongly implicating RUVBL1/2 in these processes. To further probe the effects of RUVBL1/2 loss, we performed low-level knock down (KD) of RUVBL1/2, such that viability is largely unaffected, and then measured the viability of cells in response to various drugs (n=35). These drugs target a wide variety of biological processes; however, the only drugs with increased efficacy in the presence of RUVBL1/2 KD were those that damage DNA, target ATR or its downstream partner CHEK1, or target some mitotic proteins, further implicating RUVBL1/2 in DNA replication and/or repair. Due to their roles in replication, we reasoned that RUVBL1/2 KD may enhance the effects of ionizing radiation, a treatment frequently given to NSCLC patients. Low level KD of RUVBL1/2 decreased the clonogenic potential of multiple NSCLC cell lines, which can be phenocopied by depleting independent subunits of RUVBL1/2-containing chromatin remodeling complexes. Finally, we show that RUVBL1/2 depend upon their ATPase activity to support NSCLC viability, suggesting that small molecule inhibitors of this protein may be efficacious in the treatment of NSCLC, especially when combined with radiation therapy. Citation Format: Paul Yenerall, Rahul Kollipara, Amit Das, Pamela Villalobos, Long Shan Li, Brenda Timmons, Luc Girard, Jaime Rodriguez-Canales, Ignacio Wistuba, John Minna, Ralf Kittler. The chromatin remodelers RUVBL1 and RUVBL2 are prognostic factors and therapeutic targets in non-small cell lung cancer due to their roles in DNA replication, repair, and radiosensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2017-1111

Abstract 907: Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner

Cancer-derived exosomes are intracellular signaling organelles that can act via inter-cellular communication to help cancer cells invade neighboring tissues and prime metastatic sites for cancer cell spread. Lung cancer is a highly metastatic disease, with metastases often occurring when tumors are in a clinically early stage. Little is known of how lung cancer exosomes influence the migratory/ invasive phenotype of lung cancer cells. We studied migration and invasion mechanisms by transferring cancer-derived exosomes to recipient cells including cancer cells, human bronchial epithelial cells (HBECs) cells, and HBECs that we had modified with common lung cancer oncogenic changes. Exosomes isolated from lung cancer line H1299 cells (p53null, NRAS mutant) and non-malignant, immortalized HBEC3-KT cells were characterized for exosome content and type by nanoparticle tracking analysis, electron microscopy, western blotting with antibodies directed at HSP70 and CD63, and generation of H1299 and HBEC3-KT derivatives stably transfected with CD63-GFP, releasing green fluorescence exosomes for tracking purposes. Exosomes were isolated using a variety of published methods and the amount quantified: 10 6 H1299 and HBEC-3KT cells produced 9 x 10 9 and 7 x 10 9 exosomes of similar size (125.4nm and 129.8nm). Effect of exosomes from cancer and HBECs were tested on: 1. cell motility and invasiveness of HBECs and their oncogenic derivatives determined by transwell chamber migration assay and scratch assay; 2. vascular leakiness properties in the lung by evaluating mouse lung endothelial permeability after exosomes were injected (an early event in metastatic spread for the migrated cells); and 3. xenograft tumor formation. We found CD63-GFP exosomes from H1299 and HBEC3-KT are actively incorporated by all of the cell types. However, only H1299 exosomes (but not HBEC3-KT exosomes) induced migratory/invasive phenotypic and morphologic changes and they did this in a concentration-dependent manner in lung cancer cells and HBECs with oncogenic changes (HBEC sh-p53+KRAS v12 and sh-p53+KRAS v12 +c-MYC manipulated HBECs) but not in the parental HBEC3-KT cells. Also, H1299 exosomes but not HBEC3-KT exosomes enhanced the lung endothelial permeability as evaluated by the extravasated dextran leaking. NSCLC H1299 cells when injected with its own exosomes produced better xenograft takes compared to H1299 cells alone. Lung cancer-derived exosomes compared to exosomes derived from immortalized but non-malignant human bronchial epithelial cells induced an increased migratory/invasive phenotype with lung vascular leakiness and higher xenograft tumor take rates. However, they induced these changes only in the context of key oncogenic changes. These results suggest understanding and targeting the mechanism of such lung cancer derived exosome behavior could lead to novel therapeutic strategies. Citation Format: Yoshihisa Shimada, Paul Yenerall, Kimberly Avila, Hyunsil Park, Brenda Timmons, Kenneth Huffman, Boning Gao, Dhruba Deb, Maithili Dalvi, John D. Minna. Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2017-907

Abstract 3017: Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer

The Hedgehog (Hh) signaling pathway is critical for embryonic developmental processes and its deregulation is implicated in a wide variety of tumor types. However, the role of the Hh signaling pathway in the initiation and growth of non-small cell lung cancer is largely unknown. The purpose of this study is to investigate the role of GLI1, a major Hh pathway transcription factor, in lung squamous cell carcinoma (SCC) and to test the therapeutic potential of targeting GLI1. GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. We have demonstrated that GLI1 has a critical role in SCC progression. GLI1 expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not change GLI1 expression level in SCC cell lines. However, inhibition of PI3K/mTOR and MAPK signaling pathways down regulated GLI1 expression. These results suggested that GLI1 expression is dependent on PI3K/mTOR and MAPK pathway activity rather than Hh ligand. PI3K/mTOR inhibitor, or arsenic trioxide (ATO), a direct inhibitor of GLI proteins, significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. We have also observed a significant growth reduction in SCC xenografted tumors treated with combination of PI3K inhibitor and ATO. Our findings suggest that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC. Citation Format: Sahba Kasiri, Chunli Shao, Baozhi Chen, Alexandra Wilson, Paul Yenerall, Brenda Timmons, patrick dospoy, Suzie Hight, Luc Girard, Hui Tian, Carmen Behrens, Ignacio Wistuba, Adi Gazdar, James Kim. Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3017.

Abstract 4779: RUVBL1 and RUBVL2 are chromatin remodelers that represent prognostic and novel therapeutic targets for a subset of non-small cell lung cancers (NSCLCs)

RUVBL1 and RUVBL2 (collectively referred to as RUVBL1/2) are AAA+ ATPases that function in various chromatin remodeling complexes. We found that RUVBL1/2 are overexpressed (n = 224) and prognostic of patient outcome in NSCLC patients (n = 697) who undergo surgical resection (combined TCGA, KMPlot.com, and SPORE P50CA70907 dataset analyses). To assess the importance and biological functions of RUVBL1/2 in NSCLC, we measured cell growth following depletion of RUVBL1/2 in 24 NSCLC lines, representing a spectrum of oncogenotypes and histologies, and 2 normal human bronchial epithelial cell lines (HBECs) using two independent RNAi reagents. Growth inhibitory phenotypes ranged from 19-87% and were “rescued” by exogenous expression of an RNAi-resistant cDNA construct, indicating “on-target” siRNA effects. Four of the 23 NSCLCs were very sensitive (>75% growth inhibition), while 88% of NSCLCs were more growth inhibited than HBECs, indicating a therapeutic window. To establish molecular biomarkers of RUVBL1/2 dependency, we correlated whole exome mutation status, whole transcriptome mRNA levels, and a number of other parameters to sensitivity to RUVBL1/2 depletion. Sensitivity to RUVBL1/2 knockdown did not correlate with the mutation status or expression of any genes. However, sensitivity to RUVBL1/2 depletion did correlate with doubling time. Flow cytometry analysis in NSCLC lines sensitive to RUVBL1/2 KD revealed that RUVBL1/2 depletion resulted in a G2/M arrest (but not apoptosis), and a decrease in cell cycle related transcripts, such as CDKN3, AURKA, CIT and CDC20, whereas resistant cell lines do not exhibit these changes. Initial ChIP-seq analysis suggests that RUVBL1/2 preferentially occupies cell cycle related genes and are depleted in nucleosome free regions (i.e. transcription start sites). These results, combined with results from others demonstrating the necessity of ATP hydrolysis for RUVBL1/29s function(s), indicate that the chromatin remodelers RUVBL1/2 are potential therapeutic targets in a subset of NSCLCs. (Supported by UTSW Green Center Fellowship, CPRIT RP120732, SPORE P50CA70907) Citation Format: Paul M. Yenerall, Rahul Kollipara, Ryan Carstens, Kenneth Huffman, Luc Girard, Jaime Rodriguez, Ignacio Wistuba, David Mangelsdorf, John Minna, Ralf Kittler. RUVBL1 and RUBVL2 are chromatin remodelers that represent prognostic and novel therapeutic targets for a subset of non-small cell lung cancers (NSCLCs). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4779. doi:10.1158/1538-7445.AM2015-4779

Abstract B50: Regulation of non-small cell lung cancer stem cells by STAT3 and WNT pathways.

Background: Accumulating evidence demonstrates that the malignant phenotype is driven and maintained by cancer stem cells (CSCs), which can self-renew, differentiate, and contribute to drug resistance and tumor metastasis. Our group has identified CSCs in both non-small cell lung cancer cell (NSCLC) lines and patient tumor samples by their enhanced ALDH activity and demonstrated that ALDH+ lung cancer cells are highly tumorigenic and clonogenic. We also found that ALDH1A3 is the major isozyme responsible for the ALDH activity and functionally important for lung CSCs. Aims and Methods: The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. We performed a siRNA screen of 40 stem cell pathway genes in a panel of NSCLC lines to identify potential targets related to stem cell self-renewal pathways and other regulators. qRT-PCR, western blot and flow cytometry based Aldefluor assay were used to validate the promising hits. To further explore their biological function in lung CSCs we manipulated their expression genetically or pharmacologically followed by colony formation and tumor formation assays. Results: siRNA screen revealed that knocking down ALDH1A3, Signal Transducers and Activators of Transcription 3 (STAT3), or certain components of WNT and Notch pathway caused a reduction of cell viability in many NSCLC lines. Western blot analysis confirmed that ALDH+ cells contained more activated STAT3 compared to ALDH− lung cancer cells. Inhibition of STAT3 activation by Stattic (a specific STAT3 inhibitor), GSK126 (an EZH2 inhibitor), or siRNA attenuated ALDH1A3 expression, ALDH+ lung cancer cells and tumor cell clonogenicity. In addition, FZD2, DVL3 and LBH were more commonly upregulated in ALDH+ cells across the penal of NSCLC lines. shRNA mediated knockdown of DVL3 in NSCLCs resulted in a significant reduction in ALDH activity, clonogenicity, and tumorigenicity. Conclusions: Our data suggest that ALDH1A3 expression, STAT3 pathway activation (via EZH2), and an active WNT pathway are all essential for maintenance of lung CSCs. Blocking ALDH1A3, WNT or STAT3 pathways appear to be rationale therapeutic strategies for eliminating NSCLC stem cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B50. Citation Format: Chunli Shao, Chris DeSevo, Luc Girard, Paul Yenerall, Carmen Behrens, Ignacio Wistuba, John D. Minna. Regulation of non-small cell lung cancer stem cells by STAT3 and WNT pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B50.