Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities.

The degradation tag (dTAG) system for target protein degradation can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack of reversibility, low specificity, and the inability to titrate dosage. These drawbacks can make it difficult to compare toxicity resulting from genetic and pharmacological interventions, especially in vivo. Because the dTAG system has not been studied extensively in vivo, we explored the use of this system to study the physiological sequalae resulting from CDK2 or CDK5 degradation in adult mice. Mice with homozygous knock-in of the dTAG sequence onto CDK2 and CDK5 were born at Mendelian ratios despite decreased CDK2 or CDK5 protein levels in comparison with wild-type mice. In bone marrow cells and duodenum organoids derived from these mice, treatment with the dTAG degrader dTAG-13 resulted in rapid and robust protein degradation but caused no appreciable change in viability or the transcriptome. Repeated delivery of dTAG-13 in vivo for toxicity studies proved challenging; we explored multiple formulations in an effort to maximize degradation while minimizing formulation-related toxicity. Degradation of CDK2 or CDK5 in all organs except the brain, where dTAG-13 likely did not cross the blood brain barrier, only caused microscopic changes in the testis of CDK2dTAG mice. These findings were corroborated with conditional CDK2 knockout in adult mice. Our results suggest that the dTAG system can provide robust protein degradation in vivo and that loss of CDK2 or CDK5 in adult mice causes no previously unknown phenotypes.

Structure-based classification of EGFR mutations informs inhibitor selection for lung cancer therapy.

EGFR oncogenic mutations predict sensitivity to EGFR inhibitors in NSCLC, but less is known about EGFR "variants of unknown significance." Using preclinical models, 3D structure analyses, and patient response data, Robichaux et al. show in Nature that mutations in structural regions of EGFR predict responses to different EGFR inhibitors.

Lentiviral-Driven Discovery of Cancer Drug Resistance Mutations.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis.

Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.

Mithramycin suppresses DNA damage repair via targeting androgen receptor in prostate cancer.

The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.

RUVBL1/RUVBL2 ATPase Activity Drives PAQosome Maturation, DNA Replication and Radioresistance in Lung Cancer.

RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.

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.

Taxane-Platin-Resistant Lung Cancers Co-develop Hypersensitivity to JumonjiC Demethylase Inhibitors.

Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.

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.

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.

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.

Identifying the mechanisms of intron gain: progress and trends.

Continued improvements in Next-Generation DNA/RNA sequencing coupled with advances in gene annotation have provided researchers access to a plethora of annotated genomes. Subsequent analyses of orthologous gene structures have identified numerous intron gain and loss events that have occurred both recently and in the very distant past. This research has afforded exceptional insight into the temporal and lineage-specific rates of intron gain and loss among various species throughout evolution. Numerous studies have also attempted to identify the molecular mechanisms of intron gain and loss. However, even after considerable effort, very little is known about these processes. In particular, the mechanism(s) of intron gain have proven exceptionally enigmatic and remain topics of considerable debate. Currently, there exists no definitive consensus as to what mechanism(s) may generate introns. Because many introns are known to affect gene expression, it is necessary to understand the molecular process(es) by which introns may be gained. Here we review the seven most commonly purported mechanisms of intron gain and, when possible, summarize molecular evidence for or against the occurrence of each of these mechanisms. Furthermore, we catalogue indirect evidence that supports the occurrence of each mechanism. Finally, because these proposed mechanisms fail to explain the mechanistic origin of many recently gained introns, we also look at trends that may aid researchers in identifying other potential mechanism(s) of intron gain.

Mechanisms of intron gain and loss in Drosophila.

BACKGROUND: It is widely accepted that orthologous genes have lost or gained introns throughout evolution. However, the specific mechanisms that generate these changes have proved elusive. Introns are known to affect nearly every level of gene expression. Therefore, understanding their mechanism of evolution after their initial fixation in eukaryotes is pertinent to understanding the means by which organisms develop greater regulation and complexity. RESULTS: To investigate possible mechanisms of intron gain and loss, we identified 189 intron gain and 297 intron loss events among 11 Drosophila species. We then investigated these events for signatures of previously proposed mechanisms of intron gain and loss. This work constitutes the first comprehensive study into the specific mechanisms that may generate intron gains and losses in Drosophila. We report evidence of intron gain via transposon insertion; the first intron loss that may have occurred via non-homologous end joining; intron gains via the repair of a double strand break; evidence of intron sliding; and evidence that internal or 5' introns may not frequently be deleted via the self-priming of reverse transcription during mRNA-mediated intron loss. Our data also suggest that the transcription process may promote or result in intron gain. CONCLUSION: Our findings support the occurrence of intron gain via transposon insertion, repair of double strand breaks, as well as intron loss via non-homologous end joining. Furthermore, our data suggest that intron gain may be enabled by or due to transcription, and we shed further light on the exact mechanism of mRNA-mediated intron loss.