Brad Windle

Gain-of-function mutant p53 upregulates CXC chemokines and enhances cell migration

The role of dominant transforming p53 in carcinogenesis is poorly understood. Our previous data suggested that aberrant p53 proteins can enhance tumorigenesis and metastasis. Here, we examined potential mechanisms through which gain-of-function (GOF) p53 proteins can induce motility. Cells expressing GOF p53 -R175H, -R273H and -D281G showed enhanced migration, which was reversed by RNA interference (RNAi) or transactivation-deficient mutants. In cells with engineered or endogenous p53 mutants, enhanced migration was reduced by downregulation of nuclear factor-kappaB2, a GOF p53 target. We found that GOF p53 proteins upregulate CXC-chemokine expression, the inflammatory mediators that contribute to multiple aspects of tumorigenesis. Elevated expression of CXCL5, CXCL8 and CXCL12 was found in cells expressing oncogenic p53. Transcription was elevated as CXCL5 and CXCL8 promoter activity was higher in cells expressing GOF p53, whereas wild-type p53 repressed promoter activity. Chromatin immunoprecipitation assays revealed enhanced presence of acetylated histone H3 on the CXCL5 promoter in H1299/R273H cells, in agreement with increased transcriptional activity of the promoter, whereas RNAi-mediated repression of CXCL5 inhibited cell migration. Consistent with this, knockdown of the endogenous mutant p53 in lung cancer or melanoma cells reduced CXCL5 expression and cell migration. Furthermore, short hairpin RNA knockdown of mutant p53 in MDA-MB-231 cells reduced expression of a number of key targets, including several chemokines and other inflammatory mediators. Finally, CXCL5 expression was also elevated in lung tumor samples containing GOF p53, indicating relevance to human cancer. The data suggest a mechanistic link between GOF p53 proteins and chemokines in enhanced cell motility.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

Significance Small-cell lung cancer (SCLC) is an aggressive carcinoma with few effective treatment options beyond first-line chemotherapy. BH3 mimetics, such as ABT-263, promote apoptosis in SCLC cell lines, but early phase clinical trials demonstrated no significant clinical benefit. Here, we examine the sensitivity of a large panel of cancer cell lines, including SCLC, to ABT-263 and find that high Bcl2-interacting mediator of cell death (BIM) and low myeloid cell leukemia 1 (MCL-1) expression together predict sensitivity. SCLC cells relatively resistant to ABT-263 are sensitized by TORC1/2 inhibition via MCL-1 reduction. Combination of ABT-263 and TORC1/2 inhibition stabilizes or shrinks tumors in xenograft models, in autochthonous SCLC tumors in a genetically engineered mouse model, and in a patient-derived xenograft SCLC model. Collectively, these data support a compelling new therapeutic strategy for treating SCLC. BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Gain-of-Function Activity of Mutant p53 in Lung Cancer through Up-Regulation of Receptor Protein Tyrosine Kinase Axl.

p53 mutations are present in up to 70% of lung cancer. Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 or no p53. Expression of tumor-derived mutant p53 in cells leads to up-regulated expression of genes that may affect cell growth and oncogenesis. In our study of this aggressive phenotype, we have investigated the receptor protein tyrosine kinase Axl, which is up-regulated by p53 mutants at both RNA and protein levels in H1299 lung cancer cells expressing mutants p53-R175H, -R273H, and -D281G. Knockdown of endogenous mutant p53 levels in human lung cancer cells H1048 (p53-R273C) and H1437 (p53-R267P) led to a reduction in the level of Axl as well. This effect on Axl expression is refractory to the mutations at positions 22 and 23 of p53, suggesting that p53s transactivation domain may not play a critical role in the up-regulation of Axl gene expression. Chromatin immunoprecipitation (ChIP) assays carried out with acetylated histone antibodies demonstrated induced histone acetylation on the Axl promoter region by mutant p53. Direct mutant p53 nucleation on the Axl promoter was demonstrated by ChIP assays using antibodies against p53. The Axl promoter has a p53/p63 binding site, which however is not required for mutant p53-mediated transactivation. Knockdown of Axl by Axl-specific RNAi caused a reduction of gain-of-function (GOF) activities, reducing the cell growth rate and motility rate in lung cancer cells expressing mutant p53. This indicates that for lung cancer cell lines with mutant p53, GOF activities are mediated in part through Axl.

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Analysis of The Cancer Genome Atlas sequencing data reveals novel properties of the human papillomavirus 16 genome in head and neck squamous cell carcinoma

Human papillomavirus (HPV) DNA is detected in up to 80% of oropharyngeal carcinomas (OPC) and this HPV positive disease has reached epidemic proportions. To increase our understanding of the disease, we investigated the status of the HPV16 genome in HPV-positive head and neck cancers (HNC). Raw RNA-Seq and Whole Genome Sequence data from The Cancer Genome Atlas HNC samples were analyzed to gain a full understanding of the HPV genome status for these tumors. Several remarkable and novel observations were made following this analysis. Firstly, there are three main HPV genome states in these tumors that are split relatively evenly: An episomal only state, an integrated state, and a state in which the viral genome exists as a hybrid episome with human DNA. Secondly, none of the tumors expressed high levels of E6; E6*I is the dominant variant expressed in all tumors. The most striking conclusion from this study is that around three quarters of HPV16 positive HNC contain episomal versions of the viral genome that are likely replicating in an E1-E2 dependent manner. The clinical and therapeutic implications of these observations are discussed.

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter

Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53.

Gene function classification using NCI-60 cell line gene expression profiles

Gene expression patterns from NCIs panel of 60 cell lines were used to train a Neural Network model for classifying genes to pathways. The model assigns probabilities to each gene for each of the 21 modeled pathways assigned by the Kyoto Encyclopedia of Genes and Genomes. Cross-validation of the model showed that 10 of the 21 pathways exhibited good performance in statistical significance and accuracy. The model was designed to output gene probabilities that could be screened for higher probabilities resulting in higher confidence in classification though yielding fewer genes per pathway. The model was deployed on 5798 genes and our approach allowed us to ascertain the most relevant genes above an estimated background. Eight pathways were identified with both good cross-validation and significant numbers above background, TCA Cycle, Oxidative Phosphorylation, Porphyrin Biosynthesis, Ribosome, Polymerases, Proteasome, Cell Cycle, and Cell Adhesion. Gene Ontology (GO) annotation was used for additional validation of gene classification results. A total of 551 GO annotated genes and 468 unannotated genes were classified to the 8 pathways. The primary and secondary classifications of genes revealed known pathway relationships and provide the potential for discovering new pathway relationships.

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.