Kimberly Walter

Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events

Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.

Heterogeneity and clinical significance of ESR1 mutations in ER-positive metastatic breast cancer patients receiving fulvestrant

Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study.

Loss of NAPRT1 Expression by Tumor-specific Promoter Methylation Provides a Novel Predictive Biomarker for NAMPT Inhibitors

Purpose: We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. Experimental Design: We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications. Results: Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the NAPRT1 promoter identified tumor-specific sites of NAPRT1 DNA methylation and enabled the development of a quantitative methylation-specific PCR (QMSP) assay suitable for use on archival formalin-fixed paraffin-embedded tumor tissue. Conclusions: Tumor-specific promoter hypermethylation of NAPRT1 inactivates one of two NAD salvage pathways, resulting in synthetic lethality with the coadministration of a NAMPT inhibitor. NAPRT1 expression is lost due to promoter hypermethylation in most cancer types evaluated at frequencies ranging from 5% to 65%. NAPRT1-specific immunohistochemical or DNA methylation assays can be used on archival formalin paraffin-embedded cancer tissue to identify patients likely to benefit from coadministration of a Nampt inhibitor and nicotinic acid. Clin Cancer Res; 19(24); 6912–23. ©2013 AACR.

The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

Activating Mutations in PIK3CB Confer Resistance to PI3K Inhibition and Define a Novel Oncogenic Role for p110β

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110β D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.

Integrative analysis of two cell lines derived from a non-small-lung cancer patient--a panomics approach.

Cancer cells derived from different stages of tumor progression may exhibit distinct biological properties, as exemplified by the paired lung cancer cell lines H1993 and H2073. While H1993 was derived from chemo-naive metastasized tumor, H2073 originated from the chemo-resistant primary tumor from the same patient and exhibits strikingly different drug response profile. To understand the underlying genetic and epigenetic bases for their biological properties, we investigated these cells using a wide range of large-scale methods including whole genome sequencing, RNA sequencing, SNP array, DNA methylation array, and de novo genome assembly. We conducted an integrative analysis of both cell lines to distinguish between potential driver and passenger alterations. Although many genes are mutated in these cell lines, the combination of DNA- and RNA-based variant information strongly implicates a small number of genes including TP53 and STK11 as likely drivers. Likewise, we found a diverse set of genes differentially expressed between these cell lines, but only a fraction can be attributed to changes in DNA copy number or methylation. This set included the ABC transporter ABCC4, implicated in drug resistance, and the metastasis associated MET oncogene. While the rich data content allowed us to reduce the space of hypotheses that could explain most of the observed biological properties, we also caution there is a lack of statistical power and inherent limitations in such single patient case studies.

Abstract 4978: Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival.

The immune system has multiple mechanisms by which it can eliminate cancer and yet, tumors are able to adapt resistance to host immune surveillance and continue to survive and grow. One critical resistance mechanism involves Programmed cell death 1 ligand 1 (PD-L1, CD274, B7-H1), the predominant ligand for PD-1, an inhibitory receptor expressed on T cells following activation. PD-L1 also binds to B7.1 (CD80), inhibiting its ability to provide an immune stimulatory signal. PD-L1 is expressed broadly on multiple peripheral blood mononuclear cell subtypes, placenta, and numerous cancers, including NSCLC. Tumor-specific T cells infiltrate tumors and recognize tumor cells, releasing Interferon-gamma (IFNγ), initiating signaling of the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in the tumor cells. IFNγ induces and/or greatly enhances the expression of PD-L1 in the tumor, among other cell types, allowing the tumor to become resistant to the host T cell response. Blockade of PD-L1 binding to PD-1 and B7.1 can reinvigorate the host immune response against the tumor and overcome tumor adaptive resistance. The regulation of PD-L1 expression is complex and likely involves multiple types of pre- and post-translational events. We show that basal PD-L1 expression levels can vary greatly in cancer cells, as can PD-L1 induction by IFNγ. Here we describe the different categories of PD-L1 basal expression and IFNγ–dependent regulation across multiple lung cell lines and human tumor samples. Reverse Phase Protein Array and RNA microarray data show that the JAK/STAT canonical pathways are still intact among all of these distinct categories, so alternative mechanisms of expression regulation must be active in these cell lines and tumor samples. We provide evidence that a combination of mechanisms regulate both the basal and stimulated expression levels of PD-L1 across these distinct categories. This data regarding PD-L1 expression regulation provides valuable information to better understand the PD-1/PD-L1 pathway as a therapeutic target. Citation Format: Edward (Ward) E. Kadel, Kimberly Walter, Rupal Desai, Juliet Carbon, Marigold Boe, David Shames, Marcin Kowanetz. Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2013-4978

Abstract B28: Activating mutations in PIK3CB confer resistance to PI3K inhibition in PTEN-deficient breast cancer and define a novel oncogenic role for p110β

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs commonly in breast cancers via mechanisms that include loss of function of the tumor suppressor phosphatase and tensin homolog (PTEN). Experience with other successful targeted agents suggests that clinical resistance to PI3K inhibitors is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN deficient breast cancers. PTEN null breast cancer cell lines were selected for resistance to a pan-PI3K inhibitor, GDC-0941. Comprehensive molecular and cellular profiling was conducted to identify the mechanism of resistance. We generated GDC-0941 resistant derivatives of PTEN deficient EVSA-T and ZR-75-1 cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines. We found that the PIK3CB mutation at D1067 position was recurrent in cancer patients. Stable expression of mutant PIK3CB variants in PTEN null breast cancer cells conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. We showed further that the p110β D1067Y mutant is highly activated and elevates PIP3 level at the cell membrane, promoting localization and activation of AKT and PDK1 at the cell membrane and driving PI3K signaling to a level that can overcome treatment with proximal inhibitors. Finally, we show that the PIK3CB D1067Y mutant can behave as an oncogene and transform normal cells, and that PTEN knock down enhances this activity. These novel preclinical and clinical findings implicate PIK3CB D1067 alterations as a novel oncogene that may cause resistance to selective PI3K inhibitor treatment. Taken together with previous findings that PTEN deficient cancers tend to signal through p110β rather than p110α, this work also suggests PTEN deficient breast cancers may depend on p110β and are thereby susceptible to PIK3CB mutation as an escape mechanism from PI3K inhibition. Citation Format: Yoshito Nakanishi, Kimberly M. Walter, Jill M. Spoerke, Carol O9Brien, Ling Y. Huw, Garret M. Hampton, Mark R. Lackner. Activating mutations in PIK3CB confer resistance to PI3K inhibition in PTEN-deficient breast cancer and define a novel oncogenic role for p110β. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B28.

Abstract 5155: Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter

The molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) have been well described. However, the mechanisms through which some of the clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we employed an integrative genomics approach to examine CRC progression. Transcriptional profiling of laser capture microdissected colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas, and metastases, showed distinctive patterns in the activation of developmental and oncogenic pathways, including the clinically important EGFR axis. We observed a dramatic up-regulation of the EGFR ligand EREG in primary and metastatic cancer cells as compared to normal and adenomatous tissues that was indicative of autocrine tumor production, and confirmed this pattern of gene expression by in situ hybridization. Global methylation analysis indicated that up-regulation of EREG during the adenoma-carcinoma transition was associated with de-methylation of two key sites within the EREG promoter and this was accompanied by an increase in the levels of EGFR phosphorylation, as assessed by reverse phase protein analysis. In a clinical trial setting, we observed that low levels of EREG methylation in patients who received cetuximab as part of a Phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers from the TCGA datasets, including those of the head and neck, lung, and bladder. We propose that up-regulation of EREG expression through promoter de-methylation might be an important means of activating the EGFR pathway during the genesis of CRC and, potentially, other types of cancer. Citation Format: Xueping Qu, Thomas Sandmann, Henry Frierson, Ling Fu, Eloisa Fuentes, Kimberly Walter, Kwame Okrah, Craig Rumpel, Christopher Moskaluk, Shan Lu, Yulei Wang, Richard Bourgon, Elicia Penuel, Andrea Pirzkall, Lukas Amler, Mark Lackner, Josep Tabenero, Garret Hampton, Omar Kabbarah. Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter. [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 5155.

Abstract B37: Mechanisms of acquired resistance to the PI3K inhibitor GDC-0941 in breast cancer cell lines

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K is a promising therapeutic target for cancer. There are several PI3K inhibitors in clinical trials and the current study was intended to investigate preclinical mechanisms of acquired resistance to GDC-0941, a class I selective PI3K inhibitor. GDC-0941 resistant pools and clones were generated in both EVSA-T and HCC-1954 breast cancer cell lines by treating cells with increasing concentrations of drug. We found that resistant clones from both lines demonstrated marked increase of downstream PI3K pathway signaling and upregulation of the MAPK pathway, through two different mechanisms. In HCC-1954, autocrine signaling to EGFR was observed in the resistant clones and resulted in increased dependency on the downstream factor c-Myc. EVSA-T clones with acquired resistance to GDC-0941 had elevated HER2 copy number and protein expression levels. A corresponding increase in phosphorylation of HER2 binding partner ErbB3 was also discovered in the EVSA-T resistance cells. In both cases sensitivity to PI3K inhibition was restored by blocking the activated upstream receptor tyrosine kinase, EGFR for HCC-1954 and HER2 for EVSA-T. Additionally both models were re-sensitized to GDC-0941 by blocking downstream signaling of the MAPK pathway. These preclinical data may provide rationale for combination therapy with PI3K inhibitors in the clinic. Citation Format: Kyle A. Edgar, Ling Hwu, Kimberly Walter, Mark Lackner, Lori S. Friedman, Jeffrey J. Wallin. Mechanisms of acquired resistance to the PI3K inhibitor GDC-0941 in breast cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B37.