Turgut Dogruluk

Moesin is a glioma progression marker that induces proliferation and Wnt/β-catenin pathway activation via interaction with CD44

Moesin is an ERM family protein that connects the actin cytoskeleton to transmembrane receptors. With the identification of the ERM family protein NF2 as a tumor suppressor in glioblastoma, we investigated roles for other ERM proteins in this malignancy. Here, we report that overexpression of moesin occurs generally in high-grade glioblastoma in a pattern correlated with the stem cell marker CD44. Unlike NF2, moesin acts as an oncogene by increasing cell proliferation and stem cell neurosphere formation, with its ectopic overexpression sufficient to shorten survival in an orthotopic mouse model of glioblastoma. Moesin was the major ERM member activated by phosphorylation in glioblastoma cells, where it interacted and colocalized with CD44 in membrane protrusions. Increasing the levels of moesin competitively displaced NF2 from CD44, increasing CD44 expression in a positive feedback loop driven by the Wnt/β-catenin signaling pathway. Therapeutic targeting of the moesin-CD44 interaction with the small-molecule inhibitor 7-cyanoquinocarcinol (DX-52-1) or with a CD44-mimetic peptide specifically reduced the proliferation of glioblastoma cells overexpressing moesin, where the Wnt/β-catenin pathway was activated. Our findings establish moesin and CD44 as progression markers and drugable targets in glioblastoma, relating their oncogenic effects to activation of the Wnt/β-catenin pathway.

HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome

Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFβ) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1’s ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.

Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.

Functional annotation of rare gene aberration drivers of pancreatic cancer

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets.

Systematic Functional Annotation of Somatic Mutations in Cancer

The functional impact of the vast majority of cancer somatic mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. Further, the platform is sufficiently sensitive to identify weak drivers. Our data are accessible through a user-friendly, public data portal. Our study will facilitate biomarker discovery, prediction algorithm improvement, and drug development.

Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer

Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in The Cancer Genome Atlas datasets. In addition to confirming oncogenic activity of the known fusion oncogenes engineered by our construction strategy, we validated five novel fusion genes involving MET, NTRK2, and BRAF kinases that exhibited potent transforming activity and conferred sensitivity to FDA-approved kinase inhibitors. Our fusion construction strategy also enabled domain-function studies of BRAF fusion genes. Our results confirmed other reports that the transforming activity of BRAF fusions results from truncation-mediated loss of inhibitory domains within the N-terminus of the BRAF protein. BRAF mutations residing within this inhibitory region may provide a means for BRAF activation in cancer, therefore we leveraged the modular design of our fusion gene construction methodology to screen N-terminal domain mutations discovered in tumors that are wild-type at the BRAF mutation hotspot, V600. We identified an oncogenic mutation, F247L, whose expression robustly activated the MAPK pathway and sensitized cells to BRAF and MEK inhibitors. When applied broadly, these tools will facilitate rapid fusion gene construction for subsequent functional characterization and translation into personalized treatment strategies. Cancer Res; 77(13); 3502-12. ©2017 AACR.

Abstract B106: Personalized functional screens for gene drivers of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastatingly lethal disease that remains one of the most challenging malignancies to treat successfully. The identification of oncogenic “driver” genes and their activated pathways has been the moving force behind the development of therapies for other cancers. Recognizing this, The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) are cataloging genomic aberrations in PDAC with the goal of identifying new therapeutic targets and detection biomarkers. These efforts have confirmed that the majority of PDAC tumors harbor early activating mutations in the KRAS oncogene, which is not druggable through current targeting modalities, along with other moderate to high frequency aberrations in TP53 and SMAD4 among others. We are now faced with the formidable challenge of identifying rare pathogenic aberrations from among the numerous biologically neutral “passengers” in these PDAC sequencing datasets, as identifying new drug-actionable events and understanding their mechanisms-of-action offers great promise for improving patient outcomes. However, comprehensive assessment of low frequency aberrations is difficult given their large number and the fact that they may either directly or indirectly influence tumor behavior through modifying activities of other drivers like KRAS. While RNAi-based screening platforms have successfully validated new tumor suppressors and other genetic liabilities in cancer, less progress has been made toward developing high-throughput, gain-of-function (GOF) screening systems for validating hyper-activated oncogenes that are especially attractive given the efficacy of antibody and small molecule inhibitor therapies tailored toward such factors. To address these challenges, we established a High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB) platform enabling GOF annotation of PDAC gene aberrations through (1) accurate modeling of numerous aberrations (amplifications, mutations, insertions, deletions) using our robotics-driven platform of >32,000 sequenced-verified open reading frame (ORF or “gene” clones) and (2) a molecular barcoding and sequencing-based detection strategy that permits rapid DNA tagging of wild-type and mutant ORFs for virus-based, pooled functional screens and ORF barcode quantitation. We employed HiTMMoB to build patient-specific aberration ORF sets based on ICGC sequencing data from four PDAC patients (i.e., all cloneable, uncharacterized somatic mutations reported in four separate tumors). These aberration ORFs were screened for their ability to promote in vivo tumorigenesis using a human pancreatic ductal epithelial cell line engineered with a doxycycline (DOX)-inducible KRASG12D allele, which after injection into mice on DOX diet leads to rapid tumor growth and tumor regression upon DOX withdrawal. Using this model we simultaneously screened for PDAC drivers that (1) cooperate with KRASG12D in mice on DOX, (2) drive tumor escape from DOX withdrawal (KRASG12D extinction) and (3) promote tumorigenesis in a KRAS-independent manner in the absence of DOX. This “personalized functionalization” approach coupled with our barcode detection strategy revealed two potent driver aberrations initially reported in two separate patients by the ICGC. Our screening approach, driver validation and mechanistic data support the notion that discovery of low frequency, functional aberrations may intersect or otherwise lead to important pathways representing known or novel therapeutic liabilities. When applied more broadly to aberration gene sets informed by biological importance and computational analyses, our functional screening technologies are revealing high priority PDAC targets to enroll in deep mechanistic biology studies and drug development programs with the ultimate goal of developing personalized treatment strategies critically needed for PDAC patients. Citation Format: Yiu Huen Tsang, Turgut Dogruluk, Hengyu Lu, Rosalba Minelli, Nikitha Nair, Marie-Claude Gingras, Agda Karina Eterovic, Gordon Mills, Kenneth Scott. Personalized functional screens for gene drivers of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B106.

Abstract PR06: Functional prioritization of rare gene aberration drivers of cancer

Next generation sequencing (NGS) technologies are rapidly being incorporated into the clinic to facilitate decisions on cancer patient care. However, successful translation of NGS data requires knowledge on which DNA aberrations represent actionable events, either for development or re-positioning of approved agents to target their activated pathways. Recognizing this, large-scale tumor profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are cataloging genomic aberrations across major cancer lineages. These efforts have revealed an extraordinary level of genome complexity made up of not only key “driver” events critical to pathogenesis, but also numerous biologically-neutral “passengers” that accompany unstable tumor genomes. The challenge now is to find ways to identify functional driver aberrations, as targeting such events or their activated pathways has great potential for improving patient outcomes. To do this, we have developed high-throughput approaches to construct molecularly-barcoded versions of gene aberrations for functional screens. Specifically, we developed technologies that include (1) high-throughput, accurate modeling of somatic DNA mutations (somatic missense mutations and small insertions/deletions) using our robotics-driven platform of >35,000 sequenced-verified open reading frame (ORF) clones, (2) a molecular barcoding strategy that permits rapid DNA tagging of wild-type and mutant ORFs, (3) multi-fragment recombineering methodologies allowing construction of cancer fusion genes, and (4) combining the use of these reagents for individual or pooled functional screens in vitro and in vivo using human and mouse systems. We are using these technologies, which are widely applicable to all cancer types, to identify the highest priority targets to enroll in deep mechanistic studies and drug discovery programs. We have scaled our pipelines to functionalize thousands of cancer gene aberrations. Importantly, we are now constructing entire somatically-mutated exomes from individual patients sequenced in the clinic. As an example of our “Personalized Functionalization” approach, we screened individual pancreatic ductal adenocarcinoma (PDAC) patient-derived aberration libraries for mutations capable of promoting tumorigenesis in vivo using a mouse xenograft model engineered with regulatable KRASG12D, an oncogene active in the majority of PDAC patient tumors. These studies revealed potent aberration drivers that are active as individual drivers as well as those that are contextual and are only active in the presence of KRASG12D. Based on these results, we have chosen NAD Kinase (NADK) for deep mechanistic studies and drug discovery programs. NADK catalyzes the conversion of cytoplasmic NAD+ to NADP+/NADPH and thus aids other modes of cellular NADPH production. Our validation studies indicate that the NADK mutation results in robust gain-of-function kinase activity leading to its hyper-phosphorylation of NAD+ accompanied by reduced accumulation or reactive oxygen species and increased tumor formation and growth. Interestingly, recent work by others report other mechanisms by which KRAS rewires PDAC tumors to maximize energy production and promote NADPH accumulation to maintain redox state and tumor growth. Even though NADK is mutated at low frequency in PDAC, its selection demonstrates that the discovery of rare, functional aberrations may intersect or otherwise lead us to important pathways and potential therapeutic liabilities. Our ultimate goal is to functionally annotate thousands of somatic aberrations in cancer, the vast majority of which have not been previously recognized or assayed for clinical relevance. These systems will reveal high priority edited targets to enroll in deep mechanistic biology studies, drug discovery and development programs ultimately leading to personalized treatment strategies. Citation Format: Kenneth Scott, Yiu Huen Tsang, Turgut Dogruluk, Philip Tedeschi, Joseph Bertino, Gordon Mills. Functional prioritization of rare gene aberration drivers of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR06.

Abstract C199: Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules.

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated due to the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. While basal AKT2 phosphorylation was sensitive to inhibition of PI3K, basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C199. Citation Format: Huifang Guo, Meng Gao, Yiling Lu, Jiyong Liang, Philip L. Lorenzi, Shanshan Bai, David H. Hawke, Jie Li, Turgut Dogruluk, Kenneth L. Scott, Eric Jonasch, Gordon B. Mills, Zhiyong Ding. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. [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 C199.

Abstract C10: High-throughput functional annotation of somatic driver aberrations in cancer

Tumor sequencing projects such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have revealed the high complexity of cancer genomes that are comprised of both pathogenic “driver” aberrations and many neutral “passenger” events. The cancer research community is faced with the daunting challenge of discriminating between drivers and passengers to inform the most promising therapeutic targets and diagnostic markers. Much of these efforts are focused on identifying new oncogenes given that such factors have served as successful therapeutic targets to date. To expedite oncogene discovery based on large-scale sequencing datasets, we established a target discovery pipeline involving a high-throughput mutagenesis and molecular barcoding (HiTMMoB) platform permitting (1) efficient site-directed DNA mutagenesis to model tumor somatic mutations into our collection of >32,000 human open reading frames (ORFs or genes), (2) integration of a 24 nucleotide DNA barcode sequence into ORF clones to facilitate pooled genetic screens and (3) their simultaneous recombination into lentiviral expression backbones enabling their expression in cancer cell models. Cells infected with barcoded ORF libraries can be entered into a variety of individual or pooled in vitro and in vivo genetic screens to identify ORF drivers of cancer-related activities (e.g., proliferation, tumor growth, invasion, metastasis among others). We have successfully used HiTMMoB to generate numerous wild-type and mutant ORF clones currently being used in multiple screening projects in our laboratory. One such project involves a gene focused screen designed to functionally characterize numerous lower frequency somatic tail mutations within the PIK3CA oncogene found mutated in breast and other cancers. Here, we have generated 36 mutated clones for PIK3CA that have been entered into in vitro and in vivo screens for drivers of (1) anchorage-independent growth (2) growth factor-independent growth (3) drug sensitivity/resistance and (4) orthotopic tumor formation in a pooled fashion. The goal of these screens is to functionally annotate these low frequency PIK3CA mutations, which may ultimately lead to tailored patient therapies based on the PIK3CA mutation status of an individuals tumor. In summary, we have developed a prioritization pipeline being used in a variety of target screens across diverse cancer types, and this platform promises to provide the cancer research community functional annotation on the most promising cancer aberrations for downstream biomarker and drug development. Citation Format: Turgut Dogruluk, Armel Dogruluk, Yiu-Huen Tsang, Nikitha Nair, Rosalba Minelli, Ping Wu, Kenneth L. Scott. High-throughput functional annotation of somatic driver aberrations in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C10.