KRAS-dependent Cancer Cells Research Articles

Indoloquinoline-Mediated Targeted Downregulation of KRAS through Selective Stabilization of the Mid-Promoter G-Quadruplex Structure.

KRAS is a well-validated anti-cancer therapeutic target, whose transcriptional downregulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling. G-quadruplexes (G4s) are non-canonical nucleic acid structures that mediate central dogmatic events, such as DNA repair, telomere elongation, transcription and splicing events. G4s are attractive drug targets, as they are more globular than B-DNA, enabling more selective gene interactions. Moreover, their genomic prevalence is increased in oncogenic promoters, their formation is increased in human cancers, and they can be modulated with small molecules or targeted nucleic acids. The putative formation of multiple G4s has been described in the literature, but compounds with selectivity among these structures have not yet been able to distinguish between the biological contribution of the predominant structures. Using cell free screening techniques, synthesis of novel indoloquinoline compounds and cellular models of KRAS-dependent cancer cells, we describe compounds that choose between KRAS promoter G4near and G4mid, correlate compound cytotoxic activity with KRAS regulation, and highlight G4mid as the lead molecular non-canonical structure for further targeting efforts.

Blocking the Farnesyl Pocket of PDEδ Reduces Rheb-Dependent mTORC1 Activation and Survival of Tsc2-Null Cells.

Rheb is a small GTPase member of the Ras superfamily and an activator of mTORC1, a protein complex master regulator of cell metabolism, growth, and proliferation. Rheb/mTORC1 pathway is hyperactivated in proliferative diseases, such as Tuberous Sclerosis Complex syndrome and cancer. Therefore, targeting Rheb-dependent signaling is a rational strategy for developing new drug therapies. Rheb activates mTORC1 in the cytosolic surface of lysosomal membranes. Rheb’s farnesylation allows its anchorage on membranes, while its proper localization depends on the prenyl-binding chaperone PDEδ. Recently, the use of PDEδ inhibitors has been proposed as anticancer agents because they interrupted KRas signaling leading to antiproliferative effects in KRas-dependent pancreatic cancer cells. However, the effect of PDEδ inhibition on the Rheb/mTORC1 pathway has been poorly investigated. Here, we evaluated the impact of a new PDEδ inhibitor, called Deltasonamide 1, in Tsc2-null MEFs, a Rheb-dependent overactivated mTORC1 cell line. By using a yeast two-hybrid assay, we first validated that Deltasonamide 1 disrupts Rheb-PDEδ interaction. Accordingly, we found that Deltasonamide 1 reduces mTORC1 targets activation. In addition, our results showed that Deltasonamide 1 has antiproliferative and cytotoxic effects on Tsc2-null MEFs but has less effect on Tsc2-wild type MEFs viability. This work proposes the pharmacological PDEδ inhibition as a new approach to target the abnormal Rheb/mTORC1 activation in Tuberous Sclerosis Complex cells.

AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

Recent development of the chemical inhibitors specific to oncogenic KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) mutants revives much interest to control KRAS-driven cancers. Here, we report that AIMP2-DX2, a variant of the tumor suppressor AIMP2 (aminoacyl-tRNA synthetase-interacting multi-functional protein 2), acts as a cancer-specific regulator of KRAS stability, augmenting KRAS-driven tumorigenesis. AIMP2-DX2 specifically binds to the hypervariable region and G-domain of KRAS in the cytosol prior to farnesylation. Then, AIMP2-DX2 competitively blocks the access of Smurf2 (SMAD Ubiquitination Regulatory Factor 2) to KRAS, thus preventing ubiquitin-mediated degradation. Moreover, AIMP2-DX2 levels are positively correlated with KRAS levels in colon and lung cancer cell lines and tissues. We also identified a small molecule that specifically bound to the KRAS-binding region of AIMP2-DX2 and inhibited the interaction between these two factors. Treatment with this compound reduces the cellular levels of KRAS, leading to the suppression of KRAS-dependent cancer cell growth in vitro and in vivo. These results suggest the interface of AIMP2-DX2 and KRAS as a route to control KRAS-driven cancers.

Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides.

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

Abstract LB100: Pemetrexed disodium heptahydrate and cisplatin have distinctive growth inhibitory effects in monotherapy and combination therapy on A549 cells

Abstract Background: Cisplatin combined with pemetrexed disodium heptahydrate (pemetrexed) is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, its individual growth inhibitory effects on KRAS-dependent A549 human lung cancer cells as monotherapy and combination therapy are not well understood. The aim of this study was to compare the effects of cisplatin (CDDP) and pemetrexed (PEM) on A549 cells as monotherapy and combination therapy.Methods: For in vitro studies, A549 cells were exposed to cisplatin and pemetrexed as monotherapy and combination therapy for 72 h. The results were then evaluated by a cell viability assay, apoptosis assay, ROS assay, TUNEL assay and Western blotting.Results: Our results revealed that cisplatin monotherapy was the most potent growth inhibitor; cisplatin plus pemetrexed combination therapy had an intermediate effect; and pemetrexed monotherapy induced a minimal growth inhibitory effect in the A549 cell line, which was observed by the inhibition of the RAS/RAF/MEK/ERK signaling pathway. Furthermore, apoptotic effects were examined according to ROS-mediated DNA damage and increased levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP. Interestingly, cisplatin monotherapy was the primary eliminator of cellular senescence; combination therapy with cisplatin plus pemetrexed had an intermediate eliminator effect, while pemetrexed monotherapy increased cellular senescence in A549 cells, which was assessed by the expression of the β-galactosidase protein. The results of Western blotting of proteins in the AMPK/mTOR signaling pathway indicated that the highest inducer of autophagy was pemetrexed monotherapy, while combination therapy with cisplatin plus pemetrexed had an intermediate inhibitory effect on autophagy. Cisplatin monotherapy showed the strongest autophagy inhibitory activity.Conclusion: In light of these findings, cisplatin monotherapy showed the most potent anticancer effects, suggesting that cisplatin monotherapy may be more effective than pemetrexed monotherapy or cisplatin plus pemetrexed combination therapy in A549 cells. Citation Format: Md Mohiuddin, Hideharu Kimura, Takashi Sone, Satoshi Watanabe, Hiroki Matsuoka, Keigo Saeki, Nanao Terada, Miki Abo, Kazuo Kasahara. Pemetrexed disodium heptahydrate and cisplatin have distinctive growth inhibitory effects in monotherapy and combination therapy on A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB100.

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies.

PTPN2 regulates the activation of KRAS and plays a critical role in proliferation and survival of KRAS-driven cancer cells

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function.

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

EPHA2 feedback activation limits the response to PDEδ inhibition in KRAS-dependent cancer cells.

KRAS is one of the most important proto-oncogenes. Its mutations occur in almost all tumor types, and KRAS mutant cancer is still lack of effective therapy. Prenyl-binding protein phosphodiesterase-δ (PDEδ) is required for the plasma membrane association and subsequent activation of KRAS oncogenic signaling. Recently, targeting PDEδ has provided new promise for KRAS mutant tumors. However, the therapeutic potential of PDEδ inhibition remains obscure. In this study, we explored how PDEδ inhibition was responded in KRAS mutant cancer cells, and identified KRAS mutant subset responsive to PDEδ inhibition. We first performed siRNA screen of KRAS growth dependency of a small panel of human cancer lines, and identified a subset of KRAS mutant cancer cells that were highly dependent on KRAS signaling. Among these cells, only a fraction of KRAS-dependent cells responded to PDEδ depletion, though KRAS plasma membrane association was effectively impaired. We revealed that the persistent RAF/MEK/ERK signaling seemed responsible for the lack of response to PDEδ depletion. A kinase array further identified that the feedback activation of EPH receptor A2 (EPHA2) accounted for the compensatory activation of RAF/MEK/ERK signaling in these cells. Simultaneous inhibition of EPHA2 and PDEδ led to the growth inhibition of KRAS mutant cancer cells. Together, this study gains a better understanding of PDEδ-targeted therapeutic strategy and suggests the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.

Abstract 1790: Investigation of oncogenic G-protein coupled receptor signaling pathways in Kras dependent pancreatic cancer cell lines

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the most common form of pancreatic cancer driven primarily by mutations in V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) gene. Its most common mutation, G12D, cause KRAS to remain constitutively active. KrasG12D expressing pancreatic acinar cells undergo transdifferentiation into ductal phenotype and form early tumorigenic lesions called Pancreatic Intraepithelial Neoplasm (PanIN). Coincidentally occurring inactivating mutations of tumor suppressor genes, such as Cyclin-dependent Kinase Inhibitor 2A (Cdkn2a), Tumor Protein p53 (Tp53), and Mothers Against Decapentaplegic Homolog 4 (Smad4) trigger progression of PanINs through stages from 1A to 3, eventually leading to carcinoma in situ and metastasis. G-protein Coupled Receptors (GPCR) are transmembrane receptors that transfer extracellular signals to intracellular heterotrimeric G-proteins with Gα and Gβγ subunits. Upon activation, dissociated Gα and Gβγ subunits separately induce intracellular second messengers for key cellular responses involved in various aspects of development, metabolism, and cell motility. GPCR signaling is negatively regulated by intracellular Regulator of G-protein Signaling (RGS) proteins that deactivate G-protein by mediating re-association of Gα and Gβγ subunits. Although GPCRs are the largest non-antibiotic drug targets, their specific role in pancreatic cancer is not much known. GPCR signaling can influence RTK pathways in cross-talk via various second messengers. The interplay between these two major classes of cellular pathways employing both heterotrimeric and monomeric G-proteins has therefore potential to shed new light on pancreatic cancer research. We have previously identified Rgs16 expression as a marker of pancreatic tumor formation in Rgs16::GFP o/o; Ptf1aCre/+; Lox-STOP-Lox-KrasG12D/+; Cdkn2af/f (Rgs16::GFP-KIC) mice from the onset of earliest PanINs at two weeks of age (Dis. Model Mech. 8, 1201). Using ImageJ software to quantitatively determine eGFP expression in micrographs, we have developed a rapid in vivo assay (RIVA) where we represent pancreatic tumor burden by one month of age and compare outcomes of known and novel therapeutics following two week long drug regimens. We found that just like the recently developed drug, BGB324, targeting an important RTK, called Anexelekto (Axl), the anticoagulant warfarin was more effective in reducing tumor burden than standard chemotherapy alone. Currently, my research focus is centered on understanding active GPCR pathways that stimulate cell growth, survival, and migration by enhancement of Kras downstream signaling. My aim is comparison of the effects of general G-protein signaling inhibition in Kras mutant and wild-type cell lines, such as AsPC-1 in contrast to BxPC-3, on cell behavior and making connections to active GPCR signaling components. Citation Format: Ozhan Ocal, Raymond MacDonald, Rolf A. Brekken, Thomas Wilkie. Investigation of oncogenic G-protein coupled receptor signaling pathways in Kras dependent pancreatic cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1790.

Abstract 768: Identification of signal transduction pathways specific for mutant KRas-dependent tumors

Abstract Targeting KRas directly has proven difficult. Therefore, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach to target mutant KRas-driven tumors. Towards this goal, we used phosphoproteomics to dissect key differences in signaling pathways between mutant KRas-dependent and mutant KRas-independent human cancer cells. Mass spectrometry analysis led to the identification of 25 proteins with significantly higher (up to 410 fold) phosphorylation levels in mutant KRas-dependent cells. Among these, 25 proteins- 8, 8 and 3 were tyrosine (Y), Serine (S) and Threonine (T) phosphorylated, and 6 were phosphorylated on both S and T. Presently, CRISPR-Cas9 knockout and siRNA depletion studies are ongoing to identify which of these 25 hits are required for the survival of mutant KRas-dependent human cancer cells. These studies can lead to identifying signaling circuits that can be targeted to treat mutant KRas-driven cancers. Citation Format: Oorvashi Roy Puli, Hua Yang, Bin Fang, Hong Yuan (Rays) Jiang, John Koomen, Said M. Sebti. Identification of signal transduction pathways specific for mutant KRas-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 768.

Abstract 767: Identification of cell surface proteins expressed exclusively on mutant KRas-dependent cancer cells

Abstract Mutant KRas is a major contributor to human oncogenesis and confers resistance to therapy. Identification of cell surface proteins that are expressed exclusively in mutant KRas-driven tumors could lead to novel approaches for diagnosing and treating mutant KRas-dependent tumors. To this end, we used a biotinylation/mass spectrometry approach to capture cell surface proteins from mutant KRas-dependent and -independent human cancer cells. KRas was depleted from both mutant KRas-dependent and -independent cells, and the cells were subjected to cell surface protein biotinylation using EZ-Link Sulfo-NHS-SS-Biotin reagent and captured via neutravidin column. The efficiency of cell surface protein capture was confirmed by the presence of membrane proteins EGFR, IGF1R and absence of cytosolic proteins RhoGDIa in biotin-pull down fractions (western blot analysis). Mass spectrometric and proteomics analyses of biotinylated proteins yielded 20 highly probable hits with very high expression in mutant KRas-dependent cells and very low expression in mutant KRas-independent cells. Upon KRas depletion, the level of these proteins was significantly reduced in mutant KRas-dependent cells (up to 25 fold) but not in mutant KRas-independent cells. Currently, we are analyzing these 20 hits to determine if any of these are required for the survival of mutant KRas-dependent cells. Results from these studies have the potential to lead to novel approaches for diagnosing and treating mutant KRas-driven cancers. Citation Format: Vijayendra Agrawal, Bin Fang, Eric A. Welsh, John Koomen, Said M. Sebti. Identification of cell surface proteins expressed exclusively on mutant KRas-dependent cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 767.

Targeting RAS – will GPR31 deliver us a new path forward?

ABSTRACTEffective anti-rat sarcoma viral oncogene (RAS) therapies have remained the holy grail of cancer treatment. Mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) sustains tumorigenesis when linked to the plasma membrane (PM). The G protein-coupled receptor 31 (GPR31) is now identified to mediate KRAS membrane association and is crucial for proliferation, survival and macropinocytosis of KRAS-dependent cancer cells, suggesting that GPR31 is a druggable target for anti-RAS therapy.

Abstract 4719: MYC expression correlates with PD-L1 expression and related poor clinical outcome in non-small cell lung cancer

Abstract Background: Programmed death-ligand 1 (PD-L1) is widely used biomarker for the response prediction of immune checkpoint inhibitors, but its usefulness has been challenged. MYC, a transcriptional factor which is overexpressed in various cancers, plays critical roles in preventing immune cells from attacking tumor cells by inducing PD-L1 expression. We evaluated that MYC expression is positively related that of PD-L1 in non-small cell lung cancer (NSCLC) clinical specimens and that it has potential as a predictor of response to immune checkpoint inhibitors in NSCLC. Methods: Eighty-four cases who were diagnosed as NSCLC and underwent surgical resection were evaluated by immunohistochemistry (IHC). PD-L1 expression was evaluated by FDA-approved PD-L1, 22C3 PharmDxTM protocol using the Dako Automated Link 48 platform at Clarient Diagnostic Services, Inc (Aliso Viejo, CA, USA). Its expression was determined by using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining. If TPS >= 50% of the viable tumor cells exhibit membrane staining at any intensity, it was considered positive. IHC of MYC was performed using anti-c-MYC antibody [Y69] (ab32072, Abcam, USA) and its expression was evaluated by scoring system using product of nuclear staining intensity and percentage of positive cells. Results: When MYC cDNA was transfected into KRAS-dependent lung cancer cell lines that did not express both MYC and PD-L1, the protein expression and mRNA of PD-L1 were induced in a dose-dependent manner. On the other hands, knockdown of MYC with siRNA inhibited protein expression and mRNA indicating MYC regulates PD-L1 at transcriptional level. Of 84 NSCLC tumor tissues, PD-L1 was expressed in 14 (16.7%) and MYC in 30 (35.7%) cases. Dual positive patients were 9 (10.7%) and dual negative patients were 49 (58.3%). When the relationship between PD-L1 and MYC expression was evaluated, significant positive correlation was observed between PD-L1 and MYC expression (γ=0.267, P=0.014). Dual positive patients showed significantly shorter disease free survival (16.1 vs. 62.5 months, P=0.004) and overall survival (24.9 vs. 70.9 months, P=0.002) than dual negative patients. Conclusion: MYC expression correlates with PD-L1 expression in the NSCLC and the patients with overexpression of both MYC and PD-L1 showed significantly poor survival. This suggest that it is worth of investigation of MYC expression as a surrogate predictive marker for treatment of immune checkpoint inhibitors in NSCLC. Citation Format: Eun Young Kim, Arum Kim, Se Kyu Kim, Yoon Soo Chang. MYC expression correlates with PD-L1 expression and related poor clinical outcome in non-small cell lung cancer [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 4719. doi:10.1158/1538-7445.AM2017-4719

Abstract 394: Saturation mutagenesis of KRAS reveals the functional landscape of missense variants

Abstract KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [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 394. doi:10.1158/1538-7445.AM2017-394

PKCδ regulates integrin αVβ3 expression and transformed growth of K-ras dependent lung cancer cells.

We have previously shown that Protein Kinase C delta (PKCδ) functions as a tumor promoter in non-small cell lung cancer (NSCLC), specifically in the context of K-ras addiction. Here we define a novel PKCδ -> integrin αVβ3->Extracellular signal-Regulated Kinase (ERK) pathway that regulates the transformed growth of K-ras dependent NSCLC cells. To explore how PKCδ regulates tumorigenesis, we performed mRNA expression analysis in four KRAS mutant NSCLC cell lines that stably express scrambled shRNA or a PKCδ targeted shRNA. Analysis of PKCδ-dependent mRNA expression identified 3183 regulated genes, 210 of which were specifically regulated in K-ras dependent cells. Genes that regulate extracellular matrix and focal adhesion pathways were most highly represented in this later group. In particular, expression of the integrin pair, αVβ3, was specifically reduced in K-ras dependent cells with depletion of PKCδ, and correlated with reduced ERK activation and reduced transformed growth as assayed by clonogenic survival. Re-expression of PKCδ restored ITGAV and ITGB3 mRNA expression, ERK activation and transformed growth, and this could be blocked by pretreatment with a αVβ3 function-blocking antibody, demonstrating a requirement for integrin αVβ3 downstream of PKCδ. Similarly, expression of integrin αV restored ERK activation and transformed growth in PKCδ depleted cells, and this could also be inhibited by pretreatment with PD98059. Our studies demonstrate an essential role for αVβ3 and ERK signalingdownstream of PKCδ in regulating the survival of K-ras dependent NSCLC cells, and identify PKCδ as a novel therapeutic target for the subset of NSCLC patients with K-ras dependent tumors.

Abstract A178: Structure guided development of irreversible inhibitors for TAK1

Abstract Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25 and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively non-selective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4, KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine adjacent to the ATP binding site of TAK1. Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1-dependent cell lines from 3 distinct cancer types. Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691 showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1, ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells. Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may have value as therapeutic agents and scientific tools. Citation Format: Deepak Gurbani, Li Tan, Scott Ficarro, John C. Hunter, William Singer, Faviola B. Vazquez, Ting Xie, Sang Min Lim, Jarrod Marto, Nathanael S. Gray, Kenneth D. Westover. Structure guided development of irreversible inhibitors for TAK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A178.

Abstract 3188: MEK and TAK1 signaling interactions coordinately regulate inflammation and apoptosis in KRAS dependent colon cancer cells

Abstract The KRAS protooncogene is mutated in 40 to 50% of colon cancers. In an effort to identify strategies to treat KRAS mutant colon cancers, we previously implicated the TGF-β activated kinase (TAK1) as a candidate therapeutic target that promotes the survival of KRAS dependent cancers. In follow-up studies, we have explored and investigated the detailed mechanistic basis for TAK1 mediated survival signaling in KRAS dependent cancer cells. Using proteomic and transcriptomic analyses of proinflammatory signaling mediators, we have uncovered complex autocrine/paracrine signaling loops that are constitutively activated in KRAS dependent colon cancer cells. A central mediator of these signaling loops is the BMP7-BMP receptor (BMPR1A) pathway, which functions coordinately with oncogenic KRAS to drive TAK1 and NF-κB mediated transcriptional upregulation of proinflammatory cytokines such as GM-CSF, CCL5/RANTES and IL-8. Conversely, a number of cytokines and cytokine regulators are negatively regulated by KRAS-BMPR signaling interactions, including CXCL9/MIG and IL1RN. We previously showed that TAK1 inhibition with a small molecule agent, 5Z-7-oxozeaenol promotes apoptosis in colon cancer cells. We have now determined that 5Z-7-oxozeaenol, in addition to irreversibly inhibiting TAK1 kinase activity, also transiently inhibits the MEK kinase. Therefore, combined TAK1/MEK inhibition explains the potent killing effects that we have observed in KRAS dependent colon cancer cells. To test this empirically, we have used 2 selective kinase inhibitors targeting each respective kinase, AZ-TAK1 and AZD6244 to show either single agent can induce apoptotic cell selectively in KRAS dependent cells, with TAK1 inhibition resulting in stronger killing effects. Importantly, treatment of KRAS dependent colon cancer cells with combinations of AZ-TAK1 and AZD6244 results in additive killing effects, revealing a potential therapeutic strategy for KRAS dependent cancers in the clinic. Mechanistically, MEK and TAK1 converge on the control of NF-κB and canonical Wnt-dependent transcriptional activities. Surprisingly, we find that NF-κB and Wnt signaling mutually antagonize each other in terms of cytokine expression to create a finely-tuned balance between pro-death and pro-survival signals. We hypothesize that, as a consequence, these balanced signals allow for both efficient maintenance of tumor cell survival and as well as for communication with stromal components in the tumor microenvironment. Thus, KRAS, MEK or TAK1 blockade results in a tipping of the balance to favor pro-death signals. Citation Format: Kelsey L. McNew, William J. Whipple, Anita K. Mehta, Trevor Grant, Anurag Singh. MEK and TAK1 signaling interactions coordinately regulate inflammation and apoptosis in KRAS dependent colon cancer cells. [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 3188. doi:10.1158/1538-7445.AM2015-3188