Constitutive Activation Of KRAS Research Articles

Abstract 1978: The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer

Abstract Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines. Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting. Results: N displayed nanomolar IC50 values (< 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410. Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed. Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1978.

Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib.

RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.

Effects of chloroquine and hydroxychloroquine on the sensitivity of pancreatic cancer cells to targeted therapies.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a cancer which is difficult to effectively treat as it is often detected late in the disease process. Almost all PDACs (over 90%) have activating mutations in the GTPase gene KRAS. These mutations result in constitutive KRas activation and the mobilization of downstream pathways such as the Raf/MEK/ERK pathway. Small molecule inhibitors of key components of the KRas/Raf/MEK/ERK pathways as well as monoclonal antibodies (MoAbs) specific for upstream growth factor receptors such insulin like growth factor-1 receptor (IGF1-R) and epidermal growth factor receptors (EGFRs) have been developed and have been evaluated in clinical trials. An additional key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, metabolism, cancer progression and other growth regulatory processes. Small molecule mutant TP53 reactivators have been developed which alter the structure of mutant TP53 protein and restore some of its antiproliferative activities. Some mutant TP53 reactivators have been examined in clinical trials with patients with mutant TP53 genes. Inhibitors to the TP53 negative regulator Mouse Double Minute 2 (MDM2) have been developed and analyzed in clinical trials. Chloroquine and hydroxychloroquine are established anti-malarial and anti-inflammatory drugs that also prevent the induction of autophagy which can have effects on cancer survival. Chloroquine and hydroxychloroquine have also been examined in various clinical trials. Recent studies are suggesting effective treatment of PDAC patients may require chemotherapy as well as targeting multiple pathways and biochemical processes.

Abstract 157: DUSP6 modulates migration and glycolysis in PDAC cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor and is the seventh cause of death for cancer worldwide, fifth in the US. The highly malignant profile is mainly caused by the constitutive activation of mutant KRAS - found in approximately 90% of PDAC cases. The undruggability of genetic KRAS mutations has led to efforts to find new therapeutical targets that focus on downstream molecules in this pathway. DUSP6 is a dual-specificity phosphatase that regulates ERK1/2 phosphorylation and, therefore, downstream RAS pathway activation. DUSP6 has been demonstrated to be differentially expressed during PDAC tumorigenesis, which we believe is critical for tumor progression and metastasis. In silico analysis on the TCGA dataset revealed that DUSP6 is overexpressed in primary tumor samples compared to normal pancreatic tissue. This data was confirmed in an independent dataset (GSE71729) that further revealed DUSP6 overexpression in metastatic samples compared to primary tumor samples. Additionally, patients with higher DUSP6 expression have worse overall survival than patients with low DUSP6 expression (P = 0.039), reaffirming its clinical relevance. We then assessed DUSP6 expression in tumor sections derived from KC and KPC mice using the RNAscope technology and observed that DUSP6 was strongly overexpressed in tumorigenic lesions, and largely co-localized with KRT19 expression. Using BCI - a pharmacological DUSP6 inhibitor - we observed significantly reduced viability in vitro in all the cell lines (P < 0.0001). DUSP6 inhibition also promoted ERK1/2 activation, as expected. Surprisingly, we observed that BCI-mediated DUSP6 inhibition increased migratory capacity in AsPC-1 (P < 0.0001), but not on BxPC-3 cells. Furthermore, murine AKC cells presented the completely opposite phenotype, with a significant decrease in migratory capacity (P < 0.0001), indicating a possible context dependent response. To further understand the molecular mechanisms behind DUSP6 in PDAC, we performed a gene set enrichment analysis using the TCGA and GSE15471 datasets and observed a strong correlation between DUSP6 and the glycolysis pathway. Therefore, we knocked down DUSP6 in K8484 cells and evaluated their proliferation in response to 2-DG, which was significantly decreased in comparison to treated parental cells (P < 0.001). In accordance with that, DUSP6 knockdown cells were significantly more impacted by 2-DG treatment than the parental cells regarding migratory capacity, bringing it to the basal level. Collectively, these results suggest that DUSP6 appears to modulate the metastatic process, and this phenotype correlates with the altered glycolytic capacity of pancreatic cancer cells. We are currently expanding our studies to additional cell lines and using in vivo models to further investigate if the observed phenotype is context dependent. To assess glycolytic changes in the cells, we are currently performing Seahorse-based assays. Citation Format: Mariana T. Ruckert, Bailey A. Bye, Michael N. VanSaun, Vanessa S. Silveira. DUSP6 modulates migration and glycolysis in PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 157.

The Tumor Immune Profile of Murine Ovarian Cancer Models: An Essential Tool For Ovarian Cancer Immunotherapy Research.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer with an imperative need for new treatments. Immunotherapy has had marked success in some cancer types; however, clinical trials studying the efficacy of immune checkpoint inhibitors for the treatment of EOC benefited less than 15% of patients. Given that EOC develops from multiple tissues in the reproductive system and metastasizes widely throughout the peritoneal cavity, responses to immunotherapy are likely hindered by heterogeneous tumor microenvironments (TME) containing a variety of immune profiles. To fully characterize and compare syngeneic model systems that may reflect this diversity, we determined the immunogenicity of six ovarian tumor models in vivo, the T and myeloid profile of orthotopic tumors and the immune composition and cytokine profile of ascites, by single-cell RNA sequencing, flow cytometry and IHC. The selected models reflect the different cellular origins of EOC (ovarian and fallopian tube epithelium) and harbor mutations relevant to human disease, including Tp53 mutation, PTEN suppression, and constitutive KRAS activation. ID8-p53-/- and ID8-C3 tumors were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumors were primarily infiltrated by tumor associated macrophages and were unique in MHC class I and II expression. MOE-PTEN/KRAS tumors were capable of forming T cell clusters. This panel of well-defined murine EOC models reflects some of the heterogeneity found in human disease and can serve as a valuable resource for studies that aim to test immunotherapies, explore the mechanisms of immune response to therapy, and guide selection of treatments for patient populations.

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Signaling adaptor protein Crk is involved in malignant feature of pancreatic cancer associated with phosphorylation of c-Met

Signaling adaptor protein Crk has been shown to play an important role in various human cancers. Crk links tyrosine kinases and guanine nucleotide exchange factors (GEFs) such as C3G and Dock180 to activate small G-proteins Rap and Rac, respectively. In pancreatic cancer, various molecular targeted therapies have provided no significant therapeutic benefit for the patients so far due to constitutive activation of KRAS by frequent KRAS mutation. Therefore, the establishment of novel molecular targeted therapy in KRAS-independent manner is required. Here, we investigated a potential of Crk as a therapeutic target in pancreatic cancer. Immunohistochemistry on human pancreatic cancer specimens revealed that the patients with high expression of Crk had a worse prognosis than those with low expression. We established Crk-knockdown pancreatic cancer cells by siRNA using PANC-1, AsPC-1, and MIA PaCa-2 cells, which showed decreased cell proliferation, invasion, and adhesion. In Crk-knockdown pancreatic cancer cells, the decrease of c-Met phosphorylation was observed. In the orthotopic xenograft model, Crk depletion prolonged survival of mice significantly. Thus, signaling adaptor protein Crk is involved in malignant potential of pancreatic cancer associated with decrease of c-Met phosphorylation, and Crk can be considered to be a potential therapeutic molecular target.

KRAS-Driven Metabolic Rewiring Reveals Novel Actionable Targets in Cancer.

Tumors driven by mutant KRAS are among the most aggressive and refractory to treatment. Unfortunately, despite the efforts, targeting alterations of this GTPase, either directly or by acting on the downstream signaling cascades, has been, so far, largely unsuccessful. However, recently, novel therapeutic opportunities are emerging based on the effect that this oncogenic lesion exerts in rewiring the cancer cell metabolism. Cancer cells that become dependent on KRAS-driven metabolic adaptations are sensitive to the inhibition of these metabolic routes, revealing novel therapeutic windows of intervention. In general, mutant KRAS fosters tumor growth by shifting cancer cell metabolism toward anabolic pathways. Depending on the tumor, KRAS-driven metabolic rewiring occurs by up-regulating rate-limiting enzymes involved in amino acid, fatty acid, or nucleotide biosynthesis, and by stimulating scavenging pathways such as macropinocytosis and autophagy, which, in turn, provide building blocks to the anabolic routes, also maintaining the energy levels and the cell redox potential (1). This review will discuss the most recent findings on mutant KRAS metabolic reliance in tumor models of pancreatic and non-small-cell lung cancer, also highlighting the role that these metabolic adaptations play in resistance to target therapy. The effects of constitutive KRAS activation in glycolysis elevation, amino acids metabolism reprogramming, fatty acid turnover, and nucleotide biosynthesis will be discussed also in the context of different genetic landscapes.

Abstract 3553: Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer (comprising 80-85 %), is a leading cause of cancer-related death worldwide. KRAS mutation is s major driver oncogene in NSCLC. Constitutive activation of K-Ras via oncogenic mutations plays a key role in development and progression of NSCLC, and is an important target for drug development. Indeed, drugs that target RAF-MAPK and PI3K, the well-established downstream pathways of K-Ras, are being evaluated in the clinic. However, to date, no effective treatments specifically targeting K-Ras have reached the clinic. A new direction for anti-Ras therapies targeting Ras-mediated altered cellular metabolism is implied by recent studies. Hence, as an attempt to investigate novel therapeutic targets to suppress KRAS-driven NSCLC, we assessed metabolic changes in NSCLC cell line and mouse model systems with oncogenic K-Ras mutations. We show that activation of Ras causes metabolic alterations, leading to aerobic glycolysis. Interestingly, despite the changes in the glycolytic pathway, NSCLC cell line and mouse models carrying K-Ras mutation revealed an elevated mitochondrial membrane potential, superoxide production as well as maximal oxygen consumption rate. Moreover, blockade of mitochondrial respiration effectively reduced mutant KRas-driven neoplastic phenotypes in NSCLC cell lines vitro and lung tumor formation in vivo. In contrast, blockade of glycolysis was not effective in the mutant KRas-driven tumorigenic activities. Mechanistically, mutant KRas-mediated activation of Akt2 led to increased expression of succinate dehydrogenase subunit A (SDHA) through increased protein stability by blocking proteasome-mediated degradation, resulting in mitochondrial complex II-dependent respiration. Our study suggests that induction of mitochondrial complex II-dependent respiration is an important mechanism by which oncogenic mutant KRas induces metabolic changes and ROS stress in NSCLC cells, contributing to lung carcinogenesis. Our findings elucidate potential metabolic targets that may provide novel therapeutic strategy for treating NSCLC with oncogenic KRAS mutations. Citation Format: Kwan-Hee Park, Ho-Young Lee. Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism [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 3553. doi:10.1158/1538-7445.AM2017-3553

Abstract A121: Constitutive K-Ras activity independent of K-RAS mutational status leads to resistance to anti-EGFR molecular targeting approaches and MEK-dependent reactivation of Akt following PI3K inhibition

Abstract Non-small cell lung cancer (NSCLC) cells expressing K-RAS mutation are resistant to anti-EGFR antibody cetuximab. Oncogenic Ras regulates basal effector PI3K/Akt and MAPK/ERK pathways. In this study, we investigated impact of K-RAS activity independent of its mutational status on targetibility of EGFR as well as the PI3K/Akt and MAPK/ERK pathways. A panel of five NSCLC cells (A549, H460, H661, SK-MES-1, HTB-182) and five head and neck squamous carcinoma (HNSCC) cells (SAS, FaDu, UT-SCC-5, UT-SCC-15, UT-SCC-5R (UT-SCC-5R are UT5 cells acquired resistance to cetuximab) were studied. K-Ras was constitutively activate not only in K-RAS mutated (K-RASmut) A549 and H460 cells, but also in K-RAS wild-type (K-RASwt) SAS and UT-SCC-5R cells. Enhanced K-Ras-GTP either endogenously or following overexpression of K-RAS(V12) was associated with significant shortening of population doubling time and increased clonogenic activity of tu-mor cells from both origin. Constitutive activation of K-Ras independent of K-RAS muta-tional status led to desensitization of both K-RASmut and K-RASwt cells to anti-EGFR an-tibody cetuximab and EGFR-tyrosine kinase inhibitors (TKI) erlotinib and AG1478, tested by standard clonogenic assay. Analyzing EGFR downstream pathways revealed that lack of response to the EGFR-TK inhibitors is through activation of the PI3K/Akt pathway. Thus, inhibition of PI3K was supposed to be an efficient approach to block clonogenic activity. However, PI3K inhibitor completely blocked clonogenic activity only in cells expressing low level of K-RASwt from both origins. In K-RASmut NSCLC cells or in HNSCC cells with overexpression of K-RASwt a partial response to PI-103 was observed. Analyzing pathway activity revealed that PI-103 does not inhibit Akt activity after 24 h pre-treatment while up to 4 h after post-treatment with PI-103, phosphorylation of Akt and Akt substrates (PRAS40 and GSK3αβ) are completely blocked. Lack of effect of PI103 at 24 h post-treatment was shown to be through K-RAS/MEK/ERK dependent reactivation of Akt. In line with this observation, targeting MEK by PD98059 significantly improved the anti-clonogenic activity of PI-103. Although, PD98059 blocked phosphorylation of ERK1/2, no marked effect was observed on clonogenic activity. Thus, EGFR or PI3K, but not MEK are appropriate target to inhibit clonogenic activity of tumor cells expressing low level of K-RASwt. In tumor cells with constitutive K-RAS activity, dual targeting of PI3K and MEK is an efficient approach to inhibit clonogenicity. Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/7-1 and SFB-773-TP B02] awarded to HPR, GRK 1302/2 (T11) awarded to MT/HPR. Citation Format: Mahmoud Toulany, Mohammad Saki, H. Peter Rodemann. Constitutive K-Ras activity independent of K-RAS mutational status leads to resistance to anti-EGFR molecular targeting approaches and MEK-dependent reactivation of Akt following PI3K inhibition. [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 A121.

Abstract 5102: Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway

Abstract Kras controls many cellular processes such as cell migration and survival. Mutation of Kras to a constitutively active form is a common alteration in lung cancer. Being constantly active results in a hyper activation of downstream signaling cascades, which leads to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these different functions, however, remains unclear. Using patient-derived lung cancer cell lines expressing oncogenic KrasV12 we discovered that Kras expression, not transcription, gets up-regulated under long-term culture in 1% O2. We confirmed the in vivo relevance of this finding, staining mouse lung tumors for Kras and Hif1α, as an indicator for hypoxic areas, and found heterogeneous Kras levels throughout the tumors with hot-spots that overlaid with Hif1α expression. Up-regulation of KrasV12 also correlated with a significant increase in growth of tumor-like aggregates in soft-agar assays and a morphological change from adherent single-cells into sphere-like aggregates in cell culture. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. Interestingly, the expression level of Kras correlated with clonal migration behavior. Low Kras expressers were highly motile whereas high Kras expressers remained stationary in aggregates. In agreement, exogenous expression of KrasV12 in motile, low Kras expressers converted their morphology and migration behavior to sphere-like, stationary aggregates. Using Western blot analysis we found that these changes are due to differences in Erk activation down-stream of Kras. In high Kras expressers Erk is highly phosphorylated which leads to higher Myosin II activity and thus to a contractile, sphere-like morphology with low mobility. Low Kras levels result in less Erk activation and therefore lower Myosin II activity which leads to an adherent, motile phenotype. Using a specific Erk inhibitor, we could convert high Kras expressers into adherent, motile low Kras clones. At the same time we found an adhesion-dependent feedback mechanism which regulates cellular Kras levels. Protein analysis of cells cultured in low adhesion dishes formed spheres and dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion. Additionally, blocking FAK activation in low Kras expressers resulted in up-regulation of Kras and Erk phosphorylation which went along with a switch to sphere-like morphology. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a hypoxia-sensitive, adhesion-dependent Kras switch which makes the cell very adaptable to different environments during the whole process of metastasis. Citation Format: Anette C. Schafer, Meghan Discoll, Ashwathi Mohan, Andrew Ludlow, Wesley Burford, Jerry W. Shay, Gaudenz Danuser. Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway. [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 5102. doi:10.1158/1538-7445.AM2015-5102

Abstract A48: Crosstalk between ERK2 and Akt1 in tumor cells with constitutive K-RAS activity leads to the limited response to PI3K inhibition

Abstract Constitutive K-RAS activity in K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells leads to resistance to EGFR targeting strategies. Analyzing the K-RAS activity status revealed that a constitutive high K-RAS activity is observed not only in K-RASmut NSCLC cells but also in head and neck squamous cell carcinoma (HNSCC) cells presenting K-RAS wild-type (K-RASwt) overexpression. Similar to K-RAS-mutated NSCLC cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Overexpression of mutated K-RAS in HNSCC cells expressing low level of K-RASwt stimulated Akt1 phosphorylation and increased clonogenic activity. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in both EGFR-dependent and EGFR-independent Akt1 activity. Short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in repression of Akt1 activation, whereas long-term treatment (24 h) with inhibitors led to reactivation of Akt1 and stimulated clonogenicity. Akt1 re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, inhibition of clonogenicity in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by the combination of PI3K and MAPK inhibitors. Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/5-1 and SFB-773-TP B02] and the Federal Ministry of Research and Education(BMBF grants 0258416, 03NUK006D) awarded to HPR as well as GRK 1302/2 (T11) awarded to MT/HPR. Citation Format: Mahmoud Toulany, H. Peter Rodemann. Crosstalk between ERK2 and Akt1 in tumor cells with constitutive K-RAS activity leads to the limited response to PI3K inhibition. [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 A48.

Does BRAF Mutation and Extracellular Signal Regulated Kinase Expression in Patients With Colorectal Cancer Have Any Prognostic Significance?

See Article on Page 9-15 Wild-type KRAS acts as a switch during signal transduction; however, somatic mutations that activate regulators and effectors of Ras proteins are common in tumor development and cancer [1, 2, 3]. In approximately 35%-42% of early colorectal cancer (CRC) patients, the KRAS mutation inhibits KRAS GTPase, resulting in a constitutive KRAS activation and, thus, activating a Ras/Raf signaling pathway. In CRC, 97% of KRAS mutations occur in codons 12 and 13 of exon 2, and more than 97% of changes in the protein are attributable to changes in the amino acid sequence by the substitution of seven DNA base pairs [4]. BRAF is a human gene that encodes the protein B-Raf, which is considered a proto-oncogene, encoding a serine/threonine protein kinase [5]. B-Raf is a member of the Raf kinase family that regulates the Ras/Raf/MEK/extracellular signal regulated kinase (ERK) pathway and is involved in division, differentiation, and secretion [6]. The most common BRAF mutation is a missense mutation (V600E, formally known as V599E), resulting in glutamic acid in place of valine that generates an abnormality in the MEK/ERK signaling pathway in CRC [7]. The mitogen-activated protein kinase (MAPK)/ERK signaling pathway is a highly conserved intercellular signaling system present in multicellular organisms and plays an essential role in cancer progression. MAPK/ERK activation is a common feature of tumors with KRAS, NRAS, or BRAF mutations [8, 9]. A highly activated MAPK/ERK pathway is found in approximately 30% of cancers and over 60% of melanomas, and it is associated with tumor proliferation and migration. BRAF is upstream of the MAPK/ERK pathway, and a single amino acid change, resulting in a valine-toglutamyl acid substitution at position 600 (V600E), accounts for ~90% of BRAF mutations. ERK1/2 are important kinases in the MAPK pathway. Therefore, activation of ERK1/2 could be considered as a target factor related with CRC carcinogenesis through the serrated pathway [8]. The authors of this study investigated the clinicopathologic outcomes of BRAF mutation and ERK1/2 expression in patients with CRC and the possibility of their use as prognostic indicators. The authors found that BRAF mutation and ERK1/2 expression might be associated with advanced or more aggressive CRC [10].

Abstract 3147: Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration

Abstract Cancer cell migration in vivo occurs at the single cell level or as a chain of motile cells leaving the primary tumor site. Mutation of Kras to a constituitively active form of Kras, is a common alteration in lung cancer. Kras controls many cellular processes such as cell migration and proliferation. Being constantly active results in a hyper activation of downstream signaling cascades, which lead to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these cell functions, however, has remained unclear. Using a lung cancer cell transformation model, which expresses oncogenic KrasV12, we discovered that Kras expression got substantially upregulated under long-term culture in 2% O2. This was accompanied by a significant increase in growth of tumor-like aggregates compared to the same cells cultured under atmospheric 21% O2 conditions. Up-regulation of KrasV12 also correlated with a morphological change from adherent single-cells into sphere-like aggregates. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. The expression of Kras in these two morphological different types of clones correlated with migration behavior. Low Kras expressers were mobile whereas high Kras expressers remained stationary in aggregates. In agreement, protein analysis of these cells forced to form spheres by culture in low adhesion dishes dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion and migration on collagen coated substrates. Under these conditions, cells at the periphery of the aggregate migrate onto the surrounding extracellular matrix (ECM). Curiously, they often stayed attached to a second cell that did not actively migrate. Using immunofluorescence imaging we could identify that in such pairs the actively migrating cell had a low expression of Kras whereas the passive ‘cargo cell’ retained high Kras levels. As both cells originated from the same aggregate of high Kras expressers we conclude that interaction of these lung cancer cells with the ECM negatively regulates Kras expression. This new form of combined migration was also observed in a 3D collagen matrix. By co-culturing high- and low-Kras clones we showed that low Kras expressers actively haul non-motile, high Kras expressers through a collagen scaffold. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a low O2-driven, intrinsic Kras switch. Cells expressing high levels of Kras show a more sphere-like, non-motile phenotype and can give rise to motile cells upon down-regulation of Kras in contact with ECM. These cells carry high Kras expressers with high tumorigenic potential as cargo generating a dangerous team able to migrate out, spread, and regrow elsewhere. Citation Format: Anette C. Schafer, Jerry W. Shay, Gaudenz Danuser. Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2014-3147

The influence of NFATc1 on the inflammatory tumor environment in pancreatic carcinogenesis

Introduction: Pancreatic ductal adenocarcinoma (PDAC) formation and progression requires constitutive activation of oncogenic Kras. The impact of constitutive Kras activation is not limited on epithelial cell but also participates in the modulation of the tumor environment. Latest studies revealed that even early stages of carcinogenesis are associated with a stromal reaction, characterized by a robust desmoplastic response and recruitment of immune cells.

ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition

K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors.

Abstract PR03: Selective sensitization of mutant K-Ras cancer cells to DNA damage based therapies by targeting wild type H- and N-Ras.

Abstract Mutations in Kras are highly prevalent in human cancers and are strongly predictive of resistance to chemotherapy. As the constitutive activation of K-Ras has been believed to confer functional autonomy and dominance over the wild type Ras forms, it has been thought that wild type Ras forms are dispensable to the mutant K-Ras driven tumorigenic phenotype. Here we demonstrate a functional dependence of mutant K-Ras driven tumors on wild type Ras for the activation of the DNA damage response, and that this dependence can be exploited to specifically sensitize K-Ras cancers to DNA[[Unable to Display Character: –]]damage-inducing agents. We show that in mutant K-Ras cancer cells, wild type H-Ras and N-Ras are required for the activation of Chk1, the G2 DNA damage checkpoint, and the cellular response to DNA-damage-inducing chemotherapy. Specifically, we show that targeted depletion of wild type H- and N-Ras in mutant K-Ras cancer cells leads to the hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and as a consequence, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint, and confers a specific sensitization of mutant K-Ras cancer cells to DNA damage therapeutic agents both in vitro and in vivo. Our results demonstrate a previously unappreciated role for wild type Ras in the DNA damage response of mutant K-Ras cancer cells and reveal a promising approach for targeting K-Ras-driven tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR03. Citation Format: Elda Grabocka, Yuliya Pylayeva-Gupta, Eyoel Yemanaberhan, Veronica Lubkov, Laura Taylor, Dafna Bar-Sagi. Selective sensitization of mutant K-Ras cancer cells to DNA damage based therapies by targeting wild type H- and N-Ras. [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 PR03.

Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals via K-Ras at the plasma membrane of Acute Myeloid Leukemia cells

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3 ITD) is an important oncoprotein in acute myeloid leukemia (AML). Owing to its constitutive kinase activity FLT3 ITD partially accumulates at endomembranes, a feature shared with other disease-associated, mutated receptor tyrosine kinases. Because Ras proteins also transit through endomembranes we have investigated the possible existence of an intracellular FLT3-ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-type FLT3. Ligand stimulation activated both K- and N-Ras in cells expressing wild-type FLT3. Live-cell Ras-GTP imaging revealed ligand-induced Ras activation at the plasma membrane (PM). FLT3-ITD-dependent constitutive activation of K-Ras and N-Ras was also observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells. Accordingly, specific interference with FLT3-ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potently inhibited cell proliferation and promoted apoptosis. In conclusion, Ras signaling is crucial for FLT3-ITD-dependent cell transformation and FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.

407 A Broad Tumor Cell Line Panel Screen of Ponatinib, a Pan-BCR-ABL and Multitargeted Kinase Inhibitor

constitutive KRAS activity. Our experience developing PH-domain inhibitors for other signaling proteins led us to use molecular docking and an in silico compound library to find CNKSR1 inhibitors. We identified small molecule lead compounds that using surface plasmon resonance spectroscopy we found bound to the PH-domain of CNKSR1. The most active compound was PHT-782 (Kd1.8 mM). PHT-782 showed activity similar to CNKSR1 knockdown and inhibited mut-KRAS signaling and the growth of mut-KRAS isogenic and NSCLC cell lines (IC50 ~30 mM) as effectively as siRNA knockdown of KRAS. In mice PHT-782 exhibited oral bioavailability and plasma t12 > 4 hr, and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity at doses up to 200mg/kg. To improve potency and selectivity we have undertaken iterative modeling using PHuDock a proprietary computational modeling platform and have identified additional leads with improved binding potency. The results of our study show that the PH domain of CNKSR1 can be targeted by small molecule inhibitors giving agents that block oncogenic KRAS signaling and cell growth, thus creating a therapeutic potential for patients with mut-KRAS for which there is currently no effective therapy.

409 Optimizing the Therapeutic Potential of PD-L1 Blockade as a Single Agent and Through Combination Therapy

constitutive KRAS activity. Our experience developing PH-domain inhibitors for other signaling proteins led us to use molecular docking and an in silico compound library to find CNKSR1 inhibitors. We identified small molecule lead compounds that using surface plasmon resonance spectroscopy we found bound to the PH-domain of CNKSR1. The most active compound was PHT-782 (Kd1.8 mM). PHT-782 showed activity similar to CNKSR1 knockdown and inhibited mut-KRAS signaling and the growth of mut-KRAS isogenic and NSCLC cell lines (IC50 ~30 mM) as effectively as siRNA knockdown of KRAS. In mice PHT-782 exhibited oral bioavailability and plasma t12 > 4 hr, and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity at doses up to 200mg/kg. To improve potency and selectivity we have undertaken iterative modeling using PHuDock a proprietary computational modeling platform and have identified additional leads with improved binding potency. The results of our study show that the PH domain of CNKSR1 can be targeted by small molecule inhibitors giving agents that block oncogenic KRAS signaling and cell growth, thus creating a therapeutic potential for patients with mut-KRAS for which there is currently no effective therapy.