Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Weekly EZN-2208 (PEGylated SN-38) in combination with bevacizumab in patients with refractory solid tumors.

BACKGROUND: Anti-angiogenic therapies such as bevacizumab upregulate hypoxia-inducible factor-1α (HIF-1α), a possible mechanism of drug resistance. Camptothecin analogues, including SN-38, have been shown to reduce the expression and transcriptional activity of HIF-1α in preclinical models. We hypothesized that co-administration of pegylated SN-38 (EZN-2208) may offset the induction of HIF-1α following bevacizumab treatment, resulting in synergistic antitumor effects. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled. Objectives were to evaluate the modulation of HIF-1α protein and target genes in tumor biopsies following administration of the combination of EZN-2208 administered weekly × 3 (days 1, 8, 15) and bevacizumab administered every 2 weeks, in 28-day cycles, and to establish the safety and tolerability of the combination. Tumor biopsies and dynamic contrast enhanced MRI (DCE-MRI) were obtained following bevacizumab alone (before EZN-2208) and after administration of both study drugs. RESULTS: Twelve patients were enrolled; ten were evaluable for response. Prolonged stable disease was observed in 2 patients, one with HCC (16 cycles) and another with desmoplastic round cell tumor (7 cycles). Reduction in HIF-1α protein levels in tumor biopsies compared to baseline was observed in 5 of 7 patients. Quantitative analysis of DCE-MRI from 2 patients revealed changes in K(trans) and k(ep). The study closed prematurely as further clinical development of EZN-2208 was suspended by the pharmaceutical sponsor. CONCLUSION: Preliminary proof-of-concept for modulation of HIF-1α protein in tumor biopsies following administration of EZN-2208 was observed. Two of 10 patients had prolonged disease stabilization following treatment with the EZN-2208 and bevacizumab combination.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Natural Product Graveoline Modulates Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) Membrane Association: Insights from Advanced Spectroscopic Studies.

The KRAS gene plays a pivotal role in numerous cancers by encoding a GTPase that upon association with the plasma membrane activates the MAPK pathway, promoting cellular proliferation. In our study, we investigated small molecules that disrupt KRAS's membrane interaction, hypothesizing that such disruption could in turn inhibit mutant RAS signaling. Native mass spectrometry screening of KRAS-FMe identified compounds with a preference for interacting with the hypervariable region (HVR), and surface plasmon resonance (SPR) further refined our selection to graveoline as a compound exhibiting preferential HVR binding. Subsequent nuclear magnetic resonance (NMR) analysis showed that graveoline's interaction with KRAS depends on C-terminal O-methylation. Moreover, our findings revealed multiple interaction sites, suggesting weak engagement with the KRAS G domain. Using nanodiscs as a membrane mimetic, further characterization through NMR and Förster resonance energy transfer (FRET) studies demonstrated graveoline's ability to perturb KRAS membrane interaction in a biochemical setting. Our biophysical approach sheds light on the intricate molecular mechanisms underlying KRAS-ligand interactions, providing valuable insights into understanding the KRAS-associated pathophysiology. These findings contribute to the translational aspect of our study, offering potential avenues for further research targeting KRAS membrane association with the potential to lead to a new class of RAS therapeutics.

Blockade of leukemia inhibitory factor as a therapeutic approach to KRAS driven pancreatic cancer.

KRAS mutations are present in over 90% of pancreatic ductal adenocarcinomas (PDAC), and drive their poor outcomes and failure to respond to targeted therapies. Here we show that Leukemia Inhibitory Factor (LIF) expression is induced specifically by oncogenic KRAS in PDAC and that LIF depletion by genetic means or by neutralizing antibodies prevents engraftment in pancreatic xenograft models. Moreover, LIF-neutralizing antibodies synergize with gemcitabine to eradicate established pancreatic tumors in a syngeneic, KrasG12D-driven, PDAC mouse model. The related cytokine IL-6 cannot substitute for LIF, suggesting that LIF mediates KRAS-driven malignancies through a non-STAT-signaling pathway. Unlike IL-6, LIF inhibits the activity of the Hippo-signaling pathway in PDACs. Depletion of YAP inhibits the function of LIF in human PDAC cells. Our data suggest a crucial role of LIF in KRAS-driven pancreatic cancer and that blockade of LIF by neutralizing antibodies represents an attractive approach to improving therapeutic outcomes.

Differential Effector Engagement by Oncogenic KRAS.

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies.

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

Small Cell Lung Cancer Screen of Oncology Drugs, Investigational Agents, and Gene and microRNA Expression.

BACKGROUND: Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat. METHODS: Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations. RESULTS: Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern. CONCLUSIONS: Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells.

IL-11 and its receptor, IL-11Ra, are expressed in human cancers; however, the functional role of IL-11 in tumor progression is not known. We found that IL11 is a hypoxia-inducible, VHL-regulated gene in human cancer cells and that expression of IL11 mRNA was dependent, at least in part, on HIF-1. A cooperative interaction between HIF-1 and AP-1 mediated transcriptional activation of the IL11 promoter. Additionally, we found that human cancer cells expressed a functional IL-11Ra subunit, which triggered signal transduction either by exogenous recombinant human IL-11 or by autocrine production of IL-11 in cells cultured under hypoxic conditions. Silencing of IL11 dramatically abrogated the ability of hypoxia to increase anchorage-independent growth and significantly reduced tumor growth in xenograft models. Notably, these results were phenocopied by partial knockdown of STAT1 in a human prostate cancer cell line (PC3), suggesting that this pathway may play an important role in mediating the effects of IL-11 under hypoxic conditions. In conclusion, these results identify IL11 as an oxygen- and VHL-regulated gene and provide evidence of a pathway "hijacked" by hypoxic cancer cells that may contribute to tumor progression.

Adaphostin toxicity in a sensitive non-small cell lung cancer model is mediated through Nrf2 signaling and heme oxygenase 1.

BACKGROUND: Preclinical toxicity of adaphostin has been related to oxidative stress. This study investigated the regulatory mechanism underlying adaphostin induction of heme oxygenase 1 (HMOX1) which plays a significant role in modulation of drug-induced toxicity in the non-small cell lung cancer cell line model, NCI-H522. METHODS: The transcriptional response of NCI-H522 to adaphostin prominently involved oxidative stress genes, particularly HMOX1. Reactive oxygen species (ROS) involvement was additionally established by generation of ROS prior to modulation of adaphostin-toxicity with antioxidants. To identify up-stream regulatory elements of HMOX1, immunofluorescence was used to evaluate nuclear translocation of the transcription factor, NF-E2-related factor 2 (Nrf2), in the presence of adaphostin. The PI3-kinase inhibitor, wortmannin, was employed as a pharmacological inhibitor of this process. RESULTS: Generation of ROS provided a substantial foundation for the sensitivity of NCI-H522 to adaphostin. However, in contrast to leukemia cell lines, transcriptional response to oxidative stress was associated with induction of HMOX1, which was dependent on nuclear translocation of the transcription factor, Nrf2. Pretreatment of cells with wortmannin inhibited translocation of Nrf2 and induction of HMOX1. Wortmannin pretreatment was also able to diminish adaphostin induction of HMOX1, and as a consequence, enhance the toxicity of adaphostin to NCI-H522. CONCLUSIONS: Adaphostin-induced oxidative stress in NCI-H522 was mediated through nuclear translocation of Nrf2 leading to upregulation of HMOX1. Inhibition of Nrf2 translocation by wortmannin inhibited this cytoprotective response, and enhanced the toxicity of adaphostin, suggesting that inhibitors of the PI3K pathway, such as wortmannin, might augment the antiproliferative effects of adaphostin in solid tumors that depend on the Nrf2/ARE pathway for protection against oxidative stress.