RAS-mutant Cells Research Articles

Abstract 800: Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia

Abstract FLT3 mutations (mFLT3) in acute myeloid leukemia (AML) carry a poor prognosis.The mandatory co-occurrence of other driver mutations with mFLT3 leads to suboptimal clinical responses with single agent FLT3 inhibitors (FLT3i). Pre-existing or emergent RAS mutations are one mechanism of primary and secondary resistance to single agent FLT3i. Therefore development of combinatorial strategies to overcome resistance conferring co-mutations is critical. Herein we report a novel in-vitro combination of the FLT3/SYK inhibitor (FSI) TAK-659 with Proteasome inhibitor (PI) ixazomib, to co-operatively inhibit proliferation of mFLT3 and RAS or WT1 co-mutated AML blasts, which are otherwise less sensitive to Flt3i and FSI. Peripheral blood or bone marrow derived cells from patients with AML were isolated and treated in vitro with TAK-659 and Ixazomib alone or in combination. Cytosolic/nuclear protein lysates were obtained at 12 hours of drug/control treatment and western blotting performed for proteins of interest. Tritiated thymidine proliferation assays were also performed with titrated doses of each agent alone, and combination of fixed dose of one agent with titration of the second. Patient samples were annotated for mutations by Next Generation Sequencing. Similar experiments were carried out on cell lines MV411 (Flt3ITD/MLL), THP-1 and OCI-AML3 (both NRAS). Proliferation assays on a series of primary AML samples revealed an inherent dichotomy in response patterns. mFLT-3 samples with co-mutations in DNMT3A, NPM1 or tMLL were very sensitive to TAK-659 (IC50 <75 nM) but much less sensitive to Ixazomib alone (IC50>100nM). The co-occurrence of RAS or WT1 mutations predicted for resistance to TAK-659 but interestingly sensitized cells to PI alone and in combination. In fact, presence of RAS mutations irrespective of FLT3 status predicted for sensitivity to PI (IC50<50nM). We also observed sensitivity to Ixazomib in THP-1 and OCI-AML3, while MV4-11 was sensitive to TAK-659 but relatively resistant to Ixazomib alone. Based on genome-wide CRISPR screen, overactivity of RAC-PAK signaling mediated by PREX1 is essential to RAS mutated AML (T Wang etal. Cell 168:890, 2017). Similarly, WT1 mutation in AML is known to ablate expression of a major nuclear chaperone for beta-catenin, TBL1X, and RAC has been found to be a substitute (Y Wang et al. Mol Cell. 57:662, 2015). We therefore hypothesized that PI decreased nuclear availability of RAC1 along with its chaperone partner beta-catenin - a determinant of leukemic stem cell activity, also regulated by RAC/PAK and the proteasomal system. Indeed, in primary RAS or WT1 mutated AML samples, we observed PI treatment led to significant reduction in nuclear RAC1 levels and decreased nuclear beta-catenin levels, specifically PAK phosphorylated p675 active beta-catenin. These findings suggest PI effects RAC1, PAK and beta-catenin simultaneously. Similar results were also noted in RAS mutant cell lines. siRNA knockdown experiments also linked p62 SQSTM1 in a pathway to RAS/RAC/PAK and beta-catenin. In summary, we delineate a RAS/RAC/PAK/Beta-catenin pathway to be linked to critical events in RAS or WT1 mutated AML progression and resistance. FLT3 mutated AML with these co-mutations are insensitive to single-agent activity of FLT3i or FSI alone and the addition of PI is critical to overcome them. Citation Format: Sravanti Rangaraju, Santosh Kumar Pasupuleti, Silvia Marino, Daniela Nicoleta Petrusca, Larry D. Cripe, Bhaskar Ramdas, Lakshmi Reddy Palam, Katie J. Sargent, Hamid Sayar, Heiko Konig, Reuben Kapur, David G. Roodman, Scott H. Boswell. Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 800.

Abstract 5133: Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells

Abstract Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NRAS, KRAS4A, KRAS4B). Expressed at low endogenous abundance, these four isoforms possess considerable sequence similarity, rendering the confident proteomic linkage of a given isoform with a specific mutation or post-translational modification (PTM) a formidable proposition outside of isoform-specific model cell lines. Conversely, top-down proteomic analysis (TDP), which measures intact protein molecules, can precisely identify modified protein forms, or proteoforms, and confidently localize both mutations and PTMs on each RAS isoform present. Previously, a method combining immunoprecipitation and subsequent TDP (IP-TDP) led to the identification of eleven KRAS4B proteoforms, including four mutations and two novel PTMs, within colorectal cancer cell lines and tumor samples†. These initial results demonstrated the great potential for proteoform-resolved measurements to further illuminate the role of KRAS4B mutations and modifications in human cancer. More recently, the NCI RAS Initiative has developed an enhanced RAS proteoform assay incorporating IP-TDP analysis on an Orbitrap Fusion Lumos mass spectrometer. By employing alternate antibodies, recombinant protein standards, and well-characterized model or malignant cell lines, we have obtained substantial improvements in both RAS proteoform detection and characterization, thus facilitating the identification and localization of low-abundance PTMs. Moreover, we have applied our optimized IP-TDP method to a diverse panel of malignant and RAS mutant cell lines with the goal of expanding our knowledge of the RAS proteoform landscape. In doing so, we have observed a wealth of novel RAS proteoforms, of far greater number and complexity than previously anticipated. We have also observed isoform-specific proteoform variations, most notably between KRAS4A and KRAS4B, and initial indications of proteoform context dependence, namely within a given mutation or tissue background. Our results further underscore the power of proteoform-resolved measurements and indicate that RAS-dependent signaling pathways in normal and cancer contexts may be far more complex than previously thought. † I Ntai et. al. “Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk.” Proc Natl Acad Sci U S A. 2018 Apr 17; 115(16):4140-4145. Citation Format: Caroline DeHart, Kanika Sharma, Dominic Esposito, Anna Maciag, Dwight Nissley, Frank McCormick. Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5133.

Abstract A54: Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma

Abstract RAS pathway mutations are found in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. By sequencing, we identified cell lines and PDX tumors harboring activating mutations in H- or NRAS. Further, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS cells and tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed drug-sensitizing pooled CRISPR library screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic ERK inhibition and ERMS tumor growth suppression. Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Yidong Chen, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A54.

Abstract IA32: Interplay between RAF inhibitors and RAS

Abstract Ever since RAF was shown to be a key downstream effector of RAS in the early 90s, RAF inhibition has been a goal within the drug discovery community. Sorafenib, an early entrant to the RAF armamentarium, was discovered through biochemical RAF inhibition assays with the goal of disrupting RAS signaling. Anticancer efficacy that was demonstrated through clinical trials was likely through “off-target” inhibition of VEGF receptors, and RAF is not effectively inhibited in RAS-mutant tumor cells. Furthermore, BRAF-mutant tumors were also poorly targeted by sorafenib, most likely because the “type 2” binding mode is ineffective against the activated BRAF kinase. Subsequently, compounds such as vemurafenib, dabrafenib, and encorafenib were developed as “type 1” inhibitors that target the active form of BRAF kinase: these inhibitors show efficacy in many BRAF-mutant tumors. Curiously, these RAF inhibitors cause certain side effects that have been attributed to the “RAF inhibitor paradox”: the ERK pathway is paradoxically stimulated by RAF inhibitors in cells with an activated RAS pathway. To overcome this liability, the next generation of RAF inhibitors dubbed “paradox breakers” were developed. As a BRAF-specific dimer disruptor, PLX8394 selectively inhibits ERK signaling in tumors bearing both monomer- and dimer-dependent BRAF mutants, including BRAF fusions and splice variants, while sparing RAF function in normal cells and avoiding paradoxical ERK activation in RAS-mutant cells. A converse effort seeks to take advantage of the paradox to identify RAS cell liabilities. By hyperstimulating the ERK pathway, “paradox activators” could tip the balance in favor of cellular disharmony. Future efforts exploiting this mechanism will enhance the context for thinking about RAF and near-neighbor targets in RAS tumors and guide the judicious selection of combination therapies involving RAF-targeting agents and other therapeutics. Citation Format: Ying Zhang, Gideon Bollag. Interplay between RAF inhibitors and RAS [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA32.

Abstract B06: ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression

Abstract ING2 (Inhibitor of Growth 2) is a tumor-suppressor gene involved in chromatin acetylation whose loss has been observed in nearly 60% of NSCLC. The loss of ING2 expression has been associated with the progression of tumor cells from G1 to S phase, replication stress and genomic instability. The intra-S and the G2-M phase checkpoints are mainly regulated by the cell cycle tyrosine kinase WEE1. Therefore, we hypothesized that the loss of ING2 expression could sensitize NSCLC to the WEE1 inhibitor MK-1175, according to a synthetic lethality model. In silico analysis of the cancer gene dependency map (shRNAi Achilles library v.2016) was used to explore gene interactions with ING2. Antiproliferative effects of drugs were tested in vitro with AlamarBlue and MTT assays in a panel of NSCLC cells lines with varied mutational status. Multiple functional approaches were taken to decrease ING2 expression in RAS-mutated NSCLC cell lines (si-RNA, LNA-gapmer, and CrispR-Cas9). Pharmacologic inhibition of ING2 epigenetic functions was achieved using the HDAC inhibitor SAHA. Cell cycle and apoptosis analysis were performed using flow cytometry according to a BrdU-Pi and Annexin V-PI protocols, respectively. Compusyn software was used to model MK-1775 and SAHA drug interaction. NSCLC cell lines with mutant RAS that expressed high levels of ING2 protein were more resistant to the antiproliferative effect of MK-1775 than similar lines with low levels of ING2. Colony formation assays were consistent with these results. Multiple functional assays reproducibly showed that downregulation of ING2 expression increased MK-1775 sensitivity in vitro. Similar results were obtained with pharmacologic disruption of ING2 from the HDAC-SIN3A complex using SAHA, where disruption conferred a higher antiproliferative effect to MK-1775. Interestingly, combination treatment induced cell cycle progression and synergic cell death through apoptosis in RAS-mutated cell lines nondeficient for ING2. Since the synergistic effect of SAHA and WEE1 inhibitor was shown to be dependent on CHK1 activity in a previous report, we checked whether ING2 and CHK1 expression could be linked. Using public database and functional assays, we indeed find that ING2 downregulation represses CHK1 transcription through a mechanism that remains to be elucidated. Collectively, these findings demonstrate that inhibition of WEE1 by MK-1775 could represent a therapeutic approach to more effectively target ING2-deficient RAS-mutated NSCLC. Moreover, HDAC inhibitors targeting ING2 epigenetic functions synergize with MK-1775, possibly through regulation of CHK1 transcription. Consequently, this combination therapy could also be a promising strategy in ING2-proficient RAS-mutated NSCLC. Citation Format: Charles Ricordel, Subash Thalappilly, Jérome Archambeau, Angela Chan, Nancy Nixon, Benoît Desrues, Gwyn Bebb, Karl Riabowol, Rémy Pedeux. ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B06.

Abstract B13: Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma

Abstract Recent sequencing of childhood rhabdomyosarcoma identified the presence of RAS pathway mutations in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins (HRAS, NRAS and KRAS) are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. We sequenced ERMS cell lines and PDX tumors and identified 4 cell lines harboring activating mutations in H- or NRAS and two cell lines with wild-type RAS. By siRNA mediated knockdown, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed CRISPR screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic MEK/ERK pathway inhibition and ERMS tumor growth suppression. Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozzo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B13.

Abstract B05: ASN007, an oral ERK1/2 inhibitor, shows strong antitumor activity across a panel of KRAS subtype mutant cancer models

Abstract The RAS/MAPK pathway is one of the most commonly dysregulated signaling pathways found in human cancers. KRAS, HRAS and NRAS genes, which play a critical role in transducing the signal from receptor tyrosine kinases to the downstream target proteins such as BRAF and MEK1/2, are frequently mutated in most solid tumors. The extracellular signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and RAF kinases, serve as a key node in this crucial signaling pathway in tumor cells. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. In this report, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed potent inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 showed potent antiproliferative activity that was selective for RAS/MAPK pathway dependent cancer cell lines, and did not affect the growth of cell lines harboring PI3K pathway or receptor tyrosine kinase mutations. Of note, ASN007 showed much more potent and broader activity against a panel of RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a panel of 96 KRAS mutant cell lines, ASN007 showed strong activity across all KRAS subtype mutations, including G12C, G12D, G12V and G13D. In tumor xenograft models harboring KRAS and NRAS mutations, a daily oral dosing regimen of ASN007 provided strong tumor growth inhibition and was well tolerated. ASN007 also showed strong single-agent antitumor activity in colorectal PDX models with various KRAS subtype mutations including G12C, G12D, and G12S. A clinical phase 1 trial of ASN007 in patients with BRAF and KRAS, HRAS and NRAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is currently ongoing (NCT03415126). Citation Format: Sanjeeva P. Reddy, Scott K. Thompson, Helen Usansky, Roger A. Smith, Louis Denis, Sandeeep Gupta. ASN007, an oral ERK1/2 inhibitor, shows strong antitumor activity across a panel of KRAS subtype mutant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B05.

Abstract B26: The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors

Abstract Mutual exclusivity of RAS and BRAF mutations in multiple cancer types illustrates the key role of the RAF-MEK-ERK signaling pathway in mediating RAS oncogenesis. Although MEK inhibitors in particular are potent and specific, their on-target toxicity has severely limited their clinical efficacy against RAS-driven tumors and strategies to enhance their therapeutic index are sorely needed. RAF activation is a complex process that involves multiple regulatory steps in addition to RAS binding. Key among them is the dephosphorylation of a conserved inhibitory site (S259-RAF1, S365-BRAF). We have shown that a ternary complex comprising SHOC2, MRAS and PP1 functions as a specific “S259” RAF phosphatase holoenzyme. Gain-of-function mutations at this site in RAF, and indeed SHOC2 itself, are found in the RASopathy Noonan syndrome. We have used CRISPR to knock out SHOC2 in a panel of NSCLC cell lines and show that SHOC2 inhibition strongly sensitizes RAS-mutant cells to MEK inhibitors. SHOC2 inhibition lowers the concentration of MEK inhibitor required for cytotoxicity and drives tumor regressions in xenograft models. Biochemically, we show that SHOC2 inhibition impairs the dimerization of RAF upon signaling rebound after MEK inhibitor treatment, and thereby potentiates the duration of ERK-pathway inhibition. In vivo, conditional SHOC2 ablation in the adult mouse is relatively well tolerated, and strikingly SHOC2 inactivation in the lung suppresses tumor initiation in KRAS-driven models to prolong overall survival. Taken together, our results suggest the SHOC2 complex may provide an attractive target for therapeutic intervention—both as a monotherapy and by significantly enhancing the therapeutic index of clinically available MEK inhibitors. Citation Format: Greg G. Jones, Isabel Boned Del Rio, Sibel Sari, Aysen Sekerim, Lucy C. Young, Nicole Hartig, Mona A. El-Bahrawy, Miriam Molina-Arcas, Julian Downward, Pablo Rodriguez-Viciana. The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B26.

Lactate induces PD-L1 in HRASG12V-positive oropharyngeal squamous cell carcinoma.

Intratumoral lactate production negatively correlates with survival and tumor clearance in the setting of human papillomavirus positive oropharyngeal squamous cell carcinoma (HPV-positive OPSCC). Robust anti-tumor immune activity is required for tumor clearance in human patients and animal models of this disease, and intratumoral lactate interferes with this process. While lactate is known to directly inhibit T cell activity, recent evidence has demonstrated that lactate can affect gene expression in multiple cell types. We therefore sought to determine if lactate in the tumor microenvironment could aid immune evasion by inducing the expression of immune checkpoint co-inhibitors. Using a mouse cell line transformed with HPV16 E6, E7, and HRASG12V, we determined that OPSCC cells carrying the HRASG12V mutant showed significantly increased expression of PD-L1 in the presence of extracellular lactate. Furthermore, we demonstrate here that lactate activates the MEK/ERK pathway in Ras-mutated cells.

Ferroptosis and Its Potential Role in Human Diseases.

Ferroptosis is a novel regulated cell death pattern discovered when studying the mechanism of erastin-killing RAS mutant tumor cells in 2012. It is an iron-dependent programmed cell death pathway mainly caused by an increased redox imbalance but with distinct biological and morphology characteristics when compared to other known cell death patterns. Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, neurodegenerative, and hepatic diseases. Moreover, activation or inhibition of ferroptosis using a variety of ferroptosis initiators and inhibitors can modulate disease progression in animal models. In this review, we provide a comprehensive analysis of the characteristics of ferroptosis, its initiators and inhibitors, and the potential role of its main metabolic pathways in the treatment and prevention of various diseased states. We end the review with the current knowledge gaps in this area to provide direction for future research on ferroptosis.

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

1953P - Detection of microRNAs as biomarker for anti-EGFR antibody resistance in colon cancer patients

BackgroundMonoclonal antibodies against the Epidermal Growth Factor Receptor (EGFR) such as cetuximab or panitumumab are used for the treatment of metastatic colorectal cancer (mCRC) patients. Unfortunately, most patients develop resistance against these therapies within months. Several studies have shown that aberrations in the RAS pathway are responsible for resistance. However, even in metastases that are refractory to anti-EGFR treatment a significant fraction of RAS wild-type (wt) cells remain. These findings suggest a cross-talk between RAS mutant and wt cells in mediating resistance in the wt compartment. MethodsWe used mouse and patient-derived organoids from mCRC as well as CRC cell lines to test the contribution of extracellular vesicles in mediating resistance in RAS wt cells. Using conditioned media, transfection experiments and liquid biopsies (plasma and urine) from patients differential expression of the let-7g microRNA was demonstrated in microvesicles from cetuximab sensitive and resistant cells. Changes in expression of the let-7g microRNA were further analysed by in-situ hybridization in tissues. ResultsBasal let-7g expression from pre-treatment plasma and urine samples of RAS wt patients correlated with clinical outcome and changes in let-7g circulating levels mirrored clinical behaviour. In-situ hybridization in tissues confirmed changes in expression of the let-7g microRNA observed in plasma and urine samples. ConclusionsOur data suggest that let-7g microRNA might function as a paracrine mediator of anti-EGFR resistance and might be exploited as a non-invasive biomarker of resistance to cetuximab treatment. Further work is ongoing to characterize the molecular mechanisms underpinning let-7g mediated effect on anti-EGFR sensitivity in RAS wt CRC cells. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureN. Valeri: Speaker Bureau / Expert testimony: Bayer; Speaker Bureau / Expert testimony: Eli Lilly; Speaker Bureau / Expert testimony: Pfizer; Speaker Bureau / Expert testimony: Merck. All other authors have declared no conflicts of interest.

Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme is comprised of a scaffolding subunit (PP2A Aα/β), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared to wild type Aα, suggesting a rededication of PP2A functions. Unlike wild type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.

Abstract 1164: Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines

Abstract TCGA genomic study in papillary thyroid carcinoma (PTC) samples highlights two groups of tumors: BRAF-like and RAS-like, whose genotype might influence its biological behavior. The mechanisms behind progressive loss of thyroid differentiation, observed mainly in the most aggressive histotype anaplastic thyroid carcinoma (ATC), could be associated with the reactivation of embryonic signaling and acquisition of stem cell-like phenotype. In this study we focused on Notch and Nodal embryonic signaling pathways, which are crucial for stemness maintenance in embryonic cells and has also been linked to cancer cell stemness. By assessing the expression of Notch and Nodal pathway components in BRAF- and RAS-like cell lines, we sought to investigate if the reactivation of these pathways is involved in dedifferentiation mechanisms observed in ATC. For that purpose, RNA and protein were extracted from three PTC (TPC-1, BCPAP and K1), six ATC (KTC2, SW1736, C643, Hth7, Hth74 and Hth83) and non-tumoral (Nthy-ori 3-1) cell lines. For gene expression analysis, cDNA generate from mRNA was amplified by qPCR using specific primers for NOTCH1, NOTCH4, HES1, NUMB, CER1 and LEFTY1/2. For protein expression analysis, SDS-PAGE-fractionated proteins were transferred onto nitrocellulose membrane, incubated with anti-Nodal antibody and visualized by chemoluminescence. NOTCH1 was downregulated in BRAF-like PTC cell lines, while slightly overexpressed in RET/PTC-containing TPC-1. On the other hand, NOTCH1 was overexpressed in Hth83 and C643 ATC lines, both HRAS-mutated, and also in BRAF-mutated SW1736. NOTCH4 was overexpressed in PI3K-mutated K1 and all ATC cell lines. Transcription repressor HES1, which prevents cell cycle arrest by inhibiting p27, was highly expressed in all cell lines compared to Nthy. Levels of NUMB expression were elevated in all ATC cell lines with no difference between BRAF- and RAS-mutated, but were inversely related to those of HES1, as expected for a Notch pathway inhibitor. CER1 and LEFTY1/2, which encode Nodal inhibitors, were highly expressed in ATC cell lines (4/6 and 5/6, respectively) but with no apparent distinction between BRAF- and RAS-mutated cells. At the same time, Nodal protein levels are only upregulated in TP53-mutated BCPAP and C643, suggesting that alterations in MAPK components are not sufficient to promote Nodal overexpression. Our results show re-expression of Notch embryonic signaling components in both PTC and ATC cell lines with either BRAF or RAS oncogenes. Moreover, additional genetic alterations such as in PI3K and TP53 might also interfere with the expression of both Notch and Nodal components. We therefore highlight the importance of tumor genotype, which might determine the modulation of embryonic-related genes and therefore contribute to the undifferentiated phenotype in thyroid cancer. Financial support: FAPESP and CNPq. Citation Format: Paola M. Dantonio, Cesar Seigi Fuziwara, Kelly C. Saito, Edna T. Kimura. Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid 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 1164.

Abstract LB-114: RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF

Abstract RAS mutant tumors have proven largely refractory to inhibitors of the MAPK pathway in clinic. In contrast, selective inhibitors of BRAFV600E but not wild type RAF have been successfully developed in BRAFV600E mutant melanoma, NSCLC, and thyroid cancers. Success of these drugs results largely from the therapeutic index imparted by lack of activity against wild RAFs; however, this selectivity also prevents their utility in indications beyond BRAFV600E disease, such as RAS-mutant tumors. In contrast, MEK1/2 inhibitors, while effective pre-clinically in RAS mutant models, have proven ineffective against RAS mutant tumors due at least in part to the poor therapeutic index of these molecules that results from pathway inhibition in normal cells. Thus, to inhibit effectively MAPK pathway signaling in tumors, inhibitors with improved tolerability via differential pathway inhibition in tumor vs normal tissues are required. Recent data in mice conditionally lacking different RAF paralogs suggest that inhibitors biased towards CRAF might retain anti-tumor properties, with improved tolerability. LXH254 is a RAF inhibitor with high selectivity and nanomolar potency for BRAF and CRAF. Profiling of LXH254 activity in Ras mutant lines lacking either ARAF, BRAF or CRAF by CRISPR-Cas9 modification revealed that loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. This increased sensitivity to LXH254 from ARAF knockout was rescued by over-expression of wild-type ARAF but not by either ARAF that harbored mutations that impaired kinase activity (K336M, D447A) or its ability to dimerize (R362H). Comparing the effect of LXH254 on MAPK pathway signaling in RAS mutant cells engineered to express only one of the three RAF paralogs indicated that significantly higher concentrations of LXH254 are required to inhibit MAPK signaling in cells expressing only ARAF. Furthermore, at low concentrations of LXH254, cells expressing only ARAF displayed paradoxical activation of MAPK signaling in a manner similar to the BRAFV600E selective inhibitor dabrafenib. Thus, both LXH254 and dabrafenib inhibit mutationally activated monomeric BRAF, and promote/stabilize RAS-dependent dimerization of WT RAFs. However, because dabrafenib is only able to effectively inhibit one of the two RAF protomers in each dimer it paradoxically activates MAPK signaling in a RAS-dependent manner. In contrast, LXH254 retains activity against dimerized B-and C-RAF (but not ARAF) thereby theoretically extending its utility beyond BRAF mutant disease with better tolerability relative to, e.g. MEK1/2 inhibitors. Citation Format: Kelli-Ann Monaco, Scott Delach, Paul Fordjour, Salonee Parikh, Yun Feng, Mariela Jaskelioff, Darrin Stuart, Giordano Caponigro. RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF [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 LB-114.

Innovative dual system for selective eradication of cancer cells using exosomes carrying natural bacterial toxin-antitoxin (TA).

e14635 Background: Inactivation of P53 and activation of ras are frequent genetic alterations in cancer. We have shown in vitro and in vivo, that the TA system can selectively and effectively eradicate RAS-mutated cancer cells. Aim: Selective killing of cancer cells while sparing the normal cells based on tumor genetic signature. Methods: A “first generation” ΔE1/ΔE3 human type-5 adenoviral-vectors for gene delivery were designed and constructed to specifically target cancer cells. They are designated as "PY4-mazF-mCherry" (PY4, ras responsive element), "ΔPY4-mazF-mCherry" (control viruses) and "RGC-mazE-IRES-GFP" (RGC, P53 responsive element). Their potency was tested in vitro, by the enzymatic MTT assay, microscopic observation, colony formation assay and FACS analysis, and in a xenograft model of CRC. Next, we generated, small natural vesicles, exosomes, that directly targeted cancer through specific small antibody fragments against CD24 that is expressed in most cancer cells and rarely on normal cells. Results: The TA system ("PY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") induced a massive cell death, in a dose-dependent manner in vitro, 69% as compared to 19% in control co-infected ("ΔPY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") HCT116 CRC cells (mutated RAS and p53). In vivo, growth of HCT116-/- ( KRASmutand P53mut) and HCT116+/+ ( KRASmut and P53wt) tumors were significantly inhibited (70% and 65%, respectively). Conclusions: 1. Abusing the P53 genetic status and the activated Ras pathway holds promising effective and safe strategy to target tumor cells while sparing normal tissues. 2. It is a proof of concept for personalized cancer therapy based on the tumor genetic profile.

Autophagy inhibition specifically promotes epithelial-mesenchymal transition and invasion in RAS-mutated cancer cells

ABSTRACTMacroautophagy/autophagy inhibition is a novel anticancer therapeutic strategy, especially for tumors driven by mutant RAS. Here, we demonstrate that autophagy inhibition in RAS-mutated cells induces epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor invasion. This is at least partially achieved by triggering the NFKB/NF-κB pathway via SQSTM1/p62. Knockdown of ATG3 or ATG5 increases oncogenic RAS-induced expression of ZEB1 and SNAI2/Snail2, and activates NFKB activity. Depletion of SQSTM1 abolishes the activation of the NFKB pathway induced by autophagy inhibition in RAS-mutated cells. NFKB pathway inhibition by depletion of RELA/p65 blocks this EMT induction. Finally, accumulation of SQSTM1 protein correlates with loss of CDH1/E-cadherin expression in pancreatic adenocarcinoma. Together, we suggest that combining autophagy inhibition with NFKB inhibitors may therefore be necessary to treat RAS-mutated cancer.Abbreviations: 4-OHT: 4-hydroxytamoxifen; DIC: differential interference contrast; EMT: epithelial-mesenchymal transition; ESR: estrogen receptor; MAPK/ERK: mitogen-activated protein kinase; iBMK: immortalized baby mouse kidney epithelial cells; MET: mesenchymal-epithelial transition; PI3K: phosphoinositide 3-kinase; RNAi: RNA interference; TGFB/TGF-β: transforming growth factor beta; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6.

Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy.

The interplay between p53 and RAS signaling regulates cancer chemoresistance, but the detailed mechanism is unclear. In this study, we investigated the interactive effects of p53 and RAS on ovarian cancer cisplatin resistance to explore the potential therapeutic targets.Methods: An inducible p53 and RAS mutants active in either MAPK/ERK (S35 and E38) or PI3K/AKT (C40) or both (V12) were sequentially introduced into a p53-null ovarian cancer cell line-SKOV3. Comparative microarray analysis was performed using Gene Chip Prime View Human Gene Expression arrays (Affymetrix). In vitro assays of autophagy and apoptosis and in vivo animal experiments were performed by p53 induction and/or cisplatin treatment using the established cell lines. The correlation between HDAC4 and HIF-1α or CREBZF and the association of HDAC4, HIF-1α, CREBZF, ERK, AKT, and p53 mRNA levels with patient survival in 523 serous ovarian cancer cases from TCGA was assessed.Results: We show that p53 and RAS mutants differentially control cellular apoptosis and autophagy to inhibit or to promote chemoresistance through dysregulation of Bax, Bcl2, ATG3, and ATG12. ERK and AKT active RAS mutants are mutually suppressive to confer or to deprive cisplatin resistance. Further studies demonstrate that p53 induces HIF-1α degradation and HDAC4 cytoplasmic translocation and phosphorylation. S35, E38, and V12 but not C40 promote HDAC4 phosphorylation and its cytoplasmic translocation along with HIF-1α. Wild-type p53 expression in RAS mutant cells enhances HIF-1α turnover in ovarian and lung cancer cells. Autophagy and anti-apoptotic processes can be promoted by the overexpression and cytoplasmic translocation of HDAC4 and HIF1-α. Moreover, the phosphorylation and cytoplasmic translocation of HDAC4 activate the transcription factor CREBZF to promote ATG3 transcription. High HDAC4 or CREBZF expression predicted poor overall survival (OS) and/or progression-free survival (PFS) in ovarian cancer patients, whereas high HIF-1α expression was statistically correlated with poor or good OS depending on p53 status.Conclusion: HIF-1α and HDAC4 may mediate the interaction between p53 and RAS signaling to actively control ovarian cancer cisplatin resistance through dysregulation of apoptosis and autophagy. Targeting HDAC4, HIF-1α and CREBZF may be considered in treatment of ovarian cancer with p53 and RAS mutations.

Combined treatment of ABT199 and irinotecan suppresses KRAS-mutant lung cancer cells

Lung cancer has become the most prevalent neoplasm throughout the world with 1,2 million deaths per year. Molecular genetic analyses have suggested that KRAS mutation is much more frequency in NSCLC. Significant challenges are to develop selective pharmacological inhibitors for RAS mutation to treat cancers driven. In our study, we used the combinatorial strategy to target oncogene addiction for RAS-mutant cells and the data showed that ABT199 and irinotecan leads to RAS-mutant lung cancer cell growth inhibition and enhanced apoptosis in vitro and in vivo. Furthermore, PI3K/AKT signaling was down-regulated by the combination in KRAS-mutant lung cancer cells. Importantly, the effects of ABT199 and irinotecan combination are synergistic on the RAS-mutant lung cancer cells. Therefore, the combination suggests a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers which are otherwise difficult targeted by small molecules.

Abstract 5467: Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models

Abstract Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome. Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis. Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival. Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays. Citation Format: Jens Köhler, Prafulla C. Gokhale, Jiaqi Li, Margaret K. Wilkens, Hong Tiv, Shripad V. Bhagwat, Greg Donoho, Patrick Brueck, Ramon V. Tiu, Amanda J. Redig, Emily S. Chambers, Atsuko Ogino, Jihyun Choi, Man Xu, Paul Kirschmeier, Pasi A. Jänne. Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models [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 5467.