MEK Inhibitor Treatment Research Articles

Targeting PHGDH Upregulation Reduces Glutathione Levels and Resensitizes Resistant NRAS-Mutant Melanoma to MAPK Kinase Inhibition

Melanomas frequently harbor activating NRAS mutations leading to activation of MAPK kinase (MEK) and extracellular signal-regulated kinase 1/2 signaling; however, the clinical efficacy of inhibitors to this pathway is limited by resistance. Tumors rewire metabolic pathways in response to stress signals such as targeted inhibitors and drug resistance, but most therapy-resistant preclinical models are generated in conditions that lack physiological metabolism. We generated human NRAS-mutant melanoma xenografts that were resistant to the MEK inhibitor (MEKi) PD0325901 invivo. MEKi-resistant cells showed cross-resistance to the structurally distinct MEKi trametinib and elevated extracellular signal-regulated kinase 1/2 phosphorylation and downstream signaling. Additionally, we observed upregulation of the serine synthesis pathway and PHGDH, a key enzyme in this pathway. Suppressing PHGDH in MEKi-resistant cells together with MEKi treatment decreased oxidative stress tolerance and cell proliferation. Together, our data suggest targeting PHGDH as a potential strategy in overcoming MEKi resistance.

Abstract B02: Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer

Abstract Lung cancer is the foremost cause of cancer-related deaths in the U.S. KRAS mutations are one of the most frequent oncogenic aberrations in lung adenocarcinoma patients. Despite harboring identical mutations, the response of distinct tumor cells to therapeutic agents varies due to epigenetic reprogramming of the tumor cells. Epithelial-mesenchymal transition (EMT) contributes to phenotypic heterogeneity and determines the response of tumor cells to therapeutic agents. Kras-mutant NSCLC cells have been shown to acquire EMT as a mechanism to evade therapeutic targeting. We have previously observed that murine and human epithelial lung cancer cells are highly sensitive to MEK inhibition whereas mesenchymal tumor cells remain largely resistant. We aim to identify the mechanisms conferring survival advantage and an intrinsic resistance to MEK inhibition to mesenchymal cells. Using a loss-of-function shRNA dropout screen for targets with FDA-approved drugs, we identified cyclin dependent kinase 4 (CDK4) as an important driver of growth in mesenchymal cells both in vitro and in vivo. Murine NSCLC cells are resistant to cell cycle arrest induced by serum starvation, which corresponds to higher protein levels of CDK4 and cyclin D1 allowing cells to transition from G1 to S phase. After release of cells from arrested state, the mesenchymal cells transition into S phase more rapidly than the epithelial cells, suggesting a higher dependency on CDK4-cyclin D1 complex of mesenchymal tumor cells. An Ex Vivo Tumor (EVT) system to culture lung tumors in a 3D matrix environment that retains tumor cell heterogeneity was utilized to test the therapeutic sensitivity of distinct tumor cell subpopulations. In response to treatment with a CDK4 inhibitor, we observed a depletion of mesenchymal tumor cells within heterogeneous EVTs. In combination with MEK inhibitor treatment, there was a net decrease in size and viability of EVTs. Single-agent treatment with CDK4 inhibitor upregulated pErk in mesenchymal tumor cells, increasing their sensitivity to MEK inhibition and achieving an additive response with combination treatment. Mechanistically, the effects are mediated by FOXM1, a transcriptional regulator of cell cycle. FOXM1 positively regulates CDK4-cyclin D1 activity and is negatively regulated by the miR-200 family, which is preferentially expressed in epithelial cells. Upon induction of miR-200 expression, there was a decrease in FOXM1. CDK4, cyclin D1 and pRB levels follow a similar trend, suggesting the phenotype observed in mesenchymal cells is mediated by FOXM1. Since EMT is modulated by the miR-200/Zeb1 axis, higher levels of the transcriptional repressor Zeb1 and consequently FOXM1 in mesenchymal tumor cells confer a survival advantage and an intrinsic resistance to MEK inhibition. Combinatorial treatment approaches with MEK and CDK4 inhibitors targeting different subpopulations within a tumor are an effective strategy to combat resistant outgrowth of epigenetic subsets in a heterogeneous Kras mutant tumor. Citation Format: Aparna Padhye, B. Leticia Rodriguez, Jared J. Fradette, Christin Ungewiss, Don L. Gibbons. Dissecting tumor cell heterogeneity to identify therapeutic vulnerabilities in Kras-mutant lung cancer [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 B02.

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.

Abstract B47: Systems-level dissection of tumor-macrophage crosstalk in ovarian cancer resistance to MEK inhibition

Abstract Several subtypes of ovarian cancer are associated with dysregulated MAPK signaling, including low-grade serous ovarian carcinoma (LGSOC), yet targeted inhibitors of the MAPK pathway have shown relatively limited success in clinical trials for gynecologic malignancies. Here, we investigated whether this could be explained in part by crosstalk between ovarian cancer cells and neighboring tumor-associated macrophages (TAMs) within the tumor microenvironment. Using in vitro models, human ovarian cancer cell lines were mildly protected against MEK inhibitor-induced cell killing by transwell co-culture with alternatively activated (IL-4 stimulated), but not classically activated (LPS and IFN-gamma stimulated), primary monocyte-derived macrophages. We employed multiplexed bead-based immunoassays to acquire a compendium of over 1,200 phosphoprotein, cytokine, and growth factor measurements across combinations of 3 ovarian cancer cell lines and macrophages derived from 4 healthy peripheral mononuclear blood cell (PBMC) donors. Principal components analysis (PCA) and partial least squares regression (PLSR) revealed that compensatory receptor tyrosine kinase signaling occurs in tumor cells upon MEK inhibitor treatment, and that this is amplified upon co-culture with alternatively activated macrophages. Further work will determine if targeting these tumor-intrinsic and crosstalk effects in combination with MEK inhibition represents a potential therapeutic strategy. Citation Format: Stephanie J. Wang, Miles A. Miller, Douglas A. Lauffenburger. Systems-level dissection of tumor-macrophage crosstalk in ovarian cancer resistance to MEK inhibition [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 B47.

Abstract B53: Differential response of distinct KRAS mutants to SHP2 inhibition

Abstract Introduction: RAS is mutated in 30% of tumors and is still considered “undruggable.” Among RAS genes KRAS is the most frequently mutated, mostly at residues G12, G13 and Q61, impairing its intrinsic and GAP-mediated GTP hydrolysis capacity (Prior et al., Cancer Res 2012). Nevertheless, specific mutations are differently distributed in human cancers (G12 89%, G13 9%, Q61 1%). In particular, G12D (36%) is prevalent over G12C (14%) and G12V (23%). Distinct oncogenic mutations differently affect RAS GTP hydrolysis capacity (Lu et al., Sci Rep 2016; Hunter et al., Mol Cancer Research 2015), with G12C preserving the highest GTPase activity and Q61 mutants being the most compromised. This implies that some KRAS mutants could be sensitive to upstream regulation, as suggested by the in vivo sensitivity of KRAS mutant tumors to SHP2 inhibitors (Mainardi et al. and Ruess et al., Nat Med 2018; Fedele et al., Cancer Discov 2018). In order to finely predict the response to the SHP2 inhibitor SHP099 (Chen et al., Nature 2016) in KRAS mutant patients, we used a panel of isogenic Rasless murine embryonic fibroblasts (MEFs) (Esposito et al., Semin Cancer Biol 2018) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes, and studied their response to SHP099, either alone or in combination with the MEK inhibitor AZD6244. Materials and Method: The panel of isogenic RAS-dependent MEFs was provided by the NCI RAS Initiative. In vitro, RAS-GTP loading was evaluated and dose-response curves generated based on cell viability assays. For xenograft experiments, 5x106 cells were subcutaneously injected in NSG mice. After tumor establishment (200mm3) mice were treated either with vehicle, SHP099 75mg/kg, AZD6244 25 mg/Kg or a combination of the last two. Results and Discussion: In vitro, we found that when cultured in 3% FCS, the most GTPase-proficient KRAS mutants (G13D and G12C) are more sensitive to SHP099 as compared to the most impaired (G12D and Q61R). Response to SHP099 was accompanied by a reduction in RAS-GTP loading. Surprisingly, in vivo results using xenograft models showed no correlation between residual GTPase activity of the mutants and sensitivity to SHP099. Nevertheless, synergy between SHP2 and MEK inhibitors treatment was more pronounced in the GTPase-proficent mutants, suggesting a possible correlation between residual intrinsic GTP hydrolysis capacity of the mutants and their resistance to MEK inhibitors through feedback activation of receptor tyrosine kinases (Sun et al., Cell Rep 2014). Conclusion: Our data confirm single-agent activity of SHP099 against proliferation driven by a subset of KRAS mutants, and suggest that mutants with higher residual GTPase capacity could better benefit from combined SHP2 and MEK inhibition. Citation Format: Sara Mainardi, Astrid Bosma, Antonio Mulero-Sánchez, René Bernards. Differential response of distinct KRAS mutants to SHP2 inhibition [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 B53.

A Combinatorial Strategy for Targeting BRAF V600E-Mutant Cancers with BRAFV600E Inhibitor (PLX4720) and Tyrosine Kinase Inhibitor (Ponatinib).

Most aggressive thyroid cancers are commonly associated with a BRAF V600E mutation. Preclinical and clinical data in BRAF V600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF V600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAF V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAF V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35–45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.

Abstract C089: Adaptive responses of low-grade ovarian cancer cell lines to MEK inhibitor

Abstract Low-grade ovarian serous carcinomas (OSC) account for more than 70% epithelial ovarian cancers in young women. The exact biological pathways that underlie this disease are elusive. Moreover, chemotherapy and hormonal therapy are relatively ineffective and have generally failed to reduce the high morbidity and mortality associated with this disease. Because the Ras/Raf/MEK/ERK pathway is frequently activated in low-grade OSC, targeting this pathway has been examined as a potential avenue for treatment. Results from a phase II Gynecologic Oncology Group (GOG) trial for patients with recurrent low-grade OSC indicated a majority of the patients had developed resistance to Selumetinib (a MEK inhibitor). There were one complete responder, seven partial responders and 34 patients with stable disease out of the 52 patients treated with the MEK inhibitor. In order to understand the MEK inhibitor adaptive response, we have generated two MEK inhibitor (MEKi) adaptive resistant low-grade OSC cell lines. Using functional proteomic analysis by reverse phase protein array (RPPA), we identified several signaling pathways affected by MEKi (trametinib). Our data indicated pAKT was the most up-regulated protein by MEKi. In addition, other proteins in the mTOR, NF-kB, RTK and immune suppressive pathways were up-regulated. We further validated the result by western blot analysis, phospho-ERK, CHK1, MSH2, pRB and DUSP6 were down-regulated, while pAKT, B7-H4, BIM, and p27 were significantly up-regulated. More interestingly, SERPINE1 was the only highly expressed protein in both MEKi adaptive resistance low-grade OSC cell lines, and its expression was not affected by MEKi treatment. Knocking down the expression of SERPINE1 by siRNA targeting SERPINE1 in the MEKi-resistant low-grade OSC cell line was able to attenuate its resistance to trametinib. In summary, we have deciphered several adaptive responses of low-grade ovarian cancer cell lines to MEK inhibitor and the up-regulation of the SERPINE1 pathway is partly responsible for the development of adaptive resistance. Citation Format: Kwong-Kwok Wong, Yvonne T Tsang, Michelle Chen, Eucharist H Kun, David M Gershenson. Adaptive responses of low-grade ovarian cancer cell lines to MEK inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C089. doi:10.1158/1535-7163.TARG-19-C089

Distinct Transcriptional Programming Drive Response to MAPK Inhibition in BRAF V600-Mutant Melanoma Patient-Derived Xenografts.

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAF V600-mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo, in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF-high, "epithelial-like" transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven "mesenchymal-like" transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF-high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAF V600-mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo, which in turn predicts their response to further pathway suppression using a combination of BRAF, MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAF V600-mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer.

Abstract PL06-01: Discovery of BI-3406: A potent and selective SOS1::KRAS inhibitor opens a new approach for treating KRAS-driven tumors

Abstract KRAS is the most frequently mutated oncogene with high prevalence in pancreatic, colorectal, and non-small cell lung tumors. KRAS signaling is tightly regulated and various factors, including negative feedback pathways have limited the clinical efficacy of inhibitors of downstream MAPK signaling in the KRAS mutant context. Here we report the discovery of BI-3406 and demonstrate it is a highly potent and selective, orally bioavailable SOS1::KRAS inhibitor which binds to the catalytic domain of the guanine nucleotide exchange factor (GEF) SOS1 thereby preventing the interaction with KRAS-GDP. BI-3406 does not block the interaction of KRAS with SOS2 but elicits activity on a broad panel of KRAS oncogenic variants, including all major G12 and G13 oncoproteins. In KRAS-dependent cancers, BI-3406 potently reduces the formation of GTP-loaded KRAS, and inhibits MAPK pathway signaling. Down-modulation of this signaling cascade by BI-3406 in KRAS G12 or G13 mutant cells effectively limits cell proliferation. As a monotherapy, BI-3406 modulates signaling, as assessed by p-ERK and target genes, and displays marked anti-tumor efficacy in KRAS mutant xenografts. Due to BI-3406 blocking the negative feedback relief induced by MAPK inhibition, it has the potential to sensitize KRAS-dependent cancers to MEK inhibitor treatment. Combination with MEK inhibition leads to profound pathway blockade and tumor regressions in vivo. The combination of SOS1 and MEK inhibition is a potential therapy for the majority of KRAS-driven cancers including those fuelled by the most prevalent KRAS mutant oncoproteins. Furthermore, the pharmacological properties of BI-3406 and close analogues hold the promise of a significant treatment benefit in a broad patient population that is currently lacking precision medicine options. A Phase 1 clinical trial is in preparation for patients with advanced KRAS-mutated cancers to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and preliminary efficacy of BI 1701963, a SOS1::KRAS inhibitor closely related to BI-3406. Citation Format: Marco H Hofmann, Michael Gmachl, Jürgen Ramharter, Fabio Savarese, Daniel Gerlach, Joseph R Marszalek, Michael P Sanderson, Francesca Trapani, Dirk Kessler, Klaus Rumpel, Dana-Adriana Botesteanu, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Christiane Kofink, Tobias Wunberg, Szu-Chin Fu, Jessica Teh, Christopher P. Vellano, Jonathan C. O’Connell, Rachel L Mendes, Juergen Moll, Timothy P. Heffernan, Mark Pearson, Darryl B McConnell, Norbert Kraut. Discovery of BI-3406: A potent and selective SOS1::KRAS inhibitor opens a new approach for treating KRAS-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL06-01. doi:10.1158/1535-7163.TARG-19-PL06-01

Abstract C133: Effective targeting of KRAS-driven tumors by the first-in-class, orally bioavailable SOS1::KRAS inhibitor BI-3406

Abstract KRAS is the most frequently mutated oncogene with high prevalence in pancreatic, colorectal, and non-small cell lung cancers. KRAS signaling is tightly regulated and release of negative feedback pathways have limited the clinical efficacy of inhibitors of the downstream MAPK signaling in the KRAS mutant context. Here we report the discovery of BI-3406, a highly potent and selective, first-in-class, orally bioavailable SOS1::KRAS inhibitor that binds to the catalytic domain of the guanine exchange factor SOS1 thereby preventing the interaction with KRAS-GDP. In KRAS-dependent cancers, BI 3406 potently reduces the formation of GTP-loaded KRAS, and inhibits MAPK pathway signaling. Down-modulation of this signaling cascade by BI-3406 in KRAS G12 or G13 mutant cells results in a cytostatic effect. In monotherapy, the observed pathway modulation translates into marked anti-tumor efficacy in KRAS mutant xenografts. BI-3406 blocks the negative feedback induced by MAPK inhibition and consequently sensitizes KRAS-dependent cancers to MEK inhibitor treatment resulting in complete blockade of the pathway, which translates into tumor regression in vivo. Collectively, our data suggest that SOS1 inhibition is a promising therapeutic strategy to treat KRAS dependent cancers in combination with MEK inhibition, with the aim to deliver meaningful survival benefits for cancer patients. Citation Format: Marco H Hofmann, Michael Gmachl, Jürgen Ramharter, Fabio Savarese, Michael P Sanderson, Daniel Gerlach, Francesca Trapani, Dana-Adriana Botesteanu, Dirk Kessler, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Renate Schnitzer, Christiane Kofink, Heinz Stadtmueller, Tobias Wunberg, Jonathan C. O’Connell, Rachel L Mendes, David Richard, Adrian S Saldanha, Juergen Moll, Mark Pearson, Darryl B McConnell, Norbert Kraut. Effective targeting of KRAS-driven tumors by the first-in-class, orally bioavailable SOS1::KRAS inhibitor BI-3406 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C133. doi:10.1158/1535-7163.TARG-19-C133

Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment

BackgroundPatient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment.MethodsFour microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS.ResultsWe identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment.ConclusionsOnly 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Hyperactivity of Mek in TNS1 knockouts leads to potential treatments for cystic kidney diseases

Cystic kidney disease is the progressive development of multiple fluid-filled cysts that may severely compromise kidney functions and lead to renal failure. TNS1 (tensin-1) knockout mice develop cystic kidneys and die from renal failure. Here, we have established TNS1-knockout MDCK cells and applied 3D culture system to investigate the mechanism leading to cyst formation. Unlike wild-type MDCK cells, which form cysts with a single lumen, TNS1-knockout cysts contain multiple lumens and upregulated Mek/Erk activities. The multiple lumen phenotype and Mek/Erk hyperactivities are rescued by re-expression of wild-type TNS1 but not the TNS1 mutant lacking a fragment essential for its cell–cell junction localization. Furthermore, Mek inhibitor treatments restore the multiple lumens back to single lumen cysts. Mek/Erk hyperactivities are also detected in TNS1-knockout mouse kidneys. Treatment with the Mek inhibitor trametinib significantly reduces the levels of interstitial infiltrates, fibrosis and dilated tubules in TNS1-knockout kidneys. These studies establish a critical role of subcellular localization of TNS1 in suppressing Mek/Erk signaling and maintaining lumenogenesis, and provide potential therapeutic strategies by targeting the Mek/Erk pathway for cystic kidney diseases.

Identification of Resistance Pathways Specific to Malignancy Using Organoid Models of Pancreatic Cancer.

KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells. We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities. Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies. Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Cardiovascular Adverse Events Associated With BRAF and MEK Inhibitors

Cardiovascular adverse events (CVAEs) after treatment with BRAF and MEK inhibitors in patients with melanoma remain incompletely characterized. To determine the association of BRAF and MEK inhibitor treatment with CVAEs in patients with melanoma compared with BRAF inhibitor monotherapy. PubMed, Cochrane, and Web of Science were systematically searched for keywords vemurafenib, dabrafenib, encorafenib, trametinib, binimetinib, and cobinimetinib from database inception through November 30, 2018. Randomized clinical trials reporting on CVAEs in patients with melanoma being treated with BRAF and MEK inhibitors compared with patients with melanoma being treated with BRAF inhibitor monotherapy were selected. Data assessment followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. Pooled relative risks (RRs) and 95% CIs were determined using random-effects and fixed-effects analyses. Subgroup analyses were conducted to assess study-level characteristics associated with CVAEs. The selected end points were pulmonary embolism, a decrease in left ventricular ejection fraction, arterial hypertension, myocardial infarction, atrial fibrillation, and QTc interval prolongation. All-grade and high-grade (≥3) CVAEs were recorded. Overall, 5 randomized clinical trials including 2317 patients with melanoma were selected. Treatment with BRAF and MEK inhibitors was associated with an increased risk of pulmonary embolism (RR, 4.36; 95% CI, 1.23-15.44; P = .02), a decrease in left ventricular ejection fraction (RR, 3.72; 95% CI, 1.74-7.94; P < .001), and arterial hypertension (RR, 1.49; 95% CI, 1.12-1.97; P = .005) compared with BRAF inhibitor monotherapy. The RRs for myocardial infarction, atrial fibrillation, and QTc prolongation were similar between the groups. These results were consistent when assessing high-grade CVAEs (left ventricular ejection fraction: RR, 2.79; 95% CI, 1.36-5.73; P = .005; I2 = 29%; high-grade arterial hypertension: RR, 1.54; 95% CI, 1.14-2.08; P = .005; I2 = 0%), but RRs for high-grade pulmonary embolism were similar between groups. A higher risk of a decrease in left ventricular ejection fraction was associated with patients with a mean age younger than 55 years (RR, 26.50; 95% CI, 3.58-196.10; P = .001), and the associated risk of pulmonary embolism was higher for patients with a mean follow-up time longer than 15 months (RR, 7.70; 95% CI, 1.40-42.12; P = .02). Therapy with BRAF and MEK inhibitors was associated with a higher risk of CVAEs compared with BRAF inhibitor monotherapy. The findings may help to balance between beneficial melanoma treatment and cardiovascular morbidity and mortality.

Abstract 1511: MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC

Abstract Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes between tumor and stromal cells involved in developing chemo-resistance are under-explored. Here we report studies of paired TNBC patient-derived xenografts (PDX) established before and after chemo-resistance. Despite significant genetic similarities, the chemo-resistant PDX model harbored a rare constitutively-active KRASQ61R mutation which was not present in the chemo-naive PDX. Further analysis demonstrated that the chemo-resistant KRAS-mutant model exhibited altered gene expression changes including increased expression of CXCR2-ligands CXCL1 and CXCL2, which are responsible for recruiting immune cells to tumors. These expression patterns were largely inhibited in vivo by MEK inhibitor (MEKi) treatment. Moreover, in breast cancer cell lines, CXCL1, CXCL2, and granulocyte macrophage-colony stimulating factor (CSF2, stimulates granulocyte and macrophage differentiation from hematopoietic precursor cells, including immunosuppressive myeloid cells) transcripts were also downregulated by MEKi. Notably, chemo-resistant KRAS-mutant tumors harbored increased Gr1+ and Arginase-1+ cells, consistent with recruitment of immunosuppressive M2-like macrophages and/or myeloid-derived suppressor cells (MDSCs), which was inhibited by MEKi. Further experiments demonstrate that CD45+CD11b+Ly6G+ MDSC accumulation in tumors can be inhibited by MEKi treatment alone, or by CXCR2 inhibition, suggesting that the effects of MEK inhibition on MDSC recruitment are CXCL1/2-dependent. Confirming the translational relevance of these findings, in &amp;gt;200 murine and &amp;gt;1000 human breast tumors, Ras/MAPK transcriptional activity correlated with myeloid-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11), even in the absence of activating KRAS mutations. The association with Ras/MAPK activity was also confirmed using immunofluorescence to quantify MHC-II-low myeloid cells in 80 post-chemotherapy TNBC tumors. Importantly, MEKi and chemotherapy combination treatment reversed immunosuppressive cell accumulation and metabolic phenotypes exemplified by altered optical redox ratios (NAD(P)H/FAD) in the chemo-resistant KRAS mutant tumors, resulting in tumor growth suppression in mice. MEKi treatment also reduced redox ratios in 3D cultures of breast cancer cell lines further suggesting that MEK inhibition targets multiple oncogenic processes in breast cancer. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistant disease. Citation Format: Derek A. Franklin, Joe T. Sharick, Paula I. Ericsson-Gonzalez, Violeta Sanchez, Phillip T. Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko. MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC [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 1511.

Abstract 954: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors

Abstract Introduction: FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor (MEKi) such as trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in KRAS mutant cell lines after MEKi treatment. Experimental procedures: We profiled a large panel (n&amp;gt;32) of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We then performed in vitro and in vivo combination efficacy studies and pathway analysis in various KRAS mutant cancer models. Results: We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS in KRAS G12C or G12D models. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting MAPK pathway and KRAS mutant cancer cell proliferation in vitro and in vivo. Conclusions: Our findings suggest that MAPK inhibition in KRAS mutant cancer provokes feedback re-activation of the pathway that often involves RTK activity and SHP2 inhibition may enhance the efficacy of MEKi in KRAS mutant tumors. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huaixiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors [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 954.

Abstract 4753: Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition

Abstract Background: Studies have shown that MEK inhibitors (MEKi) can reduce tumorigenic growth signaling in colorectal cancer (CRC) through inhibition of the RAS pathway which is upregulated in over 40% of CRC tumors, while HDAC inhibitors (HDACi) lead to cell cycle arrest, inhibit angiogenesis, and induce apoptosis in neoplastic cells. In addition to a direct effect on neoplastic cells, MEKi have also demonstrated the potential to increase immune response to the tumor through upregulation of MHC I on tumor cells, induction of intra-tumoral T-cell infiltration, and enhancement of anti-PDL1 activity. HDAC inhibitors lead to epigenetic alterations which have similarly shown an ability to increase tumor immunogenicity, leading to the hypothesis that these agents may be combined with immunotherapy treatments for enhanced anti-tumor effect. In this study we first analyzed the effect of MEKi and HDACi combination treatment in both resistant and sensitive CRC cell lines to assess for synergy in the attenuation of cancer cell proliferation. We then demonstrated the effect of combination MEKi and HDACi therapy on tumor immunogenicity through increased expression of tumor immune markers. Methods: We assessed the efficacy of a novel MEKi agent, HL-085, in comparison with trametinib in six different CRC cell lines. Treatment with each agent was separately administered with a maximum concentration of 10 uM. Similarly, five different HDAC inhibitors were tested in each of these cell lines, with a maximum concentration of 1 uM. Optimal combination of MEK inhibitor HL085 and HDAC inhibitor class I/II (OKI-005) were then chosen based on this data. Cellular proliferation was assessed after 72 hours with Cell Titer Glo. Flow cytometry of a panel of immunogenic markers was then performed alongside a viability stain to assess for expression level changes of MCH class I in CRC cells. Results: Combinatorial effects of dual treatment with MEK inhibitor HL085 and HDAC inhibitor OKI-005 are observed in both resistant and sensitive cell lines with synergistic effect produced in the SW620 and Lovo cell lines (MEKi sensitive) as well as the GP2D (MEKi resistant) cell line. No synergistic effect was observed in the HCT15, DLD1 and LS513 cell lines. Flow cytometry showed an increase in MHC class I expression in neoplastic cells with combined MEKi and HDACi treatment compared to individual agent treatment and control, demonstrating increased immunogenicity of CRC cells with combination treatment. Conclusions: Combination treatment with a novel MEK inhibitor HL085 and the HDAC inhibitor OKI-005 demonstrates synergy in three out of six colorectal cancer cell lines. We further show increased immunogenicity of CRC cells through enhanced expression of MCH I on tumor cell surface, indicating rationale for further investigation of combination MEKi and HDACi with immunotherapy in the treatment of metastatic colorectal cancer. Citation Format: Mihailo Miljanic, Nisha Holay, Anna Capasso, Todd Triplett, Milad Soleimani, Uma Giri, Carla Van Den Berg, Gail Eckhardt. Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition [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 4753.

Abstract LB-119: SHP2 drives adaptive resistance to ERK signaling inhibition in molecularly defined subsets of ERK-dependent tumors

Abstract Deregulated RTK/RAS/RAF/MEK/ERK signaling (MAPK signaling) drives growth of over 40% of human tumors, most commonly due to activating mutations of pathway components such as in RAS and BRAF genes. Targeting MAPK signaling using RAF and MEK inhibitors elicited remarkable responses and prolonged survival of melanoma patients with BRAF(V600E) mutation, it however showed limited efficacy in colorectal and thyroid BRAF(V600E) cancers. Similarly, MEK inhibitors showed modest effect in other MAPK-dependent tumors, such as RAS-mutant tumors or in Triple-Negative Breast Cancers (TNBC). We and others have shown that relief of negative-feedback upon RAF or MEK inhibitor treatment promotes upregulation of various Receptor Tyrosine Kinases (RTKs), which in turn activate RAS, resulting in rebound of ERK activity in the presence of the inhibitor. Individual targeting of RTKs in combination with RAF or MEK inhibitors is challenging, due to variability in RTK expression in the same tumor as well as across different tumors. SHP2 (PTPN11) is a non-receptor tyrosine phosphatase and scaffold protein that mediates signal transduction downstream of multiple RTKs promoting RAS activation. The recent development of selective small-molecule SHP2 inhibitors provides the opportunity to overcome adaptive resistance by co-targeting MAPK signaling and feedback-induced RTK-mediated RAS activation. By using the allosteric SHP2 inhibitor SHP099, we found that combinatorial targeting of SHP2 and components of MAPK signaling was broadly effective in TNBC models and molecularly defined subsets of RAS and BRAF-mutant tumors. Notably, in each MAPK-driven tumor analyzed, p(Y542)SHP2 induction in response to MAPK signaling inhibition was required for combined treatment sensitivity. In RAS-mutant tumors, sensitivity to combined SHP2 and MEK inhibition was confined to RAS(G12X) tumors, in which mutant RAS depends on RTK-mediated nucleotide exchange for its activity. In tumors expressing RAS(G13D) or RAS(Q61X) the combination was largely ineffective. We further identified BRAF(V600E) colorectal and thyroid tumors in which feedback reactivation of ERK is driven by ERBB or MET receptors and is therefore mediated by SHP2 ("SHP2-positive" tumors). In such tumors, mostly colorectal, the combination of RAF and MEK inhibitors with SHP099 showed dramatic inhibition of MAPK signaling and tumor growth, both ex vivo and in vivo. Finally, we identified a subset of BRAF(V600E) tumors, mostly thyroid, that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Collectively, our findings identify molecular determinants and predictive biomarkers of response to combined SHP2/MAPK signaling inhibition, providing a roadmap for the clinical development of this combinatorial treatment strategy for MAPK-dependent tumors, such as TNBC, and large subsets of RAS-mutant and BRAF-mutant tumors, for which there are no currently available targeted therapeutic options. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Zoi Karoulia, Xuewei Wu, Ravi Sachidanandam, Stuart A. Aaronson, Poulikos I. Poulikakos. SHP2 drives adaptive resistance to ERK signaling inhibition in molecularly defined subsets of ERK-dependent tumors [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-119.

Abstract 702: Kinetic analysis identifies determinants of sensitivity to MEK inhibitor-induced cell death

Abstract Successful identification of the genetic determinants of drug sensitivity has been a long-standing goal of cancer research and, more recently, of precision medicine. In cancer cells, drug treatment can induce both growth arrest and death. The distinction between these outcomes is important but can be overlooked in large-scale analyses that rely on traditional metabolism-based methods for quantifying cell viability. We recently demonstrated that Scalable Time-Lapse Analysis of Cell Death Kinetics (STACK) effectively quantifies cell death in response to diverse lethal stimuli. We investigated cases in which this approach captured drug-induced lethality more effectively than traditional assays. We found that inhibitors of mitogen activated protein kinase 1 and 2 (MEK1/2) arrested proliferation in diverse cancer cell lines, but induced cell death in only a subset of these lines. We sought to identify the factors that determine whether a cancer cell will undergo growth arrest or death in response to MEK inhibition. MAP kinase pathway activation has previously been reported as a determinant of MEK inhibitor sensitivity; we determined that the dynamics of Bcl-2 family proteins in response to MEK inhibitor treatment correlate with death sensitivity independent of pathway activation. In a genome-wide CRISPR screen, we identified a lipid metabolic enzyme that is a novel modulator of MEKi sensitivity. Our results suggest new approaches for predicting and enhancing sensitivity to cancer therapeutics in tumors and demonstrate that direct measurement of cell death provides clear advantages over proliferation-based metrics. Citation Format: Zintis Inde, Kyuho Han, Michael C. Bassik, Scott J. Dixon. Kinetic analysis identifies determinants of sensitivity to MEK inhibitor-induced cell death [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 702.