Effects Of MEK Inhibitors Research Articles

Trametinib inhibits lymphatic vessel invasion of bone in a mouse model of Gorham-Stout disease.

Gorham-Stout disease (GSD) is a rare lymphatic anomaly that can be caused by somatic activating mutations in KRAS. This discovery has led investigators to suggest that MEK inhibitors could be a novel treatment for GSD. However, the effect of MEK inhibitors on bone disease in animal models of GSD has not been investigated. We recently reported that Osx-tTA;TetO-Vegfc mice exhibit a phenotype that resembles GSD. Osx-tTA;TetO-Vegfc mice overexpress VEGF-C in bone, which stimulates the development of lymphatic vessels in bone and the gradual loss of cortical bone. The objective of this study was to characterize the effect of trametinib, an FDA-approved MEK1/2 inhibitor, on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Immunoblotting was performed to assess the effect of trametinib on VEGF-C-induced phosphorylation of ERK1/2, AKT, and S6 in primary human lymphatic endothelial cells (LECs). Prevention and intervention experiments were performed to determine the effect of trametinib on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. We found that trametinib blocked VEGF-C-induced phosphorylation of ERK1/2 in primary human LECs. We also found that trametinib prevented VEGF-C-induced lymphatic invasion of bone and cortical bone loss in Osx-tTA;TetO-Vegfc mice. Additionally, trametinib slowed the progression of disease in Osx-tTA;TetO-Vegfc mice with established disease. However, it did not reverse disease in Osx-tTA;TetO-Vegfc mice. Our results show trametinib impacts bone disease in Osx-tTA;TetO-Vegfc mice. These findings further support the testing of MEK inhibitors in patients with GSD and other RAS pathway-driven complex lymphatic anomalies with bone involvement.

MEK Inhibition in CAR-T Cells Prevents Exhaustion and Terminal Differentiation Via Downregulating C-Fos and JunB

Introduction: The effects of MEK inhibitors (MEKIs) in CAR-T cells are poorly understood and remain controversial. Some groups showed that MEKIs could impair CAR-T cells' function in vitro. However, another group reported that MEKIs could combine with GD2 CAR-T cells to provide additional efficacy. Yet, the mechanism for this combined effect is unclear. Considering that the CAR signaling pathway is similar to TCR, both involve the activation of MAPK signaling. We hypothesized that MEKIs might mitigate CAR-T cells' exhaustion and terminal differentiation by lessening redundant CAR signaling. The study aims to systematically evaluate the role and mechanism of MEK inhibitors on CAR-T cells. Methods and Results: To explore whether MEKIs could mitigate the unbeneficial impact of antigen-independent CAR tonic signaling, we added 3 FDA-approved MEKIs: trametinib, cobimetinib, and binimetinib, respectively, to the culture medium of CD19.28z CAR-T cells for 9 days at the concentration approaching their clinically tolerable peak blood concentration. We found that all the 3 MEKIs could reduce CAR-T cells' terminal differentiation and restrain the expression of exhaustion and activation markers. Among them, trametinib was the most potent because it could achieve the comparable effect of cobimetinib and binimetinib at the lowest concentration. Thus, we chose trametinib for further research. We confirmed that compared to concentrations of 7.5nM and 30nM, 15nM was the optimal concentration of trametinib with mild inhibition on the proliferation of CAR-T cells and a potent effect on CAR-T cells' phenotype. Pre-treatment with trametinib didn't affect the in-vitro cytotoxicity of CD19.28z CAR-T cells. The above effects of trametinib were more significant as treatment time and the dose increased. Similar results could be obtained in analogous experiments with CD19.4-1BBz and GD2.28z CAR-T cells. Intriguingly, GD2.28z CAR-T cells cultured with trametinib had better proliferation and killing capacity because they were more exhausted than CD19.28z CAR-T cells. Using the Nalm-6-bearing leukemia xenograft model, we found that compared to DMSO pre-treated CD19.28z CAR-T cells, trametinib pre-treated CD19.28z CAR-T cells exerted more potent anti-leukemia activity, showed a less exhausted and differentiated state, proliferated better, and further extended mice survival (Fig.1). Similar results could be obtained in analogous in-vivo experiments with CD19.4-1BBz CAR-T cells. To evaluate whether trametinib could protect CAR-T cells from the exhaustion and terminal differentiation triggered by antigen stimulation, we cocultured CD19.28z CAR-T cells with Nalm-6 cells in a medium with or without trametinib. We demonstrated under antigen stimulation, trametinib could effectively inhibit CAR-T cells' activation, exhaustion, apoptosis, terminal differentiation, and phosphorylation of ERK and consequently promote the proliferation of total and CD8 CAR-T cells. In addition, after repetitive antigen stimulation, trametinib could rescue CAR-T cells' functional exhaustion and improve CAR-T cells' in-vitro cytotoxicity. Mechanistically, Single-cell and bulk RNA-Seq revealed that the effect of MEK inhibition was associated with the downregulation of AP-1 and exhaustion-associated transcription factors (TFs) and the upregulation of memory-associated TFs (Fig.2). GSEA revealed the upregulation of naive/memory-associated genes and downregulation of genes involved in T cell activation/effector/exhaustion, AP-1 pathway and apoptosis in the trametinib-treated group. Additionally, single-cell transcriptional profiling demonstrated enrichment of memory and Ki67+cycling CAR-T clusters in the trametinib-treated group. Among the AP-1 TFs downregulated by trametinib, c-Fos and JunB are the direct downstream targets of the canonical MAPK signaling pathway, and both of them were reported to implicate T cell exhaustion. Thus we speculated both downregulation of c-Fos and JunB may contribute to the role of MEKIs. Consistent with our hypothesis, overexpression of c-Fos or JunB in CAR-T cells could partially, if not all, abrogated the effects of MEK inhibition. Significance: Our research provides a strategy to optimize CAR-T antitumor efficacy by MEK inhibition and shed light on the role of c-Fos and JunB in driving CAR-T cell exhaustion and terminal differentiation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 4175: Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response

Abstract To enhance the therapeutic scope of MEK inhibitors (MEKis), we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. CH5126766 (CKI27) binds the allosteric site of MEK to inhibit its kinase activity but is novel due to its interaction with MEK S218 and 228, which blocks their phosphorylation by RAF. CKI27 bound MEK binds to RAF and cannot be released by phosphorylation, thus becoming a dominant negative inhibitor of RAF activation. This prevents the induction of MEK phosphorylation observed with other MEKis. Although this results in more potent tumor control, CKI27 is also capable of inhibiting T cell function because the MAPK/ERK pathway is activated downstream of T cell receptor signaling. We aim to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. We observed that CKI27 increased MHC expression on tumor cells and improved T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Intermittent administration of CKI27 allowed T cells to recover and partially relieved these inhibitory effects. Further combination with agonist antibodies anti-OX40 and GITR completely alleviated T cell inhibition and increased combination efficacy with immune checkpoint blockade antibody anti-CTLA-4. We also observed an increase in proliferation and T cell activation markers in LLC tumor bearing mice treated with the combination of CKI27, anti-GITR, and anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind this increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in future clinical trials treating patients with RAS mutant cancers. Citation Format: Lauren Dong, Hyejin Choi, Sadna Budhu, Isabell Schulze, Svena Verma, Nezar Mehanna, Neal Rosen, Taha Merghoub, Jedd Wolchok. Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4175.

Abstract LB050: Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is a disease that affects the elderly, characterized by the accumulation of mature B cells in the bloodstream. With an estimation incidence of 21,250 new cases diagnosed and 4,320 deaths for 2021, CLL is still considered a non-curable disease despite the wide therapeutic alternatives. CLL, like other malignant diseases, involves the activity of different cells in the microenvironment that support the leukemic cells by providing survival signals, even in presence of therapeutic agents. Among these cells, nurse-like cells (NLC) are of great importance. NLCs are M2-like macrophages known to be developed from blood monocytes, which need close contact with CLL cells for both their differentiation and their protective function. They provide multiple survival signals to CLL cells, and protect them against agents like the BTK inhibitor ibrutinib. Despite their relevance, little research has been done to elucidate the program that drives NLC differentiation. Here, we addressed this question by looking at the DNA methylation signatures before and after NLC differentiation. We found that NLC differentiation produced a marked DNA hypomethylation when compared to freshly isolated monocytes, characterized by the enrichment in AP-1 transcription binding sites. AP-1 is known to be regulated by MAP kinases, so we decided to chemically inhibit these pathways and found that specific MEK inhibition lead to reduced numbers of NLCs in vitro. Further studies showed that indeed NLCs have an activated MEK signaling as seen by the basal phosphorylation of ERK in vitro by confocal microscopy. We then used the adoptive transfer Eµ-TCL1 mice model to seek the effect of MEK inhibition in vivo. By treating the mice with trametinib, an FDA-approved MEK inhibitor, we found an increase in mouse survival when compared with the vehicle control. In addition, we observed a reduced number of monocyte/macrophage populations, especially in those expressing EGR2, a known M2 marker, suggesting that MEK inhibition causes a disruption in CLL-supportive macrophages both in vitro and in vivo. In conclusion, we observed that NLC differentiation strongly depends on the MEK signaling pathway, and that its inhibition leads to reduced myeloid supportive cells for CLL cells, followed by a reduced leukemic progression and increased CLL survival in vivo. Thus, we propose that MEK inhibition could be a potential therapeutic alternative for CLL. Further research is ongoing to determine how MEK signaling is activated during NLC differentiation. Citation Format: Giovanna Merchand Reyes, Ramasamy Santhanam, Frank H. Robledo Avila, Christoph Weigel, Juan D. Ruiz Rosado, Xiaokui Mo, Santiago Partida Sanchez, Jennifer A. Woyach, Christopher C. Oakes, Susheela Tridandapani, Jonathan P. Butchar. Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB050.

An in silico analysis identifies drugs potentially modulating the cytokine storm triggered by SARS-CoV-2 infection

The ongoing COVID-19 pandemic is one of the biggest health challenges of recent decades. Among the causes of mortality triggered by SARS-CoV-2 infection, the development of an inflammatory “cytokine storm” (CS) plays a determinant role. Here, we used transcriptomic data from the bronchoalveolar lavage fluid (BALF) of COVID-19 patients undergoing a CS to obtain gene-signatures associated to this pathology. Using these signatures, we interrogated the Connectivity Map (CMap) dataset that contains the effects of over 5000 small molecules on the transcriptome of human cell lines, and looked for molecules which effects on transcription mimic or oppose those of the CS. As expected, molecules that potentiate immune responses such as PKC activators are predicted to worsen the CS. In addition, we identified the negative regulation of female hormones among pathways potentially aggravating the CS, which helps to understand the gender-related differences in COVID-19 mortality. Regarding drugs potentially counteracting the CS, we identified glucocorticoids as a top hit, which validates our approach as this is the primary treatment for this pathology. Interestingly, our analysis also reveals a potential effect of MEK inhibitors in reverting the COVID-19 CS, which is supported by in vitro data that confirms the anti-inflammatory properties of these compounds.

NGMA-3. Use of multi-omics data to initialize and validate a causal model of glioblastoma stem cell signaling

Glioblastomas and glioblastoma stem cells are heterogeneous with respect to mutations, gene expression, and response to drugs. To make predictive responses of individual GBM stem cell lines to drugs, we have constructed a causal model of glioblastoma stem cell signaling. The core model was built starting from pathways identified from TCGA mutation data with the addition of the Jak/STAT, Hedgehog, and Notch pathways. Elements and relations between them were validated and extended using the PCNet interaction database and the INDRA database which includes machine read extractions from the biomedical literature. The result is a high confidence executable model consisting of 209 element and 370 rules of interaction between the elements. Stochastic simulations of the model provide dynamic (quantile) changes in time and responses to perturbations. The output provides activity of individual nodes as well as a cellular output state of cell cycle progression, apoptosis, or differentiation. To simulate the responses of individual cell lines to kinase inhibitors, the model was initialized using DNA sequencing data, RNA-seq, and reverse phase protein array (RPPA) data from each cell line. Comparing the results of the simulations to the drug responses of 11 different kinase targets, the model was 88% accurate in predicting effects on growth and survival. The model was further tested by comparing the effects of Mek inhibition of each of the cell lines in model to the results observed in the RPPA data which overlap by 127 elements. In this case, there was 62% concordance between the model and data when binned into quintiles. Discrepancies between the model predictions and the data are being investigated to determine whether the model logic or extent needs to be revised to improve the model. This modeling approach is a step toward developing algorithms for personalized therapeutics for GBM based on multi-omics data.

Abstract 1104: Repotrectinib increases effectiveness of MEK inhibitors in KRAS mutant cancer models

Abstract KRAS is the most frequently mutated oncogene in cancers, accounting for approximately 25% of non small cell lung cancer (NSCLC), 45% of colorectal cancer (CRC), and 75% of pancreatic cancer. KRAS G12D and G12V mutations account for a large percent of mutant KRAS cancers (36% of NSCLC; 57% of CRC; 71% of pancreatic). MEK1/2 are critical downstream effectors of KRAS signaling and preclinical studies in KRAS mutant models show sensitivity to MEK inhibitors (MEKi). However, clinical studies of single agent MEKi or combinations with docetaxel in mutant KRAS NSCLC patients were associated with low response rates. Preclinical studies show that effectiveness of MEK inhibition can be limited by multiple resistance mechanisms such as compensatory upregulation of PI3K/AKT via SRC/FAK signaling, or activation of the JAK2/STAT3 pathway. Repotrectinib is a next-generation ROS1/TRK inhibitor with SRC/FAK/JAK2 inhibitory potencies which may suppress adaptive resistance to MEK inhibitors. In the current study, repotrectinib combinations with KRAS signaling network inhibitors including MEK (trametinib, selumetinib), MEK/RAF (VS-6766), ERK (LY3214996), SHP2 (TNO155) were explored. Repotrectinib/trametinib and repotrectinib/VS-6766 combinations yielded significant effects on cancer cell viability in NSCLC and pancreatic patient-derived spheroid models harboring KRASG12D and KRASG12V mutations. The repotrectinib/trametinib combination in A427 cells (NSCLC KRASG12D) exhibited increased inhibition of pAKT and pS6 compared to single agent treatments. This coincided with greater upregulation of p27 and elevated PARP cleavage, resulting in enhanced induction of apopotosis. To assess acquired MEKi resistance, several KRAS mutant cancer models were cultured under trametinib selection. Evaluation of trametinib-resistant cancer cells revealed activation of STAT3, FAK, and AKT signaling along with elevated S6 protein phosphorylation, which could be suppressed and resensitized to trametinib by combining with repotrectinib. Repotrectinib/trametinib combination in a syngeneic GEMM KRASG12D lung model had greater tumor growth inhibition than either single agent treatment. A similar combination efficacy benefit was observed in a HCT116 (CRC KRASG13D) xenograft model concomitant with suppression of SRC/FAK/STAT3/ERK activation. Taken together, repotrectinib combinations with MEK inhibitors demonstrated enhanced efficacy in both in vitro and in vivo preclinical models. Repotrectinib was shown to suppress molecular mechanisms of adaptive resistance mechanisms to MEK inhibition in preclinical models. These results suggest that the combination of repotrectinib with MEKi can repress the mutant KRAS signaling network to achieve more potent and durable anti-tumor activity and warrants clinical investigation in patients with KRASG12D and KRASG12V mutant cancers. Citation Format: Nathan V. Lee, Wei Deng, Dayong Zhai, Laura Rodon, Ana Parra, Jessica Cowell, Afsheen Banisadr, Xin Zhang, Brion W. Murray. Repotrectinib increases effectiveness of MEK inhibitors in KRAS mutant cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1104.

Abstract B072: MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer

Abstract Background: Activating mutations in BRAF, a key mediator of RAS signaling, are present in ~50% of melanoma patients. Pharmacological inhibition of BRAF or the downstream MAP kinase MEK are highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Immunotherapeutics, especially those targeting checkpoint receptors on T cells, have revolutionized treatment of many cancer types. It has been proposed that MEK inhibitors(MEKi) may help generate a tumor microenvironment that enhances response to immunotherapy. The aim of this study was to investigate a novel cross-talk mechanism between MEKi and cytokine signaling response which could be used to enhance therapeutic efficacy against cancer types that are minimally responsive to MEKi. Methods: The association between MEK inhibition, NF-κB signaling and clinical response was analyzed using publicly available RNA-sequencing data from pre-treatment and on-treatment biopsies of patients (EGA S00001000992). To identify clinical and pre-clinical compounds that enhance cytokine response, we performed drug library screening for CCL5 and CXCL10 expression. 289 different agents (0.1 mM and 1 mM) were incubated with A549 cells in 96 well plates, with or without the presence of 0.2 ng/ml TNFα and 1 ng/ml IFNγ. Chemokine levels in supernatant were detected using Bead-Based Multiplex Assays. To investigate cytokine response modulatory mechanisms of MEKi, qPCR, Western Blot and flow cytometry were used to determine NF-κB activity and targeting gene expressions in lung cancer cells lines. The anti-tumor effect of MEKi in combination with TNFα and IFNγ was assessed using viable cell counting, cell cycle assay and apoptosis marker (cleaved caspase 3) expression in vitro. For in vivo studies, we performed combinatory therapies using MEKi plus intratumoral cytokine injection, MEKi plus PD-1 blockade in LKR subcutaneous tumor model. Results: We show that treatment of melanoma patients with BRAF and MEK inhibitors activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. MEKi increased cell surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Drug screening results demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the anti-tumor efficacy of MEKi while the administration of TNFα and IFNγ in MEKi-treated mice enhanced the anti-tumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. Conclusions:These findings define a novel cytokine response modulatory function of MEKi which can be therapeutically exploited. We show that lung cancer cells are rendered sensitive to MEKi by TNFα and IFNγ, providing strong mechanistic rationale for combining MEKi with immunotherapeutics, such as checkpoint blockers, in lung cancer. Citation Format: Mengyu Xie, Hong Zheng, Ranjna Madan-Lala, Wenjie Dai, Nicholas T. Gimbrone, Zhihua Chen, Fumi Kinose, Sarah A. Blackstone, Keiran S. M. Smalley, Douglas Cress, Eric B. Haura, Uwe Rix, Amer A. Beg. MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer [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 B072. doi:10.1158/1535-7163.TARG-19-B072

MEK inhibition suppresses B regulatory cells and augments anti-tumor immunity.

Mitogen-activated protein kinase (MAPK) kinase (MEK) is an integral component of the RAS pathway and a therapeutic target in RAS-driven cancers. Although tumor responses to MEK inhibition are rarely durable, MEK inhibitors have shown substantial activity and durable tumor regressions when combined with systemic immunotherapies in preclinical models of RAS-driven tumors. MEK inhibitors have been shown to potentiate anti-tumor T cell immunity, but little is known about the effects of MEK inhibition on other immune subsets, including B cells. We show here that treatment with a MEK inhibitor reduces B regulatory cells (Bregs) in vitro, and reduces the number of Bregs in tumor draining lymph nodes in a colorectal cancer model in vivo. MEK inhibition does not impede anti-tumor humoral immunity, and B cells contribute meaningfully to anti-tumor immunity in the context of MEK inhibitor therapy. Treatment with a MEK inhibitor is associated with improved T cell infiltration and an enhanced response to anti-PD1 immunotherapy. Together these data indicate that MEK inhibition may reduce Bregs while sparing anti-tumor B cell function, resulting in enhanced anti-tumor immunity.

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 >200 murine and >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 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 3473: Investigating RAS and RTK signaling in MPNST progression and drug resistance

Abstract Neurofibromatosis, type 1 (NF1) is a tumor predisposition syndrome affecting about 1 in 2,500 live births in the United States. The protein product of the NF1 gene, neurofibromin, loses its function as a negative regulator of the RAS protein, leading to the development of painful and disfiguring benign tumors known as a neurofibromas. Eight to thirteen percent of these neurofibromas will transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are aggressive soft tissue sarcomas that do not respond to the standard sarcoma treatment of chemotherapy and surgical resection. There is a critical need for efficacious targeted therapy to increase the average life span of these patients. Current targeted therapy efforts have shown modest efficacy clinically due to the establishment of compensatory feedback mechanisms that propagate tumor progression and recurrence. These feedback mechanisms are not completely understood in MPNSTs. In lung cancer, inhibition of MEK in the RAS pathway has been shown to activate upstream receptor tyrosine kinases (RTK). Genomic studies of MPNST samples have shown that 30% of tumors have amplification of the RTKs MET and EGFR. Furthermore, in a study of the genomic progression of a NF1 patient pre- and post-treatment, we found amplification of MET and EGFR that increased following treatment. In a study on triple-negative breast cancer cells, it has been shown that MET and EGFR crosstalk may be responsible for the reactivation of the inhibited cancer pathways and drug resistance. Therefore, we hypothesize that coordinated deregulated RAS signaling and MEK inhibition leads to MET and EGFR amplification, expression, activation, and crosstalk, which drives MPNST progression, drug resistance, and tumor recurrence. To test our hypothesis, we will use both in vitro and in vivo experimental methods to pursue the following specific aims: (1) determine the effect of MEK inhibition on establishing compensatory feedback loops that increase RTK activation and downstream signaling, (2) determine the role of MET-EGFR crosstalk signaling in MPNST tumor progression and resistance to kinase inhibition, and (3) determine the efficacy of combination RTK and MEK inhibition in vivo. Citation Format: Candace N. King, Carrie Graveel, Matthew Steensma. Investigating RAS and RTK signaling in MPNST progression and drug resistance [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 3473.

Exploring effects of MEK inhibition in tumor microenvironment in non-small cell lung cancer (NSCLC) pre-clinical models.

12060Background: NSCLCs often harbor mutations in the KRAS oncogene and require improved treatment to provide durable disease control. Targeted therapy via MEK inhibition shows promising but tempor...

Nano‐Targeted Kinase Inhibitor Treatment of KRAS Mutant Lung Cancer

Lung cancer is the leading cause of mortality in the US, affecting 160,000 patients annually with a five year survival of 17%. The majority of lung cancers harbor KRAS mutations that over‐activate the RAS kinase in the RAS‐MAPK signaling pathway, RAS‐RAF‐MEK‐ERK. This leads to increased pathway activity and increased cell division and tumor growth. Kinase inhibitors (KIs), which target key effectors of cancer signaling pathways, constitute a major strategy to treat KRAS‐driven lung cancer. Although RAS itself is difficult to drug with KIs due to the lack of binding pockets, KIs inhibiting downstream MEK kinases of the RAS‐MAPK pathway have shown to be most effective in vitro. However, MEK inhibitors suppress signaling in non‐cancer cells, so their dosing is limited by toxicity and results in a narrow therapeutic index. Additionally, the anti‐tumor effects of MEK inhibitors in KRAS‐driven lung cancer are further diminished by the activation of compensatory pathways, resulting in drug resistance and tumor resurgence. Although the co‐administration of a second KI can mitigate this resistance, many such combinations of KIs are highly toxic.We recently developed a new nanomedicine platform to package kinase inhibitors with high drug loading. These indocyanine nanoparticles (INPs) alter the systemic biodistribution of the KI to improve the drug's therapeutic index via tumor targeting. The INPs target caveolin receptors overexpressed on cancer endothelium to increase the local concentration of kinase inhibitor at the tumor site and reduce the systemic exposure of non‐cancerous tissues. Thus, the selective targeting decreases the toxicity of systemic KI exposure to concomitantly improve the therapeutic index.Support or Funding InformationThis work was supported in part by the NIH New Innovator Award (DP2‐HD075698), the Cancer Center Support Grant (P30 CA008748), the Pershing Square Sohn Cancer Research Alliance, the Honorable Tina Brozman Foundation for Ovarian Cancer Research, the Expect Miracles Foundation ‐ Financial Services Against Cancer, the Anna Fuller Fund, the Louis V. Gerstner Jr. Young Investigator's Fund, the Frank A. Howard Scholars Program, Cycle for Survival, the Alan and Sandra Gerry Metastasis Research Initiative, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center, the Imaging & Radiation Sciences Program, and the Center for Molecular Imaging and Nanotechnology of Memorial Sloan Kettering Cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Efficacy of MEK inhibition in a K-Ras-driven cholangiocarcinoma preclinical model

Intrahepatic cholangiocarcinoma (iCCA) is a deadly malignancy with limited treatment options. Gain-of-function mutations in K-Ras is a very frequent alteration, occurring in ~15 to 25% of human iCCA patients. Here, we established a new iCCA model by expressing activated forms of Notch1 (NICD) and K-Ras (K-RasV12D) in the mouse liver (K-Ras/NICD mice). Furthermore, we investigated the therapeutic potential of MEK inhibitors in vitro and in vivo using human CCA cell lines and K-Ras/NICD mice, respectively. Treatment with U0126, PD901, and Selumetinib MEK inhibitors triggered growth restraint in all CCA cell lines tested, with the most pronounced growth suppressive effects being observed in K-Ras mutant cells. Growth inhibition was due to reduction in proliferation and massive apoptosis. Furthermore, treatment of K-Ras/NICD tumor-bearing mice with PD901 resulted in stable disease. At the molecular level, PD901 efficiently inhibited ERK activation in K-Ras/NICD tumor cells, mainly leading to increased apoptosis. Altogether, our study demonstrates that K-Ras/NICD mice represent a novel and useful preclinical model to study K-Ras-driven iCCA development and the effectiveness of MEK inhibitors in counteracting this process. Our data support the usefulness of MEK inhibitors for the treatment of human iCCA.

Abstract IA16: Natural history of peripheral nerve sheath tumors in NF1: Identification and characterization of malignant precursor lesions

Abstract Neurofibromatosis type 1 (NF1) is a common (incidence 1:2,700) inherited disorder characterized by the development of peripheral nerve sheath tumors, including plexiform neurofibromas (PN) and malignant peripheral nerve sheath tumors (MPNST). While PN are histologically benign they can result in substantial morbidity including pain, disfigurement, and neurologic or functional deficits. Importantly, PN are at risk for transformation to MPNST with a lifetime incidence in NF1 of 15.8%. We are evaluating the natural history of NF1 PN in children enrolled on an NCI natural history study. Patients undergo detailed clinical evaluations and longitudinal evaluation with whole body MRI to assess total body tumor burden. Our studies confirm that PN grow most rapidly in young children and that substantial PN growth in patients ≥15 years old is infrequent and should raise concern for malignant transformation. We also identified the development of distinct nodular lesions (DNL) on MRI. These lesions appear after early childhood, frequently grow more rapidly than the surrounding PN, show a growth rate independent of age, and are avid for fluorodexyglucose on positron emission tomography. On biopsy, some of these lesions were identified as atypical neurofibromas (ANF), which were recently identified as precursor lesions to MPNST, and are characterized by CDKN2A/B loss. We have confirmed that patients with ANF are at greater risk for MPNST and are developing strategies for the evaluation and management of patients with ANF. Surgical resection of ANF, when safely feasible, may reduce the risk for MPNST development. We recently identified that MEK inhibition results in shrinkage of most PN and are expanding our efforts to evaluate the effect of MEK inhibition on DNL and ANF. Citation Format: Brigitte C. Widemann. Natural history of peripheral nerve sheath tumors in NF1: Identification and characterization of malignant precursor lesions [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA16.

Post-translational Regulation of Radioactive Iodine Therapy Response in Papillary Thyroid Carcinoma.

Radioactive iodine (RAI) is the mainstay of treatment for differentiated thyroid carcinoma (DTC). Nevertheless, the mechanism of RAI resistance that occurs in many patients with DTC remains unknown. We aimed to elucidate the role of post-translational regulation of radioiodine uptake. We analyzed the expression pattern of the ribosomal glycosylphosphatidylinositol transamidase (GPIT) complex in freshly excised tumors from 10 patients with DTC. We used functional RAI uptake assays to assess the role of GPIT in iodine uptake both in vivo and in vitro. The effects of MEK inhibition on the GPIT subunit PIGU and the sodium iodide symporter (NIS) were assessed in three DTC cell lines and in four human DTC biopsies. We used a multivariable logistic regression model to study the role of PIGU in the response to RAI treatment in advanced DTC. All statistical tests were two-sided. Expression profiling of different GPIT complex subunits revealed statistically significantly lower expression of PIGU in papillary carcinomas than in matched normal thyroid tissue (P < .001). Expression of PIGU in the K1 human papillary carcinoma cell line resulted in a robust increase in NIS glycosylation and trafficking to the cell membrane, accompanied by a robust increase in I125 uptake both in vitro (465 200 ± 56 343 vs 1236 ± 156 counts per million, P < .001) and in vivo (128 945 ± 28 556 vs 7963 ± 192 counts per million, P < .001, n = 5 mice per group). Treatment with the MEK inhibitors U0126 and PD302 rescued PIGU expression. Finally, the PIGU expression levels in tumors of 18 patients with recurrent DTC were associated with a biochemical response to RAI treatment (hazard ratio = 8.06, 95% confidence interval = 3.72 to 12.3, P = .001). We showed that downregulation of PIGU in DTC determines NIS function and RAI avidity. This represents a novel mechanism for RAI resistance.

1199P - Effect of MEK inhibition on PD-L1 and MCH-1 expression and on cytokines production profile in NSCLC cells and in human lymphocites

1199P - Effect of MEK inhibition on PD-L1 and MCH-1 expression and on cytokines production profile in NSCLC cells and in human lymphocites

Abstract 652: PD-L1 and MHC class 1 expression levels are modulated by MEK inhibition on head and neck squamous cell carcinoma

Abstract Background: To overcome immunosuppression, multiple pathways can be targeted by counter strategies, including elimination of suppressive signals (such as PD-L1 and CTLA-4) and promotion of antigen presentation from tumor cells, and of T cell infiltration into tumor sites. The purpose of this study is to investigate the change in expression of PD-L1 and MHC class I as well as of T cell chemoattractants, CXCL9 and CXCL10, in human head and neck squamous cell carcinoma (HNSCC) cell lines after treatment with MEK inhibitor. In addition, for evaluation of immunologic effects of MEK inhibitor in vivo, we employed SCC VII mouse squamous cell carcinoma (SCC) model. Methods: Six human HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, Detroit 562, FaDu, and HN31) and a mouse squamous cell carcinoma cell line (SCC VII) were used. Trametinib was purchased from Selleckchem. We conducted cell viability assay using these cell lines after 72 h incubation with MEK inhibitor trametinib. PD-L1 and MHC class I expression levels were analyzed by flow cytometry after treatment with trametinib and/or interferon-gamma (IFN-γ). Expression of PD-L1, p-Erk1/2, and p-STAT1/3 were analyzed by western blot. STAT3 was knocked down by siRNA transfection. To determine the levels of CXCL9 and CXCL10 transcripts after trametinib treatment, reverse-transcription PCR was carried out. Results: The growth inhibition by trametinib treatment was variable between cell lines with moderate sensitivity (10 nM &amp;lt; IC50 &amp;lt; 100 nM in five of seven cells). IC50 values were over 10 μM in Detroit 562 and FaDu. All the cell lines have no upstream RAS or RAF mutation. Although trametinib treatment was not shown to be enough for inhibiting growth of these cell lines, it up-regulated MHC class I expression in all cells either alone (&amp;gt; 2-fold) or when combined with IFN-γ (&amp;gt; 2-fold of IFN-γ alone). At the same time, we observed PD-L1 expression was elevated by trametinib treatment when combined with IFN-γ in all cells except SNU-1066, in which trametinib treatment down-regulate IFN-γ-induced increase of PD-L1 expression. Furthermore, we observed that trametinib alone and/or with IFN-γ enhanced mRNA expression of CXCL9 and CXCL10 in all human cells. Mechanistically, trametinib treatment enhanced STAT1 phosphorylation by IFN-γ in five of six human cells, and directly phosphorylated STAT3. When we silenced STAT3, enhanced expression of MHC class I by trametinib treatment was not observed in SNU-1041 cell. Conclusion: Our results suggest that MEK pathway is closely related with modulating the expression of PD-L1 and MHC class I as well as of CXCL9 and CXCL10 in HNSCC cell lines. Up-regulated levels of MHC class I and T cell-recruiting chemokines imply that MEK inhibitor may mediate potentiating T cell responses. Moreover, simultaneous increase of PD-L1 expression suggests the possible synergistic role of MEK inhibitor when combined with anti-PD-1/PD-L1 immunotherapy in HNSCC. Citation Format: Seong-Ho Kang, Bhumsuk Keam, Soyeon Kim, Miso Kim, Yong-Oon Ahn, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. PD-L1 and MHC class 1 expression levels are modulated by MEK inhibition on head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2017-652

Abstract 2086: Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers

Abstract BRAF mutations are found in ~8% of all cancers. In addition to the commonest mutation in kinase domain at the V600, a wide range of other missense mutations (non-V600) have been reported. Non-V600 mutant BRAF protein was also shown to enhance MAPK signaling, suggesting the effectiveness of MEK inhibitors. However, the existence of feedback mechanism in MAPK singling and the roles of receptor tyrosine kinases (RTKs) in these cells are not fully understood. To interrogate this, BRAF non-V600 cell lines with intermediate/impaired kinase activity were treated with MEK inhibitor. RTKs involved in the upregulation of P-MEK following MEK inhibition in BRAF non-V600E mutant cell lines were determined by phosphor-RTK arrays. The effect of combinatorial inhibition of RTKs and MEK in BRAF non-V600 cancer was assessed in vitro and in vivo. By analyzing drug screen data, we first found cells with BRAF non-V600E mutation were less sensitive to MEK inhibition compared with BRAF V600E mutant cell lines and almost comparable to RAS/RAF wild-type cell lines. MEK inhibition did not induce P-MEK suppression and it resulted in significant upregulation of p-MEK compared to BRAF V600E mutant cell lines. Phospho-RTK arrays in the presence or absence of trametinib displayed basal phosphorylation of RTKs including EGFR, MET, and IGF1R, although the phosphorylation level was not consistently changed following MEK inhibition. Among these RTKs, treatment with the EGFR inhibitor erlotinib led to more complete suppression of P-ERK upon trametinib treatment. Furthermore, we found that erlotinib monotherapy downregulated P-ERK in BRAF non-V600 mutant cell lines in contrast that inhibition of RTKs had no effect on MAPK signaling in BRAF V600 mutant cells. While erlotinib treatment led to MAPK inhibition both in intermediate active and impaired kinase active BRAF mutant cells, p-MEK and p-ERK were more strongly suppressed in impaired type BRAF non-V600 mutant cells. The combination of EGFR inhibitor and MEK inhibitor suppressed cell growth and achieved tumor shrinkage in mice xenograft. These results suggest that RTKs, especially EGFR, are dominantly involved in the regulation of MAPK signaling in BRAF non-V600 cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Hiromichi Ebi, Hiroshi Kotani, Hidenori Kitai, Yuta Adachi, Seiji Yano. Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2086. doi:10.1158/1538-7445.AM2017-2086