C5aR-antagonism alone or in combination with MEK inhibition durably alters the plexiform neurofibroma tumor micro-environment
Niousha AhmariMelissa R. PerrinoJay PundavelaSara SzaboTrent M. WoodruffEva DombiMi‐Ok KimJörg KöhlJianqiang WuNancy Ratner
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Abstract ID 98867 Poster Board 249 Mutations in the NF1 tumor suppressor gene, which encodes a negative regulator of RAS signaling cascades, predispose affected individuals to manifestations including development of benign nerve tumors (neurofibromas). The formation and growth of plexiform neurofibroma (PNF) tumors depend on the interplay between tumor cells and the surrounding tumor microenvironment (TME), which is marked by chronic inflammation, myeloid cell expansion, and remodeling of local and systemic immune compartments. Although inhibiting mitogen-activated protein kinase (MEK) signaling downstream of RAS shrinks most established plexiform neurofibromas (PNF), not all patients respond to MEK inhibition and even in those that do the response is not durable. Our aim in this study was to identify and test a druggable target to synergize with MEK inhibitor treatment to promote efficacy and durability of treatment. Using flow cytometry and single cell anlaysis of two murine models and histological examination of human tumors, we discovered an increase in the C5a-C5a receptor (C5aR) system in PNF as characterized by an increase in C5aR1 expressing macrophages, the predominant cell in PNF tumors, which is not normalized by MEK inhibition. Thus, to test the hypothesis that C5aR can synergize with MEK inhibition to modulate inflammatory responses in PNF we treated immunocompetent PNF-bearing Nf1f/f; DhhCre mice, a model which has historically demonstrated robust translational importance, for 60 days with either a MEK inhibitor, a C5aR1 inhibitor, or a combination of the two. We found that therapeutic reduction of C5aR1 activity induced cell death in tumor macrophages and enhanced the engulfment of dying Schwann cells by macrophages but did not affect neurofibroma number or size. Complete or partial genetic depletion of C5aR1 in Nf1f/f; DhhCre mice confirmed these results, suggesting stronger therapy would not increase efficacy of response.We next tested if the combination of MEK and C5aR1 inhibition would improve durability of response. To test this idea, we treated mice for 1 month, and then maintained mice off therapy for one month. Tumors regrew in all groups, but only mice treated with a combination of MEK and C5aR1/2 inhibitors showed altered tissue cellular architecture, expansion of dendritic cells, and increased macrophage MHCII expression. We conclude that C5aRA in combination with MEK inhibition is tolerable and causes durable immunosuppressive effects on the neurofibroma microenvironment. Funding: NIH R33 NS112407 to JQ and NR, and DOD W81XWH-19-1-0816 to NRCite
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3071 Background: MEK inhibitors can be used to treat patients with mutations that affect the MAPK pathway. Several MEK inhibitors are currently FDA-approved and effectively treat BRAF-mutated tumors, but RAS-mutated cancers are considered more resistant. However, it is unclear how the many distinct RAS variants impact the MAPK pathway and are affected by MEK inhibitors. We hypothesized that the level of MAPK pathway activation induced by different RAS mutations may predict response to MEK inhibition. Methods: Thirteen RAS mutations from 34 patients treated with MEK inhibitors at UCSD were synthesized, expressed in a HeLa-derived cell line and analyzed in vitro using a functional mutational analysis assay based on assessing downstream reporters in order to measure the activity of these mutations on the MAPK pathway. Each mutation received an activity score based on known oncogenic RAS mutation. Results: The most common type of cancer was colorectal cancer (N = 13). All patients received the MEK inhibitor, trametinib, based therapy. Patients were stratified into two groups: above an activity score of 1 (14 pts) or below it (20 pts). Median progression-free survival (PFS) after MEK inhibitor treatment correlated with higher MAPK activity score (9 vs 3 months; P = 0.041). Conclusions: Using a novel functional assay methodology for characterization of MAPK activation, we show that various RAS mutations activate the MAPK pathway to different levels. Higher activity is associated with longer PFS after MEK inhibitor treatment, suggesting that the relationship between signal transduction strength and clinical relevance merits additional exploration.
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Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.
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Abstract Inhibitors of the BRAF/MEK/ERK pathway have promise in the treatment of BRAF mutant melanoma and other MAPK activated cancers. Treatment of metastatic melanoma patients with the combination of RAF + MEK inhibitor has extended progression free survival over single agent therapy. However, in a large subset of patients, resistance invariably develops. Most clinical models show reactivation of the MAPK pathway in resistant samples, suggesting further downregulation of MAPK signaling might be beneficial. Therefore, we took BRAF mutant melanoma grown to resistance by continuous culture in dabrafenib plus trametinib (RAF + MEK inhibitor) or SCH772984 (ERK inhibitor), and treated them with a triple combination of RAF+MEK+ERK inhibitors. We found cells resistant to RAF+MEK inhibitors were cross-resistant to ERK inhibitor, but sensitive to RAF+MEK+ERK inhibitors. Conversely, cells resistant to the ERK inhibitor were cross-resistant to RAF+MEK inhibitor, but sensitive to the triple combination. However, given the possible deleterious side effects of a triple combination in patients, we explored whether alternative dosing and timing strategies could delay development of resistance. We compared chronic dosing of RAF+MEK or ERK inhibitors to weekly exposures alternating between RAF+MEK and ERK inhibitors. This alternative dosing strategy delayed development of resistance in vitro over chronic dosing, while also resulting in a slower proliferation rate in resistant cells. In addition, cells exposed to a higher dose of inhibitor for a shorter period of time was equivalent to chronic exposure. These studies suggest that alternative dosing strategies (other than chronic exposure) may prevent development of resistance, and allow for treatment of patients with higher order combinations and less side effects. Citation Format: Eva Goetz, Bokang Rabasha, Levi Garraway. Alternative treatment strategies to overcome MAPK inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2150.
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The excessive activation of extracellular signal-regulated kinase(ERK) signaling pathway,which is a significant feature of rheumatoid arthritic(RA) arthropathy,plays an important role in the process of synoviocyte dysfunction and destruction of cartilage and bone.Understanding the pathomechanism of ERK signaling in RA provides a new target for developing new drug and therapeutic strategy.This review summarizes the current knowledge of the activation,regulation and function of ERK pathway,and also analyzes the role of this signaling transduction in the destruction of joints and the pathogenesis of RA.
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At present,most studies suggest that activation of ERK signaling pathway can promote cell proliferation and JNK signaling pathway are closely related with apoptosis.They are also involved in the pathophysiology change process of many diseases.JNK signal transduction pathway is closely related with neurodegenerative disease or other neurological diseases.ERK signal transduction pathway may be the main mechanism of tumorgenesis and invasiveness,which is also involved in the generation of pains.ERK intracellular localization may be closely related with neuro denaturation.
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Extracellular signal-regulated kinase (ERK) signaling pathway is one of the mitogen-activated protein kinase (MAPK) signal transduction pathways. This pathway is known as pivotal in many signaling networks that govern proliferation, differentiation and cell survival. The ERK signaling pathway comprises positive and negative feedback loops, depending on whether the terminal kinase stimulates or inhibits the activation of the initial level. In this paper, we attempt to model the ERK pathway by considering both of the positive and negative feedback mechanisms based on Michaelis-Menten kinetics. In addition, we propose a fraction ratio model based on the mass action law. We first develop a mathematical model of the ERK pathway with fraction ratios. Secondly, we analyze the dynamical properties of the fraction ratio model based on simulation studies. Furthermore, we propose a concept of an inhibitor, catalyst, and substrate (ICS) controller which regulates the inhibitor, catalyst, and substrate concentrations of the ERK signal transduction pathway. The ICS controller can be designed through dynamical analysis of the ERK signaling transduction pathway within limited concentration ranges.
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Acquired resistance to BRAF inhibitors often involves MAPK re‐activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF‐inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK‐dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.
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MEK inhibitor has been highlighted as a promising anti-tumor drug but its effect has been reported as varying over a wide range depending on patho-physiological conditions. In this study, we employed a systems approach by combining biochemical experimentation with in silico simulations to investigate the resistance mechanism and functional consequences of MEK inhibitor. To this end, we have developed an extended integrative model of ERK and PI3K signaling pathways by considering the crosstalk between Ras and PI3K, and analyzed the resistance mechanism to the MEK inhibitor under various mutational conditions. We found that the phospho-Akt level under the Raf mutation was remarkably augmented by MEK inhibitor, while the phospho-ERK level was almost completely repressed. These results suggest that bypassing of the ERK signal to the PI3K signal causes the resistance to the MEK inhibitor in a complex oncogenic signaling network. We further investigated the underlying mechanism of the drug resistance and revealed that the MEK inhibitor disrupts the negative feedback loops from ERK to SOS and GAB1, but activates the positive feedback loop composed of GAB1, Ras, and PI3K, which induces the bypass of the ERK signal to the PI3K signal. Based on these core feedback circuits, we suggested promising candidates for combination therapy and examined the improved inhibitory effects.
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Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.
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