Small Molecule Inhibitors Of MEK Research Articles

Influenza A virus replication has a stronger dependency on Raf/MEK/ERK signaling pathway activity than SARS-CoV-2.

The recent COVID-19 pandemic again highlighted the urgent need for broad-spectrum antivirals, both for therapeutic use in acute viral infection and for pandemic preparedness in general. The targeting of host cell factors hijacked by viruses during their replication cycle presents one possible strategy for development of broad-spectrum antivirals. By inhibiting the Raf/MEK/ERK signaling pathway, a central kinase cascade of eukaryotic cells, which is being exploited by numerous viruses of different virus phyla, the small-molecule MEK inhibitor zapnometinib has the potential to address this need. We here performed a side-by-side comparison of the antiviral efficacy of zapnometinib against IAV and SARS-CoV-2 to determine the concentration leading to 50% of its effect on the virus (EC50) and the concentration leading to 50% reduction of ERK phosphorylation (IC50) in a comparable manner, using the same experimental conditions. Our results show that the EC50 value and IC50 value of zapnometinib are indeed lower for IAV compared to SARS-CoV-2 using one representative strain for each. The results suggest that IAV's replication has a stronger dependency on an active Raf/MEK/ERK pathway and, thus, that IAV is more susceptible to treatment with zapnometinib than SARS-CoV-2. With zapnometinib's favorable outcome in a recent phase II clinical trial in hospitalized COVID-19 patients, the present results are even more promising for an upcoming phase II clinical trial in severe influenza virus infection.

Phase I pharmacokinetic study of an oral, small-molecule MEK inhibitor tunlametinib in patients with advanced NRAS mutant melanoma.

Background: Malignant melanoma is an aggressive disease. Tunlametinib (HL-085) is a potent, selective, and orally bioavailable MEK1/2 inhibitor. The objective of this study was to determine the pharmacokinetics (PK) of tunlametinib and its main metabolite M8 in patients with NRAS-mutant melanoma following a single dose and multiple doses in a phase I safety and PK study. Methods: A multiple-center phase I study was performed in patients with melanoma including dose-escalation phase and dose-expansion phase. PK following a single oral dose and multiple doses of 0.5-18mg twice daily was assessed. Results: A total of 30 participants were included in the dose escalation phase and then 11 patients were included in the dose-expansion phase (12mg twice daily). Tunlametinib plasma concentration rapidly increased after dosing, with a Tmax of 0.5-1h. Mean elimination half-life (t1/2) was dose-independent and had a range from 21.84 to 34.41h. Mean apparent clearance (CL/F) and distribution volume (V/F) were 28.44-51.93L/h and 1199.36-2009.26L, respectively. The average accumulation ratios of AUC and Cmax after the multiple administration of tunlametinib were 1.64-2.73 and 0.82-2.49, respectively. Tunlametinib was rapidly transformed into the main metabolite M8 and M8 reached the peak concentration about 1h after administration. Mean t1/2 of M8 was 6.1-33.54h. The body exposure of M8 in plasma was 36%-67% of that of tunlametinib. There were general dose-proportional increases in maximum concentration (Cmax) and area under the curve (AUC) of tunlametinib and M8 both in the single dose phase and in the multiple doses phase. Conclusion: Tunlametinib was absorbed rapidly and eliminated at a medium speed after drug withdrawal. Pharmacokinetic body exposure increased in general dose-proportional manner from 0.5mg up to 18mg. Slight accumulation was found after multiple oral doses. The pharmacokinetics of tunlametinib and its metabolite suggest that twice daily dosing is appropriate for tunlametinib.

PD-L1 induction via the MEK-JNK-AP1 axis by a neddylation inhibitor promotes cancer-associated immunosuppression

MLN4924 is a first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE), which is currently in several clinical trials for anti-cancer applications. However, MLN4924 also showed some off-target effects with potential to promote the growth of cancer cells which counteracts its anticancer activity. In this study, we found that MLN4924 increases the levels of PD-L1 mRNA and protein in dose- and time-dependent manners. Mechanistic study showed that this MLN4924 effect is largely independent of neddylation inactivation, but is due to activation of both ERK and JNK signals, leading to AP-1 activation, which is blocked by the small molecule inhibitors of MEK and JNK, respectively. Biologically, MLN4924 attenuates T cell killing in a co-culture model due to PD-L1 upregulation, which can be, at least in part, abrogated by either MEK inhibitor or anti-PD-L1 antibody. In an in vivo BALB/c mouse xenograft tumor model, while MLN4924 alone had no effect, combination with either MEK inhibitor or anti-PD-L1 antibody enhanced the suppression of tumor growth. Taken together, our study provides a sound rationale for effective anticancer therapy in combination of anti-PD-L1 antibody or MEK inhibitor with MLN4924 to overcome the side-effect of immunosuppression by MLN4924 via PD-L1 induction.

Direct Targeting of the Raf-MEK-ERK Signaling Cascade Inhibits Neuroblastoma Growth.

The Raf-MEK-ERK signaling network has been the subject of intense research due to its role in the development of human cancers, including pediatric neuroblastoma (NB). MEK and ERK are the central components of this signaling pathway and are attractive targets for cancer therapy. Approximately 3–5% of the primary NB samples and about 80% of relapsed samples contain mutations in the Raf-MEK-ERK pathway. In the present study, we analyzed the NB patient datasets and revealed that high RAF and MEK expression leads to poor overall survival and directly correlates with cancer progression and relapse. Further, we repurposed a specific small-molecule MEK inhibitor CI-1040 to inhibit the Raf-MEK-ERK pathway in NB. Our results show that CI-1040 potently inhibits NB cell proliferation and clonogenic growth in a dose-dependent manner. Inhibition of the Raf-MEK-ERK pathway by CI-1040 significantly enhances apoptosis, blocks cell cycle progression at the S phase, inhibits expression of the cell cycle-related genes, and significantly inhibits phosphorylation and activation of the ERK1/2 protein. Furthermore, CI-1040 significantly inhibits tumor growth in different NB 3D spheroidal tumor models in a dose-dependent manner and by directly inhibiting spheroidal tumor cells. Overall, our findings highlight that direct inhibition of the Raf-MEK-ERK pathway is a novel therapeutic approach for NB, and further developing repurposing strategies using CI-1040 is a clinically tractable strategy for effectively treating NB.

Multiplatform molecular analyses refine classification of gliomas arising in patients with neurofibromatosis type 1

Gliomas arising in the setting of neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade or high-grade, and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 inactivation and epigenetic classification of these tumors remain limited. Through next-generation sequencing, copy number analysis, and DNA methylation profiling of gliomas from 47 NF1 patients, we identified 2 molecular subgroups of NF1-associated gliomas. The first harbored biallelic NF1 inactivation only, occurred primarily during childhood, followed a more indolent clinical course, and had a unique epigenetic signature for which we propose the terminology “pilocytic astrocytoma, arising in the setting of NF1”. The second subgroup harbored additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, occurred primarily during adulthood, followed a more aggressive clinical course, and was epigenetically diverse, with most tumors aligning with either high-grade astrocytoma with piloid features or various subclasses of IDH-wildtype glioblastoma. Several patients were treated with small molecule MEK inhibitors that resulted in stable disease or tumor regression when used as a single agent, but only in the context of those tumors with NF1 inactivation lacking additional oncogenic alterations. Together, these findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

Abstract P048: A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) RAMP 202, as a single agent and in combination with defactinib (FAK inhibitor) in recurrent KRAS-mutant (KRAS-MT) non-small cell lung cancer (NSCLC)

Abstract Background: VS-6766 is a unique small molecule inhibitor of both RAF and MEK. In contrast to several other MEK inhibitors available, VS-6766 blocks both MEK kinase activity and RAF phosphorylation of MEK. This sequential blockade mechanism enables VS-6766 to block MEK signaling more consistently without the compensatory and paradoxical activation of MEK that reduces the efficacy of other small molecule inhibitors of MEK and RAF. Defactinib (VS-6063), an orally active small molecule, is a potent adenosine 5'- triphosphate (ATP) competitive, reversible inhibitor of focal adhesion kinase (FAK). Defactinib has shown synergistic activity with BRAF and MEK inhibitors in both in vitro and in vivo preclinical solid tumor models. Specifically, in several human tumor cell lines with mutations in RAS or BRAF or wildtype RAS and BRAF, defactinib has shown synergy with MEK inhibitors or VS-6766. In mouse models of KRAS mutant ovarian cancer, BRAF-mutant melanoma or uveal melanoma, FAK inhibition has been shown to induce tumor regression when combined with RAF, MEK or RAF/MEK inhibitors, while the single agents have only induced tumor stasis. The combination of VS-6766 and defactinib is currently being evaluated in the Investigator Sponsored FRAME study. Preliminary efficacy results are available for a small number of subjects with KRAS mutated NSCLC treated with a combination of VS-6766 and defactinib. Of the 10 subjects with NSCLC, 1 subject with KRAS-G12V- mutated cancer achieved PR while 3/10 subjects received treatment for ≥24 weeks. In an updated analysis of response in 15 subjects with KRAS-mutant NSCLC, there were 2 PRs and 7 SDs (ORR: 13%). The two subjects with KRAS G12V- mutated NSCLC both achieved PR (ORR: 100%). Methods: This is an adaptive, two-part, Phase 2, multicenter, parallel cohort, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 versus VS-6766 in combination with defactinib in subjects with recurrent NSCLC(NCT04620330). The study will be conducted in two parts. Part A will determine the optimal regimen, either VS-6766 monotherapy or VS-6766 in combination with defactinib based on confirmed overall response rate as assessed by independent radiology review. Part A will consist of three arms in KRAS mutated NSCLC Arm 1 monotherapy of VS-6766, Arm 2 the combination of VS-6766 and defactinib in KRAS G12V mutated and Arm 3 the combination in other KRAS mutated. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS mutated NSCLC. Subjects enrolled must have histologic or cytologic evidence of NSCLC, measurable disease according to RECIST V1.1 and known KRAS mutation. The study will enroll up to 377 subjects globally with 57 subjects (32 KRAS G12V & 25 KRAS other) in Part A and an additional 144 KRAS G12V and 176 KRAS other in Part B. This study is open for enrollment. Citation Format: D. Ross Camidge, Jonathan Pachter, Andrew Koustenis, Gloria Patrick, David R. Spigel. A phase 2 study of VS-6766 (dual RAF/MEK inhibitor) RAMP 202, as a single agent and in combination with defactinib (FAK inhibitor) in recurrent KRAS-mutant (KRAS-MT) non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P048.

Abstract 1346: Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma

Abstract Neuroblastoma is a highly heterogenous pediatric tumor that develops during the early embryonic stages, and accounts for almost 15% of pediatric cancer related deaths. Despite advanced and intensive therapeutic approaches that combine surgery, radiation, and chemotherapy, high-risk neuroblastoma (NB) patients' long-term survival is still less than 50%. Tumor metastasis, relapse, drug-resistance, and treatment related toxicities mandate the development of novel therapeutic approaches to treat NB patients. MAPK/ERK pathway plays an important oncogenic role in different types of cancers, including NB. Activation of the MAPK/ERK pathway is known to induce cancer cell proliferation, progression, metabolism, and resistance to drug-induced apoptosis. In the present study, we investigated the effect of a small-molecule MEK inhibitor that targets MEK1 and MEK2 to block MAPK/ERK pathway in NB. Cytotoxicity assays using Cell-Titer One solution in different MYCN-amplified (NGP, LAN-5, CHLA-255-MYCN) and MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) NB cell lines show that MEK inhibitor significantly reduce the NB cell proliferation in a dose-dependent manner. We further performed clonogenic assays and 3D spheroidal assays to mimic the NB tumor growth, and our results show that MEK inhibitor significantly inhibit NB colony formation, inhibit 3D spheroidal tumor size by inducing cell death in 3D spheroidal tumors, in comparison to controls. Additionally, MEK inhibitor in a dose-dependent manner induces apoptosis and blocks cell cycle progression in NB cells in comparison to control, as determined by Annexin V apoptosis assays and Click-iT EdU cell proliferation assay respectively. Furthermore, gene expression analysis show that MEK inhibitor significantly reduce the mRNA expression of specific MAPK/ERK pathway targets such as MEK1, MEK2, and ERK2. Western blot assays further confirm the efficacy of MEK inhibitor in blocking MAPK/ERK pathway by significantly inhibiting the phosphorylation of key pathway proteins. Taken together, our results highlight that: a) MAPK/ERK pathway is implicated in NB growth, and b) direct targeting of MAPK/ERK signaling pathway by using a novel small molecule MEK inhibitor inhibit NB proliferation and 3D tumor growth. We will further combine this MEK inhibitor with current chemotherapy drugs to develop effective therapeutic approaches for NB patients. Citation Format: Rameswari Chilamakuri, Bharti Sharma, Danielle C. Rouse, Saurabh Agarwal. Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma [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 1346.

Uveitis in Tumor Patients Treated with Immunological Checkpoint- and Signal Transduction Pathway-Inhibitors

ABSTRACT Purpose New tumor therapies like immune checkpoint inhibitors and small molecule inhibitors of MEK and BRAF have increased the patient’s survival rate but can be burdened with severe side-effects including uveitis. Here, we show the spectrum, treatment, and outcome of uveitis types induced by tumor treatment. Methods In this retrospective study, we have included 54 patients from different centers who were developing uveitis under tumor therapy. A 16-item questionnaire was analyzed for type, treatment, and outcome of uveitis and type of tumor treatment, which we have correlated here. Results Irrespective of the tumor treatment, most patients developed anterior uveitis. All patients received corticosteroids and some additional immunosuppressive treatments. Cessation of tumor therapy was necessary only in a minority of cases. Conclusions Ocular autoimmunity should be differentiated from toxic effects of cancer treatment and timely recognized since it can be generally well controlled by anti-inflammatory treatment, preserving the patient’s vision without cessation of the tumor treatment.

Dabrafenib Promotes Schwann Cell Differentiation by Inhibition of the MEK-ERK Pathway.

Schwann cell differentiation involves a dynamic interaction of signaling cascades. However, much remains to be elucidated regarding the function of signaling molecules that differ depending on the context in which the molecules are engaged. Here, we identified a small molecule, dabrafenib, which promotes Schwann cell differentiation in vitro and exploited this compound as a pharmacological tool to understand the molecular mechanisms regulating Schwann cell differentiation. The results indicated that dabrafenib inhibited ERK phosphorylation and enhanced ErbB2 autophosphorylation and Akt phosphorylation, and the effects of dabrafenib on ErbB2 and Akt phosphorylation were phenocopied by pharmacological inhibition of the MEK-ERK signaling pathway. However, the small molecule inhibitors of MEK and ERK had no effect on the expression of Oct6 and EGR2, which are key transcription factors that drive Schwann cell differentiation. In addition, pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K) almost completely interfered with dabrafenib-induced Schwann cell differentiation. These results suggest that the ErbB2-PI3K-Akt axis is required for the induction of Schwann cell differentiation by dabrafenib in vitro. Although additional molecules targeted by dabrafenib remain to be identified, our data provides insights into the crosstalk that exists between the MEK-ERK signaling pathway and the PI3K-Akt axis in Schwann cell differentiation.

Abstract 1906: Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer

Abstract Activating mutations in the KRAS oncogene are found in greater than 90% of pancreatic ductal adenocarcinoma (PDAC), yet KRAS remains difficult to inhibit pharmacologically. Small molecule inhibitors of RAF/MEK/ERK (MAPK) or mTOR are first line treatments for several human malignancies, however resistance to these targeted therapies continues to be a major clinical problem. We show in vitro dose dependent inhibition of proliferation and pERK1/2 in human PDAC cell lines and a KRAS-driven genetically engineered mouse model (GEMM) of pancreatic cancer in response to Trametinib, a small molecule MEK-inhibitor (MEKi). Similarly, Trametinib elicited a dose dependent inhibition of the in vivo tumor growth of a subcutaneous human PDAC xenograft, however these tumors rapidly developed resistance and contained areas of increased activation of pAkt(Ser473). We determined that genetic depletion of KRAS using doxycycline-inducible shRNA or small molecule MEKi leads to increased pAkt(Ser473) and activation of mTORC2 by western blot, implicating activation of the Akt/mTORC1/2 pathway may be a mechanism of resistance to MEKi. Interestingly, treatment of PDAC cell lines with Torin1, a small molecule inhibitor of mTORC1/2, results in the increased activation of pERK1/2, suggesting crosstalk and compensation of these two pathways as mechanisms of resistance. The combination of Trametinib and Torin1 leads to the inhibition of both pERK1/2 and pAkt(Ser473) that is accompanied by a dose dependent, synergistic increase in cytotoxicity not observed with either single agent. In human PDAC xenografts and a syngeneic GEMM, combination treatment of Trametinib and Torin1 dramatically reduced tumor burden and increased survival, without serious toxicity. These results demonstrate that combined inhibition of MEK/ERK and mTORC1/2 is a potent and viable therapeutic strategy for PDAC. Citation Format: Wells Brown, Oksana Nemirovsky, Jordan Gillespie, Paul McDonald, Shoukat Dedhar. Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1906.

MEK inhibition remodels the active chromatin landscape and induces SOX10 genomic recruitment in BRAF(V600E) mutant melanoma cells

BackgroundThe MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated.ResultsThis study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein–DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies.ConclusionsThese results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

Abstract 508: Combined MEK and PD-1 inhibition in acute myeloid leukemia: Effect on cell proliferation and phenotype

Abstract Although most patients with acute myeloid leukemia (AML) respond to standard of care chemotherapy, the majority of patients will relapse, and the 5-year overall survival rate persists at 25%. The high rates of relapse and poor survival have contributed to increased interest in the use of other therapies, including immunotherapies, in hematological malignancies in order to achieve more durable responses. In particular, immune checkpoint blockade, such as anti-PD-1, has elicited efficacious responses in Hodgkin’s lymphoma. In addition, trametinib, a small molecule MEK inhibitor, was shown to have activity in AML patients with a RAS-mutation in a phase 1/2 trial, but demonstrated the need for rational combinational therapies to produce longer lasting responses. Interestingly, in a murine model of colon carcinoma harboring a KRAS mutation, the combination of these two agents resulted in a more durable tumor response compared to either agent alone. We hypothesized that in AML, combined MEK and PD-1 inhibition would result in direct inhibition of blast proliferation while simultaneously promoting proliferation and activation of T cells with a mature/memory phenotype. Bone marrow and peripheral blood samples isolated from patients with AML were cultured in our laboratory for 5 days with CD3 stimulation to induce T-cell activation. Cultures were treated with trametinib, anti-PD-1, or both. Additional samples were cultured with interferon-gamma (IFN-γ) to induce PD-L1 expression. Mouse spleen cells from a genetically engineered mouse model of AML (FLT3-ITD/TET2+/-) were cultured under the same conditions for 3 days. Samples were evaluated by flow cytometry to measure treatment effects on T-cell and blast proliferation and phenotype. In the patient samples, trametinib consistently reduced PD-L1 expression in a dose-dependent manner on the myeloid cell population and was further able to attenuate PD-L1 expression induced by IFN-γ. A mean decrease of 36.6% was seen in PD-L1 expression after treatment with trametinib at 100 nm (p=0.0177). In most samples, trametinib decreased blast proliferation and increased blast cell death. Additionally, in a NRAS-mutant sample with high PD-L1 expression, the combination therapy best restored T cell proliferation and decreased the naive T cell population resulting in increased effector and memory T cells. We observed similar changes in our mouse model of AML. In conclusion, IFN-γ increased PD-L1 expression on cancer cells suggesting a possible mechanism of immunosuppression inhibiting autologous immune responses, and the increased PD-L1 expression could be at least partially reversed by trametinib. Our study suggests that combined MEK and PD-1 inhibition may result in greater therapeutic efficacy over each agent alone and provide a rationale for administering this combination to patients with AML. Citation Format: Kaycee B. Moshofsky, Hyun J. Cho, Yoko Kosaka, Matthew T. Newman, Jeffrey W. Tyner, Evan F. Lind. Combined MEK and PD-1 inhibition in acute myeloid leukemia: Effect on cell proliferation and phenotype [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 508.

MEK Inhibition Targets Cancer Stem Cells and Impedes Migration of Pancreatic Cancer Cells In Vitro and In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with a very poor prognosis. At the same time, its incidence is on the rise, and PDAC is expected to become the second leading cause of cancer-related death by 2030. Despite extensive work on new therapeutic approaches, the median overall survival is only 6-12 months after diagnosis and the 5-year survival is less than 7%. While pancreatic cancer is particularly difficult to treat, patients usually succumb not to the growth of the primary tumor, but to extensive metastasis; therefore, strategies to reduce the migratory and metastatic capacity of pancreatic cancer cells merit close attention. The vast majority of pancreatic cancers harbor RAS mutations. The outstanding relevance of the RAS/MEK/ERK pathway in pancreatic cancer biology has been extensively shown previously. Due to their high dependency on Ras mutations, pancreatic cancers might be particularly sensitive to inhibitors acting downstream of Ras. Herein, we use a genetically engineered mouse model of pancreatic cancer and primary pancreatic cancer cells were derived from this model to demonstrate that small-molecule MEK inhibitors functionally abrogate cancer stem cell populations as demonstrated by reduced sphere and organoid formation capacity. Furthermore, we demonstrate that MEK inhibition suppresses TGFβ-induced epithelial-to-mesenchymal transition and migration in vitro and ultimately results in a highly significant reduction in circulating tumor cells in mice.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD−/−) and limb mesenchyme (Nf1Prx1−/−) were tested.Developmental PD0325901 dosing of dams pregnant with Nf1MyoD−/− progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1−/− mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1−/− pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD−/− mice, and fibrosis higher in Nf1Prx1−/− mice.These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

Uveal Melanoma: Identifying Immunological and Chemotherapeutic Targets to Treat Metastases.

Uveal melanoma is an intraocular malignancy that, depending on its size and genetic make-up, may lead to metastases in up to 50% of cases. Currently, no therapy has been proven to improve survival. However, new therapies exploiting immune responses against metastases are being developed. The primary tumor is well characterized: tumors at high risk of developing metastases often contain macrophages and lymphocytes. However, these lymphocytes are often regulatory T cells that may suppress immune response. Currently, immune checkpoint inhibitors have shown marked efficacy in multiple cancers (eg, cutaneous melanoma) but do not yet improve survival in uveal melanoma patients. More knowledge needs to be acquired regarding the function of T cells in uveal melanoma. Other therapeutic options are related to the biochemical pathways. Targeting the RAF-MEK-ERK pathway with small molecule MEK inhibitors abrogates the growth of UM cells harboring GNAQ/GNA11 Q209 mutations, suggesting that these aberrant G-protein oncogenes mediate, at least in part, their effect through this hallmark proliferation pathway. Other pathways are also implicated, such as those involving c-Jun and YAP. Further studies may show how interference in the different pathways may affect survival.

Abstract 4785: Signaling adaptation to FLT3 inhibition in FLT3/ITD leukemia results in a reactivation of ERK signaling that can be abrogated with MEK inhibition

Abstract The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3 mutant acute myeloid leukemia (AML) has been explored as a promising strategy for over a decade. However, FLT3 TKIs have thus far shown limited clinical benefit in patients with FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response. If true, combined targeted therapeutic approaches that overcome the adaptive resistance to FLT3 inhibition could be used to maximize the efficacy of anti-leukemia treatment for those expressing this mutation. To evaluate this hypothesis, the FLT3/ITD AML cell lines Molm14 and MV4;11 were treated with FLT3 TKIs for up to 48 hours at concentrations sufficient for maximal FLT3 inhibition and downstream signaling was analyzed by immunoblotting. We identified a rebound in ERK phosphorylation discernible by six hours and continuing for the duration of treatment, despite continued drug presence. This rebound resulted in near baseline levels of phosphorylated ERK (pERK) and was coupled to a rebound in phosphorylation of elements both upstream and downstream of ERK as well as in the expression levels of ERK target genes, suggesting a global reactivation of the signaling cascade. Rebound was sensitive to a reduction in serum, suggesting a growth factor-dependent mechanism. To explore the consequences of this adaptation, the impact of combinatory targeting of ERK activity using selective small molecule inhibitors of MEK was evaluated. When Molm14 and MV4;11 cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed. Additionally, the anti-leukemia effects were more pronounced for the combination compared to either drug alone, both in vitro and in vivo. In vitro, the addition of a MEK inhibitor (PD0325901 or trametinib) to FLT3 TKI (sorafenib) treatment synergistically increased cell death and decreased cell viability. This effect was most pronounced at mid-range sorafenib combined with low dose MEK inhibitor. In vivo, the addition of low-dose PD0325901 to sorafenib treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts in a transplant model (p < 0.05). Together, these studies reveal that FLT3/ITD leukemia cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to FLT3 inhibition. This adaptation limits the efficacy of FLT3 TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to FLT3 TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML. Citation Format: J. Kyle Bruner, Hayley Ma, Christine Pratilas, Donald Small. Signaling adaptation to FLT3 inhibition in FLT3/ITD leukemia results in a reactivation of ERK signaling that can be abrogated with MEK inhibition. [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 4785.

Selective MEK Inhibition with AZD-6244 (selumetinib) in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL): A University of Chicago Phase II Consortium Trial

Introduction: The biology of DLBCL is complex, with a number of clinicopathologic and molecular features impacting outcome. MCT-1 is an oncogene that promotes lymphomagenesis via enhanced cell survival signaling, increased G1 cyclin/CDK activity and overriding cell cycle checkpoints; its expression is substantially elevated in DLBCL without a clear difference in germinal center versus non-germinal center subtypes. Our preliminary studies showed that 1) 85% of DLBCL samples have strong or increased expression of MCT-1via IHC, 2) MCT-1 knockdown induces apoptosis in cell lines, and 3) mutant MCT-1 protein expression attenuates the malignant phenotype via translational changes. (Dai Ca Res 2009 69(19): p. 7835-43) MCT-1 levels are directly regulated by MEK/ERK signaling, providing a rationale to test MEK pathway inhibition as a therapeutic target in DLBCL. AZD-6244 (ARRY-142886, selumetinib) is a novel second generation oral small molecule MEK inhibitor that induces dose dependent apoptosis in DLBCL cell lines and attenuates tumor growth in xenograft models. (Bhalla Blood 2011 118(4): p. 1052-61) In patients with solid tumors, AZD-6244 75mg twice daily has been established as the recommended phase II dose (RP2D). (Adjei JCO 2008 26(13): p. 2139-46)Methods: Eligible relapsed/refractory (R/R) DLBCL patients received AZD6244 hydrogen sulfate at a dose of 75 mg by mouth twice daily continuously for up to eight 28-day cycles or until disease progression or unacceptable toxicity. The primary objective of the study was overall response rate (ORR) with secondary objectives including safety, progression free survival (PFS), overall survival (OS) and pharmacodynamic (PD) analyses. The statistical design used a Simon two-stage design. Due to excessive early toxicity, the protocol was amended to reduce the dose to 50mg twice daily.Results: Sixteen R/R DLBCL pts (9 male, 7 female) with a median age of 70 years (range, 29-86 yrs), median 3 prior regimens (range, 0-6) were enrolled. Cell-of-origin was not prospectively determined, but only 3/10 pts were CD10 positive and 5/9 were MUM1 positive. The median Lactate Dehydrogenase (LDH) at study entry was 483 (range, 194-1128). Two patients withdrew consent prior to treatment exposure. Of 14 patients receiving at least one dose of selumetinib, two had stable disease and the remainder progressed. The median number of treatment cycles was one (range, 1-5). This was partly due to toxicity causing frequent dose interruptions of AZD-6244. Grade ³3 toxicities were observed in 37.5% (15.2 - 64.6%) and included anemia (44%), thrombocytopenia (25%), diarrhea (31%), transaminitis 44%), fatigue (44%); other infrequent toxicities included AV block, LV dysfunction, anorexia and rash. Median progression-free survival (PFS) in all patients was 34 days [95% CI 14-73 days]. Median overall survival (OS) was 129 days [CI 58-134 days]; all but 3 patients (including one patient lost to follow-up) have expired.Conclusion: Despite excellent preclinical rationale to test selective MEK antagonism as a means of modulating MCT-1 and MEK/ERK in DLBCL, AZD-6244 was not well tolerated and had limited clinical activity. Other MEK/ERK inhibitors with a more favorable toxicity profile and/or use of rational synergistic combination therapy should be considered to further evaluate the role of MEC inhibition in this heavily pretreated patient population. (Patel Cancer 2014 19(4): p. 799-805 (Supported by NCI contract N01-CM-2011-00071C) [Display omitted] DisclosuresPetrich:Seattle Genetics: Consultancy, Honoraria, Research Funding. Westin:Spectrum: Research Funding. Gordon:Northwestern University: Employment; Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending.

RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome.

The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)-specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)-specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.

Antitumor activity of the MEK inhibitor trametinib on intestinal polyp formation in Apc(Δ716) mice involves stromal COX-2.

Extracellular signal-regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using ApcΔ716 mice, a mouse model of familial adenomatous polyposis and early-stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight-week treatment of ApcΔ716 mice with trametinib, a small-molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX-2 level in ApcΔ716 tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX-2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in ApcΔ716 tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in ApcΔ716 mice, at least in part, through COX-2 induction in tumor stromal cells.

Competence for chemical reprogramming of sexual fate correlates with an intersexual molecular signature in Caenorhabditis elegans.

In multicellular organisms, genetic programs guide cells to adopt cell fates as tissues are formed during development, maintained in adults, and repaired after injury. Here we explore how a small molecule in the environment can switch a genetic program from one fate to another. Wild-type Caenorhabditis elegans XX adult hermaphrodites make oocytes continuously, but certain mutant XX adults make sperm instead in an otherwise hermaphrodite soma. Thus, puf-8; lip-1 XX adults make only sperm, but they can be switched from sperm to oocyte production by treatment with a small-molecule MEK inhibitor. To ask whether this chemical reprogramming is common, we tested six XX sperm-only mutants, but found only one other capable of cell fate switching, fbf-1; lip-1. Therefore, reprogramming competence relies on genotype, with only certain mutants capable of responding to the MEK inhibitor with a cell fate change. To gain insight into the molecular basis of competence for chemical reprogramming, we compared polyadenylated transcriptomes of competent and noncompetent XX sperm-only mutants in the absence of the MEK inhibitor and hence in the absence of cell fate reprogramming. Despite their cellular production of sperm, competent mutants were enriched for oogenic messenger RNAs relative to mutants lacking competence for chemical reprogramming. In addition, competent mutants expressed the oocyte-specific protein RME-2, whereas those lacking competence did not. Therefore, mutants competent for reprogramming possess an intersexual molecular profile at both RNA and protein levels. We suggest that this intersexual molecular signature is diagnostic of an intermediate network state that poises the germline tissue for changing its cellular fate in response to environmental cues.