Treating non-small cell lung cancer with selumetinib: an up-to-date drug evaluation.

INTRODUCTION: RAS-RAF-MEK-ERK signaling is implicated in tumor development by promoting cell proliferation and other cancer hallmarks. MEK1/2 kinases are up-regulated in the majority of human cancers due to activation of tyrosine kinase receptors, RAS proteins, BRAF kinase, or some other members of the MAPK pathway. Targeting of MEK1/2 kinases may counterbalance cancer progression. AREAS COVERED: The authors analyze the scientific publications relevant to selumetinib (AZD6244, ARRY-142886) systematically and provide their expert opinion. EXPERT OPINION: Selumetinib is an oral selective allosteric inhibitor of MEK1 and MEK2 kinases. Single-agent selumetinib is usually administered in hydrogen sulfate capsules 75 mg twice a day; combination with other therapeutic compounds may or may not require reduced dosing of this drug. The established dose for pediatric patients is 25 mg per square meter twice a day. Selumetinib was extensively evaluated in non-small cell lung cancer (NSCLC) patients. Studies utilizing this drug as a monotherapy did not confirm its efficacy toward NSCLC. A phase II trial showed that the addition of selumetinib to docetaxel improved response rates and progression-free survival (PFS) in chemotherapy-pretreated KRAS-mutated NSCLC patients; however, a subsequent phase III study did not confirm these findings. There are several highly successful non-NSCLC selumetinib trials involving, e.g., patients with neurofibromatosis type 1 related tumors and children with low-grade BRAF-driven gliomas.

Mst1 knockdown alleviates cardiac lipotoxicity and inhibits the development of diabetic cardiomyopathy in db/db mice.

Diabetic cardiomyopathy (DCM) accounts for increasing deaths of diabetic patients, and effective therapeutic targets are urgently needed. Myocardial lipotoxicity, which is caused by cardiac non-oxidative metabolic fatty acids and cardiotoxic fatty acid metabolites accumulation, has gained more attention to explain the increasing prevalence of DCM. However, whether mammalian Ste20-like kinase 1 (Mst1) plays a role in lipotoxicity in type 2 diabetes-induced cardiomyopathy has not yet been illustrated. Here, we found that Mst1 expression was elevated transcriptionally in the hearts of type 2 diabetes mellitus mice and palmitic acid-treated neonatal rat ventricular myocytes. Adeno-associated virus 9 (AAV9)-mediated Mst1 silencing in db/db mouse hearts significantly alleviated cardiac dysfunction and fibrosis. Notably, Mst1 knockdown in db/db mouse hearts decreased lipotoxic apoptosis and inflammatory response. Mst1 knockdown exerted protective effects through inactivation of MAPK/ERK kinase kinase 1 (MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway. Moreover, lipotoxicity induced Mst1 expression through promoting the binding of forkhead box O3 (FoxO3) and Mst1 promoter. Conclusively, we elucidated for the first time that Mst1 expression is regulated by FOXO3 under lipotoxicity stimulation and downregulation of Mst1 protects db/db mice from lipotoxic cardiac injury through MEKK1/JNK signaling inhibition, indicating that Mst1 abrogation may be a potential treatment strategy for DCM in type 2 diabetic patients.

MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

KDM5B Promotes Drug Resistance by Regulating Melanoma-Propagating Cell Subpopulations.

Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34+p75- (CD34+) and CD34-p75- (CD34-) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34+ and CD34- subpopulations carrying the BRAFV600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34- state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor-sensitive CD34+ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.

Update on systemic therapy for advanced cutaneous melanoma and recent development of novel drugs.

Malignant melanoma is generally chemo- and radio-resistant, and patients with advanced melanoma have a poor prognosis. However, with our increased understanding of the checkpoint immune molecules and genetic alterations of melanoma cells, more effective immunotherapy, such as anti CTLA4 antibody and anti PD-1 antibodies, and targeted drug therapy, such as BRAF inhibitors and MEK inhibitors, have been developed, resulting in improved overall survival and quality of life of patients with advanced melanoma. In addition, emerging technologies to develop prognostic and predictive biomarkers for response to systemic therapy could help clinicians make more accurate assessments of the disease and formulate more effective treatment plans. In this review, current standard systemic therapy options and recently developed novel drugs for advanced melanoma are discussed.

Assessing Therapeutic Efficacy of MEK Inhibition in a KRASG12C-Driven Mouse Model of Lung Cancer.

Purpose: Despite the challenge to directly target mutant KRAS due to its high GTP affinity, some agents are under development against downstream signaling pathways, such as MEK inhibitors. However, it remains controversial whether MEK inhibitors can boost current chemotherapy in KRAS-mutant lung tumors in clinic. Considering the genomic heterogeneity among patients with lung cancer, it is valuable to test potential therapeutics in KRAS mutation-driven mouse models.Experimental Design: We first compared the pERK1/2 level in lung cancer samples with different KRAS substitutions and generated a new genetically engineered mouse model whose tumor was driven by KRAS G12C, the most common KRAS mutation in lung cancer. Next, we evaluated the efficacy of selumetinib or its combination with chemotherapy, in KRASG12C tumors compared with KRASG12D tumors. Moreover, we generated KRASG12C/p53R270H model to explore the role of a dominant negative p53 mutation detected in patients in responsiveness to MEK inhibition.Results: We determined higher pERK1/2 in KRASG12C lung tumors compared with KRASG12D Using mouse models, we further identified that KRASG12C tumors are significantly more sensitive to selumetinib compared with KrasG12D tumors. MEK inhibition significantly increased chemotherapeutic efficacy and progression-free survival of KRASG12C mice. Interestingly, p53 co-mutation rendered KRASG12C lung tumors less sensitive to combination treatment with selumetinib and chemotherapy.Conclusions: Our data demonstrate that unique KRAS mutations and concurrent mutations in tumor-suppressor genes are important factors for lung tumor responses to MEK inhibitor. Our preclinical study supports further clinical evaluation of combined MEK inhibition and chemotherapy for lung cancer patients harboring KRAS G12C and wild-type p53 status. Clin Cancer Res; 24(19); 4854-64. ©2018 AACR.

CD271+ Cells Are Diagnostic and Prognostic and Exhibit Elevated MAPK Activity in SHH Medulloblastoma.

The extensive heterogeneity both between and within the medulloblastoma subgroups underscores a critical need for variant-specific biomarkers and therapeutic strategies. We previously identified a role for the CD271/p75 neurotrophin receptor (p75NTR) in regulating stem/progenitor cells in the SHH medulloblastoma subgroup. Here, we demonstrate the utility of CD271 as a novel diagnostic and prognostic marker for SHH medulloblastoma using IHC analysis and transcriptome data across 763 primary tumors. RNA sequencing of CD271+ and CD271- cells revealed molecularly distinct, coexisting cellular subsets, both in vitro and in vivo MAPK/ERK signaling was upregulated in the CD271+ population, and inhibiting this pathway reduced endogenous CD271 levels, stem/progenitor cell proliferation, and cell survival as well as cell migration in vitro Treatment with the MEK inhibitor selumetinib extended survival and reduced CD271 levels in vivo, whereas, treatment with vismodegib, a well-known smoothened (SMO) inhibitor currently in clinical trials for the treatment of recurrent SHH medulloblastoma, had no significant effect in our models. Our study demonstrates the clinical utility of CD271 as both a diagnostic and prognostic tool for SHH medulloblastoma tumors and reveals a novel role for MEK inhibitors in targeting CD271+ SHH medulloblastoma cells.Significance: This study identifies CD271 as a specific and novel biomarker of SHH-type medulloblastoma and that targeting CD271+ cells through MEK inhibition represents a novel therapeutic strategy for the treatment of SHH medulloblastoma. Cancer Res; 78(16); 4745-59. ©2018 AACR.

MAP3K1 and MAP2K4 mutations are associated with sensitivity to MEK inhibitors in multiple cancer models.

Activation of the mitogen-activated protein kinase (MAPK) pathway is frequent in cancer. Drug development efforts have been focused on kinases in this pathway, most notably on RAF and MEK. We show here that MEK inhibition activates JNK-JUN signaling through suppression of DUSP4, leading to activation of HER Receptor Tyrosine Kinases. This stimulates the MAPK pathway in the presence of drug, thereby blunting the effect of MEK inhibition. Cancers that have lost MAP3K1 or MAP2K4 fail to activate JNK-JUN. Consequently, loss-of-function mutations in either MAP3K1 or MAP2K4 confer sensitivity to MEK inhibition by disabling JNK-JUN-mediated feedback loop upon MEK inhibition. In a panel of 168 Patient Derived Xenograft (PDX) tumors, MAP3K1 and MAP2K4 mutation status is a strong predictor of response to MEK inhibition. Our findings suggest that cancers having mutations in MAP3K1 or MAP2K4, which are frequent in tumors of breast, prostate and colon, may respond to MEK inhibitors. Our findings also suggest that MAP3K1 and MAP2K4 are potential drug targets in combination with MEK inhibitors, in spite of the fact that they are encoded by tumor suppressor genes.

RAS-MAPK Reactivation Facilitates Acquired Resistance in FGFR1-Amplified Lung Cancer and Underlies a Rationale for Upfront FGFR-MEK Blockade.

The FGFR kinases are promising therapeutic targets in multiple cancer types, including lung and head and neck squamous cell carcinoma, cholangiocarcinoma, and bladder cancer. Although several FGFR kinase inhibitors have entered clinical trials, single-agent clinical efficacy has been modest and resistance invariably occurs. We therefore conducted a genome-wide functional screen to characterize mechanisms of resistance to FGFR inhibition in a FGFR1-dependent lung cancer cellular model. Our screen identified known resistance drivers, such as MET, and additional novel resistance mediators including members of the neurotrophin receptor pathway (NTRK), the TAM family of tyrosine kinases (TYRO3, MERTK, AXL), and MAPK pathway, which were further validated in additional FGFR-dependent models. In an orthogonal approach, we generated a large panel of resistant clones by chronic exposure to FGFR inhibitors in FGFR1- and FGFR3-dependent cellular models and characterized gene expression profiles employing the L1000 platform. Notably, resistant clones had enrichment for NTRK and MAPK signaling pathways. Novel mediators of resistance to FGFR inhibition were found to compensate for FGFR loss in part through reactivation of MAPK pathway. Intriguingly, coinhibition of FGFR and specific receptor tyrosine kinases identified in our screen was not sufficient to suppress ERK activity or to prevent resistance to FGFR inhibition, suggesting a redundant reactivation of RAS-MAPK pathway. Dual blockade of FGFR and MEK, however, proved to be a more powerful approach in preventing resistance across diverse FGFR dependencies and may represent a therapeutic opportunity to achieve durable responses to FGFR inhibition in FGFR-dependent cancers. Mol Cancer Ther; 17(7); 1526-39. ©2018 AACR.

Synergistic action of dual IGF1/R and MEK inhibition sensitizes childhood acute lymphoblastic leukemia (ALL) cells to cytotoxic agents and involves downregulation of STAT6 and PDAP1.

Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024 + U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024 + U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024 + U0126 in samples with variable basal levels of phosphorylated IGF1/Rß and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 "synergy genes" differentially targeted by IGF1/R + MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analysesrevealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key "synergy" genes, supporting their predicted role in an STAT6-ERK-nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.

Survivin knockdown induces senescence in TTF­1-expressing, KRAS-mutant lung adenocarcinomas.

Survivin plays a key role in regulating the cell cycle and apoptosis, and is highly expressed in the majority of malignant tumors. However, little is known about the roles of survivin in KRAS-mutant lung adenocarcinomas. In the present study, we examined 28 KRAS-mutant lung adenocarcinoma tissues and two KRAS-mutant lung adenocarcinoma cell lines, H358 and H441, in order to elucidate the potential of survivin as a therapeutic target. We found that 19 (68%) of the 28 KRAS-mutant lung adenocarcinomas were differentiated tumors expressing thyroid transcription factor­1 (TTF­1) and E-cadherin. Patients with tumors immunohistochemically positive for survivin (n=18) had poorer outcomes than those with survivin-negative tumors (n=10). In the H358 and H441 cells, which expressed TTF­1 and E-cadherin, survivin knockdown alone induced senescence, not apoptosis. However, in monolayer culture, the H358 cells and H441 cells in which survivin was silenced, underwent significant apoptosis following combined treatment with ABT-263, a Bcl­2 inhibitor, and trametinib, a MEK inhibitor. Importantly, the triple combination of survivin knockdown with ABT-263 and trametinib treatment, clearly induced cell death in a three-dimensional cell culture model and in an in vivo tumor xenograft model. We also observed that the growth of the H358 and H441 cells was slightly, yet significantly suppressed in vitro when TTF­1 was silenced. These findings collectively suggest that the triple combination of survivin knockdown with ABT-263 and trametinib treatment, may be a potential strategy for the treatment of KRAS-mutant lung adenocarcinoma. Furthermore, our findings indicate that the well­differentiated type of KRAS-mutant lung tumors depends, at least in part, on TTF­1 for growth.

Low Focal Adhesion Signaling Promotes Ground State Pluripotency of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFß. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p < 0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly down-regulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn2+ in the 2i and R2i cultures resulted in reduced Nanog and increased the expression of lineage marker genes. In this study, we demonstrated that reduced focal adhesion enabled mESCs to be maintained in an undifferentiated and pluripotent state.

Systemic Therapy Options for Patients With Unresectable Melanoma.

There has been a therapeutic revolution in the treatment of metastatic melanoma over the past decade. Patients presenting with inoperable disease have several therapeutic options, which can include both targeted and immune therapy. Immune checkpoint inhibitors have demonstrated an improvement in overall survival and led to some durable responses. However, toxicity, especially in combination regimens, can be severe. Adverse events should be anticipated, diagnosed as early as possible, monitored, and managed. Combination BRAF and MEK inhibition has also been shown to improve overall survival in patients with V600E-mutated melanoma. Responses to therapy are often rapid, and treatment is not associated with immune-related adverse events. Current trials are under way to determine which option is optimal as frontline therapy for patients with V600E melanoma. In patients with progressive disease despite standard therapies, clinical trials are recommended. There are several promising agents in development.

Phase Ib/II Study of the Safety and Efficacy of Combination Therapy with Multikinase VEGF Inhibitor Pazopanib and MEK Inhibitor Trametinib In Advanced Soft Tissue Sarcoma.

Purpose: Pazopanib, a multireceptor tyrosine kinase inhibitor targeting primarily VEGFRs1-3, is approved for advanced soft tissue sarcoma (STS) and renal cell cancer. Downstream of VEGFR, trametinib is an FDA-approved MEK inhibitor used for melanoma. We hypothesized that vertical pathway inhibition using trametinib would synergize with pazopanib in advanced STS.Experimental Design: In an open-label, multicenter, investigator-initiated National Comprehensive Cancer Network (NCCN)-sponsored trial, patients with metastatic or advanced STS received pazopanib 800 mg and 2 mg of trametinib continuously for 28-day cycles. The primary endpoint was 4-month progression-free survival (PFS). Secondary endpoints were overall survival, response rate, and disease control rate.Results: Twenty-five patients were enrolled. The median age was 49 years (range, 22-77 years) and 52% were male. Median PFS was 2.27 months [95% confidence interval (CI), 1.9-3.9], and the 4-month PFS rate was 21.1% (95% CI, 9.7-45.9), which was not an improvement over the hypothesized null 4-month PFS rate of 28.3% (P = 0.79). Median overall survival was 9.0 months (95% CI, 5.7-17.7). A partial response occurred in 2 (8%) of the evaluable patients (95% CI, 1.0-26.0), one with PIK3CA E542K-mutant embryonal rhabdomyosarcoma and another with spindle cell sarcoma. The disease control rate was 14/25 (56%; 95% CI, 34.9-75.6). The most common adverse events were diarrhea (84%), nausea (64%), fatigue (56%), and hypertension (52%).Conclusions: The combination of pazopanib and trametinib was tolerable without indication of added activity of the combination in STS. Further study may be warranted in RAS/RAF aberrant sarcomas. Clin Cancer Res; 23(15); 4027-34. ©2017 AACR.

Pharmacokinetics and pharmacogenetics of the MEK1/2 inhibitor, selumetinib, in Asian and Western healthy subjects: a pooled analysis.

PURPOSE: Emerging data on selumetinib, a MEK1/2 inhibitor in clinical development, suggest a possible difference in pharmacokinetics (PK) between Japanese and Western patients. This pooled analysis sought to assess the effect of ethnicity on selumetinib exposure in healthy Western and Asian subjects, and to identify any association between genetic variants in the UGT1A1, CYP2C19 and ABCG2 genes and observed differences in selumetinib PK. METHODS: A pooled analysis of data from ten Phase I studies, one in Asian subjects (encompassing Japanese, non-Japanese Asian and Indian Asian subjects) and nine in Western subjects, was conducted. Key findings were derived from the collective exposure data across doses of 25, 35, 50 and 75 mg selumetinib; primary variables were dose-normalized AUC and Cmax. RESULTS: PK data from 308 subjects (10 studies) were available for the pooled analysis; genetic data from 87 subjects (3 studies) were available for the pharmacogenetic analysis. Dose-normalized AUC and Cmax were 35% (95% CI: 25-47%) and 39% (95% CI: 24-56%) higher in the pooled Asian group, respectively, compared with Western subjects. PK exposure parameters were similar between the Japanese, non-Japanese Asian and Indian groups. There was no evidence that the polymorphisms assessed in the genes UGT1A1, CYP2C19 and ABCG2 account for observed PK differences. CONCLUSIONS: Selumetinib exposure was higher in healthy Asian subjects compared with Western subjects, and these data provide valuable insight for clinicians to consider when treating patients of Asian ethnicity with selumetinib.

Phase I trial of MEK 1/2 inhibitor pimasertib combined with mTOR inhibitor temsirolimus in patients with advanced solid tumors.

Background Dual inhibition of activated MAPK and mTOR signaling pathways may enhance the antitumor efficacy of the MEK 1/2 inhibitor pimasertib and the mTOR inhibitor temsirolimus given in combination. Methods In this phase I study, patients with refractory advanced solid tumors (NCT01378377) received once-weekly temsirolimus plus once-daily oral pimasertib in 21-day cycles in a modified 3 + 3 dose-escalation design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of pimasertib in combination with temsirolimus, safety and pharmacokinetics (PK) were investigated. Results Of 33 patients evaluated, all experienced ≥1 treatment-emergent adverse event (TEAE) and 31 had treatment-related TEAEs, most frequently stomatitis and thrombocytopenia. TEAEs were reversible. No deaths were attributed to treatment. Nine patients had dose-limiting toxicities (stomatitis, thrombocytopenia, serum creatinine phosphokinase increase, visual impairment) and the MTD was determined as 45 mg/day pimasertib plus 25 mg/week temsirolimus. However, due to overlapping toxicities no further investigations were performed and the RP2D was not defined. PK profiles of both agents were not adversely affected. Seventeen patients (17/26 patients) had a best response of stable disease; five had stable disease lasting >12 weeks. Conclusions The RP2D was not defined and the pimasertib plus temsirolimus combination investigated did not warrant further study.

Preclinical activity of EGFR and MEK1/2 inhibitors in the treatment of biliary tract carcinoma.

Biliary tract carcinomas (BTC) are malignant tumors with limited therapeutic options. Clinical experiences with anti-EGFR therapies have produced unsatisfactory results. The strategies of combined inhibition of EGFR and MEK1/2 could be a promising therapeutic option in BTC treatment. Preclinical activity of Panitumumab and Trametinib was tested in in vitro (EGI-1, MT-CHC01 and WITT cells) and in in vivo (xenograft) BTC models with different K-RAS mutational status. Trametinib reduced MAPK phosphorylation in wild type (WT) WITT cells and in both K-RAS mutated cells; in EGI-1 was also able to switch off EGFR activation. Panitumumab reduced the activation of its target only in EGI-1 cells, and of MAPK only in WITT cells. While Trametinib inhibited cell growth in K-RAS mutated cell lines, Panitumumab had no effect on proliferation independently by K-RAS status. The addition of Panitumumab to Trametinib did not significantly potentiate its anti-proliferative effect also in mutated cells. In vivo, Trametinib was able to significantly slow the tumor growth in K-RAS mutated xenograft models, but did not have effect on K-RAS WT cells; the addition of Panitumumab potentiated the Trametinib efficacy in MT-CHC01 and overcame the resistance to the anti-EGFR in WITT cells, in which the monotherapy was ineffective. Only in K-RAS mutated xenografts Trametinib alone or in combination with Panitumumab significantly decreased Ki67 positive cell fraction and CD31 angiogenesis markers. In conclusion, this preclinical study provides a rational to plan clinical trials assessing the efficacy of Trametinib in K-RAS mutated BTC patients and the combination with anti-EGFR in WT BTC patients.

Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.

PURPOSE: Though the efficacy of MEK inhibitors is being investigated in KRAS-mutant colorectal cancers (CRC), early clinical trials of MEK inhibitor monotherapy did not reveal significant antitumor activity. Resistance to MEK inhibitor monotherapy developed through a variety of mechanisms converging in ERK reactivation. Since ERK increases cyclin D expression and increases entry into the cell cycle, we hypothesized that the combination of MEK inhibitors and CDK4/6 inhibitors would have synergistic antitumor activity and cause tumor regression in vivo. RESULTS: The combination of MEK and CDK4/6 inhibitors synergistically inhibited cancer cell growth in vitro and caused tumor regression in vivo in cell line and patient-derived xenograft models. Combination therapy markedly decreased levels of phosphorylated ribosomal protein S6 both in vitro and in vivo and decreased Ki67 staining in vivo. EXPERIMENTAL DESIGN: We performed in vitro proliferation, colony formation, apoptosis, and senescence assays, and Western blots, on a panel of 11 KRAS mutant CRC cell lines treated with the MEK inhibitor MEK162, the CDK4/6 inhibitor palbociclib, or the combination. We also treated 4 KRAS mutant CRC cell line and patient-derived xenografts with the MEK inhibitor trametinib, the CDK4/6 inhibitor palbociclib, or the combination, and performed immunohistochemical and reverse phase protein array analysis. CONCLUSIONS: Combined inhibition of both MEK and CDK4/6 is effective in preclinical models of KRAS mutant CRC and justifies a planned phase II clinical trial in patients with refractory KRAS-mutant CRC.Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.