Selective MEK Inhibitor Research Articles

Tunlametinib (HL-085) plus vemurafenib in patients with advanced BRAF V600-mutant solid tumors: an open-label, single-arm, multicenter, phase I study

BackgroundTunlametinib (HL-085) is a novel, highly selective MEK inhibitor with substantial clinical activities in patients with NRAS-mutant melanoma. This phase I study evaluated the safety and preliminary efficacy of tunlametinib plus vemurafenib in patients with advanced BRAF V600-mutant solid tumors.MethodsPatients with confirmed advanced BRAF V600-mutant solid tumors who had progressed on or shown intolerance or no available standard therapies were enrolled and received tunlametinib plus vemurafenib. This study consisted of a dose-escalation phase and a dose-expansion phase. Primary end points of this study were safety, the recommended phase II dose (RP2D), and preliminary efficacy.ResultsFrom August 17, 2018 to April 19, 2022, 72 patients were enrolled. No dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. The RP2D for BRAF V600-mutant non-small cell lung cancer (NSCLC) patients was tunlametinib 9 mg plus vemurafenib 720 mg, twice daily (BID, bis in die). Until the data cut-off date of December 15, 2023, of 33 NSCLC patients with evaluable disease, the objective response rate (ORR) was 60.6% (20/33; 95% confidence interval [CI], 42.1–77.1), the median progression free survival (PFS) was 10.5 months (95%CI, 5.6–14.5) and median duration of response (DoR) was 11.3 months (95%CI, 6.8-NE). At the RP2D, ORR was 60.0% (9/15; 95% CI, 32.3–83.7), the median PFS was 10.5 months (95%CI, 5.6 -NE) and median DoR was 11.3 months (95%CI, 3.9-NE). Of 24 colorectal cancer patients with evaluable disease, the ORR was 25.0% (6/24; 95% CI, 5.6-NE). All 72 patients had treatment-related adverse events (TRAEs), and the most common grade 3–4 TRAEs were anemia (n = 13, 18.1%) and blood creatine phosphokinase increased (n = 10, 13.9%). Tunlametinib was absorbed rapidly with Tmax of 0.5–1 h. Vemurafeinib did not influence the system exposure of tunlametinib and vice versa, indicating no drug-drug interaction for this combination.ConclusionsTunlametinib (HL-085) plus vemurafenib had a favorable safety profile and showed promising antitumor activity in patients with BRAF V600-mutant solid tumors. The RP2D for NSCLC was tunlametinib 9 mg BID plus vemurafeinib 720 mg BID.Trial RegistrationClinicalTrials.gov, NCT03781219.

BRAF - a tumour-agnostic drug target with lineage-specific dependencies.

In June 2022, the FDA granted Accelerated Approval to the BRAF inhibitor dabrafenib in combination with the MEK inhibitor trametinib for the treatment of adult and paediatric patients (≥6 years of age) with unresectable or metastatic BRAFV600E-mutant solid tumours, except for BRAFV600E-mutant colorectal cancers. The histology-agnostic approval of dabrafenib plus trametinib marks the culmination of two decades of research into the landscape of BRAF mutations in human cancers, the biochemical mechanisms underlying BRAF-mediated tumorigenesis, and the clinical development of selective RAF and MEK inhibitors. Although the majority of patients with BRAFV600E-mutant tumours derive clinical benefit from BRAF inhibitor-based combinations, resistance to treatment develops in most. In this Review, we describe the biochemical basis for oncogenic BRAF-induced activation of MAPK signalling and pan-cancer and lineage-specific mechanisms of intrinsic, adaptive and acquired resistance to BRAF inhibitors. We also discuss novel RAF inhibitors and drug combinations designed to delay the emergence of treatment resistance and/or expand the population of patients with BRAF-mutant cancers who benefit from molecularly targeted therapies.

Abstract C083: SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer

Abstract While oncogenic KRAS is believed to drive pancreatic cancer by maintaining active ERK signaling, our studies suggest that ERK signaling can be also regulated in a KRAS-independent manner. We found that survival motor neuron domain containing 1 (SMNDC1), a poorly studied splicing factor is amplified or upregulated in 30% of pancreatic ductal adenocarcinomas (PDACs) and associated with lower survival rates in patients. To determine the role of SMNDC1 in PDACs, we conducted loss- and gain-of-function studies and found that this splicing factor drives tumor growth by promoting the retention of A/C-rich cassette exons, which otherwise are excluded or retained at a lower rate after RNA splicing in normal cells. Importantly, across tumors, SMNDC1 promoted the inclusion of the cassette exon 4 (E4) of MAPK3 (ERK1), which encodes the kinase-activating phosphorylation sites (Thr202/Tyr204). Forced exclusion of MAPK3-E4 using antisense oligonucleotide morpholino (ASO), inhibited tumor establishment, ERK1 phosphorylation, and expression of ERK-targets. We then tested if retention of E4 in MAPK3 by SMNDC1 is a mechanism to resist chemical inhibition of the KRAS-MEK-ERK pathway. We found that PDAC cells acutely treated with selective MEK inhibitors (MEKi), trametinib, or selumetinib increased SMNDC1 and MAPK3 E4 retention expression. In addition, PDAC cells deficient for SMNDC1 were 25-fold more sensitive to trametinib than isogenic cells expressing SMNDC1. To demonstrate that increased E4 retention drives MEKi resistance, we concomitantly treated SMNDC1 proficient PDAC cells with trametinib and E4 ASO, which led to an 8-fold increase in sensitivity compared to cells treated with trametinib and control ASO. Also, we found that resistance to standard-of-care chemotherapeutic agents to treat PDAC, Gemcitabine (GEM) and 5-fluorouracil (5-FU), was also associated with increased expression of SMNDC1 and retention of E4 in MAPK3 in multiple PDAC cells lines. To determine the biochemical mechanism that leads to E4 inclusion by SMNDC1, we performed high-throughput RNA Bind-n-Seq (RBNS) and e-CLIP of SMNDC1 in PDAC cells which revealed specific RNA-binding preference for C-rich polypyrimidine tract upstream of retained MAPK3-E4. To validate these findings, we constructed a dual-fluorescent reporter mini-gene of MAPK3-E4 splicing, with either wild-type sequences in E4 and polypyrimidine tract, or different point mutations at the predicted cis-elements. We found that retention of the E4 of MAPK3 is dependent on SMNDC1-binding to C-rich sequences at distant 3’ splice sites of PDAC cells. These data support that PDAC cells exploit a “splicing switch” to promote ERK kinase activity and resistance to therapies, independent of activating mutations in the EGFR/RAS/MAPK pathway and offer a druggable alternative to block oncogenic signaling and altered RNA splicing in PDAC. Citation Format: Md Afjalus Siraj, Yushan Zhang, Prabir Chakraborty, Robert Tseng, Li-Ting Ku, Grant Goda, Gilbert Giri, Deanne Yugawa, Mathew Wang, Shamik Das, Anindya Dey, Shailendra Dhar Dwivedi, Geeta Rao, Min Zhang, Da Yang, Md Nazir Hossen, Wei-Qun Ding, Kar-Ming Fung, Daniel Dominguez, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C083.

Preclinical characterization of tunlametinib, a novel, potent, and selective MEK inhibitor.

Background: Aberrant activation of RAS-RAF-MEK-ERK signaling pathway has been implicated in more than one-third of all malignancies. MEK inhibitors are promising therapeutic approaches to target this signaling pathway. Though four MEK inhibitors have been approved by FDA, these compounds possess either limited efficacy or unfavorable PK profiles with toxicity issues, hindering their broadly application in clinic. Our efforts were focused on the design and development of a novel MEK inhibitor, which subsequently led to the discovery of tunlametinib. Methods: This study verified the superiority of tunlametinib over the current MEK inhibitors in preclinical studies. The protein kinase selectivity activity of tunlametinib was evaluated against 77 kinases. Anti-proliferation activity was analyzed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. ERK and phospho-ERK levels were evaluated by Western blot analysis. Flow cytometry analysis was employed to investigate cell cycle and arrest. Cell-derived xenograft (CDX) and Patient-derived xenograft (PDX) models were used to evaluate the tumor growth inhibition. The efficacy of tunlametinib as monotherapy treatment was evaluated in KRAS/BRAF mutant or wild type xenograft model. Furthermore, the combination studies of tunlametinib with BRAF/KRASG12C/SHP2 inhibitors or chemotherapeutic agent were conducted by using the cell proliferation assay in vitro and xenograft models in vivo. Results: In vitro, tunlametinib demonstrated high selectivity with approximately 19-fold greater potency against MEK kinase than MEK162, and nearly 10-100-fold greater potency against RAS/RAF mutant cell lines than AZD6244. In vivo, tunlametinib resulted in dramatic tumor suppression and profound inhibition of ERK phosphorylation in tumor tissue. Mechanistic study revealed that tunlametinib induced cell cycle arrest at G0/G1 phase and apoptosis of cells in a dose-proportional manner. In addition, tunlametinib demonstrated a favorable pharmacokinetic profile with dose-proportionality and good oral bioavailability, with minimal drug exposure accumulation. Furthermore, tunlametinib combined with BRAF/KRASG12C/SHP2 inhibitors or docetaxel showed synergistically enhanced response and marked tumor inhibition. Conclusion: Tunlametinib exhibited a promising approach for treating RAS/RAF mutant cancers alone or as combination therapies, supporting the evaluation in clinical trials. Currently, the first-in-human phase 1 study and pivotal clinical trial of tunlametinib as monotherapy have been completed and pivotal trials as combination therapy are ongoing.

Reactive Oxygen Species Regulation of Chemoresistance and Metastatic Capacity of Melanoma: Role of the Cancer Stem Cell Marker CD271

BRAF mutations are present in 30-50% of cases of cutaneous melanoma, and treatment with selective BRAF and MEK inhibitors has been introduced. However, the development of resistance to these drugs often occurs. Chemo-resistant melanoma cells show increased expression of CD271, a stem cell marker that features increased migration. Concordantly, resistance to the selective inhibitor of oncogenic BRAFV600E/K, vemurafenib, is mediated by the increased expression of CD271. It has recently been shown that the BRAF pathway leads to an overexpression of the NADPH oxidase Nox4, which produces reactive oxygen species (ROS). Here, we examined in vitro how Nox-derived ROS in BRAF-mutated melanoma cells regulates their drug sensitivity and metastatic potential. We demonstrated that DPI, a Nox inhibitor, reduced the resistance of a melanoma cell line (SK-MEL-28) and a primary culture derived from a BRAFV600E-mutated biopsy to vemurafenib. DPI treatment affected the expression of CD271 and the ERK and Akt signaling pathways, leading to a drop in epithelial-mesenchymal transition (EMT), which undoubtedly promotes an invasive phenotype in melanoma. More importantly, the scratch test demonstrated the efficacy of the Nox inhibitor (DPI) in blocking migration, supporting its use to counteract drug resistance and thus cell invasion and metastasis in BRAF-mutated melanoma.

Phase Ib study of dabrafenib, trametinib, irinotecan and cetuximab in BRAF V600E-mutated metastatic colorectal cancer.

156 Background: The low efficacy of single-agent BRAF inhibitor in metastatic colorectal cancer (mCRC) is usually due to EGFR feedback upregulation caused by BRAF blockade. Therefore, multi-drug combination therapy with BRAF inhibitor has been tried. Here, we reported the preliminary results of quadruple therapy with dabrafenib, a selective BRAF inhibitor, trametinib, a selective MEK inhibitor, irinotecan and cetuximab in patients with BRAF V600E-mutated mCRC. Methods: This was an ongoing phase Ib study, patients with mCRC who relapse or refractory to standard treatments were enrolled. Patients were treated with dabrafenib (150mg/150mg doses orally twice daily), trametinib (2mg orally once daily), irinotecan (80mg/m2 weekly) and cetuximab (400mg/m2 in first dose and 250mg/m2 weekly). Treatment was continued until disease progression, development of unacceptable toxicity, or drawal of consent. The primary end-point was disease control rate (DCR), and the secondary end-points included progression-free survival (PFS) and duration of disease remission (DOR). This trial is registered with Chinese ClinicalTrials.gov, number ChiCTR2200063316. Results: From July 2018 to January 2021, 10 patients were enrolled. The median age was 53.5 (age 25-65), nine with colon cancer and one with rectal cancer. The most common adverse events (AEs) (50% or more) were fatigue, fever, rash, vomit, leukopenia, anemia, ALT elevation, and AST elevation. Four patients suffered rash which was the most common severe AE (SAE). Two patients presented with grade 3/4 anemia. Only one patient suffered grade 3/4 AEs with nausea and vomit. The DCR was 90% (9/10). After a median follow-up of 19.7 months, the median PFS was 7.5 months (range 1.8-32.4 months). The median DOR was 5.2 months (range 0-27.7 months). Conclusions: Dabrafenib, trametinib, irinotecan and cetuximab has tolerable toxicity and promising antitumor activity in BRAF V600E-mutated mCRC. This regimen warrants a further phase II clinical trial. Clinical trial information: ChiCTR2200063316 . [Table: see text]

An Inverse Agonist of Estrogen-Related Receptor Gamma, GSK5182, Enhances Na+/I- Symporter Function in Radioiodine-Refractory Papillary Thyroid Cancer Cells.

Previously, we reported that an inverse agonist of estrogen-related receptor gamma (ERRγ), GSK5182, enhances sodium iodide (Na+/I-) symporter (NIS) function through mitogen-activated protein (MAP) kinase signaling in anaplastic thyroid cancer cells. This finding helped us to further investigate the effects of GSK5182 on NIS function in papillary thyroid cancer (PTC) refractory to radioactive iodine (RAI) therapy. Herein, we report the effects of ERRγ on the regulation of NIS function in RAI-resistant PTC cells using GSK5182. RAI-refractory BCPAP cells were treated with GK5182 for 24 h at various concentrations, and radioiodine avidity was determined with or without potassium perchlorate (KClO4) as an NIS inhibitor. We explored the effects of GSK5182 on ERRγ, the mitogen-activated protein (MAP) kinase pathway, and iodide metabolism-related genes. We examined whether the MAP pathway affected GSK5182-mediated NIS function using U0126, a selective MEK inhibitor. A clonogenic assay was performed to evaluate the cytotoxic effects of I-131. GSK5182 induced an increase in radioiodine avidity in a dose-dependent manner, and the enhanced uptake was completely inhibited by KClO4 in BCPAP cells. We found that ERRγ was downregulated and phosphorylated extracellular signal-regulated kinase (ERK)1/2 was upregulated in BCPAP cells, with an increase in total and membranous NIS and iodide metabolism-related genes. MEK inhibitors reversed the increase in radioiodine avidity induced by GSK5182. Clonogenic examination revealed the lowest survival in cells treated with a combination of GSK5182 and I-131 compared to those treated with either GSK518 or I-131 alone. We demonstrate that an inverse agonist of ERRγ, GSK5182, enhances the function of NIS protein via the modulation of ERRγ and MAP kinase signaling, thereby leading to increased responsiveness to radioiodine in RAI-refractory papillary thyroid cancer cells.

Regulation of cardiac myocyte cyclic AMP production by intracellular α1-adrenergic receptors.

Sympathetic regulation of cardiac function involves β1‑adrenergic receptor (β1AR) production of cAMP. However, the effects are typically mediated by the endogenous neurotransmitter norepinephrine (NE), which is also a potent α1-adrenergic receptor (α1AR) agonist. Furthermore, activation of a mitogen-activated protein kinase (MAPK) signaling pathway by nuclear α1ARs is thought to be cardioprotective, although the exact mechanism has yet to be elucidated. Because long-term stimulation of cAMP production by β1ARs contributes to hypertrophy and apoptosis, we tested the hypothesis that the cardioprotective α1AR signaling pathway inhibits β1AR cAMP production. Changes in cAMP activity were measured in adult rat ventricular myocytes (ARVMs) using FRET-based biosensors. We found that the increase in cAMP produced by the βAR agonist isoproterenol could be antagonized by the selective α1AR agonist methoxamine (METH), and the response to METH could be antagonized by the selective α1AR antagonist prazosin (PRAZ). The effects of METH could also be antagonized by blocking the MAPK signaling pathway with non-selective tyrosine kinase inhibitors genistein and lavendustin A as well as the selective MEK inhibitors U0126 and PD98059. Furthermore, NE produced increases in cAMP activity that was enhanced in the presence of PRAZ, demonstrating α1AR inhibition of β1AR cAMP production contributes to the net response. NE stimulation of cAMP production was also significantly enhanced when the organic cation transporter OCT3 was inhibited with cortisol, and addition of PRAZ caused no further increase, suggesting the α1ARs responsible for inhibiting cAMP production are intracellular. Confocal imaging of ARVMs treated with BODIPY-labeled PRAZ confirmed α1ARs are primarily perinuclear in these cells, a result confirmed by the labeling of α1ARs on isolated nuclei. These results suggest the cardioprotective effect of α1AR stimulation may involve nuclear receptor activation of a MAPK signaling mechanism that inhibits β1AR production of cAMP.

Effect of Food (Low and High Fat) on Pharmacokinetics of FCN-159, a Selective MEK Inhibitor, in Healthy Chinese Males.

FCN-159 is a novel, oral, potent, selective MEK1/2 inhibitor in clinical development for the treatment of NRAS-mutant advanced melanoma and neurofibromatosis type 1. We investigated the effect of food on the pharmacokinetics (PK), safety, and tolerability of FCN-159. In this single-center, open-label, phase 1 study with a three-period, three-sequence, crossover design, healthy Chinese male subjects (n = 24) were randomized (1:1:1) to receive a single, oral 8mg dose of FCN-159 in the fasted state (overnight, > 10h), and with a low-fat and a high-fat meal, separated by a 10-day washout. PK parameters including time to maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC) were compared using geometric least-squares mean ratios (GLSMR), with the fasted state as the reference. A 90% CI for the GLSMR within 80-125% indicated no significant food effect. A low-fat meal (n = 23) did not affect the PK profile of FCN-159: G LSMR for AUC from time 0 to t (AUC0-t), 106.9% (90% CI 99.9-114.4%); AUC from time 0 to infinity (AUC0-∞), 106.8% (90% CI 100.0-114.0%); Cmax, 96.4% (90% CI 83.9-110.8%). A high-fat meal (n = 24) did not affect exposure to FCN-159 (GLSMR for AUC0-t, 99.4%; 90% CI 99.0-106.3%; AUC0-∞, 99.5 5%; 90% CI 93.2-106.1%), but modestly reduced Cmax by 15% (GLSMR 84.9%; 90% CI 74.0-97.3%). Both the low-fat and high-fat meals slightly prolonged the median time to Cmax by 0.5h (90% CI 0.5-1.0h). FCN-159 was generally well tolerated, with a lower incidence of treatment-emergent adverse events following administration in the fasted state than with a low-fat or high-fat meal (20.8%, 39.1%, and 37.5%, respectively). Food did not affect the PK profile of FCN-159 to a clinically meaningful extent compared with administration in the fasted state.

First-in-human phase I dose-escalation and dose-expansion trial of the selective MEK inhibitor HL-085 in patients with advanced melanoma harboring NRAS mutations

BackgroundHL-085 is a selective, orally administered MEK1/2 inhibitor. We aimed to evaluate the safety and efficacy of HL-085 in patients with advanced melanoma harboring NRAS mutations.MethodsThis was a multicenter phase 1 study. HL-085 was administered twice daily in a standard 3 + 3 dose-escalation design (10 dose cohorts; 0.5–18 mg twice daily), followed by dose expansion at the recommended phase II dose (RP2D). The primary endpoints included tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and RP2D.ResultsBetween September 13, 2017, and January 18, 2021, 42 patients were enrolled (dose escalation phase: n = 30; dose expansion phase: n = 12). No DLT was reported during dose escalation and MTD was not reached with HL-085 doses up to 18 mg twice daily. The RP2D was 12 mg twice daily. The most common all-grade drug-related adverse events (AEs) across all dose levels were rash (61.9%), increased creatine phosphokinase (CK, 59.5%), face edema (50.0%), increased aspartate aminotransferase (47.6%), peripheral edema (40.5%), diarrhea (33.3%), alanine aminotransferase (33.3%), and paronychia (19.0%), most of which were grade 1 and 2. Most frequency of grade ≥ 3 AEs were CK (14.2%), asthenia (7.1%), peripheral edema (4.8%), and acneiform dermatitis (4.8%). In the cohort of 12 mg twice daily dose (15 patients), confirmed objective response rate was 26.7%; disease control rate was 86.7%; median duration of response was 2.9 months; median progression-free survival was 3.6 months.ConclusionsThe HL-085 showed acceptable tolerability and substantial clinical activity in patients with advanced melanoma harboring NRAS mutations.Trial registrationTrial registration ClinicalTrials.gov number: NCT03973151.

BI-847325, a selective dual MEK and Aurora kinases inhibitor, reduces aggressive behavior of anaplastic thyroid carcinoma on an in vitro three-dimensional culture.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer. In this study, we used a three-dimensional in vitro system to evaluate the effect of a dual MEK/Aurora kinase inhibitor, BI-847325 anticancer drug, on several cellular and molecular processes involved in cancer progression. Human ATC cell lines, C643 and SW1736, were grown in alginate hydrogel and treated with IC50 values of BI-847325. The effect of BI-847325 on inhibition of kinases function of MEK1/2 and Aurora kinase B (AURKB) was evaluated via Western blot analysis of phospho-ERK1/2 and phospho-Histone H3 levels. Sodium/iodide symporter (NIS) and thyroglobulin (Tg), as two thyroid-specific differentiation markers, were measured by qRT-PCR as well as flow cytometry and immunoradiometric assay. Apoptosis was assessed by Annexin V/PI flow cytometry and BIM, NFκB1, and NFκB2 expressions. Cell cycle distribution and proliferation were determined via P16, AURKA, and AURKB expressions as well as PI and CFSE flow cytometry assays. Multidrug resistance was evaluated by examining the expression of MDR1 and MRP1. Angiogenesis and invasion were investigated by VEGF expression and F-actin labeling with Alexa Fluor 549 Phalloidin. Western blot results showed that BI-847325 inhibits MEK1/2 and AURKB functions by decreasing phospho-ERK1/2 and phospho-Histone H3 levels. BI-847325 induced thyroid differentiation markers and apoptosis in ATC cell lines. Inversely, BI-847325 intervention decreased multidrug resistance, cell cycle progression, proliferation, angiogenesis, and invasion at the molecular and/or cellular levels. The results of the present study suggest that BI-857,325 might be an effective multi-targeted anticancer drug for ATC treatment.

Dysregulated cystathionine-β-synthase/hydrogen sulfide signaling promotes chronic stress-induced colonic hypermotility in rats.

Hydrogen sulfide (H2 S), an important endogenous gasotransmitter, is involved in the modulation of gastrointestinal motility, but whether it mediates the intestinal dysmotility in irritable bowel syndrome (IBS) is not known. This study explored the significance of cystathionine-β-synthase (CBS)/H2 S signaling in stress-induced colonic dysmotility. A rat model of IBS was established using chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically. Intestinal motility was determined by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was assessed using the visceromotor response (VMR) to colorectal distension (CRD). Real-time PCR, Western blotting, and immunostaining were performed to identify the expression of CBS in the colon. The contractions of distal colon were studied in an organ bath system and H2 S content was measured by ELISA. The effects of SAM, a selective CBS activator, on colonic dysmotility were examined. MEK1 was tested as a potential upstream effector of CBS/H2 S loss. After 10 days of WAS, the ITT was decreased and FWC was increased, and the VMR magnitude in response to CRD was enhanced. The colonic CBS expression and H2 S levels were significantly declined in WAS-exposed rats, and the density of CBS-positive enteric neurons in the myenteric plexus in WAS-treated rats was lower than that in controls. SAM treatment relieved WAS-induced colonic hypermotility via increased H2 S production. AZD6244, a selective inhibitor of MEK1, partially reversed CBS downregulation and colonic hypermotility in WAS-treated rats. Decreased CBS/H2 S signaling through increased MEK1 signaling might be important in the pathogenesis of chronic stress-induced colonic hypermotility. SAM could be administered for disorders associated with intestinal hypermotility.

Bromodomain inhibition overcomes treatment resistance in distinct molecular subtypes of melanoma

Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior invivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Abstract 3004: Novel effect of Selumetinib-mediated autophagy via HSF1 in K-Ras mutant pancreatic cancer

Abstract Introduction: K-Ras mutant cancers such as pancreatic cancer are a major cause of cancer-related death and are difficult to treat, with a five-year survival rate of less than 6%. It is reported that oncogenic K-Ras signaling passes through RAF/MEK/ERK pathways. Selumetinib (AZD6244) is a selective MEK inhibitor for K-Ras mutant cancers dependent on mitogen-activated protein kinase (MAPK) signaling pathway and is currently in phase II trials. However, the underlying mechanisms of action are not well known. Autophagy is a self-digest pathway to degrade cellular organelles and macromolecules in maintaining proteome homeostasis, and it is suppressed by the mammalian target of rapamycin complex 1 (mTORC1), a master regulator of translation and autophagy. mTOR activity is known to be activated by AMP-activated protein kinase (AMPK) and negatively regulated by c-Jun N-terminal kinase 1/2 (JNK1/2) MAPK signaling. Autophagy is thought to play protective and suppressive roles in cancer; however, the molecular basis for the relationship between the induction of autophagy and the initiation of pancreatic malignancy is currently unknown. Heat shock factor 1 (HSF1), a key transcription factor involved in proteotoxic stress response via protein-folding. HSF1 plays a pro-oncogenic role in the development of cancer by regulating signaling transduction and translation during tumorigenesis. HSF1 is reportedly hyperactive in pancreatic cancers. AZD6244 reportedly decreases HSF1 phosphorylation at Ser326 and HSF1 expression in melanomas and therefore abates cancer development. However, the detailed mechanism by which HSF1 engages in AZD6244-mediated autophagy in K-Ras mutant human pancreatic cancer is not fully understood. Objectives: This study aims to investigate the role of HSF1 in AZD6244-mediated autophagy in K-Ras mutant human pancreatic cancer. Methods and Results: AZD6244 induces robust autophagy response in human pancreatic cells with hyperactive K-Ras signaling. Simultaneously, AZD6244 reduces HSF1 phosphorylation at Ser326 and HSF1 expression in human pancreatic cancer cells. Intriguingly, genetic deletion of HSF1 induces autophagy under starvation condition. In addition, AZD6244 induces phosphorylation of AMPK Ser172 and JNK1/2 T183/Y185 and decreases mTORC1 activity in human pancreatic cancer cells. Pharmaceutical inhibition of AMPK or genetic deletion of JNK1/2 prevents AZD6244-induced autophagy. Furthermore, HSF1 knockdown induces phosphorylation of Unc-51-like kinase 1(ULK1) Ser555 and decreases ULK Ser757, which is mediated by AMPK and mTOR, respectively. Conclusion: HSF1 plays an integral role in Selumetinib-mediated autophagy through AMPK and/or JNK1/2 signaling in K-Ras mutant human pancreatic cancer cells. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. Novel effect of Selumetinib-mediated autophagy via HSF1 in K-Ras mutant pancreatic cancer [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 3004.

Cobimetinib Sensitizes Cervical Cancer to Paclitaxel via Suppressing Paclitaxel-Induced ERK Activation

Introduction: Chemoresistance remains the main cause of treatment failure in cervical cancer and novel therapeutic strategies are required. Cobimetinib, a potent yet selective inhibitor of MEK1 and 2, is currently used to treat melanoma clinically. In this work, we identified cobimetinib as a promising candidate for treating cervical cancer. Methods: The in vitro and in vivo efficacies of cobimetinib were examined using cervical cancer cell cultures and xenograft mouse model. Its combination with paclitaxel was analyzed using the combination index. Immunoblotting was performed on MAPK and ERK pathways. Results: Cobimetinib displays a potent anti-cervical cancer activity in a panel of cell lines regardless of cellular origin and HPV presence, and its combination with paclitaxel is synergistic in inhibiting cervical cancer cells. This is achieved by the growth inhibition and caspase-dependent apoptosis induction, through inhibiting MAPK/ERK activation. In addition, paclitaxel activates ERK in cervical cancer cells, and this can be reversed by cobimetinib. We finally confirm the efficacy of cobimetinib alone and its combination with paclitaxel in the cervical cancer xenograft mouse model. Discussion/Conclusion: Our preclinical findings will accelerate the initialization of clinical trials to use combination of cobimetinib and paclitaxel for treating cervical cancer. Our work also emphasizes the therapeutic value of targeting MAPK/ERK to overcome chemoresistance in cervical cancer.

AP-1 signaling pathway promotes pro-IL-1β transcription to facilitate NLRP3 inflammasome activation upon influenza A virus infection

ABSTRACT NLRP3 inflammasome mainly controls interleukin-1β (IL-1β) secretion, leading to cell death called pyroptosis constituting a major antiviral host defense and inflammatory diseases upon viral infection. The RAF-MEK1/2-ERK1/2 cascade and downstream c-Jun/Fos and Activator protein-1 (AP1) signaling pathway control the degree of inflammatory response. Influenza A virus (IAV) infection is known to stimulate NLRP3 inflammasome activation and inflammatory responses. Nevertheless, the detailed mechanism by which IAV induces NLRP3 inflammasome activation involved in transcription of pro-IL-1β mRNA remains elusive. In our study, we found that IAV infection promotes pro-IL-1β mRNA transcription and activates NLRP3 inflammasome. Detailed studies reveal that type I interferon (IFN-α/IFN-β) as well as U0126 (a selective inhibitor of MEK-1 and MEK-2) typically inhibit IAV-mediated NLRP3 inflammasome activation via downregulating pro-IL-1β mRNA. Moreover, knock-down of c-Jun decreases pro-IL-1β mRNA and inhibits NLRP3 inflammasome activation upon IAV infection. Overall, the findings uncover that AP-1 signaling pathway promotes NLRP3 inflammasome activation upon IAV infection, which provides a new idea for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

Selumetinib: a selective MEK1 inhibitor for solid tumor treatment.

Cancer incidence is rapidly growing. Solid tumors are responsible for a majority of cancers. Recently, molecular-targeted agents have played a significant role in cancer treatment. Ras-Raf-MEK-ERK signaling pathway, is a substantial element in the survival, propagation, and drug resistance of human cancers. MEK is a specific part of the so-called cascade, and ERK proteins are its sole target. Furthermore, their downstream position in the Ras-ERK cascade, is noteworthy to direct their function in patients with upstream mutated genes. MEK1 mutations are responsible for initiating several solid tumors. Selumetinib (AZD6244) is a second-generation, selective, potent, and non-ATP competitive allosteric MEK1 inhibitor. The efficacy of selumetinib in various solid tumors such as colorectal cancer, lung cancer, neurofibroma, and melanoma is investigated. The present paper provides an overview of the MAPK cascade, the role of selumetinib as a MEK1/2 inhibitor, and the related findings of clinical trials for solid tumor treatment.

S2926 Craniopharyngioma-Induced Hypothalamic Obesity and a Red Herring Known as Binimetinib

Introduction: Craniopharyngioma suprasellar extensions damage hypothalamic nuclei resulting in obesity. Binimetinib can reduce the size of craniopharyngiomas by 30%, but also contributes to an increase in adipose and muscle mass. Here is a case of hypothalamic obesity causing weight regain post sleeve gastrectomy (LSG) being successfully treated with a GLP-1 receptor agonist to decrease her weight and Hba1c. Case Description/Methods: A 26-year-old female patient with a BMI of 29 presents to the clinic with a craniopharyngioma-induced mass effect and panhypopituitarism. She had undergone transsphenoidal resections, external beam radiation, chemotherapy, gamma knife, and a ventriculoperitoneal shunt. Due to hypothalamic nucleus injury, she developed hyperphagia with a BMI of 36 kg/m2 and NAFLD-induced cirrhosis. She had a sleeve gastrectomy and reduced her BMI to 26. Her medications include desmopressin, insulin, levothyroxine, hydrocortisone, and recently started on binimetinib. She rapidly regained weight now with a BMI of 29. Abnormal labs include an Hba1c 9.7 g/dL, PT 15.6, INR 1.4, platelets 50, AST 51 u/L and ALT 48 u/L. MRI of her brain with contrast confirmed the suprasellar extension of the craniopharyngioma impinging on the hypothalamus (Figure 1). An esophagram demonstrated the patency of her LSG. After starting 1.8 mg of liraglutide, she lost 20 lbs with a BMI of 27 and dropped her Hba1c to 6.1 g/dL in 4 months. Discussion: Carniopharyngiomas can result in obesity due to damage to regulatory medial hypothalamic nuclei, specifically the arcuate nucleus, paraventricular nucleus, and the ventromedial nucleus. This occurs secondary to tumor mass effects, surgical resection of tumor, or from exposure to radiation >51Gy. This leads to leptin resistance causing hyperphagia, hyperinsulinemia, and decreased energy expenditure causing rapid and stubborn weight gain. A selective inhibitor of MEK, binimetinib, increases the muscle mass and muscle fiber size through inhibition of C26 and IL6, leading to an average muscle gain of 2.3 kg/100 days and 4.2% increase in adipose tissue. Radiation treatment in doses ≥ 54 Gy leads to an increased visceral fat and insulin resistance. Treatment includes a roux-en-y gastric bypass, GLP-1 receptor agonists, and metformin. GLP-1s have also recently shown improvement in NASH resolution. Identification of medication side effects and incorporation of weight loss medications can non-invasively treat hypothalamic obesity weight gain and prevent weight regain.Figure 1.: MRI depicting suprasellar extension of the craniopharyngioma into the hypothalamus.

Cancer thyroïdien réfractaire/Determinants of thyroid cancer differentiation state and response to radioiodine

The use of RAI is undergoing a significant reappraisal based on its questionable efficacy in various disease contexts. Current indications for adjuvant RAI treatment are not based on randomized clinical trials, but instead on consensus expert recommendations that rely on retrospective studies, despite their methodological weaknesses and biases. Most thyroid cancers are driven by oncoproteins that activate MAPK signaling, and the transcriptional output of this pathway is inversely correlated with the expression of genes that govern many of the specialized functions of thyroid cells, including the ability to incorporate iodide into thyroid hormones. Most clinical guidelines recommend treating patients with tumors > 2 cm or with significant nodal disease with RAI after surgery. In our view this is problematic, as tumors giving rise to nodal disease are markedly enriched for BRAFV600E, which have a high MAPK pathway flux and are mostly unresponsive to RAI. We will discuss the genomic determinants of the thyroid differentiation state in thyroid cancer, their relationship to RAI avidity and the extent by which thyroid differentiation can be restored by treatment with selective RAF or MEK inhibitors, or with combinations of compounds that block the MAPK output more profoundly. We will also discuss insights arising from the analysis of patients with exceptional responses to radioactive iodine, and by contrast, the genetic lesions that determine irreversible loss of thyroid identity, primarily those associated with loss of function of genes encoding for chromatin remodeling proteins.

Trametinib enhances ATRA-induced differentiation in AML cells

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib–ATRA (tra–ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra–ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.