DataSheet1_Preclinical characterization of tunlametinib, a novel, potent, and selective MEK inhibitor.docx

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.</p

PGRMC1 Is a Novel Potential Tumor Biomarker of Human Renal Cell Carcinoma Based on Quantitative Proteomic and Integrative Biological Assessments - Fig 6

<p><b>Cell proliferation under the overexpression (A) or knockdown (B) of PGRMC1 in OS-RC-2 cells.</b> Each experiment was performed in triplicate. Control: cells transfected with Flag-containing empty plasmids; Mock: cells without treatment; NC: the nonspecific siRNA sequences. * p < 0.05.</p

Associations between PGRMC1 abundance and the overall survival period of RCC patients.

<p>The difference in overall survival was significant between RCC patients with a low-abundant PGRMC1 (with 1–4 scores) and those with a high-abundant PGRMC1 (>4 scores) (P<0.05).</p

Bioinformatic analysis of 82 differential proteins in RCC.

<p>GO enrichment analysis of differential proteins based on molecular function <b>(A)</b> and cellular component <b>(B).</b></p

A statistically higher concentration of serum PGRMC1 from RCC patients.

<p>The average serum PGRMC1 concentration was higher in 18 RCC patients compared to 12 healthy persons (P<0.05) <b>(A)</b>. The serum PGRMC1 concentration was related with the TNM stage of RCC <b>(B)</b>. Serum PGRMC1 abundance was detected in three randomly selected RCC patients <b>(C)</b>. C: a pool of sera from three healthy persons; P₁-P₃: the serum from 3 RCC patients. The total loading pretreated serum proteins were visualized by Ponceau-S staining to take as a comparison control.</p

Quantitative MS data of PGRMC1 in renal carcinoma.

<p>Quantitative MS data of PGRMC1 in renal carcinoma.</p

The relative abundance of PGRMC1 compared between RCTs and PKTs.

<p>The SILAC ratios of β-actin with isotope labeling peptides “EITALAPSTMK” (m/z 582.86/581.33, 2+) respectively from the group of protein mixture composed of PKTs and HEK293 cells <b>(A)</b>, another group of protein sample containing RCTs and HEK293 cells <b>(B)</b>. The isotope labeling peptides ‘GDQPAASGDSDDDEPPPLPR’ (m/z 1019.99/1018.49, 2+) from protein mixture containing PKTs and HEK293 cells <b>(C)</b>, RCTs and HEK293 cells <b>(D)</b> were used to quantify PGRMC1 abundance. PGRMC1 concentration was validated between 3 RCTs and corresponding PKTs by western blotting <b>(E)</b>. RCTs: renal cell carcinoma tissues; PKTs: para-cancerous kidney tissues.</p

PGRMC1 immunoreactivity between RCTs and PKTs.

<p>PGRMC1 immunoreactivity between RCTs and PKTs.</p