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PURPOSE Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the counter effect of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancers (CRCs). METHODS Plasma HGF levels were analyzed with clinical outcomes of patients with metastatic CRC (mCRC) receiving palliative first-line chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition by capmatinib. RESULTS A total of 80 patients were included: cetuximab + FOLFIRI (n = 35) and bevacizumab + FOLFIRI (n = 45). Both progression-free survival (PFS) and overall survival (OS) were significantly lesser in the high vs low HGF group: median 11.8 vs. 24.7 months, respectively, for PFS (p = 0.009), and median 21.1 months vs. not reached, respectively, for OS (p = 0.018). The difference was significantly evident in the cetuximab group. In five RAS/RAF wild-type CRC cells, the addition of HGF activated ERK1/2 and AKT via MET phosphorylation, resulting in cetuximab resistance in vitro. In cetuximab-sensitive Caco-2 and SNU-C4 cells, capmatinib overcame cetuximab resistance in the presence of HGF by attenuating HGF-induced MET signaling activation. CONCLUSION Patients with mCRC receiving cetuximab + FOLFIRI who presented with high plasma HGF levels had significantly worse PFS and OS. Cetuximab resistance induced by HGF was mediated by AKT and ERK activation and overcome by MET inhibition in vitro.

Peripheral regulatory T cells (pTregs) are a highly immunosuppressive fraction of CD4+ T cells. We aimed to evaluate the clinical significance of pTregs in patients with gastric cancer and to determine the correlation between pTregs and immune cell infiltration in tumor microenvironment. pTregs status was determined by assessing the pTreg/total T-cell ratio (ratio of Foxp3 Treg-specific demethylated region (TSDR) to CD3G/CD3D demethylation, so-called Cellular Ratio of Immune Tolerance “ImmunoCRIT”) using methylation analyses in 433 patients with gastric cancer who received curative surgery. Among 422 evaluable patients, 230 (54.5%) had high ImmunoCRIT (> 21.0). Patients with high ImmunoCRIT had significantly shorter disease-free survival (DFS) and overall survival (OS) than those with high ImmunoCRIT (p = 0.030, p = 0.008, respectively). In multivariate analysis, high ImmunoCRIT kept a prognostic role for shorter OS (hazard ratio [HR] 1.9, 95% confidence interval [CI] 1.4–2.9; p = 0.005). CD3+ cell density and CD4+ cell density was significantly higher within the tumor in high ImmunoCRIT group than those in low ImmunoCRIT group (CD3+ cell, 202.12/mm2 vs. 172.2/mm2, p = 0.029; CD4+ cell, 56.5/mm2 vs. 43.5/mm2, p = 0.007). In conclusion, the peripheral ImmunoCRIT determined by epigenetic methylation analysis provides prognostic information in resected gastric tumors.

Simple Summary Colorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer related mortality worldwide. Especially, the survival of advanced CRC patients who were failed to achieve durable remission after the anti-angiogenic and anti-epithelial growth factor receptor agents are still poor. The aim of our study was to investigate the anti-tumor activity of the CDK4/6 inhibitor, abemaciclib, as a single agent and to identify an optimal combination agent with abemaciclib in CRC cell lines. We confirmed that abemaciclib monotherapy showed anti-tumor activity and combination therapy with abemaciclib and BYL719 demonstrated synergistic effects in CRC cell lines. Moreover, our study suggested that PIK3CA mutation could be a predictive marker for efficacy of abemaciclib and BYL719 combination therapy. These findings provide novel insight into a possible therapeutic strategy for patients with relapsed and refractory CRC. Abstract Targeting cell cycle regulation in colorectal cancer has not been fully evaluated. We investigated the efficacy of the CDK4/6 inhibitor, abemaciclib, and confirmed a synergistic interaction for PI3K p110α and CDK dual inhibition in colorectal cancer cell lines. Caco-2 and SNU-C4 cell lines were selected to explore the mechanism of action for and resistance to abemaciclib. In vitro and in vivo models were used to validate the anti-tumor activity of abemaciclib monotherapy and BYL719 combination therapy. Abemaciclib monotherapy inhibited cell cycle progression and proliferation in Caco-2 and SNU-C4 cells. CDK2-mediated Rb phosphorylation and AKT phosphorylation appeared to be potential resistance mechanisms to abemaciclib monotherapy. Abemaciclib/BYL719 combination therapy demonstrated synergistic effects regardless of PIK3CA mutation status but showed greater efficacy in the PIK3CA mutated SNU-C4 cell line. Growth inhibition, cell cycle arrest, and migration inhibition were confirmed as mechanisms of action for this combination. In an SNU-C4 mouse xenograft model, abemaciclib/BYL719 combination resulted in tumor growth inhibition and apoptosis with tolerable toxicity. Dual blockade of PI3K p110α and CDK4/6 showed synergistic anti-tumor effects in vivo and in vitro in human colorectal cancer cell lines. This combination could be a promising candidate for the treatment of patients with advanced colorectal cancer.

// Koung Jin Suh 1, 3, * , Ji Hea Sung 1, * , Jin Won Kim 1 , Song-Hee Han 2 , Hye Seung Lee 2 , Ahrum Min 4 , Mi Hyun Kang 1 , Ji Eun Kim 1 , Ji-Won Kim 1 , Se Hyun Kim 1 , Jeong-Ok Lee 1 , Yu Jung Kim 1 , Keun-Wook Lee 1 , Jee Hyun Kim 1 , Soo-Mee Bang 1 , Seock-Ah Im 3, 4 and Jong Seok Lee 1 1 Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Bundang-gu, Seongnam, Korea 2 Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Bundang-gu, Seongnam, Korea 3 Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Jongno-gu, Seoul, Korea 4 Cancer Research Institute, Seoul National University, Jongno-gu, Seoul, Korea * These authors have contributed equally to this work Correspondance to: Jin Won Kim, email: jwkim@snubh.org Keywords: PD-L1, cytokine, EGFR, HER2, PI3K Received: January 04, 2017 Accepted: June 11, 2017 Published: July 12, 2017 ABSTRACT Background: Characteristics of tumor microenvironment have been suggested as predictive markers of anti-EGFR or anti-HER2 treatment response. However, the effect of EGFR/HER2 signal blockade on the tumor immune microenvironment is unclear. Methods: EGFR/HER2 pathway signaling and PD-L1 expression in gastric cancer cell lines were screened by western blot analysis. PD-L1 and HER2 expressions in 251 resected gastric tumors were determined by immunohistochemistry, and changes in EFGR, HER2, and PD-L1 expression in paired specimens between pre- and post-chemotherapy were evaluated. PD-L1 expression in HER2-amplified cell lines was evaluated by western blotting, fluorescence-activated cell sorting, reverse transcription, and real-time quantitative PCR analyses before and after afatinib, lapatinib, pictilisib and trametinib treatment. Changes in cytokines were evaluated by reverse transcription, real-time quantitative PCR, and enzyme-linked immunosorbent assay after EGFR/HER2 inhibition. Results: Cell lines with pEGFR or pHER2 overexpression showed higher PD-L1 expression. In resected gastric tumors, HER2 expression was significantly associated with PD-L1 expression ( p =0.030). PD-L1 overexpression accompanied by increased HER2 expression was identified in a post-chemotherapy specimen from a patient with an initial HER2/PD-L1-negative tumor. In HER2-overexpressing cell lines, PD-L1 expression was decreased in a dose- and time-dependent manner after afatinib and lapatinib treatment. PI3K pathway inhibition by pictilisib, but not MEK pathway inhibition by trametinib, resulted in PD-L1 suppression. After lapatinib treatment, the release of CCL2, CCL21, VEGF and CXCL1 decreased in a dose-dependent manner. Conclusions: Inhibition of the EGFR/HER2 signaling pathway, particularly of downstream PI3K activity, suppressed PD-L1 and release of cytokines, suggesting that EGFR/HER2 inhibition may create a more favorable milieu for tumor immunotherapy.

Purpose HM781-36B is a novel and irreversible pan-human epidermal growth factor receptor (HER) inhibitor with TEC cytoplasmic kinase inhibition. The aim of this study is to evaluate the antitumor activity and mechanism of action for HM781-36B in CRC cell lines. Materials and methods The CRC cell lines were exposed to HM781-36B and/or oxaliplatin (L-OHP), 5-fluorouracil (5-FU), SN-38. The cell viability was examined by Cell Titer-Glo luminescent cell viability assay kit. Change in the cell cycle and protein expression was determined by flow cytometry and immunoblot analysis, respectively. Synergism between 2 drugs was evaluated by the combination index. Results The addition of HM781-36B induced potent growth inhibition in both DiFi cells with EGFR overexpression and SNU-175 cells (IC50, 0.003 µM and 0.005 µM, respectively). Furthermore, HM781-36B induced G1 arrest of the cell cycle and apoptosis, and reduced the levels of HER family and downstream signaling molecules, pERK and pAKT, as well as nonreceptor/cytoplasmic tyrosine kinase, BMX. The combination of HM781-36B with 5-FU, L-OHP, or SN-38 showed an additive or synergistic effect in most CRC cells. Conclusion These findings suggest the potential roles of HM781-36B as the treatment for EGFR-overexpressing colon cancer, singly or in combination with chemotherapeutic agents. The role of BMX expression as a marker of response to HM781-36B should be further explored.

Purpose p21-activated kinases (PAKs) are involved in cytoskeletal reorganization, gene transcription, cell proliferation and survival, and oncogenic transformation. Therefore, we hypothesized that PAK expression levels could predict the sensitivity of pancreatic cancer cells to gemcitabine treatment, and PAKs could be therapeutic targets. Materials and methods Cell viability inhibition by gemcitabine was evaluated in human pancreatic cancer cell lines (Capan-1, Capan-2, MIA PaCa-2, PANC-1, Aspc-1, SNU-213, and SNU-410). Protein expression and mRNA of molecules was detected by immunoblot analysis and reverse transcription polymerase chain reaction. To define the function of PAK4, PAK4 was controlled using PAK4 siRNA. Results Capan-2, PANC-1, and SNU-410 cells were resistant to gemcitabine treatment. Immunoblot analysis of signaling molecules reported to indicate gemcitabine sensitivity showed higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1), a well-known predictive marker for gemcitabine activity, in the resistant cell lines. Knockdown of PAK4 using siRNA induced the upregulation of hENT1. In resistant cell lines (Capan-2, PANC-1, and SNU-410), knockdown of PAK4 by siRNA resulted in restoration of sensitivity to gemcitabine. Conclusion PAK4 could be a predictive marker of gemcitabine sensitivity and a potential therapeutic target to increase gemcitabine sensitivity in pancreatic cancer.

Background: Agents targeting HER2 or EGFR have been applied in several cancer types. Tumor microenvironment including tumor-infiltrating lymphocytes could be a predictive marker in HER2- or EGFR- targeted therapy, although there are some controversies. However, the effects of HER2 and EGFR inhibition on tumor microenvironment are unclear. Method: We screened HER2, EGFR, and PD-L1 expression in gastric cancer cell lines by western blot (SNU216, SNU668, SNU719, AGS, N87, YCC3, and YCC10). HER2 and EGFR was inhibited by using dual inhibitor (afatinib, lapatinib). After HER2 and EGFR inhibition, the change of PD-L1 expression was evaluated in HER2 overexpressed cell lines (SNU216, N87, SKBR3) at western blot, FACS, PCR, and qPCR. Selective blockade for down-steam molecules of HER2 and EGFR was conducted by using pictilisib (PI3K inhibitor) and trametinib (MEK inhibitor). The change of chemokines such as CXCL1, CCL2, and CCL21 was evaluated after HER2 and EGFR inhibition by PCR. In clinical data, PD-L1 expression and HER2 expression in resected gastric cancer were also evaluated by immunohistochemistry analysis. Results: EGFR-overexpressed or HER2-overexpressed gastric cancer cell lines (SNU216, SNU668, N87) showed higher protein expression of PD-L1. PD-L1 expression decreased with dose-dependent manner in afatinib- or lapatinib- treated cell lines (SNU216, N87, SKBR3). In pictilisib-treated cell lines, PD-L1 was also down-regulated. However, trametinib-treated cell lines did not show down-regulation of PD-L1. After lapatinib treatment in HER2 overexpressed cell lines, CXCL1, CCL21, and CCL2 decreased with dose-dependent manner. In 289 patients with resected gastric cancer, there was a significant association between HER2 and PD-L1 expression (p = 0.03). Conclusions: PD-L1 is associated with HER2 and EGFR expression. PD-L1 is regulated by inhibition of PI3K pathway. Therefore, HER2 or EGFR inhibition could interact with tumor microenvironment by down-regulation of expression of PD-L1 and chemokines. Citation Format: Jin Won Kim, Ji Hea Sung, Mi Hyun Kang, Ji-Won Kim, Se-Hyun Kim, Keun-Wook Lee, Hye Seung Lee, Jee Hyun Kim. HER2 or EGFR inhibition interacts with tumor microenvironment by downregulation of PD-L1 and chemokines. [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 5136.

Pancreatic cancer is a highly lethal disease. Although gemcitabine is activelyused in pancreatic cancer, modest activity and relativeresistanceare revealed.P21-activated kinases(PAKs) are involved in cytoskeletal reorganization, gene transcription, cellproliferation and survival, and oncogenic transformation.The gene amplification and protein overexpression of PAK1 and PAK4were reported in pancreatic cancer, which were associated with K-ras mutation, MUC13 and Smad4. Therefore, we hypothesized that PAKs could predict the drug-sensitivity of gemcitabine in pancreatic cancer and could be a therapeutic target. Using cell viability assay of human pancreatic cancer cell lines (Capan1, Capan2, MIA-paca2, PANC1, Aspc1, SNU213, SNU410) for gemcitabine, Capan-2, PANC-1 and SNU-410 cell lines was identifiedas resistant cell lines for gemcitabine. Through western blots of molecules related with down-steam pathway and drug-sensitivity, higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1) were associated with the resistance for gemcitabine. PAK4 down-regulation through siRNA induced the up-regulation of hENT1, which is well-known as predictive marker for gemcitabine activity. In resistance cell lines (Capan-2, PANC-1, SNU-410), down-regulation of PAK4 through siRNArestored the drug-sensitivity for gemcitabine. In conclusion, PAK4 can be a potential predictive marker and therapeutic target in gemcitabine chemotherapy for pancreatic cancer. Citation Format: Sung Ung Moon, Jin Won Kim, Ji Hea Sung, Mi Hyun Kang, Hyun Chang, Jeong Ok Lee, Yu Jung Kim, Keun Wook Lee, Jee Hyun Kim, Soo Mee Bang, Jong Seok Lee. P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2014-912