Cryo-EM structure of orf virus scaffolding protein orfv075.

Capsid-like poxvirus scaffold proteins self-assemble into semi-regular lattice that govern the formation of spherical immature virus particles. The scaffolding is a critical step in virus morphogenesis as exemplified by the drug rifampicin that impairs the recruitment of scaffold onto the viral membrane in vaccinia virus (VACV). Here we report cryo-electron microscopy structure of scaffolding protein Orfv075 of orf virus (ORFV) that causes smallpox-like diseases in sheep, goats and occasionally humans via zoonotic infection. We demonstrate that the regions that are involved in intertrimeric interactions for scaffold assembly are largely conserved in comparison to its VACV orthologue protein D13 whose intermediate assembly structures have been previously characterized. By contrast, less conserved regions are located away from these interfaces, indicating both viruses share similar assembly mechanisms. We also show that the phenylalanine-rich binding site of rifampicin in D13 is conserved in Orfv075, and molecular docking simulation confirms similar binding modes. Our study provides structural basis of scaffolding protein as a target for anti-poxvirus treatment across wide range of poxvirus genera.

Distinct features of B cell receptors in neuromyelitis optica spectrum disorder among CNS inflammatory demyelinating diseases.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) stands out among CNS inflammatory demyelinating diseases (CIDDs) due to its unique disease characteristics, including severe clinical attacks with extensive lesions and its association with systemic autoimmune diseases. We aimed to investigate whether characteristics of B cell receptors (BCRs) differ between NMOSD and other CIDDs using high-throughput sequencing. METHODS: From a prospective cohort, we recruited patients with CIDDs and categorized them based on the presence and type of autoantibodies: NMOSD with anti-aquaporin-4 antibodies, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) with anti-myelin oligodendrocyte glycoprotein antibodies, double-seronegative demyelinating disease (DSN), and healthy controls (HCs). The BCR features, including isotype class, clonality, somatic hypermutation (SHM), and the third complementarity-determining region (CDR3) length, were analyzed and compared among the different disease groups. RESULTS: Blood samples from 33 patients with CIDDs (13 NMOSD, 12 MOGAD, and 8 DSN) and 34 HCs were investigated for BCR sequencing. Patients with NMOSD tended to have more activated BCR features compare to the other disease groups. They showed a lower proportion of unswitched isotypes (IgM and IgD) and a higher proportion of switched isotypes (IgG), increased clonality of BCRs, higher rates of SHM, and shorter lengths of CDR3. Notably, advanced age was identified as a clinical factor associated with these activated BCR features, including increased levels of clonality and SHM rates in the NMOSD group. Conversely, no such clinical factors were found to be associated with activated BCR features in the other CIDD groups. CONCLUSIONS: NMOSD patients, among those with CIDDs, displayed the most pronounced B cell activation, characterized by higher levels of isotype class switching, clonality, SHM rates, and shorter CDR3 lengths. These findings suggest that B cell-mediated humoral immune responses and characteristics in NMOSD patients are distinct from those observed in the other CIDDs, including MOGAD. Age was identified as a clinical factor associated with BCR activation specifically in NMOSD, implying the significance of persistent B cell activation attributed to anti-aquaporin-4 antibodies, even in the absence of clinical relapses throughout an individual's lifetime.

Longitudinal follow-up of serum biomarkers in patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Recently, several serum biomarkers have been proposed in Neuromyelitis Optica Spectrum Disorders (NMOSD) to monitor disease activity. OBJECTIVE: The objective of the study is to evaluate the longitudinal clinical value of serum biomarkers in patients with NMOSD. METHODS: We prospectively recruited consecutive NMOSD patients with anti-aquaporin-4 antibody and obtained serum samples at enrollment, after 6-12 months of follow-up (main period), and at attacks. Using single-molecule array assays, we evaluated longitudinal changes of serum neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and GFAP/NfL levels. RESULTS: Overall, 64 patients (58 women) were enrolled (age: 51 years, disease duration: 6.7 years) and 133 samples were obtained. Among patients who did not develop new attacks during the main period (n = 62), serum levels of NfL, GFAP, and GFAP/NfL were significantly decreased over time in patients with attacks (<2 months) at enrollment (n = 14 (23%)), whereas serum NfL and GFAP levels gradually increased in the others (n = 48 (77%)). During the study, five (8%) patients developed new attacks; only serum GFAP levels increased consistently upon these events compared with baseline levels. To differentiate attacks from remissions, serum GFAP levels showed the largest area under the receiver operating characteristic curve (0.876, 95% confidence interval: 0.801-0.951). CONCLUSION: Among NfL, GFAP, and GFAP/NfL, serum GFAP might be the most appropriate for monitoring NMOSD longitudinally, which warrants future confirming studies.

Targeting ß-catenin overcomes MEK inhibition resistance in colon cancer with KRAS and PIK3CA mutations.

BACKGROUND: Mitogen-activated protein kinases (MEK 1/2) are central components of the RAS signalling pathway and are attractive targets for cancer therapy. These agents continue to be investigated in KRAS mutant colon cancer but are met with significant resistance. Clinical investigations have demonstrated that these strategies are not well tolerated by patients. METHODS: We investigated a biomarker of response for MEK inhibition in KRAS mutant colon cancers by LC-MS/MS analysis. We tested the MEK inhibitor in PIK3CA wild(wt) and mutant(mt) colon cancer cells. In addition, we tested the combinational effects of MEK and TNKS inhibitor in vitro and in vivo. RESULTS: We identified ß-catenin, a key mediator of the WNT pathway, in response to MEK inhibitor. MEK inhibition led to a decrease in ß-catenin in PIK3CA wt colon cancer cells but not in mt. Tumour regression was promoted by combination of MEK inhibition and NVP-TNS656, which targets the WNT pathway. Furthermore, inhibition of MEK promoted tumour regression in colon cancer patient-derived xenograft models expressing PIK3CA wt. CONCLUSIONS: We propose that inhibition of the WNT pathway, particularly ß-catenin, may bypass resistance to MEK inhibition in human PIK3CA mt colon cancer. Therefore, we suggest that ß-catenin is a potential predictive marker of MEK inhibitor resistance.

EBV-encoded EBNA1 regulates cell viability by modulating miR34a-NOX2-ROS signaling in gastric cancer cells.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a viral protein expressed in all EBV-infected cells that induces malignant transformation. EBNA1 is reported to contribute to tumor progression through an increase in reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase. However, the underlying molecular mechanism of EBNA1-induced ROS accumulation in gastric cancer is poorly understood. Here, we demonstrated that miR34a regulation by EBNA1 determined cell fate in EBV-infected gastric cancer cells. ROS content and NOX2 expression were higher in EBNA1-expressing SNU719 cells than in EBNA1-nonexpressing SNU638 cells. Downregulation of NOX2 using siRNA technology in SNU719 cells decreased cell viability and ROS content. Regulation of EBNA1 expression in EBV-associated gastric cancers modulated NOX2 expression, ROS content and cell viability. We also showed that upregulation of NOX2 by EBNA1 was mediated by downregulating miRNA34a. Finally, overexpression of miR34a in EBNA1-expressing SNU719 cells induced typical apoptosis, suggesting that reactivation of miR34a in EBNA1-expressing gastric cancer cells could be a strategy for treatment of EBV-infected gastric cancer cells.

Epstein-Barr virus-encoded latent membrane protein 1 induces epithelial to mesenchymal transition by inducing V-set Ig domain containing 4 (VSIG4) expression via NF-kB in renal tubular epithelial HK-2 cells.

The epithelial to mesenchymal transition (EMT), a hallmark of chronic kidney disease, is a key event in the conversion from tubular epithelial cells to myofibroblasts in renal fibrosis. Epstein-Barr virus (EBV) is a γ-herpes oncovirus associated with chronic kidney disease. However, the relationship between EBV and the EMT process in renal tubular epithelial cells is not well understood. Among EBV-latent genes, EBV-encoded latent membrane protein 1 (LMP1) induces EMT by regulating a variety of molecules in EBV-induced oncogenic transformation. In this study, we investigated EBV-encoded LMP1 and EMT process markers in human proximal tubule epithelial cell line HK-2. LMP1 overexpression induces cell morphological changes via the epithelial to mesenchymal process in HK-2 cells, and these changes accelerate cell proliferation, cell motility, and invasion. Furthermore, VSIG4 upregulation by EBV-LMP1 induced LMP1-mediated EMT, cell motility, and invasion. VSIG4 upregulation by LMP1 was regulated at the transcriptional level via the NF-kB signaling axis. These results suggest that EBV-encoded LMP1 regulates EMT through the NF-kB-VSIG4 axis in HK-2 cells, and VSIG4 is a potential target in EBV-induced chronic kidney diseases.

RPS27a enhances EBV-encoded LMP1-mediated proliferation and invasion by stabilizing of LMP1.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is an oncoviral protein that plays a pivotal role in EBV-induced oncogenic transformation. The function of LMP1 in EBV-induced oncogenesis has been well studied. However, the molecular mechanisms underlying LMP1 protein stability remain poorly understood. In this study, we found that ribosomal protein s27a (RPS27a) regulates LMP1 stability by a tandem affinity purification analysis. RPS27a interacts directly with LMP1 in vitro and in vivo. Furthermore, overexpression of RPS27a increases the half-life of LMP1 in 293T cells, whereas downregulation of RPS27a using lentiviral shRNA technology accelerates the decrease in LMP1 protein level in EBV-transformed B cells. We show that LMP1 ubiquitination via the proteasome is completely inhibited by overexpression of RPS27a. RPS27a also enhances LMP1-mediated proliferation and invasion, suggesting that RPS27a interacts with LMP1 and stabilizes it by suppressing proteasome-mediated ubiquitination. These results suggest that RSP27a could be a potential target in EBV-infected LMP1-positive cancer cells.

Cellular inhibitor of apoptosis protein 1 (cIAP1) stability contributes to YM155 resistance in human gastric cancer cells.

YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.

SAHA, an HDAC inhibitor, overcomes erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin.

Pancreatic cancer is one of the most lethal cancers and remains a major unsolved health problem. Less than 20 % of patients are surgical candidates, and the median survival for non-resected patients is approximately 3 to 4 months. Despite the existence of many conventional cancer therapies, few targeted therapies have been developed for pancreatic cancer. Combination therapy using erlotinib and gemcitabine is an approved standard chemotherapy for advanced pancreatic cancer, but it has marginal therapeutic benefit. To try to improve the therapeutic outlook, we studied the efficacy of another combination treatment and the relevance to E-cadherin in human pancreatic cancer cells. We treated two human pancreatic cancer cell lines with the histone deacetylase inhibitor (HDACi) SAHA. Interestingly, in these Panc-1 and Capan1 cells, we observed that the expression levels of E-cadherin and phosphorylated EGFR were gradually upregulated after treatment with SAHA. Furthermore, these cells underwent induced cell death after exposure to the combination treatment of SAHA and erlotinib. In Panc-1 cells, overexpression of E-cadherin activated the phosphorylation of EGFR and increased the cell sensitivity to erlotinib. In Capan1 cells, knocking down E-cadherin decreased the expression of phosphorylated EGFR, and these cells did not respond to erlotinib. Therefore, we demonstrated the efficacy of the combined treatment with SAHA and erlotinib in human pancreatic cancer cells, and we determined that the increased efficacy was due, at least in part, to the effects of SAHA on the expression of E-cadherin. Our studies suggest that E-cadherin may be a potent biomarker for pancreatic cancer.

TFAP2E methylation status and prognosis of patients with radically resected colorectal cancer.

OBJECTIVES: This study investigates the clinical significance of the gene encoding AP-2ε (TFAP2E) in colorectal cancer (CRC) patients undergoing curative resection. METHODS: A single-institution cohort of 248 patients who underwent curative resection of stage I/II/III CRCs between March and December 2004 was enrolled, and 193 patients whose tumors were available for the determination of the TFAP2E methylation status were included in the analysis. RESULTS: TFAP2E hypermethylation was detected in 112 patients (58%) and was significantly associated with distally located CRCs, low pathologic T stage (T1/T2), and stage I tumors. After a median follow-up of 86.3 months, the patients with TFAP2E hypermethylation tended to show better relapse-free survival (RFS) and overall survival (OS) than the patients with TFAP2E hypomethylation (5-year RFS rate: 90 vs. 80%, p = 0.063; 6-year OS rate: 88 vs. 80%, p = 0.083). Multivariate analysis showed that the pathologic nodal stage and TFAP2E methylation status were independent prognostic factors for RFS and OS, and they remained significant factors in the subgroup analysis that included 154 patients with stage II/III CRCs who had received adjuvant chemotherapy. CONCLUSIONS: TFAP2E hypermethylation is associated with good clinical outcomes and may be considered as an independent prognostic factor in patients with curatively resected CRCs.

NPS-1034, a novel MET inhibitor, inhibits the activated MET receptor and its constitutively active mutants.

The MET proto-oncogene product, which is the receptor for hepatocyte growth factor (HGF), has been implicated in tumorigenesis and metastatic progression. Point mutations in MET lead to the aberrant activation of the receptor in many types of human malignancies, and the deregulated activity of MET has been correlated with tumor growth, invasion, and metastasis. MET has therefore attracted considerable attention as a potential target in anticancer therapy. Here, we report that a novel MET kinase inhibitor, NPS-1034, inhibits various constitutively active mutant forms of MET as well as HGF-activated wild-type MET. NPS-1034 inhibited the proliferation of cells expressing activated MET and promoted the regression of tumors formed from such cells in a mouse xenograft model through anti-angiogenic and pro-apoptotic actions. NPS-1034 also inhibited HGF-stimulated activation of MET signaling in the presence or absence of serum. Furthermore, when tested on 27 different MET variants, NPS-1034 inhibited 15 of the 17 MET variants that exhibited autophosphorylation with nanomolar potency; only the F1218I and M1149T variants were not inhibited by NPS-1034. Notably, NPS-1034 inhibited three MET variants that are resistant to the MET inhibitors SU11274, NVP-BVU972, and PHA665752. Together, these results suggest that NPS-1034 can be used as a potent therapeutic agent for human malignancies bearing MET point mutations or expressing activated MET.

Rab11 suppresses neuronal stress signaling by localizing Dual leucine zipper kinase to axon terminals for protein turnover.

Dual Leucine Zipper Kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals by Rab11 for protein turnover. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling. Highlights: Wnd is highly enriched in axon terminals.Wnd protein turnover by Hiw is restricted in the axon terminals.Protein palmitoylation of Wnd and Rab11 activity is essential for Wnd axonal localization. Rab11 mutations and defective Wnd palmitoylation impair Wnd protein turnover leading to increased Wnd protein levels and neuronal loss. Inhibiting Wnd activity mitigates neuronal stress response caused by Rab11 loss-of-function.

Blood sphingolipid as a novel biomarker in patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Sphingolipids are signaling molecules and structural components of the axolemma and myelin sheath. Plasma sphingolipid levels may reflect disease status of neuromyelitis optica spectrum disorder (NMOSD). We aimed to examine plasma sphingolipids as disease severity biomarkers for NMOSD and compare their characteristics with those of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP). METHODS: We measured plasma sphingolipids, sNfL, and sGFAP levels in NMOSD cases with anti-aquaporin-4-antibody. An unbiased approach, partial least square discriminant analysis (PLS-DA), was utilized to determine whether sphingolipid profiles differ according to the disease state of NMOSD (presence, moderate-to-severe disability [Expanded Disease Severity Scale, (EDSS) > 3.0], and relapses). RESULTS: We investigated 81 patients and 10 controls. PLS-DA models utilizing sphingolipids successfully differentiated patients with EDSS > 3.0, but failed to identify the presence of disease and relapses. Ceramide-C14-a significant contributor to differentiating EDSS > 3.0-positively correlated with EDSS, while its levels were independent of age and the presence of relapses. This characteristic was unique from those of sNfL and sGFAP, which were affected by age and relapses as well as EDSS. CONCLUSION: Plasma sphingolipids may be useful NMOSD biomarkers for disability with distinct characteristics compared to sNfL and sGFAP.

Ring finger protein 149 is an E3 ubiquitin ligase active on wild-type v-Raf murine sarcoma viral oncogene homolog B1 (BRAF).

Members of the RAF family (ARAF, BRAF, and CRAF/RAF-1) are involved in a variety of cellular activities, including growth, survival, differentiation, and transformation. An oncogene encodes BRAF, the function of which is linked to MEK activation. BRAF is the most effective RAF kinase in terms of induction of MEK/ERK activity. However, the mechanisms involved in BRAF regulation remain unclear. In the present work, we used a tandem affinity purification approach to show that RNF149 (RING finger protein 149) interacts with wild-type BRAF. The latter protein is a RING domain-containing E3 ubiquitin ligase involved in control of gene transcription, translation, cytoskeletal organization, cell adhesion, and epithelial development. We showed that RNF149 bound directly to the C-terminal kinase-containing domain of wild-type BRAF and induced ubiquitination, followed by proteasome-dependent degradation, of the latter protein. Functionally, RNF149 attenuated the increase in cell growth induced by wild-type BRAF. However, RNF149 did not bind to mutant BRAF or induce ubiquitination thereof. Thus, we show that RNF149 is an E3 ubiquitin ligase active on wild-type BRAF.

Recent Advances in the Chemobiological Upcycling of Polyethylene Terephthalate (PET) into Value-Added Chemicals.

Polyethylene terephthalate (PET) is a plastic material commonly applied to beverage packaging used in everyday life. Owing to PET's versatility and ease of use, its consumption has continuously increased, resulting in considerable waste generation. Several physical and chemical recycling processes have been developed to address this problem. Recently, biological upcycling is being actively studied and has come to be regarded as a powerful technology for overcoming the economic issues associated with conventional recycling methods. For upcycling, PET should be degraded into small molecules, such as terephthalic acid and ethylene glycol, which are utilized as substrates for bioconversion, through various degradation processes, including gasification, pyrolysis, and chemical/biological depolymerization. Furthermore, biological upcycling methods have been applied to biosynthesize value-added chemicals, such as adipic acid, muconic acid, catechol, vanillin, and glycolic acid. In this review, we introduce and discuss various degradation methods that yield substrates for bioconversion and biological upcycling processes to produce value-added biochemicals. These technologies encourage a circular economy, which reduces the amount of waste released into the environment.

Disease characteristics of idiopathic transverse myelitis with serum neuronal and astroglial damage biomarkers.

Despite its close association with CNS inflammatory demyelinating disorders (CIDDs), pathogenic characteristics of idiopathic transverse myelitis (ITM) remain largely unknown. Here, we investigated serum levels of neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) in patients with ITM to unravel the disease characteristics of ITM. We prospectively recruited 70 patients with ITM, 62 with AQP4 + NMOSD and 85 with RRMS-including 31 patients with acute TM attacks-along with 30 HCs. We measured sNfL and sGFAP levels using single-molecular arrays and compared these levels per lesion volume between the disease groups during attacks. Compared to HCs, ITM patients showed higher sNfL and sGFAP during acute attacks (sNfL: p < 0.001, sGFAP: p = 0.024), while those in remission (sNfL: p = 0.944, sGFAP: p > 0.999) did not, regardless of lesion extents and presence of multiple attacks. ITM patients demonstrated lower sGFAP/volume (p = 0.011) during acute attacks and lower sGFAP (p < 0.001) in remission compared to AQP4 + NMOSD patients. These findings suggest that both neuronal and astroglial damages occur in patients with acute ITM attacks at a similar level to those with RRMS, distinct from AQP4 + NMOSD. However, active neuroinflammatory process was not remarkable during remission in this cohort.

Serum proteins for monitoring and predicting visual function in patients with recent optic neuritis.

It is unclear whether serum proteins can serve as biomarkers to reflect pathological changes and predict recovery in inflammation of optic nerve. We evaluated whether serum proteins could monitor and prognosticate optic neuritis (ON). We prospectively recruited consecutive patients with recent ON, classified as ON with anti-aquaporin-4 antibody (AQP4-ON), ON with anti-myelin oligodendrocyte glycoprotein antibody (MOG-ON), and double-seronegative ON (DSN-ON). Using ultrasensitive single-molecule array assays, we measured serum neurofilament light chain and glial fibrillary acidic protein (GFAP), and brain-derived neurotrophic factor (BDNF). We analyzed the markers according to disease group, state, severity, and prognosis. We enrolled 60 patients with recent ON (15 AQP4-ON; 14 MOG-ON; 31 DSN-ON). At baseline, AQP4-ON group had significantly higher serum GFAP levels than did other groups. In AQP4-ON group, serum GFAP levels were significantly higher in the attack state than in the remission state and correlated with poor visual acuity. As a prognostic indicator, serum BDNF levels were positively correlated with follow-up visual function in the AQP4-ON group (r = 0.726, p = 0.027). Serum GFAP reflected disease status and severity, while serum BDNF was identified as a prognostic biomarker in AQP4-ON. Serum biomarkers are potentially helpful for patients with ON, particularly those with AQP4-ON.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer.

Significant improvement in targeted therapy for colorectal cancer (CRC) has occurred over the past few decades since the approval of the EGFR inhibitor cetuximab. However, cetuximab is used only for patients possessing the wild-type oncogene KRAS, NRAS, and BRAF, and even most of these eventually acquire therapeutic resistance, via activation of parallel oncogenic pathways such as RAS-MAPK or PI3K/Akt/mTOR. The two aforementioned pathways also contribute to the development of therapeutic resistance in CRC patients, due to compensatory and feedback mechanisms. Therefore, combination drug therapies (versus monotherapy) targeting these multiple pathways may be necessary for further efficacy against CRC. In this study, we identified PIK3CA mutant (PIK3CA MT) as a determinant of resistance to SMI-4a, a highly selective PIM1 kinase inhibitor, in CRC cell lines. In CRC cell lines, SMI-4a showed its effect only in PIK3CA wild type (PIK3CA WT) cell lines, while PIK3CA MT cells did not respond to SMI-4a in cell death assays. In vivo xenograft and PDX experiments confirmed that PIK3CA MT is responsible for the resistance to SMI-4a. Inhibition of PIK3CA MT by PI3K inhibitors restored SMI-4a sensitivity in PIK3CA MT CRC cell lines. Taken together, these results demonstrate that sensitivity to SMI-4a is determined by the PIK3CA genotype and that co-targeting of PI3K and PIM1 in PIK3CA MT CRC patients could be a promising and novel therapeutic approach for refractory CRC patients.

Erlotinib Activates Different Cell Death Pathways in EGFR-mutant Lung Cancer Cells Grown in 3D Versus 2D Culture Systems.

BACKGROUND/AIM: Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutation have been shown to have a good response to erlotinib, a receptor tyrosine kinase inhibitor of EGFR. In this study, we found that the cell death pathways activated by erlotinib in 2D and 3D culture systems are different. MATERIALS AND METHODS: The cell death pathways induced by erlotinib were evaluated by flow cytometry and immunoblotting in both 2D and 3D culture systems of EGFR mutant lung cancer cells. RESULTS: Treatment with erlotinib induced caspase 8 activation and up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) expression only in 3D cultures. Knockdown of TRAIL attenuated both erlotinib-induced activation of caspase-8 and apoptosis in 3D cultures. Erlotinib also increased LC3, an autophagy marker, expression and c-Jun N terminal kinase (JNK) activation. Both 3-MA as an autophagy inhibitor and SP600125 as a JNK inhibitor, significantly inhibited erlotinib-induced cell death. CONCLUSION: Erlotinib induces apoptotic cell death in 3D cultures through an autophagy-TRAIL-JNK pathway.