Guidelines for Manufacturing and Application of Organoids: Lung.

The objective of standard guideline for utilization of human lung organoids is to provide the basic guidelines required for the manufacture, culture, and quality control of the lung organoids for use in non-clinical efficacy and inhalation toxicity assessments of the respiratory system. As a first step towards the utilization of human lung organoids, the current guideline provides basic, minimal standards that can promote development of alternative testing methods, and can be referenced not only for research, clinical, or commercial uses, but also by experts and researchers at regulatory institutions when assessing safety and efficacy.

The Significance of N6-Methyladenosine RNA Methylation in Regulating the Hepatitis B Virus Life Cycle.

N6-methyladenosine (m6A) RNA methylation has recently emerged as a significant co-transcriptional modification involved in regulating various RNA functions. It plays a vital function in numerous biological processes. Enzymes referred to as m6A methyltransferases, such as the methyltransferaselike (METTL) 3-METTL14-Wilms tumor 1 (WT1)-associated protein (WTAP) complex, are responsible for adding m6A modifications, while m6A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove m6A methylation. The functions of m6A-methylated RNA are regulated through the recognition and interaction of m6A reader proteins. Recent research has shown that m6A methylation takes place at multiple sites within hepatitis B virus (HBV) RNAs, and the location of these modifications can differentially impact the HBV infection. The addition of m6A modifications to HBV RNA can influence its stability and translation, thereby affecting viral replication and pathogenesis. Furthermore, HBV infection can also alter the m6A modification pattern of host RNA, indicating the virus's ability to manipulate host cellular processes, including m6A modification. This manipulation aids in establishing chronic infection, promoting liver disease, and contributing to pathogenesis. A comprehensive understanding of the functional roles of m6A modification during HBV infection is crucial for developing innovative approaches to combat HBV-mediated liver disease. In this review, we explore the functions of m6A modification in HBV replication and its impact on the development of liver disease.

API-2-Induced Cell Migration Is Overcome by Small Molecular Approaches Inhibiting ß-Catenin.

Frequent mutation of APC (90%) in advanced colorectal cancer (CRC) results in the simultaneous activation of Wnt/ß-catenin and AKT signaling pathways, and the current therapeutic limitations of the AKT inhibitors for treating CRC patients are nuclear ß-catenin-induced EMT and bypassing apoptosis. In this study, we discover that the combinatorial treatment of an AKT inhibitor and KY1022, a ß-catenin destabilizer, effectively overcomes the current limitations of API-2, an AKT inhibitor, by reducing nuclear ß-catenin. Taken together, we demonstrate that the simultaneous suppression of Wnt/ß-catenin with the AKT signaling pathways is an ideal strategy for suppressing the AKT-inhibitor-mediated metastasis and for maximizing the therapeutic effects of AKT inhibitors.

Review on Advanced Cancer Modeling for a Cancer Study.

Intensive efforts to develop anti-cancer agents have been made for over 60 years. However, cancer is still considered a lethal disease. To study the best anti-cancer agents for improving the survival rates of cancer patients, many researchers have focused on establishing advanced experimental applications reflecting on the biomimetics of cancer patients involving the heterogeneity of cancer cells. The heterogeneity of cancer cells, which are derived from various clones and affected by different environments, presents different genetic backgrounds and molecular characteristics attributed to the differential responses to cancer therapies, and these are responsible for the resistance to cancer therapies, as well as for recurrence following cancer treatments. Therefore, the development of advanced applications for the cancer patient is expected to help the development of more effective anti-cancer agents. The present review evaluates recently developed cancer models encompassing the heterogeneity of cancer cells, which present similar morphological architecture, genetic backgrounds, and molecular characteristics to corresponding patient tumor tissues.

A Phase I/IIa Randomized Trial Evaluating the Safety and Efficacy of SNK01 Plus Pembrolizumab in Patients with Stage IV Non-Small Cell Lung Cancer.

PURPOSE: The aim of this study is to evaluate the safety and efficacy of ex vivo activated and expanded natural killer (NK) cell therapy (SNK01) plus pembrolizumab in a randomized phase I/IIa clinical trial. MATERIALS AND METHODS: Overall, 18 patients with advanced non-small cell lung cancer (NSCLC) and a programmed death ligand 1 tumor proportion score of 1% or greater who had a history of failed frontline platinum-based therapy were randomized (2:1) to receive pembrolizumab every 3 weeks +/- 6 weekly infusions of SNK01 at either 2×109 or 4×109 cells per infusion (pembrolizumab monotherapy vs. SNK01 combination). The primary endpoint was safety, whereas the secondary endpoints were the objective response rate (ORR), progression-free survival (PFS), overall survival, and quality of life. RESULTS: Since no dose-limiting toxicity was observed, the maximum tolerated dose was determined as SNK01 4×109 cells/dose. The safety data did not show any new safety signals when SNK01 was combined with pembrolizumab. The ORR and the 1-year survival rate in the NK combination group were higher than those in patients who underwent pembrolizumab monotherapy (ORR, 41.7% vs. 0%; 1-year survival rate, 66.7% vs. 50.0%). Furthermore, the median PFS was higher in the SNK01 combination group (6.2 months vs. 1.6 months, p=0.001). CONCLUSION: Based on the findings of this study, the NK cell combination therapy may consider as a safe treatment method for stage IV NSCLC patients who had a history of failed platinum-based therapy without an increase in adverse events.

Discovery of a novel CDK7 inhibitor YPN-005 in small cell lung cancer.

In contrast to non-small cell lung cancer, there has been no significant progress in the development of therapies for the small cell lung cancer (SCLC) in recent decades. Although various targeted agents, including immunotherapies, have recently been developed for testing in clinical trials, novel therapeutic agents are still needed for SCLC. We developed a potent inhibitor of cyclin-dependent kinase 7 (CDK7), designated YPN-005, and sought to determine whether it showed any anticancer effects in SCLC cells, cisplatin or etoposide-resistant cells, or organoids derived from SCLC patients. In a panel of kinases assay, YPN-005 was highly selective for CDK7 and showed antiproliferative effects in SCLC and cells with acquired resistance to conventional anticancer drugs. Similar to other CDK7 inhibitors, YPN-005 treatment significantly decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Consistent with the in vitro results, YPN-005 treatment showed a significant inhibition of tumor growth through the suppression of RNA polymerase II phosphorylation. Finally, YPN-005 showed potent anticancer effects in organoids derived from SCLC patients compared to another CDK7 inhibitor, THZ1. Therapeutic targeting of CDK7 in SCLC might be suitable for clinical investigation, and YPN-005 may be a promising therapeutic option for primary SCLC and SCLC with acquired resistance to conventional therapy.

Establishment and Long-Term Expansion of Small Cell Lung Cancer Patient-Derived Tumor Organoids.

Differential chemo-sensitivity of cancer cells, which is attributed to the cellular heterogeneity and phenotypic variation of cancer cells, is considered to be the main reason for tumor recurrence after chemotherapy. Here, we generated small cell lung cancer patient-derived tumor organoids and subjected them to long-term expansion with the addition of WNT3A or R-spondin1. We confirmed that the organoids have similar genetic profiles, molecular characteristics, and morphological architectures to the corresponding patient tumor tissue during and after long-term expansion. Interestingly, the cellular heterogeneity of organoids is reflected in their differential response to cisplatin or etoposide. We propose to utilize the organoids as small cell lung cancer patient avatar models that would be ideal for investigating the mechanisms underlying tumor recurrence after chemotherapy, and would ultimately help to develop personalized medicine.

5-FU promotes stemness of colorectal cancer via p53-mediated WNT/ß-catenin pathway activation.

5-Fluorouracil (5-FU) remains the first-line treatment for colorectal cancer (CRC). Although 5-FU initially de-bulks the tumor mass, recurrence after chemotherapy is the barrier to effective clinical outcomes for CRC patients. Here, we demonstrate that p53 promotes WNT3 transcription, leading to activation of the WNT/ß-catenin pathway in ApcMin/+/Lgr5EGFP mice, CRC patient-derived tumor organoids (PDTOs) and patient-derived tumor cells (PDCs). Through this regulation, 5-FU induces activation and enrichment of cancer stem cells (CSCs) in the residual tumors, contributing to recurrence after treatment. Combinatorial treatment of a WNT inhibitor and 5-FU effectively suppresses the CSCs and reduces tumor regrowth after discontinuation of treatment. These findings indicate p53 as a critical mediator of 5-FU-induced CSC activation via the WNT/ß-catenin signaling pathway and highlight the significance of combinatorial treatment of WNT inhibitor and 5-FU as a compelling therapeutic strategy to improve the poor outcomes of current 5-FU-based therapies for CRC patients.

Destabilization of ß-catenin and RAS by targeting the Wnt/ß-catenin pathway as a potential treatment for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a severe and heterogeneous disease that lacks an approved targeted therapy and has a poor clinical outcome to chemotherapy. Although the RAS-ERK signaling axis is rarely mutated in TNBC, ~50% of TNBCs show an increased copy number and overexpression of epidermal growth factor receptor (EGFR). However, EGFR-targeted therapies have offered no improvement in patient survival, underscoring the need to explore downstream targets, including RAS. We found that both ß-catenin and RAS, as well as epidermal growth factor receptor (EGFR), are overexpressed and correlated with one another in tumor tissues of TNBC patients. KYA1797K, an Axin-binding small molecule reducing ß-catenin and RAS expression via degradation and suppressing EGFR expression via transcriptional repression, inhibited the proliferation and the metastatic capability of stable cell lines as well as patient-derived cells (PDCs) established from TNBC patient tissues. KYA1797K also suppressed the stemness of 3D-cultured PDCs and xenografted tumors established by using residual tumors from TNBC patients and those established by the TNBC cell line. Targeting both the Wnt/ß-catenin and RAS-ERK pathways via small molecules simultaneously reducing the levels of ß-catenin, RAS, and EGFR could be a potential therapeutic approach for TNBC.

Natural Killer Cells as a Potential Biomarker for Predicting Immunotherapy Efficacy in Patients with Non-Small Cell Lung Cancer.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors for non-small cell lung cancer (NSCLC) has emerged as an important treatment option. Although immunotherapy may significantly improve survival and quality of life, response rates are as low as 20% in NSCLC patients. OBJECTIVE: The identification of reliable biomarkers predicting response to immunotherapy is required urgently to determine patient selection guidelines. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from nine NSCLC patients were collected pre- and post-treatment with immunotherapy. The immune cell composition of PBMCs was analyzed using CyTOF with an optimized 32-marker panel. The natural killer (NK) cell activity was assessed with the measurement of interferon (INF)-γ using an NK Vue™ kit. RESULTS: We found that the percentages of NK cell populations in the immune cells of PBMCs were prominently elevated in the immunotherapy responder group when compared with non-responders. While no meaningful differences were observed in other populations of immune cells, consistent with these results, the overall activity of NK cells in responders was highly elevated compared with that of non-responders. From the analysis of NK subsets, although differences in the population of early NK cells were not observed, the functionally differentiated late NK cells were prominently high in responders. CONCLUSIONS: The overall activity or number of NK cells may be a useful biomarker to predict immunotherapy response in patients with NSCLC.

Small molecule-induced simultaneous destabilization of ß-catenin and RAS is an effective molecular strategy to suppress stemness of colorectal cancer cells.

BACKGROUND: Cancer stem cells (CSCs), the major driver of tumorigenesis, is a sub-population of tumor cells responsible for poor clinical outcomes. However, molecular mechanism to identify targets for controlling CSCs is poorly understood. METHODS: Gene Set Enrichment Analyses (GSEA) of Wnt/ß-catenin and RAS signaling pathways in stem-like subtype of colorectal cancer (CRC) patients were performed using two gene expression data set. The therapeutic effects of destabilization of ß-catenin and RAS were tested by treatment of small molecule KYA1797K using CRC patient derived cells. RESULTS: Treatment with KYA1797K, a small molecule that destabilizes both ß-catenin and RAS via Axin binding, effectively suppresses the stemness of CSCs as shown in CRC spheroids and small intestinal tumors of ApcMin/+/K-RasG12DLA2 mice. Moreover, KYA1797K also suppresses the stemness of cells in CRC patient avatar model systems, such as patient-derived tumor organoids (PDTOs) and patient-derived tumor xenograft (PDTX). CONCLUSION: Our results suggest that destabilization of both ß-catenin and RAS is a potential therapeutic strategy for controlling stemness of CRC cells. Video abstract.

A mutant KRAS-induced factor REG4 promotes cancer stem cell properties via Wnt/ß-catenin signaling.

Mutant KRAS provides a driving force for enhancement of cancer stem cells (CSCs) characteristics contributing transformation of colorectal cancer (CRC) cells harboring adenomatous polyposis coli (APC) mutations. Here, we identified the factors mediating the promotion of CSCs properties induced by KRAS mutation through microarray analyses of genes specifically induced in CRC spheroids harboring both KRAS and APC mutations. Among them, REG4 was identified as a key factor since CRISPR/Cas9-mediated knockout of REG4 most significantly affected the stem cell characteristics in which CSCs markers were effectively suppressed. We show that REG4 mediates promotion of CSCs properties via Wnt/ß-catenin signaling in various in vitro studies including tumor organoid systems. Furthermore, expression patterns of CSCs markers and REG4 correlated in intestinal tumors from Apcmin/+ /KrasG12D LA2 mice and in CRC patient tissues harboring both KRAS and APC mutations. The role of REG4 in the tumor-initiating capacity accompanied by enhancement of CSCs characteristics was also revealed by NSG mice xenograft system. Collectively, our study highlights the importance of REG4 in promoting CSCs properties induced by KRAS mutation, and provides a new therapeutic strategy for CRC harboring both APC and KRAS mutations.

WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer.

BACKGROUND: Stabilization of RAS is a key event for the hyper-activation of Wnt/ß-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. METHODS: We generated mice with deletion of Wdr76 (Wdr76-/-) and crosses of Wdr76-/- with ApcMin/+ (Wdr76-/-; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture. RESULTS: Wdr76-/- mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76-/-; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and ß-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival. CONCLUSIONS: In analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.

A Therapeutic Strategy for Chemotherapy-Resistant Gastric Cancer via Destabilization of Both ß-Catenin and RAS.

: Treatment of advanced gastric cancer patients with current standard chemotherapeutic agents frequently results in resistance, leading to poor overall survival. However, there has been no success in developing strategies to overcome it. We showed the expression levels of both ß-catenin and RAS were significantly increased and correlated in tissues of 756 gastric cancer (GC) patients and tissues of primary- and acquired-resistance patient-derived xenograft tumors treated with 5-fluorouracil and oxaliplatin modulated with leucovorin (FOLFOX). On the basis of our previous studies, where small molecules to suppress colorectal cancer (CRC) via degrading both ß-catenin and RAS were developed, we tested the effectiveness of KYA1797K, a representative compound functioning by binding axin, in the growth of GC cells. The efficacy test of the drugs using gastric tumor organoids of Apc1638N mice showed that the CD44 and ALDH1A3 cancer stem cell markers were induced by FOLFOX, but not by KYA1797K. KYA1797K also efficiently suppressed tumors generated by re-engrafting the FOLFOX-resistant patient-derived xenograft (PDX) tumors, which also showed resistance to paclitaxel. Overall, the small-molecule approach degrading both ß-catenin and RAS has potential as a therapeutic strategy for treating GC patients resistant to current standard chemotherapies.

A Ras destabilizer KYA1797K overcomes the resistance of EGFR tyrosine kinase inhibitor in KRAS-mutated non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are widely used for treatment of non-small cell lung cancer (NSCLC), but they have shown limited efficacy in an unselected population of patients. The KRAS mutations, which are identified in approximately 20% of NSCLC patients, have shown to be associated with the resistance to the EGFR tyrosine kinase inhibitors (TKIs). Currently, there is no clinically available targeted therapy which can effectively inhibit NSCLC tumors harboring KRAS mutations. This study aims to show the effectiveness of KYA1797K, a small molecule which revealed anti-cancer effect in colorectal cancer by destabilizing Ras via inhibiting the Wnt/ß-catenin pathway, for the treatment of KRAS-mutated NSCLC. While erlotinib fail to have anti-transforming effect in NSCLC cell lines harboring KRAS mutations, KYA1797K effectively inhibited the Ras-ERK pathway in KRAS-mutant NSCLC cell lines. As a result, KYA1797K treatment suppressed the growth and transformation of KRAS mutant NSCLC cells and also induced apoptosis. Furthermore, KYA1797K effectively inhibited Kras-driven tumorigenesis in the KrasLA2 mouse model by suppressing the Ras-ERK pathway. The destabilization of Ras via inhibition of the Wnt/ß-catenin pathway is a potential therapeutic strategy for KRAS-mutated NSCLC that is resistant to EGFR TKI.

A small molecule approach to degrade RAS with EGFR repression is a potential therapy for KRAS mutation-driven colorectal cancer resistance to cetuximab.

Drugs targeting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, have been prescribed for metastatic colorectal cancer (CRC), but patients harboring KRAS mutations are insensitive to them and do not have an alternative drug to overcome the problem. The levels of ß-catenin, EGFR, and RAS, especially mutant KRAS, are increased in CRC patient tissues due to mutations of adenomatous polyposis coli (APC), which occur in 90% of human CRCs. The increases in these proteins by APC loss synergistically promote tumorigenesis. Therefore, we tested KYA1797K, a recently identified small molecule that degrades both ß-catenin and Ras via GSK3ß activation, and its capability to suppress the cetuximab resistance of KRAS-mutated CRC cells. KYA1797K suppressed the growth of tumor xenografts induced by CRC cells as well as tumor organoids derived from CRC patients having both APC and KRAS mutations. Lowering the levels of both ß-catenin and RAS as well as EGFR via targeting the Wnt/ß-catenin pathway is a therapeutic strategy for controlling CRC and other types of cancer with aberrantly activated the Wnt/ß-catenin and EGFR-RAS pathways, including those with resistance to EGFR-targeting drugs attributed to KRAS mutations.

ß-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3ß.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of ß-catenin signaling.

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/ß-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both ß-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of ß-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/ß-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-RasG12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the ß-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of ß-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

Long-term functional outcomes after resection of tongue cancer: determining the optimal reconstruction method.

The appropriate tongue reconstruction method is critical for better functional outcomes. The aim of this study was to determine the optimal reconstructive method for restoring postoperative function based on the extent of resection. We retrospectively reviewed 43 patients with lateral oral tongue cancer who underwent glossectomy between January 2010 and October 2014. Tongue mobility, articulation, verbal diadochokinesis, speech intelligibility and swallowing outcomes were assessed 2-3 years postoperative and were analyzed according to resected tongue volume and the method of reconstruction. In partial glossectomy cases, the secondary intention group had better function in tongue mobility, articulation, and speech intelligibility (p < 0.001 for all) than the free flap reconstruction group. In contrast, in hemi-glossectomy cases, the free flap reconstruction group had better tongue mobility, articulation, verbal diadochokinesis and speech intelligibility (p < 0.05 for all) than the secondary intention group. There was no significant difference in swallowing outcome between the secondary intention and flap reconstruction groups in both partial glossectomy and hemi-glossectomy cases. In conclusion, secondary intention appears to be the most appropriate option after partial glossectomy. However, flap reconstruction is necessary to restore tongue volume and function in patients who undergo a resection of more than half of the tongue volume.

KY1022, a small molecule destabilizing Ras via targeting the Wnt/ß-catenin pathway, inhibits development of metastatic colorectal cancer.

APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both ß-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Therefore, targeting both the Wnt/ß-catenin and Ras pathways could be an ideal therapeutic approach for treating mCRC patients. In this study, we characterized the roles of KY1022, a small molecule that destabilizes both ß-catenin and Ras via targeting the Wnt/ß-catenin pathway, in inhibiting the cellular events, including EMT, an initial process of metastasis, and apoptosis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. A small molecular approach degrading both ß-catenin and Ras via inhibition of the Wnt/ß-catenin signaling would be an ideal strategy for treatment of mCRC.