Switching from tenofovir disoproxil fumarate to tenofovir alafenamide in virologically suppressed patient with chronic hepatitis B.

BACKGROUND AND AIM: Our study evaluated the outcomes of switching from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF) in patients with chronic hepatitis B (CHB). We assessed viral and biochemical responses as well as changes in the estimated glomerular filtration rate (eGFR) and bone mineral density (BMD). METHODS: This retrospective multicenter study included CHB patients who achieved virologic response (VR) (HBV DNA < 20 IU/mL) while on TDF and were subsequently switched to TAF between April 2018 and October 2021. RESULTS: This study included 309 patients with a median age of 59 years, and 42.1% were male. The mean duration of TDF and TAF administration were 54.0 and 37.5 months, respectively. All patients maintained VR after switching to TAF. Alanine aminotransferase (ALT) normalization rate significantly increased 6 months after switching (74.8%-83.5%; P = 0.008). Adjusted eGFR significantly improved at 6 months (+5.55 ± 10.52 mL/min/1.73 m2; P < 0.001) and 12 months (+6.02 ± 10.70 mL/min/1.73 m2; P < 0.001) after switching. In the subgroup of patients with renal impairment (eGFR < 60 mL/min/1.73 m2), significant improvement in renal function was observed at 6 months (+0.6 ± 10.5 mL/min/1.73 m2; P < 0.001) and 12 months (+1.0 ± 10.7 mL/min/1.73 m2; P < 0.001) after switching to TAF. In patients with osteoporosis (n = 182), switching to TAF resulted in significant improvement in spine and hip BMD at 12 months, with increases of 9.7% (95% CI: 7.0-12.5) and 9.4% (95% CI: 7.0-11.8), respectively. CONCLUSION: In this real-world study, switching to TAF was effective and safe in patients, with notable improvements in ALT levels, renal function, and BMD.

A Machine Learning Algorithm Facilitates Prognosis Prediction and Treatment Selection for Barcelona Clinic Liver Cancer Stage C Hepatocellular Carcinoma.

BACKGROUND: Given its heterogeneity and diverse clinical outcomes, precise subclassification of BCLC-C hepatocellular carcinoma (HCC) is required for appropriately determining patient prognosis and selecting treatment. METHODS: We recruited 2,626 patients with BCLC-C stage HCC from multiple centers, comprising training/test (n=1,693) and validation cohorts (n=933). The XGBoost was chosen for maximum performance among the machine learning (ML) models. Patients were categorized into low-/intermediate-/high-/very high-risk subgroups which were based on the estimated prognosis, and this subclassification was named the CLAssification via Machine learning of BCLC-C (CLAM-C). RESULTS: The areas under the receiver operating characteristic curve of the CLAM-C for predicting the 6-/12-/24-month survival of patients with BCLC-C were 0.800/0.831/0.715, respectively-significantly higher than those of the conventional models, which was consistent in the validation cohort. The four subgroups had significantly different median overall survivals, and this difference was maintained among various patient subgroups and treatment modalities. Immune-checkpoint inhibitors and transarterial therapies were associated with significantly better survival than tyrosine kinase inhibitors (TKIs) in the low- and intermediate-risk subgroups. In cases with first-line systemic therapy, the CLAM-C identified atezolizumab-bevacizumab as the best therapy particularly in the high-risk group. In cases with later-line systemic therapy, nivolumab had better survival than TKIs in the low-to-intermediate-risk subgroup, whereas TKIs had better survival in the high-to-very high-risk subgroup. CONCLUSIONS: ML modeling effectively subclassified patients with BCLC-C HCC, potentially aiding treatment allocation. Our study underscores the potential utilization of ML modeling in terms of prognostication and treatment allocation in patients with BCLC-C HCC.

A refined prediction model for survival in hepatocellular carcinoma patients treated with transarterial chemoembolization.

Background/Aims: Transarterial chemoembolization (TACE) is widely performed as a major treatment for hepatocellular carcinoma (HCC) patients, and there is a need to stratify patients for whom the most benefit from the treatment. This study aimed to develop a refined prediction model for overall survival (OS) in patients undergoing TACE as a first-line treatment in a large cohort and validate its performance. Methods: A total of 2,632 patients with HCC of Barcelona Clinic Liver Cancer stage A or B who underwent TACE between 2008 and 2017 were enrolled. The patients were randomly assigned to a training cohort (n = 1,304) or a validation cohort (n = 1,328). Independent predictors of OS were used to develop a prediction model. Results: The median age of patients in the entire cohort was 63 years, with the majority having hepatitis B virus (56.6%) and being classified as Child-Pugh class A (82.4%). We developed a new prognostic model, called the TACE-prognostic (TP) score, based on tumor burden (sum of the largest tumor diameter and tumor number), alpha-fetoprotein, and Albumin-Bilirubin grade. Patients were classified into five risk groups according to TP scores, with median survival significantly differentiated in both training and validation cohorts (P < 0.001). The new model consistently outperformed other currently available models in both the training and validation cohorts. Conclusion: This newly developed TP scoring system has the potential to be a useful tool in identifying ideal candidates of TACE and predicting OS with favorable performance and discrimination. However, further external validation is needed to confirm its effectiveness.

Differential liver function at cessation of atezolizumab-bevacizumab versus lenvatinib in HCC: a multicenter, propensity-score matched comparative study.

Background: Atezolizumab+bevacizumab (AB) and lenvatinib have been proposed as first-line treatment options for patients with advanced hepatocellular carcinoma (HCC), but comparative efficacy and associated factors are controversial. Materials and methods: This real-world multicenter study analysed patients with HCC who received AB (n=169) or lenvatinib (n=177). Results: First, 1:1 propensity score matching (PSM) was performed, resulting in 141 patients in both the AB and lenvatinib groups. After PSM, overall survival (OS) was better in the AB group than in the lenvatinib group [hazard ratio (HR)=0.642, P=0.009], but progression-free survival (PFS) did not vary between the two groups (HR=0.817, P=0.132). Objective response rate (ORR) was also similar between AB and lenvatinib (34.8% vs. 30.8%, P=0.581). In a subgroup of patients with objective responses (OR, n=78), OS (HR=0.364, P=0.012) and PFS (HR=0.536, P=0.019) were better in the AB group (n=41) than in the lenvatinib group (n=37). Time-to-progression from time of OR was also better in the AB group (HR=0.465, P=0.012). Importantly, residual liver function was a significant factor related to OS in both treatments. Child-Pugh score following cessation of the respective treatments was better in the AB group (n=105) than in the lenvatinib group (n=126) (median 6 versus 7, P=0.008), and proportion of salvage treatment was also higher in the AB group (52.4% versus 38.9%, P=0.047). When we adjusted for residual liver function or salvage treatment, there was no difference in OS between the two treatments. Conclusion: Our study suggests that residual liver function and subsequent salvage treatments are major determinants of clinical outcomes in patients treated with AB and lenvatinib; these factors should be considered in future comparative studies.

Navigating the microbial community in the trachea-oropharynx of breast cancer patients with or without neoadjuvant chemotherapy (NAC) via endotracheal tube: has NAC caused any change?

Background: We compare the diversity and niche specificity of the microbiome in the trachea-oropharynx microbiome of malignant breast neoplasm with or without neoadjuvant chemotherapy (NAC) via NGS analysis. Methods: We prospectively collected a total of 40 endotracheal tubes intubated from subjects, of whom 20 with NAC treated breast cancer (NAC group) and 20 with breast cancer without NAC (Non-NAC group). We generated 16S rRNA-based microbial profiles in IlluminaTM platform and alpha diversity indices were compared between groups. For the comparison of taxa abundance, linear discriminant analysis effect size method with Kruskal-Wallis test was used. The distribution of variables between the two groups was compared using the Mann-Whitney test. For beta diversity analysis, PERMANOVA was used. Results: Among the diversity indices, the NAC group showed significantly lower Chao1, Inverse Simpson, and Shannon indices than the Non-NAC group. The three most frequent taxa of all two groups were Streptococcus (20.4%), followed by Veillonella (11.9%), and Prevorella (10.4%). This order was the same in NAC and non-NAC groups. Conclusion: Here, we provide the first comparison data of the respiratory tract microbiome of breast cancer patients with or without NAC via NGS analysis. This study ultimately seeks to contribute to future studies on the lower respiratory tract in cancer patients with cytotoxic chemotherapy by establishing reliable control data.

A Real-World Comparative Analysis of Atezolizumab Plus Bevacizumab and Transarterial Chemoembolization Plus Radiotherapy in Hepatocellular Carcinoma Patients with Portal Vein Tumor Thrombosis.

This study aimed to compare the treatment outcomes of atezolizumab-plus-bevacizumab (Ate/Bev) therapy with those of transarterial chemoembolization plus radiotherapy (TACE + RT) in hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) and without metastasis. Between June 2016 and October 2022, we consecutively enrolled 855 HCC patients with PVTT. After excluding 758 patients, 97 patients (n = 37 in the Ate/Bev group; n = 60 in the TACE + RT group) were analyzed. The two groups showed no significant differences in baseline characteristics and had similar objective response and disease control rates. However, the Ate/Bev group showed a significantly higher one-year survival rate (p = 0.041) compared to the TACE + RT group, which was constantly displayed in patients with extensive HCC burden. Meanwhile, the clinical outcomes were comparable between the two groups in patients with unilobar intrahepatic HCC. In Cox-regression analysis, Ate/Bev treatment emerged as a significant factor for better one-year survival (p = 0.049). Finally, in propensity-score matching, the Ate/Bev group demonstrated a better one-year survival (p = 0.02) and PFS (p = 0.01) than the TACE + RT group. In conclusion, Ate/Bev treatment demonstrated superior clinical outcomes compared to TACE + RT treatment in HCC patients with PVTT. Meanwhile, in patients with unilobar intrahepatic HCC, TACE + RT could also be considered as an alternative treatment option alongside Ate/Bev therapy.

Veratramine Inhibits the Cell Cycle Progression, Migration, and Invasion via ATM/ATR Pathway in Androgen-Independent Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer-related death among men. Treatment of PC becomes difficult after progression because PC that used to be androgen-dependent becomes androgen-independent prostate cancer (AIPC). Veratramine, an alkaloid extracted from the root of the Veratrum genus, has recently been reported to have anticancer effects that work against various cancers; however, its anticancer effects and the underlying mechanism of action in PC remain unknown. We investigated the anticancer effects of veratramine on AIPC using PC3 and DU145 cell lines, as well as a xenograft mouse model. The antitumor effects of veratramine were evaluated using the CCK-8, anchorage-independent colony formation, trans-well, wound healing assays, and flow cytometry in AIPC cell lines. Microarray and proteomics analyses were performed to investigate the differentially expressed genes and proteins induced by veratramine in AIPC cells. A xenograft mouse model was used to confirm the therapeutic response and in vivo efficacy of veratramine. Veratramine dose dependently reduced the proliferation of cancer cells both in vitro and in vivo. Moreover, veratramine treatment effectively suppressed the migration and invasion of PC cells. The immunoblot analysis revealed that veratramine significantly downregulated Cdk4/6 and cyclin D1 via the ATM/ATR and Akt pathways, both of which induce a DNA damage response that eventually leads to G1 phase arrest. In this study, we discovered that veratramine exerted antitumor effects on AIPC cells. We demonstrated that veratramine significantly inhibited the proliferation of cancer cells via G0/G1 phase arrest induced by the ATM/ATR and Akt pathways. These results suggest that veratramine is a promising natural therapeutic agent for AIPC.

Association between Posttreatment Serum Platelet-to-Lymphocyte Ratio and Distant Metastases in Patients with Hepatocellular Carcinoma Receiving Curative Radiation Therapy.

BACKGROUND: We sought to investigate whether serum immune and inflammatory parameters can help to predict distant metastasis (DM) in patients with unresectable hepatocellular carcinoma (HCC) undergoing curative radiation therapy (RT). METHODS: A total of 76 RT courses were analyzed. The following variables were included in the analysis: systemic inflammation index, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio (PLR), prognostic nutritional index (PNI), absolute lymphocyte count, lymphocyte-to-monocyte ratio, albumin, albumin-to-alkaline phosphatase ratio, RT-related parameters, and levels of total protein, hemoglobin, α-fetoprotein, and PIVKA-II. Distant control (DC) and overall survival (OS) rates were calculated and compared. RESULTS: The mean age was 61.4 years, and most patients were men (n = 62, 81.6%). The median RT fraction number and fractional doses were 12 (range, 4-30) and 5 (range, 2-12) Gy, respectively. With a median follow-up of 12 (range, 3.1-56.7) months, the 1-year DC and OS rates were 64.4% and 55.2%, respectively. The development of DM significantly deteriorated OS (p = 0.013). In the multivariate analysis, significant independent prognostic indicators for DC and OS rates were the highest posttreatment PLR (≤235.7 vs. >235.7, p = 0.006) and the lowest posttreatment PNI (≤25.4 vs. >25.4, p < 0.001), respectively. CONCLUSIONS: Posttreatment serum PLR might be helpfully used as a predictive biomarker of DM in unresectable HCC patients undergoing RT. Future research is necessary to confirm our findings.

Methods to detect AUTOphagy-Targeting Chimera (AUTOTAC)-mediated Targeted Protein Degradation in Tauopathies.

Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic. Graphical abstract Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper.

Matrix Metalloproteinase 1 as a Marker of Tonsil-Derived Mesenchymal Stem Cells to Assess Bone Marrow Cell Migration.

BACKGROUND: To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient's bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. METHODS: To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. RESULTS: Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow-derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. CONCLUSION: These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.

NOLC1 knockdown suppresses prostate cancer progressions by reducing AKT phosphorylation and ß-catenin accumulation.

Although several studies have focused on cancer diagnosis and therapy, prostate cancer (PC) remains an intractable disease. Androgen deprivation therapy (ADT), which is used to treat early stage PC can lead to the development of castration-resistant prostate cancer (CRPC), which is highly associated with androgen receptor (AR) mutations. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a chaperone that shuttles between the nucleus and the cytoplasm. Studies suggest that NOLC1 regulates PC progression; however, the underlying mechanisms remain unclear. Herein, we showed that NOLC1 knockdown suppresses PC cell proliferation by altering the signaling pathways and the expression of various proteins involved in DNA replication, amino acid metabolism, and RNA processing. Mechanistically, NOLC1 knockdown suppressed cell cycle progression by inhibiting AKT phosphorylation and ß-catenin accumulation. Finally, we showed that NOLC1 expression is higher in human PC than in human hyperplastic prostate tissues. Altogether, we demonstrated that NOLC1 knockdown suppresses the progression of both AR-positive and AR-negative PC cells by inducing changes in the expression of several genes leading to cell cycle arrest. Thus, NOLC1 might be a novel and promising therapeutic target for PC.

Cholesterol Synthesis Is Important for Breast Cancer Cell Tumor Sphere Formation and Invasion.

Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA+CD44+CD24- population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

Targeted protein degradation via the autophagy-lysosome system: AUTOTAC (AUTOphagy-TArgeting Chimera).

Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several types of degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux remains unavailable. In this study, we developed a general chemical tool by which given intracellular proteins are targeted to macroautophagy for lysosomal degradation. This platform technology, termed AUTOTAC (AUTOphagy-TArgeting Chimera), employs bifunctional molecules composed of target-binding ligands (TBLs) linked to autophagy-targeting ligands (ATLs). Upon binding to targets via the TBL, the ATL binds the ZZ domain of the otherwise dormant autophagy receptor SQSTM1/p62 (sequestosome 1), which activates SQSTM1 associated with targets and sequesters them into oligomeric species for autophagic targeting and lysosomal degradation. AUTOTACs were used to degrade various oncoproteins or aggregation-prone proteins in neurodegeneration both in vitro and/or in vivo. We suggest that AUTOTAC provides a platform for selective proteolysis as a research tool and in drug development.

Knockdown of Maged1 inhibits cell cycle progression and causes cell death in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are characterized by self-renewal and pluripotency and can undergo differentiation into the three germ layers (ectoderm, mesoderm, and endoderm). Melanoma-associated antigen D1 (Maged1), which is expressed in all developing and adult tissues, modulates tissue regeneration and development. In the present study, we examined the expression and function of Maged1 in mESCs. Maged1 protein and mRNA expression increased during mESC differentiation. The pluripotency of mESCs was significantly reduced through extracellular signal-regulated kinase 1/2 phosphorylation upon knockdown of Maged1, and through G1 cell cycle arrest during cell division, resulting in significantly reduced mESC proliferation. Moreover, the diameter of the embryoid bodies was significantly reduced, accompanied by increased levels of ectodermal differentiation markers and decreased levels of mesodermal and endodermal differentiation markers. Maged1-knockdown mESC lines showed significantly reduced teratoma volumes and inhibition of teratoma formation in nude mice. Additionally, we observed increased ectodermal markers but decreased mesodermal and endodermal markers in teratoma tissues. These findings show that Maged1 affects mESC pluripotency, proliferation, cell cycle, and differentiation, thereby contributing to our understanding of the basic molecular biological mechanisms and potential roles of Maged1 as a regulator of various mESC properties.

Hypofractionated Radiotherapy for Early-Stage Breast Cancer: A Propensity Score Matched Analysis.

BACKGROUND: In patients with early-stage breast cancer, the treatment results of hypofractionated radiation therapy (RT) and conventional RT are evaluated in efficacy and cost. METHODS: We retrospectively evaluated 280 patients with early-stage (Tis-2N0M0) breast cancer (including 100 hypofractionated RT patients) with regards to treatment outcomes according to the RT schedule. The median whole-breast RT dose was 42.56 Gy/16 fractions for hypofractionated RT and 50.4 Gy/28 fractions for conventional RT. Most patients (n = 260, 92.9%) additionally received a tumor bed boost RT. We used propensity score matching (PSM) analysis to balance the baseline risk factors for recurrence. The co-primary endpoints of this study were disease-free survival (DFS) and ipsilateral breast tumor recurrence (IBTR). DFS or IBTR was analyzed using the Kaplan-Meier survival curve and log-rank test. RESULTS: Total 89 pairs of matched patients (1:1 matching, n = 178) were finally evaluated. The median follow-up was 23.6 months. After matching, the 3-year DFS was 100% in the hypofractionated RT group and 98.4% in the conventional RT group; there was no significant difference in DFS between the groups (P = 0.374). Furthermore, the IBTR did not differ between the hypofractionated RT and conventional RT groups (P = 0.374) after matching. The 3-year overall survival was not different between two groups (both 100%). Hypofractionated RT saved 26.6% of the total cost of RT compared to conventional RT. Additionally, the acute skin toxicity rate (≥ grade 2) was also not significantly different between the groups (hypofractionated RT: 10.1% vs. conventional RT: 2.2%). CONCLUSION: Hypofractionated RT showed good IBTR and DFS, which were compatible to those in conventional RT in breast cancer. Hypofractionated RT is expected to be used more widely because of its low cost and convenience.

The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system.

Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux is still missing. In this study, we develop a general chemical tool and platform technology termed AUTOphagy-TArgeting Chimera (AUTOTAC), which employs bifunctional molecules composed of target-binding ligands linked to autophagy-targeting ligands. AUTOTACs bind the ZZ domain of the otherwise dormant autophagy receptor p62/Sequestosome-1/SQSTM1, which is activated into oligomeric bodies in complex with targets for their sequestration and degradation. We use AUTOTACs to degrade various oncoproteins and degradation-resistant aggregates in neurodegeneration at nanomolar DC50 values in vitro and in vivo. AUTOTAC provides a platform for selective proteolysis in basic research and drug development.

Combined treatment with anti-HER2/neu and anti-4-1BB monoclonal antibodies induces a synergistic antitumor effect but requires dose optimization to maintain immune memory for protection from lethal rechallenge.

Human epidermal growth factor receptor type 2 (HER2)-positive breast cancer that is treated with anti-HER2/neu monoclonal antibody (mAb) is not free from late recurrences. Addition of anti-4-1BB mAb to anti-HER2/neu mAb has been demonstrated to strengthen the cytotoxic antitumor response. Our study expands on this by revealing the influence of anti-4-1BB mAb addition on the immune memory of anti-HER2/neu mAb. We designed murine breast cancer models by implanting TUBO and TUBO-P2J cell lines in mice, which were then treated with anti-HER2/neu and/or anti-4-1BB mAb. After complete surgical and/or chemical regression of the tumor, the mice were rechallenged with a second injection of cancer cells. Notably, anti-HER2/neu and anti-4-1BB mAb combination therapy had a synergistic antitumor effect at the initial treatment. However, the combination therapy did not evoke immune memory, allowing the tumors to thrive at rechallenge with reduced CD44+ expression in CD8+ T cells. Immune memory was also impaired when anti-4-1BB mAb was administered to naive CD8+ T cells but was sustained when this was administered to activated CD8+ T cells. In an attempt to resist the loss of immune memory, we controlled the dose of anti-4-1BB mAb to optimize the stimulation of activated CD8+ T cells. Immune memory was achieved with the dose regulation of anti-4-1BB mAb to 1 mg/kg in our model. Our study demonstrates the importance in understanding the adaptive immune mechanism of anti-HER2/neu and anti-4-1BB mAb combination therapy and suggests a dose optimization strategy is necessary to ensure development of successful immune memory.

Synergism of a novel MCL­1 downregulator, acriflavine, with navitoclax (ABT­263) in triple­negative breast cancer, lung adenocarcinoma and glioblastoma multiforme.

Myeloid cell leukemia sequence 1 (MCL­1), an anti­apoptotic B­cell lymphoma 2 (BCL­2) family molecule frequently amplified in various human cancer cells, is known to be critical for cancer cell survival. MCL­1 has been recognized as a target molecule for cancer treatment. While various agents have emerged as potential MCL­1 blockers, the present study presented acriflavine (ACF) as a novel MCL­1 inhibitor in triple­negative breast cancer (TNBC). Further evaluation of its treatment potential on lung adenocarcinoma and glioblastoma multiforme (GBM) was also investigated. The anticancer effect of ACF on TNBC cells was demonstrated when MDA­MB­231 and HS578T cells were treated with ACF. ACF significantly induced typical intrinsic apoptosis in TNBCs in a dose­ and time­dependent manner via MCL­1 downregulation. MCL­1 downregulation by ACF treatment was revealed at each phase of protein expression. Initially, transcriptional regulation via reverse transcription­quantitative PCR was validated. Then, post­translational regulation was explained by utilizing an inhibitor against protein biosynthesis and proteasome. Lastly, immunoprecipitation of ubiquitinated MCL­1 confirmed the post­translational downregulation of MCL­1. In addition, the synergistic treatment efficacy of ACF with the well­known MCL­1 inhibitor ABT­263 against the TNBC cells was explored [combination index (CI)<1]. Conjointly, the anticancer effect of ACF was assessed in GBM (U87, U251 and U343), and lung cancer (A549 and NCI­H69) cell lines as well, using immunoblotting, cytotoxicity assay and FACS. The effect of the combination treatment using ACF and ABT­263 was estimated in GBM (U87, U343 and U251), and non­small cell lung cancer (A549) cells likewise. The present study suggested a novel MCL­1 inhibitory function of ACF and the synergistic antitumor effect with ABT­263.

O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells.

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.

Comparative Analysis of Lenvatinib and Hepatic Arterial Infusion Chemotherapy in Unresectable Hepatocellular Carcinoma: A Multi-Center, Propensity Score Study.

The comparative efficacy and safety between lenvatinib and hepatic artery infusion chemotherapy (HAIC) in patients with unresectable hepatocellular carcinoma (HCC) is still unclear. This multicenter historical cohort study enrolled 244 patients who were treated with HAIC (n = 173) or lenvatinib (n = 71) between 2012 and 2020. Propensity score matching (PSM) was performed, and 52 patients were selected per group. Clinical outcomes and safety were compared. Objective response rate (ORR) was not different between the two groups (26.0% vs. 23.1%, p = 0.736). Before PSM, the HAIC group had a higher proportion of Child-Pugh B and portal vein tumor, whereas the lenvatinib group had more patients with extrahepatic metastases, which was adjusted after PSM. There were no differences in progression-free survival (PFS) and overall survival (OS) after PSM (HAIC vs. lenvatinib, median PFS, 3.6 vs. 4.0 months, p = 0.706; median OS 10.8 vs. 7.9 months, p = 0.106). Multivariate Cox-regression showed that alpha-fetoprotein ≤1000 ng/mL was only an associated factor for OS after PSM in all patients (hazard ratio = 0.421, p = 0.011). Subgroup analysis for patients with a high tumor burden beyond the REFLECT eligibility criteria revealed that the HAIC group (n = 29) had a significantly longer OS than did the lenvatinib group (n = 30) (10.0 vs. 5.4 months, p = 0.004). More patients in the HAIC group achieved better liver function than those in the lenvatinib group at the time of best responses. There was no difference in the incidence of grade 3 and 4 adverse events between the two groups. Therefore, lenvatinib is comparable to HAIC in terms of ORR and OS in unresectable HCC meeting REFLECT eligibility criteria.