Semi-Synthesis and Biological Evaluation of 25(R)-26-Acetoxy-3ß,5α-Dihydroxycholest-6-One.

Previously, we identified a series of steroids (1-6) that showed potent anti-virus activities against respiratory syncytial virus (RSV), with IC50 values ranging from 3.23 to 0.19 µM. In this work, we first semi-synthesized and characterized the single isomer of 5, 25(R)-26-acetoxy-3ß,5α-dihydroxycholest-6-one, named as (25R)-5, in seven steps from a commercially available compound diosgenin (7), with a total yield of 2.8%. Unfortunately, compound (25R)-5 and the intermediates only showed slight inhibitions against RSV replication at the concentration of 10 µM, but they possessed potent cytotoxicity activities against human bladder cancer 5637 (HTB-9) and hepatic cancer HepG2, with IC50 values ranging from 3.0 to 15.5 µM without any impression of normal liver cell proliferation at 20 µM. Among them, the target compound (25R)-5 possessed cytotoxicity activities against 5637 (HTB-9) and HepG2 with IC50 values of 4.8 µM and 15.5 µM, respectively. Further studies indicated that compound (25R)-5 inhibited cancer cell proliferation through inducing early and late-stage apoptosis. Collectively, we have semi-synthesized, characterized and biologically evaluated the 25R-isomer of compound 5; the biological results suggested that compound (25R)-5 could be a good lead for further anti-cancer studies, especially for anti-human liver cancer.

Distinct roles of programmed death ligand 1 alternative splicing isoforms in colorectal cancer.

Although anti-programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has achieved great success in some cancers, most colorectal cancer (CRC) patients remain unresponsive. Therefore, further clarification of the underlying mechanisms is needed to improve the therapy. In this study, we explored the distinct functions of different PD-L1 alternative splicing isoforms in CRC. We investigated the biological functions in PD-L1 knocked down/knockout cells, which were verified through overexpression of PD-L1 isoforms a, b, and c. The roles of PD-L1 isoforms in immune surveillance resistance was also analyzed. Meanwhile, we performed RNA-seq to screen the downstream molecules regulated by PD-L1 isoforms. Finally, we detected PD-L1 and PD-L1 isoforms levels in a cohort of serum samples, two cohorts of CRC tissue samples, and analyzed the correlation of PD-L1 isoforms with PD-1 blockade therapy response in two clinical CRC cases. The results indicated that PD-L1 knockout inhibited proliferation, migration, and invasion, and isoform b exerted a more significant inhibitory effect on T cells than the other two isoforms. Moreover, isoform c could promote CRC progression through regulating epithelial-mesenchymal transition. Clinical data showed that CRC patients with positive PD-L1 expression were associated with poorer overall survival. High serum PD-L1 level was associated with poor prognosis. The level of isoform b or c was negatively associated with prognosis, and a higher level of isoform b was associated with a good response to anti-PD-1 therapy. In conclusion, isoform b should be considered as a biomarker for clinical responsiveness to anti-PD-1/PD-L1 immunotherapy; isoform c had a prometastatic role and is a new potential target for CRC therapy.

Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration.

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation. We next constructed a SUMO isoform- and E3 ligase-dependent enzyme-substrate relationship network. Protein kinases were significantly enriched among SUMOylation substrates, suggesting crosstalk between phosphorylation and SUMOylation. Cell-based analyses of tyrosine kinase, PYK2, revealed that SUMOylation at four lysine residues promoted PYK2 autophosphorylation at tyrosine 402, which in turn enhanced its interaction with SRC and full activation of the SRC-PYK2 complex. SUMOylation on WT but not the 4KR mutant of PYK2 further elevated phosphorylation of the downstream components in the focal adhesion pathway, such as paxillin and Erk1/2, leading to significantly enhanced cell migration during wound healing. These studies illustrate how our SUMO E3 ligase-substrate network can be used to explore crosstalk between SUMOylation and other PTMs in many biological processes.

MeDReaders: a database for transcription factors that bind to methylated DNA.

Understanding the molecular principles governing interactions between transcription factors (TFs) and DNA targets is one of the main subjects for transcriptional regulation. Recently, emerging evidence demonstrated that some TFs could bind to DNA motifs containing highly methylated CpGs both in vitro and in vivo. Identification of such TFs and elucidation of their physiological roles now become an important stepping-stone toward understanding the mechanisms underlying the methylation-mediated biological processes, which have crucial implications for human disease and disease development. Hence, we constructed a database, named as MeDReaders, to collect information about methylated DNA binding activities. A total of 731 TFs, which could bind to methylated DNA sequences, were manually curated in human and mouse studies reported in the literature. In silico approaches were applied to predict methylated and unmethylated motifs of 292 TFs by integrating whole genome bisulfite sequencing (WGBS) and ChIP-Seq datasets in six human cell lines and one mouse cell line extracted from ENCODE and GEO database. MeDReaders database will provide a comprehensive resource for further studies and aid related experiment designs. The database implemented unified access for users to most TFs involved in such methylation-associated binding actives. The website is available at http://medreader.org/.

Tenascin-C Function in Glioma: Immunomodulation and Beyond.

First identified in the 1980s, tenascin-C (TNC) is a multi-domain extracellular matrix glycoprotein abundantly expressed during the development of multicellular organisms. TNC level is undetectable in most adult tissues but rapidly and transiently induced by a handful of pro-inflammatory cytokines in a variety of pathological conditions including infection, inflammation, fibrosis, and wound healing. Persistent TNC expression is associated with chronic inflammation and many malignancies, including glioma. By interacting with its receptor integrin and a myriad of other binding partners, TNC elicits context- and cell type-dependent function to regulate cell adhesion, migration, proliferation, and angiogenesis. TNC operates as an endogenous activator of toll-like receptor 4 and promotes inflammatory response by inducing the expression of multiple pro-inflammatory factors in innate immune cells such as microglia and macrophages. In addition, TNC drives macrophage differentiation and polarization predominantly towards an M1-like phenotype. In contrast, TNC shows immunosuppressive function in T cells. In glioma, TNC is expressed by tumor cells and stromal cells; high expression of TNC is correlated with tumor progression and poor prognosis. Besides promoting glioma invasion and angiogenesis, TNC has been found to affect the morphology and function of tumor-associated microglia/macrophages in glioma. Clinically, TNC can serve as a biomarker for tumor progression; and TNC antibodies have been utilized as an adjuvant agent to deliver anti-tumor drugs to target glioma. A better mechanistic understanding of how TNC impacts innate and adaptive immunity during tumorigenesis and tumor progression will open new therapeutic avenues to treat brain tumors and other malignancies.

Major depression but not minor to intermediate depression correlates with unfavorable prognosis in surgical colorectal cancer patients underwent adjuvant chemotherapy.

This study aimed to investigate the correlation of anxiety/depression with clinicopathological features, and the association of anxiety/depression degrees with overall survival (OS) in surgical colorectal cancer (CRC) patients. Three hundred and eighty-one surgical CRC patients were consecutively enrolled and underwent adjuvant chemotherapy accordingly. Anxiety and depression were assessed by the Hospital Anxiety and Depression Scale (HADS). Patients with no, minor, intermediate and major anxiety were 249 (65.4%), 79 (20.7%), 37 (9.7%) and 16 (4.2%), respectively, and patients with no, minor, intermediate and major depression were 231 (60.6%), 73 (19.2%), 44 (11.5%) and 33 (8.7%), respectively. Younger age, increased the pathological grade, elevated tumor, node and metastasis (TNM) stage, as well as higher education correlated with raised HADS-A score, while younger age, drinking history, increased pathological grade, elevated TNM stage and higher education associated with raised HADS-D score. There was no difference in OS among CRC patients with no anxiety, minor anxiety, intermediate anxiety and major anxiety. And CRC patients with major depression were of worse OS compared to those with no depression, minor depression or intermediate depression. Younger age, higher pathological grade, TNM stage and education associates with anxiety and depression, and major depression is negatively correlated with OS in surgical CRC patients underwent adjuvant chemotherapy.

Krüppel-like factor 4 (KLF4) induces mitochondrial fusion and increases spare respiratory capacity of human glioblastoma cells.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critical for the regulation of many cellular functions in both normal and neoplastic cells. Here, using human glioblastoma cells, we investigated KLF4's effects on cancer cell metabolism. We found that forced KLF4 expression promotes mitochondrial fusion and induces dramatic changes in mitochondrial morphology. To determine the impact of these changes on the cellular functions following, we analyzed how KLF4 alters glioblastoma cell metabolism, including glucose uptake, glycolysis, pentose phosphate pathway, and oxidative phosphorylation. We did not identify significant differences in baseline cellular metabolism between control and KLF4-expressing cells. However, when mitochondrial function was impaired, KLF4 significantly increased spare respiratory capacity and levels of reactive oxygen species in the cells. To identify the biological effects of these changes, we analyzed proliferation and survival of control and KLF4-expressing cells under stress conditions, including serum and nutrition deprivation. We found that following serum starvation, KLF4 altered cell cycle progression by arresting the cells at the G2/M phase and that KLF4 protected cells from nutrition deprivation-induced death. Finally, we demonstrated that methylation-dependent KLF4-binding activity mediates mitochondrial fusion. Specifically, the downstream targets of KLF4-mCpG binding, guanine nucleotide exchange factors, serve as the effector of KLF4-induced mitochondrial fusion, cell cycle arrest, and cell protection. Our experimental system provides a robust model for studying the interactions between mitochondrial morphology and function, mitochondrial dynamics and metabolism, and mitochondrial fusion and cell death during tumor initiation and progression.

Krüppel-like factor 9 and histone deacetylase inhibitors synergistically induce cell death in glioblastoma stem-like cells.

BACKGROUND: The dismal prognosis of patients with glioblastoma (GBM) is attributed to a rare subset of cancer stem cells that display characteristics of tumor initiation, growth, and resistance to aggressive treatment involving chemotherapy and concomitant radiation. Recent research on the substantial role of epigenetic mechanisms in the pathogenesis of cancers has prompted the investigation of the enzymatic modifications of histone proteins for therapeutic drug targeting. In this work, we have examined the function of Krüppel-like factor 9 (KLF9), a transcription factor, in chemotherapy sensitization to histone deacetylase inhibitors (HDAC inhibitors). METHODS: Since GBM neurosphere cultures from patient-derived gliomas are enriched for GBM stem-like cells (GSCs) and form highly invasive and proliferative xenografts that recapitulate the features demonstrated in human patients diagnosed with GBM, we established inducible KLF9 expression systems in these GBM neurosphere cells and investigated cell death in the presence of epigenetic modulators such as histone deacetylase (HDAC) inhibitors. RESULTS: We demonstrated that KLF9 expression combined with HDAC inhibitor panobinostat (LBH589) dramatically induced glioma stem cell death via both apoptosis and necroptosis in a synergistic manner. The combination of KLF9 expression and LBH589 treatment affected cell cycle by substantially decreasing the percentage of cells at S-phase. This phenomenon is further corroborated by the upregulation of cell cycle inhibitors p21 and p27. Further, we determined that KLF9 and LBH589 regulated the expression of pro- and anti- apoptotic proteins, suggesting a mechanism that involves the caspase-dependent apoptotic pathway. In addition, we demonstrated that apoptosis and necrosis inhibitors conferred minimal protective effects against cell death, while inhibitors of the necroptosis pathway significantly blocked cell death. CONCLUSIONS: Our findings suggest a detailed understanding of how KLF9 expression in cancer cells with epigenetic modulators like HDAC inhibitors may promote synergistic cell death through a mechanism involving both apoptosis and necroptosis that will benefit novel combinatory antitumor strategies to treat malignant brain tumors.

Single-Cell Co-expression Analysis Reveals Distinct Functional Modules, Co-regulation Mechanisms and Clinical Outcomes.

Co-expression analysis has been employed to predict gene function, identify functional modules, and determine tumor subtypes. Previous co-expression analysis was mainly conducted at bulk tissue level. It is unclear whether co-expression analysis at the single-cell level will provide novel insights into transcriptional regulation. Here we developed a computational approach to compare glioblastoma expression profiles at the single-cell level with those obtained from bulk tumors. We found that the co-expressed genes observed in single cells and bulk tumors have little overlap and show distinct characteristics. The co-expressed genes identified in bulk tumors tend to have similar biological functions, and are enriched for intrachromosomal interactions with synchronized promoter activity. In contrast, single-cell co-expressed genes are enriched for known protein-protein interactions, and are regulated through interchromosomal interactions. Moreover, gene members of some protein complexes are co-expressed only at the bulk level, while those of other complexes are co-expressed at both single-cell and bulk levels. Finally, we identified a set of co-expressed genes that can predict the survival of glioblastoma patients. Our study highlights that comparative analyses of single-cell and bulk gene expression profiles enable us to identify functional modules that are regulated at different levels and hold great translational potential.

Multi-echo length and offset VARied saturation (MeLOVARS) method for improved CEST imaging.

PURPOSE: The aim of this study was to develop a technique for rapid collection of chemical exchange saturation transfer images with the saturation varied to modulate signal loss transfer and enhance contrast. METHODS: Multi-echo Length and Offset VARied Saturation (MeLOVARS) divides the saturation pulse of length Tsat into N = 3-8 submodules, each consisting of a saturation pulse with length of Tsat /N (∼0.3-1 s), one or more low flip-angle gradient-echo readout(s) and a flip back pulse. This results in N readouts with increasing saturation time from Tsat /N to Tsat without extra scan time. RESULTS: For phantoms, eight images with Tsat incremented every 0.5 s from 0.5-4 s were collected simultaneously using MeLOVARS, which allows rapid determination of exchange rates for agent protons. For live mice bearing glioblastomas, the Z-spectra for five different Tsat values from 0.5 to 2.5 s were acquired in a time normally used for one Tsat . With the additional Tsat -dependence information, LOVARS phase maps were produced with a more clearly defined tumor boundary and an estimated 4.3-fold enhanced contrast-to-noise ratio (CNR). We also show that enhancing CNR is achievable by simply averaging the collected images or transforming them using the principal component analysis. CONCLUSIONS: MeLOVARS enables collection of multiple saturation-time-weighted images without extra time, producing a LOVARS phase map with increased CNR.

Construction of human activity-based phosphorylation networks.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity.

BACKGROUND: Targeting cell metabolism offers promising opportunities for the development of drugs to treat cancer. We previously found that the fatty acyl-CoA synthetase VL3 (ACSVL3) is elevated in malignant brain tumor tissues and involved in tumorigenesis. This study investigates the role of ACSVL3 in the maintenance of glioblastoma multiforme (GBM) stem cell self-renewal and the capacity of GBM stem cells to initiate tumor xenograft formation. METHODS: We examined ACSVL3 expression during differentiation of several GBM stem cell enriched neurosphere cultures. To study the function of ACSVL3, we performed loss-of-function by using small interfering RNAs to target ACSVL3 and examined stem cell marker expression, neurosphere formation and tumor initiation properties. RESULTS: ACSVL3 expression levels were substantially increased in GBM stem cell enriched neurosphere cultures and decreased after differentiation of the neurospheres. Down-regulating ACSVL3 with small inhibiting RNAs decreased the expression of markers and regulators associated with stem cell self-renewal, including CD133, ALDH, Musashi-1 and Sox-2. ACSVL3 knockdown in neurosphere cells led to increased expression of differentiation markers GFAP and Tuj1. Furthermore, ACSVL3 knockdown reduced anchorage-independent neurosphere cell growth, neurosphere-forming capacity as well as self-renewal of these GBM stem cell enriched neurosphere cultures. In vivo studies revealed that ACSVL3 loss-of-function substantially inhibited the ability of neurosphere cells to propagate orthotopic tumor xenografts. A link between ACSVL3 and cancer stem cell phenotype was further established by the findings that ACSVL3 expression was regulated by receptor tyrosine kinase pathways that support GBM stem cell self-renewal and tumor initiation, including EGFR and HGF/c-Met pathways. CONCLUSIONS: Our findings indicate that the lipid metabolism enzyme ACSVL3 is involved in GBM stem cell maintenance and the tumor-initiating capacity of GBM stem cell enriched-neurospheres in animals.

c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype.

The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met-responsive reprogramming transcription factors.

Effects of ESPCS mode nursing on the surgical tolerance, gastrointestinal tract recovery and self­management efficacy of patients with colon cancer.

Colon cancer is a common gastrointestinal malignant tumor. In addition to conventional treatment, thoughtful and comprehensive aftercare should be given to patients. The present study aimed to explore the effects of explain-simulate-practice-communication-support (ESPCS) model nursing on the surgical tolerance, gastrointestinal recovery and self-management efficacy of patients with colon cancer. The clinical data of 136 patients with colon cancer diagnosed and treated at the Second Affiliated Hospital of Harbin Medical University (Harbin, China) from June 2020 to April 2022 were retrospectively analyzed and a total of 84 patients met the inclusion criteria. A total of 42 patients who underwent conventional nursing were included in the conventional nursing group and 42 patients who underwent ESPCS model nursing were included in the ESPCS model nursing group. Surgical tolerance, gastrointestinal recovery, self-management efficacy (Cancer Self-Management Efficacy Scale), quality of life (Comprehensive Quality of Life Inventory-74) and nursing satisfaction were analyzed. Slightly higher proportions of excellent and good surgical tolerance were found in the ESPCS model nursing group (97.62%) compared with those in the conventional nursing group (85.71%); however, no significant difference was shown (P>0.05). Compared with the conventional nursing group, the time needed for gastric tube removal, bowel sound recovery, anal exhaust, first defecation, general food intake and the time until getting out of bed was significantly shorter in the ESPS model nursing group (all P<0.05). Before the intervention, no statistically significant difference was found between the indicators in the Cancer Self-Management Efficacy Scale of the two groups (all P>0.05). After the intervention, the ESPCS model nursing group had significantly higher scores for positive attitude, stress relief and self-determination than the conventional nursing group (all P<0.05). Before intervention, there was no statistically significant difference in the indicators of CQOLI-74 between the two groups (P>0.05). After the intervention, the ESPCS model nursing group also had significantly higher scores for social function, psychological function, life state and somatic function compared with the conventional nursing group (all P<0.05). Higher satisfaction of patients was found in the ESPCS mode group (95.24%) compared with that in the conventional nursing group (78.57%) (P<0.05). Overall, ESPCS mode nursing could effectively elevate the surgical tolerance of patients with colon cancer, promote the recovery of gastrointestinal function, increase self-management efficacy, and improve the quality of life and nursing satisfaction, which is certainly worthy of clinical promotion.

Effect of cognitive behavioral stress management on anxiety, depression, and quality of life in colorectal cancer patients post tumor resection: a randomized, controlled study.

OBJECTIVE: Cognitive behavioral stress management (CBSM) is a psychotherapy helping individuals develop adaptive behaviors, whose application in colorectal cancer (CRC) is rare. This randomized, controlled study intended to explore the effect of CBSM on anxiety, depression, and quality of life in CRC patients post tumor resection. METHODS: One hundred and sixty CRC patients who received tumor resection were randomized (1:1) to receive weekly CBSM or usual care (UC) for 10 weeks after discharge (120 min for each session). Hospital Anxiety and Depression Scale (HADS) and Quality of Life Questionnaire-Core 30 (QLQ-C30) of each patient were assessed after randomization (M0), one month (M1), three months (M3), and six months (M6). RESULTS: CBSM realized decreased HADS-anxiety scores at M1 (P = 0.044), M3 (P = 0.020), M6 (P = 0.003) compared to UC, so did anxiety rates at M3 (28.0% vs. 43.6%, P = 0.045), M6 (25.7% vs. 42.5%, P = 0.035), HADS-depression scores at M3 (P = 0.017), M6 (P = 0.005), and depression rates at M3 (25.3% vs. 41.0%, P = 0.040), M6 (22.9% vs. 41.1%, P = 0.020). Concerning the quality of life, CBSM achieved elevated QLQ-C30 global health status scores at M6 (P = 0.008), QLQ-C30 functions scores at M3 (P = 0.047), M6 (P = 0.031), and decreased QLQ-C30 symptoms scores at M3 (P = 0.048) and M6 (P = 0.039) compared with UC. By subgroup analyses, CBSM had a better utility on relieving anxiety, depression and improving quality of life in patients with higher education level and patients receiving adjuvant chemotherapy. CONCLUSION: CBSM program alleviates anxiety, depression, and elevates quality of life in CRC patients post tumor resection.

Research Note: Identification of breeding-related candidate genes in Tianjin-monkey chickens by transcriptome analysis.

Tianjin-monkey Chicken is a locally bred dual-purpose naked neck poultry with high tolerance to heat stress and poor reproductive ability. We aim to explore breeding-related genes to promote its growth, reproduction, meat, and egg performances. In this study, purebred, crossbred neck-naked and crossbred neck-feathered Tianjin-monkey Chickens (male = 5 and female = 5 in each group) were sampled for transcriptome analysis. Differential gene expression analysis was performed to identify candidate genes based on mRNA expression profiles. Functional enrichment analyses including GO, KEGG and GSEA analysis were conducted. Forty-five candidate breeding-related genes were identified, which were significantly enriched in 5 KEGG pathways and 37 GO terms. Some of the candidate genes were considered to be valuable in guiding breeding in the future, including SPRY3, CPXM2, FST, HDDC2, TLR1B, CYBB, and EHHADH.

Effectiveness and Safety of Apatinib Plus Programmed Cell Death Protein 1 Blockades for Patients with Treatment-refractory Metastatic Colorectal Cancer: A Retrospective Exploratory Study.

This study aimed to investigate the efficacy and safety of apatinib plus programmed cell death protein 1 (PD-1) blockades for patients with metastatic colorectal cancer (CRC) who were refractory to the standard regimens. In this retrospective study, patients with metastatic CRC who received apatinib plus PD-1 blockades in clinical practice were included. The initial dosage of apatinib was 250 mg or 500 mg, and PD-1 blockades were comprised of camrelizumab, sintilimab and pembrolizumab. Efficacy and safety data were collected through the hospital's electronic medical record system. From October 2018 to March 2022, a total of 43 patients with metastatic CRC were evaluated for efficacy and safety. The results showed an objective response rate of 25.6% (95% CI, 13.5%-41.2%) and a disease control rate of 72.1% (95% CI, 56.3%-84.7%). The median progression-free survival (PFS) of the cohort was 5.8 months (95% CI, 3.81-7.79), and the median overall survival (OS) was 10.3 months (95% CI, 5.75-14.85). The most common adverse reactions were fatigue (76.7%), hypertension (72.1%), diarrhea (62.8%), and hand-foot syndrome (51.2%). Multivariate Cox regression analysis revealed that Eastern Cooperative Oncology Group (ECOG) performance status and location of CRC (left or right-side) were independent factors to predict PFS of patients with metastatic CRC treated with the combination regimen. Consequently, the combination of apatinib and PD-1 blockades demonstrated potential efficacy and acceptable safety for patients with treatment-refractory metastatic CRC. This conclusion should be confirmed in prospective clinical trials subsequently.

VERY LONG-CHAIN ACYL-CoA SYNTHETASE-3 (ACSVL3) PROMOTES THE MALIGNANT GROWTH BEHAVIOR OF U87 GLIOMA CELLS VIA CHANGES IN CELL CYCLE WITHOUT AFFECTING APOPTOSIS.

Decreasing the expression of very long-chain acyl-CoA synthetase 3 (ACSVL3) in U87MG glioblastoma cells by either RNA interference or genomic knockout (KO) significantly decreased their growth rate in culture, as well as their ability to form rapidly growing tumors in mice. U87-KO cells grew at a 9-fold slower rate than U87MG cells. When injected subcutaneously in nude mice, the tumor initiation frequency of U87-KO cells was 70% of that of U87MG cells, and the average growth rate of tumors that did form was decreased by 9-fold. Two hypotheses to explain the decreased growth rate of KO cells were investigated. Lack of ACSVL3 could reduce cell growth either by increasing apoptosis, or via effects on the cell cycle. We examined intrinsic, extrinsic, and caspase-independent apoptosis pathways; none were affected by lack of ACSVL3. However, significant differences in the cell cycle were seen in KO cells, suggesting arrest in S-phase. Levels of cyclin-dependent kinases 1, 2, and 4 were elevated in U87-KO cells, as were regulatory proteins p21 and p53 that promote cell cycle arrest. In contrast, lack of ACSVL3 reduced the level of the inhibitory regulatory protein p27. γ-H2AX, a marker of DNA double strand breaks, was elevated in U87-KO cells, while pH3, a mitotic index marker, was reduced. Previously reported alterations in sphingolipid metabolism in ACSVL3-depleted U87 cells may explain the effect of KO on cell cycle. These studies reinforce the notion that ACSVL3 is a promising therapeutic target in glioblastoma.

Depleting glioblastoma cells of very long-chain acyl-CoA synthetase 3 (ACSVL3) produces metabolic alterations in non-lipid pathways.

Knockout (KO) of the fatty acid-activation enzyme very long-chain acyl-CoA synthetase 3 (ACSVL3; SLC27A3) in U87MG glioblastoma cells reduced their malignant growth properties both in vitro and in xenografts. These U87-KO glioma cells grew at a slower rate, became adherence-dependent, and were less invasive than parental U87 cells. U87-KO cells produced fewer, slower-growing subcutaneous and intracranial tumors when implanted in NOD-SCID mice. Thus, depleting U87MG cells of ACSVL3 restored these cells to a phenotype more like that of normal astrocytes. To understand the mechanisms underlying these beneficial changes, we investigated several possibilities, including the effects of ACSVL3 depletion on carbohydrate metabolism. Proteomic and metabolomic profiling indicated that ACSVL3 KO produced changes in glucose and energy metabolism. Even though protein levels of glucose transporters GLUT1 and GLUT3 were reduced by KO, cellular uptake of labeled 2-deoxyglucose was unaffected. Glucose oxidation to CO2 was reduced nearly 7-fold by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Surprisingly, lactate production and the levels of lactate dehydrogenase isozymes LDHA and LDHB were elevated by ACSVL3 KO. The activity of the pentose phosphate pathway was found to be lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria were elongated in KO cells, but had a more punctate morphology in U87 cells. The mitochondrial potential was unaffected by lack of ACSVL3. We conclude that the beneficial effects of ACSVL3 depletion in human glioblastoma cells may result in part from alterations in diverse metabolic processes that are not directly related to role(s) of this enzyme in fatty acid and/or lipid metabolism. (Supported by NIH 5R01NS062043 and KKI institutional funds.).

Isocitrate dehydrogenase 1 mutation drives leukemogenesis by PDGFRA activation due to insulator disruption in acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.