A novel carboxyl polymer-modified upconversion luminescent nanoprobe for detection of prostate-specific antigen in the clinical gray zonebase by flow immunoassay strip.

Prostate specific antigen (PSA) is a widely-used biomarker for the diagnosis, screening, and prognosis of prostate cancer (PCa). It is critical to develop a rapid and convenient method to accurately detect PSA levels, especially when the PSA levels are in the clinical gray area of 4-10 ng/mL. We developed a novel upconversion nanoparticle (UCNP)-based fluorescence lateral flow test strip for qualitatively and quantitatively detecting PSA. The carboxyl group-modified UCNPs (UCNP-COOH) were labeled with anti-PSA antibodies via 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as labeling probes to recognize PSA. The fluorescence intensity of the UCNP-probe was then measured with a laser fluorescence scanner. A total of 1397 serum and 20 fingertip blood samples were collected to validate the UCNP strip. A reliable correlation between the area ratio (TC), reflecting the fluorescence intensity of the test/control line, and the PSA concentration was observed (r = 0.9986). The dose-dependent luminescence enhancement showed good linearity in the PSA concentration range from 0.1 to 100.0 ng/mL with a detection limit of 0.1 ng/mL. Our UCNP POCT strip demonstrated excellent accuracy, anti-interference and stability in the gray zone (4-10 ng/mL) of PSA clinical application and outperformed other PSA test strips. The UCNP strip showed good consistency with the Roche chemiluminescence assay in 1397 serum samples. It also showed good performance for PSA detection using fingertip blood samples. This novel UCNP-based test strip could be a sensitive and reliable POCT assay to detect PSA, facilitating the diagnosis and surveillance of PCa.

GNF-7, a novel FLT3 inhibitor, overcomes drug resistance for the treatment of FLT3­ITD acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation accounts for a large proportion of AML patients and diagnosed with poor prognosis. Although the prognosis of FLT3-ITD AML has been greatly improved, the drug resistance frequently occurred in the treatment of FLT3 targeting drugs. GNF-7, a multitargeted kinase inhibitor, which provided a novel therapeutic strategy for overriding leukemia. In this study, we explored the antitumor activity of GNF-7 against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML. METHODS: Growth inhibitory assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutants to evaluate the antitumor activity of GNF-7 in vitro. Western blotting was used to examine the inhibitory  effect of GNF-7 on FLT3 and its downstream pathways. Molecular docking and cellular thermal shift assay (CETSA) were performed to demonstrate the binding of FLT3 to GNF-7. The survival benefit of GNF-7 in vivo was assessed in mouse models of transformed Ba/F3 cells harboring FLT3-ITD and FLT3-ITD/F691L mutation. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of GNF-7. RESULTS: GNF-7 inhibited the cell proliferation of Ba/F3 cells expressing FLT3-ITD and exhibited potently anti-leukemia activity on primary FLT3-ITD AML samples. Moreover, GNF-7 could bind to FLT3 protein and inhibit the downstream signaling pathway activated by FLT3 including STAT5, PI3K/AKT and MAPK/ERK. In vitro and in vivo studies showed that GNF-7 exhibited potent inhibitory activity against FLT3-ITD/F691L that confers resistant to quizartinib (AC220) or gilteritinib. Importantly, GNF-7 showed potent cytotoxic effect on leukemic stem cells, significantly extend the survival of PDX model and exhibited similar therapy effect compared with gilteritinib. CONCLUSIONS: Our results show that GNF-7 is a potent FLT3-ITD inhibitor and may become a promising lead compound applied for treating some of the clinically drug resistant patients.

Clinical significance of flow cytometry in detection of minimal residual disease in cerebrospinal fluid. / è„‘è„Šæ¶²æµå¼ç»†èƒžæœ¯æ£€æµ‹å¾®å°æ®‹ç•™ç— çš„ä¸´åºŠæ„ä¹‰.

OBJECTIVES: Central nervous system leukemia (CNSL) is one of the main causes of recurrence and death in patients with acute leukemia. This study aims to dynamically monitor minimal residual disease (MRD) in cerebrospinal fluid and bone marrow of patients with different types of acute leukemia by flow cytometry (FCM), and to compare the timeliness and consistency of MRD detection between the 2 methods to further explore the application value of monitoring MRD in cerebrospinal fluid. METHODS: A total of 199 patients with acute leukemia admitted to the Guangdong Provincial people's Hospital between October 2018 and January 2022 were retrospectively analyzed, and multiparametric FCM method was adopted to summarize and analyze MRD in cerebrospinal fluid of patients with different types of leukemia and MRD in cerebrospinal fluid and bone marrow specimens of the same patients, and its role in assessing the prognostic value of patients was discussed. RESULTS: Among the 199 acute leukemia cases, a total of 31 cases (15.58%) were positive MRD in the cerebrospinal fluid, of which 18 cases (58%) were detected earlier than the corresponding bone marrow specimens. Among the 19 patients with acute T lymphoblastic leukemia, 134 patients with acute B lymphoblastic leukemia, and 46 patients with acute myeloid leukemia counted, there were 4, 18, and 9 patients with positive MRD in the cerebrospinal fluid. The Kappa value of the concordance test between the results of cerebrospinal fluid MRD and bone marrow MRD in different types of acute leukemia was only 0.156, demonstrating a low concordance between them. CONCLUSIONS: Dynamic monitoring of cerebrospinal fluid MRD by FCM can be used as a monitoring index for central nervous system leukemia, and monitoring cerebrospinal fluid can detect MRD earlier compared with bone marrow, which complements each other as a sensitive index for evaluating prognosis with significant guidance in clinic.

Parthenolide reveals an allosteric mode to inhibit the deISGylation activity of SARS-CoV­2 papain-like protease.

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.

Targeting deubiquitinating enzymes in cancer stem cells.

Cancer stem cells (CSCs) are rare but accounted for tumor initiation, progression, metastasis, relapse and therapeutic resistance. Ubiquitination and deubiquitination of stemness-related proteins are essential for CSC maintenance and differentiation, even leading to execute various stem cell fate choices. Deubiquitinating enzymes (DUBs), specifically disassembling ubiquitin chains, are important to maintain the balance between ubiquitination and deubiquitination. In this review, we have focused on the DUBs regulation of stem cell fate determination. For example, we discuss deubiquitinase inhibition may lead stem cell transcription factors and CSCs-related protein degradation. Also, CSCs microenvironment is regulated by DUBs activity. Our review provides a new insight into DUBs activity by emphasizing their cellular role in regulating stem cell fate and illustrates the opportunities for the application of DUBs inhibitors in the CSC-targeted therapy.

TMPRSS2 Serves as a Prognostic Biomarker and Correlated With Immune Infiltrates in Breast Invasive Cancer and Lung Adenocarcinoma.

TMPRSS2 is a transmembrane serine protease and plays a pivotal role in coronavirus disease 2019 (COVID-19). However, the correlation of TMPRSS2 with prognosis and immune infiltration in tumors has not yet been explored. Here, we analyzed the expression of TMPRSS2 in Oncomine and TIMER databases, the correlation between TMPRSS2 and overall survival in the PrognoScan, Kaplan-Meier plotter, and GEPIA databases. The association between TMPRSS2 and immune infiltration levels was investigated in the TIMER database. In addition, the prognosis of TMPRSS2 related to immune cells in cancers was analyzed. Quantitative real-time PCR (qRT-PCR) confirmed that TMPRSS2 was upregulated in lung adenocarcinoma (LUAD) and downregulated in breast invasive carcinoma (BRCA). We demonstrated that high TMPRSS2 expression was associated with favorable prognosis in LUAD, but it was associated with poor prognosis in BRCA. Interestingly, we found that TMPRSS2 expression was significantly correlated with immune infiltration of B cells, CD4+ T cells, macrophages, and dendritic cells in LUAD, and it was positively correlated with the infiltrating levels of CD8+ T cells, CD4+ T cells, neutrophils, and dendric cells in BRCA. Consistent with the prognosis of TMPRSS2 in LUAD and BRCA, the high expression level of TMPRSS2 has a favorable prognosis in enriched immune cells such as B cells, macrophages, and CD4+ T cells in LUAD, and it has a poor prognosis in CD4+ T cells and CD8+ T cells in BRCA. In conclusion, our results indicate that the prognosis of TMPRSS2 in LUAD and BRCA is significantly correlated with immune cells infiltration. Our study comprehensively revealed the relationship between the prognosis of TMPRSS2 in pan-cancers and tumor immunity.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway.

INTRODUCTION: Despite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. METHODS: We investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. RESULTS: We found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DISCUSSION: We found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma.

Rationale: In multiple myeloma (MM), the activities of non-homologous end joining (NHEJ) and homologous recombination repair (HR) are increased compared with healthy controls. Whether and how IKZF1 as an enhancer of MM participates in the DNA repair pathway of tumor cells remains elusive. Methods: We used an endonuclease AsiSI-based system and quantitative chromatin immunoprecipitation assay (qChIP) analysis to test whether IKZF1 is involved in DNA repair. Immunopurification and mass spectrometric (MS) analysis were performed in MM1.S cells to elucidate the molecular mechanism that IKZF1 promotes DNA damage repair. The combination effect of lenalidomide or USP7 inhibitor with PARP inhibitor on cell proliferation was evaluated using MM cells in vitro and in vivo. Results: We demonstrate that IKZF1 specifically promotes homologous recombination DNA damage repair in MM cells, which is regulated by its interaction with CtIP and USP7. In this process, USP7 could regulate the stability of IKZF1 through its deubiquitinating activity. The N-terminal zinc finger domains of IKZF1 and the ubiquitin-like domain of USP7 are necessary for their interaction. Furthermore, targeted inhibition IKZF1 or USP7 could sensitize MM cells to PARP inhibitor treatment in vitro and in vivo. Conclusions: Our findings identify USP7 as a deubiquitinating enzyme for IKZF1 and uncover a new function of IKZF1 in DNA damage repair. In translational perspective, the combination inhibition of IKZF1 or USP7 with PARP inhibitor deserves further evaluation in clinical trials for the treatment of MM.

Hsp90/C terminal Hsc70-interacting protein regulates the stability of Ikaros in acute myeloid leukemia cells.

The stability of Ikaros family zinc finger protein 1 (Ikaros), a critical hematopoietic transcription factor, can be regulated by cereblon (CRBN) ubiquitin ligase stimulated by immunomodulatory drugs in multiple myeloma. However, other stabilization mechanisms of Ikaros have yet to be elucidated. In this study, we show that the pharmacologic inhibition or knockdown of Hsp90 downregulates Ikaros in acute myeloid leukemia (AML) cells. Proteasome inhibitor MG132 but not autophagy inhibitor chloroquine could suppress the Hsp90 inhibitor STA-9090-induced reduction of Ikaros, which is accompanied with the increased ubiquitination of Ikaros. Moreover, Ikaros interacts with E3 ubiquitin-ligase C terminal Hsc70 binding protein (CHIP), which mediates the STA-9090-induced ubiquitination of Ikaros. In addition, the knockdown of Ikaros effectively inhibits the proliferation of leukemia cells, but this phenomenon could be rescued by Ikaros overexpression. Collectively, our findings indicate that the interplay between HSP90 and CHIP regulates the stability of Ikaros in AML cells, which provides a novel strategy for AML treatment through targeting the HSP90/Ikaros/CHIP axis.

Directly targeting c-Myc contributes to the anti-multiple myeloma effect of anlotinib.

Despite the significant advances in the treatment of multiple myeloma (MM), this disease is still considered incurable because of relapse and chemotherapy resistance, underscoring the need to seek novel therapies with different mechanisms. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor (TKI), has exhibited encouraging antitumor activity in several preclinical and clinical trials, but its effect on MM has not been studied yet. In this study, we found that anlotinib exhibits encouraging cytotoxicity in MM cells, overcomes the protective effect of the bone marrow microenvironment and suppresses tumor growth in the MM mouse xenograft model. We further examined the underlying molecular mechanism and found that anlotinib provokes cell cycle arrest, induces apoptosis and inhibits multiple signaling pathways. Importantly, we identify c-Myc as a novel direct target of anlotinib. The enhanced ubiquitin proteasomal degradation of c-Myc contributes to the cell apoptosis induced by anlotinib. In addition, anlotinib also displays strong cytotoxicity against bortezomib-resistant MM cells. Our study demonstrates the extraordinary anti-MM effect of anlotinib both in vitro and in vivo, which provides solid evidence and a promising rationale for future clinical application of anlotinib in the treatment of human MM.

Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia.

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin-Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

Overexpression of Fbxo6 inactivates spindle checkpoint by interacting with Mad2 and BubR1.

The spindle assembly checkpoint prevents chromosome mis-segregation during mitosis by delaying sister chromatid separation. Several F-box protein members play critical roles in maintaining genome stability and regulating cell cycle progress via ubiquitin-mediated protein degradation. Here, we showed that Fbxo6 critically regulated spindle checkpoint and chromosome segregation. Fbxo6 was phosphorylated during mitosis. Overexpression of Fbxo6 lead to faster exit from nocodazole-induced mitosis arrest through premature sister chromatid separation. Moreover, we found substantially more binuclear and multilobed nuclei cells accompanied with impaired cell viability in Fbxo6-overexpressed HeLa cells. Mechanistically, Fbxo6 interacted with spindle checkpoint proteins including Mad2 and BubR1 leading to the premature exit from mitosis. Overall, we revealed a novel role of Fbxo6 in regulating spindle checkpoint, which may shed light on the regulation of genome instability of cancer cells.

USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive leukemia that is primarily caused by aberrant activation of the NOTCH1 signaling pathway. Recent studies have revealed that posttranslational modifications, such as ubiquitination, regulate NOTCH1 stability, activity, and localization. However, the specific deubiquitinase that affects NOTCH1 protein stability remains unestablished. Here, we report that ubiquitin-specific protease 7 (USP7) can stabilize NOTCH1. USP7 deubiquitinated NOTCH1 in vivo and in vitro, whereas knockdown of USP7 increased the ubiquitination of NOTCH1. USP7 interacted with NOTCH1 protein in T-ALL cells, and the MATH and UBL domains of USP7 were responsible for this interaction. Depletion of USP7 significantly suppressed the proliferation of T-ALL cells in vitro and in vivo, accompanied by downregulation of the NOTCH1 protein level. Similarly, pharmacologic inhibition of USP7 led to apoptosis of T-ALL cells. More importantly, we found that USP7 was significantly upregulated in human T-ALL cell lines and patient samples, and a USP7 inhibitor exhibited cell cytotoxicity toward primary T-ALL cells, indicating the clinical relevance of these findings. Overall, our results demonstrate that USP7 is a novel deubiquitinase that stabilizes NOTCH1. Therefore, USP7 may be a promising therapeutic target in the currently incurable T-ALL.