Discovery of a Highly Potent, Selective and Efficacious USP7 Degrader for the Treatment of Acute Lymphoblastic Leukemia.

USP7 is an attractive therapeutic target for cancers, especially for acute lymphoblastic leukemia (ALL) with wild-type p53. Herein, we report the discovery of XM-U-14 as a highly potent, selective and efficacious USP7 proteolysis-targeting chimera degrader. XM-U-14 achieves DC50 values of 0.74 nM and Dmax of 93% in inducing USP7 degradation in RS4;11 cell lines, and also significantly inhibits ALL cell growth. XM-U-14 even at 5 mg/kg dosed daily effectively inhibits RS4;11 tumor growth with 64.7% tumor regressions and causes no signs of toxicity in mice. XM-U-14 is a promising USP7 degrader for further optimization for ALL treatment.

Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.

Current therapy for acute myeloid leukemia (AML) is largely hindered by the development of drug resistance of commonly used chemotherapy drugs, including cytarabine, daunorubicin, and idarubicin. In this study, we investigated the molecular mechanisms underlying the chemotherapy drug resistance and potential strategy to improve the efficacy of these drugs against AML. By analyzing data from ex vivo drug-response and multi-omics profiling public data for AML, we identified autophagy activation as a potential target in chemotherapy-resistant patients. In THP-1 and MV-4-11 cell lines, knockdown of autophagy-regulated genes ATG5 or MAP1LC3B significantly enhanced AML cell sensitivity to the chemotherapy drugs cytarabine, daunorubicin, and idarubicin. In silico screening, we found that chloroquine phosphate mimicked autophagy inactivation. We showed that chloroquine phosphate dose-dependently down-regulated the autophagy pathway in MV-4-11 cells. Furthermore, chloroquine phosphate exerted a synergistic antitumor effect with the chemotherapy drugs in vitro and in vivo. These results highlight autophagy activation as a drug resistance mechanism and the combination therapy of chloroquine phosphate and chemotherapy drugs can enhance anti-AML efficacy.

Isolation of feline panleukopenia virus from Yanji of China and molecular epidemiology from 2021 to 2022.

BACKGROUND: Feline panleukopenia virus (FPV) is a widespread and highly infectious pathogen in cats with a high mortality rate. Although Yanji has a developed cat breeding industry, the variation of FPV locally is still unclear. OBJECTIVES: This study aimed to isolate and investigate the epidemiology of FPV in Yanji between 2021 and 2022. METHODS: A strain of FPV was isolated from F81 cells. Cats suspected of FPV infection (n = 80) between 2021 and 2022 from Yanji were enrolled in this study. The capsid protein 2 (VP2) of FPV was amplified. It was cloned into the pMD-19T vector and transformed into a competent Escherichia coli strain. The positive colonies were analyzed via VP2 Sanger sequencing. A phylogenetic analysis based on a VP2 coding sequence was performed to identify the genetic relationships between the strains. RESULTS: An FPV strain named YBYJ-1 was successfully isolated. The virus diameter was approximately 20-24 nm, 50% tissue culture infectious dose = 1 × 10-4.94/mL, which caused cytopathic effect in F81 cells. The epidemiological survey from 2021 to 2022 showed that 27 of the 80 samples were FPV-positive. Additionally, three strains positive for CPV-2c were unexpectedly found. Phylogenetic analysis showed that most of the 27 FPV strains belonged to the same group, and no mutations were found in the critical amino acids. CONCLUSIONS: A local FPV strain named YBYJ-1 was successfully isolated. There was no critical mutation in FPV in Yanji, but some cases with CPV-2c infected cats were identified.

Targeting C/EBPα overcomes primary resistance and improves the efficacy of FLT3 inhibitors in acute myeloid leukaemia.

The outcomes of FLT3-ITD acute myeloid leukaemia (AML) have been improved since the approval of FLT3 inhibitors (FLT3i). However, approximately 30-50% of patients exhibit primary resistance (PR) to FLT3i with poorly defined mechanisms, posing a pressing clinical unmet need. Here, we identify C/EBPα activation as a top PR feature by analyzing data from primary AML patient samples in Vizome. C/EBPα activation limit FLT3i efficacy, while its inactivation synergistically enhances FLT3i action in cellular and female animal models. We then perform an in silico screen and identify that guanfacine, an antihypertensive medication, mimics C/EBPα inactivation. Furthermore, guanfacine exerts a synergistic effect with FLT3i in vitro and in vivo. Finally, we ascertain the role of C/EBPα activation in PR in an independent cohort of FLT3-ITD patients. These findings highlight C/EBPα activation as a targetable PR mechanism and support clinical studies aimed at testing the combination of guanfacine with FLT3i in overcoming PR and enhancing the efficacy of FLT3i therapy.

Design, synthesis, and biological evaluation of Wee1 kinase degraders.

Proteolysis targeting chimera (PROTAC) technology has received widespread attention in recent years as a promising strategy for drug development. Herein, we report a series of novel Wee1 degraders, which were designed and synthesized based on PROTAC technology by linking AZD1775 with CRBN ligands through linkers of different lengths and types. All degraders could effectively and completely degrade cellular Wee1 protein in MV-4-11 cell line at IC50 concentrations. Preliminary assessments identified 42a as the most active degrader, which possessed potent antiproliferative activity and induced CRBN- and proteasome-dependent degradation of Wee1. Moreover, 42a also exhibited a time- and concentration-dependent depletion manner and inducing cell cycle arrest in G0/G1 phase and cancer cell apoptosis. More importantly, 42a showed acceptable in vitro and in vivo pharmacokinetic properties and displayed rapid and sustained Wee1 degradation ability in vivo. Taken together, these findings contribute to understanding the development of PROTACs and demonstrate that our Wee1-targeting PROTAC strategy has potential novel applications in cancer therapy.

Development and Evaluation of NanoPCR for the Detection of Goose Parvovirus.

Gosling plague (GP) is an acute and hemorrhagic infectious disease caused by goose parvovirus (GPV). The goose industry suffers significant economic losses as a result of GP, which is found to be widespread worldwide, with high rates of morbidity and mortality. Our group developed a novel technique for detecting GPV nanoparticle-assisted polymerase chain reaction (nanoPCR) and the characterization of its specificity and sensitivity. It was developed by using the traditional polymerase chain reaction (PCR) and nanoparticles. The findings of this study revealed that GPV nanoPCR products were 389 bp in length, and the lower limit of the nanoPCR assay was 4.68 × 102 copies/µL, whereas that of the conventional PCR assay was 4.68 × 104 copies/µL. A total of 230 geese suspected of GPV were detected using nanoPCR, with a positive rate of 83.0% and a specificity of 73%, respectively. Overall, we present a hitherto undocumented method for identifying GPV by using nanoPCR to aid in the evaluation of subclinical illness.

Molecular epidemiology of canine parvovirus 2 from 2014, 2019, and 2021 shows CPV2 circulating and CPV2c increasing in Yanbian, China.

Canine parvovirus 2 (CPV2) causes one of the most serious canine viral infections, with high mortality in young dogs. In 2014, 2019, and 2021, we determined genetic sequences of CPV2 strains obtained from 39 fecal samples collected from the Yanbian Korean Autonomous Prefecture in the Jilin Province of China. Sequence alignments were performed using the major capsid protein (VP2) gene; protein sequences of these samples had high nucleotide (>97.4%) and amino acid (>95.6%) identity. All of the amino acid sequences contained Ser297Ala and Tyr324Ile mutations. Our survey indicated a high prevalence of CPV2 variants in Yanbian Prefecture, with the new CPV2a variant (26 of 39; 67%) being the most frequent. CPV2c, identified in 9 of 39 (23%) samples, had not been detected in this region previously, indicating the potential risk of CPV2 mutation. The sequences of our 39 CPV2 samples were more highly homologous to the published Chinese strains than to the CPV2 variant strains found in other countries.

Discovery of a Potent and Selective Degrader for USP7.

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.

Comparative studies of senescence-related enzymes in the cotyledon of chlorophyll b-deficient mutant and its wild type oilseed rape during senescence.

The change patterns of senescence-related enzymes during cotyledon senescence were studied in a chlorophyll (Chl) b-deficient mutant type (MT, Cr3529) and its wild type (WT) of Brassica napus L. seedlings. The fresh weight on the basis of cotyledon number initially increased till 20 days after planting (DAP) and then kept relative constant. The protein content decreased sharply since 20 DAP and Chl content reduced since 10 DAP in both types; however the rate of degradation in protein and Chl in the MT was slower than that in the WT since 20 DAP. Superoxide dismutase (SOD; E.C.1.15.1.1) activity declined in the WT but increased in the MT since 20 DAP. Activity of peroxidase (POD; E.C.1.11.1.7) increased markedly after 20 DAP in both types. Esterase (EST; E.C.3.1.1.1) activity increased in both types since 10 DAP, whereas at 40 DAP it was much lower in the MT than that in the WT. In addition, bands patterns of SOD, POD and EST isozymes were changed during cotyledon development in both types, but some differences were observed. Cu/ZnSODs activities were higher in the MT at 40 DAP as compared with the WT. These results showed that day 20 was the turning point during the cotyledon development and the senescence in the MT cotyledon was slower than that in the WT.