Cereblon-Targeting Ligase Degraders in Myeloma: Mechanisms of Action and Resistance.

Cereblon-targeting degraders, including immunomodulatory imide drugs lenalidomide and pomalidomide alongside cereblon E3 ligase modulators like iberdomide and mezigdomide, have demonstrated significant anti-myeloma effects. These drugs play a crucial role in diverse therapeutic approaches for multiple myeloma (MM), emphasizing their therapeutic importance across various disease stages. Despite their evident efficacy, approximately 5% to 10% of MM patients exhibit primary resistance to lenalidomide, and resistance commonly develops over time. Understanding the intricate mechanisms of action and resistance to this drug class becomes imperative for refining and advancing novel therapeutic combinations.

The importance of routine genetic testing in pediatric epilepsy surgery.

Genetic variants in relevant genes coexisting with MRI lesions in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. Still, presurgical evaluation does not include genetic diagnostics routinely. Here, we report our presurgical evaluation algorithm that includes routine genetic testing. We analyzed retrospectively the data of 68 children with DRE operated at a mean age of 7.8 years (IQR: 8.1 years) at our center. In 49 children, genetic test results were available. We identified 21 gene variants (ACMG III: n = 7, ACMG IV: n = 2, ACMG V: n = 12) in 19 patients (45.2%) in the genes TSC1, TSC2, MECP2, DEPDC5, HUWE1, GRIN1, ASH1I, TRIO, KIF5C, CDON, ANKD11, TGFBR2, ATN1, COL4A1, JAK2, KCNQ2, ATP1A2, and GLI3 by whole-exome sequencing as well as deletions and duplications by array CGH in six patients. While the results did not change the surgery indication, they supported counseling with respect to postoperative chance of seizure freedom and weaning of antiseizure medication (ASM). The presence of genetic findings leads to the postoperative retention of at least one ASM. In our cohort, the International League against Epilepsy (ILAE) seizure outcome did not differ between patients with and without abnormal genetic findings. However, in the 7/68 patients with an unsatisfactory ILAE seizure outcome IV or V 12 months postsurgery, 2 had an abnormal or suspicious genetic finding as a putative explanation for persisting seizures postsurgery, and 3 had received palliative surgery including one TSC patient. This study highlights the importance of genetic testing in children with DRE to address putative underlying germline variants as genetic epilepsy causes or predisposing factors that guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning. PLAIN LANGUAGE SUMMARY: Genetic variants in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. However, presurgical evaluation does not include genetic diagnostics routinely. This retrospective study analyzed the genetic testing results of the 68 pediatric patients who received epilepsy surgery in our center. We identified 21 gene variants by whole-exome sequencing as well as deletions and duplications by array CGH in 6 patients. These results highlight the importance of genetic testing in children with DRE to guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning.

Circ ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 increases the stability of G9a and ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 messenger RNA through recruiting eukaryotic translation initiation factor 4A3, transcriptionally inhibiting PDZ and homeobox protein domain protein 1, and promotes the metastasis of hepatocellular carcinoma.

BACKGROUND AND AIM: Circular ubiquitin-like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1-G9a-ubiquitin-like, containing PHD and ring finger domains 1 (UHRF1) mRNA-eukaryotic translation initiation factor 4A3 (EIF4A3)-PDZ and LIM domain 1 (PDLIM1) network in HCC. METHODS: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial-mesenchymal transition (EMT)-related proteins, and Hippo-Yap pathway components was determined by quantitative polymerase chain reaction (Q-PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit-8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull-down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co-immunoprecipitation was performed to examine the binding between UHRF1 and G9a. RESULTS: Circular ubiquitin-like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription. CONCLUSION: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo-Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1-G9a-UHRF1 mRNA-EIF4A3-PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment.

ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma.

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Predictive Impact of RNF43 Mutations in Patients With Proficient Mismatch Repair/Microsatellite Stable BRAFV600E-Mutated Metastatic Colorectal Cancer Treated With Target Therapy or Chemotherapy.

PURPOSE: Target therapy (TT) with encorafenib plus cetuximab is a standard option in patients with BRAFV600E-mutated (mut) pretreated metastatic colorectal cancer (mCRC). Recently, mutations in RNF43, encoding a negative regulator of the WNT pathway, were associated with longer progression-free survival (PFS) and overall survival (OS) in patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) BRAFV600E-mut mCRC treated with TT. Here, we explored the effect of RNF43 mutations on the efficacy of second-line TT versus standard chemotherapy (CT). METHODS: A retrospective cohort of patients with pMMR/MSS BRAFV600E-mut tumors, available RNF43 mutational status, and treated with second-line TT or oxaliplatin- and/or irinotecan-based CT was analyzed. RESULTS: One hundred thirty-two patients with pMMR/MSS BRAFV600E-mut mCRC were included. RNF43 was found mut in 34 (26%) cases. Among RNF43 mutants, TT was associated with longer PFS (7.7 v 3.0 months; P = .002) and higher overall response rate (ORR; 45% v 0%; P = .009) compared with CT. Conversely, among RNF43 wild-type (wt) patients, only a trend for longer PFS (4.5 v 3.7 months; P = .064) favoring TT, with no differences in ORR (P = .14), was observed. After excluding 36 patients receiving TT in third line or beyond, a longer OS (19.4 v 10.1 months; P = .022) and a numerically OS advantage (10.6 v 6.6 months; P = .068) were reported for TT both in the RNF43-mut and in the RNF43 wt groups. However, no interaction effect was reported between RNF43 mutational status and treatment in ORR (Pinteraction = .96), PFS (Pinteraction = .13), and OS (Pinteraction = .44). CONCLUSION: Patients with pMMR/MSS BRAFV600E-mut mCRC achieve benefit from TT versus CT independently of RNF43 mutational status, although a higher magnitude of benefit from TT is observed in RNF43-mut tumors. These findings deserve confirmation in concluded and ongoing randomized trials.

Effects of UBE3A on the insulin resistance in polycystic ovary syndrome through the ubiquitination of AMPK.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a reproductive hormonal abnormality and a metabolic disorder, which is frequently associated with insulin resistance (IR). We aim to investigate the potential therapeutic effects of Ubiquitin-protein ligase E3A (UBE3A) on IR in the PCOS rats via Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. METHODS: The PCOS and IR rats model was established by dehydroepiandrosterone (DHEA) and high fat diet (HFD) treatment, and the fat rate, glucose tolerance and insulin tolerance were measured. The IR rats numbers were calculated. Besides, the mRNA levels of glucose transporter 4 (GLUT4) and UBE3A were detected by RT-qPCR. Furthermore, the relationship between was demonstrated by co-IP assay. The phosphorylation and ubiquitination of AMPK were analyzed by western blot. RESULTS: UBE3A was up-regulated in the PCOS rats. UBE3A knockdown significantly decreased the fat rate, glucose tolerance and insulin tolerance in the PCOS and IR rats. Additionally, the GLUT4 levels were significantly increased in PCOS + IR rats. Besides, after UBE3A knockdown, the IR rats were decreased, the p-IRS1 and p-AKT levels were significantly up-regulated. Furthermore, UBE3A knockdown enhanced phosphorylation of AMPK through decreasing the ubiquitination of AMPK. AMPK knockdown reversed the role of UBE3A knockdown in the PCOS + IR rats. CONCLUSIONS: UBE3A knockdown inhibited the IR in PCOS rats through targeting AMPK. Our study indicated that UBE3A might become a potential biological target for the clinical treatment of PCOS.

Association between SYVN1 and SEL1 genetic polymorphisms and remission in rheumatoid arthritis patients treated with TNF-α inhibitors: a machine learning approach.

Rheumatoid arthritis (RA) is a severe chronic inflammatory condition that affects joint synovium. Suppressor/enhancer of lin-12-like (SEL1L)-Synoviolin 1 (SYVN1)-mediated endoplasmic reticulum-associated degradation (ERAD) is highly associated with RA development. Although targeting SEL1L-SYVN1-mediated ERAD can be beneficial, studies that evaluate the association between polymorphisms in their genes and remission from the disease in RA patients taking tumor necrosis factor (TNF)-α inhibitors have yet to be carried out. Hence, the purpose of this study was to investigate the association between SYVN1 and SEL1L polymorphisms and TNF-α inhibitor response using various machine learning models. A total of 12 single-nucleotide polymorphisms (SNPs), including 5 SNPs in SYVN1 and 7 SNPs of SEL1L were investigated. Logistic regression analysis was used to examine the relationship between genetic polymorphisms and response to treatment. Various machine learning methods were employed to evaluate factors associated with remission in patients receiving TNF-α inhibitors. After adjusting for covariates, we found that sulfasalazine and rs2025214 in SEL1L increase the remission rates by approximately 3.3 and 2.8 times, respectively (95% confidence intervals 1.126-9.695 and 1.074-7.358, respectively). Machine learning approaches showed acceptable prediction estimates for remission in RA patients receiving TNF-α inhibitors, with the area under the receiver-operating curve (AUROC) values ranging from 0.60 to 0.65. A polymorphism of the SEL1L gene (rs2025214) and sulfasalazine were found to be associated with treatment response in RA patients receiving TNF-α inhibitors. These preliminary data could be used to tailor treatment for RA patients using TNF-α inhibitors.

MDM2 for the practicing pathologist: a primer.

The mouse double minute 2 (MDM2) gene is located on the long arm of chromosome 12 and is the primary negative regulator of p53. The MDM2 gene encodes an E3 ubiquitin-protein ligase that mediates the ubiquitination of p53, leading to its degradation. MDM2 enhances tumour formation by inactivating the p53 tumour suppressor protein. The MDM2 gene also has many p53-independent functions. Alterations of MDM2 may occur through various mechanisms and contribute to the pathogenesis of many human tumours and some non-neoplastic diseases. Detection of MDM2 amplification is used in the clinical practice setting to help diagnose multiple tumour types, including lipomatous neoplasms, low-grade osteosarcomas and intimal sarcoma, among others. It is generally a marker of adverse prognosis, and MDM2-targeted therapies are currently in clinical trials. This article provides a concise overview of the MDM2 gene and discusses practical diagnostic applications pertaining to human tumour biology.

BRAF V600E and RNF43 Co-mutations Predict Patient Outcomes With Targeted Therapies in Real-World Cases of Colorectal Cancer.

Anti-BRAF/EGFR therapy is approved for metastatic colorectal cancer (mCRC) with BRAFV600E mutations, although not all patients respond. Novel recent findings indicate the potential of RNF43 mutations to predict outcomes in patients with BRAF-mutated microsatellite stable (MSS) mCRC treated with anti-BRAF/EGFR therapy. This study aimed to independently and rapidly validate BRAFV600E/RNF43 co-mutations as predictive biomarkers of benefit to anti-EGFR/BRAF therapy. Clinical data were derived from electronic health record data from ~280 US cancer clinics between January 2011 and March 2022 from the Flatiron Health-Foundation Medicine real-world clinico-genomic mCRC database. Real-world cases of BRAFV600E-mutated mCRC, with patients receiving anti-BRAF/EGFR therapy (n = 49), were included. Patients who were MSS, with RNF43 mutations, had favorable progression-free survival (hazard ratio [HR] 0.29; 95% CI [CI], 0.13-0.65) and overall survival (HR 0.32, 95% CI, 0.12-0.84) compared with wild type. No difference in outcomes was observed between patient groups with RNF43-mutant versus wild-type receiving standard-of-care chemotherapy. BRAFV600E/RNF43 co-mutations predict mCRC anti-BRAF/EGFR outcomes in diverse clinical settings.

Effect of CPP-related genes on GnRH secretion and Notch signaling pathway during puberty.

Puberty is a complex biological process of sexual development, influenced by genetic, metabolic-nutritional, environmental and socioeconomic factors, characterized by the development of secondary sexual characteristics, maturation of the gonads, leading to the acquisition of reproductive capacity. The onset of central precocious puberty (CPP) is mainly associated with the early activation of the hypothalamic-pituitary-gonadal (HPG) axis and increased secretion of gonadotropin-releasing hormone (GnRH), leading to increased pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and activation of gonadal function. Due to the expense and invasiveness of current diagnostic testing and drug therapies for CPP, it would be helpful to find serum and genetic markers to facilitate diagnosis. In this paper, we summarized the related factors that may affect the expression of GnRH1 gene and the secretion and action pathway of GnRH and related sex hormones, and found several potential targets, such as MKRN3, DLK1 and KISS1. Although, the specific mechanism still needs to be further studied, we would be encouraged if the insights from this review could provide new insights for future research and clinical diagnosis and treatment of CPP.

The role of E3 ubiquitin ligase in multiple myeloma: potential for cereblon E3 ligase modulators in the treatment of relapsed/refractory disease.

INTRODUCTION: Insights into the mechanisms of protein homeostasis and proteasomal degradation have led to new strategies of redirecting the ubiquitin-proteasome system (UPS) to reduce or eliminate proteins or survival factors key to malignant pathobiology, multiple myeloma (MM) in particular. These strategies have enabled researchers to target proteins that were previously considered difficult to modulate by pharmacological means. AREAS COVERED: This review provides a brief overview of UPS biology, particularly the role of the CRL4CRBN E3 ubiquitin ligase complex, and summarizes current strategies for co-opting the UPS, including CELMoD compounds, SNIPERs, PROTACs, and degronimids. A detailed discussion is provided on lead CELMoD compounds iberdomide and mezigdomide, which are currently being evaluated in clinical trials in patients with MM. EXPERT OPINION: Since a high proportion of patients develop drug resistance, it is vital to have novel therapeutic agents for treating relapsed patients with MM more effectively. It is encouraging that the expanding pathophysiological insight into cellular signaling pathways in MM increasingly translates into the development of novel therapeutic agents such as targeted protein degraders. This holds promise for improving outcomes in MM and beyond.

Evaluation of the Prognostic Significance of Cereblon Protein Expression in Multiple Myeloma.

BACKGROUND: Considerable progress has been made in the treatment of multiple myeloma (MM) patients with the development of various new agents that increased survival rates over the past fifteen years. Cereblon (CRBN) plays an important role in mediating the antitumor effects of immunomodulatory drugs (IMiDs) among these new agents. The aim of our study is to investigate immunohistochemically (IHC) cereblon protein expression status in MM. METHODS: Immunohistochemically, CRBN expression and its relationship with various prognostic factors were evaluated in bone marrow biopsies of 96 patients with MM in a single centre. RESULTS: Cytoplasmic and nuclear CRBN expression was detected in all neoplastic cells. While a complete or partial response to treatment was obtained in 45 patients, the disease was stable in 13 and progressive in 17 patients. Survival was longer in those treated with IMiD-containing regimens (p = 0.044). Both the survival rate (p = 0.013) and the survival time were significantly increased (p = 0.023) in those who received the treatment protocol containing protease inhibitors. A significant relationship was found between the treatment protocol and treatment response in the chi-squared analysis (p = 0.008). Although the longest survival time - though not statistically significant - was detected in the group treated with protease inhibitors (log rank, p = 0.217). The survival analysis revealed the presence of a relationship between IgG and IgA positivity and survival. CONCLUSIONS: In this study, the survival time of the patients who received treatment regimens containing protease inhibitors and IMiD was longer, independent of the presence of strong nuclear CRBN expression. The survival rate was significantly higher in those who used IMiD and protease inhibitors in combination. Since the survival rate was found to be increased in IgG positive cases and we thought that evaluation of immunoglobulin tissue expression in MM cases can provide prognostic prediction.

Novel 11ß-substituted estradiol conjugates: Transition from ERα agonizts to effective PROTAC degraders.

Endocrine therapy is widely used in clinic for breast cancer treatment, but long-term treatment inevitably causes drug resistance. Most of endocrine therapy-resistant breast cancers continue to depend on ERα signaling for growth and survival. In this regard, small molecule-induced ERα degradation, i.e. proteolysis targeting chimeras (PROTACs), represents an effective strategy to overcome endocrine resistance. Herein, we describe the design, synthesis, and biological evaluation of novel ERα-targeting PROTACs, wherein a E3 ligase ligand was attached to the 11ß-position of estradiol via various linkers. Our efforts have identified a potent ERα PROTAC 15b that achieved excellent ERα degradation activity (DC50 = 67 nM) and induced comparable inhibition of cell growth to that of fulvestrant in MCF-7 cells. Besides, 15b displayed antagonistic effects in uterine cells and favorable physicochemical properties, making it as a good lead compound for further development as anti-breast agents.

[Characteristics and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia patients].

Objective: To explore the characteristics, clinical features and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia (T-ALL) patients. Methods: The clinical data of 61 T-ALL patients who underwent second-generation gene sequencing in Henan Provincial People's Hospital from March 2016 to March 2021 were retrospectively analyzed. There were 46 males and 15 females, with a median age [M (Q1, Q3)] of 18 (11, 30) years. The relationship between NOTCH1/FBXW7 gene mutation characteristics, clinical and laboratory parameters and their impact on event free survival (EFS) and overall survival (OS) were analyzed. Results: NOTCH1 gene mutations were found in 34 cases (55.7%, 34/61), including 22 cases of heterodimer domain (HD) mutations (64.7%), 7 cases of proline/glutamate/serine/threonine (PEST) mutations (20.6%), and 5 cases of both HD and PEST mutations (14.7%). FBXW7 gene mutations were detected in 9 cases (14.8%, 9/61), of which 5 cases had both NOTCH1 and FBXW7 gene mutations. Twenty-three (37.7%, 23/61) cases were wild type. The median white blood cell count of patients in NOTCH1/FBXW7 gene mutations group and wild-type group was 76.4×109/L (8.3×109/L, 149.2×109/L), 54.1×109/L (5.3×109/L, 156.6×109/L), respectively. Moreover, the hemoglobin was (89.1±27.1) g/L and (99.5±23.1) g/L, respectively, and the median proportion of bone marrow primordial cells was 84.5% (69.0%, 91.3%) and 60.0%(35.0%, 80.0%), respectively. The gene expression rate of SIL-TAL1, Hox11 and Hox11L2 was 7.9% (3/38) vs 17.4% (4/23), 18.4% (7/38) vs 4.3% (1/23), 5.3% (2/38) vs 13.0% (3/23), respectively (all P>0.05). However, the median platelet level in the NOTCH1/FBXW7 gene mutations group was 60.5×109/L (36.8×109/L, 100.3×109/L), which was lower than that in the wild-type group [116.0×109/L (63.0×109/L, 178.0×109/L)] (P=0.018). The median number of gene mutations in the group with NOTCH1/FBXW7 gene mutations group was 2.5 (1.8, 4.0), which was more than that in the group without NOTCH1/FBXW7 gene mutations group [0 (0, 1.0)] (P<0.001). The median EFS and OS of adult NOTCH1/FBXW7 gene mutations group were 28.0 (95%CI: 7.3-48.7) months and 30.0 (95%CI: 8.9-51.1) months, respectively, which were better than those of adult wild-type group [4.5 (95%CI: 0-11.6) months and 9.0 (95%CI: 0-19.1) months] (P=0.008 and 0.014).The median EFS and OS of children NOTCH1/FBXW7 gene mutations group were 12.0 (95%CI: 10.4-13.6) months and 19.0 (95%CI: 13.6-24.4) months, respectively, and those of wild-type group were 10.0 (95%CI: 8.9-11.1) months and 21.0 (95%CI: 0-51.4) months, respectively (P=0.673 and 0.434). Conclusions: The mutation rate of NOTCH1/FBXW7 gene is higher in T-ALL patients. Patients with NOTCH1/FBXW7 gene mutations group have lower platelet count and better EFS and OS. NOTCH1/FBXW7 gene mutation may be used as a hierarchical basis for individualized treatment of adult T-ALL patients.

Genome-wide CRISPR screen identified Rad18 as a determinant of doxorubicin sensitivity in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated. METHODS: Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy. RESULTS: We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin. CONCLUSIONS: Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.

GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma.

BACKGROUND: Aberrant TAK1 (transforming growth factor ß-activated kinase 1) activity is known to be involved in a variety of malignancies, but the regulatory mechanisms of TAK1 remain poorly understood. GRAMD4 (glucosyltransferase Rab-like GTPase activator and myotubularin domain containing 4) is a newly discovered p53-independent proapoptotic protein with an unclear role in HCC (hepatocellular carcinoma). RESULTS: In this research, we found that GRAMD4 expression was lower in HCC samples, and its downregulation predicted worse prognosis for patients after surgical resection. Functionally, GRAMD4 inhibited HCC migration, invasion and metastasis. Mechanistically, GRAMD4 interacted with TAK1 to promote its protein degradation, thus, resulting in the inactivation of MAPK (Mitogen-activated protein kinase) and NF-κB pathways. Furthermore, GRAMD4 was proved to recruit ITCH (itchy E3 ubiquitin protein ligase) to promote the ubiquitination of TAK1. Moreover, high expression of TAK1 was correlated with low expression of GRAMD4 in HCC patients. CONCLUSIONS: GRAMD4 inhibits the migration and metastasis of HCC, mainly by recruiting ITCH to promote the degradation of TAK1, which leads to the inactivation of MAPK and NF-κB signalling pathways.

Therapeutic potential of parkin as a tumor suppressor via transcriptional control of cyclins in glioblastoma cell and animal models.

Parkin (PK) is an E3-ligase harboring tumor suppressor properties that has been associated to various cancer types including glioblastoma (GBM). However, PK is also a transcription factor (TF), the contribution of which to GBM etiology remains to be established. Methods: The impact of PK on GBM cells proliferation was analyzed by real-time impedance measurement and flow cytometry. Cyclins A and B proteins, promoter activities and mRNA levels were measured by western blot, luciferase assay and quantitative real-time PCR. Protein-protein and protein-promoter interactions were performed by co-immunoprecipitation and by ChIP approaches. The contribution of endogenous PK to tumor progression in vivo was performed by allografts of GL261 GBM cells in wild-type and PK knockout mice. Results: We show that overexpressed and endogenous PK control GBM cells proliferation by modulating the S and G2/M phases of the cell cycle via the trans-repression of cyclin A and cyclin B genes. We establish that cyclin B is regulated by both E3-ligase and TF PK functions while cyclin A is exclusively regulated by PK TF function. PK invalidation leads to enhanced tumor progression in immunocompetent mice suggesting an impact of PK-dependent tumor environment to tumor development. We show that PK is secreted by neuronal cells and recaptured by tumor cells. Recaptured PK lowered cyclins levels and decreased GBM cells proliferation. Further, PK expression is decreased in human GBM biopsies and its expression is inversely correlated to both cyclins A and B expressions. Conclusion: Our work demonstrates that PK tumor suppressor function contributes to the control of tumor by its cellular environment. It also shows a key role of PK TF function in GBM development via the control of cyclins in vitro and in vivo. It suggests that therapeutic strategies aimed at controlling PK shuttling to the nucleus may prove useful to treat GBM.

"All Our Wisdom is Stored in the Trees" - Degrading BCR-ABL with Berberis Vulgaris.

Treating BCR-ABL-positive chronic myeloid leukemia remains impeded by the development of clinical resistance to imatinib. It has been demonstrated that berberine, a plant alkaloid, has activity against imatinib-resistant BCR-ABL mutants by inducing autophagic degradation of BCR-ABL, thereby preventing the acquisition of drug-resistant mutations.See related article by Yin et al., p. 4040.

Increased survivability of ischemic skin flap tissue in Flk-1+/- mice by Pellino-1 intervention.

OBJECTIVE: Reduced skin flap survival due to ischemia is a serious concern during reconstructive cosmetic surgery. The absence of VEGF and its receptors during ischemia may lead to flap failure. We identified Peli1, a 46-kDa protein, as a proangiogenic molecule and is directly regulated by VEGF. Therefore, we hypothesized that Peli1 acts downstream of Flk-1/VEGFR2 and aids in skin flap survival during ischemia. METHODS: Scratch and matrigel assays were performed to observe cell proliferation, migration, and tube formation in vitro. Western blot analysis was carried out to detect the phosphorylation of Akt (p-Akt) and MAPKAPK2 (p-MK2) in HUVECs. The translational potential of Peli1 pretreatment in the rescue of skin flap tissue was studied in vivo using Flk-1+/- mice. Animals underwent dorsal ischemic skin flap surgery, and the tissue was collected on day 12 for analysis. RESULTS: Western blot analysis revealed a direct relationship between Peli1 and VEGF, as demonstrated by loss-of-function and gain-of-function studies. In addition, pretreatment with Ad.Peli1 restored the phosphorylation of Akt and MK2 and improved the migration potential of Flk-1-knockdown cells. Ad.Peli1 pretreatment salvaged the ischemic skin flap of Flk-1+/- mice by increasing blood perfusion and reducing the inflammatory response and the extent of necrosis. CONCLUSION: Our findings reveal that Peli1 is a proangiogenic molecule that acts downstream of VEGF-Flk-1 and restores angiogenesis and enhances skin flap survivability.