E2F1 rs3213150 polymorphism influences cytarabine sensitivity and prognosis in patients with acute myeloid leukemia.

Cytarabine (Ara-C) plays an irreplaceable role in the treatment of acute myeloid leukemia (AML). However, there are significant differences in efficacy among patients. Our previous studies found that E2F1 rs3213150 polymorphism was associated with remission rate of Ara-C chemotherapy, but the specific mechanism is not clear. This study aimed to further confirm the correlation between E2F1 rs3213150 polymorphism and Ara-C resistance and prognosis in AML patients, and to provide valuable information for elucidating the molecular mechanisms involved. METHODS: Rs3213150 genotyping was performed in 922 AML patients by Sanger sequencing, and the effects of different genotypes on chemosensitivity and prognosis were analyzed by Logistic regression and Cox regression. Meanwhile, a prediction model of Ara-C chemotherapy resistance was established. The impact of rs3213150 polymorphism on E2F1 expression level was determined by luciferase reporter gene assay, and differentially expressed genes between patients with different genotypes were identified by RNA sequencing. RESULTS: Compared with rs3213150 G allele carriers, patients with AA genotype had more obvious Ara-C resistance (41.94% vs. 27.94%, P = 0.002), shorter overall survival (529 d vs. 644 d, P = 0.008) and disease-free survival (519 d vs. 556 d, P = 0.023). Rs3213150G > A mutation resulted in decreased E2F1 expression. CONCLUSION: E2F1 rs3213150 polymorphism influences the chemosensitivity and prognosis of Ara-C in Chinese AML patients.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

OBJECTIVE: Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. METHODS: CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). RESULTS: Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. CONCLUSIONS: Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

Chemosensitization of tumor cells: inactivation of nuclear factor-kappa B associated with chemosensitivity in melanoma cells after combination treatment with E2F-1 and doxorubicin.

Combination chemotherapy has been shown to be more effective than single-agent therapy for many types of cancer, but both are known to induce drug resistance in cancer cells. Two major culprits in the development of this drug resistance are nuclear factor-kappaB (NF-kappaB) and the multidrug resistance (MDR) gene. For this reason, chemogene therapy is emerging as a viable alternative to conventional chemotherapy combinations. We have shown that transduction of the E2F-1 gene in melanoma cells markedly increases cell sensitivity to doxorubicin, thereby producing a synergistic effect on melanoma cell apoptosis. Our microarray results show that the NF-kappaB pathway and related genes undergo significant changes after the combined treatment of E2F-1 and doxorubicin. In fact, inactivation of NF-kappaB is associated with melanoma cell apoptosis induced by E2F-1 and doxorubicin, providing a link between the NF-kappaB signaling pathway and the chemosensitivity of melanoma cells after this treatment.

Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo.

Overexpression of the transcription factor E2F-1 induces apoptosis in a variety of carcinoma cells and inactivates murine double minute protein 2, a factor associated with poor prognosis in soft tissue sarcomas. We have shown previously that the double-stranded RNA-activated protein kinase PKR plays an important role in mediating this apoptotic response in carcinoma cells to E2F-1. We sought to evaluate the potential of E2F-1 gene therapy in soft tissue sarcomas and to study the involvement of PKR in the response to E2F-1 overexpression in mesenchymal cells. A replication-deficient adenovirus carrying the E2F-1 gene (Ad5E2F) was used to induce E2F-1 overexpression in the p53 mutated leiomyosarcoma cell line, SKLMS-1. Western blot analysis confirmed E2F-1 overexpression and up-regulation of the antiapoptotic factor Bcl-2 48 hours following infection with Ad5E2F. Apoptosis in Ad5E2F-treated cells was confirmed by fluorescence-activated cell sorting analysis and by poly(ADP-ribose) polymerase cleavage and DNA fragmentation assays. Vector-dependent up-regulation of PKR correlated with the amount of Ad5E2F-induced apoptosis. In vivo treatment of SKLMS-1 tumor-bearing BALB/c mice with intratumoral injections of Ad5E2F at a dose of 2 x 10(10) viral particles resulted in significant inhibition in tumor growth compared with control-treated animals (P < 0.016). Complete disappearance of all tumors was seen in two of seven mice in the Ad5E2F-treated animals. Immunohistochemical analysis of tumor specimens showed overexpression of E2F-1 and up-regulation of PKR in Ad5E2F-treated tumors. These findings show that adenovirus-mediated overexpression of E2F-1 results in up-regulation of PKR and significant growth suppression of leiomyosarcomas in vivo. Taken together, these data suggest that E2F-1 gene therapy and PKR modulation might be a promising treatment strategy for these tumors that are highly resistant to conventional therapies.