Wee1 promotes cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1.

BACKGROUND: The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA damage repair mechanism plays an essential role not only in the initiation and progression of hematological malignancies but also links to the development of TKI resistance. Deciphering the abnormally regulated DNA damage repair and proteins involved brings new insights into the therapy of leukemias. As a G2/M phase checkpoint kinase and a DNA damage repair checkpoint kinase engaged in the DNA damage response (DDR), along with an oncogenic driver present in various cancers, the particular involvement of Wee1 in DNA damage is far from clear. Deciphering its function and targeting it via modulating DNA repair pathways is important for improving our understanding of cancer treatment. METHODS: Wee1 expression was assessed in cell lines using RT-qPCR and western blot, and Wee1 knockdown efficacy was validated using RT-qPCR, western blot, and immunofluorescence. Wee1 function was investigated by CCK-8, colony formation, and flow cytometry assay in vitro. Wee1 role in DNA repair and its interactions with other proteins were then studied using western blot, immunofluorescence, and double plasmid-repair studies. Finally, the CCK-8 and flow cytometry assay was utilized to investigate Wee1 and imatinib's synergistic effect, and a CML mouse model was constructed to study Wee1's role in carcinogenesis in vivo. RESULTS: Wee1 was reported to respond quickly to DDR in an ATM-γH2AX-MDC1-dependent way upon DNA double-strand breaks (DSBs) occurrence, and it regulated homologous recombination by stimulating the recruitment of critical proteins RAD51/BRCA1 upon DSB sites. Wee1 was also revealed to be abnormally upregulated in CML cells. Further suppression of Wee1 not only causes cell cycle arrest and inhibits the proliferation of cancer cells but also enhances CML cell sensitivity to Imatinib in vitro and in vivo, possibly through an excessive accumulation of overall DSBs. CONCLUSION: Wee1 is extensively involved in the DRR signaling and DSB repair pathway. Inhibiting abnormally elevated Wee1 benefits CML therapy in both IM-resistant and IM-sensitive cells. Our data demonstrated that Wee1 participated in promoting cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1. In the fight against CML, Wee1's dysregulation in the DNA damage repair mechanism of CML pathogenesis makes it a viable therapeutic target in clinical applications.

Targeting MDC1 promotes apoptosis and sensitizes Imatinib resistance in CML cells by mainly disrupting non-homologous end-joining repair.

The first-line drug Imatinib (IM) has achieved a curative effect in most chronic myeloid leukemia (CML) patients, but drug resistance remains a problem. More alternative therapeutic strategies need to explore. In recent years, targeting dysregulated DNA repair mechanisms provided promising options for cancer treatment. Here, we discovered the versatile Mediator of DNA Damage Checkpoint 1 (MDC1) interacted with γ-H2AX and 53BP1 in the early stage of the DNA damage response of cells. MDC1 overexpressed in CML cell lines and patients' bone marrow mononuclear cells. By knocking down MDC1, non-homologous end-joining pathways were mainly inhibited, leading to an intense accumulation of unrepaired intracellular DNA damage and an apparent cell apoptosis promotion. Notably, targeting MDC1 further enhanced drug sensitivity in IM-resistant CML cells. Our work revealed that MDC1 is a prospective target for CML treatment through regulating DNA damage repair mechanism, and also an alternative option for IM resistance dilemma. This study extends the understanding of regulating dysfunctional DNA repair mechanisms for cancer treatment.

circCRKL, a circRNA derived from CRKL, regulates BCR-ABL via sponging miR-877-5p to promote chronic myeloid leukemia cell proliferation.

BACKGROUND: The BCR-ABL fusion protein is the key factor that results in the occurrence of chronic myeloid leukemia (CML). Imatinib (IM) is a targeted inhibitor of BCR-ABL to achieve complete remission. However, remission failure occurs due to acquired resistance caused by secondary BCR-ABL mutations, underlining the need for novel BCR-ABL-targeting strategies. Circular RNAs (circRNAs) derived from tumor-related genes have been revealed as possible therapeutic targets for relevant cancers in recent investigations. In CML, the roles of this kind of circRNA are yet obscure. METHODS: Firstly, RT-qPCR was used for determining circCRKL expression level in cell lines and clinical samples, RNase R and Actinomycin D were employed to verify the stability of circCRKL. Then shRNAs were designed to specifically knockdown circCRKL. The function of circCRKL in vitro was investigated using CCK-8, colony formation assay, and flow cytometry, while a CML mouse model was constructed to explore the function in vivo. Finally, a dual-luciferase reporter assay, RNA pull-down, RNA immunoprecipitation, and rescue experiments were conducted to investigate the mechanism of circCRKL functioning. RESULTS: Here, we determined circCRKL, which derives from CML-relevant gene CRKL, is over-expressed in BCR-ABL+ cells. Then we noticed knocking down circCRKL using shRNA lentivirus dampens the proliferation of BCR-ABL+ cells both in vitro and in vivo, and augments susceptibility of resistant cells to IM. Intriguingly, we observed that circCRKL has a considerable impact on the expression level of BCR-ABL. Mechanistically, circCRKL could behave like a decoy for miR-877-5p to enhance the BCR-ABL level, allowing BCR-ABL+ cells to maintain viability. CONCLUSIONS: Overall, the current study uncovers that circCRKL is specifically expressed and regulates BCR-ABL expression level via decoying miR-877-5p in BCR-ABL+ cells, highlighting that targeting circCRKL along with imatinib treatment could be utilized as a potential therapeutic strategy for CML patients.

ACY-1215 suppresses the proliferation and induces apoptosis of chronic myeloid leukemia cells via the ROS/PTEN/Akt pathway.

Chronic myeloid leukemia (CML) is a hematological tumor marked by the bcr-abl fusion gene formed by t (9;22) (q34; q11), which translated into the BCR-ABL protein. Tyrosine kinase inhibitors (TKIs) have been widely used to cure CML patients. Nevertheless, the emergence of TKI resistance has become the problem to the outcome of CML patients. Histone deacetylase 6 (HDAC6), a kind of Hsp90α deacetylase, was detected to be overexpressed in chronic myeloid leukemia stem cells. Besides, the loss of HDAC6 enzymatic activity can result in the degradation of Hsp90α's client proteins, such as BCR-ABL, the oncoprotein of CML. Here, we explored the expression of HDAC6 and discovered that it was upregulated compared with control in CML. Then we explored the effect of Rocilinostat (ACY-1215), a specific HDAC6 inhibitor, on CML cells. Our results proved that ACY-1215 could induce apoptosis and cell cycle arrest in a ROS-dependent manner. Moreover, we detected a downregulation of the BCR-ABL signaling pathway in the ACY-1215 treatment group. Mechanistically, we noted that the upregulation of PTEN was induced after being treated by ACY-1215 and its downstream protein p-Akt was decreased. The Akt activator SC79 can partially reverse the influence of ACY-1215 on CML cells. Besides, our results also proved that ACY-1215 can synergize with imatinib to suppress chronic myeloid leukemia in vitro and in vivo. On the whole, our study revealed that HDAC6 is a possible therapeutic target in CML, and the combination therapy of TKI and HDAC6 inhibitor may improve the outcome of CML patients.