The FABD domain is critical for the oncogenicity of BCR/ABL in chronic myeloid leukaemia.

BACKGROUND: Abnormally expressed BCR/ABL protein serves as the basis for the development of chronic myeloid leukaemia (CML). The F-actin binding domain (FABD), which is a crucial region of the BCR/ABL fusion protein, is also located at the carboxyl end of the c-ABL protein and regulates the kinase activity of c-ABL. However, the precise function of this domain in BCR/ABL remains uncertain. METHODS: The FABD-deficient adenovirus vectors Ad-BCR/ABL△FABD, wild-type Ad-BCR/ABL and the control vector Adtrack were constructed, and 32D cells were infected with these adenoviruses separately. The effects of FABD deletion on the proliferation and apoptosis of 32D cells were evaluated by a CCK-8 assay, colony formation assay, flow cytometry and DAPI staining. The levels of phosphorylated BCR/ABL, p73, and their downstream signalling molecules were detected by western blot. The intracellular localization and interaction of BCR/ABL with the cytoskeleton-related protein F-actin were identified by immunofluorescence and co-IP. The effect of FABD deletion on BCR/ABL carcinogenesis in vivo was explored in CML-like mouse models. The degree of leukaemic cell infiltration was observed by Wright‒Giemsa staining and haematoxylin and eosin (HE) staining. RESULTS: We report that the loss of FABD weakened the proliferation-promoting ability of BCR/ABL, accompanied by the downregulation of BCR/ABL downstream signals. Moreover, the deletion of FABD resulted in a change in the localization of BCR/ABL from the cytoplasm to the nucleus, accompanied by an increase in cell apoptosis due to the upregulation of p73 and its downstream proapoptotic factors. Furthermore, we discovered that the absence of FABD alleviated leukaemic cell infiltration induced by BCR/ABL in mice. CONCLUSIONS: These findings reveal that the deletion of FABD diminished the carcinogenic potential of BCR/ABL both in vitro and in vivo. This study provides further insight into the function of the FABD domain in BCR/ABL.

Jak2/STAT6/c-Myc pathway is vital to the pathogenicity of Philadelphia-positive acute lymphoblastic leukemia caused by P190BCR-ABL.

BACKGROUND: The Philadelphia chromosome encodes the BCR-ABL fusion protein, which has two primary subtypes, P210 and P190. P210 and P190 cause Philadelphia-positive chronic myeloid leukemia (Ph+ CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), respectively. The Ph+ ALL is more malignant than Ph+ CML in disease phenotype and progression. This implies the key pathogenic molecules and regulatory mechanisms caused by BCR-ABL in two types of leukemia are different. It is reported that STAT6 was significantly activated only in P190 transformed cells. However, the potential role and the mechanism of STAT6 activation in Ph+ ALL and its activation mechanism by P190 are still unknown. METHODS: The protein and mRNA levels of STAT6, c-Myc, and other molecules were measured by western blot and quantitative real-time PCR. The STAT6 inhibitor AS1517499 was used to specifically inhibit p-STAT6. The effect of p-STAT6 inhibition on Ph+ CML and Ph+ ALL cells was identified by CCK-8 and FCM assay. Dual luciferase reporter and ChIP assay were performed to confirm the direct binding between STAT6 and c-Myc. The impact of STAT6 inhibition on tumor progression was detected in Ph+ CML and Ph+ ALL mouse models. RESULTS: Our results demonstrated that P210 induced CML-like disease, and P190 caused the more malignant ALL-like disease in mouse models. STAT6 was activated in P190 cell lines but not in P210 cell lines. Inhibition of STAT6 suppressed the malignancy of Ph+ ALL in vitro and in vivo, whereas it had little effect on Ph+ CML. We confirmed that p-STAT6 regulated the transcription of c-Myc, and STAT6 was phosphorylated by p-Jak2 in P190 cell lines, which accounted for the discrepant expression of p-STAT6 in P190 and P210 cell lines. STAT6 inhibition synergized with imatinib in Ph+ ALL cells. CONCLUSIONS: Our study suggests that STAT6 activation plays an essential role in the development of Ph+ ALL and may be a potential therapeutic target in Ph+ ALL. Video abstract.

mTOR inhibition by AZD2014 alleviates BCR::ABL1 independent imatinib resistance through enhancing autophagy in CML resistant cells.

Chronic myeloid leukemia (CML) is a common hematopoietic malignancy in adults. Great progress has been made in CML therapy with imatinib. However, resistance to imatinib may occur during treatment. BCR::ABL1 dependent imatinib resistance has been well resolved with more potent tyrosine kinase inhibitors, but BCR::ABL1 independent resistance still remains to be resolved. This study is devoted to find novel targets for BCR::ABL1 independent imatinib-resistant patients. It is reported BCR::ABL1 independent resistance is mainly related to the activation of alternative survival pathway, and mTOR is an important regulator for cell growth especially in tumor cells. Hence, we explored the role of mTOR in BCR::ABL1 independent resistance, the possibility of mTOR to be a therapeutic target for imatinib resistant patients and the related mechanism. We found mTOR was upregulated in imatinib-resistant cells. mTOR inhibition by AZD2014 led to growth inhibition and synergized with imatinib in apoptosis induction in K562/G01. AZD2014 exerted its anti-leukemia effect through enhancing autophagy. mTOR signal pathway is poorly inhibited by imatinib and AZD2014 shows little effect on BCR::ABL1 signal pathway, which indicates that mTOR is involved in imatinib resistance via a BCR::ABL1 independent manner. Taken together, mTOR represents a potential target to overcome BCR::ABL1 independent imatinib resistance.

The HSP90 inhibitor KW-2478 depletes the malignancy of BCR/ABL and overcomes the imatinib-resistance caused by BCR/ABL amplification.

BACKGROUND: With the widespread clinical application of tyrosine kinase inhibitors (TKIs), an increasing number of chronic myeloid leukaemia (CML) patients have developed resistance or intolerance to TKIs. BCR/ABL is the oncoprotein of CML. HSP90 is an essential chaperone of BCR/ABL and plays an important role in protein folding and the function of BCR/ABL. Therefore, inhibiting the chaperone function of HSP90 may be an effective strategy for CML treatment and to overcome TKI resistance. METHODS: The effect of KW-2478 on CML cell viability, apoptosis and cell cycle progression was detected by CCK-8 assay or flow cytometry. The levels of BCR/ABL, HSP90 and other signalling proteins were detected by western blots. The mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The interaction between BCR/ABL and HSP90α was detected by coimmunoprecipitation. The effect of KW-2478 on BCR/ABL carcinogenesis in vivo was investigated in CML-like mouse models. RESULTS: KW-2478 inhibited growth and induced apoptosis of CML cells. KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. This treatment also caused an increase in p27 and p21 expression and a decrease in cyclin B1 expression, which led to G2/M phase arrest. The mitochondrial pathway was primarily responsible for KW-2478-induced apoptosis. KW-2478 had a synergistic effect with imatinib in growth inhibition. Notably, KW-2478 had a stronger effect on growth inhibition, apoptosis induction and cell cycle arrest of K562/G01 cells than K562 cells. KW-2478 could effectively prolong the mouse lifespan and alleviate disease symptoms in CML-like mouse models. CONCLUSIONS: This finding demonstrated that KW-2478 had anticancer properties in imatinib-sensitive and imatinib-resistant CML cells and illustrated the possible mechanisms. This study provides an alternative choice for CML treatment, especially for TKI-resistant patients with BCR/ABL amplification and TKI-intolerant patients.