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 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.

Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation.

BACKGROUND: Tyrosine kinase inhibitors have achieved quite spectacular advances in the treatment of chronic myeloid leukemia (CML), but disease progression and drug resistance that related to the T315I mutation, remain major obstacles. Dendritic cell-derived exosomes (Dex) induce NK cell immunity, but have yet to achieve satisfactory clinical efficacy. An approach to potentiate antitumor immunity by inducing both NK- and T-cell activation is urgently needed. Retinoic acid early inducible-1γ (RAE-1γ), a major ligand of natural killer group 2 member D (NKG2D), plays an important role in NK-cell and T-lymphocyte responses. We generated RAE-1γ enriched CML-specific Dex (CML-RAE-1γ-Dex) from dendritic cells (DCs) pulsed with lysates of RAE-1γ-expressing CML cells or T315I-mutant CML cells, aiming to simultaneously activate NK cells and T lymphocytes. METHODS: We generated novel CML-RAE-1γ-Dex vaccines, which expressed RAE-1γ, and were loaded with CML tumor cell lysates. NK cells or T lymphocytes were coincubated with CML-RAE-1γ-Dex vaccines. Flow cytometry was performed to evaluate the activation and proliferation of these immune cells. Cytokine production and cytotoxicity toward CML cells with or without the T315I mutation were detected by ELISPOT, ELISA and LDH assays. CML models induced by BCR-ABL or BCR-ABLT315I were used to determine the immunological function of Dex in vivo. RESULTS: Herein, CML-RAE-1γ-Dex were prepared. CML-RAE-1γ-Dex effectively enhanced the proliferation and effector functions of NK cells, CD4+ T cells and CD8+ T cells, which in turn produced strong anti-CML efficacy in vitro. Moreover, CML-RAE-1γ-Dex-based immunotherapy inhibited leukemogenesis and generated durable immunological memory in CML mouse models. Similar immune responses were also observed with imatinib-resistant CML cells carrying the T315I mutation. CONCLUSIONS: This approach based on CML-RAE-1γ-Dex vaccines may be a promising strategy for CML treatment, especially for cases with the T315I mutation.

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.

LncRNA OIP5-AS1 Promotes the Autophagy-Related Imatinib Resistance in Chronic Myeloid Leukemia Cells by Regulating miR-30e-5p/ATG12 Axis.

Background: Resistance to tyrosine kinase inhibitors (TKIs) in patients with chronic myeloid leukemia (CML) remains a problem in clinical treatment, and the mechanism has not been fully clarified. Autophagy can protect cancer cells under chemotherapeutic stimulation. Long noncoding RNAs (lncRNAs) are critical in drug resistance of CML. The role of lncRNAs in autophagy and drug resistance of CML needs to be further explored. Methods: Western blot and immunofluorescence were used to evaluate the autophagy activity in the drug-resistant CML cell line K562/G01 and its parental cell line K562. Then the sensitivity of K562/G01 cells to the first generation TKI imatinib (IM) after autophagy inhibition was determined by CCK-8 assays. The lncRNA OIP5-AS1 related to the drug resistance of CML cells was determined by Gene Expression Omnibus database analysis. Western blot and drug-sensitivity assays were used to detect changes in autophagy and sensitivity to the IM in resistant CML cells after OIP5-AS1 knockdown. The interactions of OIP5-AS1, miR-30e-5p, and ATG12 were explored by RNA immunoprecipitation and dual-luciferase reporter assays. Results: In this study, we found that autophagy was associated with drug resistance in CML cells. Moreover, the upregulation of OIP5-AS1 in K562/G01 cells was related to the enhancement of autophagy. Knockdown of OIP5-AS1 suppressed autophagy and enhanced the sensitivity of K562/G01 cells to IM. Furthermore, OIP5-AS1 regulated ATG12 by competitively binding miR-30e-5p, thereby affecting autophagy-related drug resistance. Conclusion: Our study reveals that OIP5-AS1 promotes the autophagy-related IM resistance in CML cells by regulating miR-30e-5p/ATG12 axis, providing new insights into the drug resistance mechanism of CML.

Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells.

BACKGROUND: The pathogenesis of chronic myeloid leukemia (CML) is the formation of the BCR/ABL protein, which is encoded by the bcr/abl fusion gene, possessing abnormal tyrosine kinase activity. Despite the wide application of tyrosine kinase inhibitors (TKIs) in CML treatment, TKIs drug resistance or intolerance limits their further usage in a subset of patients. Furthermore, TKIs inhibit the tyrosine kinase activity of the BCR/ABL oncoprotein while failing to eliminate the pathologenic oncoprotein. To develop alternative strategies for CML treatment using therapeutic antibodies, and to address the issue that antibodies cannot pass through cell membranes, we have established a novel intracellular delivery of anti-BCR/ABL antibodies, which serves as a prerequisite for CML therapy. METHODS: Anti-BCR/ABL antibodies were encapsulated in poly(D, L-lactide-co-glycolide) nanoparticles (PLGA NPs) by a double emulsion method, and transferrin was labeled on the surface of the nanoparticles (Ab@Tf-Cou6-PLGA NPs). The characteristics of nanoparticles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cellular uptake of nanoparticles was measured by flow cytometry (FCM). The effect of nanoparticles on the apoptosis and proliferation of CML cells was testified by FCM and CCK-8 assay. In addition, the anti-cancer impact of nanoparticles was evaluated in mouse models of CML. RESULTS: The results demonstrated that the Ab@Tf-Cou6-PLGA NPs functioned as an intracellular deliverer of antibodies, and exhibited an excellent effect on degrading BCR/ABL oncoprotein in CML cells via the Trim-Away pathway. Treatment with Ab@Tf-Cou6-PLGA NPs inhibited the proliferation and induced the apoptosis of CML cells in vitro as well as impaired the oncogenesis ability of CML cells in vivo. CONCLUSIONS: In conclusion, our study indicated that this approach achieved safe and efficient intracellular delivery of antibodies and degraded BCR/ABL oncoprotein via the Trim-Away pathway, which provides a promising therapeutic strategy for CML patients, particularly those with TKI resistance.

RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport.

Abnormal subcellular localization of proteins is an important cause of tumorigenesis and drug resistance. Chromosome region maintenance 1 (CRM1), the nuclear export regulator of most proteins, has been confirmed to be over-expressed in various malignancies and is regarded as an efficient target. But the potential role of the CRM1 cofactor RanBP3 (Ran Binding Protein 3) is left unrevealed in chronic myeloid leukemia (CML). Here, we first detected the level of RanBP3 in CML and found an elevated RanBP3 expression in CML compared with control. Then we used shRNA lentivirus to down-regulated RanBP3 in imatinib sensitive K562 cells and resistant K562/G01 cells and found RanBP3 silencing inhibited cell proliferation by up-regulating p21, induced caspase3-related cell apoptosis, and enhanced the drug sensitivity of IM in vitro. Notably, we observed that RanBP3 silencing restored imatinib sensitivity of K562 cells in NOD/SCID mice. Mechanistically, the nuclear aggregation of SMAD2/3 revealed that tumor suppressor axis (TGF-ß)-SMAD2/3-p21 was the anti-proliferation program related to RanBP3 knockdown, and the decrease of cytoplasmic ERK1/2 caused by RanBP3 interference leaded to the down-regulation of anti-apoptosis protein p(Ser112)-BAD, which was the mechanism of increased cell apoptosis and enhanced chemosensitivity to imatinib in CML. In summary, this study revealed the expression and potential role of RanBP3 in CML, suggesting that targeting RanBP3 alone or combined with TKIs could improve the clinical response of CML.

A novel fusion circular RNA F-circBA1 derived from the BCR-ABL fusion gene displayed an oncogenic role in chronic myeloid leukemia cells.

The BCR-ABL fusion gene plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). The BCR-ABL oncoprotein encoded by this fusion gene has been extensively studied. However, research on whether BCR-ABL also affects circular RNAs (circRNAs) is limited. This study aimed to explore the new fusion circRNAs produced by BCR-ABL and their role in CML cells. In this study, we identified a novel fusion circRNA, named F-circBA1, originating from BCR-ABL in K562 and K562/G01 cells using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Sanger sequencing. qRT-PCR of the nuclear RNA and cytoplasmic RNA were separated, indicating that F-circBA1 was mainly localized in the cytoplasm. Cell counting kit-8 assay and flow cytometry showed that F-circBA1 knockdown by shRNA prevented the proliferation of K562 and K562/G01 cells, and the cell cycle was arrested at G2/M. Mechanically, dual-luciferase reporter assay and western blotting assay showed that F-circBA1 sponged miR-148-3p and F-circBA1 silencing decreased CDC25B expression in vitro. Furthermore, the results of the murine leukemogenesis model showed that F-circBA1 knockdown suppressed leukemogenesis in vivo. Besides, we found the existence of F-circBA1 in some patients with BCR-ABL-positive CML. In conclusion, these results demonstrate the presence of F-circBA1 and its oncogenic role in CML cells.

The effect of Gö6976 on chronic myeloid leukemia in vitro and in vivo.

Objectives: Chronic myeloid leukemia (CML) is a malignant tumor of the blood system. Gö6976, as a type of indolocarbazole and shows strong antitumor effects, but there have been no reports on the effect of Gö6976 on CML. The objectives of this research were: (1) to explore the impact of Gö6976 on CML in vitro and in vivo; and (2) to explore the drug toxicity of Gö6976 to normal cells and animals.Methods:K562 cells and CML mice were used to explore the effect of Gö6976 on CML. Peripheral blood mononuclear cells (PBMCs), CD34+ cells, and healthy mice were used to explore the drug toxicity of Gö6976.Results: Cell experiments showed that Gö6976 could inhibit the proliferation of K562 cells and enhance the inhibitory effects of imatinib at 5 µM and 10 µM, but it had little effect on CD34+ cells or PBMCs at concentrations less than 5 µM. Animal experiments showed that 2.5 mg/kg Gö6976 could effectively inhibit the development of CML in mice, and it had almost no effects on healthy mice at 2.5 mg/kg and 10 mg/kg.Discussion: Because of the direct inhibitory effect of Gö6976 on CML and its pharmacological enhancement effect on imatinib, it is foreseeable that Gö6976 could become a new type of anti-CML medicine. And the further research is needed.Conclusion: Our findings verified that Gö6976 could effectively inhibit CML in vitro and in vivo, and it is almost nontoxic to hematopoietic cells, immune cells, and healthy mice.

Effect of HSP90AB1 and CC domain interaction on Bcr-Abl protein cytoplasm localization and function in chronic myeloid leukemia cells.

BACKGROUND: The fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear. METHODS: Herein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis. RESULTS: HSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells. CONCLUSION: Our study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies. Video abstract.

TCF7 knockdown inhibits the imatinib resistance of chronic myeloid leukemia K562/G01 cells by neutralizing the Wnt/ß­catenin/TCF7/ABC transporter signaling axis.

Clinical resistance to ABL tyrosine kinase inhibitor (TKI) imatinib remains a critical issue in the treatment of chronic myeloid leukemia (CML). Transcription factor 7 (TCF7) is one of the main Wnt/ß­catenin signaling mediators. Previous studies have shown that TCF7 is vital for tumor initiation, and targeting TCF7 can reduce drug resistance in many types of cancer. However, the role of TCF7 in CML imatinib­resistant cells is unclear. In the present study, we analyzed the transcriptomic data from CML clinical samples in the Gene Expression Omnibus (GEO) and performed experimental verification in the CML imatinib­resistant cell line K562/G01. We found that the expression of TCF7 was independent of BCR­ABL1 activity. Silencing of TCF7 downregulated the expression levels of CTNNB1, CCND1, and ABCC2, and therefore inhibited proliferation, weakened colony formation, and increased the drug sensitivity of imatinib­resistant cells. After analyzing the transcriptomic data of four groups (Scramble, TCF7_KD, Scramble+imatinib, and TCF7_KD+imatinib) using bioinformatics, we noted that Wnt/ß­catenin and ATP­binding cassette (ABC) transporter signaling pathways were upregulated in imatinib­resistant cells under conventional dose of imatinib, and TCF7 knockdown could neutralize this effect. Next, using ChIP­qPCR, we demonstrated that TCF7 was recruited to the promoter region of ABCC2 and activated gene transcription. In summary, our results highlight that the upregulation of Wnt/ß­catenin and ABC transporter signaling pathways induced by imatinib treatment of resistant cells confers imatinib resistance, and reveal that targeting TCF7 to regulate the Wnt/ß­catenin/TCF7/ABC transporter signaling axis may represent an effective strategy for overcoming imatinib resistance.

Induction of CML-specific immune response through cross-presentation triggered by CTP-mediated BCR-ABL-derived peptides.

Although targeted therapy using tyrosine kinase inhibitors (TKIs) has made remarkable progress in treating chronic myeloid leukemia (CML), this disease remains largely incurable, warranting further investigation of new therapeutic strategies. BCR-ABL is a highly specific tumor antigen in CML and provides an attractive opportunity for vaccination therapy. Exogenous antigens must be presented on MHC class I molecules-via a process termed cross-presentation-to activate specific cytotoxic T lymphocyte response. The relative efficiency of cross-presentation is determined in part by the ability of dendritic cells (DCs) to internalize and present antigens. Here, we present a novel tool that uses cytoplasmic transduction peptide (CTP) to facilitate the internalization of antigens by DCs in an endocytosis-independent manner, which greatly enhances the efficiency of antigen presentation, thereby inducing stronger cytotoxic activity to ensure the elimination of CML cells. The data suggest that CTP-fused CML-specific peptides can be applied in vaccination therapies for CML patients.

Inhibition of KPNB1 Inhibits Proliferation and Promotes Apoptosis of Chronic Myeloid Leukemia Cells Through Regulation of E2F1.

BACKGROUND: Karyopherin-ß1 (KPNB1) belongs to the karyopherin superfamily, which functions as shuttling proteins from the cytoplasm to nuclear. A high level of KPNB1 has been reported in various cancers which promotes cell proliferation and inhibits apoptosis. However, the role of KPNB1 in chronic myeloid leukemia (CML) remains uncertain. METHODS: Expression level of KPNB1 in CML patient samples and cell lines was analyzed by Western blotting. The proliferation assays and colony formation assay were used to study the CML cell proliferation when KPNB1 knockdown in vitro. Next, Western blotting was used to evaluate the effects of KPNB1 on E2F1 and other cell cycle regulators. Then, the location of E2F1 was detected by immunofluorescence. Finally, flow cytometry was used to detect the effect of KPNB1 inhibitor importazole (IPZ) on CML cells. RESULTS: In this study, we firstly showed that KPNB1 is over-expressed in CML cells. Targeting KPNB1 with small interfering RNA (siRNA) and IPZ reduced proliferation and induced apoptosis of CML cells. The underlying mechanisms were also investigated that E2F1 nuclear transport was blocked after inhibiting KPNB1 with siRNA, suggesting KPNB1 over-expression mediates the excessive nuclear transport of E2F1 in CML cells. Moreover, the expression of the E2F1 targeted molecule such as c-Myc and KPNA2 was markedly reduced. The IPZ arrested CML cells at G2/M phase and induced cell apoptosis. CONCLUSION: In summary, our results clearly showed that KPNB1 is over-expressed in CML cells and mediates the translocation of E2F1 into the nucleus of CML cells, thereby inhibition of KPNB1 reduced proliferation and induced apoptosis of CML cells which provides new insights for targeted CML therapies.

Disrupting myddosome assembly in diffuse large B­cell lymphoma cells using the MYD88 dimerization inhibitor ST2825.

Diffuse large B­cell lymphoma (DLBCL), the most common type of non­Hodgkin's lymphoma, is classified into germinal center and activated B cell (ABC) subtypes. The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is the most prevalent oncogenic mutation among patients with ABC DLBCL, the subtype that has the more inferior outcome. MYD88 oligomerization driven by the L265P mutant augments myddosome assembly and triggers the activation of nuclear factor kappa­light­chain­enhancer of activated B cells (NF­κB) signaling, highlighting MYD88 oligomerization as a potential therapeutic target for this malignancy. The synthetic peptidomimetic compound ST2825, which has previously been used as an anti­inflammatory agent, has been reported to inhibit MYD88 dimerization. In the present study, the anticancer effects of ST2825 were investigated using L265P­expressing ABC DLBCL cell lines. Using confocal microscopy and high­molecular­weight fraction experiments, it was revealed that L265P­associated myddosome assembly was disrupted by ST2825. The results also revealed that disrupting myddosome assembly promoted the death of ABC DLBCL cells harboring the L265P mutation, as well as downregulating survival signals, including the inhibition of NF­κB and the suppression of IL­10 and interferon­ß production. Further co­immunoprecipitation studies demonstrated that MYD88 bound to BTK in L265P­DLBCL cells, and that this binding was abrogated following ST2825 treatment. Furthermore, the combination of myddosome­assembly disruption and BTK or BCL­2 signaling inhibition led to synergistic ABC DLBCL cell death, and more robust inhibition of NF­κB activity or increased apoptosis, respectively. The results of the present study provide evidence that the synthetic peptidomimetic compound ST2825, which targets myddosome assembly, may serve as a pharmacological inhibitor. ST2825 has the potential for clinical use in patients with L265P DLBCL, and other B­cell neoplasms driven by activated MYD88 signaling.

Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells.

BACKGROUND: The bcr-abl fusion gene encodes BCR-ABL oncoprotein and plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). Current therapeutic methods have limited treatment effect on CML patients with drug resistance or disease relapse. Therefore, novel therapeutic strategy for CML is essential to be explored and the CRISPR RNA-guided FokI nucleases (RFNs) meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML. The RFNs provide us a new therapeutic direction to obliterate this disease. METHODS: Guide RNA (gRNA) expression plasmids were constructed by molecular cloning technique. The modification rate of RFNs on bcr-abl was detected via NotI restriction enzyme digestion and T7 endonuclease 1 (T7E1) assay. The expression of BCR-ABL and its downstream signaling molecules were determined by western blotting. The effects of RFNs on cell proliferation and apoptosis of CML cell lines and CML stem/progenitor cells were evaluated by CCK-8 assay and flow cytometry. In addition, murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of bcr-abl. RESULTS: The RFNs efficiently disrupted bcr-abl and prematurely terminated its translation. The destruction of bcr-abl gene suppressed cell proliferation and induced cell apoptosis in CML lines and in CML stem/progenitor cells. Moreover, the RFNs significantly impaired the leukemogenic capacity of CML cells in xenograft model. CONCLUSION: These results illustrate that the RFNs can target to disrupt bcr-abl gene and may provide a new therapeutic option for CML patients affiliated by drug resistance or disease relapse.

Synergistic antitumor effect of suberoylanilide hydroxamic acid and cisplatin in osteosarcoma cells.

Cisplatin, as a first-line chemotherapy drug, has been widely applied for therapy of osteosarcoma. However, its application is limited by drug resistance and serious side effects, including nephrotoxicity and ototoxicity. Suberoylanilide hydroxamic acid (SAHA) is a newly developed histone deacetylase (HDAC) inhibitor, which is the first Food and Drug Administration-approved HDAC inhibitor for the treatment of cutaneous manifestations of T-cell lymphoma. However, SAHA as a monotherapy was revealed to be limited, particularly in solid tumors. In the present study, 143B osteosarcoma cells were treated with multiple concentrations of SAHA or cisplatin, either alone or combined. The morphological characteristics of the treated cells were observed using an inverted microscope. The cytotoxicity effects of the combination of SAHA and cisplatin on 143B cells were analyzed by MTT assay, colony formation assay, wound healing cell migration assay, cell apoptosis assay and cell cycle analysis. Western blot analysis was performed to detect the protein expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2, cleaved-caspase-3, cleaved-caspase-8 and cleaved-poly (ADP-ribose) polymerase (PARP). The experimental data indicated that the inhibition of cell proliferation in the combination group was significantly increased compared with that in single drug groups. Expression levels of pro-apoptotic protein were upregulated, whereas anti-apoptotic Bcl-2 was downregulated significantly in 143B cells following SAHA/cisplatin treatment. Taken together, the results revealed that the combination of SAHA and cisplatin inhibited the proliferation of 143B cells and induced their apoptosis synergistically, and this effectiveness may be mediated by caspase activation.

Colorimetric determination of BCR/ABL fusion genes using a nanocomposite consisting of Au@Pt nanoparticles covered with a PAMAM dendrimer and acting as a peroxidase mimic.

A colorimetric assay is described for the detection of BCR/ABL fusion genes. Polyamidoamine (PAMAM) dendrimers were placed on peroxidase (POx) mimicking Au@Pt nanoparticles to form a nanocomposite of type Au@Pt-PAMAM. Capture DNA probe is a designed nucleic acid strand that specifically binds target DNA to the surface of the electrode. The capture probe was attached to magnetic beads via biotin and avidin interaction. The hairpin structure of the capture probe can only be opened by the complementary BCR/ABL DNA. This results in a highly specific assay. The POx-mimicking property of the Au@Pt-PAMAM causes the formation of a blue dye by reaction of H2O2 and 3,3,3',3'-tetramethylbenzidine (TMB) which is measured by a microplate reader. Under optimum conditions, the absorbance increases linearly the 1 pM to 100 nM BCR/ABL concentration range, and the detection limit is as low as 190 fM. The method is highly selective and was successfully applied to the determination of fusion genes in spiked real samples. Conceivably, it possesses a large potential in clinical testing of patients suffering from chronic myeloid leukemia. Graphical abstract Au@PtNP, an efficient catalyst, is bound with polyamidoamine (PAMAM) dendrimer to amplify the colorimetric signal. With the introduction of streptavidin-magnetic beads to remove non-specific signals, a novel colorimetric sensor is constructed to detect BCR/ABL fusion genes.