High Prevalence of the BIM Deletion Polymorphism in Young Female Breast Cancer in an East Asian Country

Background

A rapid surge of female breast cancer has been observed in young women in several East Asian countries. The BIM deletion polymorphism, which confers cell resistance to apoptosis, was recently found exclusively in East Asian people with prevalence rate of 12%. We aimed to evaluate the possible role of this genetic alteration in carcinogenesis of breast cancer in East Asians.

Method

Female healthy volunteers (n = 307), patients in one consecutive stage I-III breast cancer cohort (n = 692) and one metastatic breast cancer cohort (n = 189) were evaluated. BIM wild-type and deletion alleles were separately genotyped in genomic DNAs.

Results

Both cancer cohorts consistently showed inverse associations between the BIM deletion polymorphism and patient age (≤35 y vs. 36-50 y vs. >50 y: 29% vs. 22% vs. 15%, P = 0.006 in the consecutive cohort, and 40% vs. 23% vs. 13%, P = 0.023 in the metastatic cohort). In healthy volunteers, the frequencies of the BIM deletion polymorphism were similar (13%-14%) in all age groups. Further analyses indicated that the BIM deletion polymorphism was not associated with specific clinicopathologic features, but it was associated with poor overall survival (adjusted hazard ratio 1.71) in the consecutive cohort.

Conclusions

BIM deletion polymorphism may be involved in the tumorigenesis of the early-onset breast cancer among East Asians.     

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.     

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL

The pro-apoptotic BH3 only protein BIM_EL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM_EL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM_EL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM_EL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM_EL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM_EL and may be relevant to the therapeutic use of agents such as paclitaxel.     

Correlation of glucocorticoid-mediated E4BP4 upregulation with altered expression of pro- and anti-apoptotic genes in CEM human lymphoblastic leukemia cells

In Caenorhabditiselegans, motorneuron apoptosis is regulated via a ces-2–ces-1–egl-1 pathway. We tested whether human CEM lymphoblastic leukemia cells undergo apoptosis via an analogous pathway. We have previously shown that E4BP4, a ces-2 ortholog, mediates glucocorticoid (GC)-dependent upregulation of BIM, an egl-1 ortholog, in GC-sensitive CEM C7-14 cells and in CEM C1-15mE#3 cells, which are sensitized to GCs by ectopic expression of E4BP4. In the present study, we demonstrate that the human ces-1 orthologs, SLUG and SNAIL, are not significantly repressed in correlation with E4BP4 expression. Expression of E4BP4 homologs, the PAR family genes, especially HLF, encoding a known anti-apoptotic factor, was inverse to that of E4BP4 and BIM. Expression of pro- and anti-apoptotic genes in CEM cells was analyzed via an apoptosis PCR Array. We identified BIRC3 and BIM as genes whose expression paralleled that of E4BP4, while FASLG, TRAF4, BCL2A1, BCL2L1, BCL2L2 and CD40LG as genes whose expression was opposite to that of E4BP4.     

Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells

The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd₂(R₍₊₎)C², N-dmpa)₂(μ-dppe)Cl₂], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy.     

A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.     

IGF-IR determines the fates of BCR/ABL leukemia

Background

The tyrosine kinase receptor insulin-like growth factor 1 receptor (IGF-IR) contributes to the initiation and progression of many types of malignancies. We previously showed that IGF-2, which binds IGF-IR, is an extrinsic factor that supports the ex vivo expansion of hematopoietic stem cells (HSCs). We also demonstrated that IGF-IR is not required for HSC activity in vivo.

Methods and results

Here we investigated the role of IGF-IR in chronic myeloid leukemia (CML) using the retroviral BCR/ABL transplantation mouse model. Existing antibodies against IGF-IR are not suitable for flow cytometry; therefore, we generated a fusion of the human IgG Fc fragment with mutant IGF-2 that can bind to IGF-IR. We used this fusion protein to evaluate mouse primary hematopoietic populations. Through transplantation assays with IGF-IR⁺ and IGF-IR⁻ cells, we demonstrated that IGF-IR is expressed on all mouse HSCs. The expression of IGF-IR is much higher on CML cells than on acute lymphoblastic leukemia (ALL) cells. The depletion of IGF-IR expression in BCR/ABL⁺ cells led to the development of ALL (mostly T cell ALL) but not CML. Lack of IGF-IR resulted in decreased self-renewal of the BCR/ABL⁺ CML cells in the serial replating assay.

Conclusion

IGF-IR regulates the cell fate determination of BCR/ABL⁺ leukemia cells and supports the self-renewal of CML cells.     

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.     

Analysis of the expression pattern of the BCL11B gene and its relatives in patients with T-cell acute lymphoblastic leukemia

Introduction

The development of Philadelphia chromosome (Ph) negative acute leukemia/myelodysplastic syndrome (MDS) in patients with Ph-positive chronic myeloid leukemia (CML) is very rare. The features of restrictive usage and absence of partial T cell clones have been found in patients with CML. However, the T-cell clonal evolution of Ph-negative malignancies during treatment for CML is still unknown.

Objective

To investigate the dynamic change of clonal proliferation of T cell receptor (TCR) Vα and Vβ subfamilies in one CML patient who developed Ph-negative acute lymphoblastic leukemia (ALL) after interferon and imatinib therapy.

Methods

The peripheral blood mononuclear cells (PBMC) samples were collected at the 3 time points (diagnosis of Ph-positive chronic phase (CP) CML, developing Ph-negative ALL and post inductive chemotherapy (CT) for Ph-negative ALL, respectively). The CDR3 size of TCR Vα and Vβ repertoire were detected by RT-PCR. The PCR products were further analyzed by genescan to identify T cell clonality.

Results

The CML patient who achieved complete cytogenetic remission (CCR) after 5 years of IFN-α therapy suddenly developed Ph-negative ALL 6 months following switch to imatinib therapy. The expression pattern and clonality of TCR Vα/Vβ T cells changed in different disease stages. The restrictive expression of Vα/Vβ subfamilies could be found in all three stages, and partial subfamily of T cells showed clonal proliferation. Additionally, there have been obvious differences in Vα/Vβ subfamily of T cells between the stages of Ph-positive CML-CP and Ph-negative ALL. The Vα10 and Vβ3 T cells evolved from oligoclonality to polyclonality, the Vβ13 T cells changed from bioclonality to polyclonality, when Ph-negative ALL developed.

Conclusions

Restrictive usage and clonal proliferation of different Vα/Vβ subfamily T cells between the stages of Ph-positive CP and Ph-negative ALL were detected in one patient. These changes may play a role in Ph- negative leukemogenesis.     

The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome-Positive Acute Lymphatic Leukemia

The hallmark of Philadelphia chromosome positive (Ph⁺) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph⁺ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph⁺ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL. The co-expression of p96^ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185^BCR/ABL. Targeting p96^ABL/BCR by RNAi inhibited growth of Ph⁺ ALL cell lines and Ph⁺ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96^ABL/BCR and p185^BCR/ABL in hematopoietic stem cells. Co-expression of p96^ABL/BCR abolished the capacity of p185^BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL.     

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors—imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.     

An ent‐Kaurane Diterpenoid Isolated from Rabdosia excisa Suppresses Bcr‐Abl Protein Expression in Vitro and in Vivo and Induces Apoptosis of CML Cells

Chronic myelogenous leukemia (CML) is a disease of the blood stem cells that features the oncoprotein Bcr‐Abl. Tyrosine kinase inhibitors (TKIs) are used to treat CML patients, but these have limited efficacy due to the emergence of resistance via genetic mutation. Kamebakaurin is an ent‐kaurane diterpenoid that has been isolated from Rabdosia excisa (Maxim .) H.Hara . Herein, we investigate the potential of kamebakaurin as a chemotherapy reagent for the treatment of CML. We conducted in vitro and in vivo biological experiments and found that kamebakaurin potently inhibits cell proliferation, mainly by enhancing cell apoptosis and down‐regulating Bcr‐Abl protein levels. In addition, kamebakaurin was found to inhibit tumor growth and has no side effects on five internal organs for in vivo experiment. These results suggest that kamebakaurin is a potential anticancer agent and is a key compound for further investigations.     

More acute lymphoid leukemia than acute myeloid leukemia blasts are killed by rabbit antithymocyte globulin

Rabbit antithymocyte globulin (ATG, thymoglobulin), a polyclonal antibody, is used to prevent graft-versus-host disease (GVHD) and graft failure in the setting of allogeneic hematopoietic cell transplantation (HCT). Recent in vitro studies suggest that ATG also has anti-leukemic activity. Whether acute lymphoid leukemia (ALL) or acute myeloid leukemia (AML) is more sensitive to ATG is not known. We used primary cells from 12 B-ALL and 38 AML patients and measured ATG-induced complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC) at clinically relevant ATG concentrations (10 and 50 mg/L). At 50 mg/L, ALL blasts were killed to a greater degree than AML blasts by CDC (median 96% vs 50% dead cells, P = 0.001) as well as CIC (median 23% vs 11% apoptotic cells, P = 0.049). At 10 mg/L, the difference was significant for CDC but not CIC. In conclusion, the anti-leukemic activity of ATG, particularly CDC, is more potent for ALL than AML in vitro. If this applies in vivo, ATG-based GVHD prophylaxis may be particularly advantageous for ALL.