[Bleeding due to acquired factor V inhibitor successfully treated with platelet transfusion during treatment for recurrent prostate squamous cell carcinoma].

A 78-year-old man with prostate squamous cell carcinoma recurrence in his pelvis was admitted to our hospital. Rectal obstruction led to creation of an artificial anus on the transverse colon. Then, docetaxel and radiation therapies were started. A week later, severe hematuria and melena occurred. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were extremely prolonged. Cross-mixing test for APTT and PT revealed an inhibitor pattern, which was diagnosed as acquired factor V inhibitor. Fresh frozen plasma and vitamin K infusions were ineffective, but platelet transfusion successfully stopped the bleeding. Platelet factor V derived from megakaryocytes may affect local hemostasis. The patient received prednisolone (PSL), and the inhibitor disappeared on day 70 and was in remission. PSL could be stopped on day 100. Later, we demonstrated APTT and PT shortening of factor V deficient plasma by the supernatant of activated platelets with collagen.

Feasibility of the imatinib stop study in the Japanese clinical setting: delightedly overcome CML expert stop TKI trial (DOMEST Trial).

BACKGROUND: Treatment-free remission (TFR), the ability to maintain a molecular response (MR), occurs in approximately 50% of patients with chronic myelogenous leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). METHODS: A multicenter phase 2 trial (Delightedly Overcome CML Expert Stop TKI Trial: DOMEST Trial) was conducted to test the safety and efficacy of discontinuing imatinib. Patients with CML with a sustained MR of 4.0 or MR4.0-equivalent for at least 2 years and confirmed MR4.0 at the beginning of the study were enrolled. In the TFR phase, the international scale (IS) was regularly monitored by IS-PCR testing. Molecular recurrence was defined as the loss of MR4.0. Recurrent patients were immediately treated with dasatinib or other TKIs including imatinib. RESULTS: Of 110 enrolled patients, 99 were evaluable. The median time from diagnosis to discontinuation of imatinib was 103 months, and the median duration of imatinib therapy was 100 months. Molecular recurrence-free survival rates were 69.6%, 68.6% and 64.3% at 6, 12, and 24 months, respectively. After discontinuation of imatinib therapy, 26 patients showed molecular recurrence, and 25 re-achieved deep MR after dasatinib treatment. Molecular response MR4.0 was achieved in 23 patients within 6 months and 25 patients within 12 months. Multivariate analysis revealed that a longer time from diagnosis to discontinuation of imatinib therapy (p = 0.0002) and long duration of imatinib therapy (p = 0.0029) predicted a favorable prognosis. CONCLUSIONS: This DOMEST Trial showed the feasibility of TKI discontinuation in a Japanese clinical setting.

Establishment and characterization of a novel vincristine-resistant diffuse large B-cell lymphoma cell line containing the 8q24 homogeneously staining region.

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU-ML2, from a patient with diffuse large B-cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high-resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU-ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC-shRNA-mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU-ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment.

INTRODUCTION: We previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second- or subsequent-line dasatinib treatment safely at a median follow-up of 20 months. However, the results from longer follow-up periods would be much more useful from a clinical perspective. PATIENTS AND METHODS: The DADI trial was a prospective, multicenter trial conducted in Japan. After confirming a stable DMR for ≥ 1 year, dasatinib treatment subsequent to imatinib or nilotinib was discontinued. After discontinuation, the loss of DMR (even of 1 point) was defined as stringent molecular relapse, thereby triggering therapy resumption. The predictive factors of treatment-free remission (TFR) were analyzed. RESULTS: The median follow-up period was 44.0 months (interquartile range, 40.5-48.0 months). The estimated overall TFR rate at 36 months was 44.4% (95% confidence interval, 32.0%-56.2%). Only 2 patients developed a molecular relapse after the 1-year cutoff point. The presence of imatinib resistance was a significant risk factor for molecular relapse. Moreover, high natural killer cell and low γδ+ T-cell and CD4+ regulatory T-cell (CD25+CD127low) counts before discontinuation correlated significantly with successful therapy discontinuation. CONCLUSION: These findings suggest that discontinuation of second- or subsequent-line dasatinib after a sustained DMR of ≥ 1 year is feasible, especially for patients with no history of imatinib resistance. In addition, the natural killer cell count was associated with the TFR.

Bone Marrow Adipocytes Facilitate Fatty Acid Oxidation Activating AMPK and a Transcriptional Network Supporting Survival of Acute Monocytic Leukemia Cells.

Leukemia cells in the bone marrow must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the bone marrow is a novel therapeutic approach. In this study, we investigated the metabolic role of bone marrow adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation-induced apoptosis of leukemic cells by bone marrow adipocytes, as well as the metabolic and molecular mechanisms involved in this process, was investigated using various analytic techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by bone marrow adipocytes was associated with an increase in fatty acid ß-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, bone marrow adipocyte coculture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone HSPs. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, and induced the integrated stress response mediator ATF4 and apoptosis in AMoL cells cocultured with bone marrow adipocytes. Our results suggest that bone marrow adipocytes support AMoL cell survival by regulating their metabolic energy balance and that the disruption of FAO in bone marrow adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy. Cancer Res; 77(6); 1453-64. ©2017 AACR.

Overexpression of salivary-type amylase reduces the sensitivity to bortezomib in multiple myeloma cells.

Amylase-producing myeloma exhibits refractoriness to chemotherapy and a dismal prognosis. In this study, we established a human myeloma cell line, 8226/AMY1, in which a lentivirally transfected AMY1 gene was stably expressed and explored its biological characteristics. 8226/AMY1 showed a survival advantage over mock control when treated with dexamethasone, bortezomib, and lenalidomide in vitro partly through inhibition of apoptosis induced by these reagents. In a xenograft murine model, 8226/AMY1 showed rapid tumor growth and reduced sensitivity to bortezomib compared with mock. A microarray gene expression analysis identified TCL1A, which functions as a coactivator of the cell survival kinase Akt, differentially up-regulated in 8226/AMY1. The expression of phosphorylated Akt was increased in the 8226/AMY1 cells following bortezomib treatment, but not in the mock cells. In addition, treatment with perifosine, an inhibitor of Akt phosphorylation, enhanced the anti-myeloma effect of bortezomib in the 8226/AMY1 cells. Our data suggest that amylase-producing myeloma reduced the sensitivity to bortezomib in vitro and in vivo, and the up-regulation of TCL1A may influence the drug susceptibility of 8226/AMY1 via the phosphorylation of Akt. These findings provide clues for developing treatment approaches for not only amylase-producing myeloma, but also relapsed and refractory myelomas.

Importance of glutamine metabolism in leukemia cells by energy production through TCA cycle and by redox homeostasis.

Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

Adaptation of leukemia cells to hypoxic condition through switching the energy metabolism or avoiding the oxidative stress.

BACKGROUND: Like normal hematopoietic stem cells, leukemia cells proliferate in bone marrow, where oxygen supply is limited. However, the growth and energy metabolism of leukemia cells under hypoxia have not been well understood. Although it has been known that reactive oxygen species (ROS) is generated under hypoxic conditions, normal and leukemia stem cells were characterized by relatively low levels of ROS. Roles of ROS on leukemia cells under hypoxia also have not been well understood. METHODS: Four Leukemia cell lines were cultured under normoxia (21% O2) or hypoxia (1% O2), where NB4 and THP-1 were most extensively studied. To evaluate energy metabolism, we estimated whole cell number or apoptotic cells with or without a glycolysis inhibitor or an oxidative phosphorylation (OXPHOS) inhibitor. Glucose consumption and lactate production were also measured. To evaluate oxidative stress in hypoxic condition, the ROS level and GSH (reduced glutathione) / GSSG (oxidized glutathione) ratio was measured. In addition, pyruvate dehydrogenase kinase 1 (PDK1) and cytochrome c oxidase subunit 4 (COX4) were examined by western blotting or RT-PCR. RESULTS: NB4, which grows well under normoxia depending on glycolysis, demonstrated prominent apoptosis and growth suppression after 48 hours culture under hypoxia. NB4 cells cultured under hypoxia showed significantly increased ROS. Culture with a ROS scavenger resulted in decrease of apoptotic cell death of NB4 under hypoxia. NB4 cells cultured for longer period (7 days) under hypoxia did not come to extinction, but grew slowly by upregulating GSH synthesis to protect from ROS generated in hypoxic condition. By contrast, THP-1, which largely depends on OXPHOS in mitochondria under normoxia, demonstrated more growth under hypoxia by changing metabolism from OXPHOS to glycolysis through upregulating PDK1. Moreover, THP-1 avoided ROS generation by substituting COX 4 subunit (from COX 4-1 to COX 4-2) through upregulation of LON, a mitochondrial protease under hypoxia. CONCLUSIONS: We showed that leukemia cells survive and adapt to the hypoxic condition through various pathways. Our results will help understanding energy metabolism of leukemia cells and creating novel therapeutics.

Class IA PI3K inhibition inhibits cell growth and proliferation in mantle cell lymphoma.

BACKGROUND/AIMS: Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells. METHODS: The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis. RESULTS: Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells. CONCLUSION: These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL.

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment.

UNLABELLED: Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. KEY MESSAGE: Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

TGF-ß-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment.

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFß1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFß1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFß-neutralizing antibody 1D11 abrogated rhTGFß1 induced cell cycle arrest. Blocking TGFß with 1D11 further enhanced cytarabine (Ara-C)-induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFß by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.

Growth of xenotransplanted leukemia cells is influenced by diet nutrients and is attenuated with 2-deoxyglucose.

We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid ß-oxidation.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose.

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid ß-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

Establishment of a novel human myeloid leukemia cell line, AMU-AML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation.

For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC(50): 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a "glycolytic" leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (-) medium showed more growth and survival than glucose (-) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC(50): 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (-) FCS (+) medium showed more growth and survival than glucose (+) FCS (-) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid ß-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.

Growth inhibition of AML cells with specific chromosome abnormalities by monoclonal antibodies to receptors for vascular endothelial growth factor.

By using neutralizing monoclonal antibodies to vascular endothelial growth factor receptor type 1 (VEGFR1) and VEGFR2, we have shown that acute myelogenous leukemia (AML) cells with specific chromosome abnormalities are dependent on VEGF/VEGFR system. AML with t(8;21) is the most dependent subtype on VEGF with both VEGFR1 and VEGFR2. t(15;17)AML cells depend on VEGF with VEGFR1. AML cells with 11q23 abnormalities showed variable dependence on VEGF. The growth of t(11;19)AML cells are most extensively inhibited by anti-VEGFR1 antibody. Then, the growth of Kasumi-1, a t(8;21) cell line was suppressed by either anti-VEGFR1 antibody (p=0.0022) or anti-VEGFR2 antibody (p=0.0029) in a dose-dependent manner. The growth of NB4, a t(15;17) cell line was more potently suppressed by anti-VEGFR1 antibody (p=0.0111) than by anti-VEGFR2 antibody (p=0.0477). These results are quite concordant with the results of clinical samples with t(8;21) or t(15;17). In addition, anti-VEGFR2 monoclonal antibody significantly potentiated the growth inhibitory effect of idarubicin for Kasumi-1. As for downstream signals, we have shown that VEGFR2 transduce growth and survival signals through phosphorylation of Akt and MEK in leukemia cells (Kasumi-1). However, VEGFR1 transduce growth and survival signals through pathways other than MEK and Akt (NB4), although Akt phosphorylation may account for some of the VEGFR1 signals (Kasumi-1). Finally, our data suggested that autocrine pathway of VEGF and VEGFRs observed in AML cells with specific chromosomal translocations have contributed to leukemogenesis as activated signaling of receptor tyrosine kinase.

Cyclin-dependent kinase 1 inhibitor RO-3306 enhances p53-mediated Bax activation and mitochondrial apoptosis in AML.

Cyclin-dependent kinase (CDK) 1 and the murine double minute 2 homolog (MDM2)-p53 interaction are potential therapeutic targets in cancer, and their inhibition has been reported to be more proapoptotic in malignant cells compared to normal cells. We investigated the effect of CDK1 inhibition on p53 signaling after simultaneous dual blockade using the CDK1 inhibitor RO-3306 and the MDM2 inhibitor Nutlin-3 in AML. Treatment of growing AML cells with RO-3306 induced G2/M-phase cell cycle arrest and apoptosis in a dose- and time-dependent manner. We found that RO-3306 acts cooperatively with Nutlin-3 to induce mitochondrial apoptosis in a cell cycle-independent fashion. RO-3306 downregulated expression of the antiapoptotic proteins Bcl-2 and survivin and blocked p53-mediated induction of p21 and MDM2. CDK1 siRNA experiments showed that reduced CDK1 expression affects p53-induced p21 transactivation. We suggest that RO-3306 actively enhances downstream p53 signaling to promote apoptosis and that a combination strategy aimed at both inhibiting CDK1 and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

t(8;21) acute myeloid leukaemia cells are dependent on vascular endothelial growth factor (VEGF)/VEGF receptor type2 pathway and phosphorylation of Akt.

Several anti-angiogenic drugs have recently been clinically tested for haematological malignancies. To improve the efficacy of molecular target therapy against angiogenic molecules in acute myeloid leukaemia (AML), we examined the dependency of AML cells on the vascular endothelial growth factor (VEGF)/VEGF receptor type2 (VEGFR2) system by using VEGFR2 kinase inhibitor. Nineteen patient AML samples were cultured with or without VEGFR2 kinase inhibitor. All four t(8;21) viable AML cells showed significant reductions when treated with VEGFR2 kinase inhibitor, although VEGFR2 kinase inhibitor did not affect the cell proliferation of five t(15;17) AML samples. Other AML cases showed variable responses. VEGFR2 kinase inhibitor greatly suppressed the growth of Kasumi-1, a t(8;21) cell line in a dose-dependent manner through induction of apoptosis, but did not show any significant influence on NB4, a t(15;17) cell line. In addition, VEGFR2 kinase inhibitor potentiated the growth inhibitory effect of cytarabine in Kasumi-1. Finally, it was shown that the Akt phosphorylation was augmented by VEGF(165) in Kasumi-1, which was abrogated by VEGFR2 kinase inhibitor. NB4 showed undetectable Akt phosphorylation even with VEGF(165). These data demonstrated that t(8;21) AML cells are dependent on VEGF through VEGFR2, resulting in the phosphorylation of Akt.

Low p53 expression of acute myelocytic leukemia cells with t(8;21) chromosome abnormality: association with low p14(ARF) expression.

In this study, the mRNA expression of p14(ARF) in t(8;21)AML cells was found to be significantly lower than acute myelocytic leukemia (AML) cells without t(8;21) chromosome abnormality, which was concordant with previous observation by Linggi et al. that AML1-MTG8 represses the transcription of p14(ARF). Although p53 mRNA expression level of t(8;21)AML cells was not low, p53 protein expression was reduced in t(8;21)AML cells. Genotoxic damage by ionizing radiation did not induce p53 upregulation in t(8;21)AML cells. Since p14(ARF) has been demonstrated to inhibit p53 degradation by binding to MDM2, repression of p14(ARF) expression in t(8;21)AML may facilitate the degradation of p53 by MDM2. Low p14(ARF) in t(8;21)AML may also account for the absence of upregulation of p53 by ionizing radiation. Then, we have shown that p53 expression level was inversely correlated with S/G2/M population of cell cycle in AML cells. Most of the t(8;21)AML are considered to be in p53(low) S/G2/M(high). It is now widely known that formation of AML1-MTG8 by t(8;21) translocation is a very early event in leukemogenesis, and AML1-MTG8 alone might have limited proliferative potential. Then, secondary oncogenic events such as activated receptor tyrosine kinase (like c-kit mutation), is necessary to become full-blown leukemia. Low p53 protein expression and insufficient induction of p53 by genotoxic damage might increase the opportunity to obtain additional oncogenic events, since genome guard function of p53 does not work in t(8;21)AML cells.