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Introduction Invasive fungal infections have been attracting attention as significant fatal complications in patients with febrile neutropenia (FN) who undergo intensive chemotherapy or hematopoietic stem cell transplantation to treat hematological malignancies. Although clinical trials are already underway in other countries, evidence supporting the use of caspofungin (CAS) in FN patients in Japan is still insufficient. Methods A retrospective study of patients treated with CAS for FN associated with hematological diseases between April 2015 and March 2018 was conducted to determine the treatment efficacy and safety. The study was conducted as a multicenter collaboration, and the data of 52 patients who met all of the inclusion criteria were analyzed. A five-composite-endpoint method was used, and the treatment was judged to be effective when all five endpoints (defervescence during neutropenia; no breakthrough fungal infections; resolution of baseline fungal infections; a survival for seven days or more after the completion of therapy; and no discontinuation of therapy due to side effects or invalidity) were met. Results The efficacy rate was 53.8% (28/52), which is close to the average reported efficacy rate. Adverse events included liver dysfunction and electrolyte abnormalities, but no renal dysfunction or serious events were seen. Conclusion These results suggest that the use of CAS in FN patients with hematological diseases is effective and well-tolerated, and we believe that the use of CAS could become a significant treatment in Japan.

Background Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs. Methods ASMCs were stimulated for the indicated time by pH 6.3 or pH 7.4-adjusted Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM). As a control stimulant, pH 7.4-adjusted 0.1% BSA-DMEM containing 10 ng/mL tumor necrosis factor-α (TNF-α) was used. Interleukin-8/C-X-C motif chemokine ligand 8 (CXCL8) mRNA expression in ASMCs was quantified by RT-PCR using real-time TaqMan technology. CXCL8 secreted from ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylation at serine 536 of NF-κB p65 and binding of p65 to oligonucleotide containing an NF-κB consensus binding site were analyzed by Western blotting and an ELISA-based kit. Results Acidic pH induced a significant increase of CXCL8 mRNA expression and CXCL8 protein secretion in ASMCs. ASMCs transfected with small interfering RNA (siRNA) targeted for OGR1 produced less CXCL8 compared with those transfected with non-targeting siRNA. Protein kinase C (PKC) inhibitor, MEK1/2 inhibitor, and the inhibitor of IκB phosphorylation reduced acidic pH-stimulated CXCL8 production in ASMCs. Dexamethasone also inhibited acidic pH-stimulated CXCL8 production of ASMCs in a dose-dependent manner. Dexamethasone did not affect either phosphorylation or binding to the consensus DNA site of NF-κB p65. Conclusions CXCL8 released from ASMCs by extracellular acidification may play a pivotal role in airway accumulation of neutrophils. Glucocorticoids inhibit acidic pH-stimulated CXCL8 production independent of serine 536 phosphorylation and the binding to DNA of NF-κB p65, although NF-κB activity is essential for CXCL8 production in ASMCs.

Critically ill patients in the intensive care unit (ICU) develop muscle atrophy and decreased physical function. Though neuromuscular electrical stimulation (NMES) therapy has been shown to be effective in preventing this, but its effect on older patients is unknown. To examine the course of critically ill older patients treated with NMES in the ICU and to define the impact of its use. A retrospective cohort study was conducted using older ICU patients (≥65 years) categorized into a control group (n = 20) and an NMES group (n = 22). For subgroup analysis, each group was further classified into pre-old age (65-74 years) and old age (≥75 years). The control group showed significant decrease in muscle thickness during ICU and hospital stay. The NMES group showed lower reduction in muscle thickness and showed decrease in muscle echo intensity during hospital stay, compared to the control group. NMES inhibited decrease in muscle thickness in the pre-old age group versus the old age group. The decreasing effect of NMES on echo intensity during hospital stay manifested only in the pre-old age group. We did not find much difference in physical functioning between the NMES and control groups. Lower limb muscle atrophy reduces in critically ill older patients (≥65 years) with NMES and is pronounced in patients aged 75 years. The impact of NMES on the physical functioning of older patients in ICU needs to be further investigated.

Background Cytarabine (ara-C) is the major drug for the treatment of acute myeloid leukemia (AML), but cellular resistance to ara-C is a major obstacle to therapeutic success. The present study examined enhanced anti-apoptosis identified in 3 newly established nucleoside analogue-resistant leukemic cell line variants and approaches to overcoming this resistance. Methods HL-60 human AML cells were used to develop the ara-C– or clofarabine (CAFdA)-resistant variants. The Bcl-2 inhibitor venetoclax and the Mcl-1 inhibitor alvocidib were tested to determine whether they could reverse these cells’ resistance. Results A 10-fold ara-C-resistant HL-60 variant, a 4-fold CAFdA-resistant HL-60 variant, and a 30-fold CAFdA-resistant HL-60 variant were newly established. The variants demonstrated reduced deoxycytidine kinase and deoxyguanosine kinase expression, but intact expression of surface transporters (hENT1, hENT2, hCNT3). The variants exhibited lower expression of intracellular nucleoside analogue triphosphates compared with non-variant HL-60 cells. The variants also overexpressed Bcl-2 and Mcl-1. Venetoclax as a single agent was not cytotoxic to the resistant variants. Nevertheless, venetoclax with nucleoside analogs demonstrated synergistic cytotoxicity against the variants. Alvocidib as a single agent was cytotoxic to the cells. However, alvocidib induced G1 arrest and suppressed the cytotoxicity of the co-administered nucleoside analogs. Conclusions Three new nucleoside analogue-resistant HL-60 cell variants exhibited reduced production of intracellular analogue triphosphates and enhanced Bcl-2 and Mcl-1 expressions. Venetoclax combined with nucleoside analogs showed synergistic anti-leukemic effects and overcame the drug resistance.

Excessive activation and proliferation of inflammatory cell and uncontrolled release of cytokines and chemokines, also known as cytokine storm, is considered to be the main cause of sepsis. Accumulating evidence has indicated that autophagy may play an important role in regulating immune response and controlling excessive inflammation. Recent studies have showed that minocycline has immunomodulatory effects on cytokine and chemokine production. It has also been reported that minocycline can induce autophagy, suggesting that autophagy may be involved in the process of minocycline regulating inflammation and immune response. However, the precise mechanism is unclear. In the present study, we used enzyme-linked immunosorbent assays (ELISA) to measure the production of cytokines following minocycline treatment of lipopolysaccharide- (LPS-) stimulated THP-1 cells. Western blotting analysis was performed to confirm autophagy and the mTOR signal pathway. Cell proliferation was measured by WST-1 cell proliferation assay. We demonstrated that LPS induced autophagy in a tumor necrosis factor- (TNF-) α-mediated manner, and simultaneously, LPS induced the release of TNF-α to trigger inflammation and activated mammalian target of rapamycin (mTOR) to potentiate cell proliferation. Minocycline, which induces autophagy by inhibiting mTOR, suppresses cytokine production and cell proliferation and protects THP-1 cells from LPS toxicity. Further study demonstrated that there might be an intimate crosstalk between the inhibitor kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) signaling pathway and autophagy flux in modification of inflammatory responses. In addition, rapamycin, the mTOR inhibitor, has cooperative effect with minocycline on suppression of TNF-α release and induction of autophagy by repressing mTOR. Our data brought a novel clue to evaluate minocycline using as a potential therapeutic medicine for sepsis.

YM155, a novel small molecule inhibitor of survivin, shows broad anticancer activity. Here, we have focused on the cytotoxic activity of YM155 against multiple myeloma (MM) including cytokinetically quiescent (G0/G1) cells and bortezomib resistant cells. YM155 strongly inhibited the growth of MM cell lines with the IC50 value of below 10 nM. YM155 also showed potent anti-myeloma activity in mouse xenograft model. YM155 suppressed the expression of survivin and rapidly directed Mcl-1 protein for proteasome degradation. YM155 abrogated the interleukin-6-induced STAT3 phosphorylation, subsequently blocked Mcl-1 expression and induced apoptosis in MM cells. Triple-color flow cytometric analysis revealed that YM155 potently induced cell death of MM cells in G0 phase. Quiescent primary MM cells were also sensitive to YM155. We established bortezomib-resistant MM cell line, U266/BTZR1, which possess a point mutation G322A. YM155 exhibited similar cytotoxic potency against U266/BTZR1 compared with parental cells. Interestingly, survivin expression was markedly elevated in U266/BTZR1 cells. Treatment with YM155 significantly down-regulated this increased survivin and Mcl-1 expression in U266/BTZR1 cells. In conclusion, our data indicate that YM155 exhibits potent cytotoxicity against quiescent (G0/G1) MM cells and bortezomib-resistant cells. These unique features of YM155 may be beneficial for the development of new therapeutic strategies to eliminate quiescent MM cells and overcome bortezomib resistance.

BackgroundThe early response to treatment with immune-checkpoint inhibitors is difficult to evaluate. We determined whether changes in integrated [18F]-fluoro-2-deoxy-D-glucose positron emission tomography/MRI (18F-FDG PET/MRI) parameters after the first 2 weeks of antiprogrammed death-1 antibody nivolumab therapy could predict the response of patients with non-small cell lung cancer (NSCLC).MethodsTwenty-five patients with previously treated NSCLC were enrolled prospectively and underwent18F-FDG PET/MRI before and at 2 weeks after nivolumab therapy. Changes in maximal standardized uptake value, total lesion glycolysis (ΔTLG) and apparent diffusion coefficient (ΔADC) between the two scans were calculated and evaluated for their associations with the clinical response to therapy.ResultsThe disease control rate was 64%. Patients with non-progressive disease (non-PD) had significantly decreased TLG, increased ADCmean(ie, negative ΔADCmean) and lower ΔTLG+ΔADCmeanthan patients with PD. Among the parameters tested, receiver operating characteristic curve analysis revealed that a cut-off value of 16.5 for ΔTLG+ΔADCmeanhad the highest accuracy (92%) for distinguishing between patients with non-PD and PD. A ΔTLG+ΔADCmeanvalue meanvalue ≥16.5. A multivariate Cox model revealed that ≥16.5 ΔTLG+ΔADCmeanwas an independent predictor of shorter progression-free survival (HR 37.7) and overall survival (HR 9.29).ConclusionsA combination of ΔTLG and ΔADCmeanmeasured by integrated18F-FDG PET/MRI may have value as a predictor of the response and survival of patients with NSCLC following nivolumab therapy.Trial registration numberUMIN 000020707.

Gemtuzumab ozogamicin (GO) consists of the cluster of differentiation 33 (CD33) antibody linked to calicheamicin. The binding of GO to the CD33 antigen on leukemic cells results in internalization and subsequent release of calicheamicin, thereby inducing DNA strand breaks. We hypothesized that the poly (ADP-ribose) polymerase inhibitor olaparib might inhibit DNA repair initiated by GO-induced DNA strand breaks, thereby increasing cytotoxicity.The human myeloid leukemia cell line HL-60 and a GO-resistant variant (HL/GO20) were used.The 50% growth-inhibitory concentrations (IC50) were 24 ng/ml for HL-60 cells and 550 ng/ml for GO-resistant variant HL/GO20 cells. HL/GO20 cells were also refractory to GO-induced apoptosis. CD33 positivity was reduced in HL/GO20 cells. Olaparib-alone did not inhibit the cell growth and did not induce apoptosis in either HL-60 cells or HL/GO20 cells at concentrations of up to 10 μM. When cells were treated with different concentrations of GO in the presence of 10 μM olaparib, the IC50 of GO for HL-60 cells was 13 ng/ml. The combination index was 0.86, indicating synergistic cytotoxicity of GO and olaparib in combination. Such a combination was ineffective for HL/GO20 cells.GO and olaparib exerted synergistic cytotoxicity in CD33-positive myeloid leukemia cells in vitro.

Clofarabine is transported into leukemic cells via the equilibrative nucleoside transporters (hENT) 1 and 2 and the concentrative nucleoside transporter (hCNT) 3, then phosphorylated by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) to an active triphosphate metabolite. Cytarabine uses hENT1 and dCK for its activation. We hypothesized that cytarabine-resistant leukemia cells retain sensitivity to clofarabine.Human myeloid leukemia HL-60 cells and cytarabine-resistant variant HL/ara-C20 cells were used in the present study.Despite 20-fold cytarabine resistance, the HL/ara-C20 cells exhibited only a 6-fold resistance to clofarabine compared to HL-60 cells. The intracellular concentration of the triphosphate metabolite of cytarabine was reduced to 1/10, and that of clofarabine was halved in the HL/ara-C20 cells. hENT1 and dCK were reduced, but hCNT3 and dGK were not altered in the HL/ara-C20 cells, which might contribute to their retained capability to produce intracellular triphosphate metabolite of clofarabine.Clofarabine was cytotoxic to leukemia cells that were resistant to cytarabine.

s Background Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death. Methods HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression. Results HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO. Conclusions The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

Barasertib, an aurora B inhibitor, terminates cell division, introduces polyploidy, and consequently causes apoptosis. In the present study, we evaluated the effect of the combination of barasertib and cytarabine (ara-C), a key agent for leukemia chemotherapy, on leukemic cells in vitro. Human leukemia HL-60 cells and HL-60/ara-C20 cells, a 20-fold ara-C-resistant variant, were used. The 50% growth inhibitory concentrations of an active metabolite of barasertib, barasertib-hydroxyquinazoline-pyrazol-aniline (Barasertib-HQPA), and ara-C were 51 nM and 300 nM for HL-60 cells and 70 nM and 5300 nM for HL-60/ara-C20 cells, respectively. Barasertib-HQPA induced polyploidy with a subsequent induction of sub-G1 phase apoptosis, indicating the M-phase specific cytotoxicity. Cells treated with the S-phase specific ara-C accumulated in S phase and subsequently died through apoptosis. When HL-60 cells were treated with barasertib-HQPA and ara-C in combination, a greater-than-additive apoptosis was induced. This enhancement was obtained when the cells were treated with barasertib-HQPA prior to ara-C (37.9% sub-G1) or with both concurrently (31.2% sub-G1), but not with ara-C prior to barasertib-HQPA (17.8% sub-G1). The combination effects were similarly obtained in HL-60/ara-C20 cells with 19.7% sub-G1 for barasertib-HQPA→ara-C, 18.4% sub-G1 for both concurrently, and 13.8% sub-G1 for ara-C→barasertib-HQPA, and another leukemic U937 cells with 25.4% sub-G1 for barasertib-HQPA→ara-C, 28.2% sub-G1 for both concurrently, and 16.0% sub-G1 for ara-C→barasertib-HQPA. Barasertib-HQPA inhibited aurora B autophosphorylation and histone H3 phosphorylation in all the cell lines. Barasertib-HQPA did not inhibit DNA synthesis, allowing ara-C incorporation into DNA for its cytotoxicity. Thus, barasertib-HQPA and ara-C provided a greater-than-additive cytotoxicity in leukemic cells in vitro.

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 μM CAFdA was 20 pmol/10(7) cells in HL/CAFdA20 and 3 pmol/10(7) cells in HL/CAFdA80 compared with 63 pmol/10(7) cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.

Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

A 77-year-old man treated with prednisolone for pemphigus developed severe sepsis by Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Several antibiotics were administered. A peripheral blood smear showed growth of a large number of yeast extending pseudohyphae which could be seen both inside and outside of leucocytes. Antifungal agents were added immediately; however, he did not recover. Several days later, blood culture showed Candida albicans septicemia. The autopsy revealed microabscesses in the lung, heart, liver and kidney. A large amount of neutrophil invasion and yeast with pseudohyphae were also detected.

Current first-line treatment options for chronic myeloid leukemia (CML) include imatinib (IM) and the second-generation agents nilotinib and dasatinib. Despite the effectiveness of these tyrosine kinase inhibitors, a small percentage of chronic-phase CML patients are primarily refractory or acquire secondary resistance to these agents. Moreover, the prognosis of patients in blast crisis is still poor despite the use of the current treatment modalities because of drug resistance. The major mechanism of drug resistance is the re-activation of ABL kinase either through mutations in the BCR-ABL gene or by BCR-ABLgene amplification. A novel pan-histone deacetylase inhibitor, panobinostat (formerly LBH589), induces the acetylation of heat shock protein 90, thereby inhibiting its chaperone function in association with its client proteins BCR-ABL, leading to the degradation of BCR-ABL. A new pan–ABL tyrosine kinase inhibitor, ponatinib, is a promising therapeutic option in patients with all kinds of BCR-ABLmutation including T315I. It was thus hypothesized that the combination of panobinostat and ponatinib exerts synergistic cytotoxicity against IM-resistant cells through a mechanism of action different from that of each agent. To test this hypothesis, K562/IM-R1 and Ba/F3/T315I cell lines were evaluated for the cytotoxicity of panobinostat and ponatinib in vitro. K562/IM-R1 cells, established in our previous study, showed BCR-ABL overexpression due to BCR-ABL gene amplification. The Ba/F3/T315I cell line showed BCR-ABL with a T315I mutation. The XTT proliferation assay revealed that K562/IM-R1 cells were 12-fold more IM-resistant (50%-inhibitory concentration (IC50), 7.6 µM) than K562 cells (IC50, 0.6 µM). Ba/F3/T315I cells were refractory to IM treatment (IC50, 34.1 µM) compared with Ba/F3 cells (IC50, 3.4 µM). Panobinostat overcame IM-resistance and inhibited similarly the growth of K562, K562/IM-R1, Ba/F3, and Ba/F3/T315I cells, with IC50 values of around 50 nM (40.0 - 51.0 nM). Ponatinib inhibited the growth of both K562/IM-R1 cells (IC50, 4 nM) and Ba/F3/T315I cells (25 nM) as potently as parental K562 cells (IC50, 2 nM) and Ba/F3 cells (IC50, 5 nM). Importantly, the combination of panobinostat and ponatinib exhibited enhanced growth inhibition effects on all cell lines. The IC50 values for this combination were 0.7 nM for K562 cells, 1.3 nM for K562/IM-R1 cells, 3.7 nM for Ba/F3 cells, and 10 nM for Ba/F3/T315I cells. The combination index clearly showed synergism, with values of 0.5 for K562 cells, 0.28 for K562/IM-R1 cells, 0.9 for Ba/F3 cells, and 0.4 for Ba/F3/T315I cells. When the cells were treated with 10 nM panobinostat or 10 nM ponatinib for 48 h alone or in combination, the combination of the 2 agents led to greater-than-additive apoptotic cell death than each agent alone in all cell lines, evaluated by annexin V-positivity. Western blotting was used to evaluate the protein expression levels of BCR-ABL and phospho-BCR-ABL in cells after treatment with panobinostat or ponatinib or both in combination. BCR-ABL expression was greater in K562/IM-R1 than K562 cells. Phospho-BCR-ABL expression was not inhibited by IM in K562/IM-R1 or Ba/F3/T315I cells. However, ponatinib inhibited the autophosphorylation of BCR-ABL in these cell lines. Treatment with panobinostat reduced the BCR-ABL and phospho-BCR-ABL expression levels in K562/IM-R1 and Ba/F3/T315I cells. The combination of the 2 agents augmented inhibition of the autophosphorylation of BCR-ABL in these IM-resistant cell lines. The activity of histone deacetylase, determined using the HDAC assay Kit (Active Motif, Carlsbad, CA, USA), was inhibited by panobinostat in all cell lines regardless of IM sensitivity. In comparison, IM did not alter cellular histone deacetylase activity. Upon treatment of K562 cells with panobinostat, the protein expression levels of acetylated histone H3 and H4 were increased, suggesting the consequence of the inhibition of histone deacetylase. In conclusion, we firstly reported that panobinostat and ponatinib demonstrated synergistic cytotoxicity against IM-resistant cell lines not only due to BCR-ABL gene amplification but also BCR-ABL T315I mutation. The synergism is attributable to the greater inhibition of ABL kinase activity through a mechanism of action different from that of each agent. Disclosures: No relevant conflicts of interest to declare.

Abstract 1356 Clofarabine(2-Chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine,2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, CAFdA) is a relatively new purine nucleoside analog. Upon administration, CAFdA is incorporated into leukemic cells by human Equilibrative Nucleoside Transporters (hENT) 1 and 2, and human Concentrative Nucleoside Transporter (hCNT) 3. Inside the cell, the agent is phosphorylated to CAFdA monophosphate by cytosolic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK), and then to an intracellular active metabolite CAFdA triphosphate (CAFdATP). CAFdATP inhibits ribonucleotide reductase and is incorporated into DNA, thereby terminating DNA synthesis as an antimetabolite. Moreover, CAFdA induces apoptosis via direct mitochondrial damage. Clinical studies suggest that CAFdA is effective against both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). CAFdA therapy should be optimized based on the mechanistic understanding, because pharmacological determinants that correlate to the drug sensitivity may predict clinical efficacy of CAFdA as biological surrogate markers. Here, we have established two novel leukemic cell line variants that were resistant to CAFdA, and elucidated the mechanism of the drug resistance. The study focused on factors that were involved in the intracellular CAFdATP production and in the induction of apoptosis. To develop resistant variants, HL-60 cells were treated with escalating concentrations of CAFdA with the initial concentration at 1/100 of the concentration that inhibited 50% cell growth. After 7 months of the repeated passage, one cell line resistant to CAFdA (HL/CAFdA20) was cloned by the limiting dilution method. A part of this clone was further maintained with the drug for the subsequent 4 months to develop another variant (HL/CAFdA80). The 2 variants were 20- and 80-fold more CAFdA-resistant than HL-60 cells, respectively. They were cross-resistant to similar nucleoside analogs such as cladribine, gemcitabine, and cytarabine. Compared with HL-60 cell line, mRNA levels of the transporters (hENT1, hENT2, hCNT3) and protein levels of kinases (dCK, dGK), and the subsequent production of intracellular CAFdATP were all reduced in both CAFdA-resistant variants. Real time RT-PCR demonstrated that mRNA levels of hENT1, hENT2, and hCNT3 were 53.9%, 41.8%, 18.3% in HL/CAFdA20 cells, and 30.8%, 41.6%, 31.5% in HL/CAFdA80 cells, respectively, compared to the parental cells. The values of the initial uptake of CAFdA into the cell at 40 seconds after administration of tritiated CAFdA are 0.2 pmol/107cells in HL/CAFdA20 cells, and 0.1 pmol/107cells in HL/CAFdA80 cells, compared with 0.6 pmol/107 cells in parental HL60 cells. Western blotting revealed that protein levels of kinases were also reduced in these resistant variants with the greater reduction in HL/CAFdA80 cells. The subsequent production of CAFdATP after 4-h incubation with 10 μM CAFdA was 20 pmpl/107 cells in HL/CAFdA20 cells, 3 pmol/107cells in HL/CAFdA80 cells, and 63 pmol/107cells in HL-60 cells. The decreased CAFdATP production led to the attenuated incorporation of the drug into both mitochondrial and nuclear DNA. Concerning apoptosis, antiapoptotic Bcl2 protein overexpressed in the 2 resistant variants. The two variants were resistant to mitochondria-related apoptosis induced by CAFdA, in part due to the enhanced Bcl2 expression. A Bcl2 inhibitor ABT737 synergized the cytotoxic effect and the growth inhibition effect of CAFdA in both variants and HL-60, but the synergism was more profound in the resistant cell lines. The Combination Index values were 0.27 in HL/CAFdA20 cells, and 0.21 in HL/CAFdA80 cells, compared with 0.63 in HL-60 cells. This suggested the contribution of the enhanced Bcl2 protein to the mechanism of drug resistance. In conclusion, the mechanism of cellular resistance to CAFdA in the 2 variants was multifactorial, but primarily includes the reduced CAFdATP production and the increased antiapoptotic factor. It is noted that the decreased dGK level and Bcl2 overexpression were not reported previously in the context of CAFdA resistance. We suggest combination of CAFdA and ABT737 might be effective to CAFdA resistant and refractory leukemia. (This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. No.23501307) Disclosures: No relevant conflicts of interest to declare.

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course., Highlights • Tetracyclines inhibit cytokine and chemokine production in LPS-induced THP-1 cells. • Tetracyclines inhibit NF-κB, p38 and activate ERK phosphorylation. • Tetracyclines regulate these pathways and NF-κB pathway has a pivotal role.

Background Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Methods In the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting. Results CEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells. Conclusions An ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.