Using Fludarabine to Reduce Exposure to Alkylating Agents in Children with Sickle Cell Disease Receiving Busulfan, Cyclophosphamide, and Antithymocyte Globulin Transplant Conditioning: Results of a Dose De-Escalation Trial

AbstractHigh-dose busulfan, cyclophosphamide, and antithymocyte globulin (BU-CY-ATG) is the most commonly used conditioning regimen in HLA-matched related hematopoietic cell transplantation for children with sickle cell disease. Disease-free survival with this regimen is now approximately 95%; however, it produces significant morbidity. We hypothesized we could create a less toxic regimen by adding fludarabine (FLU) to BU-CY-ATG and reduce the dosages of busulfan and cyclophosphamide. We conducted a multicenter dose de-escalation trial with the objective of decreasing the doses of busulfan and cyclophosphamide by 50% and 55%, respectively. Using day +28 donor-predominant chimerism as a surrogate endpoint for sustained engraftment, we completed the first 2 of 4 planned levels, enrolling 6 patients at each and reducing the total dose of cyclophosphamide from 200 mg/kg to 90 mg/kg. On the third level, which involved a reduction of i.v. busulfan from 12.8 mg/kg to 9.6 mg/kg, the first 2 patients had host-predominant T cell chimerism, which triggered trial-stopping rules. All 14 patients survive disease-free. No patients suffered severe regimen-related toxicity. Our results suggest BU-FLU-CY-ATG using lower dose CY could be a less toxic yet effective regimen. Further evaluation of this regimen in a full-scale clinical trial is warranted

Using Fludarabine to Reduce Exposure to Alkylating Agents in Children with Sickle Cell Disease Receiving Busulfan, Cyclophosphamide, and Antithymocyte Globulin Transplant Conditioning: Results of a Dose De-Escalation Trial

AbstractHigh-dose busulfan, cyclophosphamide, and antithymocyte globulin (BU-CY-ATG) is the most commonly used conditioning regimen in HLA-matched related hematopoietic cell transplantation for children with sickle cell disease. Disease-free survival with this regimen is now approximately 95%; however, it produces significant morbidity. We hypothesized we could create a less toxic regimen by adding fludarabine (FLU) to BU-CY-ATG and reduce the dosages of busulfan and cyclophosphamide. We conducted a multicenter dose de-escalation trial with the objective of decreasing the doses of busulfan and cyclophosphamide by 50% and 55%, respectively. Using day +28 donor-predominant chimerism as a surrogate endpoint for sustained engraftment, we completed the first 2 of 4 planned levels, enrolling 6 patients at each and reducing the total dose of cyclophosphamide from 200 mg/kg to 90 mg/kg. On the third level, which involved a reduction of i.v. busulfan from 12.8 mg/kg to 9.6 mg/kg, the first 2 patients had host-predominant T cell chimerism, which triggered trial-stopping rules. All 14 patients survive disease-free. No patients suffered severe regimen-related toxicity. Our results suggest BU-FLU-CY-ATG using lower dose CY could be a less toxic yet effective regimen. Further evaluation of this regimen in a full-scale clinical trial is warranted

Inhibition of MDM2 by nilotinib contributes to cytotoxicity in both Philadelphia-positive and negative acute lymphoblastic leukemia.

Nilotinib is a selective BCR-ABL tyrosine kinase inhibitor related to imatinib that is more potent than imatinib. Nilotinib is widely used to treat chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The present study identifies Mouse double minute 2 homolog (MDM2) as a target of nilotinib. In studying ALL cell lines, we found that the expression of MDM2 in both Philadelphia positive (Ph+) and Philadelphia negative (Ph-) ALL cells was remarkably inhibited by nilotinib, in a dose- and time-dependent manner. Further studies demonstrated that nilotinib inhibited MDM2 at the post-translational level by inducing MDM2 self-ubiquitination and degradation. Nilotinib-mediated MDM2 downregulation did not result in accumulation and activation of p53. Inhibition of MDM2 in nilotinib-treated ALL cells led to downregulation of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP), a translational target of MDM2, resulting in activation of caspases. Inhibition of XIAP following nilotinib-mediated downregulation of MDM2 resulted in apoptosis of MDM2-expressing ALL; however, similar nilotinib treatment induced stronger apoptosis in Ph+/MDM2+ ALL than in Ph-/MDM2+ or Ph+/MDM2- ALL. The ALL cells that were Ph-/MDM2- were totally resistant to nilotinib. These results suggested that nilotinib can inhibit MDM2 and induce a p53-independent apoptosis pathway by downregulating XIAP; thus, nilotinib can treat not only Ph+, but also Ph- ALL patients whose cancer cells overexpress MDM2

Cytotoxic and apoptotic effects of nilotinib on ALL cells.

<p><b>A</b>, dose-dependent cytotoxic response to nilotinib in the six ALL cell lines, as tested for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100960#pone-0100960-g001" target="_blank">Fig. 1A</a>. The viability of cells incubated with different concentrations of nilotinib for 24 h was determined by WST assay. Data from three independent experiments is represented by the mean percentage ± SD of surviving cells, as compared to the untreated controls. *p<0.01; **p<0.05 (as compared with SUP-B15) <b>B</b>, dose-response of apoptosis of the six cell lines treated with the nilotinib doses indicated for 24 h, as quatitatively detected by flow cytometry. *p<0.01; **p<0.05 (as compared with SUP-B15). <b>C</b>, the effect of p53 knockdown on sensitivity of BCR-ABL+ cells to nilotinib. SUP-B15 cells, transfected with 200 nM of either p53 siRNA or control siRNA, were treated with different dose of nilotinib for 24 h, and cell viability was detected by WST assay, *<i>p</i>>0.5. The expression of p53 in SUP-B15 cells transfected with p53 siRNA was determined by Western blot assay (<i>insert</i>). <b>D</b>, the effect of enforced overexpression of XIAP on sensitivity of ALL cells to nilotinib. SUP-B15 cells, transfected with pCDNA3-6myc-XIAP plasmid and vehicle control, were treated with different dose of nilotinib for 24 h, and cell viability was detected by WST assay, *<i>p</i><0.05. Western blot showing the expression of ectopic XIAP (as detected by Myc antibody) in SUP-B15 cells (<i>insert</i>).</p

The effect of nilotinib-mediated downregulation of MDM2 and XIAP on activation of p53 and caspases.

<p><b>A</b>, SUP-B15 (BCR-ABL+) and EU-1 (BCR-ABL–) were treated with 2 µM nilotinib for different times and different doses for 24 h, respectively. The expression of p53, p21 and PARP was detected by western blotting. <b>B</b>, cell-cycle analysis in SUP-B15 cells performed 4 h post-treatment with 2 µM nilotinib, as compared with untreated cells. <b>C</b> and <b>D</b>, activation of caspase-9 (C) and -3 (D) in nilotinib-treated SUP-B15 and EU-1 cells was detected by enzyme-linked immunosorbent assay (ELISA), *p>0.05.</p

Evaluation of the mechanism by which nilotinib inhibits MDM2.

<p><b>A</b>, RT-PCR for MDM2 mRNA expression in SUP-B15 cells, treated with 2 µM nilotinib and 0.2 µM triptolide (as a control) for the different times, as indicated. <b>B</b>, SUP-B15 cells were treated with or without 2 µM nilotinib for 4 h; then their cytoplasmic lysates were fractionated on a sucrose gradient. RNA was extracted from each of the fractions and subjected to quantitative RT-PCR for analysis of the distribution of MDM2 and actin mRNAs. Data represent the percentage of the total amount of corresponding mRNA in each fraction. <b>C</b>, CHX pulse-chase assay for detection of MDM2 turnover in SUP-B15 cells treated with or without 2 µM nilotinib for 4 h. Numerical labels under each protein band represent protein expression levels after normalization for GAPDH, compared with the untreated (0) samples (defined as 1 unit). <b>D</b>, SUP-B15 cells with or without nilotinib exposure were treated with 10 µM MG32 for 4 h and then the expression of proteins as indicated was assessed by Western blot. <b>E</b>, co-IP and western blot assay to detect the effect of nilotinib on the interaction between MDM2 and MDM4. Cell lysates from SUP-B15 treated with or without nilotinib were immunoprecipitated with the indicated antibodies. Normal mouse IgG served as a control. Proteins in immune complexes were separated on denaturing gels, transferred to filters, and then detected by western blotting with antibodies, as indicated. Antibodies for western blotting were from different species than those used in the IP. <b>F</b>, IP-western blot assay for detection of MDM2 ubiquitination after nilotinib treatment. SUP-B15 cells were treated with 2 µM nilotinib for the indicated length of time. Cellular lysates were immunoprecipitated with anti-MDM2 antibody, and then the MDM2 ubiquitination (MDM2-Ub) was detected by Western blot using anti-ubiquitin antibody.</p

Downregulation of MDM2 by nilotinib in ALL.

<p><b>A</b>, western blot assay for the expression of BCR-ABL, MDM2, p53 and XIAP in six cultured ALL cell lines. The p53 status of these lines (EU-1, UOC-B1 and Sup-B15: wt-p53; EU-6: mutant-p53; EU-5 and EU-9: p53-null) was previously characterized <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100960#pone.0100960-Zhou1" target="_blank">[28]</a>. <b>B</b>, changes of the expression levels of MDM2 and XIAP by nilotinib treatment. Representative cell lines, as indicated, were treated (T) with 2 µM nilotinib for 24 h. Untreated cells served as a control (C). The expression of proteins was detected by western blot, as indicated. <b>C</b> and <b>D</b>, western blot assays showed the dose-response and time-course of MDM2 and XIAP inhibition by nilotinib, in both BCR-ABL positive (SUP-B15) and negative (EU-1) ALL cell lines.</p

Diterpene Glycosides and Polyketides from <i>Xylotumulus gibbisporus</i>

Four new tetracyclic diterpene glycosides, namely, sordarins C–F (<b>1</b>–<b>4</b>), and three new γ-lactone polyketides, namely, xylogiblactones A–C (<b>5</b>–<b>7</b>), along with sordarin were isolated from the ethyl acetate extracts of the fermented broths of <i>Xylotumulus gibbisporus</i> YMJ863. The structures of <b>1</b>–<b>7</b> were elucidated on the basis of spectroscopic data analyses. The configurations of <b>1</b>–<b>4</b> were deduced by NOESY, molecular modeling, and comparison with the literature. The relative configurations of <b>5</b>–<b>7</b> were deduced by X-ray crystallographic analysis of <b>5</b>. Compounds <b>1</b>–<b>5</b> and sordarin were evaluated in an antifungal assay using <i>Candida albicans</i> ATCC 18804, <i>C. albicans</i> ATCC MYA-2876, and <i>Saccharomyces cerevisiae</i> ATCC 2345, and only sordarin exhibited significant antifungal activities against these fungal strains, with MIC values of 64.0, 32.0, and 32.0 μg/mL, respectively. The effect of compounds <b>1</b>–<b>7</b> and sordarin on the inhibition of NO production in lipopolysaccharide-activated murine macrophages was also evaluated. Compounds <b>2</b> and sordarin inhibited NO production with IC₅₀ values of 327.2 ± 46.6 and 157.1 ± 24.1 μM, respectively