To evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the moleclular pathways responsible for antiproliferative effects on leukemia cells during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. The inhibitory concentration (IC 50 ) of deferasirox was 17-50 mM in three human myeloid cell lines (K562, U937, and HL60), while those in fresh leukemia cells obtained from four patients it varied from 88 to 172 mM. Gene expression profiling using Affymerix GeneChips (U133 Plus 2.0) revealed up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) encoding p21 (1) Evidence suggests that iron is required for cell survival and proliferation, and perturbation in cellular iron uptake can arrest cell growth both in vitro and in vivo.(2) As a part of ribonucleotide reductase, the enzyme responsible for deoxyribonucleotides synthesis, iron is an essential growth factor and ratelimiting trace element in DNA synthesis.(3) Dysregulaton of iron metabolism leads to iron overloading associated with deleterious effects on cells and tissues.(3) Numerous iron chelators have been synthesized in order to treat iron overload diseases, especially thalassemia. Evidence suggests the hyperproliferative effect of iron overload in a subset of cancer cells and iron depletion by chelators inhibits the proliferation of cancer cells, including leukemia cells.(4-8) Among the different molecules synthesized, hexadenate deferoxamine (DFO) is the major molecule used for the treatment of iron overload. However, it is highly hydrophilic, and inactive if taken perorally. For this reason, the perorally active iron chelator, deferasirox, is of special interest, since recent reports demonstrated that it acts as a potent nuclear factor kappa-lightchain-enhancer of activated B cell (NF-kappa-B) inhibitor and improves hematological data in a subset of patients with myelodysplastic syndromes (MDS). (9,10) To evaluate the effect of deferasirox (also knows as ICL670, Novartis, Basel, Switzerland), and to identify molecular pathways responsible for the observed reduced transfusion requirement during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. Materials and MethodsReagents and cell cultures. The oral iron chelator, deferasirox was donated by Novartis. We purchased three human myeloid leukemia cell lines, K562, U937, and HL-60 from Health Science Research Resources Bank (Osaka, Japan) for this study. Cells were grown in RPMI1640 with 10% fetal bovine serum. After obtaining written informed consent, peripheral blood mononuclear cells (PBMCs) were isolated from four patients with acute myeloid leukemia (AML) by the Ficoll-Hypaque technique. This study was approved by our institutional medical ethics committee.Cell viability and apoptosis assay. The inhibitory effect of deferasirox on cell growth was assessed by a Cell Counting Kit-8 (Wako Chemicals, Tokyo, Japan)....

Background aims To develop a treatment option for Philadelphia chromosome—positive acute lymphoblastic leukemia (Ph+ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR). Methods A CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15–containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph+ALL cell lines including three TKI-resistant (T315I–mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines. Results We obtained ~ 1.3 × 108 CART cells (CD4+, 25.4%; CD8+, 71.3%), co-expressing CD45RA and CCR7 up to ~80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CART cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor–related apoptosis-inducing ligand and interleukin-2. Conclusions We generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph+ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph+ALL.

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