Daiki Hirano

YM155 induces apoptosis through proteasome-dependent degradation of MCL-1 in primary effusion lymphoma

&NA; Primary effusion lymphoma (PEL) is a lymphoma that shows malignant effusion in body cavities without contiguous tumor masses and has a very poor prognosis. We recently developed a novel drug screening system using patient‐derived xenograft (PDX) cells that maintained the primary cell phenotype better than cell lines. This screening is expected to discover anti‐tumor drugs that have been overlooked by conventional screening using cell lines. We herein performed this screening to identify new therapeutic agents for PEL. We screened 3518 compounds with known pharmaceutical activities based on cytotoxic effects on PDX cells of PEL and selected YM155, a possible survivin inhibitor. It exerted strong anti‐tumor effects in PDX cells and three cell lines of PEL; the GI50 of YM155 was 1.2–7.9 nM. We found that YM155 reduced myeloid cell leukemia‐1 (MCL‐1) protein levels prior to decreasing survivin levels, and this was inhibited by a proteasome inhibitor. The knockdown of MCL‐1 by siRNA induced cell death in a PEL cell line, suggesting the involvement of decreased MCL‐1 levels in YM155‐induced cell death. YM155 also induced the phosphorylation of ERK1/2 and MCL‐1, and a MEK1 inhibitor inhibited the phosphorylation of ERK1/2, degradation of MCL‐1, and YM155‐induced apoptosis. These results indicate that YM155 induces the proteasome‐dependent degradation of MCL‐1 through its phosphorylation by ERK1/2 and causes apoptosis in PEL cells. Furthermore, a treatment with YM155 significantly inhibited the development of ascites in PEL PDX mice. These results suggest the potential of YM155 as an anti‐cancer agent for PEL. Graphical abstract Figure. No caption available.

The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, particularly from the viewpoints of microenvironmental independence. Patient-derived xenografts (PDX) are established by the transfer of primary tumor cells directly from patients into immunodeficient mice and can provide primary-like tumor cells of the amount needed at the desired time. We developed a high-throughput drug screening system using PDX cells and performed drug screening using the PDX cells of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). We established four Ph+ ALL PDX mice and performed high-throughput screening of 3440 compounds using leukemia cells from the PDX mice (PDX-cell screening). The profiles of drugs selected by PDX-cell screening were markedly different from those by screening using the Ph+ ALL cell line. We found that verteporfin, an FDA-approved drug, exhibited strong PDX cell-specific cytotoxicity. In the validation assay, its GI50 was 228 nM, 395 nM, and 538 nM in three PDX cells and 3.93 μM, 2.11 μM, and 5.61 μM in three cell lines. Although verteporfin is a photosensitizer activated by photoirradiation, its cytotoxic effects were mediated by the light-independent production of reactive oxygen species; therefore, its anti-leukemic effects were also exerted in vivo without photoirradiation. Furthermore, it exhibited synergistic effects with dasatinib, an ABL kinase inhibitor. These results indicated the potential of verteporfin as a new anti-leukemic reagent.

High incidence of extensive chronic graft-versus-host disease in patients with the REG3A rs7588571 non-GG genotype

Regenerating islet-derived protein 3 alpha (REG3A) is a biomarker of lower gastrointestinal graft-versus-host disease (GVHD); however, the biological role of REG3A in the pathophysiology of GVHD is not understood. Here, we examined the association between a single nucleotide polymorphism in the REG3A gene, rs7588571, which is located upstream and within 2 kb of the REG3A gene, and transplant outcomes including the incidence of GVHD. The study population consisted of 126 adult Japanese patients who had undergone bone marrow transplantation from a HLA-matched sibling. There was no association between rs7588571 polymorphism and the incidence of acute GVHD. However, a significantly higher incidence of extensive chronic GVHD was observed in patients with the rs7588571 non-GG genotype than in those with the GG genotype (Odds ratio 2.6; 95% confidence interval, 1.1–6.0; P = 0.029). Semi-quantitative reverse transcription PCR demonstrated that the rs7588571 non-GG genotype exhibited a significantly lower REG3A mRNA expression level than the GG genotype (P = 0.032), and Western blot analysis demonstrated that the rs7588571 non-GG genotype exhibited a trend toward lower REG3A protein expression level than the GG genotype (P = 0.053). Since REG proteins have several activities that function to control intestinal microbiota, and since intestinal dysbiosis is in part responsible for the development of GVHD, our findings lead to the novel concept that REG3A could have some protective effect in the pathogenesis of GVHD through the regulation of gut microbiota.

Salvage therapy with single agent L-asparaginase followed by local irradiation in an elderly patient with CD56-positve primary isolated extramedullary T-cell lymphoblastic lymphoma of the sinus.

Dear Editor, A 68-year-old Japanese man presented with rapid swelling of the right cheek and a gingival enlargement in the right upper anterior region. A computed tomography scan (CT) demonstrated a bulky tumor of maxillary sinus which invaded surrounding tissues (Fig. 1). He was diagnosed as clinical stage IA T-cell lymphoblastic lymphoma (T-LBL) and offered a Hyper-CVAD regimen [1]. A complete radiological remission was achieved according to CT scan performed after 3 cycles of the regimen; however, therapy was discontinued owing to grade 3 neuropathy and arrhythmias, and poor performance status. We decided to choose irradiation as consolidation therapy after 4 cycles of the regimen. During radiation therapy was planned, rapid progression at the site of the original tumor was observed. He was diagnosed as recurrent stage IA T-LBL (Fig. 1). Chemotherapy consisting of single agent L-asparaginase 3,000 U/m was given intravenously on days 1, 3, 5, 7, 9, and 11, and after that, local radiation therapy of 45 Gy in 25 fractions was performed with dynamic conformational technique. A second complete remission was confirmed. At present, 1 year after treatment, he is in good physical condition without evidence of disease. T-LBL represents 1.7 % of adult non-Hodgkin lymphoma and is thought to much less frequently occur in the elderly [2]. While common sites of involvement are the mediastinum, bone marrow, and central nervous system, primary isolated T-LBL of the sinus is very rare. The prognosis of relapsed or refractory lymphoblastic lymphoma (LBL)/acute lymphoblastic leukemia (ALL) is usually poor in both children and adults, because of extremely low remission rates of reinduction therapy. Data on the effective therapy for elderly patients with relapsed LBL are scarcely available. Few reports of clinical outcomes in elderly patients with newly diagnosed ALL have been published and only some of them were prospective trials [3–5]. Intensive chemotherapy is usually intolerable to elderly patients. Early deaths during induction therapy are reported to occur up to approximately 40 %, and the results of clinical outcomes of ALL in elderly patients are discouraging with median overall survival duration from 1 to 14 months. Nevertheless, a proportion of elderly patients could achieve long-term remission. Some study suggests that the choice of optimal post-remission consolidation therapy could be an important factor [4]. D. Hirano :H. Kato (*) :N. Ueda : S. Murakami :Y. Higuchi : H. Taji :K. Yamamoto : T. Kinoshita Department of Hematology and Cell Therapy, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan e-mail: hkato@aichi-cc.jp

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.