Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development.
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 GI 50 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 antileukemic 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.
Despite improved clinical outcomes of diffuse large B-cell lymphoma, a certain proportion of patients still develop a primary refractory disease. To overcome these lymphomas that are intractable to existing treatment strategies, the tumor microenvironment has been identified as a potential therapeutic target. Here we describe our search for effective drugs for primary refractory lymphoma cells with MYC rearrangement. Through the drug screening of 3,440 known compounds, we identified a unique compound, emetine. This compound was effective against lymphoma cells with MYC rearrangement from two different patients that were co-cultured with cancer associated fibroblasts. Emetine induced the death of these cells with a half maximal inhibitory concentration of 312 nM and 506 nM, respectively. Subsequent analyses of the mechanism of action of emetine showed that the drug induced apoptosis of tumor cells via alteration of glucose metabolism through inhibition of hypoxia inducible factor-1α. Moreover, emetine inhibited the potential of cancer associated fibroblasts to support tumor cell viability in vitro and demonstrated significant inhibition of tumor growth in in vivo analyses. Emetine also induced cell death in other primary refractory lymphoma cells with MYC rearrangement. Our combined data indicate that emetine is a potential promising drug for the treatment of intractable lymphomas, which targets both the tumor and its microenvironment.
PAX5 is a transcription factor that is required for the development and maintenance of B cells. Promyelocytic leukemia (PML) is a tumor suppressor and proapoptotic factor. The fusion gene PAX5-PML has been identified in acute lymphoblastic leukemia with chromosomal translocation t(9;15)(p13; q24). We have reported previously that PAX5-PML dominantnegatively inhibited PAX5 transcriptional activity and impaired PML function by disrupting PML nuclear bodies (NBs). Here we demonstrated the leukemogenicity of PAX5-PML by introducing it into normal mouse pro-B cells. Arrest of differentiation was observed in PAX5-PML-introduced pro-B cells, resulting in the development of acute lymphoblastic leukemia after a long latency in mice. Among the transactivation targets of PAX5, B cell linker protein (BLNK) was repressed selectively in leukemia cells, and enforced BLNK expression abrogated the differentiation block and survival induced by PAX5-PML, indicating the importance of BLNK repression for the formation of preleukemic state. We also showed that PML NBs were intact in leukemia cells and attributed this to the low expression of PAX5-PML, indicating that the disruption of PML NBs was not required for the PAX5-PML-induced onset of leukemia. These results provide novel insights into the molecular mechanisms underlying the onset of leukemia by PAX5 mutations.PAX5 is a member of the highly conserved paired box (PAX) 2 domain family of transcription factors. PAX5 is expressed exclusively from the pro-B to mature B cell stage and is downregulated during terminal differentiation into plasma cells (1). PAX5 is indispensable for B lineage commitment by the transcriptional activation of B lineage-specific genes (2), such as CD19 (3), CD79A (4), and B cell linker protein (BLNK) (5), and its target disruption has been shown to cause B lymphoid maturation arrest at the pro-B cell stage (6). Previous studies have identified the PAX5 gene as the most frequent target of somatic mutations in childhood and adult B-progenitor acute lymphoblastic leukemia (ALL), being altered in 38.9% and 34% of cases, respectively (7,8), and these findings further emphasized the essential role of PAX5 in the proper development of B cells. Somatic mutations consist of partial or complete hemizygous deletions, homozygous deletions, partial or complete amplifications, point mutations, or fusion genes (7). These aberrations in the PAX5 gene are considered to impair PAX5 function and play a role in blocking B cell differentiation. PAX5 fusion proteins such as PAX5-TEL, PAX5-ENL, PAX5-PML, and PAX5-C20S have been shown previously to have dominant-negative effects on PAX5 transcriptional activity and have been suggested to be mainly responsible for the differentiation disorder of ALL with these fusion genes (9 -12). Consistently, a previous study has reported that PAX5 haploinsufficiency cooperated with the constitutive activation of STAT5 to initiate ALL in mice (13). However, the oncogenicity of PAX5 mutations, including fusion genes, has yet to be demonstrated...
Cell type-specific gene expression programs in mammals are mainly governed by a small number of transcription factors (TF) that collectively control each other's gene expression to form highly interconnected autoregulatory loops, termed as core regulatory circuitry (CRC; 1-4). Identifying the CRC in cancer can help reveal the cell of origin ( 5) and critical TFs required to maintain an oncogenic cell state (6-11), thus providing valuable insights into cancer biology. However, such efforts in leukemia have been limited (6,11). Although fusion
Objective Conditioning regimens for hematopoietic stem cell transplantation (HSCT) are well known to cause severe gastrointestinal toxicities that often disturb the oral intake of the patients followed by poor nutrition and life-threatening infection. An oral elemental diet (ED) is an easily consumed and assimilated form of liquid nutrients mainly composed of amino acids. It alleviates the digestive loading from the intestine and is mainly used for enteral nutritional support in patients with Crohn's disease. We herein report, for the first time, the efficacy of ED for patients undergoing HSCT. Methods We evaluated the efficacy of ED in a prospective cohort study. The primary endpoint for this study was the hospitalization period. The secondary endpoint was the occurrence of oral mucositis, nausea, diarrhea and fever. Patients A total of 73 patients were consecutively enrolled between March 2011 and March 2013. Twenty-three patients underwent autologous HSCT and 50 patients underwent allogeneic HSCT. The first 21 patients did not receive ED (non-ED group; NEG) while in the successive 52 patients (ED group; EG), oral ED was started before conditioning and was continued until 28 days after transplantation. Results The patient characteristics were similar between the two groups. The mean duration of ED administration for EG was 28.7 days (range, 3-37 days), and the mean total-dose of ED administration was 1904 g (range, 240-2,960 g). The median hospitalization period was significantly shorter in EG compared to NEG, (34 days vs. 50 days; p=0.007). Grade 3-4 oral mucositis occurred less in EG than NEG (25% vs. 48%; p=0.06). Conclusion Oral ED may promote an early mucosal recovery and thereby shorten the duration of hospitalization.
Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.
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