Takanobu Morishita

Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

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.

Emetine elicits apoptosis of intractable B-cell lymphoma cells with MYC rearrangement through inhibition of glycolytic metabolism

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.

Long-Term Remission After Multiple Relapses in an Elderly Patient With Lymphomatoid Granulomatosis After Rituximab and High-Dose Cytarabine Chemotherapy Without Stem-Cell Transplantation

Introduction Use of the anti-CD20 monoclonal antibody rituximab has markedly improved clinical outcomes for patients with diffuse large B-cell lymphoma (DLBCL) over the last decade. However, there are some histologic subtypes of DLBCL for which prognoses have not fully improved, even after rituximab therapy. Epstein-Barr virus (EBV) –positive B-cell lymphoproliferative disorders (LPDs) have been well documented over the last decade, and one disorder that is representative of this group is lymphomatoid granulomatosis (LYG), as defined in the 2008 WHO classification guidelines. LYG, a rare subtype of DLBCL, is an angiocentric and angiodestructive lymphoproliferative disease involving extranodal sites, especially bilateral lungs. This disease involves development of lesions comprising EBV-positive B cells admixed with reactive T cells, which are usually predominant. Some cases of EBV-positive LPDs have morphologic and histologic overlap with classic Hodgkin’s lymphoma (cHL), including the presence of Hodgkin’s and ReedSternberg (HRS) –like giant cells. Most LYG is aggressive; the median survival is less than 2 years. The proportion of EBV-positive B cells relative to the reactive lymphocyte background is related to the grading of LYG. Patients with low-grade lesions usually achieve a durable response to interferon alfa-2b (IFN-2b) therapy because of its antiviral, antiproliferative, and immunomodulatory properties, whereas those with grade 3 lesions have been treated with chemotherapy in combination with rituximab, in a similar manner to management of patients with DLBCL. However, the optimal treatment strategy for patients with LYG has not yet been established. Although case reports of efficacious rituximab monotherapy, rituximabcontaining chemotherapy, and high-dose chemotherapy with autologous stem-cell transplantation have been published, to our knowledge, there has been no previous report of successful treatment resulting in a long-term complete remission (CR) of more than 10 years. Here we present the case of an elderly patient who experienced long-term CR, after previous multiple recurrences, following the administration of a regimen containing rituximab and high-dose cytarabine.

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.

B Cell Linker Protein (BLNK) Is a Selective Target of Repression by PAX5-PML Protein in the Differentiation Block That Leads to the Development of Acute Lymphoblastic Leukemia

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 dominant-negatively 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.

Iodine staining and p16 immunohistochemistry as a novel screening for secondary esophageal neoplasm after hematopoietic stem cell transplantation

Regarding allogeneic hematopoietic stem cell transplantation (HSCT) survivors, several studies have reported a twofold to fourfold increased risk of secondary solid tumors, and incidence ratios of oral, pharyngeal and esophageal cancers were significantly higher than the general population [1–3]. Notably, increased risk of oral squamous cell carcinoma (SCC) has been reported in patients with chronic graft-versus-host disease (GVHD) [4–6]. Considering the dismal prognosis of esophageal cancer found in the advanced stage, detection in the asymptomatic early stages is highly desirable. Human papillomavirus (HPV) infection is suspected as an associated risk factor for oral and esophageal SCC in patients with immunodeficiency caused by the human immunodeficiency virus, solid organ transplantation, or allogeneic HSCT [5, 7, 8]. A case report has assessed the usefulness of p16 immunohistochemistry, a surrogate marker for HPV, for the early diagnosis of secondary oral and esophageal malignancy after HSCT [4]. Indeed, p16 immunohistochemistry is known as a pathologically good tool for the detection of oral and esophageal squamous cell neoplasm (SCN). The HPV-associated oncoprotein E6 and E7 interfere with cell cycle and inactivate tumor-suppressor protein p53 and pRb (retinoblastoma protein), which result in increased p16 expression levels caused by negative feedback control [9]. In patients with oral and pharyngolaryngeal SCC, the esophagus should be examined and screened for another SCN [4, 5]. In addition, esophagogastroduodenoscopy (EGD) with iodine staining and narrow band imaging (NBI) of the esophagus is a powerful tool for the detection of early esophageal SCN. In this study, we screened esophageal SCN using EGD examination with iodine staining, as well as pathological examination with p16 immunostaining, in patients after allogeneic HSCT. We performed EGD screening in patients who survived at least 1 year after the first allogeneic HSCT and who visited Japanese Red Cross Nagoya First Hospital from January 2009 to January 2017. Patients with oral mucosal lesions, primarily oral GVHD, but without gastrointestinal symptoms were selected. All patients provided informed consent in accordance with the Declaration of Helsinki. This study was designed as a prospective observational survey, and approved by the ethical committee of our institutional review board. EGD was performed by using an Olympus GIF-H290Z, GIF-H260Z, or GIF-H260 scope (Olympus Corporation, Tokyo, Japan). All patients were examined with normal white light, NBI, and iodine staining, respectively. Endoscopic biopsies were performed for abnormal endoscopic findings, even if it were minor changes. Biopsy specimens were stained with hematoxylin and eosin (HE) and p16 mouse monoclonal antibody (Ventana, Arizona, USA) immunostaining by using an autostainer BOND Max (Leica Biosystems, Newcastle, UK) according to the instruction manual, and then we compared p16 staining status with patient’s clinical characteristics. Each patient had a single-time EGD screening; however, annual EGD examinations were recommended for patients that were p16 positive. * Masafumi Ito itom@nagoya-1st.jrc.or.jp

Higher Peak Tacrolimus Concentrations after Allogeneic Hematopoietic Stem Cell Transplantation Increase the Risk of Endothelial Cell Damage Complications

Noninfectious transplantation-related complications (TRCs) such as graft-versus-host disease (GVHD) and endothelial cell damage (TRC-EC) are critical after allogeneic hematopoietic stem cell transplantation. Tacrolimus (TAC) is used to control GVHD. Hypertension and renal failure are common adverse events after TAC treatment. Higher blood concentrations of TAC would be expected to reduce the risk of GVHD but may increase TRC-EC. TRC-EC often develops in patients with GVHD; thus, it is difficult to clinically determine the proper intensity of immunosuppression. We therefore evaluated the impact of weekly mean/peak TAC blood concentrations (PTCs) on TRC-EC occurrence and prognosis. Patients (N = 295) who received TAC as a GVHD prophylaxis at our institute from 2009 to 2016 were eligible for this retrospective study. Forty-three patients were diagnosed with TRC-EC: 8 with sinusoidal obstructive syndrome, 28 with transplant-associated microangiopathy, and 7 with idiopathic pneumonia syndrome. The cumulative incidence of TRC-EC at 12 months was 13.8% (95% confidence interval [CI] 10.1% to 18.1%). After multivariate analysis high PTCs during days 22 to 28 (hazard ratio [HR] 2.47; 95% CI, 1.37 to 4.45; P < .01) and grades II to IV acute GVHD (HR, 5.61; 95% CI, 2.99 to 10.53; P < .01) were associated with TRC-EC occurrence. The probability of overall survival (OS) at 12 months was 67.7% (95% CI, 61.7% to 73.0%). After multivariate analysis TRC-EC diagnosis (HR, 2.47, 95% CI, 1.59 to 3.83; P < .01) and high-risk disease (HR, 1.75; 95% CI, 1.17 to 2.61; P < .01) were significantly associated with poor OS. In conclusion, higher PTC during days 22 to 28 increased the risk of TRC-EC. TRC-EC development was associated with poor OS.

Safety and Efficacy of Once-Daily Intravenous Busulfan in Allogeneic Transplantation: A Matched-Pair Analysis

Compared with 4-times-daily infusion of intravenous busulfan (ivBU4), the safety and efficacy of once-daily infusion of ivBU (ivBU1) has not been fully clarified. We have been routinely using ivBU1 in a conditioning regimen in adult patients with myeloid malignancy who undergo allogeneic hematopoietic stem cell transplantation. In this study, a total of 91 patients who received ivBU1 for 2 days (n = 18) or 4 days (n = 73) in our institutions were compared with 273 control patients who received ivBU4, who were matched for age, sex, performance status, disease risk, conditioning regimen, and donor type, selected from the database of the Japanese Society for Hematopoietic Cell Transplantation using optimal matching algorithms. One-year overall survival (56.8% versus 57.1%, P = .94), disease-free survival (51.6% versus 50.8%, P = .73), relapse rate (28.5% versus 26.2%, P = .94), nonrelapse mortality (19.9% versus 23.0%, P = .71), and the incidence of graft-versus-host disease were not significantly different between the ivBU1 and ivBU4 groups. In patients who received ivBU1, neutrophil recovery was slower (median days: 22 versus 17, P = .001), and the incidence of veno-occlusive disease was lower (2.6% versus 17.4%, P = .04). In conclusion, ivBU1 can be safely administered with clinical outcomes similar to those with ivBU4.

Emetine Elicits Apoptosis of Intractable B-Cell Lymphoma Cells with MYC Rearrangement through Inhibition of Glycolytic Metabolism

In this issue of Blood, Aljabri and colleagues report on their analysis of the cost-effectiveness of fondaparinux for the treatment of heparin-induced thrombocytopenia (HIT) in the United States.1 HIT is a relatively uncommon but serious complication of the use of heparin-containing products.2 Treatment of HIT requires use of an alternate anticoagulant such as the direct thrombin inhibitors argatroban and bivalirudin.3