ALBERT

Description: Background: Pretreatment status assessment at diagnosis in patients with chronic myeloid leukemia in chronic phase (CML-CP) is more important for successful treatment in the tyrosine kinase inhibitors (TKIs) era. For instance, the Charlson comorbidity index (CCI) score is considered the useful tool for predicting overall survival (OS) in patients with CML treated with imatinib (IM) (Saussele S et al., 2015). However, the effect of CCI in patients with CML treated with second-generation TKI (2nd TKI) and clinical practice setting was unknown. This study aimed to evaluate the effect of pretreatment statuses, including CCI, on OS of participants of the New TARGET observational study 1 conducted by the Japanese Society of Hematology (JSH). Methods: Patients newly diagnosed with CML-CP were registered to the New TARGET observational study 1 from April 2010 to March 2013. They were treated with TKIs (IM, nilotinib [NIL], or dasatinib [DAS]) after registration. Exclusion criteria were defined as follows: (1) accelerated phase/blast crisis (AP/BC) CML and (2) pretreatment with interferon-alfa, any TKIs, or hydroxyurea for more than 3 months, or allogeneic hematopoietic stem cell transplantation before registration. Other details of the protocol were previously reported (Kizaki et al., 2019). Patients were classified into CCI risk groups of 2, 3, and ≥4 for analysis. The New TARGET observational study 1 was supported by research fundings from Novartis Pharmaceuticals and Bristol-Myers Squibb to JSH. This subgroup analysis was approved by the institutional review board of the Hamamatsu University School of Medicine. The chi-square test was used to compare clinical characteristics for categoricaldata and the Wilcoxon rank-sum test for continuous data. OS was calculated using the Kaplan-Meier method and compared by the log-rank test. Gray's test was used to compare cumulative incidence curves. Cox proportional hazard analyses were performed to determine prognostic indicators of OS. The Wald test was used to assess the prognostic significance of a candidate variable. Statistical analyses were performed using EZR, a graphical user interface for R (Kanda Y, 2013). Results: Among 506 enrolled patients, 475 with a median age of 56 years were assessable. The median follow-up period was 5.4 years. In total, 103 patients (21.7%) had various types of comorbidities, with diabetes mellitus, mild liver disease, peripheral vascular disease, myocardial infarction, renal disease, and peptic ulcer disease (7.3%, 4.1%, 3.2%, 2.6%, 2.2%, and 2.2%, respectively) as the most common. The lowest CCI score was 2 owing to CML. CCI scores were stratified as follows: 2, 372 patients (78%); 3, 74 patients (16%); and ≥4, 29 patients (6%). Higher CCI scores were significantly associated with older age (P

Description: Abstract 1743 Poster Board I-769 Background Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. Aims Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. Results Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. Conclusions Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Description: Abstract 1661 Most cancer drug developments are focusing either in vitro target-based screening or cell-based phenotypic screening to identify potential compounds. The target-based screenings are powerful screening methods if the cancer relay on a single driver mutation. On the other hand, cell-based phenotypic screenings measure cell phenotypes such as growth inhibitory effect on established cancer cell lines. However, these established cell lines do not recapitulate human cancer in some aspects. For instance, most cell lines have the microenvironment-independent growth ability, while primary cancer cells can not survive in ex vivo culture. In this study, we established a new drug screening system targeting microenvironment-dependent primary lymphoma cells. First of all, we established primary lymphoma xenograft models, including three diffuse large B cell lymphoma (DLBCL), one follicular lymphoma, one intravascular large cell lymphoma (IVL), and one mantle cell lymphoma. Interestingly, lymphoma cells from the patient with severe extranodal invasion invaded extranodal organ also in mice. And pattern of invasion in tissue such as intravascular tumor invasion of IVL was maintained in the xenograft models. In addition, mRNA expression profiles were similar between primary lymphoma cells and the lymphoma cells obtained from NOG mice. Furthermore, the heterogeneity of primary tumor such as heterogenous expression of CD20 was maintained in the xenograft models. These data suggested that the lymphoma cells propagated in NOG mice kept the original patient's phenotype. Next, we investigated the microenvironment of lymphoma in primary lymphoma xenograft models. Fibroblastic reticular cell (FRC) and follicular dendritic cell (FDC) are reported to be important to support the survival of lymphoma cells in their microenvironment. In the spleen of DLBCL model mice, lymphoma cells colocalized with FRC, but FDC was not detected in NOG mouse. FRC is known to produce reticular fiber and forms fibroblastic reticular network (FRN). Lymphoma induced reticular fiber production and tumor formation on FRN were also observed in our system. Furthermore, co-culture with FRC cell line, BLS4, significantly enhanced viability of 2 out of 3 DLBCL cells obtained from primary lymphoma xenograft mice and enabled more than three weeks long-term ex vivo culture of them. These results indicated that FRC played an important role for lymphoma cell survival. Finally, we established a new drug screening system using this co-culture system. Primary DLBCL cells transplanted to NOG mice were collected and seeded on pre-seeded BLS4 in 96-well plates (lymphoma cell co-culture). Monoculture of BLS4 in 96-well plates was also prepared. Both lymphoma co-cultured cell and BLS4 monoculture cell were treated by 2613 kinds of pharmacologically active compounds for 72 h. DAPI-stained dead cells of lymphoma cells were counted by image analyzer, and the proliferation of BLS4 was measured by MTT assay. We calculated Drug Effect Index (DEI) by multiplication of dead cell number of lymphoma cells and MTT value of BLS4 and compared. The compound with the highest DEI was pyrvinium pamoate, oxyuricide. Pyrvinium pamoate aborogated the survival of lymphoma cells co-cultured with BLS4 dose-dependently. And subcutaneous tumor of primary lymphoma cells and BLS4 were diminished by single intratumoral injection of 20 mg/kg pyrvinium pamoate. These results indicated that our screening system could be performed in large scale and select drugs with anti-tumor activity to primary lymphoma cells. Screening against primary lymphoma cells sheds new light on lymphoma drug development. Disclosures: Sugimoto: Otsuka Pharmaceutical co ltd: Employment.

Description: Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

Description: Abstract 3227 PAX5 is a transcription factor expressed in B lymphoid lineage from pro-B cell to mature B cell, and is required for B-cell development and maintenance. De-regulated and reduced PAX5 activity has been implicated in B cell malignancies both in human disease and mouse models. Furthermore, the PAX5 gene is 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. These mutations consist of partial or complete hemizygous deletions, homozygous deletions, partial or complete amplifications, point mutations or fusion genes. These aberrations of the PAX5 gene will impair PAX5 function more or less, and will be involved in the block of B-cell differentiation. Chromosomal translocation t(9;15)(p13;q24) was found in 2 cases of childhood ALL and resulted in an in-frame fusion of PAX5 to PML gene. PAX5 moiety of PAX5-PML retains its DNA binding domain but loses its transactivation domain suggesting PAX5-PML will be a dominant negative form of PAX5. PML is originally found as a fusion partner of RARα in PML-RARα, an oncoprotein found in acute promyeloid leukemia (APL), and is now thought to be a tumor suppressor and a pro-apoptotic factor. PML-RARα dominant-negatively affects PML function by disrupting PML nuclear bodies (NBs) where PML exerts its function and gives APL cells survival advantage. These findings give rise a speculation that PAX5-PML not only causes differentiation block by transcriptional repression of PAX5 target genes but also confers a survival advantage by inhibition of PML function. However, no functional analysis has been done for PAX5-PML. Here, we demonstrate that PAX5-PML had a dominant negative effect on both PAX5 and PML. PAX5-PML inhibited transcriptional activity of PAX5 in luciferase reporter assay. PAX5-PML expression also suppressed expression of CD19, one of the transcriptional targets of PAX5, in B-lymphoid cell line. Surprisingly, PAX5-PML hardly showed DNA binding activity in electro mobility shift assay although it retains DNA binding domain of PAX5. Further detailed analyses including, luciferase assay using PAX5-PML with DNA-binding-dead mutations, co-IP assay, and ChIP assay, suggested that transcriptional repression by PAX5-PML was independent of its DNA binding ability, that PAX5 and PAX5-PML formed a heterodimer, and that PAX5-PML bound to CD19 promoter through the association with PAX5 on the promoter. On the other hand, co-expression of PAX5-PML inhibited PML sumoylation, an essential post-translational modification for PML to form NBs, and altered PML localization from NB pattern to diffuse nuclear pattern. Furthermore, treatment with arsenic trioxide, a therapeutic reagent for APL that induces enhancement of PML sumoylation, reconstitution of PML NBs, and apoptosis in APL cells, induced recovery of PML sumoylation and reconstitution of PML NBs also in cells expressing PAX5-PML. More importantly, PAX5-PML expressing HeLa cells showed resistance to PML dependent apoptosis such as apoptosis induced by irradiation and histone deacetylace inhibitor. And arsenic trioxide treatment overcame these apoptosis resistance conferred by PAX5-PML. These data suggest the involvement of this fusion protein in the leukemogenesis of B-ALL in dual-dominant negative manner and the possibility that some cases of ALL can be treated with arsenic trioxide. Disclosures: Hayakawa: Otsuka: Research Funding. Naoe:Chugai: Research Funding; Zenyaku: Research Funding; Kyowa-Kirin: Research Funding; Dainippon-Sumitomo: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Otsuka: Research Funding.

Description: Abstract 3277 Poster Board III-1 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells. Disclosures: Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.