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// Shuang Liu 1 , Wenting Lu 1 , Shouyun Li 1 , Saisai Li 1 , Jia Liu 1 , Yuanyuan Xing 2 , Shuzu Zhang 2 , Joe Zhongxiang Zhou 2 , Haiyan Xing 1 , Yingxi Xu 1 , Qing Rao 1 , Chengjun Deng 2 , Min Wang 1 , Jianxiang Wang 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China 2 Fujian Jinler Pharmaceuticals, Jiangle County, Fujian 353300, China Correspondence to: Chengjun Deng, email: cjdeng@yahoo.com Min Wang, email: wangjxm@ihcams.ac.cn Jianxiang Wang, email: wangjx@ihcams.ac.cn Keywords: LSD1 inhibitor, leukemia, proliferation inhibition, apoptosis, autophagy Received: December 09, 2016 Accepted: March 15, 2017 Published: March 29, 2017 ABSTRACT Lysine-specific demethylase 1 (LSD1) has been recognized as a potential therapeutic target for acute myeloid leukemia (AML). Herein, we identified a novel LSD1 inhibitor, JL1037, via Computer Aided Drug Design technology. JL1037 is a potent, selective and reversible LSD1 inhibitor with IC50s of 0.1 μM and >1.5 μM for LSD1 and monoamine oxidases A/B (MAO-A/B), respectively. Treatment of THP-1 and Kasumi-1 cell lines with JL1037 resulted in dose dependent accumulation of H3K4me1 and H3K4me2, the major substrates of LSD1, as well as inhibition of cell proliferation, blockade of cell cycle and induction of apoptosis. Further investigations demonstrated that JL1037 could upregulate cell cycle-related proteins P21, P57, pro-apoptotic protein Bax and downregulate anti-apoptosis proteins Bcl-2 and Bcl-XL. JL1037 appeared to activate autophage response in AML cell lines as well as primary cells from AML patients by increasing LC3-II expression and the formation of autophagosomes and autolysosomes in cytoplasm. Co-treatment with autophagy inhibitor chloroquine (CQ) enhanced JL1037-induced cell apoptosis. Moreover, daily intravenous administration of JL1037 tended to reduce tumor burden and prolong the survival of t(8;21) leukemia mice. In conclusion, JL1037 exhibited potent anti-leukemia effect and could be a potential therapeutic agent for AML treatment.

// Wenting Lu 1 , Tengteng Yu 1 , Shuang Liu 1 , Saisai Li 1 , Shouyun Li 1 , Jia Liu 1 , Yingxi Xu 1 , Haiyan Xing 1 , Zheng Tian 1 , Kejing Tang 1 , Qing Rao 1 , Jianxiang Wang 1 and Min Wang 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China Correspondence to: Jianxiang Wang, email: wangjx@ihcams.ac.cn Min Wang, email: wangjxm@ihcams.ac.cn Keywords: FHL2, iASPP, biological functions, leukemia Received: December 23, 2016 Accepted: March 09, 2017 Published: March 28, 2017 ABSTRACT iASPP is an inhibitory member of apoptosis-stimulating proteins of p53 (ASPP) family, which inhibits p53-dependent apoptosis. iASPP was highly expressed in acute leukemia, inhibited leukemia cells apoptosis and promoted leukemogenesis. In order to clarify its mechanism, a yeast two-hybrid screen was performed and FHL2 was identified for the first time as one of the binding proteins of iASPP. FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phase, and increase cell apoptosis. Western blot analysis showed that the level of p21 and p27 increased, CDK4, E2F1, Cyclin E and anti-apoptotic proteins Bcl-2 and Bcl-xL reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. These results indicated that FHL2 might be a novel potential target for acute myelocytic leukemia treatment.

The majority of acute promyelocytic leukemia (APL) cases are characterized by the PML-RARα fusion gene. Although the PML-RARα fusion gene can be detected in >98% of APL cases, RARα is also found to be fused with other partner genes, which are also related to all-trans retinoic acid (ATRA)-dependent transcriptional activity and cell differentiation. In this study, we identified a novel RARα fusion gene, TBLR1-RARα (GenBank KF589333), in a rare case of APL with a t(3;17)(q26;q21),t(7;17)(q11.2;q21) complex chromosomal rearrangement. To our knowledge, TBLR1-RARα is the 10th RARα chimeric gene that has been reported up to now. TBLR1-RARα contained the B-F domains of RARα and exhibited a distinct subcellular localization. It could form homodimers and also heterodimers with retinoid X receptor α. As a result, TBLR1-RARα exhibited diminished transcriptional activity by recruitment of more transcriptional corepressors compared with RARα. In the presence of pharmacologic doses of ATRA, TBLR1-RARα could be degraded, and its homodimerization was abrogated. Moreover, when treated with ATRA, TBLR1-RARα could mediate the dissociation and degradation of transcriptional corepressors, consequent transactivation of RARα target genes, and cell differentiation induction in a dose- and time-dependent manner.

A20, also known as tumor necrosis factor-α (TNFα)-induced protein 3 (TNFAIP3), has been identified as a key regulator of cell survival in many solid tumors. However, little is known about the protein expression level and function of A20 in acute lymphoblastic leukemia (ALL). In this study, we found that A20 is up-regulated in ALL patients and several cell lines. Knockdown of A20 in Jurkat, Nalm-6, and Reh cells resulted in reduced cell proliferation, which was associated with cell cycle arrest. Phospho-ERK (p-ERK) was also down-regulated, while p53 and p21 were up-regulated in A20 knockdown cells. In addition, A20 knockdown induced apoptosis in Jurkat and Reh cells and enhanced the sensitivity of these cell lines to chemotherapeutic drugs. These results indicate that A20 may stimulate cell proliferation by regulating cell cycle progression. A20 inhibited apoptosis in some types of ALL cells, thereby enhancing their resistance to chemotherapy. This effect was abolished through A20 silencing. These findings suggest that A20 may contribute to the pathogenesis of ALL and that it may be used as a new therapeutic target for ALL treatment.

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL. Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice. Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn't survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions. Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn't benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What' more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. Disclosures No relevant conflicts of interest to declare.

Introduction iASPP played an important role in leukemogenesis in our previous study. In order to clarify its mechanism, a yeast two-hybrid screen was performed to find the binding partner of iASPP. In this study, we reported FHL2 as a novel binding partner of iASPP. The biological functions of the communication between FHL2 and iASPP were detected, and its possible mechanisms were investigated in human leukemia cell lines. Methods A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP. Immunofluorescence, Co-IP and Western blot analysis were used to confirm the communication between FHL2 and iASPP. After FHL2 or iASPP was knocked down in K562 and Kasumi-1 cells by lentiviral, MTT assay and flow cytometry were performed to detect the proliferation, cell cycle distribution and apoptosis rate of leukemic cells, meanwhile Western blot analysis was used to analyze the level of cell cycle- and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when iASPP and FHL2 were overexpressed or FHL2 was knocked down. Results FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phases, and increase cell apoptosis. Western blot analysis showed that the level of p21 increased and anti-apoptotic protein Bcl-2 was reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax, furthermore, when FHL2 was knocked down at the same time, the transcriptional activity of p53 was rescued. Conclusions The interaction between FHL2 and iASPP in AML was observed for the first time. Cell proliferation reducing, cell cycle arresting at G0/G1 phases, and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown. Moreover, iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for AML treatment. Disclosures No relevant conflicts of interest to declare.