bluuebunny/crossref_metadata_2025_split huggingface dataset:
Germline Genetic Variations in Methotrexate Candidate Genes Are Associated with Pharmacokinetics and Outcome in Pediatric Acute Lymphoblastic Leukemia in China
Authors: Liu Shuguang , Gao Chao , Zhang Ruidong , Zhao Xiaoxi , Cui Lei , Li Weijing , Zheng Huyong , Li Zhigang 
Published: October 2019

Abstract
BackgroundMethotrexate (MTX) is a key chemotherapeutic agent in the treatment of pediatric acute lymphoblastic leukemia (ALL). Nevertheless, MTX can cause severe adverse effects and toxicities. The aim of the present study was to identify genetic polymorphisms in candidate genes of the MTX pathway associated with MTX pharmacokinetics, toxicity, and outcome in ALL in China.
MethodsThree hundred and twenty-two Chinese children with ALL in the standard-risk and medium-risk treatment branches from the Beijing Children's Hospital-2003 and Chinese Childhood Leukemia Group-2008 protocols were enrolled in this study. Sequenom MassARRAY was used to genotype 12 single nucleotide polymorphisms (SNPs) in 4 candidate genes of the MTX/folate pathway. A total of 1268 high-dose MTX (HD-MTX) courses were analyzed. The plasma MTX levels were evaluated at 48 h after the first dose of HD-MTX infusion. Oral mucositis during the consolidation therapy period was recorded.
Results No polymorphism was associated with clinical features, prednisone response, and minimal residual disease (MRD) at day 33 and 78. Long-term outcome was better in SLCO1B1 rs4149056 T and TC allele carriers than patients with C allele (5-year RFS 92.3±1.6% vs. 27.8±23.2%,P<0.0001), in ABCB1 rs1128503 T and TC allele carriers than patients with C allele (92.7±1.6% vs. 78.2±6.9%, P=0.020), and in SCL19A1 rs2838958 AG and G allele carries than patients with A allele (93.9±1.6% vs. 83.0±4.2%, P =0.010). Multiple Cox regression analyses revealed an association of MRD at day 33 (hazard ratio 3.356; P=0.018), MRD at day 78 (hazard ratio 2.843; P=0.034), and SLCO1B1 rs4149056 (hazard ratio 8.073; P=0.002) with RFS in the study population.
As to MTX pharmacokinetics, ABCB1 rs1128503 showed a significant association with serum MTX levels (P=0.004). SNPs (rs3788200, rs1131596, rs1051266) of the SLC19A1 gene were also associated with the plasma levels of MTX (P=0.003, 0.004, and 0.003, respectively). No association was found between oral mucositis with any polymorphism.
Conclusions Genetic variations substantially influence the kinetics and response to HD-MTX therapy in childhood ALL.

Disclosures
No relevant conflicts of interest to declare.
Inhibition of c-Myc By Apto-253 As an Innovative Therapeutic Approach to Induce Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukemia
Authors: Zhang Hongying , Local Andrea , Benbatoul Khalid , Folger Peter , Sheng Susan , Esquivies Luis , Lightfoot Jeff , Vellanki Avanish , Rice William G. 
Published: October 2019

Abstract
The c-Myc multifunctional transcription factor protein, a product on the c-myc proto-oncogene, contributes to the pathogenesis of many types of human cancers through mechanisms of proliferation, apoptosis, cell cycle progression and cellular senescence. c-Myc is frequently overexpressed in acute myeloid leukemia, yet strategies to effectively modulate c-Myc function do not exist. We evaluated inhibition of c-myc gene expression by APTO-253, a small molecule anticancer agent that is being developed clinically for the treatment of acute myelogenous (myeloid) leukemia (AML) and high risk myelodysplastic syndromes (MDS). We first confirmed that c-Myc mRNA level were significantly higher in AML cell lines as compared to peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. However, the c-Myc expression in AML cells was inhibited by APTO-253 in dose-dependent and time-dependent manners at both the mRNA and protein levels. Likewise, APTO-253 was found to induce AML cell apoptosis in dose-dependent and time-dependent manners as demonstrated by positive Annexin-V staining and increases in cleaved poly (ADP-ribose) polymerase (c-PARP). APTO-253 induced AML cells arrest at G1/G0 phase of cell cycle by increasing p21 expression and decreasing expression of cyclin D3 and cyclin-dependent kinases 4/6 (CDK4/6). For the p53 positive cell lines MV4-11 and EOL-1, p53 was also increased by APTO-253 at early time points (less than 6-hour treatment), suggesting that p53-dependent cell cycle arrest and apoptosis is mechanistically operative as a consequence of treatment with APTO-253. Importantly, we demonstrated that APTO-253 selectively targeted tumor cells but not normal healthy cells, with MV4-11 AML cells and normal PBMCs having IC50s of 0.25±0.03µM and more than 100µM, respectively. Our previous studies (56th ASH abstract #4813) showed that APTO-253 induces the Krüppel-like Factor 4 (KLF4) transcription factor and was effective and well tolerated as a single agent in multiple AML xenograft models without causing bone marrow suppression. Taken together, our results suggested that APTO-253 may serve as an effective and safe agent for AML chemotherapy, and that APTO-253 mechanistically inhibits c-Myc expression in AML cells and subsequently induces cell cycle arrest and apoptosis.

Disclosures
No relevant conflicts of interest to declare.
Functional Analysis of BTG1 and BTG2 Variants in Non-Hodgkin Lymphoma
Authors: Almasmoum Hibah , Russell Nigel H. 
Published: October 2019

Abstract
Non-Hodgkin's lymphoma (NHL) represents a highly heterogeneous group of lympho-proliferative disorders and accounts for more than half of the cases of haematological malignancies. These disorders are characterised by multiple steps of accumulating genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative genes B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in approximately 10-15% of NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation.
BTG1 and BTG2 are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the Caf1 catalytic subunit of the Ccr4-Not deadenylase complex. In addition, the BTG domain binds to the poly(A)-binding protein PABPC1. This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression.
It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, we used a combination of sequence analysis and molecular modelling to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) prediction tool was used to predict amino acidic residues potentially interfering with the protein function. In total we assessed 45 mutations in BTG1 and BTG2 derived from NHL samples, including diffuse large B-cell lymphoma, germinal centre B-cell-like, activated B-cell-like lymphoma, follicular lymphoma and Burkitt'slymphoma. Of the mutations analysed, 25 were predicted to interfere with the function of BTG1 and BTG2. We therefore analysed the ability of these protein variants to interact with known partners and functionally assessed the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation. Using a yeast two-hybrid system, fourteen mutations were shown to affect the interaction of BTG1 or BTG2 with the Caf1catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when we transfected into mammalian cells, these BTG1 and BTG2 variants, unlike the wild-type proteins, were not able to inhibit cell cycle progression. Interestingly, our findings show that several mutations (M11I, F25C, P58L, I115V) on BTG1 do not require interaction with the Caf1 deadenylase enzyme to reduce reporter activity as established using 3' UTR tethering assays. This suggests that BTG1 and BTG2 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not-independent mechanisms.
The data show that mutations in BTG1 and BTG2, commonly found in NHL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow.

Disclosures
Almasmoum: Umm Al-Qura University: Employment, Research Funding.
CRLF2 Overexpression Demonstrates Enhanced Leukemogenicity in Down Syndrome Hematopoietic Cells
Authors: Junco Jacob J. , Liang Shan , Gokani Vishal , Gant Vincent Ulysses , Munoz Jaime , Mayle Allison , Park Chun Shik , Lacorazza H. Daniel , Goodell Margaret A , Rabin Karen R. 
Published: October 2019

Abstract
Introduction: Children with Down syndrome (DS) are 10-20 times more likely than children without DS to develop acute lymphoblastic leukemia (ALL), and they demonstrate a distinctive spectrum of genetic alterations. Approximately 50% of DS-ALL cases demonstrate CRLF2 rearrangements (CRLF2-R), an approximately 10-fold higher frequency than in non-DS ALL. We sought to identify the functional basis for the increased incidence of ALL, and specifically CRLF2-R ALL, in children with DS.
Methods: We created retroviral vectors which induce overexpression of CRLF2 and green fluorescent protein (GFP) for transduction into bone marrow (BM) cells isolated from the Dp16(1)Yey (Dp16) mouse model of DS, which is trisomic for the approximately 115 human chromosome 21 gene orthologs present on mouse chromosome 16. Transduced BM cells from Dp16 and wild-type (WT) control mice were co-cultured with OP9 stromal cells for one week to promote B-lymphoid lineage development, and then characterized by flow cytometric Hardy fraction analysis, or grown in B-lymphoid-promoting methylcellulose medium for colony growth assays.
Results: We achieved efficient transduction (80-95%) of Dp16 and WT BM enriched for hematopoietic stem cells (HSCs) with CRLF2-GFP+ and control GFP+ viruses. Following OP9 co-culture, transduced HSCs were characterized by Hardy fraction analysis. CRLF2-GFP+ Dp16 lymphoid cells demonstrated significantly higher percentages of immature Fraction A (pre-pro-B) cells compared with GFP+ Dp16 cells (39.9% vs 15.7%, p=0.004, Fig. 1A). This CRLF2-GFP-induced immature immunophenotype was more pronounced in Dp16 versus WT HSCs, with a significantly higher percentage of Fraction A cells (39.9% in Dp16 vs 24.0% in WT, p=0.0002) and a significantly lower percentage of more mature Fraction B (pro-B) cells (24.3% in Dp16 vs 49.1% in WT, p=0.02, Fig. 1A,B). In methylcellulose colony assays, CRLF2-GFP+ Dp16 cells yielded a 36-fold increase in B cell colonies compared to GFP+ Dp16 cells (Fig 1C). Again, the effect of CRLF2 transduction was magnified in the Dp16 versus WT background. CRLF2-GFP+ WT cells demonstrated only a 2.9-fold increase in B cell colonies (Fig 1C).
Conclusions: Here we demonstrate that CRLF2 overexpression results in a more immature B-lineage immunophenotype and increased lymphoid colony growth in vitro, and that these effects are significantly greater in a murine DS versus WT genetic background. Experiments to investigate the pathways involved and to evaluate these effects in vivo are ongoing. This work provides functional evidence of the enhanced leukemogenicity of CRLF2 overexpression in DS-ALL, and creates a tractable model system for additional future genetic studies.

Disclosures
No relevant conflicts of interest to declare.
Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia
Authors: Goyama Susumu , Shrestha Mahesh , Schibler Janet , Rosenfeldt Leah , Miller Whitney , O'Brien Eric , Mizukawa Benjamin , Kitamura Toshio , Palumbo Joseph S. , Mulloy James C. 
Published: October 2019

Abstract
Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Disclosures
No relevant conflicts of interest to declare.
The Role of Notch in Vascular Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy
Authors: Le Quy , Meshinchi Soheil , Hadland Brandon Kenneth , Walter Roland B. , Bernstein Irwin D. 
Published: October 2019

Abstract
Despite advances in the treatment of acute myeloid leukemia (AML), disease relapse following an initial period of remission remains a significant risk, suggesting the existence of resistant leukemic precursors. Recent studies indicate resistance may be imparted to the leukemic precursors by the hematopoietic niche within the bone marrow. Previously, we have shown that a key component of the niche, endothelial cells (EC), can support long-term growth of AML precursors in culture. Here, we show that Notch is a novel target to disrupt the protective AML-EC interactions and enhance treatment effectiveness.
In preliminary studies of AML with FLT3 mutations, we demonstrated that leukemic precursors from primary human AML patient samples are protected from treatment with a small molecule FLT3 inhibitor, AC220, when cultured in the presence of EC. We assayed cell viability of AML cells following 3 days of treatment with AC220 (100nM) or control (DMSO) in EC co-culture or liquid culture, and found that cells survived AC220 treatment better in EC co-culture compared to liquid culture (average percent cell viability from 3 primary FLT3-ITD+ AML patient samples relative to DMSO control: liquid culture (3 ± 3%) vs. EC co-culture (35 ± 19%), p=0.04). To identify genes involved in conferring protection to AML cells, we performed genome-wide transcriptional analysis of AML cells following 2 days of treatment with AC220 or DMSO in EC co-culture. EdgeR was used to assess differences in gene enrichment across cell populations. We found 1171 and 555 genes were increased and decreased, respectively, in AC220-treated population compared to DMSO control-treated population AML based on a 2-fold change and FDR<0.01. Amongst the differentially expressed genes, we found an enrichment of genes involved in Notch signaling pathway, including HES1, CDKN1A, and CCND1, in AML cells that survived treatment with AC220 compared to control. To evaluate a role for Notch signaling in EC-mediated growth of AML cells, we incubated AML cells in EC co-culture with inhibitory antibodies against Notch 1 and 2 receptors or control antibody for 2 weeks, and assayed the cells for colony-forming cell (CFC) activity. We found that inhibition of Notch signaling led to an increase in CFC formation. In contrast, when we combined AC220 with Notch 1 and 2 blocking antibodies in AML/EC co-culture, we found that inhibition of Notch signaling significantly reduced the number of total CFC and FLT3-ITD+ CFC compared to AC220 treatment combined with control antibody (p<0.005). These results suggest a role for Notch in EC-mediated protection of resistant AML precursors and thus offer a potential therapeutic strategy for sensitizing resistant precursors to targeted therapies.

Disclosures
No relevant conflicts of interest to declare.
Oncogenic ZEB2 Activation Drives Sensitivity Towards LSD1 Inhibition in T-Cell Acute Lymphoblastic Leukemia
Authors: Goossens Steven , Peirs Sofie , Berx Geert , Van Vlierberghe Pieter , Haigh Jody J. 
Published: October 2019

Abstract
T-cell acute lymphoblastic leukemias (T-ALLs) are rare aggressive hematologic tumors resulting from the malignant transformation of T-cell progenitors. The prognosis of T-ALL has gradually improved with the introduction of intensified chemotherapy. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains extremely poor. Therefore, current research efforts are focused on the development of more effective and less toxic anti-leukemic drugs, which will likely require an improved understanding of the molecular biology of chemotherapy resistant residual tumor cells that eventually drive disease recurrence.
Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of an immature subtype of T-ALL with increased leukemia-initiating properties. ZEB2 is a large multidomain homeobox transcription factor that recognizes bipartite E-box motifs through its amino- and carboxyterminal Zinc finger domains. The domains outside the Zn-finger clusters have been shown to be essential for the recruitment of various tissue-specific co-activators/repressors. Identification and targeting of these interaction partners that are essential for the oncogenic properties of ZEB2 could be a feasible option for the development of novel therapeutics to treat this aggressive leukemia.
In this study, we performed ZEB2 protein pull down experiments and subsequent mass spectrometry to explore the ZEB2 interactome in the context of T-ALL. We identified the Lysine-specific demethylase KDM1A/LSD1 as a novel interaction partner of ZEB2. The ZEB2-LSD1 interaction was confirmed via bi-directional co-immunoprecipitation experiments in both mouse and human T-ALL cell lines. KDM1A/LSD1 is a flavin-containing amino oxidase that specifically catalyzes the demethylation of mono- and di-methylated lysines on histone 3. LSD1 has been reported to regulate the balance between self-renewal and differentiation of pluripotent stem cells and its expression is upregulated in various cancers. Pharmacological inhibition of LSD1 has been proposed as a novel promising therapy to treat and kill cancer stem cells and novel potent inhibitors are being tested in clinical trials. We demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on LSD1 activity for their leukemic survival. In addition, doxycycline induced ZEB2 overexpression significantly increased sensitivity to LSD1 inhibition in the MLL-AFX positive T-ALL cell line KARPAS-45.
We conclude that targeting the ZEB2 protein complex through direct disruption of the ZEB2-LSD1 interaction or pharmacological inhibition of the LSD1 demethylase activity itself, could serve as a novel therapeutic strategy for this aggressive subtype of T-ALL, and possibly other ZEB2-driven malignancies.

Disclosures
No relevant conflicts of interest to declare.
Role of Daunorubicin Dose Intensification for Induction Therapy in Acute Myeloid Leukemia Patients with FLT3-ITD Mutants
Authors: Choi Eun-Ji , Lee Je-Hwan , Lee Jung-Hee , Park Han-Seung , Ko Sun-Hye , Seol Miee , Woo Ji Min , Lee Kyoo-Hyung 
Published: October 2019

Abstract
Background
Patients with FLT3-ITD mutated acute myeloid leukemia (AML) have generally poor survival. Recent update of ECOG trial comparing standard- vs. high-dose daunorubicin showed that daunorubicin dose intensification improved survival in AML with FLT3-ITD mutants (Blood 2016;127:1551). In subgroup analysis of our previous randomized trial, high-dose daunorubicin seemed to be more effective than idarubicin in AML patients with FLT3-ITD mutants (ASH abstract No. 2535, 2015). In this retrospective investigation, we aimed to evaluate the role of daunorubicin dose intensification for induction therapy in AML patients with FLT3-ITD mutants who were treated at a single institute.
Methods
We analyzed data from 120 patients of newly diagnosed FLT3-ITD mutated AML patients who received induction chemotherapy between January 2002 and March 2016. The regimens consisted of high-dose daunorubicin (HD-DN, 90 mg/m2/d x 3d, n=39), standard-dose daunorubicin (SD-DN, 45 mg/m2/d x 3d, n=48), or idarubicin (IDA, 12 mg/m2/d x 3d, n=33) in combination with cytarabine (200 mg/m2/d x 7d). Patients with acute promyelocytic leukemia were not included.
Results
After the first round of induction chemotherapy, 53 patients had persistent leukemia; 50 received the second round of induction chemotherapy consisting of daunorubicin (45 mg/m2/d x 2d) or idarubicin (8 mg/ m2/d x 2d) in addition to cytarabine (200 mg/m2/d x 5d) and 3 received other regimens. A total of 81 patients achieved CR, and the CR rates were 76.9%, 58.3%, and 69.7% in HD-DN, SD-DN, and IDA, respectively (P=0.175). The 4-year cumulative incidence of relapse (CIR) of these 81 patients was 48.8%. With the median follow-up duration of survivors of 59.9 months (range, 4.6-170.7), 4-year overall survival (OS) and event-free survival (EFS) were 57.1%/27.7%/35.7% (P=0.025) and 45.2%/23.9%/36.0% (P=0.042) in HD-DN, SD-DN, and IDA, respectively. HD-DN showed statistically higher OS (hazard ration [HR], 0.424; P=0.005) and EFS (HR, 0.497; P=0.01), and lower CIR (P=0.036) than SD-DN, while OS and EFS differences between HD-DN and IDA were not statistically significant.
Conclusion
Daunorubicin dose intensification for induction therapy seemed to be effective in AML patients with FLT3-ITD mutants. Further studies are needed to investigate whether HD-DN is superior to IDA in this population. Considering high relapse rate, combination strategies of daunorubicin dose intensification and targeted agents such as FLT3 inhibitors should be developed.

Disclosures
No relevant conflicts of interest to declare.