bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of Pleiotrophin in LepR+ Perivascular Cells Depletes Hematopoietic Stem Cells
Authors: Himburg Heather A , Chang Vivian Y. , Sasine Joshua , Kan Jenny , Zhao Liman , Chute John P.
Published: October 2019

Pleiotrophin (PTN) is a heparin binding growth factor which is expressed by bone marrow vascular endothelial cells (BM ECs) and perivascular stromal cells. Treatment of murine or human HSCs ex vivo promotes HSC expansion (Nat Med. 2010 Apr;16(4):475-82) and constitutive deletion of PTN depletes LT-HSCs in steady state and markedly impairs HSC regeneration following myeloablation (Cell Rep. 2012 Oct 25;2(4):964-75; JCI. 2014;18(7):1123-1129). Here, we sought to determine which BM microenvironment cell is responsible for PTN-mediated maintenance of the HSC pool. Utilizing the Cre-loxP system, we deleted PTN from VE-cadherin+ ECs, leptin receptor+ (lepR+) perivascular stromal cells, osteocalcin+ osteoblasts, and vav1+ hematopoietic cells and examined the effects on hematopoiesis. We observed no differences in steady state hematopoiesis or HSC content as measured by long-term competitive repopulation assays in mice lacking PTN expression in osteocalcin+ cells, vav1+ hematopoietic cells or VE-cadherin+ BM ECs. However, deletion of PTN from lepR+ BM perivascular cells caused a significant decrease in BM c-kit+sca-1+lin- cells (KSL cells) and BM SLAM+KSL HSCs, and colony forming cell (CFC) content compared to PTN+/+ controls (*p = 0.04, 0.04, and 0.001, respectively). Importantly, deletion of PTN in lepR+ cells, caused a significant reduction in long-term HSC content as measured by primary and secondary competitive repopulation assays (*P<0.01 for all time points through 20 weeks). These data suggest that LepR+ BM perivascular cells, rather than VE-cadherin+ ECs are the primary source of PTN in the BM niche which contributes to the maintenance of the HSC pool.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Chronic Pancytopenia and Increased Bone Density Due to TBXAS1 Deficiency
Authors: Sharma Richa , Schwartz Jennifer E. , Nalepa Grzegorz
Published: October 2019

Non-dysmorphic 32-year old Caucasian female developed malaise, fluctuating body aches and progressive transfusion-dependent pancytopenia with peripheral blood morphology consistent with myelodysplastic syndrome (MDS). Reportedly, she had an episode of "low blood counts" of unclear etiology 23 years prior. Multiple attempts at bone marrow biopsy were unsuccessful due to technical difficulties with penetrating the bone, necessitating surgical biopsy by an orthopedic surgeon. Bone densitometry revealed increased bone density with Z-score of 4.5, consistent with mild thickening of long bone diaphysis noted via x-ray. Strikingly, the bone marrow biopsy revealed haphazard ossification, immature cartilage formation and thickened trabeculae replacing hematopoiesis within the marrow cavity. Next-generation sequencing revealed two novel biallelic mutations within the thromboxane synthase gene TBXAS1 (c.266T>C; c.989T>C). Both mutations affected conserved amino acid residues and were predicted to be disruptive to the protein function by two independent bioinformatics algorithms (Provean and PolyPhen-2).

Thromboxane cascade regulates pathways of inflammation, coagulation and osteoclast activation, suggesting a mechanistic explanation of how thromboxane synthase malfunction causes abnormal bone remodeling and bone marrow failure. TBXAS1 disruption had been described in a rare autosomal recessive syndrome Ghosal hematodiaphyseal dysplasia (GHDD) associated with increased bone density and bone marrow failure. GHDD due to TBXAS1 mutations was genetically confirmed only in four families of Northern African and Middle Eastern descent (Nat Genet 2008; 40: 284-286). To our knowledge, this is the first genetically validated case of TBXAS1-/- Ghosal syndrome in the Caucasian population.

Consistent with the well-established role of thromboxane in platelet function, our patient's platelets displayed impaired aggregation in response to arachidonic acid and structural abnormalities by transmission electron microscopy, revealing a functional platelet defect. Steroid therapy rendered the patient transfusion-independent and alleviated chronic pain and inflammation. A long-term follow-up will be needed to determine whether steroid treatment will result in stable improvement in hematopoiesis and decreased bone density, similar to other GHDD patients. This case illustrates how establishing the diagnosis of an underlying rare inherited bone marrow failure syndrome allows personalized, evidence-based treatment with lower risk of morbidity compared to cytotoxic chemotherapy and hematopoietic stem cell transplantation.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
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

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.
Specific Inhibition of AKT with ARQ 092, an Orally-Available Selective AKT Inhibitor, Attenuates Acute Vaso-Occlusive Events in Sickle Cell Disease
Authors: Li Jing , Kim Kyungho , Tseng Alan , Barazia Andrew , Youn Seock-Won , Abbadessa Giovanni , Yu Yi , Schwartz Brian , Andrews Robert K , Gordeuk Victor R. , Cho Jaehyung
Published: October 2019

Previous studies have identified the Ser/Thr protein kinase, AKT, as a therapeutic target for thromboinflammatory diseases. Recently, we have shown that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014; Barazia et al. Blood 2015). Although our results suggest that AKT2 inhibition may be a novel therapy to treat a vaso-occlusive crisis in SCD patients, no AKT2 specific inhibitor is available in clinic. Using ARQ 092, an orally-available, allosteric AKT inhibitor that is currently in Phase I clinical trials in oncology indications and AKT-driven overgrowth disease, Proteus syndrome, here we report that specific inhibition of AKT attenuates the adhesive function of neutrophils and platelets from sickle cell disease (SCD) patients in vitro and heterotypic cell-cell interactions in the Berkeley mouse model of SCD in vivo. Studies using neutrophils and platelets isolated from SCD patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Consistently, treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Using in vivo real-time fluorescence intravital microscopy, we demonstrated that short-term oral administration of ARQ 092 (100 mg/kg body weight) or hydroxyurea (HU, 250 mg/kg body weight), a main therapy for the treatment of SCD, diminishes neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley mice challenged with TNF-α to induce a vaso-occlusive crisis. Co-administration of HU and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in the cremaster vessel wall, and improved survival in these mice. Ex vivo studies in Berkeley mice suggested that co-administration of HU and ARQ 092 efficiently blocks neutrophil and platelet activation and reactive oxygen species generation. The synergistic effects of both inhibitors resulted from increased NO production by HU and specific inhibition of AKT by ARQ 092. Our results provide important evidence that ARQ 092 has potential as a novel drug to be developed to prevent or treat the acute vaso-occlusive complications in SCD patients.

Disclosures

Abbadessa: ArQule, Inc: Employment. Yu:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment.
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

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

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

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

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

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

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

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.
Specific Immune Responses for Leukemia-Associated Antigens Against Myeloid Leukemic Cells Are Increased By Immune Checkpoint Inhibition
Authors: Greiner Jochen , Götz Marlies , Schneider Vanessa , Schrezenmeier Hubert , Wiesneth Markus , Bullinger Lars , Döhner Hartmut , Hofmann Susanne
Published: October 2019

Immunotherapy in cancer treatment has gained importance in the last few years. The efficacy of immunotherapeutic approaches such as immune-checkpoint inhibitors, chimeric antigen receptor T cells (CARs) or bi-specific T cell activating antibodies becomes more and more obvious. However, mechanisms of these immune responses and responsible antigen structures have to be further elucidated. Leukemia-associated antigens (LAA) represent immunogenic antigens that are candidates for specific immunotherapy since they are target structures relevant for elimination of malignant cells by cytotoxic T cells (CTL).

In this work, we investigated the influence of PD-1 antibody Nivolumab and CTLA-4 antibody Ipilimumab on the antigen-specific immune responses by specific T cells against leukemic myeloid blasts in functional T cell assays using ELISpot Assays, tetramer-analysis and colony-forming immunoassays. We investigated T cell responses stimulated against known LAAs like RHAMM, PRAME, WT-1, SSXIP2, Proteinase 3, Survivin, Aurorakinase A and NPM1 against several AML cell lines and samples of AML patients. Expression of different LAAs were measured and correlated to functional T cell assays.

Colony forming unit immunoassays displayed a significant inhibition of CFU (colony forming units) in AML patient samples when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. The LAAs PRAME, RHAMM and WT1 showed highest frequency and intensity of immunogenic reactions: PRAME stimulated CTL induced an immune response in 83% of tested samples in CFU, stimulated with WT1 in 75% and with RHAMM in 58% of colonies from AML samples. Specific immune responses of cytotoxic T cells were additionally detected by ELISpot assays and correlated to results detected in CFU. Immune effects increased adding nivolumab to the CTL for several days before starting CFU immunoassays whereas no effect was measured when CTL were incubated with Ipilimumab. The combination of Nivolumab and Ipilimumab showed no additional effect of immune responses compared to Nivolumab alone.

Taken together, the immune checkpoint inhibitor Nivolumab increases specific T cell responses of LAA-stimulated cytotoxic T cells and the cytotoxic effect of T cells against blasts of AML patients. No additional effect was detected with Ipilimumab. These data suggest that PD-1 antibodies could be an immunotherapeutic approach in AML and combination with LAA-directed vaccination strategies might open interesting application possibilities.

Disclosures

Greiner: BMS: Research Funding.
Bone Marrow Macrophage Galectin-3 Regulates Platelet Production through Recognition of O-Glycans on Megakaryocytes
Authors: Lee-Sundlov Melissa M , Grozovsky Renata , Giannini Silvia , McGrath Martina , Ramsey Haley E , Sola-Visner Martha , Hoffmeister Karin M.
Published: October 2019

Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies.

The TF antigen is a disaccharide presented in cryptic form on O-glycans and covered by a sialic acid moiety. The sialyltransferase ST3Gal1 transfers sialic acid onto the TF antigen. To investigate the role of O-glycans in thrombopoiesis, we generated mice with increased TF antigen in MKs by generating St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the MK lineage. As expected, St3gal1loxP/PF4+ circulating platelets and BM MKs had increased TF antigen expression, compared to controls, as evidenced by peanut agglutinin (PNA) binding. Other blood cell lineages had no increase in TF antigen expression. St3gal1loxP/PF4+ mice developed mild thrombocytopenia, but surprisingly had virtually normal platelet clearance. BM MK colony forming units and in vitro proplatelet production were normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+BM environment affected platelet production. St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, indicative of an increase in phagocytic macrophages. In vivo macrophage ablation by injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages, thereby normalizing blood platelet counts and size. Flow cytometric phenotypic analysis of BM-derived macrophages showed an increased population of activated macrophages in St3gal1loxP/PF4+ mice, compared to controls, specifically macrophages with increased galectin-3 expression, a ligand for the TF antigen. Immunofluorescence staining of BM sections using a specific antibody towards the TF antigen showed that MK progenitors and pro-platelet-like structures expressed TF antigen in control BMs, which is significantly increased in St3gal1loxP/PF4+ mice and co-localized with galectin-3 expressing macrophages, supporting the notion that MK O-glycans and macrophage galectin-3 play a role in thrombopoiesis under steady state and pathological conditions. Consistent with this notion, galectin-3 deficient mice have slightly, but significantly increased blood platelet counts. We conclude that galactin-3 plays a minor role in normal thrombopoiesis. Activation of galectin-3 expressing macrophages by the MK TF antigen leads to MK phagocytosis, inhibition of platelet formation and thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
The G-Protein Coupled Receptors and G-Proteins Alpha Expression in Mantle Cell Lymphoma
Authors: Wasik Agata M , Mundt Filip , Christensson Birger , Sander Birgitta
Published: October 2019

Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin lymphoma comprising approximately 8% of malignant lymphomas. MCL is considered to be an incurable disease even though survival has improved with novel biological therapies that interrupt survival signaling from the lymphoma microenvironment. The signaling pathways that are deregulated and the factors leading to systemic dissemination of the disease are still poorly understood. There is also a clear need to identify additional druggable targets for therapy (Saba and Wiestner, Curr Opin Hematol, 2014). Druggable targets may be G-protein coupled receptors (GPCRs) and G-proteins. GPCRs belong to the family of seven transmembrane receptors, which upon activation by extracellular ligands transfer the signals inside the cell for activation of the specific pathway. The signaling involves G-proteins. G-proteins consist of the subunits: alpha, beta and gamma and their subtypes. During GPCR activation the G alpha subunit is released and the subtype of G-protein subunit determines which downstream signaling pathways (including Akt, PI3K, ERK, JNK, MAPK, NFkappaB and others) is then activated. In the immune cells different subtypes of G-proteins alpha are instrumental for leukocyte migration, architecture of B cells compartments in spleen, thymus, lymph nodes, and gastrointestinal tract (Hwang et al., PlosOne, 2013) and BCR-mediated cell death (Misra et al., J Exp Med, 2010). G alpha13 signaling is involved in development of germinal centre-derived lymphomas (Muppidi et al., Nature, 2014).

In order to map the aberrant expression of GPCR and G-proteins in MCL, the expression of genes encoding for GPCRs and G-protein alpha subunits was analyzed by Affymetrix U133A Array of seventeen MCL biopsies and eight non-malignant lymph nodes. Among investigated GPCR and G-protein alpha subunit genes 25/298 were differently expressed in MCL compared to reactive lymph nodes (moderated t-test, adj. p=or< than 0.05). The four most significantly different genes expressed were: LPAR6, GPRC5C, OPN3 and GNAZ (all adj.p<0.003). The latter, GNAZ, encodes for G-protein alpha z. The analysis of GNAZ by qPCR in 77 additional MCL biopsies from a population-based Stockholm MCL cohort showed varied expression of GNAZ. Interestingly, the MCL cases, which expressed the highest levels of GNAZ, had significantly worse survival (Kaplan-Meier, log rank, p<0.003) suggesting that GNAZ has, a yet unknown, role in MCL biology. Further analysis showed that GNAZ expression was correlated to lymphocytosis (Mann-Whitney test, p=0.023) and leukocytosis (Mann-Whitney test, p=0.011) in MCL patients but not to: high tumor cell proliferation, blastoid morphology nor to p53 immunoreactivity. Our results suggest that GNAZ-signaling is of importance for cell-cell interaction in the tumor microenvironment and/or for egress of lymphoma cells to the blood.

Disclosures

No relevant conflicts of interest to declare.
Expression and Function of Tigit in B-Cell Non-Hodgkin Lymphoma
Authors: Yang Zhi-Zhang
Published: October 2019

Background:T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and a novel member of CD28 family. TIGIT is expressed on NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the expression and function of TIGIT in the blood and tumor microenvironment of patients with B-cell non-Hodgkin lymphoma (NHL) is completely unknown.

Goal: To phenotypically and functionally characterize TIGIT+T cell subsets in B-cell NHL and compare expression of TIGIT on intratumoral T cells to normal controls.

Results: In peripheral blood, TIGIT expression was not detected on resting T cells. In contrast, when we analyzed biopsy specimens from B-cell NHL patients, we observed that TIGIT is variably but highly expressed on a subset of intratumoral T cells. A median of 41.1% (range: 24.9-56.1, n=5) of CD4+ or 22.2% (range: 13.4-33.3, n=5) of CD8+ T cells from tumor specimens expressed TIGIT on the cell surface. In normal tonsil tissue, expression level of TIGIT was modest or negligible. A median of 12.0% (range: 9.1-15.8, n=5) of CD4+ or 3.9% (range: 1.3-5.7, n=5) of CD8+ T cells express TIGIT on the cell surface, which is significantly lower than that in B-cell NHL (p=0.005 and p=0.003 for CD4+ and CD8+, respectively). We observed that both CD45RA+ and CD45RA- T cells express TIGIT with the greatest expression on the CD45RA- population in B-cell NHL specimens. Furthermore, we found that TIGIT+ T cells coexpressed other immune checkpoint molecules including PD-1 and TIM-3. Functionally, TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and IL-2-producing cells was lower in the TIGIT+ population than in TIGIT-T cells. Unlike TIM-3 that is coexpressed with PD-1 and whose expression is upregulated by IL-12, TIGIT expression is not upregulated by IL-12, suggesting that a different mechanism is involved in TIGIT upregulation in B-cell NHL.

Conclusion: Taken together, these results indicated that TIGIT is highly expressed on intratumoral T cells in B-cell NHL and is expressed on a population of T-cells with suppressed immune function. TIGIT, while co-expressed with PD-1 and TIM-3, is not upregulated by cytokines such as IL-12, suggesting that it is regulated by an alternate pathway. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell NHL.

Disclosures

No relevant conflicts of interest to declare.
Ocular MALT Lymphoma Free from the Chlamydia Psittaci Infection ; Analysis of 150 Cases
Authors: Park Gyeongsin , Lee Hye Won , Cho Uiju , Yang Suk Woo , Cho Seok-Goo , Lee Youn Soo , Kang Chang Suk
Published: October 2019

Background: Clamydophila psittaci (C. psittaci) has been proposed as an etiologic factor for extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in the ocular region. However, previous studies showed varied association rates ranging 0% to 87%, not constant even in the series of same geographical areas, which suggest that there could be some technical variance in detecting methods for C. psittaci. The authors validated five sets of primers for detecting C. psittaci DNA and investigated its association with ocular MALT lymphomagenesis.

Material and Methods: Five sets of PCR primers including 4 previously reported and one newly designed were evaluated with positive control C. psittaci DNA (acquired from Korea Centers for Disease Control and Prevention). One hundred fifty cases of confirmed ocular MALT lymphomas were collected from pathology archives of our institutes from 2008 to 2014, and entered into this study. DNA was extracted from archival paraffin block sections with Qiagen DNA extraction kits. Quality of DNA was assessed with NanoDrop and beta-globin PCR. Standard PCR was performed together with negative (H2O and tonsil DNA) and positive control C. psittaci DNA.

Results: In all five primer sets for C. psittaci DNA, each PCR product showed clear positive band and negative with appropriate positive and negative controls, respectively. All 150 ocular MALT lymphoma cases showed positive for Beta-globin DNA control, but negative for C. psittaci DNA in any of PCR product with five primer sets.

Conclusion: These results suggest that possibility of the pathogenetic role of C. psittaci in ocular MALT lymphoma would have been overestimated so far, at least in Korean people.

Disclosures

No relevant conflicts of interest to declare.
Outcome of Bone Marrow Biopsy in Patients with Diffuse Large B-Cell Lymphoma Patients Staged by Combined 18 FGD-PET/CT Scan
Authors: Hamed Rasha Abdel Tawab , Kotp Rami , Turecotte Eric , Sawan Baseem , Morine Annie
Published: October 2019

DLBCL represents 1/3 of non-Hodgkin lymphoma, in 60% of the cases the disease presented in advanced stage (III-IV). Extranodal organs are involved in 40% of cases; BM involvement in 11% to 27% of cases. BMB is an invasive procedure, & it also could represent false negative result in patients with patchy pattern of involvement or if involvement else where the routine biopsy site. Study included 102 patients, above the age of 18 years, confirmed to have newly diagnosed DLBCL with no any other malignancy. Every patient had a baseline PET-Ct, bone marrow biopsy. PET-CT detected BM infiltration in 23 patients, i.e 22.5%. BMB were positive in 20 patients, while PET-CT showed BM involvement in 23 patients.- 94 patients had concordant negative ( 75 patients ) or positive ( 19 patients), PET-CT & BMB results. One patient had positive BMB and negative PET-CT. All patients with stage I & II had concordant PET-CT & BMB results One patient graded stage III by PET-CT showed 1-2% BM infiltration & upstaged to stage IV. Of the 49 patients graded stage IV by PET-CT, 3 had positive BM involvement PET-CT and negative BMB. the sensitivity of PET-CT was 95%, the specificity of PET-CT was 96.2%. the PPV was 86.4% & the NPV was 98.7%. PET-CT showed 95.9% accuracy. Our results suggest PET-CT as a powerful tool to evaluate BM infiltration in patients with DLBCL, with overall concordance exceeding 94% (100%for stage I & II ). For patients with advanced stage IV disease, PET-CT was able to retrieve patients with BM involvement & false negative BMB.

Disclosures

No relevant conflicts of interest to declare.