bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal and Sequential Expression of EBF1 and PAX5 Restricts the Non-B Cell Fate In Early Lymphopoiesis
Authors: Zandi Sasan , Zetterblad Jenny , Tsapogas Panagiotis , Månsson Robert , Sigvardsson Mikael
Published: October 2019

Abstract 3867

The generation of B cells from multipotent hematopoietic stem cells involves activation of B cell specific and repression of alternative lineage programs. This process to a large extent is under the control of transcription factors such as EBF1, E2A, IKAROS and PAX5. Inactivation of PAX5 and EBF1, by point mutations or deletions can be found in 30% of the acute lymphocytic leukemia (ALL) cases, probably due to a disturbance of differentiation in early B cell development. Conditional deletion of PAX5 and EBF1 leads to a complete block in B cell production and subsequent accumulation of early immature B cell progenitors. Although the roles of these transcription factors have been established in B cell development, the presence of a feedback loop between PAX5 and EBF1 as well as lack of well defined early B cell developmental events and cell populations, lead to some discrepancies in the current literature regarding the role of these factors in restricting non-B cell lineages and the establishment of B cell fate. By using a combination of RAG1/EBF1 reporter mice and newly identified surface markers i.e Ly6D, we have shown the exact point of myeloid (M), natural killer (NK) and T cells restriction within conventional common lymphoid progenitors (CLP) population. The Lin-IL7r+flt3+kitloSca1lo(CLP)Ly6D-λ5- population retains the residue of myeloid potential together with dendritic (D), Nk, T and B potentials. Upon expression of Ly6D, these cells lose the residual myeloid, NK and D potentials, and eventually the expression of λ5, associated with loss of T cell potential, marks the B-cell committed cells. Based on this new model of lymphoid development we have revisited the functional roles of EBF1 and PAX5 in B-cell commitment. This work suggests that EBF1 restricts the myeloid, NK and D potentials in the transition from CLPLy6D- to CLPLy6D+ stage while the expression of PAX5 in the transition from CLPLy6D+λ5- to CLPLy6D+λ5+ restricts the T cell potential by possibly by counteracting Notch1 signaling in the bone marrow.

Considering the high prevalence of PAX5 and EBF1 mutations in ALL and diffuse large B cell lymphoma (DLBL) cases and the fact that inactivation of PAX5 and EBF1 leads to accumulation of B cell progenitors in mice models, understanding the temporal and spatial expression of EBF1 and PAX5 in regard to restriction of non-B cell fates in early stages of lymphoid development will allow us to identify the leukemia-initiating cells and the mechanisms of leukemogenesis in these diseases.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Profiling of Epigenetic and Transcription Factor Regulation In Human Macrophage Differentiation
Authors: Pham Thu-Hang , Lichtinger Monika , Benner Chris , Pape Sabine , Schwarzfischer Lucia , Klug Maja , Gogol-Doering Andreas , Hu Yuhui , Andreesen Reinhard , Chen Wei , Rehli Michael
Published: October 2019

Abstract 3875

The differentiation of human macrophages is accompanied by distinctive phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are still poorly understood. Using ChIP-Seq technology we studied epigenetic changes as well as alterations in transcription factor occupancy during human monocyte differentiation and correlated these events with gene expression levels in hematopoietic cell types. We show that putative enhancer regions marked by histone H3 lysine4 monomethylation (H3K4me1) at different developmental stages (human progenitor cells, peripheral blood monocytes and in vitro differentiated macrophages) are enriched in distinct sets of transcription factor motifs corresponding to lineage-determining factors. Cell stage-specific histone methylation at promoter-distal sites corresponds with increased mRNA expression levels of neighboring genes. We generated global DNA-binding maps in monocytes and macrophages for two transcription factors (PU.1 and C/EBPβ) with a well established role in monocyte/macrophage differentiation. Comparison of human binding sites with corresponding mouse data revealed a surprisingly low level of conservation (∼10-15%) of PU.1-or C/EBPβ -bound sites between man and mouse, despite a highly conserved binding preference for both transcription factors. During monocytic differentiation, human macrophages primarily gained additional binding sites for both transcription factors (as well as promoter-distal H3K4me1). Interestingly, only neighboring genes with multiple binding events showed significantly increased, macrophage-specific mRNA expression as compared to monocytic as well as lymphocytic cell types. Human macrophage-specific H3K4me1-marked regions as well as macrophage-specific PU.1- and C/EBP-bound sites were characterized by overlapping sets of novel sequence motifs, suggesting that the combinatorial interaction of corresponding DNA-binding factors with PU.1 and C/EBPβ may be required for the establishment of human macrophage-specific enhancers. These data provide novel insights into PU.1 and C/EBPβ mediated gene regulation during human macrophage differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Early Interim 18f-FDG PET In Hodgkin's Lymphoma: Evaluation on 304 Patients
Authors: Zinzani Pier Luigi , Rigacci Luigi , Stefoni Vittorio , Broccoli Alessandro , Puccini Benedetta , Gandolfi Letizia , Fanti Stefano , Castagnoli Antonio , Pellegrini Cinzia , Montini Gian Carlo , Quirini Federica , Antognoli Giulia , Vaggelli Luca , Derenzini Enrico , Argnani Lisa , Ambrosini Valentina , Agostinelli Claudio , Pileri Stefano A. , Bosi Alberto , Baccarani Michele
Published: October 2019

Abstract 3879

Purpose.

The use of early (interim) positron emission tomography (PET) restaging during front-line therapy in Hodgkin's lymphoma (HL) has considerably increased in clinical practice as an early recognition of treatment failure allows patients to be addressed to more intensive treatment regimens.

Patients and Methods.

Between June 1997 and June 2009, 304 newly-diagnosed Hodgkin's lymphoma patients (147 early-stage and 157 advanced-stage) were treated with the ABVD regimen at two Italian institutions. Patients underwent to a PET staging and restaging at baseline, after 2 cycles of therapy and at the end of the treatment.

Results.

53 patients showed a positive interim PET and only 13/53 (24.5%) achieved a complete response (CR), whereas 251 patients showed a negative PET and 231/251 (92%) remained in CR. Comparison between interim PET-positive and interim PET-negative patients indicated a significant association between PET findings and 9-year progression-free survival (p=0.0000) and 9-year overall survival (p=0.0000), with a median follow-up of 31 months.

Among the early-stage patients, 19 had a positive interim PET and only 4 (21%) achieved a CR; among the 128 negative interim PET patients, 122 (97.6%) obtained a CR. In the advanced-stage subset, 34 patients showed a persistently positive PET (with only 9/34, 26.4% in CR), whereas 123 showed a negative interim PET, with 109 (88.6%) remaining in CR.

Conclusions.

Our results confirm the role of early PET as a significant step forward for the management of both early and advanced-stage HL patients, offering the potential for an immediate switch to high-dose treatments, if required.

Disclosures:

No relevant conflicts of interest to declare.
Direct Sensing of Toll-Like Receptor Agonists by Dendritic Cell Progenitors Leads to Their Homing to Inflamed Lymph Nodes Via Regulation of CXCR4 and CCR7
Authors: Schmid Michael A , Baumjohann Dior , Manz Markus G
Published: October 2019

Abstract 3895

Dendritic cells (DCs), the key antigen-presenting cell population, continuously need to be regenerated from bone marrow (BM) hematopoietic stem and progenitor cells. Common dendritic progenitors (CDP) were previously shown to efficiently generate DCs in lymphoid and non-lymphoid tissues. How the dissemination of bone marrow (BM) DC-progenitors to peripheral tissues is regulated upon demand remains elusive to date. Acute microbial infections are sensed via Toll-like receptors (TLR). Recent studies showed that stem and progenitor cells express TLRs. We found that CDPs in the BM of mice express relative high levels of Tlr2, Tlr4 and Tlr9, and hypothesized that these might be involved in regulating CDP migration. CDPs in steady-state expressed high levels of Cxcr4, but no, or low Ccr7. Upon direct stimulation with the respective TLR-agonists in vitro, CDPs rapidly down-regulated Cxcr4 and up-regulated Ccr7 mRNA and protein. CDPs that were stimulated with TLR-agonists for only 2 h preferentially homed to the lymph nodes (LN) in expense of BM in steady-state recipients. When TLR-agonists were injected subcutaneously, CDPs gave rise to increased numbers of plasmacytoid DCs, classical DCs, and DCs with a skin-derived migratory phenotype in inflamed LNs on day 4. This was not due to increased proliferative activity. Injecting the CXCR4 antagonist AMD3100 demonstrated that the retention of CDPs in the BM depends on CXCR4. Furthermore, CCR7 was important for the engraftment of CDP-derived DCs into LNs in steady-state and during inflammation. In conclusion, DC progenitors in the bone marrow are capable to directly sense TLR-agonists via their cognate receptors in systemic infections. This results in differential expression of chemokine receptors and consecutive migration of DC-progenitors to inflamed LNs. This mechanism helps to restore DC subsets during ongoing immune responses and to return to DC homeostasis once the inflammation ceases.

Disclosures:

No relevant conflicts of interest to declare.
Lestaurtinib (CEP701) Inhibits Proliferation and Activates Apoptosis In Hodgkin Lymphoma Cells through Inhibition of the JAK/STAT Signaling Pathway
Authors: Navarro Alfons , Díaz Tania , Martinez Antonio , Gaya Anna , Monzó Mariano
Published: October 2019

Abstract 3919

Background:

The constitutive activation of the JAK/STAT pathway plays an important role in the pathogenesis and proliferation of Hodgkin Lymphoma (HL). Although somatic activating point mutations in the JAK2 gene have been reported in myeloproliferative disorders (MPD), they are rarely described in HL, where JAK2 amplification is associated with mutations of regulator genes such as SOCS-1, constitutive activation of STAT proteins or miRNA deregulation. Recently, many JAK2 inhibitors, including Lestaurtinib (CEP701), have been reported to have clinical efficacy in MPD. CEP701 is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 at nanomolar concentrations. Recent studies in MPD have further shown that CEP701 inhibitory activity is not limited to FLT3 and can suppress JAK2/STAT5 signaling through JAK2 inhibition. As a first step towards elucidating the potential role of CEP701 in HL therapy, we have analyzed its efficacy in vitro.

Methods:

Four HL cell lines, L-428, L-1236, HDMYZ and L-540, were assayed for proliferation, apoptosis and levels of proteins in the JAK2/STAT pathway (pJAK2, JAK2, pSTAT5, STAT5, Bcl-xL) after CEP701 treatment. 100,000 cells were plated in a 96-well plate in 100 ml culture medium with CEP701 or DMSO (vehicle control) at concentrations of 30–300 nM. After 1 or 24 hours of incubation with CEP701, the levels of the proteins and of FLT3 were analyzed by Western blot. Proliferation was analyzed with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) and apoptosis by CaspaseGlo 3/7 after 48 hours of treatment.

Results:

The proliferation analysis showed an effective dose-dependent inhibition of cell growth in the 4 HL cell lines after treatment with increasing concentrations of CEP701. At 48h, in comparison to cells treated with DMSO alone (normalized to 100%), in cells treated with 100nM of CEP701, we observed a marked inhibition of 35% in L-428, 55% in L-1236, 15% in HDMYZ and 77% in L-540. Moreover, apoptosis increased by 38%, 31%, 21% and 25%, respectively. The protein analysis showed that after one hour, CEP701 inhibited phosphorylation of JAK2 (pJAK2) and its downstream target STAT5 (pSTAT5) in a dose-dependent manner, with no changes in the non-phosphorylated proteins. The downstream target Bcl-xL also decreased.

Conclusions:

Taken together, these data demonstrate that growth inhibition and apoptosis activation by CEP701 in HL cells correlates with the inhibition of the JAK2/STAT5-dependent signal transduction pathway. Here we present the first biological evidence that Lestaurtinib could be a promising new agent in the treatment of patients with HL.

Supported by a FIS grant (PS09/00547).

Disclosures:

No relevant conflicts of interest to declare.
Mimetic Peptide to CDK Inhibitor p16 INK4a Induces Cell Death In Mantle Cell Lymphoma Cells: A New Strategy to Eradicate Minimal Residual Disease
Authors: Liu Hongtao , Koval Greg , Malnassy Greg , Ihonor Pamela , Liu Hui , Luo Roger , Wickrema Amittha , Dowdy Steven , Thirman Michael , van Besien Koen , Stock Wendy
Published: October 2019

Abstract 3921

Autologous stem cell transplantation (ASCT) is an effective treatment strategy for Mantle Cell Lymphoma (MCL) that has been shown to improve disease free survival. However, data from recent trials suggest that the presence of minimal residual disease (MRD) contributes to relapse following current intensive treatment strategies, including ASCT. Thus, we sought to test a highly selective approach to eradication of MRD in stem cell collections from MCL patients using targeted small peptides that disrupt the aberrant cyclin/CDKs interactions that are involved in the pathogenesis of MCL. In the current study, the efficacy of a mimetic peptide to the CDK inhibitor p16INK4a to eliminate MRD was tested. Using (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) cell viability assays, we demonstrated that a transducible TAT-p16 mimetic peptide was able to induce cell death in the MCL cell line, Jeko-1, in a dose and time-dependent fashion (see Figure). A TAT-p16 mimetic was able to induce cell death in approximately 60% of Jeko-1 cells after 4 hours of incubation, and approximately 80% of Jeko-1 cells at 24 hours. In contrast, the same dose of TAT-p16 mimetic had no effect on cell survival when it was incubated with CD34+ rich apheresis samples from healthy donors. Furthermore, CFU-GM, BFU-E colony assays showed that there was no toxicity of the TAT-p16 mimetic when compared to untreated cells or cells treated with a control, scrambled TAT-p16 peptide. TAT-p16 mimetic did not impair erythroblast differentiation but induced mild apoptotic cell death in the erythroblast cells. The strategy was further tested using a CD34+ enriched apheresis sample collected from a patient with MCL with documented MRD who had been treated on a clinical trial of ASCT for MCL. Using Real-time quantitative PCR for IgH and BCL1 gene rearrangements, we demonstrated that the TAT-p16 mimetic was able to reduce MRD level by 40% compared with the scrambled TAT-p16 peptide. These data suggest that the use of a peptide-mimetic to p16 selectively and effectively can reduce MRD in MCL cells and in an apheresis (stem cell) sample from a patient with MCL. Treatment with the peptide allowed differentiation of CD34+ progenitor cells. A combinational approach using additional targeted cell cycle regulatory peptides that block p21CIP1/WAF1 and cyclin D2 is being explored to optimize the efficacy of this purging technique. Thus, this novel strategy may be an effective, selective and non-toxic method for eradication of MRD in MCL.

Disclosures:

No relevant conflicts of interest to declare.
A Small Molecule PLK1 Inhibitor, MLN0905, Induces An Anti-Tumor Response In Human Models of Diffuse Large B-Cell Lymphoma
Authors: Shi Judy , Lasky Kerri , Shinde Vashali , Duffey Matt O , Stringer Bradley , Qian Mark , Liao Debra , Liu Ray , Rao Youlan , Vos Tricia J , D'Amore Natalie Roy , Hyer Marc L
Published: October 2019

Abstract 3922

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30 percent of newly diagnosed cases. Current treatment options for this disease are effective, but not always curative; therefore experimental therapies are being investigated. Diffuse large B-cell lymphoma cells typically over-express the serine/threonine mitotic kinase PLK1, which plays a key role in mitotic cell cycle progression, and has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL lines. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in mouse models harboring human DLBCL disease. MLN0905 has drug-like pharmacokinetic properties and an acceptable toxicity profile making it a good clinical candidate. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. The anti-tumor activity of MLN0905 was evaluated in three human subcutaneous xenograft models OCI-LY10, OCI-LY19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant anti-tumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The anti-tumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI-LY19) setting to better mimic DLBCL in humans. In this disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with Rituximab in the disseminated OCI-LY19 model. Combining Rituximab and MLN0905 provided both a synergistic anti-tumor effect and a synergistic survival advantage. Our findings indicate for the first time that PLK1 inhibition leads to pharmacodynamic pH3 modulation and significant anti-tumor activity in multiple models of DLBCL. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anti-cancer agents in the clinic.

Disclosures:

Shi: Millennium: The Takeda Oncology Company: Employment. Lasky:Millennium: The Takeda Oncology Company: Employment. Shinde:Millennium: The Takeda Oncology Company: Employment. Duffey:Millennium: The Takeda Oncology Company: Employment. Stringer:Millennium: The Takeda Oncology Company: Employment. Qian:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Liu:Millennium: The Takeda Oncology Company: Employment. Rao:Millennium: The Takeda Oncology Company: Employment. Vos:Millennium: The Takeda Oncology Company: Employment. D'Amore:Millennium: The Takeda Oncology Company: Employment. Hyer:Millennium: The Takeda Oncology Company: Employment.
Rituximab Plus Chlorambucil Compared with Chlorambucil and Prednisone In Patients with Untreated Follicular Lymphoma
Authors: Bassi Simona , Sammassimo Simona , Gigli Federica , Pruneri Giancarlo , Bertazzoni Paola , Quarna Jessica , Cocorocchio Emilia , Laszlo Daniele , Negri Mara , Lionetti Maria , Vanazzi Anna , Alietti Alessandra , Agazzi Alberto , Gardellini Angelo , Andreola Giovanna , Preda Lorenzo , Pastano Rocco , Martinelli Giovanni
Published: October 2019

Abstract 3956

Background:

Recently in large randomized studies, the addition of Rituximab to standard chemotherapy in the first-line treatment of follicular lymphoma (FL) demonstrated improvements in long-term outcome in FL.

Methods:

We compared the outcome of 111 naive FL patients (pts) treated at our institution from 1995 to 2009 with a single alkylating agent in combination or not with Rituximab: 58 pts received Chlorambucil plus Rituximab (R-Chl) and 53 pts Chlorambucil and prednisone (Chl+PDN). The 2 schedules included an induction phase, where Chlorambucil was given at 6mg/mq for 6 consecutive weeks in both groups and the Rituximab in 4 weekly administrations. The maintenance phase was longer in the Chl+PDN group: Chlorambucil was administered 6mg/mq daily 14 days each month for a total of 12-months of treatment versus 14 days with monthly-Rituximab for 4 consecutive months in the R-Chl group.

Results:

The demographic and prognostic factors are reported in Table 1.

At the end of treatment the ORR was 98% in the R-Chl pts and 77% in the Chl+PDN group, with a percentage of complete response of 79% and 55% respectively. No significant incidence of adverse events were reported and only one HBsAg positive patient in R-Chl group discontinued the therapy. One case of myelodysplastic syndrome was described in a Chl+PDN relapsed patient. With a median follow up of 34 months, the OS and the EFS in R-Chl pts is 95% and 76% respectively. Otherwise, with a median observation time of 82 months in the Chl+PDN group 73% are alive and only 28% of pts maintained the response. The median time to subsequent therapy (TTST) is 21 and 15 months respectively in the 2 groups.

Conclusions:

The addition of Rituximab to alkylating in first-line treatment in previously untreated FL pts improves significantly the EFS and prolongs the time to next antilymphoma therapy compared with alkylating alone. Because of its low toxicity profile, our combination with Rituximab and Chlorambucil could be considered a first-line therapy, especially in FL patients not eligible for aggressive chemotherapy regimens.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia
Authors: Suppipat Koramit , Zhu Xiao , Park Chun Shik , Lacorazza H. Daniel
Published: October 2019

Abstract 3974

Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4.

Disclosures:

No relevant conflicts of interest to declare.
A Polymorphism In the 3'UTR Region of ABCB1 Is Associated with Increased Allele Activity with Corresponding Increases In P-Glycoprotein Expression and Function
Authors: Bajuaifer Nada , Grundy Martin , Hills Robert , Russell Nigel H. , Pallis Monica , Seedhouse Claire
Published: October 2019

Abstract 3990

Multidrug resistance is one of the major prognostic factors in acute myeloid leukemia (AML). Expression of p-glycoprotein (p-gp), a member of the ABC transporter family and encoded by the ABCB1 gene, is associated with failure to respond to chemotherapy. Control of MDR1 expression is multifactorial and to-date emphasis has been on the 5'-promoter region of the gene. Evidence is now accumulating regarding a role for the 3'-untranslated region (3' UTR) of genes in controlling expression. We therefore studied a single nucleotide polymorphism (SNP) in the 3'UTR of ABCB1 (rs3842) and determined its effect on p-gp expression.

Samples were available on 450 AML patients who had been entered into the UK LRF AML14 and NCRN AML15 trials. The distribution of the rs3482 A/G SNP was determined using the SNaPshot method; 10% of samples had their genotype confirmed by sequencing. P-gp protein expression and function were analysed by flow cytometry of CD45-gated cells; expression using MRK-16 and function by assessing the modulation of R123 accumulation by PSC833. To determine the activity of the two polymorphic alleles the 3'UTR of ABCB1 was cloned into a modified pGL3 luciferase vector and the normalised luciferase activity was assessed for both the A and G allele.

The distribution of the rs3482 SNP in our AML patient cohort was 71% AA, 27% AG and 1.5% GG. These results are comparable with the published prevalence of the SNP in European populations. Approximately 60% of AML patients do not express p-gp so we therefore focused upon the 40% of patients who expressed p-gp. We found that in the p-gp-expressing patients a higher p-gp protein and function was associated with patients with the wild–type homozygous AA genotype when compared to the GA+GG genotypes. Furthermore when the ABCB1 3'UTR was cloned downstream of the firefly luciferase gene and transfected into HL-60 cells the ‘A’ construct demonstrated significantly increased activity when compared to the G construct (normalized ratio (firefly luciferase/renilla luciferase): A 7.7, G 1.9; n=4, p<0.05).

Outcome data was available on 414 of the intensively treated trial patients. Whilst the ABCB1 3'UTR polymorphism had no impact on overall survival or relapse risk there was a trend towards an increased relapse free survival in patients with the wildtype AA genotype (RFS AA 37%; AG+GG 28%, p=0.08). This may correspond to higher p-gp expression in immunocompetent cells post-remission.

In conclusion this work demonstrates that the ABCB1 rs3482 3'UTR SNP is functionally significant with the A allele associated with higher activity and a concomitant increase in p-gp expression and function in p-gp expressing patient samples.

Disclosures:

No relevant conflicts of interest to declare.
Profiling of Multiple Gene Mutations In Myelodysplastic Syndromes Using High-Throughput Resequenceing Combined with Barcode Labeling
Authors: Nagata Yasunobu , Sanada Masashi , Yoshida Kenichi , Nakaya Takeo , Matsubara Aiko , Obara Naoshi , Ishiyama Ken , Miyawaki Shuichi , Takita Jyunko , Chiba Shigeru , Shih Lee-Yung , Koeffler H.Phillip , Ogawa Seishi
Published: October 2019

Abstract 4011

Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect-system® (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with >20 depths of reading. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations.

Disclosures:

No relevant conflicts of interest to declare.
Promoter Methylation and Decreased Expression of Mir-124 In Myelodysplastic Syndromes
Authors: Meng Yuesheng , Xia Qiao , Hu Jun
Published: October 2019

Abstract 4014

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders of hematopoietic stem/progenitor cells. Although demethylating agents such azacytidine and decitabine have been widely used to treat MDS, the underlying molecular mechanisms remain obscure. Abnormalities of microRNAs (miRNA) have been recently associated with hematological malignancies including MDS. The miR-124 was initially demonstrated to modulate neurogenesis. It was recently shown that EVI1-induced methylation and silencing of miR-124 were present in murine MDS cells. In the retrospective study we evaluated methylation status and expression levels of miR-124 in fifteen MDS patients (subtypes included RCUD, RCMD, RAEB-1, RAEB-2 and CMML). Genomic DNA samples were modified with bisulfite and methylation at three promoter regions of miR-124 was examined with methylation-specific real time quantitative polymerase chain reaction analysis (MQPCR). In general, we observed an increased methylation levels of miR-124 in MDS patients than that in normal bone marrow (NBM, P<0.01). In accordance with this, marked depression of miR-124 was seen in six patients when compared with NBM (more than 2 times lower), as determined with quantitative reverse-transcriptive PCR assay. Moreover, there were higher degrees of promoter methylation in cases with depressed miR-124 than that in remaining cases. A negative correlation between the expression and methylation levels was statistically significant (R= -0.498, P<0.01). The change of miR-124 was not directly related to short-term clinical response or prognosis, possibly due to limited size of the sample. However, the miR-124 amount returned to basal levels in two cases (RCMD and CMML subtypes respectively) after low-dose decitabine therapy and DNA methylation of all three loci disappeared. Continued work is underway to accumulate more cases and make long-term clinical follow-up. In conclusion, this primary work suggested a possible role of the methylation-mediated silencing of miR-124 in the pathogenesis or disease progression of MDS.

Disclosures:

No relevant conflicts of interest to declare.
Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma
Authors: Zheng Yuhuan , Yang Jing , Zhang Liang , Qian Jianfei , Matthews Jairo , Wang Michael , Kwak Larry , Yi Qing
Published: October 2019

Abstract 4080

Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Remission Maintenance on Weekly Imatinib Dosage In Patients with FIP1L1-PDGFRA-Positive Chronic Eosinophilic Leukaemia
Authors: Helbig Grzegorz , Hus Marek , Moskwa Andrzej , Zawilska Krystyna , Molendowicz-Portala Lucyna , Świderska Alina , Urbanowicz Alina , ˙Zuk Ewa , Raźny Małgorzata , Rodzaj Marek , Seferyńska Ilona , Całbecka Małgorzata , Wichary Ryszard , Kyrcz-Krzemien Slawomira
Published: October 2019

Abstract 4097

A small subset of patients with hypereosinophilic syndrome (HES) presents an interstitial deletion in chromosome 4q12, which leads to the expression of an imatinib -responsive fusion gene- called FIP1L1-PDGFRA (F/P). These patients have chronic eosinophilic leukemia (CEL). Here, we treated twenty five F/P-positive CEL patients (22 male, 2 female; median age of 50 years) with imatinib using initial daily doses ranging from 100 – 400 mg. At diagnosis a median peripheral blood eosinophilia and eosinophil marrow infiltration were 12×109/L (range 2.5–40.8) and 39% (range 7–80), respectively. Splenomagaly was the most frequent clinical manifestation in this patient subgroup. All imatinib-treated patients achieved clinical and molecular response. A complete haematological remission (CHR) was demonstrated after median of 13 days (range 3–90) whereas molecular response (MR) was confirmed after median of 9 months (range 3–24). In a remission maintenance phase, imatinib doses were de-escalated and they were following: 100mg once weekly (n=11), 100mg twice weekly (n=6), 100mg daily (n=5), 200mg once weekly (n=2) and 400mg once weekly (n=1). Plasma imatinib level was measured 24 hours after the last drug intake in 7 patients treated in once weekly schedule and it remained extremely low, ranging between 44–167 ng/ml. Molecular studies performed at the same time points confirmed molecular remission. With a median follow-up of 40 months all patients remained in CHR and FIP1L1-PDGFRA expression was undetectable in all treated patients. These data indicate that even very low imatinib doses are highly effective in remission maintenance of patients with F/P-positive CEL.

Disclosures:

No relevant conflicts of interest to declare.
UCH-L1, a Potent Oncogene That Drives the Development of B-Cell Lymphoma, Promotes Akt Signaling by Regulating Mtor Complex Assembly
Authors: Hussain Sajjad , Galardy Paul J.
Published: October 2019

Abstract 4182

De-ubiquitinating enzymes (DUBs) are important regulators of many cellular processes through their ability to reverse the modifications installed by ubiquitin ligases. Several members of the DUB family are implicated in human cancers, where they may act as oncogenes or tumor suppressors. There is little direct in vivo evidence, however, for their roles in cancer. We recently reported that the DUB UCH-L1 is a potent oncogene that drives the development of spontaneous B-cell lymphoma in transgenic mice, and is required for the survival of malignant B-cells in vitro. The mechanism of these effects involves the strong induction of Akt signaling that is observed in culture and in lymphoid tissues of Uchl1 transgenic mice. Akt is phosphorylated on two distinct sites by the kinases PDK1 and mTOR, with both phosphorylation events required for maximal signaling. The activity of mTOR towards its substrates is dependent on the binding partners to which it is bound. When mTOR binds to raptor, it forms mTORC1, an important regulator of nutrient sensitive protein translation, whereas when complexed with rictor and SIN1, it forms mTORC2, a critical regulator of Akt signaling. It is unclear how the cell regulates the formation of mTORC1/2. Here we show a novel mechanism by which UCH-L1 regulates mTOR-dependent Akt signaling. By co-immunoprecipitating mTOR and its binding partners, we find that UCH-L1 expression promotes the binding of mTOR with rictor at the expense of raptor, without changing the level of either protein. These data suggest a novel ubiquitin-dependent regulatory step in mTOR complex assembly. In addition, UCH-L1 expression leads to a reduction in the levels of the Akt antagonist phosphatase PHLPP1 in vitro and in vivo. This decline is completely dependent on UCH-L1 DUB activity as expression of a catalytic mutant does not affect levels of PHLPP1. We find that the mechanism of this decline is not due to proteasome degradation, nor transcriptional control. We therefore hypothesize that the UCH-L1 associated shift in cellular mTOR activities leads to reduced mTORC1-dependent translation of PHLPP1. To test this, we inhibited mTORC1 activity by depleting raptor using shRNA and found a dramatic decline in PHLPP1 levels, whereas depletion of rictor had no effect on PHLPP1. We conclude that UCH-L1 boosts Akt signaling through a novel two-step mechanism by which it promotes the generation of mTORC2, reduces the levels of mTORC1, and thereby impairs the translation of PHLPP1. These data have important implications for understanding the roles of ubiquitin, mTOR, and Akt, in lymphoma biology and may lead to more intelligent use of next generation mTOR inhibitors in the treatment of lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
The Ubiquitination-Dependent Interaction of p210 BCR/ABL with β-Catenin Contributes to Chronic Myeloid Leukemogenesis
Authors: Chen Ru , Gwen Mahon , Tala Ilona , Whitehead Ian
Published: October 2019

Abstract 4186

Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with the kinase activity of BCR/ABL. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified a ubiquitin binding domain (UBD) in p210 BCR/ABL which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This UBD does not conform to any known consensus sequence, and is unique to BCR and BCR/ABL. Deletion of the UBD does not impair the auto- or trans-kinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β. Treatment with an E1 inhibitor impairs the interaction between β-catenin and p210 BCR/ABL suggesting that the interaction is ubiquitination-dependent. Consistent with this possibility, previous studies have identified a glycine residue in β-catenin that is required for the BCR interaction. Based on these observations we propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation. In a murine bone marrow transplantation model for CML, mice transplanted with hematopoietic cells that express the ubiquitin binding mutant have significantly increased life spans compared to mice transplanted with p210 BCR-ABL expressing cells. This delayed disease progression may be due to a decrease in levels of β-catenin and less granulocyte macrophage progenitors (GMPs), which are thought to function as leukemic stem cells in the blast phase of CML. The role of ubiquitin-mediated b-catenin binding in p210 BCR/ABL-mediated leukemogenesis is currently being explored.

Disclosures:

No relevant conflicts of interest to declare.
Intrinsic Requirement of MicroRNA In Hox-Based Leukemia Initiating Cell Maintenance
Authors: Chaubey Aditya , Velu Chinavenmeni Subramani , Horman Shane , Jegga Anil , Guzman Monica L. , Zeleznik-Le Nancy , Jordan Craig T. , Carroll Martin , Gebelein Brian , Grimes H. Leighton
Published: October 2019

Abstract 4192

Mouse models of MLL-Af9 leukemia have been exploited to roughly determine leukemia initiating cell (LIC) characteristics and biology. Each colony formed by MLL-Af9 leukemic splenocytes is capable of initiating leukemia upon transplantation. MLL-fusion oncoproteins signal through elevated expression of HoxA9, Meis and Pbx proteins. The HoxA9 transcription factor is of critical interest in human AML because it is; 1) directly affected by chromosomal translocations, 2) upregulated by leukemia oncoproteins, and 3) the level of HoxA9 in cytogenetically normal human AML predicts outcome. However, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. We have shown that Hox and Gfi1 orthologs antagonize each other during Drosophila anterior posterior patterning, and that in mammalian myeloid progenitors Gfi1 directly represses the expression of Hoxa9, Pbx1 and Meis1. Moreover, Gfi1 regulates the expression of miR-21 and miR-196b, and forced expression of these miR blocks G-CSF instructed granulopoiesis. Here we demonstrate that microRNA genes are the targets of endogenous HoxA9 versus Gfi1 antagonism and that this antagonism is relevant in the context of human leukemia. Moreover, we also show that miR-196b and miR-21 are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts colony replating potential of Lin- bone marrow cells transformed by HoxA9, Nup98-HoxA9 and MLL-Af9, as well as bona fide MLL-Af9 leukemias. In contrast, cells transformed by AML-ETO (which does not signal through HoxA9) are not affected. In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia (but not AML-ETO leukemia) lethality. Finally, limiting dilution analyses demonstrate that antagomirs inhibit Hox-based transformation by targeting the LIC. Antagomir treatment induces the re-expression of a histone lysine demethylase, downregulation of gene expression associated with maintenance of MLL-Af9 leukemia and associated H3K4me3 marks on these genes. Our data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins in LIC maintenance.

Disclosures:

Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.