bluuebunny/crossref_metadata_2025_split huggingface dataset:
MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,
Authors: Zhou Beiyan 
Published: October 2019

Abstract
Abstract 3461
The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein.
We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells.

Disclosures:
No relevant conflicts of interest to declare.
Daunorubicin -TiO2 Nanocomposites As ‘smart' pH-Responsive Drug Delivery System,
Authors: Zhang Hai-jun , Chen Bao-An 
Published: October 2019

Abstract
Abstract 3479
Daunorubicin (DNR) with a broad spectrum of anti-tumor activity is limited due to the serious side-effects in the clinical application. The aim of this study was to explore the novel pH-responsive drug delivery system (DDS) based on titanium dioxide (TiO2) nanoparticles (Nps) for its potential roles to enable more intelligently controlled release, enhance chemotherapeutic efficiency, and reduce the side-effects of DNR. DNR was loaded onto the TiO2 Nps by forming (six-membered chelate) complexes with transition metal Ti to contract DNR-TiO2 nanocomposites as DDS. The encapsulation efficiency and loading efficiency of DNR loaded TiO2 Nps were assessed and calculated as 65.46±6.82% and 20.63±3.55%, respectively.The DNR was released from the DDS much more rapidly at pH 5.0 and 6.0 than at pH 7.4. The release behavior is a desirable characteristic for tumor-targeted drug delivery. Most DNR will remain in the carrier for a considerable time period at normal physiological conditions (pH 7.4), indicating the potential for the prolonged DNR retention time in the blood circulation and thereby greatly reducing the side effects to the normal tissues. On the other hand, once the DNR loaded TiO2 Nps are taken up by tumor cells via endocytotic process, a faster release may occur at lower local pH, i.e, inside the endosome and lysosome of cancer cells ((pH 4.5∼6.5), leading to the significant improvement in cancer treatment efficacy. The DNR- TiO2 nanocomposites as DDS induced the remarkable improvement in the anti-tumor activity, which were demonstrated by the flow cytometry, MTT assay and nuclear DAPI staining. Furthermore, the possible signaling pathway was explored by Western blot. For instance, in human leukemia cells (K562 cells), our observations demonstrated that the DDS could obviously increase the intracellular concentration of DNR and enhance its potential anti-tumor efficiency through inducing apoptosis in a caspase-dependent manner, indicating that DNR-TiO2 nanocomposites could act as an efficient DDS importing DNR into target cancer cells. These findings revealed that such ‘smart' DNR delivery strategy represent a promising approach in hematologic malignancy therapy.

Disclosures:
No relevant conflicts of interest to declare.
Combining Tetrahydrouridine with Decitabine Addresses Malignant Cell Sanctuary in the Liver, a Tissue That Expresses High Levels of Cytidine Deaminase,
Authors: Ebrahem Quteba , Mahfouz Reda Z , Peng Kwok Ng , Saunthararajah Yogen 
Published: October 2019

Abstract
Abstract 3509
Non-cytotoxic DNA methyl-transferase 1 (DNMT1) depletion by decitabine induces p53-independent differentiation mediated cell cycle exit in acute myeloid leukemia (AML) cells and spares normal hematopoietic stem cells, providing a potentially important alternative to conventional apoptosis-based therapy. However, there are pharmacologic barriers that impede optimal clinical translation of pre-clinical observations. One major barrier is the enzyme cytidine deaminase (CDA), which drastically reduces the half-life of decitabine from many hours in vitro to a few minutes in vivo. This problem is significant since decitabine is S-phase specific in its mechanism of action, and for effective DNMT1 depletion, low peak levels but extended half-life are optimal. Furthermore, CDA is highly expressed in the liver and intestines, generating potential sanctuary sites from the effects of decitabine. Finally, upregulation of CDA expression is a potential mechanism by which AML cells may resist decitabine at the cellular level. To overcome the multiple CDA-mediated barriers to effective therapy, we evaluated the combination of very low dose decitabine with the CDA inhibitor tetrahydrouridine (THU) in a murine xenotransplantation model of aggressive human AML (THP1 AML cells – p53 and p16-null). Treatment groups (n=5 per group) received equal volume vehicle (PBS), low dose cytarabine (0.1 mg/kg subcutaneous [SC]) with (THU) (4mg/kg intraperitoneal [IP]), decitabine (0.1 mg/kg SC) with THU (4mg/kg IP), and decitabine alone (0.2 mg/kg SC). Median survival in decitabine (61 days) and THU-decitabine (70 days) treated mice was significantly extended compared to the PBS (38 days) and THU-cytarabine (50 days) treatment groups (Log Rank p=0.00156 (figure 1). In decitabine treated mice, eventual emergence of disease was concentrated in the liver, consistent with a sanctuary function of this organ. Combination of THU with decitabine largely eliminated liver infiltration by AML, reducing liver tumor load from average 5.2g in PBS treated mice to 0.8g in THU-Decitabine treated mice (t-test p<0.001, livers were harvested at different time-points corresponding to the Kaplan-Meier curve). In separate experiments without AML cell xenotransplantation, the non-cytotoxic, DNMT1 depleting character of the decitabine therapy was confirmed by hematologic and bone marrow evaluation for DNA damage by phospho-H2AX staining. In conclusion, combining THU with decitabine can address malignant cell sanctuary in the liver, an organ that expresses high levels of cytidine deaminase. The ability of this combination to address other aspects of CDA-mediated resistance to therapy is also being evaluated.

Disclosures:
No relevant conflicts of interest to declare.
AML Depends on VEGFR1 and Not VEGFR2 for Proliferation and Protection From Chemotherapy,
Authors: Bais Swarna , Wise Elizabeth , Harris Eric M. , Meacham Amy , Cogle Christopher R 
Published: October 2019

Abstract
Abstract 3598

Background:
Vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs) are known to serve important roles in normal hematopoiesis, but the importance of the VEGF/VEGFR axis in malignant hematopoiesis, namely acute myeloid leukemia (AML), is poorly defined.

Methods:
Bone marrow from AML patients were analyzed for VEGFRs on the malignant myeloblast populations. Human myeloid leukemia cell lines, KG-1, HL60 and K562, were also evaluated for VEGFR expression. Knockdown of VEGFR1 and VEGFR2 receptors in AML cells expressing these receptors were performed using lentivirus transfection of shRNA. Cell proliferation was quantified using XTT colorimetric assay and apoptosis was evaluated by Annexin V and PI staining.

Results:
Bone marrow from 12 consecutive AML patients showed that 33% of patients had malignant myeloblasts that expressed VEGFR1. Of the AML cell lines tested, only KG-1 cells expressed VEGFR1 and VEGFR2. Knockdown of VEGFR1 and VEGFR2 in KG-1 cells were confirmed by Western blot. When VEGFR1 was knocked down in KG-1 leukemia cells, cell proliferation was significant decreased (Figure 1). In addition, knocking down VEGFR1 resulted in enhanced sensitivity to cytarabine chemotherapy (Figure 1). In contrast, knockdown of VEGFR2 did not result in changes in leukemia cell proliferation or sensitivity to cytarabine chemotherapy (Figure 2). When KG-1 leukemia cells knocked down for VEGFR1 expression were transplanted into sublethally, irradiated NOD/scid/IL2R□−/− (NSG) mice (n=10), none of the mice showed engraftment of VEFR1 deficient cells.

Conclusions:
The VEGF/VEGFR axis is an important determinant in AML pathobiology. In specific, AML cells depend upon VEGFR1 and this receptor represents a promising target for future therapeutic intervention.

Disclosures:
No relevant conflicts of interest to declare.
Synergistic Depression of Wnt Pathway by Sequential Combination of Decitabine and Idarubicin in Human Myeloid Leukemic Cell Lines,
Authors: Tong Hongyan , Li Kongfei , Mei Chen , Jin Jie 
Published: October 2019

Abstract
Abstract 3628

PURPOSE:
In the preliminary experiment, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin cytarabine, thalidomide, homoharringtonine) in combination with decitabine of inhibiting growth of myeloid leukemic cell lines. The result showed notable synergistic effect of sequential combination of decitabine and idarubicin. In searching the associated mechanism, we assessed the effect of sequential combination of decitabine and idarubicin in apoptosis, cell cycle arrest, and depression of wnt pathway. Methods: Myeloid leukemia cells (U937, HEL and SKM-1) were treated with decitabine alone, idarubicin alone, and decitabine sequential with idarubicin to determine their impact on cellular proliferation, cell cycle regulation, apoptosis and the regulation of wnt pathway by detecting DNA methylation status, mRNA level and protein expression. Results: The sequential combination of decitabine and idarubicin produced synergistic inhibition of myeloid leukemia cells growth by apoptosis and cell cycle arrest. Sequential treatment with both agents induced synergistic effect on decreasing methylation status of wnt antagonists (SFRP1, HDPR1 and DKK3) which result in re-expression or up-expression of these genes. Furthermore, we found that the down-steam genes of wnt pathway including c-myc and β-catenin were down-regulated. Conclusion: Taken together, these findings showed that decitabine sequentially combined with idarubicin has synergistic anti-leukemia effect, which suggest clinical potential in the treatment of myeloid leukemias.

Disclosures:
No relevant conflicts of interest to declare.
The Functional Role of TLR9 in Human Platelets
Authors: Thon Jonathan Noah , Peters Christian G. , Aslam Rukhsana , Rowley Jesse , Weyrich Andrew S. , Semple John W. , Flaumenhaft Robert C. , Italiano Joseph E. 
Published: October 2019

Abstract
Abstract 366
Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation in the Context of Daily Practice of Follicular Lymphoma patients' Outcome Before and After Approval of Rituximab in First Line Therapy in This Indication: Results of a Retrospective Analysis of 247 Unselected Patients with a 5-Year Median of Follow-up,
Authors: Virelizier Emmanuelle Nicolas , Segura-Ferlay Celine , Domnisoru Izabela-Irina , Rey Philippe , Gargi Thérèse , Chassagne-Clément Catherine , Favier Bertrand , Biron Pierre , Belhabri Amine , Sebban Catherine , Blay Jean-Yves , Ghesquieres Herve 
Published: October 2019

Abstract
Abstract 3698

Background:
Randomized trials had demonstrated that rituximab improved outcome in newly diagnosed follicular lymphoma (FL) and this anti-CD20 monoclonal antibody was approved in France since 2004 in combination of chemotherapy (CT) for LF in first line therapy. As these results were obtained in selected patients with strict inclusion and exclusion criteria used in clinical trials, whether the improvement of patient's outcome is valuable in unselected population of FL patients with various medical history, different CT regimens is questionable. We performed a retrospective analysis to evaluate if the management and the outcome of FL have significantly changed in our institution before and after rituximab approval in 2004.
Patients and Methods: all adult patients referred for first line LF were included and separated into cohort 1 diagnosis between 1996 and 2003 (103 patients) and cohort 2 between 2004 and 2010 (144 patients). Baseline clinical and biological datas, Follicular Lymphoma International prognostic Index (FLIPI), type of therapy, treatment response, progression free survival (PFS) and overall survival (OS) were compared.

Results:
There were no statistical differences in patients' characteristics between the 2 cohorts, including FLIPI score. In cohort 2, 71% received in first line therapy a rituximab based regimen (19% in monotherapy and 52% with CT). In cohort 1, 58% of patients were treated with CT and 10% with rituximab, 2% in monotherapy and 8% in combination with CT. Response rate were significantly improved in cohort 2 compared to cohort 1: complete response rate was 74.3% versus 57.8%, P <.001. With a median follow-up of 115.5 and 46.5 months in cohort 1 and 2, the 2-year PFS was 67% for cohort 1 and 82% for cohort 2 (P =.0039), respectively. The 2-year OS was also improved between the two periods (98% versus 87%, P =.0007). We could confirm the prognostic value on OS of the FLIPI score in whole series, in cohort 1 before the rituximab era but not in cohort 2. In fact, the 2-year OS was respectively 100% in low risk, 98% in intermediate risk and 95% in high risk (P =.14)/

Conclusions:
These datas confirms the improvement of FL patients' outcome observed in clinical trials after the approval of rituximab in first line therapy prescript in a context of daily practice whatever age, medical history, and type of CT.

Disclosures:
No relevant conflicts of interest to declare.
Rituximab Followed by Involved Fields Radiotherapy (IF-RT) in Stage I-II Follicular Lymphoma (FL): Long Term Results,
Authors: Ruella Marco , Filippi Andrea , Russo Anna DI , Caracciolo Daniele , Matteucci Paola , Magni Michele , Di Nicola Massimo , Ricardi Umberto , Montefusco Vittorio , Parvis Guido , Tarella Corrado , Gianni Alessandro M. , Devizzi Liliana 
Published: October 2019

Abstract
Abstract 3699

Background:
Radiotherapy is considered the standard approach in stage I-II non bulky FL but no consensus of the required radiation field has been reached and failure-free survival (FFS) reported ranged between 41 to 49%. Published randomized trials failed to demonstrate the superiority of non adriamycin-containing regimens plus RT over RT alone and only one single arm study with COP-B plus IF-RT reported superior FFS (73%) however complicated by an high rate of treatment related toxicity. Since the monoclonal anti CD-20 antibody Rituximab has shown to be effective in systemic therapy of FL as primary treatment as well at relapse, the combination of the anti-CD20 Rituximab with IF-RT may be considered a valid approach, allowing an effective systemic disease control while sparing the toxicity of chemotherapy. We reported here the long-term outcome of a series of stage I-II FL pts treated front-line with Rituximab followed by IF-RT. Patients and Methods: From July 1999 to April 2009, 36 consecutive stage I-II FL WHO grade I-II, have been enrolled in this phase II study and treated with 4 weekly doses of Rituximab followed by IF-RT. Median age: 49 yrs (range 34–82), M/F: 20/16; FLIPI 0–1: 36; Bulky:0. BM PCR analysis to detect minimal residual disease (MRD) was carried out in 25 pts with a molecular probe: PCR+/PCR-: 10/15. Treatment consisted of 4 weekly doses of Rituximab (375 mg/sqm) followed by external bean RT on involved fields. Median radiation dose was 30.6 Gy, (range 20–40). Results: Rituximab followed by IF-RT was well tolerated without any additional toxicity. All but one pts reached a complete remission (CR). With a median follow-up of 7 years (range: 2–12), 34 pts are alive and 24 in continuous complete remission. Of the 12 relapsed pts, 6 were PCR+ versus 4 PCR- at baseline BM analysis; 2 pts had no molecular probe. Two smokers pts died of lung cancer at 3 and 4 years after immuno-radiotherapy. The OS and FFS were 94% and 67% respectively. These results compare favorably with those observed in similar historical control group treated at our Institution with IF-RT alone (OS:87%,FFS:46.8%). Conclusion: Rituximab followed by IF-RT is a well tolerated and effective front-line treatment for limited stage FL. Long-term results are comparable to those reported for non-anthracycline-containing chemo-radiotherapy and higher when compared to radiotherapy alone, making Rituximab-IF-RT a potential option as initial therapy of limited stage FL.

Disclosures:
No relevant conflicts of interest to declare.
Cycling Toward Leukemia Stem Cell Elimination Wtih a Selective Sonic Hedgehog Antagonist,
Authors: Shih Alice Y , Schairer Annelie , Barrett Christian L , Geron Ifat , Recart Angela C Court , Goff Daniel , Prashad Sacha , Wu Jerry , Jiang Qingfei , Gotlib Jason , Balaian Larisa , Minden Mark D. , Leu Heather , Wall Russell , Ma Wenxue , Shazand Kamran , McPherson John D , Kornblau Steven M. , Deichaite Ida , Pu Minya , Bao Lei , Martinelli Giovanni , Reya Tannishtha , Morris Sheldon R , VanArsdale Todd , Hudson Thomas J , Messer Karen , Mikkola Hanna , Levin Wendy J. , Frazer Kelly A , Sadarangani Anil , Jamieson Catriona 
Published: October 2019

Abstract
Abstract 3776
Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse.

Disclosures:
Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.