bluuebunny/crossref_metadata_2025_split huggingface dataset:
An EVI1 Point Mutant Unable To Bind GATA1 Rescues EVI1−Induced Defects in Erythroid Differentiation.
Authors: Laricchia-Robbio Leopoldo , Rinaldi Ciro R. , Fazzina Raffaella , Nucifora Giuseppina 
Published: October 2019

Abstract
EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes.
Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.
Tribbles Homolog 2 (Trib2) Inactivates C/EBPalpha and Causes Acute Myelogenous Leukemia.
Authors: Keeshan Karen , He Yiping , Wouters Bas J. , Shestova Olga , Xu Lanwei , Sai Hong , Rodriguez Carlos G. , Maillard Ivan , Valk Peter , Carroll Martin , Aster Jon C. , Delwel Rudd , Pear Warren S. 
Published: October 2019

Abstract
Trib2 is a member of the Trib family of serine/threonine kinase-like proteins (Trib1, Trib2, Trib3), whose function in hematopoiesis is not documented. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML), with a median survival of 179 days. Retroviral Trib2 expression in hematopoietic stem cells perturbed myeloid development, enhanced progenitor proliferation, and directly inhibited the function of C/EBPα, a critical transcription factor that is frequently dysregulated in AML. Furthermore, an analysis of microarray data generated from 285 AML patient samples identified elevated Trib2 expression in a distinct subset of patients in a cluster with a high frequency of C/EBPα mutations. In mechanistic studies, we found that Trib2 associated with and led to the proteasomal-dependent degradation of C/EBPα. To determine the structural requirements for Trib2 to inhibit C/EBPα, and to induce AML, we have initiated a structure/function analysis of Trib2. Together, our data identify Trib2 as a novel oncogene that induces AML through a mechanism involving inactivation of C/EBPα. The identification of Trib2 as potent leukemogen points to new pathogenic mechanisms and possible therapeutic opportunities in this aggressive cancer, which is not currently curable in the majority of patients.
Xanthohumol: A New Potential Proteasome Inhibitor That Activates the Pro-Apoptotic Arm of the Unfolded Protein Response in B-CLL Cells.
Authors: Lust Sofie , VanHoecke Barbara , Van Gele Mireille , Boelens Jerina , Philippé Jan , Vincken Petra , Bracke Marc , Poelman Martine , Offner Fritz 
Published: October 2019

Abstract
B-Chronic Lymphocytic Leukemia (B-CLL) is incurable by current methods because of increasing resistance to chemotherapy. Therefore, new options are needed for the treatment of CLL. The proteasome-inhibitor bortezomib was described to promote apoptosis in CLL cells and to induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in refractory multiple myeloma. The UPR is mainly a self-protective mechanism activated when protein folding is disrupted and misfolded proteins are accumulating in the ER. However, sustained ER stress eventually leads to cell death. Three major ER sensors are involved during UPR, namely ATF6, PERK and IRE1. In inactivated state, they are bound to the ER-chaperone BiP/GRP78. Upon ER stress, BiP is released from the luminal domain of the ER stress transducers resulting in their activation and downstream signaling. Pro-survival signals are delivered by IRE1 (splicing of XBP1) and ATF6 (cleavage), while pro-apoptotic signals are generated by PERK (upregulation of CHOP). Previously, we demonstrated that the xanthohumol (X) is able to kill B-CLL cells in a dose- and time-dependent way as evidenced by PARP cleavage and Annexin V staining (Lust et al., 2005). In this study, we first demonstrated that X induces apoptosis of B-CLL cells in part via activation of ER stress and the UPR and identified the associated molecular markers. Treatment of freshly isolated B-CLL cells with X, stimulated the expression BiP and Hsp70 (suggestive for ER stress), the phosphorylation of eIF2a (suggestive for PERK activation), and the splicing of XBP1 mRNA (indicative for IRE activation). In contrast, ATF6 activation seemed not to be implicated since no cleaved ATF6 could be demonstrated. Induction of UPR was associated with a pro-apoptotic outcome evidenced by upregulation of CHOP, generation of ROS, downregulation of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, cleavage of PARP, and processing of caspase-3. Next, we showed that X inhibited the 20S proteasome activity in reticulocyte lysates but also functionally in B-CLL as demonstrated from the accumulation of polyubiquitin-conjugates under X treatment. The activation of UPR and influence on the anti-apoptotic proteins can mostly be explained by this proteasome inhibitory activity. In conclusion, we identified proteasome inhibitory capacities of xanthohumol in B-CLL in vitro. Proteasome-inhibition was accompanied by ER stress, UPR and apoptosis. Our results further suggest that tackling organelles like the proteasome and the ER is a valuable strategy in treatment of B-CLL.
APcK 110, a novel Inhibitor of Kit, Extends Survival of NOD-SCID Mice in An AML Xenograft Model.
Authors: Faderl Stefan , Bueso-Ramos Carlos , Liu Zhiming , Pal Ashutosh , Bornmann William , Harris David , Hazan-Halevy Inbal , Kantarjian Hagop M. , Estrov Zeev 
Published: October 2019

Abstract
Abstract 1045
Poster Board I-67
Kit is a type III receptor protein tyrosine kinase and membrane receptor for stem cell factor (SCF). Tight regulation of the activity of kit is a crucial component of normal hematopoiesis. Mutations of the KIT gene have been identified in blasts of patients with AML predominantly those with core-binding factor leukemias. As the presence of mutations of KIT has been associated with higher likelihood of relapse and shorter survival, targeting Kit may be of therapeutic benefit. Therapeutic activity may extend to cases with Kit overexpression. We have recently described the activity of APcK110, a novel inhibitor of Kit, in vitro where we have shown potent proapoptotic and antiproliferative effects in AML cell lines and primary AML samples irrespective of mutation status but with measurable expression of Kit (Faderl S et al. Cancer Res 2009; 69: 3910-3917). Here we extend our investigations to the activity of APcK110 in vivo in a xenograft mouse model. After exposure of 8-weeks-old female NOD-SCID mice (NOD.CB17-PRKDS SCID\J) to 30cGy sub-lethal WBXRT, 1 × 105 OCI/AML3 cells (human AML cell line) were injected I.V. Ten days later, the mice were injected I.P. every other day with the same volume of either APcK110 or PBS. The mice were sacrificed according to experimental protocol or when they became moribund, unable to obtain food or water, or if they lost > 20% of their body weight. Necropsy was performed on all animals. Marrow, blood and organs were preserved in formalin for histological analysis. Kaplan-Meier estimates were applied to calculate survival of the mice. We could show that 1) all mice injected with OCI/AML3 cells developed a clinical and histological picture consistent with AML. Wright stains of blood and marrow confirmed presence of a large, heterogeneous blast population, which was negative for myeloperoxidase, but stained positive for butyrate and anti-human CD45. Histological analysis of lung and liver tissue revealed dense infiltration with AML blasts as confirmed by anti-human CD45-positive sheets of cells in these tissues; 2) survival of APcK110-treated mice was significantly improved compared with mice which were injected with PBS (p=.02); 3) Marrow histology showed residual AML following injection of mice with APcK110. In conclusion, APcK110 is a novel kit kinase inhibitor with anti-AML activity in vitro and in vivo. Further evaluation in toxicology and clinical studies should be pursued.

Disclosures:
Pal: Patent Office: patent application pending. Bornmann:Patent office: Patent application pending. Estrov:Patent Office: Patent application pending.
The Cyclin Dependent Kinase 2, 7 and 9 Inhibitor SNS-032 Has Single Agent Activity in Acute Myeloid Leukaemia Cells and Is Highly Synergistic with Cytarabine.
Authors: Walsby Elisabeth J , Pepper Chris , Knapper Steven , Burnett Alan K 
Published: October 2019

Abstract
Abstract 1059
Poster Board I-81
Cyclin dependent kinases (CDKs) are a family of serine/threonine kinases that require activation by binding to cyclin partners. These complexes of CDK-cyclin play an essential role in the regulation of cell cycle progression (e.g. CDK2) and some have also been implicated in the regulation of mRNA transcription (e.g. CDK9). CDK7 is involved in both these processes. Cyclins, CDK complexes and other cell cycle regulators have been shown to be mechanistically involved in the development of human tumours making them tractable drug targets. SNS-032 (BMS-387032) is a 2-aminothiazole derivative that has been shown to function as a selective inhibitor of CDK2, 7 and 9. Blocking the action of CDK2 and 7 is thought to block cell cycle progression in tumour cells. Additionally blocking CDK7 and 9 may also reduce transcription of survival factors in the tumour cells by abrogating their ability to promote transcription. Primary mononuclear cells isolated from acute myeloid leukaemia patients at diagnosis (n=87) were treated with SNS-032 for 48h resulting in a mean LD50 of 0.14μM ± 0.2 as measured by MTS assay. By comparison, cytarabine (AraC) was more than 35 times less potent in the same cohort (mean LD50 = 4.88μM ± 5.02). In combination with AraC (ratio SNS-032 1:1.9 cytarabine) showed a mean combination index (CI) of 0.13 in the 25 primary samples tested indicating strong synergism between SNS-032 and AraC. SNS-032 induces apoptosis as evidenced by dose-dependent increases in AnnexinV/propidium iodide (PI) staining and caspase-3 activation. SNS-032 affects activity of CDK 2 and 9 at the expression level as quantitative PCR showed that levels of CDK 2 and 9 mRNA were decreased at six hours of treatment to SNS-032 (CDK2 to 20% untreated levels (0.26μM), CDK9 to 36% untreated levels) but CDK7 mRNA levels were not significantly altered. However, CDK7 phosphorylation was reduced at 6 hours suggesting the SNS-032 modulates CDK7 at the post-translational level by suppressing activation. Treatment of the cell lines with the combination of SNS-032 and AraC resulted in reduced mRNA levels of CDK2 and 9. In contrast, AraC alone did not induce these reductions in mRNA expression. Additionally, the pro-survival proteins XIAP and Bcl-2 and Mcl-1 mRNA levels were decreased in SNS-032 treated cells, increased in AraC treated cells and reduced in cells treated with the combination. In conclusion, SNS-032 is effective in AML cells in vitro as a single agent and has also demonstrated a remarkable degree of synergy with AraC. One putative mode of action of this synergy is the SNS-032-induced decrease in pro-survival proteins. This may sensitise AML cells to the actions of cytotoxic agents LIKE AraC leading to the levels of synergy seen in this study.

Disclosures:
No relevant conflicts of interest to declare.
High Dose Cytarabine, Clofarabine and Gemtuzumab Ozogamicin (CLAC-MYL) in Relapsed or Refractory AML Patients.
Authors: Scappini Barbara , Gianfaldoni Giacomo , Salvatore Palmieri , Susini Maria Chiara , Izzo Teresa , Mannelli Francesco , Longo Giovanni , Fanci Rosa , Ferrara Felicetto , Bosi Alberto 
Published: October 2019

Abstract
Abstract 1060
Poster Board I-82
Clofarabine has been shown to be effective in AML patients, either as single agent or, mainly, in association with intermediate dose cytarabine. On the basis of these reports, we conducted a preliminary study combining clofarabine, intermediate dose cytarabine and gemtuzumab ozogamicin (Mylotarg) in AML patients who relapsed or failed to respond to at least two induction therapies.
We treated 20 patients affected by relapsed/refractory AML with a regimen including clofarabine at 22,5 mg/m2 daily on days 1-5, followed after three hours by cytarabine at 1 gr/m2 daily on days 1-5, with the addition of gemtuzumab ozogamicin 6 mg/m2 on day 6 (CLAC-Myl). Six patients received a further consolidation cycle with clofarabine at 22,5 mg/m2 and cytarabine at 1 gr/m2 day 1-4.
Among the twenty patients, six were in first relapse, six in second relapse, eight with resistant disease. The mean age was 52 years (range 33-68 years), the white blood count at the accrual was 31.500 mcc (range 2140-153.000). 10/20 (50%) patients achieved a complete remission, 1/20 a partial response, 7/20 had resistant disease, 2/20 died of complications during the aplastic phase (a case of multiorgan failure an a septic shock caused by Pseudomonas Aeruginosa).
The most frequent non hematologic adverse events were vomiting, diarrhea, transient liver toxicity (2/20 grade 3-4), febrile neutropenia (7/20), infections microbiologically documented (2/20 Pseudomonas Aeruginosa sepsis).
Comparing with other salvage strategies, in this small cohort of patients we did not observe a significant delay in bone marrow recovery (median time to ANC recovery 31.5 days), except in a patient (female, 34 years old, relapsed after ABMT) that experienced an unexpected, irreversible aplasia after the consolidation course, complicated by an unusual HHV6 reactivation.
Among the ten responding patients, three underwent allogeneic bone marrow transplantation, one patient still in CR after 7 months died for complications of an acute myocardial infaction occurred during the consolidation course, one relapsed after 6 months, and five not eligible for transplant procedures are still in complete remission with a median follow up of 6 months.
These very preliminary results suggest that the CLAC-Myl regimen is effective in this particularly poor prognosis category of patients, with safety data consistent with previously reported salvage therapies. Further studies are warranted.

Disclosures:
Off Label Use: Clofarabine in relapsed/refractory AML.
Lenalidomide and Dexamethasone Promote Erythropoiesis in Both Normal and RPS14 and RPS19 shRNA Infected Human CD34+ Cells: Implications for Therapy of Myelodysplastic Syndrome and Diamond-Blackfan Anemia.
Authors: Narla Anupama , Dutt Shilpee , McAuley J. Randall , Ebert Benjamin L. 
Published: October 2019

Abstract
Abstract 1092
Poster Board I-114
Lenalidomide, a derivative of thalidomide, induces erythroid responses in patients with myelodysplastic syndrome while dexamethasone induces erythroid responses in patients with Diamond Blackfan anemia. In vitro, both lenalidomide and dexamethasone have been shown to stimulate erythropoiesis. In the present study, we explored the activity of lenalidomide and dexamethasone in stimulating erythroid differentiation of primary hematopoietic progenitor cells and CD34+ cells expressing RPS19 or RPS14 shRNAs. Both agents increase the absolute number of erythroid cells produced from normal human CD34+ bone marrow cells by 1.3 to 1.6-fold. Importantly, both lenalidomide and dexamethasone increased the diminished erythroid proliferative capacity of CD34+ cells expressing either RPS19 or RPS14 shRNAs. However, the drugs have distinct effects on the production of erythroid progenitor colonies, Blast Forming Units – Erythroid (BFU-E) and Colony Forming Units – Erythroid (CFU-E). Dexamethasone caused a dose-dependent and statistically significant increase in the number of BFU-E colonies by 2-fold, while it had little effect on CFU-E. In marked contrast, lenalidomide caused a dose-dependent and statistically significant increase in the number of CFU-E colonies by 2.5-fold but had no effect on BFU-E colonies. In addition, the combination of lenalidomide and dexamethasone had an additive effect on the production of erythroid cells and on erythroid colony formation. Our findings imply that lenalidomide and dexamethasone regulate different stages of erythropoiesis and lend support to the concept that combination therapy may be effective in minimizing the potential toxicity of these two drugs in the clinical management of patients. These findings have important implications for the treatment of patients with Myelodysplastic Syndrome as well as with Diamond Blackfan Anemia.

Disclosures
Off Label Use: Lenalidomide is approved for 5q- MDS. We are investigating its possible use in Diamond Blackfan Anemia.
Deletion of IKZF1 in Pediatric Precursor-B ALL Is a Strong Prognostic Marker for Relapse.
Authors: Kuiper Roland P. , Waanders Esmee , Van der Velden Vincent H.J. , van Reijmersdal Simon V , Scheijen Blanca , Hehir-Kwa Jayne , Keijzers-Vloet Suzanne T.M. , Sonneveld Edwin , van Dongen Jacques JM , van Leeuwen Frank N. , van Kessel Ad Geurts , Hoogerbrugge Peter M. 
Published: October 2019

Abstract
Abstract 1104
Poster Board I-126
Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse.

Disclosures
No relevant conflicts of interest to declare.
Proteomic Screening Applied to the Diagnosis of Chronic Graft-Versus-Host-Disease.
Authors: Mischak-Weissinger Eva M. , Stadler Michael , Holler Ernst , Schleuning Michael , Greinix Hildegard , Kolb Hans-Jochem , Dickinson Anne M , Krons Annika , Diedrich Helmut , Kontsendorn Julia , Hahn Norbert , Metzger Jochen , Ganser Arnold 
Published: October 2019

Abstract
Abstract 1164
Poster Board I-186
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes, but the application is still limited due to major complications, such as severe graft versus host disease (GvHD) and infectious complications. Diagnosis chronic GvHD is based on clinical features and biopsies, a non invasive, unbiased laboratory test does not exist.
We used the urine collected from 20 patients (10 with limited cGvHD, 10 with extensive cGvHD) to establish a proteomic pattern that allowed the diagnosis of cGvHD development and tested the resulting set of polypeptide markers (27 differentially excreted peptides) on more than 200 patients prospectively and blinded for the correct classification of cGvHD samples. The majority of the patients included were transplanted for hematological malignancies (n=209), 6 for hematopoietic failure syndromes. Conditioning regimens included dose reduced conditioning regimens (FLAMSA and ClaraC for the majority of the patients of MHH), as well as standard conditioning regimens (TBI+Cy or Busulfan+Cy) for about 35% of the patients, with GvHD-prophylaxis including cyclosporine A and mycophenolate (MMF) or metothrexate (MTX) as appropriate. Eighty percent of the patients received ATG (antithymocyte globulin) prior to HSCT.
A peptide pattern of 27 peptides, differentiating chronic from acute GvHD was developed. Controls were patients at least 100 days post HSCT, with no GvHD in the history, no infections and without relapse at the time of sampling. Prospective and blinded evaluation of the patients revealed the correct classification of patients developing cGvHD with a sensitivity of 85% and specificity of 95%.Further evaluation of the cGvHD patterns specific for particular organ manifestations of cGvHD are currently ongoing. Interestingly, the cGvHD pattern seems to be predictive for GvHD developing post DLI, while the aGvHD-specific proteomic pattern only predicts GvHD of the intestine, which may be more similar to “late acute GvHD”.

Disclosures
Krons: mosaiques-diagnostics GmbH: Employment. Metzger:mosaiques-diagnostics GmbH: Employment.