bluuebunny/crossref_metadata_2025_split huggingface dataset:
PRL2 Maintains Hematopoietic Stem and Progenitor Cells Through Regulating SCF/KIT Signaling
Authors: Kobayashi Michihiro , Dong Yuanshu , Yu Hao , Bai Yunpeng , Chen Sisi , Gao Rui , Yoder Mervin C. , Kapur Reuben , Zhang Zhong-Yin , Liu Yan 
Published: October 2019

Abstract
The phosphatase of regenerating liver family of phosphatases, consisting of PRL1, PRL2 and PRL3, represents an intriguing group of proteins implicated in cell proliferation and tumorigenesis. However, the role of PRLs in normal and malignant hematopoiesis is largely unknown. While SCF/KIT signaling plays an important role in hematopoietic stem and progenitor cell (HSPC) maintenance, how SCF/KIT signaling is regulated in HSPCs remains poorly understood. We here report that PRL2 regulates HSPC maintenance through regulating SCF/KIT signaling. To define the role of PRL2 in hematopoiesis, we analyzed the hematopoietic stem cell (HSC) behavior in Prl2 deficient mice generated by our group. Prl2 deficiency results in ineffective hematopoiesis and impairs the long-term repopulating ability of HSCs. In addition, Prl2 null HSPCs are less proliferative and show decreased colony formation in response to SCF stimulation. Furthermore, Prl2 null HSPCs show reduced activation of the PI3K/AKT and ERK signaling in steady state and following SCF stimulation. Importantly, we found that PRL2 associates with KIT and the ability of PRL2 to enhance SCF signaling depends on its enzymatic activity, demonstrating that PRL2 mediates SCF/KIT signaling in HSPCs. Thus, PRL2 plays a critical role in hematopoietic stem and progenitor cell maintenance through regulating SCF/KIT signaling. Furthermore, loss of Prl2 decreased the ability of oncogenic KITD814V mutant in promoting hematopoietic progenitor cell proliferation and in activation of signaling pathways. We also checked the expression of PRL2 proteins in human AML cell lines and found increased level of PRL2 proteins in some acute myeloid leukemia (AML) cells compared with normal human bone marrow cells, indicating that PRL2 may play a pathological role in AML. Our results suggest that the PRL2 phosphatase may be a druggable target in myeloproliferative disease (MPD) and acute myeloid leukemia (AML) with oncogenic KIT mutations.

Disclosures:
No relevant conflicts of interest to declare.
NIPA Deficiency Leads To Acceleration Of The Lymphoma Development In EµMyc Mice
Authors: Illert Anna Lena , Klingeberg Cathrin , Albers Corinna , Morris Stephan w. , Peschel Christian , Keller Ulrich , Duyster Justus 
Published: October 2019

Abstract
Timely degradation of proteins that control cell proliferation and apoptosis is an essential mechanism in keeping normal growth from turning into runaway malignancy. We previously reported the cloning of NIPA (Nuclear-Interaction-Partner-of-ALK) and characterized it as a F-Box-protein that defines an oscillating E3-ubiquitin-ligase. Using a conditional-knockout strategy we inactivated Nipa and found Nipa-/- animals to be viable, but sterile due to a block of spermatogenesis. Our studies demonstrate that loss of Nipa has no substantive effect on physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, Nipa checkpoint control can be unmasked by oncogenic c-Myc-transformation. Here we show significant differences in c-Myc-induced transformation: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced. Moreover, Nipa-deficiency leads to premature senescence in cultured primary MEFs. Ectopic reexpression of Nipa resulted vice versa in delayed senescence of knockout MEFs. Next, we sought to know, whether increased apoptosis in Nipa-/- c-Myc-transduced MEFs is dependent on a functional p53-Axis. Interestingly, the effect of Nipa deficiency on c-Myc-mediated transformation was totally abolished by p53-knockdown. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53, suggesting the importance of p53 in Nipa-induced cell death. Looking in more detail on the c-myc-p53 axis we detected a substantial increase in Arf-p19 levels in Nipa-/- cells. Moreover, Nipa-knockdown in Zn-inducible-Arf-NIH/3T3 cells lead to stabilization of Arf p19. To test the impact of these findings in a relevant in-vivo model we intercrossed Nipa-/- animals with a transgene EµMyc-Strain. Nipa-/-EµMycTG/wt animals developed lymphomas within a significantly shorter latency than Nipa+/+EµMycTG/wt animals. Furthermore, lymphomas of knockout animals were more aggressive. FACS- and biochemical-analyses showed no gross differences between Nipa-/- and wt lymphomas except highly elevated Arf-p19 levels in Nipa-/- lymphomas, pointing to an important role of Nipa in Myc-p19-signalling. Taken together our results highlight the functional importance of the Nipa-p53-axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis.

Disclosures:
No relevant conflicts of interest to declare.
Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase
Authors: Zhang Jingfang , Liu Yangang , Kong Guangyao , Chang Yuan-I , Ranheim Erik A. , Wang Jinyong , Du Juan , Damnernsawad Alisa , Zhang Jing 
Published: October 2019

Abstract
Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation.
Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro.
We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma.
Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells.

Disclosures:
No relevant conflicts of interest to declare.
Increased Expression Of Fgf2 Induces Cytokine Hypersensitivity In Leukemia Stem Cells With Translocations Involving The MLL Gene
Authors: Shah Chirag , Wang Hao , Bei Ling , Eklund Elizabeth 
Published: October 2019

Abstract
Leukemias with chromosomal translocation or partial tandem duplications involving the MLL (mixed lineage leukemia) gene have exceptionally poor prognosis (referred to as 11q23-leukemias). At the molecular level, 11q23-leukemias are characterized by increased expression of a set of homeodomain transcription factors in CD34+ hematopoietic stem cells (HSC) and differentiating CD34- myeloid progenitor cells. This transcription factor set includes HoxB3, B4, A7-11 and Meis1. Although many elegant studies have identified the precise molecular mechanisms by which MLL-fusion proteins increase HOX gene transcription, less is known about the downstream effects of increased Hox expression and potential implications for leukemogenesis. In prior studies, we determined that the gene encoding fibroblast growth factor 2 (Fgf2 or basic Fgf) is a HoxA10 target gene, and production of Fgf2 is increased in HoxA10 overexpressing primary bone marrow cells. In the current studies, we determine that FGF2 is also activated by HoxA9; identifying the only common target gene for HoxA9 and HoxA10 where both proteins activate transcription. We also find Mll-Ell (an MLL-fusion protein) increases FGF2 transcription and Fgf2 expression in a HoxA9 and HoxA10 dependent manner. Importantly, autocrine production of Fgf2 by Mll-Ell expressing HSC and myeloid progenitor cells increases cytokine-induced proliferation and enhances cell survival. In additional studies, we determine that these effects of Fgf2 are mediated by phospho inositol 3 kinase (PI3K) dependent stabilization of beta catenin. We find that autocrine production of Fgf2 results in increased expression of key beta catenin target genes in Mll-Ell expressing cells, including c-myc, survivin and cyclinD1. Beta catenin target genes also include the genes encoding CDX4 and HOXA10. Additionally, Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates the CDX4 gene. Therefore, increased Fgf2 also establishes a positive feedback loop that enhances transcriptional activation of HOXA9 and HOXA10 by MLL-fusion proteins. 11q23-AML is generally refractory, but the molecular mechanisms for this are unknown. Our studies suggest that increased Fgf2 expression in 11q23-AML contributes to cytokine hypersensitivity and sustained survival of leukemia stem cells. Targeting the effects of Fgf2 might increase chemotherapy sensitivity in this form of leukemia. Small molecule inhibitors of Fgf-receptors are available, but have not yet been tested in this form of AML. We suggest that this would be a rational therapeutic approach to this disease.

Disclosures:
No relevant conflicts of interest to declare.
Pharmacological Validation Of Potentiating Targets From SAHA RNA-Interference Modifier Screens In Acute Myeloid Leukemia
Authors: Bogenberger James M , Rudd James , Chow Donald , Kassner Michelle , Fauble Veena , Mesa Ruben A. , Yin Hongwei , Greene Casey , Tibes Raoul 
Published: October 2019

Abstract
The lysine deacetylase inhibitor suberoylanilide acid (SAHA) exhibits promising but limited activity in the treatment of acute myeloid leukemia (AML). To identify potential targets for rational combination therapies that increase the efficacy of SAHA in AML, we performed a functional RNA-interference (RNAi) drug modifier screen to identify genes that, when inhibited, potentiate or antagonize the in vitro anti-leukemic activity of SAHA. A total of 901 kinase, phosphatase and closely associated signaling genes were silenced in TF-1, HEL and THP-1 cells, with four different siRNA sequences per gene, both alone and in combination with SAHA treatment. Screen hit lists for each cell line were over-laid on an integrated functional relationship network. A community detection algorithm was then applied to this sub-network and siRNA sensitive modules were identified. Each module represents a highly connected set of genes in the integrated network. To identify pathways represented by each module, enrichment was evaluated using the National Cancer Institute (NCI) Protein Interaction Database (PID) pathways. Both cell line-specific and universal sensitizing targets, grouped into a small number of pathways, emerged from these screens. Seven pharmacologic inhibitors interfering with these pathways have been assessed in a panel of four or five AML cell lines, including SET-2, TF-1, HEL, THP-1, and OCI-AML3. Of the seven tested inhibitors targeting SAHA sensitizing networks, one combination exhibiting universal SAHA sensitization in all AML cell lines tested was further assessed in drug dose response assays using ex vivo cultures of bone marrow mononuclear cell populations derived from patients with various myeloid malignancies. The combination demonstrated “one-sided” SAHA EC50 fold-enhancement, as well as strong synergy by “two-sided” CalcuSyn combination index determination in all ex vivo specimens examined to date (N=6), including polycythemia vera (PV), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndrome, MDS transformed to AML, and de novo AML samples. Thus, this first reported large-scale SAHA RNAi modifier screen in AML has identified a specific pathway/target whose inhibition broadly synergizes with HDAC inhibition in myeloid malignancies. A novel compound putatively targeting this respective pathway/target is currently in early clinical development and has shown limited single-agent activity. With the full dataset presented, we will propose a novel rational combination to enhance the activity of HDAC inhibition in AML and other myeloid malignancies that could be translated into design of a clinical trial.

Disclosures:
No relevant conflicts of interest to declare.
Genomic Signature Predicts Resistance To Busulfan In AML Cell Lines
Authors: Patel Pritesh R. , Mahmud Dolores , Zhang Wei , Garcia Joe GN , Rondelli Damiano 
Published: October 2019

Abstract
The alkylating agent Busulfan (Bu) is commonly used in the pre-transplant conditioning regimen for patients with acute myeloid leukemia (AML). Relapse after transplant is in part due to resistance to Bu. Our study was aimed at identifying a genomic signature that could predict resistance to Bu in AML cells.
In order to obtain a list of candidate genes which may correlate with increasing resistance to Bu we performed a linear regression analysis controlled for cancer type on NCI60 Stanford cDNA data and GI50 values. This produced a list of 111 genes correlating with increasing Busulfan GI50 (p<0.01, FDR<50%). The ability of these 111 genes to predict GI50 was subsequently tested based upon their expression in a second independent gene expression platform (Affymetrix U133 plus 2.0). This analysis yielded 6 genes (KCNH2, CD74, CD53, HCLS1, ERC2 and HLA-DQB2) predicting higher GI50 values (p<0.05). We then tested the ability of these genes to predict Bu GI50 in a panel of 6 AML cell lines (K562, HL60, HL60-MX1, KG1, THP1 and NB4). To obtain GI50 values on AML cell lines we treated the cells with doses of Bu ranging from 0-200mcg/ml. At 24 hours, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. GI50 values varied from 13.9 - 70.4 micromoles/ml. RNA quantitation for the 6 identified genes was performed using the Quantigene assay (Panomics, Santa Clara, CA). Expression was normalized to control gene levels (RPLP0). Two genes (ERC2 and HLA-DQB2) showed low or undetectable expression and were excluded from further analysis. Logistic regression analysis showed that a combined overexpression of any 2 of the remaining 4 genes was significantly associated with increasing resistance to Bu GI50 in AML cells (p=0.006). Of the 6 cell lines tested, the 2 most resistant (THP1 and K562) both displayed this pattern of gene overexpression.
To our knowledge, this is the first attempt to obtain a list of genomic biomarkers to predict Bu resistance in AML using a genome wide approach. This data provides a rationale for validation of these 4 genes in a clinical dataset with the potential future application of predicting relapse after Bu based high dose therapy.

Disclosures:
No relevant conflicts of interest to declare.
Expression Of Tissue Transglutaminase Is Associated With Glucocorticoid Resistance In T-Cell Acute Lymphoblastic Leukemia
Authors: Jung Hyun Joo , Lee Seong Wook , Park Jun Eun 
Published: October 2019

Abstract
Glucocorticoids are core drugs in the treatment of T-cell acute lymphoblastic leukemias (T-ALL) because they induce apoptosis and cell-cycle arrest in leukemia lymphoblasts. The treatment response to glucocorticoid is the most important predictive factor for treatment outcome of T-ALL. Development of secondary glucocorticoid resistance is lethal to the patients at relapse. To improve survival for glucocorticoid-resistant T-ALL patients, it is necessary to develop new therapeutic strategies to overcome glucocorticoid resistance. In the present study, we used biochemical and molecular methodologies to demonstrate that tissue transglutaminase (TG2) confers glucocorticoid resistance in T-ALL. TG2 is a calcium-dependent protein crosslinking enzyme reported to be overexpressed in various metastatic or chemotherapy-resistant cancer cells including lymphoma cells. We found that T-ALL cells expressed elevated levels of TG2. We generated a model of glucocorticoid-adapted subclones of T-ALL cell lines that were extremely less sensitive to glucocorticoid than the parent cells. Glucocorticoid-adapted subclones not only showed the change of ABC transporters activity but also increased TG2 expression and activity than the parent cells in T-ALL. Inhibition of TG2 suppressed glucocorticoid resistance and improved the cytotoxicity of glucocorticoid in glucocorticoid-adapted subclones of T-ALL. Our study is the first to show the expression of TG2 and the contribution of TG2 to glucocorticoid resistance in T-ALL. The modulation of TG2 expression could be a new therapeutic target to overcome glucocorticoid resistance in T-ALL. Furthermore, change of TG2 expression could serve as markers of secondary glucocorticoid resistance in T-ALL.

Disclosures:
No relevant conflicts of interest to declare.
Loss Of IKZF1 Function Mediates Resistance Towards Glucocorticoid-Induced Apoptosis
Authors: Havinga Jorn , Yuniati Laurensia , Demkes Marc , Schenau Dorette van Ingen , Kuiper Roland P. , Hoogerbrugge Peter M. , van Leeuwen Frank N. , Scheijen Blanca 
Published: October 2019

Abstract

Background
Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids.

Results
We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death.
In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL.

Disclosures:
No relevant conflicts of interest to declare.
Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation
Authors: Staudinger Matthias , Kellner Christian , Peipp Matthias , Schub Natalie , Humpe Andreas , Gramatzki Martin 
Published: October 2019

Abstract
Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented.
To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays.
As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml).
Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations.

Disclosures:
No relevant conflicts of interest to declare.