bluuebunny/crossref_metadata_2025 huggingface dataset:
Albuminuria As a Risk Factor for Development of Chronic Kidney Disease and Short-Term Mortality Following Hematopoietic Stem Cell Transplantation
Authors: Takahashi Yukie , Morito Taku , Ando Minoru , Ohashi Kazuteru , Sakamaki Hisashi
Published: October 2019

Abstract 1998

Background:

Microalbuminuria is a risk factor for the development of chronic kidney disease (CKD), cardiovascular disease and mortality in the general population. We studied if the incidence of microalbuminuria following hematopoietic stem cell transplantation (SCT) could be a warning sign of the future CKD and all-cause mortality.

Method:

A 1-year prospective cohort study was conducted in 29 patients (46.3±12.5 years) receiving allogeneic myeloablative SCT. Patients with prevalent CKD and those who died by 1 month after SCT were excluded from the cohort. Albumin to creatinine ratio (ACR) in a morning urine sample was consecutively measured before conditioning therapy (baseline), at the time of SCT, days 7 and 14, and 1 month after SCT. Microalbuminuria was defined as ACR ≥30mg/girl. CKD was defined as a decrease in estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73m2, sustained at least for 3 months. Cumulative incidence of CKD or mortality or both was assessed by the Kaplan-Meier method. Multivariate Cox regression analysis was used to calculate the hazard ratio (HR) of developing these outcomes within 1 year, adjusted for covariates with clinical and statistical significance.

Results:

The prevalence of microalbuminuria was 6.9% at baseline, and increased to 62.1% at the day of SCT. It varied among the time points; 62.1% on day 7, 48.3% on day 14 and 51.7% on 1 month, respectively. Mortality within 1 year was 27.6%. Cumulative incidence of CKD or mortality or both was significantly higher in the group with □emicroalbuminuria on 1 month' than in the opposite. [figure1] In addition, the presence of □emicroalbuminuria on 1 month' was significantly associated with each outcome: for CKD, HR 14.5, 95% confidence interval (CI) 2.88 to 121, P=0.0006; for death, HR 11.7, 95% CI 1.58 to 252, P=0.0138; for either CKD or death, HR 16.6, 95% CI 3.79 to 151, P=0.0001, respectively.

Conclusion:

The incidence of microalbuminuria following SCT is a promising predictor of the development of CKD and short-term mortality.

Disclosures:

No relevant conflicts of interest to declare.
Residual Plasmatic Activity of ADAMTS13 in Congenital Thrombotic Thrombocytopenic Purpura Correlates with Disease Phenotype
Authors: Lotta Luca A , Wu Haifeng M , Scully Marie A , Noris Marina , Veyradier Agnes , Mackie Ian J , Remuzzi Giuseppe , Coppo Paul , Liesner Ri , Donadelli Roberta , Loirat Chantal , Gibbs Richard A , Horne April , Yang Shangbin , Palla Roberta , Peyvandi Flora
Published: October 2019

Abstract 2219

Congenital thrombotic thrombocytopenic purpura (TTP) (OMIM #274150) is a rare, recessively inherited thrombotic microangiopathy characterized by the congenital deficiency of ADAMTS13 due to mutations in the corresponding gene. The clinical phenotype of congenital TTP is variable, encompassing neonatal-onset disease and adult-onset disease, forms with a single disease episode and chronic-relapsing forms. A review of the ∼100 published cases of congenital TTP showed similar age of first disease episode in patients carrying the same ADAMTS13 gene suggesting that different ADAMTS13 mutations might influence the severity of clinical phenotype, conceivably by determining different patterns of residual plasmatic activity of ADAMTS13. However, the quantification of residual ADAMTS13 activity in severely deficient patients was blunted by the relatively high limit of detection (LOD) of commonly used ADAMTS13 activity assays (varying between 3% and 6%). We report 29 cases of congenital TTP from 4 European cohorts, with 32 mutations of ADAMTS13 including 8 not previously published. To assess if residual ADAMTS13 activity was detectable in these patients and correlated with their clinical history, ADAMTS13 activity was measured by SELDI-TOF mass spectrometry (LOD=0.5%).

ADAMTS13 activity above 0.5% was measurable in 26 (90%) patients, and lower levels of activity correlated with an earlier age of disease onset (r=0.51; p=0.006) and with the need for regular fresh frozen plasma prophylaxis (2.24% vs 3.88%; p=0.03). Mutations at the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity (1.48% vs 4.89%, p=0.02) and earlier disease onset (3.2 years vs 24.2 years, p=0.003). Our results show that residual ADAMTS13 activity correlates with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

Disclosures:

No relevant conflicts of interest to declare.
The Earliest Thymic T Cell Progenitors Sustain B Cell and Myeloid Lineage Potentials
Authors: Luc Sidinh , Macaulay Iain C , Buza-Vidas Natalija , Bouriez-Jones Tiphaine , Lutteropp Michael , Woll Petter , Mead Adam J. , Boukarabila Hanane , Luis Tiago C , Mizukami Takuo , Matsuoka Sahoko , Brown John , Ferry Helen , Anderson Kristina , Duarte Sara , Atkinson Deborah , Soneji Shamit , Domanski Aniela , Farley Alison , Sanjuan-Pla Alejandra , Carella Cintia , Patient Roger , de Bruijn Marella , Enver Tariq , Nerlov Claus , Blackburn Clare , Godin Isabelle , Jacobsen Sten Eirik W
Published: October 2019

Abstract 2335

The stepwise lineage-commitment from hematopoietic stem cells (HSCs) in the bone marrow (BM) to T-lymphocyte-restricted progenitors in the thymus represents a paradigm for how distinct stages of lineage restriction from a multipotent to a lineage-restricted progenitor require different extrinsic cues. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Previous studies demonstrated the existence of adult early thymic progenitors (ETPs) restricted to T lymphocyte and granulocyte-monocyte (GM) fates (Bell and Bhandoola, Nature 2008; Wada et al., Nature 2008). The challenge remains to couple this thymic T–GM restricted progenitor to candidate thymus seeding progenitors (TSPs) identified in the BM, such as common lymphoid progenitors, lymphoid-primed multipotent progenitors (LMPPs) and HSCs, which all also possess B cell potential.

Using high resolution FACS and sensitive clonal assays, we have identified the most primitive ETPs in the neonatal thymus and demonstrate at the single cell level that rare Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs possess combined granulocyte-monocyte (GM), T and B lymphocyte but not megakaryocyte-erythroid (MkE) lineage potentials, identical to closely molecularly related thymus-seeding progenitors in the BM.

Moreover, global molecular profiling demonstrates that neonatal Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs cluster closest to multipotent progenitors in the BM, rather than subsequent (DN1 and DN2) progenitor stages in the thymus. Finally, in support of Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs with combined T, B and GM lineage potentials, being the most multipotent progenitors in the thymus, phenotypic and functional studies demonstrate that the neonatal thymus is not seeded by HSCs or MPPs with MkE potential. These findings establish for the first time a distinct lineage commitment stage for the transition of T-lineage commitment from the BM to the remote thymus.

Disclosures:

No relevant conflicts of interest to declare.
Src Kinases Act Downstream Akt and Are Required for the Transforming Properties of Constitutively Activated Forms of STAT5A and STAT5B
Authors: Nyga Remy , Savay Lea , Marcq Ingrid , Regnier Aline , Damaj Gandhi , Rochette Jacques , Gouilleux Fabrice , Lassoued Kaiss
Published: October 2019

Abstract 2378

Background:

Stat5A and Stat5B transcription factors play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia. Moreover, expression of constitutively active Stat5 (caStat5) mutants in primary hematopoietic cells can lead to leukaemia. The transforming properties of CaStat5 have been previously shown to require Akt activation. However, the mechanisms by which CaStat5 mutants and Akt are activated remain enigmatic. In this study we aimed to determine the roles of src kinases in these processes,

Methods:

For this purpose, we used murine Ba/F3 cells transfected or not with CaSta5A (Stat5A1*6) or CaStat5B (Stat51*B6) or with the oncogenic fusion proteins Tel-JAK2 and Tel-Abl as controls. Primary hematopoietic progenitors obtained from mouse bone marrow were also used in this sutdy. Cells were treated or not with the pan-src inhibitors PP1, PP2, and SU 6656 (and with PP3 an inactive analogue of PP1 as a control), imatinib (Abl inhibitor) and AG490 (JAK inhibitor). In some experiments cells were treated with U0126 (MEK inhibitor) or with the highly transducible TaT-dominant negative (dn) Akt fusion protein. Cell proliferation was assessed by cell counting. Cell death was evaluated using the trypan blue dye exclusion test. Cell apoptosis was assessed by analysing annexin V staining using flow cytometry and PARP cleavage using western blot. Expression of kinases and their phosphotylated forms were examined by western blot.

Results:

CaStat5A and CaStat5B cells were shown to express constitutively phosphorylated Lyn, Hck, Src kinases as well as Akt, IKK and ERK1/2. Stat1, Stat3, and JAK family members (TYK2 and JAK1-3) were not phosphorylated in these cells. Use of pan-Src inhibitors inhibited proliferation of CaStat5 cells and induced apoptosis of a fraction of these cells, in a dose-dependent manner. By contrast, imatinib and AG490 had no effect on the growth and survival of CaStat5 cells. Moreover, pan-src inhibitors prevented Erk but not Akt, IKK or STAT5 phosphorylation. By contrast, inhibition of Akt by means of TaT-dnAkt abolished Lyn, Src and ERK1/2 phosphorylation. Importantly, inhibition of MAPK did not interfere with Akt phosphorylation and did not interfere with CaStat5 cell survival.

Conclusion:

Altogether our data indicate that Akt acts upstream in the CaStat5-associated signalling cascade, then activates src kinases and thereby promotes MAPK-dependent cell proliferation and MAPK-independent cell survival. These results shed light onto a hitherto undescribed role of Src kinases acting downstream Akt in the mechanisms of action of CaStat5.

Disclosures:

No relevant conflicts of interest to declare.
Survivin Is Highly Expressed in AML Stem Cells and Predicts Poor Clinical Outcome
Authors: Carter Bing Z , Qiu Yihua , Huang Xielin , Diao Lixia , Zhang Nianxiang , Coombes Kevin R , Mak Duncan H , Konopleva Marina , Cortes Jorge E. , Kantarjian Hagop M. , Andreeff Michael , Kornblau Steven M.
Published: October 2019

Abstract 238

Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure).

These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy.

Disclosures:

No relevant conflicts of interest to declare.
Selective Damage to Antioxidant Gene Promoters in FA Cells
Authors: Du Wei , Rani Reena , Sipple Jared , Schick Jonathan , Pang Qishen
Published: October 2019

Abstract 2413

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Here we show that several major anti-oxidant defense genes, including Glutathione peroxidase 1, Peroxiredoxin 3, Thioredoxin reductase 1, Superoxide dismutases 1, NAD(P)H:quinone oxireductase and Catalase, are down-regulated in bone marrow cells of FA patients. This gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of these anti-oxidant defense genes. Further, we show that both increased initial damage and reduced repair rate contribute to augmented oxidative DNA damage in FA cells. Using cell-based assays to assess promoter activity and damage repair kinetics, we demonstrate that FA proteins function to protect the promoter DNA from oxidative damage. Mechanistically, FA proteins appeared to act in concert with Brg1, a chromatin-remodeling ATPase subunit of the BAF complex. Specifically, Brg1 binds to the promoters of the anti-oxidant defense genes in steady state. Upon challenge with oxidative stress, FANCA and FANCD2 proteins are recruited to the promoter DNA, which correlates with significant increase in the binding of Brg1 within the promoter regions. Intriguingly, the formation of the FA-Brg1-promoter complex results in a marked decrease in nuclease hypersensitivity and oxidative damage in the promoter DNA in normal cells compared to FA cells. Finally, disassociation of the FA proteins from the Brg1-promoter complex parallels Pol II loading, suggesting a regulatory role for the FA proteins in transcription. Taken together, the study identifies a role of FA proteins in protecting anti-oxidant genes from oxidative damage.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA Expression in Acute Myeloid Leukaemia and Their Effects on the Differentiation of Acute Myeloid Leukaemia Cells
Authors: Palma Catalina A , Tonna Elise , Bryant Adam J , Jayaswal Vivek , Agapiou David , Yang Yee Hwa , Ma David , Lutherborrow Mark
Published: October 2019

Abstract 2430

Acute myeloid leukaemia (AML) is typified by an aberrant halt in maturation of myeloid progenitor cells, leading to uncontrolled proliferation of immature blasts. In the majority of cases of AML with normal karyotype, the underlying cause of the maturation arrest remains unclear. MicroRNAs, small inhibitory RNAs, are known to be dysregulated in cancers and have been postulated to play a causative role in leukaemogenesis.

We aimed to investigate the contribution of aberrant microRNA expression to the inhibition of maturation in AML cells. MicroRNA expression profiling was performed on the bone marrow of 28 AML patients with normal karyotype and 8 normal controls. Differential expression was confirmed by qRT-PCR. We found that the expression of a panel of 12 microRNAs was able to accurately separate AML-M1 and AML-M5 subtypes. The AML-M1 subtype represents a more immature cell population when compared to the monoblast morphology observed in AML-M5. Four candidate microRNAs, miR-181a, -146a, -130a and -135b were selected for further investigation based in their putative targeting of key monocytic transcription factors as determined by in silico modelling followed by luciferase assays.

In vitro monocyte and macrophage differentiation of HL60 and NB4 cell lines with 1,25-dihydroxyvitamin D3 and/or phorbol-12-myristate-13-acetate treatment resulted in a significant decrease in all four candidate microRNAs (measured by qRT-PCR), supporting the hypothesis that candidate microRNA over-expression in AML-M1 may contribute to its maturation arrest. Over-expression of the candidate microRNAs by transfection with Pre-miR microRNA precursor molecules (Ambion) into the HL60 and NB4 monocyte differentiation model, resulted in the significant suppression of CD14 (Figure, *p <0.05 compared to Scrambled control) and CD15 expression, markers of monocytes and granulocytes respectively. Conversely, knockdown of miR-181a, -146a, -130a and -135b with miRCURY LNA microRNA knockdown probes (Exiqon) induced an increase in CD14 expression in HL60 cells compared to non-targeting Scrambled control.

An important regulatory role of these microRNAs in myeloid/monocytic differentiation is strongly suggested by their targeted suppression of the key transcription factors KLF4, MAFB, IRF8, HOXA10, MCL1 and PU.1 which was confirmed by luciferase reporter assay. This study provides evidence that the over-expressed microRNAs discovered in our profiling work between AML-M1 and AML-M5 are biologically relevant microRNAs, which have the potential to affect the maturation potential of AML cells.

Disclosures:

No relevant conflicts of interest to declare.
Reduced c-FOS Is Associated with Poor Prognosis and Clinical Course in Chronic Lymphocytic Leukemia
Authors: Oakes Christopher C , Claus Rainer , Schmidt Christopher , Park Yoon Jung , Boutros Michael , Stilgenbauer Stephan , Plass Christoph
Published: October 2019

Abstract 2433

We have previously shown that silencing of the tumor suppressor gene, DAPK1, by genetic and epigenetic mechanisms is associated with the pathogenesis of CLL. To elucidate genes and pathways involved in DAPK1 transcriptional regulation, genome-wide siRNA screens were performed using a novel transgene reporter system involving a stably-integrated BAC construct expressing luciferase under the control of the DAPK1 locus. c-FOS and other members of the FOS gene family were identified as positive regulators, implicating the AP-1 pathway in DAPK1 transcription. A subsequent comprehensive examination of the FOS, JUN and ATF gene families in CLL and healthy B cells was performed. As the expression of the AP-1 gene family is highly subject to fluctuations in stress and mitogenic stimuli, freshly isolated cells were cultured using autologous serum conditions, allowing for a uniform establishment of baseline gene expression. This work reveals an alteration of the relative composition of AP-1 transcription factors in CLL, demarked by a substantial reduction in c-FOS gene expression. We find that the inducibility of c-FOS in CLL following MAPK activation by TPA (ERK-MAPK) is impaired 7.0-fold relative to healthy B cells and is completely abrogated following anisomycin (p38-MAPK) stimulation. The level of c-FOS induction following TPA activation can be used to clearly segregate CLL patients into two non-overlapping groups, those with low (mean=4.0-fold reduced expression; range=3.0–7.2; n=9) and very low (mean=21.7-fold reduced expression; range=17.6–36.7; n=8) following one hour induction versus healthy B cells. The very low c-FOS induction cases are characterized by poor prognostic indicators (IGHV unmutated (0/9 in low vs. 5/8 in very low); 17p, 6q deletion (0/9 in low vs. 3/8 in very low); and a substantially shorter time to progression (102.7±40.4 months in low vs. 10.8±8.6 in very low). Patients with very low c-FOS induction also demonstrate elevated c-MYC induction following activation. TPA-dependent ERK phosphorylation and activation of other immediate early genes, such as EGR1 and c-JUN, is intact in both CLL groups, absolving MAPK pathway dysfunction as a relevant c-FOS silencing mechanism. Reduced c-FOS expression can be partially explained, in the majority of very low c-FOS inducible cases, by elevated levels of miR-155 and miR-221 targeting the c-FOS transcript. Reduced expression of c-FOS and the resulting reconfiguration of AP-1 transcription factor composition may be involved in the pathogenesis of CLL and the subsequent silencing of DAPK1.

Disclosures:

No relevant conflicts of interest to declare.
CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway
Authors: Polzer Harald , Janke Hanna , Hiddemann Wolfgang , Eick Dirk , Spiekermann Karsten
Published: October 2019

Abstract 2447

We examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants.

RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands.

Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells.

CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants.Figure:Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.Figure:. Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.

Disclosures:

No relevant conflicts of interest to declare.
The Functional Link Between CDK1 and Retinoic Acid Receptor γ (RARγ) in Response to Treatment with All-Trans Retinoic Acid
Authors: Hedblom Andreas , Laursen Kristian B , Miftakhova Regina R , Sarwar Martuza , Mongan Nigel P , Gudas Lorraine J , Persson Jenny L
Published: October 2019

Abstract 2485

Alterations in cell cycle pathways and retinoic acid signaling are implicated in the pathogenesis of leukemia and treatment response. The retinoic acid receptors (RARα, RARβ and RARγ) mediate the differentiation, cell cycle arrest and cellular response to all-trans retinoic acid (ATRA). However, it is largely unknown whether the cyclin dependent kinases (CDK1 and CDK2) mediate the ATRA effects, also whether and how CDK1 and CDK2 are functional linked to RARs. Therefore in the present study, we examined the function of CDKs and their regulation in relationship to the RAR α, β and γ.

The use of siRNA mediated knockdown of CDK1 in human leukemic U-937 cells affected cell cycle distribution, resulting in a decreased fraction of G0/G1 cells which coincided with the reduced expression of p27 protein. Treatment of U-937 si-CDK1 cells with ATRA demonstrated reduced terminal differentiation and decreased cell cycle arrest at G0/G1 phases in comparison to U-937 siRNA control cells. We observed a reduced responsiveness to ATRA treatment due to the loss of CDK1 function, and this was accompanied with the alterations in the expression of RARγ and the increased phosphorylation of Akt. However, loss of CDK2 function showed no effects on ATRA-mediated growth arrest and differentiation in U-937 cells.

We also found that nuclear CDK1 was actively phosphorylated and was rapidly degraded upon the induction of ATRA, in contrast to its cytoplasmic counterpart in human leukemic U-937 cells. When examining the functional link between RARs and the regulation of CDKs in response to ATRA, we found that RARβ and RARγ, but not RARα, participated in ATRA-mediated protein degradation of CDK1 and CDK2. Moreover, we compared the effects of ATRA on ubiquitin-mediated protein degradation in the F9 teratocarcinoma cells and in F9 cells harboring targeted deletions of the RARα, RARβ and RARγ genes. ATRA induced ubiquitin conjugates showed striking changes in patterns in cells lacking RARγ. This indicates that the blockage in protein degradation of CDK1 may be due to the loss of RARγ function. Among the RAR isoforms, RARγ co-localized with CDK1 predominantly in the nuclear bodies. There was no detectable complex formation between RARβ and CDK1. This suggests that the phosphorylation and degradation of the nuclear CDK1 and its interaction with RARγ may be important events in mediating ATRA-induced effects on cell cycle and differentiation.

Our study has revealed a novel mechanism by which CDK1 cooperates with RARγ to mediate the effects of all-trans retinoic acid.

Disclosures:

No relevant conflicts of interest to declare.
RNA-Sequencing of the Transcriptome of Ara-C Resistant Murine AML Cell Lines Identifies Potential Drug Targets
Authors: Rathe Susan K. , Stoltenberg Christopher B. , Largaespada David A.
Published: October 2019

Abstract 2489

One frequently reported change to acute myeloid leukemia (AML) cells, as they become resistant to arabinoside cytarabine (Ara-C), is the downregulation of deoxycytidine kinase (Dck) (Veuger et al, Blood, 2000, Shi et al, Pharmacogenetics, 2004, Cai et al, Cancer Res, 2008, Song et al, Int J Oncol, 2009, Rathe et al, Leukemia, 2010). Dck is the rate limiting enzyme in the phosphorylation and activation of Ara-C. Transcriptome deep sequencing (RNA-seq) was performed on the transcriptome of two Ara-C resistant murine leukemia cell lines (B117H and B140H), as well their Ara-C sensitive parental cell lines (B117P and B140P). Previously published microarray analysis identified the downregulation of Dck as the primary common change in the B117H and B140H cell lines, when compared to their parental cell lines (Rathe et al, Leukemia, 2010). Analysis of the RNA-seq results identified an alternatively spliced form of Dck in the B117H cell line secondary to a genomic deletion/rearrangement, as well as a loss of heterozygosity. Indeed, knockdowns of Dck in the parental cell lines demonstrated that downregulation of Dck can result in a significant increase in the IC50 of Ara-C, consistent with the increase in Ara-C resistance displayed by the B117H and B140H cells. The RNA-seq analysis showed a number of significant expression changes that did not appear on the microarray analysis. These changes include the significant upregulation of specific isoforms of deoxyguanosine kinase (Dguok) and protein phosphatase 2 (formerly 2A), regulatory subunit A (PR65), beta isoform (Ppp2r1b). It has already been shown that downregulation of Dguok results in the upregulation of Dck (Franco et al, Exp Cell Res, 2007), thereby demonstrating an important balance between these two molecules. Ppp2r1b is a putative deactivator of Dck (Smal et al, Biochem Pharmacol, 2004), which may assist in the prevention of Ara-C metabolic activation and thereby contribute to resistance. This effort demonstrates the power of using RNA-seq transcriptome analysis to identify drug targets for the treatment of AML and suggests multiple genetic/epigenetic mechanisms contribute to alterations in Dck activity during the acquisition of Ara-C resistance.

Disclosures:

Largaespada: NeoClone Biotechnology, Inc.: Co-founder, Consultancy, Equity Ownership; Discovery Genomics Inc.: Co-founder, Consultancy, Equity Ownership.
Incidence, Characteristics, and Outcome of FLT3-ITD Mutations in AML Arising from an Antecedent Hematologic Disorder
Authors: Kadia Tapan , Ravandi Farhad , Borthakur Gautam , Jabbour Elias , Pierce Sherry , Garcia-Manero Guillermo , Faderl Stefan , Cortes Jorge E. , Kantarjian Hagop M.
Published: October 2019

Abstract 2522

Internal tandem duplication (ITD) mutations in FLT3 (fms-like tyrosine kinase III) are common in acute myeloid leukemia (AML) and are associated with increased relapse risk and adverse outcome. A subset of AML, arising from an antecedent hematologic disorder (AHD) such as myelodysplastic syndrome (MDS), is also associated with an adverse outcome. We sought to review our experience and define the incidence, characteristics, and outcome of FLT3-ITD in AML arising from AHD. We reviewed all cases of newly diagnosed AML, excluding acute promyelocytic leukemia from 2001–2010 at our institution. FLT3-ITD mutational analysis was performed using PCR-based DNA sequencing on 1254 cases. Overall, 187 (15%) patients (pts) had FLT3-ITD mutations. Of the 1254 pts, 545 (43%) were classified as having an AHD and 709 (57%) were not. The incidence of FLT3-ITD in pts with and without AHD was 11% (125/709) and 18% (62/545), respectively (p=0.002). As a separate analysis, among pts who had received any prior chemotherapy or radiation, the incidence of FLT3-ITD was 8% (17/201) vs. 16% (170/1053) in those who had not (p=0.005). Baseline characteristics of the patients with FLT3-ITD positive AML with or without AHD are shown in table 1. The 187 pts with FLT3-ITD were treated with several different therapies as their initial treatment for AML. These included: high dose cytarabine-based (HiDAC) in 131 (70%), conventional dose cytarabine-based in 28 (15%), hypomethylating agents (HMA) in 12 (6%), and other in 16 (9%). The response rates to HiDAC and HMA in each of the two cohorts is shown in table 1. The median overall survival of patients with FLT3-ITD and AHD was 40 weeks vs. 54 weeks in those without AHD (p=0.046, Figure 1). In conclusion, FLT3-ITD is relatively less common in AML arising from AHD and in therapy-related AML compared to de novo AML. AML pts with FLT3-ITD and AHD tend to be older, have a lower bone marrow and peripheral blasts, a lower serum LDH, and significantly shorter overall survival. Compared to those without AHD, the response rate to HiDAC-based therapy in patients with FLT3-ITD and AHD are similar, but the response rates to HMA-based therapy are higher. The role of HMA in the treatment of FLT3-ITD patients with and without AHD should be investigated further.Table 1.Figure 1.Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNAs Mediate Resistance to Tyrosine Kinase Inhibitors in Philadelphia-Positive B-ALL by Down-Regulating Key Tumor Suppressors
Authors: Schatz Jonathan H. , Mavrakis Konstantinos , Wolfe Andrew L , Wendel Hans-Guido
Published: October 2019

Abstract 2553

The Philadelphia (Ph) chromosome (t(9;22)(q34;q11)) is the most common recurrent cytogenetic abnormality in adult B-cell acute lymphoblastic leukemia (B-ALL). Resulting expression of the BCR-ABL fusion gene product, a constitutively active tyrosine kinase, leads to an extremely poor prognosis when the disease is treated with chemotherapy alone. In recent years targeted therapy with tyrosine kinase inhibitors (TKIs) in combination with chemotherapy has improved outcomes. Therapeutic resistance remains a major clinical problem, however, with less than half of patients surviving three years from initial therapy in most series even with up-front TKI-chemotherapy regimens. TKI resistance in B-ALL may result from acquired mutations of the BRC-ABL protein or from BCR-ABL-independent causes, including feedback activation of BCL6 or activation of other oncogenic signaling pathways through poorly understood mechanisms. In this study, we screened a whole-genome library of microRNAs for ability to produce resistance to TKI therapy in murine pro-B cells transformed with BCR-ABL. Several initial candidates from the screen were individually confirmed to confer TKI resistance, including miR 148/152 and 19, known oncomirs in T-ALL. Computational analysis of the gene targets of validated miRs shows significant overlap at several tumor-suppressor pathways, including down-regulation of PTEN, a known mechanism of TKI resistance in Ph+ B-ALL cell lines. In sum, we have identified a novel mechanism of TKI resistance in Ph+ B-ALL mediated by miRs and leading to down-regulation of specific tumor suppressive intermediates. These findings will allow development of new therapeutic combinations to improve outcomes for B-ALL patients.

Disclosures:

No relevant conflicts of interest to declare.
Homoharringtonine in Combination with Cytarabine and Aclarubicin in the Treatment of Refractory/Relapsed Acute Myeloid Leukemia: a Single-Center Experience
Authors: Yu Wenjuan , Mao Liping , Qian Jiejing , Qian Wenbin , Meng Haitao , Mai Wenyuan , Tong Hongyan , Tong Yin , Jin Jie
Published: October 2019

Abstract 2595

Patients with refractory and relapsed acute myeloid leukemia (AML) have relatively unfavorable prognoses and require innovative therapeutic approaches. We evaluated the efficacy and toxicity profiles of homoharringtonine (HHT), cytarabine (Ara-c) and aclarubicin combination chemotherapy in 46 refractory and/or relapsed AML patients between February 2004 and December 2009.

Eleven cases were primary refractory disease and rest of them had relapsing disease. After one course of HAA treatment, complete remission (CR) was achieved in 35 of 46 patients (76.1%), while 3 cases showed partial remission (PR) (6.5%) and 8 cases (17.4%) had resistant to this regimen. One PR and one NR patient obtained CR after second course of HAA, yielding a total CR rate was 80.4%. Hematological toxicity was the most prominent toxicity of this regimen. Grade IV hematologic toxicity occurred in 91.3% cases. Neutrophil recovery time was 13 (5–60) days. Nonhematologic complications were mild to moderate nausea, vomiting, and mucositis and could be controlled by routine measures. Early death (ED) did not occur in these 46 patients.

There was no correlation between response rate and leukemia subtype (AML FAB subtype), leukocyte, blast count in bone marrow, platelet count and sex (P >0.05). However, poor karyotype and age (≥45 years old) were factors associated with increased risk of treatment failure. Of the 46 patients, one patient, who upon achieving CR after the first re-induction therapy refused further treatment, was ultimately lost to follow-up. The median overall survival rate (OS) was 29 (2–86) months in 45 cases. Relapse-free survival (RFS) was 36 (0.5–85) months in 36 CR cases. By the time of closeout, the estimated 3-year OS rate was 42% for all 45 cases, and 43% estimated RFS at 3 years for the 36 cases of CR. OS and RFS were influenced by leukemia-related factors and disease status: Hb <10g/dl and poor karyotype in both univariate and multivariate analyses.

Allogeneic stem cell transplantation was performed in 7 patients and all achieved CR, but 1 patient died of GVHD, 2 died of pneumonia, 1 relapsed followed by death and another 3 are still alive with CR. At present, 19 patients are still alive and 18 of these are in continuous remission with one alive with leukemia. The rest (26/45) of the patients are all deceased.

In conclusion, HAA is a good choice in cases with refractory/relapsing AML for salvage chemotherapy, preferably with a high efficacy and low-toxicity profile.

Disclosures:

No relevant conflicts of interest to declare.
Transcriptional Deregulation of Homeobox Gene ZHX2 in Hodgkin Lymphoma
Authors: Nagel Stefan , Schneider Björn , Meyer Corinna , Kaufmann Maren , MacLeod Roderick AF , Drexler Hans G.
Published: October 2019

Abstract 2629

Hodgkin/Reed-Sternberg (HRS) cells represent the malignant fraction of infiltrated lymph nodes in Hodgkin lymphoma (HL). Although HRS cells display multiple chromosomal aberrations, few are recurrent and the targeted genes unknown. However, understanding the pathology of HL and developing rational therapies may well require identification and characterization of these genes and their deregulating mechanisms. Recently, we have identified a novel chromosomal rearrangement in HL cell line L-1236, t(4;8)(q27;q24), which targets ZHX2 at the recurrent breakpoint 8q24. ZHX2 encodes a transcription factor of the homeobox gene family and possesses tumour suppressor activity connected with a worse prognosis in HL-related multiple myeloma. Comparative expression analysis in HL cell lines and primary hematopoietic cells indicated aberrant downregulation in HL as well. In L-1236 the translocation breakpoint at 8q24 deletes the regulative far upstream region of ZHX2, indicating loss of activating elements. Therefore, we have looked for potential binding sites within this deleted region to analyze deregulation of this B-cell tumour suppressor gene. Accordingly, we identified two transcription factors, homeodomain protein MSX1 and bZIP protein XBP1, regulating ZHX2 expression. SiRNA-mediated knockdown and reporter gene analyses indicated that both factors activated transcription of ZHX2 directly via their corresponding binding sites. Furthermore, MSX1-cofactor histone H1C mediated repression of ZHX2 and showed enhanced expression levels in L-1236. As analyzed by fluorescence in situ hybridization (FISH) and genomic arrays, the gene loci of MSX1 at 4p16 and H1C at 6p21 were rearranged in several HL cell lines, correlating with their altered expression activity. As compared to primary hematopoietic cells, XBP1 expression was reduced in 6/7 HL cell lines while one cell line showed elevated levels, corresponding to a chromosomal rearrangement therein. Interestingly, the neighbouring pro-apoptotic genes EDEM1 and BHLHE40 were removed by del(3p26) in this cell line and activated by XBP1 in other HL cells. Taken together, our results demonstrate multiple mechanisms decreasing expression of tumour suppressor gene ZHX2 in HL cells: loss of enhancing binding sites, reduced expression of activators MSX1 and XBP1, and overexpression of MSX1-corepressor H1C. Moreover, chromosomal deregulations of genes involved in this regulative network highlight their role in development and malignancy of B-cells.

Disclosures:

No relevant conflicts of interest to declare.
R-ACVBP Benefits to Younger Patients with Non-Germinal Centre Diffuse Large B-Cell Lymphoma As Compared to R-CHOP in the GELA Trial LNH03-2B
Authors: Molina Thierry Jo , Canioni Danielle , Copie-Bergman Christiane , Recher Christian , Fournier Marion , Haioun Corinne , Briere Josette , Berger Francoise , Fermé Christophe , Copin Marie-Christine , Casasnovas Rene-Olivier , Thieblemont Catherine , Leroy Karen , Salles Gilles Andre , Jardin Fabrice , Jais Jean-Philippe , Gaulard Philippe , Coiffier Bertrand , Tilly Herve
Published: October 2019

Abstract 2632

Hans algorithm using immunohistochemistry correlates well with gene expression data in Diffuse large B-cell lymphomas (DLBCL) (Meyer PN, 2011) and has demonstrated in some studies clear survival differences in favor of germinal-centre (GC) vs non-germinal centre (n-GC) B-cell among DLBCL treated with R-CHOP. We undertook an immunohistochemical study among patients aged 18 to 59 years with aaIPI 1 included in the GELA trial LNH 03-2B that compared R-ACVBP intensified immunochemotherapy to standard R-CHOP. This trial demonstrated an improvement of EFS, PFS and overall survival (OS) of patients treated with R-ACVBP (C Recher et al, in press). Our goal was to evaluate survival of patients with GC and n-GC DLBCL according to treatment regimens. We analyzed by immunohistochemistry the expression of CD10, BCL6 and MUM1 and classified patients as GC or n-GC according to the Hans algorithm. Among the 380 patients enrolled in this study, 229 patients were available for Hans algorithm classification. There was no differences considering clinical characteristics of these 229 patients (age, sex, B symptoms, PS, Stage, LDH, number of extranodal sites, bulky mass, bone marrow involvement) compared to the whole LNH03-2B population. 175 DLBCL cases were present on a tissue microarray (TMA) and 54 other cases were analyzed using unstained slides. 101 patients were classified as GC and 128 as n-GC. 107 patients were treated by R-ACVBP and 122 by R-CHOP. EFS, PFS and OS were not different between the GC and n-GC profile among the whole population (P=.82, P=.90, P=.68, respectively). There was no statistical difference in EFS, PFS and OS between R-ACVBP and R-CHOP in GC patients (P=.78; P=.84, P=.33, respectively). Interestingly, EFS, PFS and OS were significantly much longer among n-GC patients treated by R-ACVBP compared to R-CHOP (P=.02; P=.007, P=.007, respectively). Results were similar considering only TMA population (P=.02, P=.001, P=.001, respectively). This subgroup analysis suggests that the survival benefit related to R-ACVBP over R-CHOP in the LNH 03-2B is in large part linked to a survival improvement in the n-GC population. This algorithm, easy to apply on routine paraffin-embedded tissue, might be useful in the future to select patients that can primarily benefit from this intensive regimen.

Disclosures:

No relevant conflicts of interest to declare.
Gene Expression Profiling Identifies the JAK/STAT and NFκB Pathways to Be Important in Peripheral T-Cell Lymphomas and Natural-Killer T-Cell Lymphomas
Authors: Tang Tiffany , Allen George , Koo Ghee Chong , Tay Kevin , Tan Daryl , Quek Richard , Tao Miriam , Tan Soo Yong , Teh Bin Tean , Lim Soon Thye
Published: October 2019

Abstract 2658

Background:

Peripheral T-cell lymphomas (PTCL) carry a poorer prognosis compared to their B-cell counterparts and the molecular pathogenesis of PTCL is still largely unknown. The aims of this study are to characterize the molecular signatures and identify signaling pathways involved in the different subsets of PTCL and NKTCL.

Materials and Methods: RNA was extracted from tumors of 60 patients with newly diagnosed PTCL and NKTCL: 21 with angioimmunoblastic T-cell lymphoma (AITL), 12 anaplastic large-cell lymphoma (ALCL), 15 peripheral T-cell lymphoma not-otherwise-specified (PTCL-NOS) and 12 with natural-killer T-cell lymphoma (NKTCL). Comparisons were made using published gene expression data files of normal T and NK T-cells. Gene expression profiling was performed using the Affymetrix HG-U133 Plus 2.0 GeneChip platform.

Results:

The Affymetrix expression profiling distinguishes the 48 PTCL samples from normal T-cell controls (p<0.005). Similarly, the molecular signature of 12 NKTCL significantly differs from normal NK cells. The JAK/STAT pathway is significantly upregulated in AITL and a subset of PTCL-NOS compared to normal T cells and ALCL (p<0.005). [Figure 1] Pathways related to NFκB significantly distinguish PTCL from normal T-cells and it also distinguishes NKTCL from normal NK T-cells. Unsupervised hierarchical clustering by gene expression profiles is able to separate the subsets of PTCL (AILT, ALCL, NKTCL) in concordance with their immmunohistochemical diagnosis. PTCL-NOS however, are dispersed across the clusters reflecting its molecular heterogeneity. [Figure 2]

Discussion:

Gene expression profiling identifies distinct molecular pathways in PTCL. In particular, the JAK-STAT pathway is upregulated in AITL and the NFκB pathway is dysregulated in PTCL and NKCL. Our results suggest that targeting the key kinases in these pathways may be effective in the treatment for this subset of lymphomas with poor prognosis.

Disclosures:

Tan: Janssen: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Research Funding.
Excellent Long-Term Survival of Patients with Waldenström‘s Macroglobulinemia (WM) treated in Private Oncology Practices and a University hospital with different Treatment Strategies
Authors: Hensel Manfred , Brust Jürgen , Plöger Christoph , Schuster Dieter , Memmer Marie-Luise , Rieger Michael , Hensel Lara , Mosthaf Solveigh , Franz-Werner Judith , Feustel Hans-Peter , Mosthaf Franz , Karcher Andreas , Fuxius Stefan , Ho Anthony D. , Witzens-Harig Mathias
Published: October 2019

Abstract 2671

Background:

WM is a rare type of malignant B-cell lymphoma, defined as a lymphoplasmocytic lymphoma in bone marrow and a monoclonal gammopathy of immunoglobulin-M (IgM) type. Due to the paucity of large clinical trials, a standard treatment regimen is still controversial. The purpose of this study was to compare the standard treatment and outcome of patients (pts) treated in private oncology practices (PP) and a university hospital (UH) in a region of southern Germany (Nordbaden).

Methods:

We retrospectively reviewed the charts of all pts with WM of the last 2 decades of 4 PP in Mannheim, Heidelberg, Karlsruhe and Speyer and the department of hematology of the University of Heidelberg.

Results:

170 patients with WM could be identified, 74 from PP, 96 from the UH. The median age of the 170 pts was 63.3 years (range 29.1–88.5). The clinical symptoms were splenomegaly in 21.2%, hyperviscosity in 15.9%, lymphadenopathy in 15.9%, and polyneuropathy in 11.2%. 12.9% had a previous phase of IgM-MGUS. Pts from PP were older (median 65.3 vs. 62.5, p=0.01). Mean hemoglobine level at diagnosis was lower in pts from UH (11.4 vs. 12.2, p=0.04). There was no significant difference in mean platelet count, IgM and ß2-microglobuline level at diagnosis. 54% of pts from PP have received treatment during the observation time, as compared to 78.1% of the UH (p<0.001). The most common first line treatment protocols of PP were Chlorambucil (17/40, 42.5%), Bendamustin (17.5%) and Rituximab(R)-Bendamustin (17.5%). The UH most commonly administered R-Pentostatin-Cyclophosphamide (28%), Chlorambucil (17.3%) and COP (16%). 35% of the treated pts of PP have received R, as compared to 62.6% of the pts of the UH (p<0.001). 60% of the treated pts of PP have received Bendamustin, as compared to 8% of the pts of the UH (p<0.001). The time to first treatment was significantly shorter in pts from the UH (median 13.7 mo.) as compared to PP (52.9 mo.) (p=0.05). Median overall survival of all pts was 25.0 yrs and did not differ between PP and UH.

Conclusion:

Pts with WM treated in PP were older and had higher initial hemoglobin levels as compared to pts from the UH. Treatment differs significantly between PP and UH. Time to first treatment was significantly shorter in pts from UH. Overall survival was excellent, and better than reported previously.

Disclosures:

Hensel: Roche: Honoraria, Research Funding; Mundipharma: Research Funding.