bluuebunny/crossref_metadata_2025 huggingface dataset:
Tandem Autologous Transplant Versus Reduced Intensity Conditioned Allogeneic Transplant (Allo-RIC) as Second Intensification in Chemosensitive Patients with Multiple Myeloma (MM) Not Achieving Complete Remission (CR) or Near-CR with a First Autologous Transplant. Results from a Spanish PETHEMA/GEM Study.
Authors: Blade Joan , Rosinol Laura , Lahuerta Juan Jose , Sureda Anna , de la Rubia Javier , Garcia-Larana Jose , Hernandez-Garcia Miguel , Hernandez-Ruiz Belen , Perez-Simon Jose Antonio , Bello Jose Luis , Carrera Dolores , Penarrubia Maria Jesus , Abella Eugenia , Leon Angel , Poderos Concepcion , Garcia-Ruiz Juan Carlos , Besalduch Joan , Martinez-Martinez Rafael , Perez-Fernandez Isabel , Ribas Paz , Miguel Jesus San
Published: October 2019

One randomized trial showed that tandem transplant resulted in a significantly longer EFS and OS in patients failing to achieve CR or near-CR with a single transplant. However, other studies failed to show benefit from a second transplant. The aim of our study was to investigate the efficacy in terms of response improvement and survival from a second transplant intensification in patients with chemosensitive disease who failed to achieve CR or near-CR with a first high-dose procedure. Patients diagnosed with MM from Oct 1999 to Dec 2004 younger than 70 years received 6 courses of VBMCP/VBAD and responding patients were intensified with busulphan/melphalan or MEL-200 followed by stem cell support. Patients not achieving CR or near-CR were planned to undergo a second transplant (second auto with CVB - cyclophosphamide, etoposide and BCNU - or MEL-200 intensification or an allo-RIC with Fludarabine/MEL-140 conditioning, if sibling donor available). Eighty-eight patients received a second autologous transplant while 26 underwent an allo-RIC. Thirty-seven percent of the patients given a second autologous transplant improved their response status (CR 11%, near-CR: 6%, PR: 9% and MR 11%) while 63% showed “no change”, progressive disease or early death. A response was observed in 45% of patients undergoing the allo-RIC (CR: 33%, PR: 4%, MR: 8%). The CR rate was significantly higher with allo-RIC (33% vs. 11%, p= 0.02). There was a trend towards a higher TRM with the allo-RIC (5% vs. 16%, p=0.09). Although the median EFS (26 vs 19 m) and OS (57 m vs not reached in allo-RIC) from the second high-dose procedure were not significantly different, there is a plateau in the “allo-RIC” group beyond 3 years of the second procedure not observed in the autologous arm.

Conclusions:

an allo-RIC transplant after an autologous procedure results in a significantly higher CR rate than a second autologous transplant, and despite the lack of significant differences in survival between the two transplant modalities, there is a plateau in the allogeneic group not observed in the autologous arm.
Induction of Raf-1 Kinase Inhibitory Protein (RKIP) by the Proteasome Inhibitor NPI-0052 and Reversal of B-NHL Resistance to Apoptosis.
Authors: Baritaki Stavroula , Yeung Kam , Chatterjee Devasis , Palladino Michel , Bonavida Benjamin
Published: October 2019

Patients with B-cell malignancies initially respond to conventional cytotoxic therapies. However, many patients experience relapses and recurrences. Novel therapeutics are being explored in the reversal of resistance such as TRAIL. TRAIL has been shown to be selectively cytotoxic to few tumors and both TRAIL and agonist TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies are currently being explored clinically in unresponsive cancer patients. We have recently found that treatment of TRAIL resistant B-NHL cell lines such as Ramos with the novel proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals) sensitizes tumor cells to TRAIL-induced apoptosis. The concentration of NPI-0052 achieving optimal sensitization to TRAIL was in the range of 1–5 nM. In contrast 400 fold more VelcadeTM (bortezomib) was necessary to achieve a similar level of apoptosis. The mechanism of NPI-0052- mediated sensitization to TRAIL was examined. Treatment of Ramos with NPI-0052 inhibited significantly the constitutively activated NF-κB concomitantly with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) resulting in up-regulation of DR5 expression. The role of YY1 in NPI-0052-induced sensitization to TRAIL was corroborated by the use of YY1siRNA as treatment with the YY1siRNA mimicked NPI-0052 induced DR5 up-regulation and sensitization to TRAIL. Studies examining the inhibition of the NF-κB pathway by NPI-0052 revealed a novel finding demonstrating the induced expression of the metastatic suppressor gene, Raf-1 kinase inhibitory protein (RKIP). RKIP has been reported to inhibit NF-κB activity and, thus, its induction by NPI-0052 contributed to the inhibition of NF-κB activity. The role of RKIP in NPI-0052 induced sensitization to TRAIL was corroborated by the use of RKIPsiRNA and transfectants mimicked NPI-0052 mediated inhibition of YY1, DR5 upregulation and sensitization to TRAIL mediated apoptosis. The above findings suggest that NPI-0052-induced inhibition of NF-κB results at least from two distinct mechanisms, namely, inhibition of p-IκBα degradation by the proteasome and by the induction of RKIP expression. The apoptosis induced by combination of NPI-0052 and TRAIL was the result of the activation of the type II mitochondrial pathway. It has been reported that the RKIP levels are reduced in many tumors (e.g. breast, prostate, ovarian) and the expression levels are of prognostic significance. Hence, the present findings demonstrating NPI-0052 induced RKIP expression may be therapeutically relevant in the prevention of metastasis. In addition, NPI-0052 should facilitate the reversal of tumor resistance when used in combination with subtoxic levels of cytotoxics.
Maintenance Treatment with Azacytidine for Patients with High Risk Myelodysplastic Syndromes or Acute Myeloid Leukaemia in Complete Remission after Intensive Chemotherapy.
Authors: Grövdal Michael , Khan Rasheed , Aggerholm Anni , Antunovic Petar , Astermark Jan , Bernell Per , Engström Lena-Maria , Kjeldsen Lars , Linder Olle , Nilsson Lars , Olsson Anna , Wallvik Jonas , Tangen Jon Magnus , Öberg Gunnar , Jacobsen Sten Eirik , Hokland Peter , Porwit Anna , Hellström-Lindberg Eva
Published: October 2019

Patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) secondary to MDS may reach complete remission (CR) following intensive chemotherapy but the CR-duration is usually very short. Promoter hypermethylation of e.g. tumour suppressor genes is considered to be important in cancerogenesis and a negative risk factor for survival in patients with MDS. We designed a prospective clinical trial to assess the impact of methylation status on the outcome of induction chemotherapy and to evaluate the effect of maintenance treatment with 5-azacytidine (aza) on CR duration. Sixty patients with a median age of 68 years (54–83) with high risk MDS (n=23) or AML (n=37) following MDS were treated with standard doses of daunorubicin and ara-C. Standard prognostic variables, and methylation status of the P15ink4b (P15), E-cadherin (CDH) and Hypermethylated in Cancer 1 (HIC) genes were analyzed before treatment. Patients in CR were given low dose aza subcutaneously for 5 days/4 weeks until relapse. The CR rate was 40%; significantly lower in patients with high white blood cell counts (P=0.03) and high CD34 expression on bone marrow cells (P=0.02). While P15 status alone was not associated with CR rate (P=0.25) patients with CDH methylation showed a lower CR rate (P=0.008). Moreover, no patient with hypermethylation of all three genes achieved CR (P=0.03). CDH methylation retained its prognostic value in the multivariate analysis. Hypermethylation was associated with increased CD34 expression but not with blast count or cytogenetic risk group. Median duration of CR was 13 months (2 to +37) and 7/ 23 patients (30%) had a CR duration >20 months. Three of four patients with trisomy 8 had CR >20 months. The overall survival of patients reaching CR was 17 months (6 to +40). Methylation status before treatment did not correlate with CR duration. We demonstrate for the first time a significant impact of methylation status on the outcome of conventional chemotherapy in high-risk MDS and MDS-AML and propose that this variable should be further evaluated in prospective studies. CR duration is promising, with a substantial portion of patients showing durable responses.
ATR Dependent Phosphorylation of FANCM Is Required for the Fanconi Anemia Pathway.
Authors: Singh Thiyam R. , Ali Abdullah M. , Du Chang-hu , Meetei Ruhikanta A.
Published: October 2019

Fanconi anemia (FA) is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. We have shown that FANCM which is one of the FA core complex proteins is hyperphosphorylated in response to DNA damage suggesting that it may serve as a signal transducer through which the activity of the FA-core complex is regulated. The cell cycle checkpoint kinase, ATR has been shown to act upstream of the FA pathway, however, its substrate within the FA-core complex has not been identified yet. FANCM contains multiple predicted ATR phosphorylation sites suggesting that FANCM could be a direct ATR target. In this study, we examined the roles of ATR in regulating FANCM phosphorylation in response to DNA damage:

by kinetics study we found that phosphorylation of FANCM is concurrent with FANCD2 monoubiquitination; siRNA mediated suppression of ATR activity abrogates both phosphorylation of FANCM and monoubiquitination of FANCD2; and ATR knock out HCT116 cells display defective phosphorylation of FANCM as well as defective monoubiquitination of FANCD2 indicating that DNA damage induced phosphorylation of FANCM is ATR dependant.

Furthermore, we used mass spectrometry to identify the in vivo phosphorylation sites of FANCM and found a novel DNA damage-inducible phosphorylation site (S-1045; one of the potential ATR phosphorylation sites) within FANCM protein. Using ATR knock out HCT116 cells and the anti-p-S1045 antibody, we show that phosphorylation of FANCM at S-1045 is ATR dependant. The biological relevance of phosphorylation of FANCM at S1045 in FA pathway will be investigated by functional complementation analysis with non phosphorylatable FANCM mutants in FANCM deficient cells.
Phase I Study of Triciribine Phosphate Monohydrate, a Specific Inhibitor of AKt Phosphorylation, in Adult Patients with Advanced Hematologic Malignancies.
Authors: Ravandi Farhad , Lancet Jeffrey , Giles Francis , Plunkett William , Williams Betsy , Burton Michelle , Faderl Stefan , Estrov Zeev , Borthakur Gautam , Akinsanmi Lawrence , Kantarjian Hagop
Published: October 2019

Activation of Akt, a serine/thronine protein kinase downstream of PI3 kinase, promotes cell survival through phosphorylation of Bad as well as activation of IKKa-NFkB pathway. Aberrant activation of Akt, by its amplification and overexpression, as well as through the loss of the upstream tumor suppressor PTEN, has been reported in many human cancers and is associated with poor prognosis, resistance to chemotherapy and shortened survival; disruption of Akt pathways inhibits tumor cell growth, angiogenesis and metastasis, and induces apoptosis. Through high through put screening of a library of small molecules, it has been determined that Triciribine Phosphate Monohydrate (TCN-PM), a nucleoside analog, is an inhibitor of Akt activation, and inhibition of Akt activation may be one of its antiproliferative mechanisms. We have conducted a phase I study of TCN-PM in patients with advanced hematological malignancies. Cohorts of 3 patients receive escalating doses of TCN-PM at 15, 25, 35, and 45 mg/m2 administered IV on days 1, 8, and 15 of a 28 day cycle. Twenty four (19M, 5F) patients have been enrolled in 2 institutions and have received a median of 1 cycle (range, 1 – 3) of TCN-PM including 4 in cohort I, 4 in cohort II and 16 in cohort III. Median age of the patients is 60 (range, 30 – 79). Twenty two patients had AML, 1 CLL, and 1 MDS. Median number of prior therapy before inclusion in the study was 3 (range, 1 – 7). Ten patients were inevaluable including 1 in cohort I, 1 in cohort II, and 8 in cohort III due to disease progression (in 7) and other (in 3). Dose limiting toxicity of mucositis was observed in a patient on the third cohort resulting in expansion of that cohort. No further incidences of mucositis were seen in the expanded cohort. No other severe adverse events related to treatment have been observed. 2 patients have achieved major improvements in platelet count lasting 7 and 36 days. One patient achieved a minor improvement in platelet count. Four patients have achieved major improvements in neutrophil count lasting a median of 19 (range, 8 – 40) days while on therapy. Cell death, measured by annexin-5 staining was examined in pre and post treatment samples in 14 patients; in 2 patients there was a significant increase in apoptosis to 25% and 27% after TCN-PM but this did not correlate with response. Pharmacodynamic studies evaluating inhibition of Akt phosphorylation as well as pharmacokinetic data will be presented. Accrual to higher dose levels is continuing.
Purinergic Signaling Differentially Modulates Normal and Leukemic Hematopoiesis.
Authors: Salvestrini Valentina , Gulinelli Sara , Zini Roberta , Caione Luisa , Migliardi Giorgia , Piacibello Wanda , Ferrari Davide , Virgilio Francesco Di , Ferrari Sergio , Manfredini Rossella , Lemoli Roberto M
Published: October 2019

Abstract 1436

Poster Board I-459

Extracellular nucleotides adenosine and uridine triphosphates (ATP and UTP, respectively) are emerging as ubiquitous molecules involved in a wide variety of biological responses and their biological effects are mediated by specific plasma membrane receptors, P2 receptors (P2R). Previously, we showed that extracellular nucleotides stimulate the proliferation and engraftment potential of normal human hematopoietic stem cells. In this study, we assessed whether P2R are expressed on acute myeloblastic leukemia (AML) cells and whether their engagement modulates leukemic cell functions. We show that AML cells express several functional P2R subtypes at the molecular and protein level. Stimulation of AML cells by extracellular nucleotides induced intracellular Ca2+ concentration increases. Furthermore, gene expression profiling revealed that leukemic cells stimulated with ATP underwent a down-regulation of genes involved in cell proliferation and migration whereas those involved in cell cycle inhibition were strongly up-regulated. At the functional level, the clonogenic efficiency of leukemic blasts and CD34+CD38− leukemic stem/progenitor cells was significantly inhibited by the addition of ATP and, to a higher extent, by the stable analogs INS415 and INS973. We also observed a pronounced inhibitory effect of triphosphate nucleotides on spontaneous migration of AML cells and in response to CXCL12.In vivo, xenotransplant experiments demonstrated that the homing and the engraftment capacity of human AML blasts and leukemic stem cells to NOD/SCID/IL-2RGamma-Null mice was significantly inhibited by pre-treatment with ATP, UTP and INS415 and INS973 analogues. Thus, our data show that purinergic signaling modulates leukemic cells in a opposite way than normal cells. Characterization of purinergic signalling in leukemia may help the better understading of the mechanism of neoplastic transformation and tumor progression.

Disclosures

Di Virgilio: F. Di Virgilio serves as a consultant for Cordex Pharma Inc. (USA) involved in the development of “P2 receptor-based drugs”.: Consultancy; F. Di Virgilio serves as a consultant for Affectis Pharmaceuticals AG (Germany), involved in the development of “P2 receptor-based drugs”.: Consultancy.
Quantitative Imaging of Femoral Bone Marrow Microenvironments Reveals a Heterogenous Distribution of Hematopoietic Stem and Progenitor Cells.
Authors: Nombela-Arrieta Cesar , Harley Brendan , Pivarnik Gregory , Mahoney John E , Levantini Elena , Protopopov Alexei , Tenen Daniel G. , Silberstein Leslie E.
Published: October 2019

Abstract 1455

Poster Board I-478

Sustained production of all mature blood cell types relies on the continuous proliferation and differentiation of a rare population of self-renewing, multipotent hematopoietic stem cells (HSCs). HSC maintenance and lineage differentiation are thought to be regulated by spatially confined niches, defined by cellular components, soluble regulators, and the extracellular matrix immediately surrounding stem cells. Identification of these microenvironments in which endogenous and transferred HSCs reside within the BM is a major challenge in stem cell biology with relevant clinical implications. Yet the extreme rarity of HSCs, their dynamic nature, and the lack of specific markers to identify them, have precluded an accurate definition of HSC niches to date. Quantitative imaging technologies such as Laser Scanning Cytometry (LSC) are designed for the automated analysis of large cell numbers at a single cell level with high resolution while preserving the morphological information lost in flow cytometry, therefore providing data of statistical significance even for rare cell populations such as HSCs. We have employed LSC to analyze the localization of both adoptively transferred and endogenous hematopoietic stem and progenitor cell (HSPC) populations inside whole longitudinal sections of murine femoral BM cavities.

Our results indicate that, as previously suggested, purified HSPC (Lin−c-kit+Sca-1+) significantly accumulate in endosteal regions (ER) of BM cavities (within 100μm of inner bone surface) upon transplantation. Nevertheless, analysis of sufficient numbers of more differentiated cell subsets (Lin−c-kit+Sca-1− progenitors, pro B cells and mature B cells) indicated that these areas serve as homing sites for most hematopoietic cells, highlighting the limitations of any conclusions drawn on HSC niche identity from studies performed with transferred HSPC populations. Immunofluorescent staining of endogenous cell populations revealed a gradient in distribution of early hematopoietic progenitors (c-kit+), which accumulated in but were not restricted to ER regions. Of note, a vast majority (>80%) of HSPC (Bmi-GFPhic-kit+, or Lin−c-kit+Sca-1+),were found inside ER, although not directly adjacent to endosteal surfaces. Our studies define endosteal areas as tissue regions where HSPC reside in close proximity, but not necessarily in direct contact with a dense vascular network, osteoblastic cells and other potential niche cell types and growth factors currently under investigation.

Disclosures

No relevant conflicts of interest to declare.
Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.
Authors: Andre-Schmutz Isabelle , Ditadi Andrea , Boudil Amine , Ezine Sophie , Cavazzana-Calvo Marina
Published: October 2019

Abstract 1475

Poster Board I-498

We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells.

Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression.

Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications.

1. Ditadi 2009

Disclosures:

No relevant conflicts of interest to declare.
Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.
Authors: Shinnick Kathryn M. , Barry Kelly A. , Eklund Elizabeth A. , McGarry Thomas J.
Published: October 2019

Abstract 1478

Poster Board I-501

Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.
Authors: Merchant Akil , Singh Anju , Joseph Giselle , Wang Qiuju , Zhang Ping , Brennan Sarah , Biswal Shyam , Matsui William
Published: October 2019

Abstract 1492

Poster Board I-515

Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling.

Disclosures:

No relevant conflicts of interest to declare.
NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.
Authors: Bungartz Gerd , Garrett Russell , Emerson Stephen G.
Published: October 2019

Abstract 1507

Poster Board I-530

Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion.

Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status.

To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function.

Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature.

Disclosures

No relevant conflicts of interest to declare.
FcγRIIa Rescues Normally-Poor αIIbβ3-Mediated Outside-in Signaling in Mouse Platelets.
Authors: Boylan Brian , McKenzie Steven E. , Newman Debra K. , Newman Peter J.
Published: October 2019

Abstract 153

Outside-in signal transduction is one of several amplification pathways that platelets employ following their adhesion to components of the extracellular matrix or to multivalent ligands like fibrinogen and von Willebrand factor. Previous studies have shown that outside-in signal transduction initiated by the binding of fibrinogen to its receptor, the integrin αIIbβ3, results in the activation of β3-associated Src family kinases (J Cell Biol 157:265, 2002; J Cell Sci 121:1641, 2008) that phosphorylate key tyrosine residues within the cytoplasmic domain of the transmembrane immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, Fcγ;RIIa (Blood 112:2780, 2008). “Activation” of FcγRIIa sets off a cascade of events that facilitate assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2. PLCγ2, through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. Curiously, mice lack the gene encoding FcγRIIa, and murine platelets are notoriously poor at sending αIIbβ3-mediated outside-in signals into the cell. The purpose of the current investigation, therefore, was to determine whether enforced expression of FcγRIIa in mouse platelets might serve to amplify murine platelet activation downstream of ligand binding to αIIbβ3. We found that the spreading of platelets derived from FcγRIIa transgenic mice was more robust and extensive compared to that of their wild-type counterparts. ADP- and collagen related peptide-induced granule secretion, as measured by P-selectin exposure, was also enhanced, as was the degree of integrin of αIIbβ3 activation, as reported by the binding of the activation-dependent monoclonal antibody, JON/A. Taken together, these data provide further support for importance of FcγRIIa in transmitting αIIbβ3-mediated amplification signals into the cell, and help to explain the often-observed defect in αIIbβ3-mediated signal transduction in murine platelets.

Disclosures:

Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.
Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).
Authors: Uhrmacher Sabrina , Hertweck Magdalena , Paesler Julian , Erdfelder Felix , Filipovich Alexandra , Poll-Wolbeck Simon Jonas , Razavi Regina , Gandhirajan Rajesh Kumar , Gehrke Iris , Hallek Michael , Kreuzer Karl Anton
Published: October 2019

Abstract 1587

Poster Board I-613

In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced.

Disclosures

Hallek: Roche: Consultancy, Honoraria, Research Funding.
Mutation in the Nucleophosmin Gene (NPM1) Is a Stable Marker for Minimal Residual Disease Monitoring in Acute Myeloid Leukemia Patients with Increased Sensitivity and Specificity Compared to Expression of the Wilms Tumor (WT1) Gene.
Authors: Kristensen Thomas , Preiss Birgitte Strange , Friis Lone , Møller Michael B.
Published: October 2019

Abstract 1602

Poster Board I-628

Mutation in exon 12 of the nucleophosmin (NPM1) gene occurs in approximately 60% of acute myeloid leukemia (AML) patients with normal karyotype. To date, molecular minimal residual disease (MRD) monitoring in this patient group has primarily been based on expression of the Wilms tumor gene (WT1), although expression of WT1 in non-leukemia cells limits the specificity of this marker. Mutation in the NPM1 gene is potentially a superior MRD marker compared to WT1 gene expression by being specific to the malignant clone. The use of NPM1 mutation as a MRD marker would furthermore be in line with the widespread use of leukemia cell specific fusion-genes as MRD markers in AML patients with balanced translocations. In the present study, we therefore evaluated NPM1 mutation as a MRD marker with respect to stability, sensitivity and specificity in direct comparison to WT1 gene expression. A total of 13 relapsed AML patients with normal karyotype that were positive for mutation in NPM1 and WT1 gene expression at the time of diagnosis were included in the study. The NPM1 mutational load and WT1 gene expression was analyzed by real-time qPCR in up to 22 peripheral blood mononuclear cell samples per patient from the time of primary diagnosis to latest follow-up to compare the kinetics of the two markers during periods of morphological remission and relapse events. The 13 patients experienced a total of 18 morphological relapses which were all accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of NPM1 mutation during relapse in the present material. During periods of complete morphological remission, the NPM1 mutational load was below detection limit (< 1/1000 cells) in all samples. In contrast, WT1 gene expression was detectable in 70% of these samples, thus demonstrating the limited specificity of this marker. This background WT1 expression in non-leukemia cells reached levels of up to 1% of the levels detected at the time of diagnosis thus limiting the de facto MRD marker sensitivity of WT1. All samples with detectable levels of NPM1 mutation after a period of complete molecular remission were followed by a morphological relapse within weeks. The present study therefore shows that mutation in NPM1 is a stable and more sensitive and specific, and therefore superior, molecular MRD marker than WT1.

Disclosures

No relevant conflicts of interest to declare.
Excellent Prognosis of Children with ETV6-RUNX1 Positive (+) Acute Lymphoblastic Leukemia (ALL) in the FRALLE 2000 Protocol.
Authors: Gandemer Virginie , Auclerc Marie-Françoise , Petit Arnaud , Brethon Benoît , Ballerini Paola , Michel Gerard , Perel Yves , Auvrignon Anne , Mechinaud Francoise , Schmitt Claudine , Schneider Pascale , Vermylen Christiane , Lejars Odile , Leblanc Thierry , Gabert Jean , Macintyre Elizabeth , Cayuela Jean-Michel , Leverger Guy , Baruchel Andre
Published: October 2019

Abstract 1628

Poster Board I-654

Since the cloning of the t(12 ;21) in 1995 the prognosis of children with ETV6-RUNX1(+)ALL seems to further increase in the current era. From december 2000 to july 2008, 1461 children and adolescents aged from 1 to 20 years with B cell lineage ALL have been treated according the FRALLE protocols: one for standard-risk (SR) ALL (age 1-9 y, WBC < 50 G/L, no extra medullary involvement) F2000-A; the other one FRALLE 2000-B for high and very high risk ALL (HR) (all other pts). The two protocols mainly differ by a larger use of dexamethasone and a reduced use of anthracyclins in F2000A and the use of HDMTX and a double delayed intensification in FRALLE 2000B. ETV6-RUNX1 presence has been assessed by RT-PCR in 1392 pts (i.e. 96%) and found positive in 321 pts, i.e. 23%. Initial features include a median age of 4.2 y (1.2-15.6), a sex ratio (M/F) of 1.2, a median WBC of 10 G/L(1-293). Both peripheral blood D8 response to prednisone (PRED) and D21 marrow response to chemotherapy were evaluable in 316 pts: 94% of the pts have a rapid early response at D8 and D21. Only 3% of the pts were qualified as D8 poor PRED responders and 3% slow marrow responders at D21. The D35-42 CR rate is 100%. End of induction (EOI) minimal residual disease (MRD) using Ig-TCR methods is known in 259 out of 321 pts (81%) pts. Only 4 pts (2%) had a very high (≥10-2) EOI-MRD. Five year EFS, DFS and OS are 95±2%, 95±2%, 98±1%, respectively. The 5y EFS is 96±2% for the 252 SR pts and 90±7% for the 69 HR pts, p=0.30. The 5y EFS is significantly better for children with ETV6-RUNX1(+)ALL compared to those with ETV6-RUNX1(-) B-lineage ALL: 95±2% vs 84±2%, respectively (p=.001). Nine relapses have been reported in the bone marrow (4) the testis (4), the CNS (1) after a median time of 44 m (25-68), i.e. significantly longer than in the ETV6-RUNX1(-) cases (28m (1-92), p=.01). More testis relapses are observed in boys with ETV6-RUNX1(+)ALL (4 out 7 relapses vs 2 out of 62 in boys with ETV6-RUNX1(-) ALL). These results represent a significant progress compared to the previous protocol F 93 (191 children) both in terms of EFS and overall survival (5y EFS: 95±2% vs 78±3%, p=.001; 5 y OS: 98±1% vs 92±2%, p=.001).

Conclusion

an excellent prognosis of children with t(12;21)/ETV6-RUNX1 positive acute lymphoblastic leukemia is now observed in the FRALLE 2000 protocol. The question of a cautious de-escalation in this subgroup will be envisaged in the next protocol.

Disclosures

No relevant conflicts of interest to declare.
C-Myc Is Required for Acute but Not for Chronic Hematopoietic Malignances in Pten-Null Mice.
Authors: Guo Yinshi , Seet Christopher , Niu Chao , Breslin Peter , Zhang Shubin , Wei Wei , Diaz Manuel , Stiff Patrick , Zhang Jiwang
Published: October 2019

Abstract 1632

Poster Board I-658

c-Myc, the product of an oncogene, is a common target of most leukemic oncoproteins. Deregulation of c-Myc is commonly found in human leukemic blasts. Transgenic over-expression of c-Myc induces myeloid, erythroid, and lymphocytic leukemia in mice, however whether c-Myc is absolutely required for leukemogenesis has not yet been addressed.

Pten, a tumor-suppressing phosphatase, inhibits cell proliferation and promotes apoptotic cell death through repression of PI3K-Akt signaling. Inactivating mutations of Pten and deregulation of PI3K/Akt signaling are both involved in the development of both chronic and acute hematopoietic malignances. Mice with Pten deletions in hematopoietic stem cells (HSCs) develop myeloproliferative disorders (MPD) followed by acute T lymphocytic or myeloid leukemia, reminiscent of disease progression in human chronic myelogenous leukemia. Mice with Pten deletions in lymphocytes develop lymphadenopathy (due to a chronic polyclonal lymphoproliferative disorder) as well as CD4+ T lymphocytic lymphoma. The appearance of these diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously.

To study whether c-Myc is required for the development of these hematopoietic disorders in Pten-mutant mice, we generated inducible Pten and c-Myc double-knockout mice (Pten-/-c-Myc-/-). By comparing the hematopoietic phenotypes of the Pten-/-c-Myc-/- mice with those of Pten-mutant (Pten-/-) mice, we found that both sets of mice developed MPD and lymphadenopathy, however none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPD which developed in Pten-/- mice, which is dominated by granulocytes, megakaryocytes predominate in the MPD of Pten-/-c-Myc-/- mice.

We have concluded that c-Myc is required for the development of both T lymphocytic lymphoma and the acute leukemic transformation of Pten-/- MPD, but is not essential for the development of chronic myeloid or lymphoid proliferative disorders. Our study suggests that deregulation of PI3K/Akt signaling in Pten-mutant hematopoietic cells protects these cells from apoptotic cell death, which results in chronic proliferative disorders, while the deregulation of c-Myc resulting from additional mutations promotes hematopoietic cell proliferation and blockage of maturation, and is absolutely required for the development of acute hematopoietic malignances.

Disclosures

No relevant conflicts of interest to declare.
Characterization of Treg Subpopulations in Chronic Lymphocytic Leukemia Using 15 Color Flow Cytometry.
Authors: Biancotto Angelique , Fuchs John Chris , Dagur Pradeep , Wiestner Adrian , McCoy J. Philip
Published: October 2019

Abstract 1644

Poster Board I-670

CD4+ regulatory T cells (Treg) act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have been shown to be increased in the peripheral circulation of patients with various types of cancer including CLL and have been postulated to play a role in inhibition of anti-tumor immune responses. 15 color staining was performed on mononuclear cells isolated from peripheral blood from 33 healthy controls and 12 patients with chronic lymphoid leukemia (CLL). Treg cells were defined as viable CD45+ CD3+ CD8- CD4+ CD25+ Forkhead box P3+ (FoxP3)+ mononuclear cells. Among the Treg population we defined 4 sub-populations; Naïve Treg (CD45-RA+ CD27+ CD197+), Effector Treg (CD45-RA- CD27- CD197-), Central memory Treg (CD45-RA- CD27+ CD197+) and Effector memory Treg (CD45-RA- CD27+ CD197-). We report level of expression of activation markers CD39, HLA-DR and CD38 and IL-7 receptor CD127 on Treg subsets as defined above. The percentage of Treg was increased significantly in CLL patients compared with healthy controls 12.8±1.3% vs 5.7±0.3% (p<0.0001). In healthy controls, the majority of Treg are CD45-RA- (80.2±2.9 %) whereas in CLL patients, we observed Treg cells in both CD45-RA- and CD45-RA+ populations (62.6±4.9% and 40.4±5% respectively). Inside the CD45-RA+ populations, we showed that there was a significant higher proportion of naïve Treg among CLL patients compare to healthy controls ((83.8±5% and 41.8±5% respectively (p<0.0001)). Among the CD45-RA- populations, there was no significant difference in the proportion of each memory subtypes. We observed a different pattern of activation among the Treg in CLL patients, naïve Treg expressed more CD39 and HLA-DR compare to healthy controls (2x to 3x fold increase, p<0.0001), but less CD38. In central memory subpopulation, CD39 and CD127 expressions were increased in Treg isolated from CLL patients (p=0.04 and p=0.01 respectively), whereas CD38 was decreased (p=0.01). In effector memory population, HLA-DR expression was lower in CLL patients compared to healthy control (3±0.9% and 9.8±0.8% respectively, p<0.0001). Finally, in the effector subpopulation, HLA-DR expression was also decreased in Treg from CLL patients (p<0.0001). In summary, in these studies, we showed proportions of naïve and memory Treg as well as a different activation pattern of Treg cells present in CLL patients compared to healthy controls. The assessment of complex patterns of sub-phenotype expression in diseases vis a vis healthy individuals should expand our ability to understand the intricacies of both adaptive and innate cellular immune responses and their dysregulation in cancer patients.

Disclosures

No relevant conflicts of interest to declare.