bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells
Authors: Doulatov Sergei , Vo Linda T , Macari Elizabeth R , Chou Stephanie S , Gupta Manav , Godfrey Michelle , McGrath Katherine , Schlaeger Thorsten M , Zon Leonard I. , Daley George Q.
Published: October 2019

Human induced pluripotent stem cells (iPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells has limited their use for modeling of hematological diseases. We previously reported a strategy to respecify lineage-restricted CD34+hematopoietic precursors derived from iPSCs into multilineage progenitors that can be expanded in vitro and transplanted in vivo. Five transcription factors, HOXA9, ERG, RORA, SOX4 and MYB, enable expansion and maintenance of primitive CD34+CD38- cells, and allow their differentiation upon transgene silencing. Respecified iPSC-derived hematopoietic progenitors give rise to robust short-term engraftment with myeloid and erythroid lineages. Notably, the erythrocytes undergo definitive maturation and hemoglobin switching to express β-globin in vivo. This system presents a useful platform for modeling hematological disorders due to its capacity to generate large numbers of engraftable disease cells for in vitro and in vivo screens.

Congenital anemias, such as Diamond Blackfan anemia (DBA) represent a defined system for understanding red blood cell development and more common idiopathic anemias. To model this disease in vitro, we combined factor-induced respecification with stepwise erythroid maturation. We show that respecified iPSCs from DBA patients recapitulate the defect in erythroid differentiation. Consistent with clinical observations, early erythroblasts show impaired proliferation marked by increased apoptosis and p21 expression, while terminal maturation is unaffected. Interestingly, while early passage iPSC lines display a profound erythroid defect, continuous passage restores a near-normal capacity for erythroid differentiation. Furthermore, transplanted DBA iPSC-derived progenitors give rise to normal myeloid, but fail to generate erythroid, engraftment. This validates this system as a powerful tool for disease modeling and drug discovery. Mice transplanted with DBA iPSC-derived progenitors treated with conventional anemia drugs, such as dexamethasone, show a modest improvement in human erythroid output suggesting the need for novel treatments. Using high-throughput chemical screens, we have identified several signaling pathways which may be attractive therapeutic targets in DBA and other anemias. Further characterization is currently underway to better target defective erythropoiesis and dissect underlying disease mechanisms.

Disclosures

Zon: FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Syk and Btk Transduce Survival and Proliferation-Inducing Signals By Activating Distinct Signaling Pathways and Transcriptional Programs in AML Cells
Authors: Oellerich Thomas , Mohr Sebastian , Beck Julia , Bohnenberger Hanibal , Doebele Carmen , Corso Jasmin , Bug Gesine , Schuetz Ekkehard , Urlaub Henning , Serve Hubert
Published: October 2019

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple driver mutations coincide in branching hematopoietic subclones leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as FLT3, thereby helping AML cells to overcome the physiological communication with their microenvironment. To investigate, if also non-mutated signaling molecules might be potential drug targets in AML, we performed an unbiased proteomic kinase activity profiling. Our (phospho)proteomic and functional analyses revealed spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) as novel non-mutated drug targets in AML. By immunohistochemical analyses we identified expression of both kinases in more than 70% of analyzed AML cases. Oncogenic SYK signaling in AML cells turned out to be critically regulated by beta-integrins upon intercellular contact formation between AML cells and bone marrow stroma cells. BTK, however, turned out to act downstream of FLT3-ITD to activate MYC-dependent oncogenic transcriptional programs. FLT3-ITD expression induced activation of BTK in cell culture models and patient-derived AML cultures and sensitized them towards BTK inhibitors. This newly identified FLT3-ITD/BTK signaling axis can be therapeutically exploited by combining FLT3 and BTK inhibitors resulting in additive apoptosis-inducing effects. As in some cases also FLT3-ITD-negative AML cells showed response to BTK inhibition, we characterized the BTK interactome and its downstream signaling networks by quantitative proteomic techniques and identified the apoptosis sensor Toll-like receptor 9 (TLR9) as an upstream activator of BTK in FLT-ITD-negative cells. In the absence of FLT3-ITD, TLR9 activates BTK to induce cell survival through NFkB as revealed by transcriptome sequencing and cell biological studies. While TLR9 stimulation enhanced AML cell proliferation, knock-down of TLR9 induced a partial cell cycle arrest and apoptosis. Thus, we here describe a novel microenvironment-derived signaling axis that may represent an important mechanism of AML maintenance in various important clinical settings including efficient FLT3-ITD inhibition and cytotoxic therapy. Taken together, these data highlight the importance of interactions between AML cells and their microenvironment and unravel cooperative dependencies between mutant oncoproteins and wild-type kinases in AML pathogenesis. They also provide a rationale for the clinical evaluation of SYK- and BTK inhibitors in AML.

Disclosures

Beck: Chronic Biomedical: Employment. Schuetz:Chronix Biomedical: Employment.
Reversal Multidrug Resistance and Biodistribution of Targeted Nanoparticles Tf-Peg-PLL-PLGA Combined with Daunorubicin and Tetrandrine in Tumor-Bearing Nude Mice
Authors: Guo Liting , Liu Ping , Wang Fei , Xia Guohua , Wang Yonglu , Chen Wei , Yin Haixiang , Chen Baoan
Published: October 2019

The study was to investigate the reversal multidrug resistance treatment and biodistribution of nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) (PEG-PLL-PLGA) modified by transferrin (Tf) combined with daunorubicin (DNR) and tetrandrine (Tet) in leukemia bearing nude mice model. Anti-tumor effect of nanoparticles in nude mice with K562/A02 resistant leukemia cell lines xenografts was observed. Subsequently, transcription and protein expression levels of transferrin receptor (TfR), P-glycoprotein (P-gp), MRP and NF-κB were determined by quantitative real-time polymerase chain reaction (quantitative real-time PCR) and Western blot analysis. DNR concentration in plasma, tumor tissue and major organs was detected by high performance liquid chromatography (HPLC). DNR-Tet-Tf-PEG-PLL-PLGA nanoparticles group significantly inhibited tumor, while the immunohistochemistry of tumor tissue displayed that the nanoparticles can induce apoptosis of tumor cells. Furthermore, DNR-Tet-Tf-PEG-PLL-PLGA group markedly increased TfR expression, and noticeably decreased P-gp, MRP and NF-kB expression. DNR concentration of tumor tissues in DNR-Tet-Tf-PEG-PLL-PLGA group injected by the tail vein was significantly higher than DNR-Tet-PEG-PLL-PLGA group. DNR-Tet-Tf-PEG-PLL-PLGA nanoparticles have the good ability to active targeting and reverse drug resistance, therefore provide a new initiative targeted therapy for cancer.

Disclosures

No relevant conflicts of interest to declare.
Lymphoproliferative Disease in Wiskott-Aldrich Syndrome: Analysis of the French National Registry of Primary Immunodeficiencies
Authors: Cheminant Morgane , Mahlaoui Nizar , Canioni Danielle , Desconclois Celine , Hermine Olivier , Pasquet Marlène , Malphettes Marion , Bertrand Yves , Blanche Stephane , Fischer Alain , Suarez Felipe
Published: October 2019

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency (PID) characterized by persistent thrombocytopenia, eczema and immunodeficiency. It has been suggested that these patients had an increased risk of lymphoproliferative diseases (LPD).

We conducted a retrospective study in order to describe LPD occurring in patients with WAS/XLT (X Linked Thrombocytopenia with WAS gene mutation) enrolled in the French national registry for PIDs. Post- hematopoietic stem cell transplantation (HSCT) LPD were excluded. Methodology of the French National Registry for PIDs has been previously described (Clin. Immunol, 2010, ASH 2009 abstract#23950).

Eight patients (4%) with LPD were identified among the 197 patients of our cohort. All patients had a WAS and presented first symptoms of WAS before the age of one year. One patient developed a LPD before the clinical diagnosis of WAS.

All patients for whom histological charts were available (6/8) developed a B-cell LPD. Three of them had diffuse large B-cell lymphomas and three patients had polymorphic B-cell LPD associated with EBV and without detection of clonal IGHV rearrangement. The median age of cancer diagnosis was 18.3 years (range: 2.0-50.7). All LPD were disseminated diseases, mostly with a central nervous system involvement (4/6 patients). All patients received intensive chemotherapy, except one patient with an EBV-associated LPD who only received rituximab. Three patients obtained a complete remission (CR), two patients had a stable disease (SD) and the last one progressed on therapy. Four patients, of whom two were in CR and one in SD, received an allogeneic HSCT during the year following the diagnosis of LPD. Two patients received peripheral blood-derived stem cells and the two other patients received bone marrow-derived stem cells. There were three matched unrelated donor (MUD) and one haplotype-mismatched family donor, with no ex vivo graft manipulation. Three of these four patients were long-term survivors after LPD (2.7, 7.7 and 9.8 years).

The median life expectancy for the entire cohort is 19.1 years (range: 7.8-52.0). Three patients, two of which have received an HSCT, are still alive 2.4, 2.7 and 9.8 years after the cancer onset. The median overall survival among the five remaining patients after diagnosis of LPD was 0.5 years (from 0.3 to 7.7 years).

Additional details on genetic, clinical characteristics and a centralized histopathology review are currently being studied and will be presented.

In conclusion, the occurrence of LPD is less frequent than expected, may occur only in patients with WAS phenotype rather than XLT. As it impairs the prognosis of patients, HSCT could be a therapeutic option for these patients, even in partial remission of LPD.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Natural Killer Cell Responses By Dendritic Cells Via Lymphotoxin-Alpha, Interleukin-12, and Tumor Growth Factor-Beta
Authors: Sarhan Dhifaf , Palma Marzia , Mao Yumeng , Adamson Lars , Kiessling Rolf , Mellstedt Håkan , Österborg Anders , Lundqvist Andreas
Published: October 2019

Dendritic cells (DC) induce T-cell responses in cancer patients. However, less is known about the role of DC-vaccines in shaping natural killer (NK) cell responses. In this study, we sought to investigate the activity of NK cells following vaccination with DC. Patients with chronic lymphocytic leukemia (CLL) and melanoma were vaccinated with tumor-antigen loaded monocytes-derived DC and NK cell activity was analyzed before and two weeks after vaccination. Compared with pre-vaccination samples, NK cells expressed reduced levels of several activation markers and produced less IFNγ (p=0.03). To further explore the mechanism of the reduced NK cell responses, DC were differentiated from monocytes in GM-CSF, IL-4 and LPS and thereafter co-cultured with autologous NK cells in vitro. In these experiments, DC-mediated inhibition of NK cells was independent of cell-contact but dependent on the phosphorylation status of STAT3 in DC. Compared with untreated DC, STAT3-inhibited-DC produced higher amounts of lymphotoxin-alpha (LTα, 5.5±5.0 vs. 38.1±25.9 pg/ml; p=0.005) and IL-12 (29.6±39.0 vs. 176.8±96.8 pg/ml; p=0.005) and reduced amounts of tumor growth factor-beta (TGFβ, 495.0±138.7 vs. 300.4±141.8 pg/ml; p=0.03) and did not suppress NK cell proliferation (p=0.03), cytotoxicity (p=0.0003), and IFNγ production (p=0.02). Neutralization of TGFβ resulted in elevated production of LTα (2.25±4.5 vs. 251.5±12.0 pg/ml; p=0.03) and IL-12 (0.25±0.5 vs. 15.0±1.82 pg/ml; p=0.02) by DC and addition of recombinant LTα or IL-12 in co-cultures of DC and NK cells was indispensable to maintain NK cell activity. Compared with LPS, DC matured with a cocktail of R848, poly I:C, and IFNγ showed reduced levels of pSTAT3 and produced higher levels of LTα and IL-12 and did not inhibit NK cell activity. These results show that LTα, IL-12 and TGFβ are involved in the cross-talk between NK cells and DC. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.

Disclosures

Österborg: Celgene: DMC memeber of Celgene-initiated phase 3 trials Other.
The Role of CD11b+CX3CR1+ Cells in Neovascularization
Authors: Kim Jeong-A , Kwak Jae-Yong , Noh Kyoungmi , Lee Joon , Park Youngrok , Broxmeyer Hal E.
Published: October 2019

There are growing evidences that myelo-monocytic cells play a key role in mediating tissue repair and stimulating angiogenesis in response to ischemia. However, the subpopulations of myelo-monocytic cells participating in vascularization and their underlying mechanisms have not been well identified. We previously demonstrated that CD11b+CX3CR1+ cells, a type of proangiogenic myelo-monocytic cells, promotes angiogenesis in a C57BL/6 hind-limb ischemic mouse model. We now provide insight into mechanisms of CD11b+CX3CR1+ cells during recovery of blood perfusion after ischemia.

Ischemic injuries were induced by dissecting the femoral artery of C57BL/6 mice. Single-cell suspensions were made from ischemic muscles, and ischemic muscle-derived CD11b+CX3CR1+ cells were isolated with a FACS Sorter. Extracted proteins (50ug) from the cells were loaded on nitrocellulose membranes, containing 53 different capture antibodies in proteome profiler mouse angiogenesis Kits (R&D systems, ARY105). For aortic ring sprouting assay, thoracic aortas were embedded in Matrigel.

Proangiogenic effects of CD11b+CX3CR1+ cells were confirmed by enhanced blood perfusion upon injection of additional CD11b+CX3CR1+ cells into ischemic tissues after femoral artery dissection in C57BL/6 mice. Sprouting effects were specifically confirmed through purified CD11b+CX3CR1+ cells co-cultured with aortic ring sprouting assay. Also, medium conditioned by CD11b+CX3CR1+ cells was as efficient as CD11b+CX3CR1+ cells in supporting sprouting, suggesting that vascular endothelial cells (ECs) are likely activated by the proteins secreted by CD11b+CX3CR1+ cells in a paracrine fashion. To investigate whether muscle-derived CD11b+CX3CR1+ cells could secrete angiogenic proteins, we examined the angiogenic proteins in CD11b+CX3CR1+ cells isolated from ischemic muscles after 4 days of surgery. Contrary to our expectations, VEGF proteins were not detected from CD11b+CX3CR1+ cells. However, MCP-1, MIP-1α, platelet factor-4 (PF-4) and MMP-9 were expressed at a higher level in CD11b+CX3CR1+ cells than those of CD11b+CX3CR1- and CD11b-CX3CR1- cells. MCP-1, MIP-1α and MMP-9 have been reported to promote angiogenesis, while PF-4 is recognized as an angiostatic factor. However, it has recently been reported that PF-4 combined with VEGF is essential for the initial separation of ECs from vessels prior to angiogenic sprouting. It is postulated that, after ischemia, collaboration of VEGF with PF-4 secreted from CD11b+CX3CR1+ cells dissociates the ECs from vessels and promotes vascular branching processes.

In summary, CD11b+CX3CR1+ cells are a newly identified myelo-monocytic subset for vessel formation, capable of vascular sprouting via secreting PF-4.

Disclosures

Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.
Similar Levels of Complement Activation in Both Patients with Thrombotic Thrombocytopenic Purpura and Atypical Hemolytic Uremic Syndrome: The Report from the Korean TTP Registry
Authors: Oh Doyeun , Huh Ji Young , Chong So Young , Kim In-Ho , Bang Soo-Mee , Jung Chul-Won , Lee Jong-Wook , Lee Hong Ghi , Lee Sun Min , Kim Yeo-Kyeoung , Park Yong , Jang Seongsoo , Kim Jin-Seok , Jo Deog-Yeon , Zang Dae Young , Yoon Hwi-Joong , Yhim Ho-Young , Sohn Sang Kyun , Hong Junshik , Lee Won-Sik , Baker Ross I
Published: October 2019

Background: Uncontrolled complement activation has a major role in the pathogenesis of atypical HUS (aHUS) and the restraint of this process by eculizumab is life saving. However, the evidence of complement dysregulation in the pathogenesis of Thrombotic Thrombocytopenic Purpura (TTP) is still unclear. In this study we examined the presence of complement activation biomarkers in patients with aHUS and TTP and the levels were compared to normal healthy controls .

Patients and Methods: Patients with thrombotic microangiopathic thrombocytopenia diagnosed either as TTP with low ADAMTS13 activity less than 10% or aHUS with impaired renal function, Cr> 2mg/dL and normal ADAMTS13 activity were chosen from the Korean TTP registry from February 2012 to June 2014. Prospective plasma and serum samples prior to intervention were collected from newly diagnosed patients with TTP (n=20), aHUS (n=20), and 20 healthy controls and frozen at -700C. Complement activation products (C3a, Bb as alternative pathway; C4d as classic pathway; C5a, C5b-9; terminal pathway) were measured by ELISA.

Results: Significantly increased levels of Bb and C5b-9 were observed in TTP (median [range], ng/mL; Bb, 1220 [540.0 – 16560], p=0.048; C5b - 9, 390.1 [238.5 - 938.7], p<0.0001) when compared with controls (Bb, 870.0 [630.0 - 2070]; C5b - 9, 190.8 [77.96 - 458.9]). Increased levels of C3a, C5a, C5b - 9, and Factor Bb were observed in HUS (C3a, 231.3 [80.70 - 791.8], p<0.0001; C5a, 21.38 [5.590 - 34.96], p= 0.006; C5b - 9, 0.49 [0.21 - 1.41], p<0.0001; Bb, 1490 [540.0 – 11800], p= 0.0003) as compared with controls (C3a, 108.7 [30.98 - 425.1]; C5a, 8.620 [2.660 - 26.93]; C5b - 9, 0.49 [0.21 - 1.41]; Bb, 870.0 [630.0 - 2070]). These suggested alternative and terminal complement pathways were activated in initial episodes of TTP or HUS. However levels of C4d were not different in HUS and TTP as compared with controls which suggested classic complement pathways were not important in this process. There were no significant differences in complement levels between TTP and HUS although levels of C3a, C4d, C5b - 9 in HUS (C3a, 231.3 [80.70 - 791.8]; C4d, 2140 [10.00 - 960.0]; C5b - 9, 488.4 [212.7 – 1414]) tended to be increased as compared with TTP (C3a, 134.5 [61.97 - 378.4]; C4d, 1330 [2.000 - 699.0]; C5b - 9, 390.1 [238.5 - 938.7]).

Conclusion: Complement biomarkers are activated to a similar level in both newly diagnosed cases of TTP and aHUS. Complement activation product levels did not differentiate aHUS from TTP.

Disclosures

No relevant conflicts of interest to declare.
Activated Protein C Prevents Peritoneal Fibrosis
Authors: Dinarvand Peyman , Hassanian Mehr Seyed Mahdi , Rezaie Alireza R.
Published: October 2019

Patients with chronic kidney disease require dialysis (hemodialysis or peritoneal dialysis) for treatment. Peritoneal dialysis is an alternative to hemodialysis for the treatment of end-stage renal disease and is based on the use of the peritoneum as a permeable membrane where ultrafiltration and diffusion between dialysate and blood can take place across the peritoneum. Peritoneal fibrosis is one of the main complications of peritoneal dialysis and affects up to 20% of patients undergoing continuous ambulatory peritoneal fibrosis. The exact mechanism of this process has yet to be elucidated and no effective therapy for the problem has been established. APC is a natural vitamin-K dependent anticoagulant protease, which also has potent antiinflammatory activity. We hypothesized that the antiinflammatory function of APC may inhibit dialysis-mediated peritoneal fibrosis. To test this hypothesis, we generated a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model by injecting CG to male C57BL/6 mice for 21 days with or without intraperitoneal administration of a low dose of recombinant APC (50 µg/kg/day bodyweight) one hour before injecting CG. At 10 and 21 days after injection, the mice were sacrificed and the parietal peritoneum and omentom were dissected for histological evaluation and for analysis of expression of inflammatory molecules. Strikingly, we discovered that APC inhibits the thickness of peritoneal fibrosis and potently inhibits the mRNA expression of TGF-b1, cytokeratins, a2-integrin, MMP-2 and 9 and also significantly increases the mRNA expression of TIMP-2 in peritoneal tissues of the experimental animals. We also discovered that APC significantly decreases the concentration of TGF-b1 and dramatically increases the concentration of tPA in peritoneal fluids. Taken together, our findings suggest that APC may have therapeutic potential in ameliorating dialysis-mediated peritoneal fibrosis.

Disclosures

No relevant conflicts of interest to declare.
Cancer-Associated Deep Vein Thrombosis: The Role of Residual Vein Thrombosis for Assessing the Duration of Low Molecular Weight Heparin (the EXTENDED Cancer-DACUS)
Authors: Napolitano Mariasanta , Saccullo Giorgia , Raso Simona , Mancuso Salvatrice , Casuccio Alessandra , Siragusa Sergio
Published: October 2019

Introduction. The optimal duration of Low Molecular Weight Heparin (LMWH) after cancer associated deep vein thrombosis (DVT) is unknown; current guidelines suggest to prolong anticoagulation until cancer is active. We have recently demonstrated, in a randomized trial, that detection of Residual Vein Thrombosis (RVT) after 6 months of LMWH identify patients who require or not extension of therapy with anticoagulants (JCO in press). Now we present data of a prospective study evaluating a RVT-based management of patients with cancer-associated DVT, in whom LMWH has been extended up to 2 years in patients considered at high-risk for recurrent DVT because of persistence of RVT.

Material and Methods. Patients were included at the time of a first diagnosis of cancer-associated DVT of the lower limbs. All received LMWH at therapeutic dosage for the first month (approximately 100 UI anti-FXa b.i.d.) then reduced at 75% for the following months. After 6 months of heparin, the presence of RVT was detected: those without RVT (no-RVT group) suspended treatment, while those with RVT (RVT group) continued LMWH for up to 2 years. Recurrent thrombosis and/or bleeding events were recorded during treatment and one year after LMWH withdrawal. Baseline differences between groups were assessed by the chi-square test (Yates’ correction) for categorical variables and ANOVA test or Kruskal-Wallis test for parametric and nonparametric analyses. Relative risks (RRs) and 95% confidence intervals (CIs) were evaluated.

Results. Between January 2009 and April 2011, 211 cancer patients were enrolled; RVT was detected in 129 patients (61.1%). Recurrent VTE occurred in 19 (14.7%); 4 episodes (3.1%) occurred while on heparin. Among patients without RVT (82), 3 (3.6%) developed recurrent VTE (after LMWH therapy). Adjusted HR for RVT vs no-RVT group was 5.8 (95% CI, 1.9 to 19.2; p 0.0003). Three major bleeding events occurred in RVT group and one in no-RVT group (during LMWH administration). The HR for major bleeding (RVT vs no-RVT group) was 2.58 (95% CI, 0.66 to 12.43;p 0.103). Overall, 44 patients (20.8%) died during follow-up as a result of cancer progression.

Conclusions. These results indicate that in patients without RVT, a short period of treatment with a LMWH is sufficient; in those with persistent RVT, treatment extended to 2 years substantially reduces, but does not eliminate, the risk of recurrent thrombosis. However, when still on LMWH, the risk for recurrent VTE is low.

Disclosures

No relevant conflicts of interest to declare.
Protein Tyrosine Phosphatase-Sigma (PTPσ) Regulates Hematopoietic Stem Cell Repopulating Capacity
Authors: Quarmyne Mamle , Doan Phuong L. , Himburg Heather A. , Yan Xiao , Zhao Liman , Chao Nelson J. , Chute John P.
Published: October 2019

Hematopoietic stem cell (HSC) proliferation, differentiation and self-renewal are regulated by signaling through receptor tyrosine kinases (RTKs) such as c-kit, Flt-3 and Tie2. The functions of receptor protein tyrosine phosphatases (RPTPs) in counterbalancing RTK signaling in HSCs remain incompletely understood. Among 9 examined RPTPs, we found that PTP-sigma (PTPσ) was significantly overexpressed in mouse and human HSCs compared to more mature hematopoietic cells. PTPσ-/- mice displayed no difference in mature blood counts or phenotypic HSC frequency compared to PTPσ+/+ mice. However, competitive transplantation of BM cells from PTPσ-/- mice resulted in greater than 8-fold increased multilineage hematopoietic repopulation in primary and secondary recipient mice compared to mice transplanted with BM from PTPσ+/+ mice. While HSCs from PTPσ-/- mice displayed no differences in cell cycle status or homing capability compared to PTPσ+/+ mice, PTPσ-/- HSCs expressed significantly increased levels of activated Rac1, a RhoGTPase which regulates HSC engraftment capacity, compared to PTPσ+/+ BM cells. PTPσ-/- BM cells also displayed significantly increased transendothelial migration capacity and cobblestone area forming cells (CAFCs), consistent with increased Rac1 activation. Furthermore, Rac inhibition abrogated the increased migration capacity of PTPσ-/- BM cells, suggesting that the augmented engraftment capacity of PTPσ-/- BM cells was mediated via Rac activation. Translationally, we demonstrate that negative selection of human cord blood CD34+CD38-CD45RA-Lin- cells for PTPσ expression yielded a 15-fold enrichment for human long-term repopulating HSCs compared to CD34+CD38-CD45RA-Lin- cells or CD34+CD38-CD45RA-lin-PTPσ+ cells. These data suggest that PTPσ regulates HSC repopulating capacity via inhibition of Rac1 and that selection for human PTPσ - negative HSCs is a translatable strategy to significantly enrich human HSCs for transplantation.

Disclosures

No relevant conflicts of interest to declare.
Mitochondrial Activity Determines Hematopoietic Stem Cell Fate Decisions
Authors: Vannini Nicola , Girotra Mukul , Naveiras Olaia M. , Campos Vasco , Williams Evan , Roch Aline , Auwerx Johan , Lutolf Matthias
Published: October 2019

A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation.

Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture.

Disclosures

No relevant conflicts of interest to declare.
Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice
Authors: Scholl Amanda , Lam Kentson , Muselman Alex , Tang Tingdong , Matsuura Shinobu , Zhang Dong-Er
Published: October 2019

RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function.

A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result.

GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis.

Disclosures

No relevant conflicts of interest to declare.
The Duration of Erk1/2 Signaling Determines Neutrophil Versus Monocyte Cell Fates in Response to G-CSF and M-CSF
Authors: Hu Nan , Qiu Yaling , Dong Fan
Published: October 2019

Neutrophils and monocytes/macrophages are derived from hematopoietic stem cells that, through progressive commitment, give rise to granulocyte-monocyte progenitors that in turn develop into either neutrophils or monocytes/macrophages. Although it is well known that cell fate specification in the hematopoietic system depends on the expression of lineage specific transcription factors, the roles of cytokines in lineage commitment are less clear and two models have been proposed. According to the stochastic model, cell fate choice is stochastic and cytokines simply provide signals for the survival and proliferation of committed cells. The instructive model, on the other hand, proposes that cytokines stimulate intracellular signaling pathways that dictate cell fate decisions. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. Recent studies lend strong support to the roles of G-CSF and M-CSF in instructing lineage commitment. However, the signaling pathways that determine neutrophil versus monocyte cell fate following stimulation with G-CSF and M-CSF are unknown. Here we show that tyrosine (Y) 729 of the G-CSFR is involved in transducing signals that specify neutrophil cell fate. Substitution of Y729 with phenylalanine (F) results in monocytic differentiation in response to G-CSF in murine myeloid 32D and multipotent FDCP-mix A4 cells. G-CSF stimulated activation of Erk1/2 was prolonged in cells expressing G-CSFR Y729F mutant. Significantly, treatment of cells with Mek1/2 inhibitors U0126 or PD0325901 rescued neutrophilic differentiation. M-CSF has been shown to induce prolonged activation of Erk1/2, which is required for monocytic differentiation. Interestingly, the Mek1/2 inhibitors also promoted neutrophil cell fate at the expense of monocytic development in lineage marker negative (Lin-) primary bone marrow cells cultured in M-CSF. We further demonstrate that prolonged activation of Erk1/2 was associated with augmented activation of c-Fos and Egr1, both of which have previously been shown to promote monocytic development. Consistent with this, knockdown of c-Fos or Egr1 redirected 32D cells expressing G-CSFR Y729F mutant to develop into neutrophils in response to G-CSF. We propose that M-CSF stimulates more sustained activation of Erk1/2 than G-CSF does and that the duration of Erk1/2 signaling regulates neutrophil versus monocyte cell fate choices, likely through altering the activation statuses of c-Fos and Egr1.

Disclosures

No relevant conflicts of interest to declare.
Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice
Authors: Saito Yasuyuki , Ellegast Jana M. , Müller Rouven , Flavell Richard A. , Manz Markus G.
Published: October 2019

Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014).

To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice.

Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals.

Disclosures

No relevant conflicts of interest to declare.
Advanced Stage Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) in Children and Adolescents : Retrospective Study from Sfce and Ukccssg
Authors: Gaelle Roques , Shankar Ananth , Leblanc Thierry , Schmitt Claudine , Hayward Janis , Pacquement Helene , Lambilliotte Anne , Aladjidi Nathalie , gorde-Grosjean Stephanie , Martin Hewitt , Haouy Stephanie , Abbou Samuel , Plantaz Dominique , Plat Genevieve , Hall Georgina , Landman-Parker Judith
Published: October 2019

Background: Advanced stage nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) in children and adolescents is a very rare entity and therefore few data about their treatment and outcome are available.

Aim of the study: To describe the clinical characteristics and evolution of advanced stage NLPHL in children and adolescents

Methods:A retrospective analysis of clinical data of patients treated in the SFCE and UK CCLG centers

Patients and Results: 32 patients with NLPHL according to the REAL/WHO classification with disease stage ≥ IIB were recorded between 1998 and 2013. Two patients had composite NLPHL and B cell non-Hodgkin lymphoma at diagnosis. Median age was 12 years (range 4-17) and the majority were boys [n=28; 88%]. Stage distribution was: IIB =2 ( 6%), III =25 (71%) & stage IV =5 (15%). Twenty-seven patients had cervical node involvement while mediastinum involvement was seen in 11 (34% ) patients. B symptoms were seen in 2 and eight patients had ESR >30 mm. Sites of extra nodal localisation were bone, bone marrow, liver and lungs.Standard Hodgkin chemotherapy regimens (CT) were used in 26 patients (81%), combined with rituximab (R) in 5 children (3 to 6 doses) and along with 20Gy involved field radiotherapy in 6 children. NHL chemotherapy regimens were used in 5 patients; R-CHOP [n=3], LMB 01 regimen [n=1] and other regimen [n=1]. One patient received rituximab alone (4 doses) and 1 child had no treatment [watch and wait strategy]. Median follow up is 45 months (5 – 162).

Outcome: Overall survival is 96% and cumulative incidence of an event is 25%. Eight patients relapsed with a median delay of 15 months (5-73), and 3 had a second relapse 9,11 and 48 months respectively after first relapse. One patient treated with chemotherapy alone developed Ewing's sarcoma and died 2 years later. The only patient treated with rituximab alone relapsed at 22 months. Of note, no relapses were observed in patients who had chemotherapy and rituximab.

Conclusion: Our report shows that while advanced stage NLPHL in children and adolescents is rare, their clinical outcome is very good. Additionally, this report may inform the basis of future treatment of advanced stage NLPHL in children and adolescents.

Disclosures

No relevant conflicts of interest to declare.
Systematic Coronary Risk Evaluation (SCORE) Chart Identify Chronic Myeloid Leukemia Patients at Risk of Cardiovascular Diseases during Nilotinib Treatment
Authors: Breccia Massimo , Molica Matteo , Zacheo Irene , Alimena Giuliana
Published: October 2019

Nilotinib is currently approved for the treatment of chronic myeloid leukemia (CML) in chronic (CP) and accelerated phase (AP) after failure of imatinib and in newly diagnosed patients. Atherosclerotic events were retrospectively reported in patients with baseline cardiovascular risk factors during nilotinib treatment. We estimated the risk of developing atherosclerotic events in patients treated with second or first line nilotinib, with a median follow-up of 48 months, by retrospectively applying the SCORE chart proposed by the European Society of Cardiology (ESC) and evaluating risk factors at baseline (diabetes, obesity, smoking and hypertension). Overall, we enrolled in the study 82 CP patients treated frontline (42 patients, at the dose of 300 mg BID) or after failure of other tyrosine kinase inhibitors (40 patients, treated with 400 mg BID). The SCORE chart is based on the stratification of sex (male vs female), age (from 40 to 65 years), smoker vs non-smoker, systolic pressure (from 120 to 180 mm Hg) and cholesterol (measured in mmol/l, from 150 to 300 mg/dl). For statistical purposes we considered patients subdivided in low, moderate, high and very high risk. There were 48 males and 34 females, median age 51 years (range 22-84). According to WHO classification, 42 patients were classified as normal weight (BMI < 25), 26 patients were overweight (BMI 26- <30) and 14 were obese (BMI > 30). Retrospective classification according to the SCORE chart revealed that 27 patients (33%) were in the low risk category, 30 patients (36%) in the moderate risk category and 24 patients (29%) in the high risk category.

As regards risk factors, we revealed that 17 patients (20.7%) had a concomitant type II controlled diabetes (without organ damage), 23 patients (28%) were smokers, 29 patients (35%) were receiving concomitant drugs for hypertension, 15 patients (18%) had concomitant dyslipidaemia. Overall, the cumulative incidence of atherosclerotic events at 48 months was 8.5% (95% CI: 4.55-14.07): none of the low-risk patients according to the SCORE chart experienced atherosclerotic events compared to 10% in the moderate risk and 29% in the high risk category (p=0.002). Atherosclerotic-free survival was 100%, 89% and 69% in the low, moderate and high-risk population, respectively (p=0.001). SCORE chart evaluation at disease baseline could be a valid tool to identify patients at high risk of atherosclerotic events during nilotinib treatment.

Disclosures

Breccia: novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy.
NADPH Oxidase 2 Is Critical for Heterotypic Neutrophil-Platelet Interactions during Vascular Inflammation
Authors: Li Jing , Kim Kyungho , Andrews Robert K. , Cho Jaehyung
Published: October 2019

Intravital microscopic studies have shown that neutrophil-platelet interactions on activated endothelial cells are the major determinant of vascular occlusion under thromboinflammatory conditions. Heterotypic neutrophil-platelet interactions are mainly mediated by two crucial receptors and counter receptors, P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. Although the major receptors and counter receptors are well identified, it remains unclear how heterotypic cell-cell interactions are regulated during vascular disease. Recently, we demonstrated that neutrophil AKT2 is a key regulator for neutrophil-platelet interactions during vascular inflammation. Since it is known that AKT2 is important for NADPH oxidase 2 (NOX2) activity and reactive oxygen species (ROS) generation during neutrophil activation, we investigated whether NOX2 regulates the heterotypic neutrophil-platelet interaction during vascular inflammation. Using real-time fluorescence intravital microscopy, we found that platelet-neutrophil interactions are nearly completely inhibited during TNF-alpha-induced cremaster venular inflammation in NOX2 KO mice, compared with WT mice. Studies with bone marrow chimera further demonstrated that both hematopoietic and endothelial cell NOX2 are crucial for neutrophil-platelet interactions on the inflamed venules. In vitro studies with neutrophils and platelets isolated from WT and NOX2-deficient mice indicated that both neutrophil and platelet NOX2 are required for neutrophil-platelet aggregation under shear conditions. Using ROS scavengers and NOX2 KO cells, we observed that neutrophil NOX2-generated ROS regulate the activation and ligand-binding activity of alphaMbeta2 integrin during neutrophil activation and that platelet NOX2-produced ROS are important for P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Ibalpha. Interestingly, neutrophil NOX2 was crucial for store-operated calcium entry (SOCE), but not calcium release from the endoplasmic reticulum, whereas platelet NOX2 modulated intracellular calcium release, but not SOCE. We further demonstrated that the differential regulation of platelet and neutrophil NOX2 in calcium signaling correlates with the differences in the phosphorylation of AKT, ERK, and p38MAPK, suggesting the importance of AKT and its downstream molecules for NOX2-mediated calcium signaling. Our results indicate that neutrophil and platelet NOX2-produced ROS play critical roles in regulating the function of surface receptors required for heterotypic neutrophil-platelet interactions during vascular inflammation.

Disclosures

No relevant conflicts of interest to declare.
STAT3 Mediates C6-Ceramide-Induced Cell Death in Chronic Lymphocytic Leukemia
Authors: Doshi Ushma Atul , Loughran Thomas P. , Kester Mark
Published: October 2019

Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in adults in Western countries. Despite high incidence, its pathogenesis is still poorly understood and it remains incurable with current therapies. Ceramide is a tumor suppressor' lipid known to regulate differentiation, senescence and cell cycle arrest. Previous work from our laboratory has demonstrated the therapeutic efficacy of nanoliposomal C6-ceramide in several solid and non-solid tumor models. We have shown that nanoliposomal C6-ceramide selectively induces cell death in CLL cells by targeting the Warburg effect with no detrimental effects on normal peripheral blood mononuclear cells. Additionally, nanoliposomal C6-ceramide treatment results in tumor regression in an in vivo murine model of CLL. However, the mechanism and key molecular players mediating C6-ceramide-induced cell death in CLL remains unknown. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in many cancers. Several reports in the literature have shown that STAT3 is constitutively phosphorylated on serine 727 (S727 p-STAT3) in CLL and STAT3 might be a therapeutic target in this disease. We hypothesize that STAT3 is a target of C6-ceramide in CLL cells.

We first validated STAT3 as a therapeutic target in CLL. STAT3 knockdown in CLL cells induced cell death. C6-ceramide treatment also induces necrotic cell death in these cells. Examination of STAT3 phosphorylation state demonstrates constitutive phosphorylation at S727 and Y705 residue in CLL cell lines and patient cells. C6-ceramide treatment suppressed the levels of Y705 p-STAT3 and S727 p-STAT3, while having no effect on total STAT3 levels. The downregulation of phosphorylation was specific to STAT3 and did not affect the other STATs. C6-ceramide treatment also led to the downregulation of several downstream targets of STAT3 like p21, cyclin D1, Mcl-1 and survivin. Furthermore, the suppression in STAT3 phosphorylation preceded induction of C6-ceramide-mediated cell death in CLL cells, suggesting that this event might be critical for cell death induction. Analysis of the upstream kinases revealed that C6-ceramide-induced suppression of S727 p-STAT3 might be, in part, a consequence of suppression of MEK1/2 kinase and protein kinase C (PKC). Ongoing studies will focus on identifying the specific phosphorylation site(s) that mediates cell death and examine the effect of C6-ceramide on upstream regulators of p-STAT3 in an effort to delineate the molecular mechanism of C6-ceramide-induced cell death in CLL. In conclusion, our data suggests that targeting STAT3 with nanoliposomal C6-ceramide is a potential therapy for CLL. This work is the first body of evidence demonstrating an effect of C6-ceramide on STAT3 phosphorylation.

Figure 1 Figure 1.

Disclosures

Kester: Keystone Nano, Inc: Employment, Equity Ownership.