bluuebunny/crossref_metadata_2025 huggingface dataset:
Side Population of Multiple Myeloma and Multiple Myeloma Stem Cell
Authors: Kim Kyoung Ha , Cheong Hee-Jeong , Kim Sook-Ja , Kim Se Hyung , Yoon Jina , Kim Han Jo , Kim Hyun Jung , Lee Sang-Cheol , Bae Sang Byung , Kim Chan-Kyu , Lee Namsu , Lee Kyu Taeg , Park Sung Kyu , Hong Dae Sik , Won Jong-Ho , Jun Hyun Jung , Chang Myung Hee
Published: October 2019

Background

Side population (SP) cells are known as enriched source of cancer initiating cells with stem cell properties. The SP cells show a distinct low-staining pattern with the Hoechst 33342 dye. Our aim was to identify SP cells in multiple myeloma (MM) cell lines as well as characteristics of SP cells. Furthermore, we investigated drugs that are effective against SP cells of MM.

Methods

SP (side population), MP (main population) cells from human multiple myeloma cell line (RPMI8226) were sorted by flow cytometry based Hoechst 33342 staining. We performed proliferative capacity in vitro, colony-forming cell assay, cell cycle analysis, drug toxicity (doxorubicin, dexamethasone, bortezomib, melphalan). Expression of stem cell markers (ABCG2, Oct4, Sox2, and Nanog) was observed by western blotting assay.

Results

Several established studies have shown a clonogenic MM cell population with CD 138-status. Therefore, we assessed whether there is correlation between CD 138 - subpopulations and SP cells. CD 138- subpopulations of MM cell lines were not consistent with SP cells. And we have found no correlation between expression of CD 19, CD20, and CD 27 and the proportion of SP cells. No difference was observed for expression of stem cell marker between the SP cells and MP cells. We examined cell cycle analysis and proliferation ability of SP and MP cells. SP cells showed a capacity for higher proliferation abilities and clonogenecity than MP cells. (Fig1,2)

We investigated target SP cells with several drugs (doxorubicin, dexamethasone, melphalan, bortezomib). Importantly, bortezomib was the most effective drug for SP cells.(Table1)

Conclusion

In our study, there was lack of evidence that SP cells was consistent with MM stem cells. No difference was observed for any other marker between SP cells and MP cells. However SP cells had higher proliferation index and clonogenicity compared with MP cells. We suggest that SP cells are rather related to resistance of drugs. Furthermore, bortezomib reduced the SP cells more effectively compared to other drugs.

Fig 1. Colony assay Fig 1. Colony assay

Fig 2. Cell growth ( X 105 ) Fig 2. Cell growth ( X 105 )

Disclosures

No relevant conflicts of interest to declare.
Mice Deficient in Urokinase-Type Plasminogen Activator (uPA) or uPA Receptor Develop Significantly Diminished Collagen-Induced Arthritis
Authors: Thornton Sherry , Raghu Harini , Jone Alice , Cruz Carolina , Rewerts Cheryl L. , Frederick Malinda D. , Degen Jay L. , Flick Matthew J.
Published: October 2019

Rheumatoid arthritis (RA) is a common and debilitating autoimmune disease characterized by chronic inflammation, synovial hyperplasia, edema, cartilage and bone erosion and loss of joint function. Increasing evidence suggests that the plasminogen activation (PA) system plays a fundamental role in the mechanisms mediating inflammatory joint disease pathogenesis. However, analysis of the precise contribution of PA system components to arthritis pathogenesis has been complicated by the use of gene-targeted mice on non-susceptible genetic backgrounds or experimental models that simultaneously induce wound trauma in conjunction with arthritis induction. To rigorously define the contribution of the urokinase-type plasminogen activator system to arthritis pathogenesis, previously generated genetic deficiencies in both uPA and uPA receptor (uPAR) were inbred for 7 generations (99% inbred) to the well-characterized, collagen-induced arthritis (CIA)-susceptible strain, DBA/1J. Our results indicate a near complete amelioration of joint disease in uPA-deficient mice that was also observed in uPAR-deficient mice. Limited disease development in both uPA- and uPAR-deficient mice correlated with significantly reduced local mRNA levels of key inflammatory mediators (e.g., TNFα, IL-1β, and IL-6) in these animals. To determine if development of inflammatory joint disease in CIA-challenged mice was dependent on the expression of uPAR by non-hematopoietic- or hematopoietic-derived cells, reciprocal bone marrow transplant studies were performed. Mice in which uPAR deficiency was limited to the bone marrow compartment elicited significantly reduced macroscopic and histopathological disease in the paws and knees compared to wild-type mice or mice in which only hematopoietic-derived cells express uPAR. Our results are the first to report in the context of the highly CIA susceptible DBA/1 background that both uPA and uPAR are key determinants of inflammatory joint disease pathogenesis. Furthermore, our findings indicate a fundamental role for uPAR expression by hematopoietic cells in driving arthritis incidence and progression. Thus, these findings suggest that cell-surface associated uPA/uPAR-mediated proteolysis and/or uPAR-mediated signaling events from bone-marrow derived cells are important in promoting inflammatory joint disease, and that disrupting this key proteolytic/signaling system may provide a novel therapeutic strategy to limit clinical arthritis.

Disclosures

No relevant conflicts of interest to declare.
Lower Doses of Plerixafor Use Is Sufficient to Effectively Mobilize PBSC (Peripheral Blood Hematopoetic Stem Cells) When Compared to Standard Administration
Authors: Goker Hakan , Eliacik Eylem , Altuntas Fevzi , Tekgunduz Emre , Ozdemir Evren , Isik Ayse , Kayikci Omur , Kaya Ali Hakan , Aksu Salih , Haznedaroglu Ibrahim C. , Sayinalp Nilgun , Demiroglu Haluk , Ozcebe Osman Ilhami , Buyukasik Yahya
Published: October 2019

Background: Plerixafor (CXCR4 chemokine receptor antagonist), is indicated to mobilize hematopoietic stem cells (HSCs) to the peripheral blood for collection and subsequent autologous transplantation in patients who are poor mobilizers. In this retrospective analysis, we compared the efficacy of plerixafor 0.24 mg/kg by subcutaneous (SC) injection (1-2 doses) versus plerixafor (3-4 doses) 0.24 mg/kg by SC injection in terms of their effect on the hematopoietic recovery in hematopoietic cell transplantation (HCT).

Methods: We retrospectively reviewed the peripheral blood stem cell (PBSC) collections of 40 consecutive lymphoma and multiple myeloma patients who underwent autologous HCT, who had previously mobilized poorly and were given plerixafor for mobilization. Twenty-six patients received 1-2 doses of plerixafor and 14 patients received 3-4 doses. All patients gave written informed consent for the procedure. Successful engraftment time was defined as neutrophils >0.5x 109 cells/L, platelets>20 x109 cells/L for three consecutive days. SPSSincver. 20 is used for statistics.

Results: All of the 40 patients were mobilized promptly after plerixafor administration without any problem. The mean amount of autologous CD34+ PBSC was 3,8 x 10(6)/kg versus 3,2 x 10(6)/kg, for 1-2 doses of plerixafor administration ve3-4 doses, respectively(p=0,4). Median times to the neutrophil engraftment to >0,5x 109/L were 12 and 10 days for the 1-2 doses and 3-4 doses, respectively (p=0,2). Additionally, median times to the platelet engraftment to >20x 109/L were 14 and 15 days for the 1-2 doses and 3-4 doses, respectively (p=0,6).

Conclusions: These data show that the lower doses, i.e. 1-2 days of plerixafor administration evaluated in this study are sufficient to mobilize HSCs. This retrospective comparison suggest that larger prospective studies comparing two days vs. four days of plerixafor should be performed in order to further elucidate the role of two days of plerixafor use in HSC mobilization, and hence would a relatively cost effective way of mobilization in poor mobilizing patients.

Disclosures

No relevant conflicts of interest to declare.
Early Apoptotic Cells (ApoCell) As Prophylaxis of Graft-Versus-Host Disease in Myeloablative HLA-Matched Allogeneic Bone Marrow Transplantation Is Safe and Effective: 1 Year Follow-up
Authors: Mevorach Dror , Tzila Zuckerman , Nagler Arnon , Rowe Jacob M. , Or Reuven
Published: October 2019

Apoptotic cells infusion (ApoCell) is a novel cellular therapy that was found to be effective in a variety of mouse models of autoimmune diseases. ApoCell was tested in humans in addition to cyclosporine and methotrexate, as prophylaxis of graft-versus-host disease (GVHD) in HLA-matched myeloablative allogeneic bone marrow transplantation (alloBMT) from a related donor. We conducted a phase I//IIa clinical trial where we treated 13 patients (median age, 37 years; range, 20-59 years) with advanced hematologic malignancies that received conventional myeloablation with 35, or 70, or 140, or 210x106 cell/kg of donor ApoCell, on day -1 of transplantation (Mevorach et al. BBMT 2014).

The non-relapse mortality at day 100 and 180 after transplantation was the same, 7.7%. The overall survival at 100 and 180 days after transplantation was 92% and 85%, respectively. We now report 1 year overall survival rate which was 62% with a non-relapse survival rate of 89% (100% for the two higher cohorts).

These results are in agreement with additional safety observations that included: ten serious adverse effects with all being not related (7) or unlikely to be related (3) to ApoCell infusion, and only three out of hundreds adverse effects, that were reported as possibly related to ApoCell infusion (with no definite or probable adverse effects).

The incidences of acute grades II through IV GVHD for all 13 patients and for the two higher doses were 23% and 0% (six patients), respectively. GvHD clinical grade was supported by biomarker ratio levels including tumor necrosis factor receptor I (TNFR1), hepatocyte growth factor (HGF), interleukin 2 receptor alpha (IL-2Ra), and interleukin 7 (IL-7).

These results suggest that single-infusion high-dose donor early apoptotic cells in HLA-matched myeloablative alloBMT is a safe and effective prophylaxis for acute GVHD (clinicaltrials.gov no. NCT00524784).

Disclosures

Mevorach: Enlivex: Consultancy, Equity Ownership.
Recovery of IgG Production Following Rituximab Therapy after Allogeneic Stem Cell Transplantation
Authors: Morris Christopher L. , Morris Joan
Published: October 2019

Rituximab is a monoclonal antibody to the CD20 antigen expressed on B lymphocytes. Several recent reports suggest that rituximab may be used to treat complications of SCT including GVHD and EBV reactivation. The long term consequences on IgG recovery aren’t well studied. This is a retrospective analysis of allogeneic pediatric SCT patients treated with rituximab at Loma Linda University from 2004 to 2013. We treated 23 patients with rituximab following allogeneic SCT (11 MUD, 7 MSD, 3 Cord) for post-transplant EBV reactivation alone (4), cGVHD alone (4), cGVHD in combination with thrombocytopenia (9), plus hemolysis (4) and EBV reactivation (2). Most patients required IVIG monthly (18) or more frequently (4) to maintain serum IgG levels >500mg/dl. We examined the length of time from end of rituximab therapy to recovery of endogenous IgG production. The effect on recovery of IgG production of cGVHD therapy, EBV infection, age, type of stem cell graft, and number of doses of rituximab were evaluated. Those treated for cGVHD plus any other indication (n=19) received a median of 11 doses of rituximab (range 4 to >30). Among these patients 11 of 14 whose cGVHD resolved and came off immune suppression recovered the ability to produce IgG completely (9) or partially (2, defined as decreasing frequency of IVIG infusions to maintain IgG>500mg/dl). Median time to recovery was 14 months (range 1-50 months) after last dose of rituximab. For patients who continued to require immune suppression for cGVHD only 1 of 5 recovered IgG production 13 months after last dose of rituximab. The remaining 4 patients required monthly or greater IVIG for >3 years after the last dose of rituximab (all still requiring IVIG). There were 4 patients treated with 2-4 doses of rituximab for EBV reactivation early after SCT, and 1 treated with 11 doses for cGVHD and EBV. Only 1 of these 5 has completely recovered IgG production. The 4 other patients remain dependent on IVIG a median of >39 months since end of rituximab therapy.

We found no relationship between age of patient or duration of rituximab therapy on the time interval between end of rituximab therapy and onset of endogenous IgG production. Recipients of MUD grafts had a non-significant increase in failure to recover endogenous IgG production, but when the impact of therapy for cGVHD was taken into account the type of stem cell graft lost significance.

Ability to recover endogenous IgG production following treatment with rituximab after SCT depends on successful treatment of cGVHD and in this group of patients requires about 1 year from end of therapy. Among those treated with rituximab whose cGVHD does not resolve and in those with early EBV reactivation, loss of IgG production persists at least several years, and this may be unrelated to rituximab therapy.

Disclosures

Off Label Use: rituximab for cGVHD.
Treatment Approaches in Acute Leukemia Patients Relapsing Following Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Topcuoglu Pervin , Gunduz Mehmet , Ozen Mehmet , Civriz Bozdag Sinem , Kocak Toprak Selami , Arslan Onder , Ozcan Muhit , Demirer Taner , Ilhan Osman , Akan Hamdi , Beksac Meral , Konuk Nahide , Gurman Gunhan
Published: October 2019

Aim: We would like to present our center experiences in the approaches of relapsing and/or progressive acute leukemia patients after allogeneic hematopoietic stem cell transplantation (AHSCT).

Patients: We have retrospectively evaluated 133 patients (86 Male; 47 Female) with acute leukemia who has been detected relapse and/or progression after AHSCT between June 1990 and October 2013. Median age was 30 years (16-63 years) at the transplant time. Their diagnosis were 89 AML (12 secondary AML), 41 ALL and 3 biphenotipic leukemia. Median time of relapse and/or progression was 165 days (13-4713 days) after ASCT. Relapse types were as extramedullary relapse (EMR) alone in 11, extramedullary with hematological relapse in 30 and hematological relapses (HR) alone in 92 patients.

Results: Our treatment approaches were chemotherapy and/or donor lymphocyte infusion in 109, second ASCT from same or different donor in 16 and radiotherapy alone in two patients. However, six patients were not able to receive any therapy. We have observed that the numbers of donor lymphocyte infusion or second transplantation increased after the year of 1999 (Figure 1). The probability of three-month, six-month or one-year survival after the relapse&progression were calculated as 53.7%±4.4%, 35.1 %±4.3% and 24.2%±3.9%, respectively. Survival after the relapse or progression was median 102 days (ranging between 68.7 days and 135.3 days, 95% CI). The type of relapse (EMR alone, EMR plus HR versus HR alone) and leukemic types (AML vs ALL) did not effect survival (p>0.5). When we compared second transplantation with other approaches, survival after relapse did not change (p=0.8) (Figure 2). In addition, survival was not affected by the year of relapses as 1990-1999, 2000-2009 versus 2010-2014 (p=0.7).

Conclusion: Treatment approaches in acute leukemia relapsing after transplantation vary from the cellular therapy to chemotherapy. But our experience was not able to show a difference between second transplantation and others. Moreover each center should be approached to the patients according to their experience and type of relapse until an effective treatment option will be defined.

Figure 1. Treatment approaches according to years Figure 1. Treatment approaches according to years

Figure 2. Overall survival according to treatment approaches Figure 2. Overall survival according to treatment approaches

Disclosures

No relevant conflicts of interest to declare.
Molecular Mechanisms of EML in AML1/ETO+ AML and Its Targeting Treatments
Authors: Meng Fan Yi , Jiang Ling , Zhong Qingxiu , Wang Li Chun , Yu Guopan , Zhu Xinhua
Published: October 2019

Our previous study has been reported that AML1/ETO positive patients with highly expressed of APP were much easier to extramedullary infiltration, and got poor prognosis£¨followed-up median 35 ( 6-96 ) months, RFS in the high expression APP group was significantly lower than the low expression group £¬40.0% vs 80.0%£¬P = 0.001). In vitro study, we constructed a cell model kasumi-1 which was consistent with AML1/ETO positive and high expressed of APP gene. The cell migration was significantly reduced after interferce of APP expression. This study was designed to investigate the molecule mechanism of extramedullary leukemia (EML) in kasumi-1 cell model and to invent a strategy for treatment in clinic.

In this study, we found p-ERK, c-Myc and MMP-2 were significantly decreased after APP expression knockdown in kasumi-1 cell. Meanwhile, we added the inhibitors to block p-ERK and c-Myc expression. The results showed that protein expression of p-ERK and c-Myc was significantly decreased after p-ERK inhibitor performance, which was proportional to the time and concentration. c-Myc and MMP-2 protein expression was significantly reduced after c-Myc inhibitor was used, but p-ERK didn't change (Fig.1B). So, we concluded that APP might regulated the AML cell migration via APP/p-ERK/c-Myc/MMP-2 pathway.

Also, we found that kasumi-1 cell was resistant to adriamycin (ADM) and Ara-C, which meant APP may be related with drug resistance. So, we detected cell surviving fraction after ADM and Ara-C performance via MTT assay. The results showed that there was no difference in control group and siAPP group. But, when compared with controls groups, cell surviving fraction in siEZH2 group was significantly decreased after ADM and Ara-C performance respectively. Furthermore, we found protein expression of EZH2 was significantly reduced after LBH589 treatment in cell culture. So, we concluded that LBH589 or SiEZH2 could increase sensitivity of kasumi-1 cell to ADM and Ara-C.

In sum, in AML1/ETO positive leukemia cells, we first report that APP gene regulates cell migration via APP/p-ERK/c-Myc/MMP-2 pathway and EZH2 gene induces drug resistantence. Interference or blocking of EZH2 and APP expression may be helpful in treating AML1/ETO positive leukemia.

Disclosures

No relevant conflicts of interest to declare.
DJ-1 Plays an Important Role in Homoharringtonine-Induced Apoptosis of Imatinib-Resistant Chronic Myeloid Leukemia Cells
Authors: Zhou Xuan , Xu Na , Li Rong , Li Lin , Ding Li , Lu Qisi , Li Yulin , Xiaoli Liu
Published: October 2019

Background and Objective: Although the treatment of chronic myeloid leukemia (CML) has improved since the introduction of tyrosine kinase inhibitors (TKI), cases of resistance have been reported and resulted in challenges to the treatment. Recent studies have suggested that Homoharringtonine (HHT), a cephalotaxine ester, has demonstrated a clinical activity in imatinib-resistant CML patients, however, the molecular mechanisms underlying this phenomenon are unknown. Our previous study found that treatment with HHT significantly increased apoptosis of K562 cells. Moreover, the protein DJ-1, identified by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, was demonstrated to decrease after HHT treatment. Therefore, we performed the experiment to address the hypothesis that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cells

Methods and Results: To find the pivotal protein by HHT, Imatinib-resistant K562 cells were treated with HHT (10 ug/ml) for 5 h, 12 h, 24 h respectively and the control group without HHT were harvested to assess apoptosis with Annexin V-FITC and propidium iodide per the manufacturer’s protocol and analyzed by ﬂow cytometry. The data indicated a time dependent induction of apoptosis by HHT, with the number of apoptotic cells (FITC-Annexin-V and PI double-positive cells) signiﬁcantly increasing from 2.2± 1.5 % in control to 35.9 ± 6.7% in cells treated with HHT for 24 h (P<0.01). The protein DJ-1 expression change upon HHT treatment which were analyzed with western blot, found that the protein level of DJ-1 had significantly decreased after the treatment of HHT for 24 h.

Furthermore, primary cells from six CML patients and three healthy donors were obtained with informed consent and divided into three groups: the CML-CP group (three newly diagnosed patients in the chronic phase), the imatinib-resistant CML group(three imatinib-resistant patients in the blastic phase) and the control group. Mononuclear cells were all cultured in vitro in the absence and presence of 10 mg/ml HHT for 5 h and 24 h. The results showed that DJ-1 expression in primary leukemia cells (both CML-CP group and imatinib-resistant CML group) were found to be decreased after HHT treatment and the expression level of DJ-1 seemed lower in the healthy donor as compared to primary CML cells, moreover, the protein changes induced by HHT were significantly different among three groups and the protein changes were not as significant in CML-CP cells as in imatinib-resistant CML cells (P<0.05).

Conclusions: These findings indicated that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cells. Further study may help to assess a promising potential of this protein to be used as a target for a molecular therapy.

Disclosures

No relevant conflicts of interest to declare.
Forewarned Is Forearmed- Comparison of Southern Network on Adverse Reactions (SONAR) Versus an FDA-Mandated Prospective Observational Registry Assessment of Gemtuzumab Ozogamycin-Associated Sinusoidal Obstructive Syndrome
Authors: Magwood Jametta , Georgantopoulos Peter , Norris LeAnn B , Bennett Charles L.
Published: October 2019

Gemtuzumab ozogamycin (GO) received initial approval in 2000 from the Food and Drug Administration (FDA) for patients age 60 and older suffering with relapsed acute myeloid leukemia. After 10 years of administering GO-containing combination chemotherapy regimens, the drug was voluntarily removed from the commercial market in 2010 amid reports of unexpected development of sinusoidal obstructive syndrome (SOS); which has the potential to be fatal. Four months after the initial approval for marketing in 2000, SONAR initiated an investigation of GO-associated SOS. SONAR reported an incidence rate of 38% among allogeneic stem-cell transplants and 16% among autologous stem-cell transplant (SCT) patients. Ultimately, there was an overall incidence rate of 3% when single-agent GO was administered as directed by the FDA. In 2007, as a result of the examination of the reports from clinical trials and observational studies; data mining of interim reports from the manufacturer’s Prospective Observational Registry and the FDA’s Adverse Event Reporting System was undertaken. In 2013, the pharmaceutical manufacturer reported results from its FDA-mandated prospective observational study. Major differences between the SONAR report and the mnaufacturer’s report include: timing of publication (2007 versus 2013); inclusion of information on allogeneic stem cell versus autologous stem cell transplantation prior to GO administration (in our reports only) and identification of risk factors associated with higher rates of SOS (SCT within 3 months of GO administration, off-label administration of high doses of GO, and concomitant administration of 6-TG in our analysis versus no identified risk factors in the manufacturer’s analysis). The incidence of SOS was 9.1% among 482 patients at 54 centers in the manufacturer’s registry.

Table 1: Comparison of gemtuzumab ozogamycin (GO) safety reports analyses between SONAR and manufacturer SONAR Pharmaceutical Investigation # of pts who received GO 1071 482 # of pts with SOS 99 44 DATA METHODS # of centers involved 50 centers (US and UK) 54 centers (US only) Interim analysis Not done Yes (2002 and 2005) Concomitant chemo given 67% 41% # of pts with concomitant HCT 26% 0% % with prior alloSCT 2.6% Not reported % with prior autoSCT 3.9% Not reported % with prior HSCT 6.5% 18.3% % with subsequent alloSCT 1.2% 0% % with subsequent autoSCT 2.0% 0% Publication Date* 2007 2013

* Gemtuzumab ozogamycin voluntarily removed from market in 2010 by the pharmaceutical manufacturer

Disclosures

No relevant conflicts of interest to declare.
Inhibition of ADAMTS13 Activity By Human Neutrophil Peptide 1 (HNP-1): Potential Link Between Inflammation, Onset of Thrombotic Thrombocytopenic Purpura, and Other Thrombosis
Authors: Bao Jialing , Bdeir Khalil , Siegel Don L. , Cines Douglas B. , Zheng X. Long
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome associated with severe deficiency of plasma ADAMTS13 activity resulting from either mutations or autoantibodies. However, patients with severe ADAMTS13 deficiency do not always develop TTP. Rather, a trigger, such as infection or inflammation, often precedes the onset of the TTP syndrome. We hypothesized that antimicrobial human neutrophil peptides 1-3 (HNPα1-3) or α-defensins, the most abundant proteins in the granules of neutrophils, which are released at site of inflammation, activate platelets, and inhibit fibrinolysis, might help to initiate TTP. This question arose because we noted that the amino acid sequences of HNPα1-3, which are nearly identical except for one residue at the N-terminus, all contain a motif (RRY) similar to exosite 3 (659RRYGEEY665) in the spacer domain of ADAMTS13 (Fig. 1) that was shown to be critical for recognition of von Willebrand factor (VWF). Here, we found that both purified and synthetic HNPα1 bind to FRETS-VWF73 and plasma-derived VWF and inhibit proteolytic cleavage of these substrates in a concentration-dependent manner. At the final concentrations of 10 micro mol/L and 150 micro mol/L, HNPα1 completely abolished the cleavage of FRETS-VWF73 (IC50=3.5 micro mol/L) (Fig. 2) and VWF (IC50=75 micro mol/L) (not shown), respectively. Such concentrations are readily attained locally after systemic infection. Deletion or alanine substitution within the RRY motif of HNPα1 completely abolished its ability to inhibit ADAMTS13 activity assessed by FRETS-VWF73 and VWF multimer analysis. This suggests that an interaction of the RRY motif in HNPα1 with the central A2 domain of VWF is required to mediate its inhibition. In support of this hypothesis, HNPα1 interacts with a human monoclonal antibody against ADAMTS13 scFv (the single chain fragment of variable region) designated 4-20, but not scFv3-1, both isolated by phage display from patients with acquired autoimmune TTP. Hydrogen-deuterium exchange mass spectrometry has shown that the binding site for scFv4-20, but not scFv3-1, contains the RRY sequence. These results suggest that HNPα1-3 released from neutrophils following infection or inflammation may inhibit residual plasma ADAMTS13 activity in vivo similar to anti-ADAMTS13 autoantibodies by interfering with its interaction with VWF, thereby triggering the onset of hereditary and acquired autoimmune TTP. Our findings suggest a potential novel link between systemic inflammation and the pathogenesis of TTP and possibility other thrombotic sequelae.

Figure 2 Figure 2.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Kit Deficiency Regulates Stable Human Hematopoietic Stem Cell Engraftment in Mice
Authors: Waskow Claudia , Rahmig Susann , Cosgun Nehir
Published: October 2019

Humanized mice are required for in-depth analysis of human hematopoietic stem cell (HSC) function and immunobiology. The currently available options are problematic because stable engraftment of substantial numbers of human HSCs and the continuous generation of human myeloid cell types remain difficult to achieve. We generated three novel recipient mouse strains that combine immune deficiency with a functionally impaired endogenous HSC compartment mediated by a defective Kit receptor: BALB/c Rag2- Il2rg-KitWv/Wv (BRgWv), NOD/SCID Il2rg- (NSG) KitWv/Wv (NSGWv) and NSG KitW41/W41 (NSGW41). We find that the mutant Kit receptor opens up stem cell niches across species barriers and allows for robust and sustained engraftment of human HSCs after transfer into adult mice without the necessity for irradiation conditioning prior to transplantation. Following stable engraftment in the mouse bone marrow niches, human HSCs give rise to lymphoid cells and to robust numbers of erythroid and myeloid lineage cells over extended periods of time in primary and secondary recipient mice. Particularly in NSGW41 mice, we observe improved reconstitution of human myeloid cell types compared to control irradiated NSG mice. In the bone marrow, endogenous hematopoietic progenitor cells with a defective Kit receptor are largely replaced by human Kit-proficient hematopoietic progenitor cells because progenitor cell expansion requires normal signaling by Kit. Thus, human Kit-proficient donor cells have an advantage over endogenous murine Kit-mutant cells. Increased numbers of myeloid cells are found in the bone marrow and spleen of NSGW41 transplanted mice compared with grafts established in irradiated NSG mice. We conclude that Kit-signaling regulates HSC engraftment across the human-mouse species barrier and that Kit deficient mice show great potential for the study of human HSC functions including self-renewal, differentiation and mechanisms of innate immunity.

Disclosures

No relevant conflicts of interest to declare.
Eradication of Chronic Myelogenous Leukemia By Inactivation of the Polycomb Group Protein EZH2
Authors: Xie Huafeng , Peng Cong , Qi Jun , Cheloni Giullia , Das Partha , Huang Jialiang , Nguyen Minh , Li Shaoguang , Bradner James E. , Orkin Stuart H.
Published: October 2019

Chronic myelogenous leukemia (CML) is a stem-cell driven malignancy caused by BCL/ABL, the oncoprotein derived from the Philadelphia chromosome 9/22 translocation. Although tyrosine kinase inhibitors (TKIs) are highly effective in managing chronic phase CML, challenges remain, including acquisition of TKI resistance, toxicities of long-term TKI administration and third-generation TKIs, and persistence of leukemia initiating cells (LICs). Reliable curative chemotherapy remains elusive. Alternative approaches to definitive treatment of CML would enhance current management.

We report here that the Polycomb group protein EZH2, a catalytic subunit of Polycomb repressive complex 2 (PRC2), is a potential target for curative chemotherapy for CML. EZH2 is overexpressed in LICs, compared to normal hematopoietic stem cells (HSCs), and is sustained by BCR/ABL signaling, as TKIs suppress EZH2 expression in CML cells. Removal of EZH2 in an engineered mouse model prevents CML initiation and development. Moreover, Ezh2 is also required for maintenance of established CML. In a tamoxifen-inducible EZH2 deletion mouse model, conditional inactivation of EZH2 eradicates existing disease induced by wild-type BCR/ABL or the gatekeeper T315I mutant, and leads to long-term survival. Importantly, EZH2 inactivation also eliminates phenotypic and functional LICs. Relevance of the genetic studies in mice is supported by shRNA knockdown and EZH2-inhibitor treatment of human CML cell lines and primary samples, which leads to cell cycle inhibition, apoptosis, and reduced colony formation. Effects of EZH2 loss in CML are context-specific, as EZH2 is dispensable for HSCs, in part due to decreased EZH1 expression in CML LICs compared to normal HSCs. EZH1 expression decreases even further in LICs upon the loss of EZH2. Thus, loss of EZH2 in CML LICs creates a scenario resembling complete inactivation of PRC2, which is essential for the maintenance of HSCs.

EZH2-dependence of CML LICs raises prospects for curative chemotherapy based on addition of EZH2 inhibitors to conventional TKI administration.

Disclosures

No relevant conflicts of interest to declare.
CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia
Authors: Chae Hee-Don , Mitton Bryan , Sakamoto Kathleen
Published: October 2019

CREB (cAMP Response Element Binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication Factor C3 (RFC3), a 38 kDa subunit of the RFC complex, is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in DNA replication and repair processes. CREB1 expression was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line, suggesting that RFC3 and CREB1 might be target genes of E2F, a key transcriptional regulator of the G1/S progression. Though there were two potential E2F binding sites in the RFC3 promoter region, chromatin immunoprecipitation assays provided no evidence for E2F1 binding to the RFC3 promoter, whereas E2F1 could directly act on the CREB1 expression. Treatment with the cyclin-dependent kinase (CDK) inhibitor AT7519 decreased expression of CREB1 and RFC3 as well as well-known E2F target genes such as CCNE1, CCNA2 and CCNB1 in KG-1 cells. These results indicate that CREB1 overexpression, a potentially important prognostic marker in leukemia patients, may be associated with dysregulated CDK-E2F activity in leukemia. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in AML cells from patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB knockdown primarily inhibited G1/S cell cycle transition, decreasing expression of RFC3 as well as PCNA loading onto chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading [Chromatin-bound PCNA-positive cells (%), control vs. CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 8h after release from mitotic arrest: 66.87 +/– 0.90 vs. 24.77 +/– 0.99 vs. 79.17 +/– 0.12, n=3, p< 0.01, mean +/– SEM] caused by CREB knockdown. Taken together, our results suggest that RFC3 may play a role in neoplastic myelopoiesis by promoting the G1/S progression, and its expression is regulated by CREB. These data provide new insight into CREB-driven regulation of the cell cycle in AML cells, and may contribute to leukemogenesis associated with CREB overexpression.

Disclosures

No relevant conflicts of interest to declare.
Genome Engineering to Prospectively Investigate the Pathogenesis of MLL-AF9 Acute Leukemia
Authors: Buechele Corina , Breese Erin H. , Schneidawind Dominik , Porteus Matthew H. , Cleary Michael L.
Published: October 2019

Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias, and are associated with a poor prognosis. While animal models of MLL-AF9 translocations have improved our understanding of the role of MLL oncogenes in leukemia pathogenesis, in this study a more representative approach based on generation of endogenous activated oncogenes is used to more faithfully model the genomic events on human leukemia. This system allows for prospective investigation of the downstream effects of the initiating event on gene expression, epigenetic regulation, stem cell biology, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. We designed a set of TALENs that cut in intron 11 of the MLL gene (MLL-11 TALENs) to facilitate insertion of sequences encoding for AF9 and the fluorescent marker gene coding NeonGreen (knock-in approach). This resulted in MLL-AF9 expression controlled through the endogenous MLL promoter. Co-expression of MLL-11 TALENs with the AF9 knock-in template resulted in AF9 NeonGreen insertion both in K562 cells and in primary human hematopoietic stem cells isolated from umbilical cord blood. This approach showed a knock-in efficiency of ~20% (range 2-40%) in K562 and CD34+ cells based on NeonGreen expression detected by flow cytometry and confocal microscopy. Long-term culture of primary human hematopoietic cells showed increased frequency of knock-in cells suggesting a survival advantage. Remarkably, further enrichment of knock-in cells was promoted by colony-forming assays in semi-solid medium. Subsequent transplantation into NSG mice was performed to assess their leukemogenic potential. Strikingly, leukemia was induced within 8-14 weeks post transplantation. All mice presented with a similar disease profile that included peripheral blood blasts and splenomegaly. Histologic examination confirmed extensive replacement of bone marrow cells and splenic infiltration by leukemic blasts expressing MLL-AF9 detected by RT-PCR. The leukemic blasts showed increased levels of common MLL target genes (HoxA9, Meis1) compared to non-MLL leukemias and comparable levels to known MLL-AF9 cell lines (Mono Mac 6, THP-1). The blast cells displayed a pre-lymphoid phenotype with CD10+/CD19++/CD38++/CD34+ and were negative for mature B cell markers CD20 and IgM as measured by flow cytometry. Our studies establish a novel experimental model to generate MLL leukemia deriving from primary human hematopoietic stem cells expressing the fusion oncogene under control of the endogenous promoter. The model will allow for further prospective study of leukemia initiating and stem cell biology for a genetic subtype of poor prognosis leukemia.

Disclosures

No relevant conflicts of interest to declare.
Acquired Aplastic Anemia: New Genetics, New Genomics
Authors: Young Neal S. , Dumitriu Bogdan , Ogawa Seishi
Published: October 2019

Clinical and laboratory data have established that aplastic anemia is an immune-mediated disease in which T lymphocytes destroy hematopoietic stem and progenitor cells. Immunosuppressive therapies are effective in the majority of patients. Addition of the thrombopoietin mimetic eltrombopag increases the overall and complete response rates to anti-thymocyte globulin and as a single agent can rescue patients refractory to immunosuppressive therapy (IST). Multi-lineage robust blood count improvements with increased marrow cellularity suggest activity of eltrombopag on the hematopoietic stem cell. Recently, genetic factors have been identified that increase the risk of bone marrow failure: adults may present in the clinic with late manifestations of a pediatric syndrome (dyskeratosis congenita), but more typically there are no physical anomalies, often no family history, and earlier blood counts may be normal. In the telomeropathies, which in later life are almost always due to mutations in TERT (which encodes the telomerase) or TERC (which encodes the RNA template), there may be personal or familial multi-organ involvement, especially of liver and lung; early hair greying is a useful clinical clue. Detection of extremely short telomeres of leukocytes, accompanied by gene sequencing, is used to establish the diagnosis. In the syndrome defined by GATA2 mutations, there may be a history of unusual or persistent mycobacterial and viral infections, and monocytopenia preceding pancytopenia. Diagnosis requires sophisticated interpretation of gene sequencing. Large genomic data sets are now available acquired (somatic) mutations in aplastic anemia. For almost 300 NIH aplastic anemia patients treated with IST, candidate gene sequencing of myeloid cells obtained six months after treatment revealed somatic mutations in about one-third of cases. PIGA was most frequently abnormal, followed by BCOR1, DNMT3A, and ASXL1. Initial variant allele frequency was usually low. Mutations in a subset of genes predicted poor survival and progression to myelodysplastic syndrome and acute myeloid leukemia, while mutations in BCOR and PIGA correlated favorably with outcomes. When we also examined a subset of patients at the time of progression to monosomy 7 with pancytopenia and/or incipient leukemia by whole exome sequencing.DNMT3A and ASXL1 were implicated in only two cases, RUNX1 in another, and there were no novel recurring mutations. Telomere attrition was markedly accelerated in these monosomy 7 cases. A good model of evolution from bone marrow failure to myeloid malignancy centers on haploinsufficiency of specific genes, by mutations or chromosome loss, both of which would be favored in a stressed, regenerative environment. The failed marrow environment may select for defective cells, as, for example, in differentiation capability. Malignant evolution should be predictable in the clinic and amenable to therapeutic intervention.

Disclosures

Off Label Use: Eltrombopag in bone marrow failure syndromes, research protocols.
Post-Transcriptional Defects and Erythroid Pathobiology
Authors: Sankaran Vijay G.
Published: October 2019

A great body of work has focused on understanding the role that gene regulation at the transcriptional level plays in blood cell production and diseases that disrupt this process. However, until recently there has been limited insight on the role that post-transcriptional gene regulation has in both normal and pathological disorders of human hematopoiesis. Specifically, the regulation of messenger RNA translation can have a significant impact upon gene expression, and how this process affects hematopoiesis has only been explored in limited studies. In this talk, the role of ribosomal protein gene mutations in the specific disorder of red blood cell production, Diamond-Blackfan anemia, will be discussed. Recent findings from our laboratory show that mutations in the key hematopoietic transcription factor gene, GATA1, can result in Diamond-Blackfan anemia in rare cases. We have gone on to show that more common mutations in ribosomal protein genes can disrupt translation of GATA1 and thereby suggest a common underlying mechanism for the impaired erythropoiesis observed in Diamond-Blackfan anemia. We discuss both the mechanistic underpinnings of our observations and how these findings have important therapeutic implications. Other recent examples of how disordered translation can impair human hematopoiesis will also be examined. This talk will provide a cohesive framework to understand the implications of these recent findings for both normal and disordered human hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Erythron to the Liver: Send Iron
Authors: Nemeth Elizabeta , Ganz Tomas , Kautz Léon
Published: October 2019

For successful expansion of erythropoiesis, the activity of the hormone erythropoietin (EPO) must be coordinated with the supply of iron to erythroid precursors. Increased iron supply for erythropoiesis is ensured by the suppression of hepcidin, the iron-regulatory hormone produced by the liver. Low hepcidin levels allow greater absorption of dietary iron and greater mobilization of iron from the stores in the spleen and the liver. The mechanisms coordinating erythropoietic activity with iron delivery are not well understood. We recently identified erythroferrone as a new mediator of hepcidin suppression during stress erythropoiesis1. Erythroferrone (ERFE) is a member of the C1q/TNF-related protein (CTRP) family of metabolic mediators. ERFE is produced in response to EPO by erythroblasts of the bone marrow and spleen of mice. The induction of ERFE by EPO was dependent on Jak2/Stat5 signaling. Ex vivo treatment of human erythroblasts with EPO also resulted in a dramatic induction of ERFE expression. The essential role of ERFE in acute hepcidin suppression by erythropoiesis was demonstrated in ERFE-deficient mice. In contrast to wild-type mice which suppressed hepcidin ~10-fold within hours after hemorrhage or erythropoietin injection, no hepcidin suppression was observed in ERFE knockout mice within 24 h. As a consequence, ERFE-deficient mice exhibited delayed recovery of hemoglobin after hemorrhage or severe inflammation. Treatment of mice or hepatocytes with recombinant ERFE protein confirmed the hepcidin-suppressive activity of the protein. It remains to be seen whether administration of ERFE protein would be useful for the treatment of anemia of inflammation mediated by elevated hepcidin. In iron-loading anemias including β-thalassemia, hepcidin is chronically suppressed by the exuberant but ineffective erythropoietic activity. This is the cause of iron overload in untransfused thalassemia patients and may contribute to iron loading even in transfused patients. We found that ERFE expression is greatly increased in the bone marrow and spleen of mice with β-thalassemia intermedia (th3 model). Transgenic ablation of ERFE in th3 mice normalized hepcidin and partially corrected their iron overload. Although human studies of the role of ERFE in health and disease are clearly needed, ERFE is a promising candidate for the pathological suppressor of hepcidin in anemias with ineffective erythropoiesis.

References:

1. Kautz L, Jung G, Valore EV, et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014; 46: 678-684.

Disclosures

Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Merganser Biotech: Equity Ownership. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.
Maintenance of Pluripotent Stem Cells is Influenced by Ser/Thr Metabolism
Authors: Cantley Lewis C.
Published: October 2019

Recent studies have suggested not only that stem cells have different metabolic requirements than terminally differentiated cells, but also that metabolic intermediates may play a role in the maintenance of stem cells. It has long been clear that changes in acetylation and methylation of histones, as well as methylation of DNA play critical roles in deciding cell fate. The availability of critical intermediates in metabolism, especially S-adenosylmethionine (SAM), acetyl-CoA, nicotinamide adenine dinucleotide (NAD) and a-ketoglutarate play critical roles in modulating acetylation and methylation of histones and methylation of DNA. In the course of evaluating an unusual requirement of threonine (Thr) for the growth of murine embryonic stem cells, we found that metabolism of Thr to glycine (Gly) and the subsequent use of the methyl group of Gly for converting homocysteine to methionine is critical for maintaining high levels of SAM and low levels of S-adenosyl-homocysteine. Importantly, depletion of Thr from the media resulted in decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. Demethylation of histones and DNA can also be controlled by metabolic intermediates. Mutated forms of isocitrate dehydrogenase 1 (IDH1) and IDH2 that drive acute myeloid leukemia (AML) and other cancers, produce an oncometabolite (2-hydrogyglutarate) that can compete with the a-ketoglutarate requirement for enzymes involved in hydroxy-methylation and subsequent demethylation of DNA and histones. Recent studies indicate that 2-hydroxyglutarate plays a role in blocking differentiation of cancer cells. These and other observations linking intermediates in metabolism to stem cell maintenance will be discussed.

Disclosures

Cantley: Agios Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.