bluuebunny/crossref_metadata_2025 huggingface dataset:
GLI Transcription Factors Modulate IgM Secretion In Waldenström Macroglobulinemia
Authors: Amarsaikhan Nansalmaa , Dennison Joel R. , Boi Shannon K. , Neil Matthew S. , Elsawa Sherine F.
Published: October 2019

Elevated immunoglobulin (Ig) secretion by malignant cells is a characteristic feature of several malignancies including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we defined a novel pathway involving the oncogenic GLI transcription factors modulating IgM secretion by WM malignant cells. Initial screening of molecules involved in Ig secretion identified components of the Hedgehog-GLI pathway as modulators of this cellular process. Inhibition of smoothened (SMO), the signaling component of the Hedgehog receptor, with the pharmacological inhibitor cyclopamine had no effect on cell proliferation by XTT assay. Cyclopamine inhibition had no effect on cell viability by Annexin V/Propidium Iodide staining, but had a modest in IgM secretion by ELISA in BCWM.1 but not in MWCL-1 and RPCI-WM1 cells. Interestingly, pharmacological inhibition of GLI proteins (GLI1, 2 and 3), downstream effectors of the pathway, using GANT61 reduced the expression of GLI1 and GLI2 and had no effect on cell proliferation or viability. However, it resulted in a significant reduction in IgM secretion (40-60%) in BCWM.1 (p=0.0030), MWCL (p=0.0206) and RPCI-WM1 (p=0.0303) cells. Validation of these results using RNAi showed that out of the three GLI transcription factors, only GLI2 knockdown impaired IgM secretion by BCWM.1 (p=0.0415) and MWCL-1 (p=0.0075) cells. To further characterize the mechanism of regulation of IgM secretion by GLI2, we screened for pathways that are known to modulate Ig secretion. We identified the IL-6 receptor (IL-6R) alpha subunit as a potential mechanism mediating GLI2-IgM axis. Treatment of B-cells with GANT61 resulted in reduced IL-6R expression. Furthermore, overexpression of GLI2 in B-cells increased IL-6R expression. Conversely, GLI2 knockdown reduced IL-6R expression suggesting the involvement of IL-6R in GLI2 mediated regulation of IgM secretion. Taken together, these results identify a novel role for GLI proteins, particularly GLI2, as modulators of IgM secretion by malignant B-cells. Therefore therapies targeting this signaling axis may provide efficacy in patients with Ig-mediated diseases.

Disclosures:

No relevant conflicts of interest to declare.
NF10 Project: An International, Prospective, Observational Study Of Patients With Indolent Non-Follicular Lymphoma. Analysis Of First 215 Patients
Authors: Arcaini Luca , Luminari Stefano , Cesaretti Marina , Gaidano Gianluca , Rattotti Sara , Merli Michele , Cabras Maria Giuseppina , Spina Michele , Baldini Luca , Tedeschi Alessandra , Orsucci Lorella , Gomes da Silva Maria , Marcheselli Luigi , Marcia Torresan Delamein , Federico Massimo
Published: October 2019

Background

Indolent non follicular B-Cell Lymphomas (INFL) are a heterogeneous group of lymphomas and include small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphomas (LPL), and marginal zone lymphomas (MZL). In 2010 the NF10 study was proposed by the Fondazione Italiana Linfomi as a prospective registry specifically devised for investigating the prognosis of this group of lymphomas with particular emphasis on splenic MZL.

Methods

The purpose of the study is to verify whether a prospective collection would provide more accurate data to better define prognosis of INFL. The registration of consecutive patients with newly diagnosed INFL satisfying entry criteria is ongoing at a dedicated website via secure HTTP protocols. Each patient should be followed for up to 5 years. So far the study has been activated in several centers in Europe, South America and Asia. Patients who have a locally established diagnosis of SLL, LPL, or MZL and have never received anti-lymphoma therapy are eligible for inclusion in the study. All ages and disease stages were allowed.

Results

Between September 1st 2011 and August 1st 2013, 215 cases from 28 European and South American Institutions have been registered. At time of current analysis, data on 186 cases were available. Based on local pathology report 12% of cases were registered as SLL, 19% as LPL and 57% as MZL, including splenic (20%), nodal (15%), and extranodal (22%) subtypes; 12% of cases were classified has CD5-negative B-cell chronic lymphoproliferative disorders. Median age was 66 years (rage 28-90), 54% of patients were males; Ann Arbor stage was I-II in 23% and III-IV in 77%; 13% had B symptoms, 10% had ECOG performance status > 1, LDH and Beta2-microglobulin were elevated in 19%, and 49% of cases, respectively. Six percent of cases were HCV positive. Regarding HBV infection, 20% of patients were HBcAb-positive and only 3% of patients were HbsAg-positive. Among baseline characteristics, also details on initial treatment plan were collected; an immediate systemic therapy was planned in 48% of cases and included the use of anti-CD20 monoclonal antibody in 94% of cases, the use of alkylating agents-based regimens in 57%, CHOP/like in 15% and bendamustine in 15%. Patient enrollment and data collection on actual therapy, response and follow-up and histological revision are currently ongoing.

Conclusions

The NF10 confirms that a web based world-wide cooperation allows the collection of a relevant and quite complete set of data in a limited period of time, providing a platform for future prognostic and therapeutic studies.

Disclosures:

Federico: MedImmune: Research Funding.
Acquired Factor VIII Inhibitor: A Single Institution Experience With 62 Patients
Authors: Wysokinska Ewa M. , Mehta Ramila , Grill Diane , Pruthi Rajiv K.
Published: October 2019

Background

Acquired Factor VIII inhibitor or autoimmune hemophilia A (AHA), has an estimated incidence of up to 1.5 cases per million/year and may result in severe hemorrhagic complications and death. Approximately 50% of cases have an underlying condition such as malignancies, autoimmune disorders and post-partum state. AHA should be suspected in any patient presenting with unexplained bleeding and an inhibited aPTT. Management consists of maintaining hemostasis and elimination of the inhibitor, however management is not standardized. We present 62 cases of AHA managed at Mayo Clinic Rochester, over the course of 36 years. We also analyzed whether aPTT at presentation correlated with the strength of inhibition measured by Bethesda Titer.

Methods

After IRB approval, medical records of patients with AHA were reviewed and all clinical data collected. Cumulative incidence of death was estimated by Kaplan-Meier analysis. Spearman correlation was used to calculate relation of APTT to Bethesda titer.

Results

Between 1976 and 2012, we identified 62 patients (male: 35), with a median age at diagnosis of AHA 69 years (mean 64, range 20-86). Clinical presentation consisted of extensive ecchymoses (n=40, 64%) in majority of cases. 29/62 (47%) patients had at least 1 identifiable predisposing condition with 12/62 (19%) patients with an underlying malignancy and 16/62 (26%) with underlying autoimmune conditions. Median Bethesda titer was 29 (range 1 to 1178). Bethesda titer was not related to the number or duration of hospitalizations. Most (69%) patients had at least one hospitalization and 12 (19%) had more than one hospitalization for bleeding complication. Inpatient therapy for bleeding consisted most commonly of FEIBA in 21 pts (34%) and rFVIIa in 6 pts (10%). Prednisone was the most common immunosuppressant used in 54 (87%) patients while Rituximab was used in 11 (18%) patients. Of 32 patients with available follow up labs most (69%) achieved remission. There was no difference in remission rates between patients treated or not treated with Rituximab (p=0.1735).

Conclusion

Acquired Hemophilia A is a rare condition with very heterogenous presentation. It affects mostly older male patients who present with ecchymoses and elevated APTT. The degree of APTT prolongation at the time of diagnosis does not correlate with the strength of the Bethesda titer and should not guide choice of therapy in a patient presenting with an acute bleed. Rituximab use in the 11 patients treated at Mayo did not seem to influence remission rates or survival.

Disclosures:

No relevant conflicts of interest to declare.
Predictors Of Cancer Associated Thrombosis
Authors: Tafur Alfonso , Dale George L. , Rathbun Suman , Mansfield Aaron S. , Cherry Mohamad , Comp Philip C. , Wren Jonathan , Stoner Julie
Published: October 2019

Introduction

Cancer associated thrombosis is the second most common cause of mortality among patients with malignancy. While preventive low molecular weight heparin is effective in preventing venous thromboembolism (VTE), lack of agreement on risk stratification methods have precluded its widespread use. The most studied VTE prediction tool is the Khorana score, but it does not capitalize on the use of novel biomarkers. We aimed to evaluate the value of biomarkers in prediction of VTE among patients with active cancer receiving outpatient chemotherapy.

Methods

We prospectively collected pre-chemotherapy blood samples on adult patients with solid organ tumors. We excluded patients on anticoagulation and those unable or unwilling to consent. Along with demographics and cancer specific variables, potential novel predictors of VTE were selected a priori and included: coated platelets (CP), D dimer (DD), protein C (PC), fibrin monomers (FM), mean platelet volume (MPV), platelet microparticles (PMP). Type of cancer was grouped according to the Khorana score (Very high risk: Stomach, Pancreas; High risk: Lung, Gynecologic, Bladder, Testicular; Low/Moderate risk: all other sites). VTE events were adjudicated at 6 months of follow up. For all statistical analyses we used SAS 9.2 (SAS Institute Inc., Cary, NC, USA). A p value of <0.05 was considered as statistically significant. We only performed univariate analysis at this point of our study due to the stage of recruitment and period of follow-up.

Results

We have recruited a total of 101 patients (mean + standard deviation age 59.7+11 years, 68% female), 20% of the 75 patients with complete follow up have developed a VTE. We found no significant association between the incidence of VTE and age, gender, or baseline Khorana score. The variables associated with incident VTE were: BMI (odds ratio [OR] 8.5 for BMI<25 kg/m2, p<0.001), type of cancer (OR 14.5 for very high risk compared to high risk, p<0.01) and protein C (OR 3.3 for protein C<110ug/mL the lowest tertile, p=0.05). The rest of the tested potential biomarkers did not have a strong association with incident VTE in the current analysis (Table). Twenty percent of the patients were on aspirin and none of them developed VTE compared to 23% of those not taking aspirin who developed VTE, (p=0.2).

Conclusion

Pre Chemotherapy level of Protein C is a potential biomarker for the incidence of VTE. We confirm that cancer type and BMI are predictors of cancer associated VTE. In addition, lower BMI is not usually recognized as a potential predictor of VTE in other prediction scores. We were underpowered to detect differences in the VTE incidence according to Khorana score. Finally, aspirin may have a role in outpatient cancer associated VTE prevention and deserves further investigation.

Disclosures:

Tafur: Stago Diagnostica: Research Funding. Rathbun:Stago Diagnostica: Research Funding.
Symptomatic and Incidental Venous Thromboembolic Disease Are Both Associated With Mortality In Patients With Prostate Cancer
Authors: Chaturvedi Shruti , Sidana Surbhi , Khorana Alok A. , McCrae Keith R.
Published: October 2019

Introduction

The association between malignancy and venous thromboembolism (VTE) is well established. Less is known about the independent impact of VTE, both symptomatic and incidental, on survival in patients with prostate cancer.

Methods

We conducted a retrospective cohort study of 457 consecutive patients with prostate cancer who received outpatient clinical care at the Cleveland Clinic from January 2006 to June 2006. Data were collected regarding clinical characteristics, treatment and outcomes. Fisher exact (for categorical variables) and t-test (continuous variables) were utilized to test associations with VTE and mortality. Survival functions were estimated using the Kaplan Meier method and a Cox regression model was used to model the mortality hazard ratio (HR) with date of diagnosis as time origin.

Results

The mean age of our cohort of patients was 65.86 ± 8.64 years. Three hundred and fifty eight (78.3%) had clinically localized disease, 76 (16.7%) had locally advanced disease and 41 (8.9%) had metastatic cancer at diagnosis. One hundred twenty four (27.1%) men underwent surgery, 315 (68.9%) received radiotherapy, 201 (44%) received hormonal therapy and 71 (15.5%) received chemotherapy. Complete follow up was available on 403 patients of which 109 (27%) died during the period of follow up. VTE occurred in 42 (9.2%) patients (33 deep vein thrombosis, 5 pulmonary embolism, and 4 patients with both DVT and PE), of which 27 (64.3%) were symptomatic and 15 (35.7%) were incidentally diagnosed. Twenty-three were outpatients and 17 were hospitalized when VTE was diagnosed. Metastatic disease (p<0.0021), and chemotherapy (p=0.002) were associated with VTE. There was no association of VTE with more traditional risk factors such as obesity, renal disease, rheumatologic disease, hip fracture, COPD, and smoking.

Metastatic disease (p=0.002), ECOG score of 3 or 4 (p<0.001), and VTE (p<0.001) were predictors of mortality. Adjustment for stage of disease was included in the multivariate analysis. Mean survival of patients with and without VTE was 91.8±11.3 months and 144.8±3.6 months respectively (p<0.01). Five-year survival rates were also significantly lower for patients with VTE compared to those without (45.2% vs. 87.8%, p<0.001). Patients with both symptomatic and incidental VTE had significantly poorer survival than those without however there was no significant difference in survival between patients with symptomatic or incidental VTE (70.78±44.82 years versus 77.87±63.07, p=0.674).

Conclusion

VTE, both symptomatic and incidental, is a predictor of poor survival in patients with prostate cancer, especially those with advanced disease. This finding has implications for the clinical care of patients with prostate cancer. Further studies are needed to evaluate the benefit of prophylactic and therapeutic anticoagulation in this population.

Disclosures:

No relevant conflicts of interest to declare.
Increased Capacity Of Monocytes From Patients With Multiple Myeloma Treated With Thalidomide Or Lenalidomide To Generate Tissue Factor
Authors: Kornberg Abraham , Izak Marina , Cohen Yossi
Published: October 2019

Treatment of multiple myeloma (MM) with thalidomide or lenalidomide is associated with increased incidence of thrombosis in contrast to treatment with bortezomib. The mechanism of thrombosis is unknown. Monocytes generate potent tissue factor (TF), the main activator of coagulation, in response to various stimuli (endotoxin, cytokins and others) and in diseases with increased incidence of thrombosis. We investigated the capacity of monocytes from patients with MM to generate TF in relation to different modalities of treatments and activation of coagulation. Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Hypaque centrifugation and monocytes by adhesion. TF activity was assayed by modified PT using the cells as source of TF and TF antigen by ELISA, in endotoxin stimulated (10 ug/ml) and unstimulated PBMC ( all patients) and purified monocytes (50% of patients), before and after 3-4 courses of treatment. Monocytes were identified by anti CD14 and Plasma Fragment1.2 was assayed by ELISA.

No TF activity@ (X10-5 U/monocyte) TF antigen@ (X10-5 pg/monocyte) Plasma fragment 1.2@ (nM) Endotoxin - + - + Bortezomib based treat* 12  before 2.2 4.8 22 64 0.28  after 1.8 4.2 18 57 0.36 Thalidomide based treat* 4  before 2.4 5.5 26 60 0.24  after 1.8 34.8# 20 440# 2.48# Lenalidomide based treat^ 6  before 2.1 6.2 28 62 0.30  after 4.1 58.6# 16 312# 3.42# @Mean *First line treatment ^Second line treatment # P<0.05

One patient on thalidomide and 2 on lenalidomide developed thrombosis. The results show that TF was low in unstimulated monocytes from all patients before treatment. It was increased mildely in stimulated monocytes from all patients before treatment and also after treatment with bortezomib (X 2-3). TF in stimulated monocytes and also plasma fragment1.2 were increased significantly after treatment with thalidomide and lenalidomide (x 14-22 and X 10). The phenomenon of enhanced capacity of monocytes to generate potent TF after treatment with thalidomide and lenalidomide but not bortezomib, and the correlation with activation of coagulation suggest a role of monocyte TF in thrombus formation by these drugs. The mechanism of the enhanced capacity is unknown and may be attributed to the immunomodulatory effect of the drugs.

Disclosures:

No relevant conflicts of interest to declare.
Tanshinone IIA has anti-platelet effect and induces apoptosis in megakaryocytes via TNFR and Caspases
Authors: Yang Mo , Wu Fu qun , Zhou Min , Ye Jie yu , Li Xiao jing , Chong Beng , Liu Chang
Published: October 2019

Tanshinone IIA (TIIA) isolated from Danshen (Radix Salviae Miltiorrhiza Bge) is a derivative of phenanthrenequinone, which has been used for centuries to treat hypercoagulation related diseases. Our previous study showed that TIIA has neural protective effect (Xia et al, Pediatric Research, 2005). In this study, we investigated the effect of TIIA on platelets and megakaryocytes in a rabbit model, and its molecular mechanisms. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin with TIIA treatment (5 mg/kg/day, iv). The platelet count, platelet aggregation, and inflammatory cytokine IL-1β level were significantly increased in vasculitis model compared with normal group at day 7. There were positive correlations between platelet count and IL-1β level. The number of megakaryocytes and CFU-MK formation in bone marrow were significantly increased in this model. Treatment with TIIA significantly decreased all these parameters, and also significantly reduced the injury vessel in immune vasculitis. Furthermore, the effect of IIA on in-vitro megakaryopoiesis was investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation (p<0.05, n=6). TIIA also induced the apoptosis of megakaryocytic cell line CHRF. The cell viability was reduced to 81% after 72hrs of TIIA treatment. Using Annexin V/PI staining, TIIA induced apoptosis on CHRF cells in a dose dependent manner. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5% to 16% after TIIA treatment. Using JC-1 assay, we found that the depolarized cells increased from 9% to 17% after TIIA treatment suggesting the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated gene (6.0±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was up-regulated to 2.0±0.167 folds. These results suggest that TIIA has an anti-platelet effect in this model. Its mechanism may be related to its inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes via TNFR and caspases.

Disclosures:

No relevant conflicts of interest to declare.
Development of HemSol-P(TM), a Novel Platelet Storage Solution Permitting Storage at 4o C
Authors: Ho David H. , Stefanson Steingrimur , Ershler William B
Published: October 2019

The current method of storing platelets as platelet rich plasma (PRP) limits storage life to five days. As many as 10% of platelet units are discarded at a cost of approximately $500 million dollars annually in the U.S. alone. Platelets stored at 22°C remain metabolically active and thus require nutrients supplied in PRP. These nutrition rich platelet preparations promote bacterial growth, demonstrable in approximately one of 3000 platelet units and one of six contaminated units result in clinical sepsis. Also of critical importance, the transfusion of plasma has been associated with transfusion-related acute lung injury (TRALI), which is now the leading cause of transfusion related deaths.

We have developed an innovative preservation technology to address these issues. This process involves mixing cells with a non-toxic proprietary mixture of simple and complex carbohydrates that protect cells from oxidative damage, reduce metabolic activity and allow extended storage. Preliminary studies demonstrate that platelets preserved in this solution (HemSol-P) for up to 14 days at 22°C respond normally to platelet agonist in aggregation assays and had low active GPIIb-IIIa expression and demonstrated low Annexin V binding.

Furthermore, platelets stored in HemSol-P can be maintained at 4oC for at least 15 days and remain in circulation in a mouse transfusion model. Mouse platelets were isolated from 0.1 ml of blood collected by retro-orbital puncture. Washed platelets were biotinylated and then resuspended in either plasma or HemSol-P to a final concentration of 2x109 platelets/mL and stored at 4°C for up to 15 days. Our results show that platelet survival in the circulation 24 hours after transfusion held steady at 65%, even after 15 days of cold storage in HemSol-P.

HemSol-P has excellent potential for clinical application in transfusion medicine. The potential for cold storage extends shelf life of donated platelets beyond five days. The absence of a plasma component in HemSol-P will likely reduce the incidence of contamination/infection and TRALI, and also reduce overall transfusion costs by increasing the platelet storage time and simplifying procurement and preparation logistics.

Disclosures:

Ho: HeMemics Biotechnologies Inc: Employment, Equity Ownership, Patents & Royalties. Stefanson:HeMemics Biotechnologies Inc: Employment, Equity Ownership, Patents & Royalties. Ershler:HeMemics Biotechnologies Inc: Consultancy, Equity Ownership.
Glucocorticoids Reduce Expression Of p53 Target Genes and Reverse Anemia In a Mouse Model Of Diamond-Blackfan Anemia
Authors: Sjögren Sara , Jaako Pekka , Karlsson Stefan , Flygare Johan
Published: October 2019

Diamond-Blackfan anemia (DBA) is a rare congenital disease of macrocytic anemia, increased cancer predisposition and progressive bone marrow failure. Mutations in ribosomal protein genes are responsible for most cases and the most commonly mutated gene, RPS19 is affected in around 25% of DBA patients. Although glucocorticoid (GC) administration has been used to stimulate erythropoiesis in DBA patients for decades, the therapeutic mechanism of GC treatment is poorly understood. Since it has been shown that bone marrow failure in DBA is caused by p53 activation we hypothesized the therapeutic mechanism of GC involves reduced p53 activity. To assess possible connections between p53 activity and GC administration in DBA we used a doxycycline-inducible mouse model for RPS19-deficient DBA (Jaako et al, Blood 2011). In these mice, doxycycline administration leads to reduced Rps19 levels, defective ribosomal biogenesis, proliferative arrest, increased apoptosis of erythroid progenitors and p53-dependent bone marrow failure.

In this study we show that Rps19-deficient mice respond to treatment with the GC prednisolone, with a significant increase in red blood cell count as well as in hemoglobin concentration and hematocrit compared to Rps19-deficient controls (p-values<0.05). Also, prednisolone treated Rps19-deficient mice show a significant increase in the Ter119+ erythroid cell fraction in the bone marrow compared to Rps19-deficient controls. Gene expression analysis of c-Kit+ fetal liver cells from E14.5-15.5 of Rps19-deficient embryos cultured in vitro in the presence of doxycycline for 3 days, showed an increase in p53 responsive gene expression of multiple p53 targets such as p21, Bax, Ccng1, Phlda3 and Prl-3. However, upon administration of the synthetic GC dexamethasone all these genes significantly failed to up-regulate, even in the presence of doxycycline (p-values< 0.02). Also, dexamethasone treated RPS19-deficient c-Kit+ fetal liver cells show an increase in proliferation compared to RPS19-deficient controls (p-value< 0.05), that starts already after 2 days of in vitro culture, being more pronounced over time. These results indicate that administration of GC dampens p53 responsive gene expression of genes known to promote cell cycle arrest as well as apoptosis. Although GCs are known to regulate many functions within an organism, our results indicate that the therapeutic effect of GC in DBA patients at least in part is explained by inhibition of p53 responsive gene activation, leading to increased proliferation and survival of erythroid progenitors.

Disclosures:

No relevant conflicts of interest to declare.
BMI1 Promotes Erythropoiesis Through Regulating Ribosome Biogenesis
Authors: Gao Rui , Chen Sisi , Kobayashi Michihiro , Yoder Mervin C. , Kapur Reuben , Zhu Xiaofan , Liu Yan
Published: October 2019

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome of childhood manifested as macrocytic anemia with insufficient erythroid precursors in the bone marrow. Within the decade following the demonstration that mutations in the ribosomal protein gene RPS19 can lead to DBA, this disease has become a paradigm for an emerging group of pathologies (ribosomopathies) linked to defects in ribosome biogenesis. Mutations in ribosomal protein genes impair ribosome biogenesis, resulting in activation of the p53 tumor suppressor pathway, cell cycle arrest and defective erythropoiesis. While mutations in ribosomal protein genes have been found in 50-60% of DBA patients, genetic abnormalities in the remaining patients are largely unknown. Despite improvements in our understanding of the pathophysiology of DBA, the molecular basis for selective impairment of the erythroid lineage in this disorder is not understood. In particular, how ribosome biogenesis is regulated in erythroid precursors remains elusive. Our laboratory has been investigating the role of Polycomb group protein Bmi1 in regulating hematopoietic stem cell (HSC) self-renewal and lineage commitment. Recently, we found that Bmi1 is a critical downstream target of AKT signaling and AKT-mediated phosphorylation of Bmi1 inhibits HSC self-renewal (Liu et al., Science Signaling, 2012). Upon more detailed analysis of the hematopoietic phenotype of the Bmi1 knockout mice, we have observed that these mice develop macrocytic anemia and show delayed recovery following phenylhydrazine (PHZ)-induced hemolytic anemia. This phenotype suggests defective erythropoiesis and we identified that loss of Bmi1 expression results in a block in erythroid differentiation and decreased erythroid colony formation. Gene expression profiling indicated that multiple ribosomal protein genes were downregulated in Bmi1 null erythroid precursors. Moreover, we discovered that the p53 pathway is activated in Bmi1 null erythroid progenitor cells and genetic inhibition of p53 activity rescued the erythroid defects in the Bmi1 deficient mice. Thus, Bmi1 null mice recapitulate many critical features of human DBA. Furthermore, we demonstrated that BMI1 plays a critical role in human erythropoiesis as knockdown of BMI1 in human CD34+ cells decreases ribosomal protein gene expression, activates the p53 pathway, and blocks erythroid differentiation. Importantly, we observed that BMI1 expression is downregulated in bone marrow cells from some DBA patients. Thus, BMI1 plays a critical role in regulating ribosome biogenesis in erythroid precursors and BMI1 deficiency may contribute to the pathogenesis of DBA. Understanding how BMI1 regulates ribosome biogenesis and erythroid development will provide novel insight into the processes by which BMI1 and ribosomopathies contribute to the pathogenesis of DBA and potentially new targets for therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
FANCD2 and RAD51 Regulate PCNA Monoubiquitination and Translesion Synthesis
Authors: Kupfer Gary M. , Chen Xiaoyong
Published: October 2019

FANCD2 is a key player in Fanconi anemia (FA) pathway. It has been shown that PCNA and Rad18 can interact with FANCD2 and regulate FANCD2 monoubiquitination. Rad18 is the E3 ubiquitin ligase responsible for PCNA monoubiquitination, which is critical for PCNA function in translesion synthesis (TLS). Previous work by us and others has shown that the FA pathway interacts with PCNA and RAD18. Even though FA cells demonstrate unequivocal sensitivity to crosslinkers such as mitomycin C (MMC), we find surprisingly that they are largely resistant to hydroxyurea (HU), except for cells containing no expression of FANCD2. In order to understand the mechanism for this finding, we explored the interaction of FANCD2, Rad51, Rad18, and PCNA, which form a complex and is enhanced upon HU exposure. While FANCD2 is required, monoubiquitination of FANCD2 is not. In addition, PCNA monoubiqutination follows exactly the same pattern. The monoubiquitination of PCNA in response to HU in FANCA deficient mutant confirms that non-ubiquitinated FANCD2 can still support PCNA monoubiquitination. Rad51, but not BRCA1 is also required in regulating PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion polymerase Pol H chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knock down cells and Rad51 siRNA knock down cells. Our data suggest that FANCD2 and Rad51 play an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with mitomycin C (MMC) treatment, indicating that DNA repair systems differentially respond to different types of DNA damage.

Disclosures:

No relevant conflicts of interest to declare.
NSGS Mice Develop a Progressive, Myeloid Cell Dependent Aplastic Anemia and Bone Marrow Failure Upon Engraftment With Human Umbilical Cord Blood CD34+ Cells
Authors: Wunderlich Mark , Sexton Christina , Mizukawa Benjamin , Mulloy James C.
Published: October 2019

One key limitation of hematopoietic xenograft mouse models is the relative lack of effective myelopoiesis compared with human hematopoiesis. NSGS mice expressing human myelo-supportive cytokines allow for improved myeloid cell output upon human hematopoietic cell transfer. Surprisingly, we found that NSGS mice developed a severe, eventually fatal anemia upon humanization with umbilical cord blood (UCB) CD34+ cells. Anemia was characterized by a consistent, progressive drop in red blood cell numbers and hemoglobin that was correlated with higher MCV and an increased reticulocyte production at early time points. Eventually total white blood cell and platelet counts were also affected and reticulocyte numbers declined to or below normal. These effects were only apparent in mice with human grafts, and were also not observed in NSG mice with human grafts. Bone marrow cellularity was 40% of control humanized NSG mice and spleens were significantly larger, implying bone marrow dysfunction and extra medullary hematopoiesis. Bone marrow sections from anemic mice clearly showed a general hypocellularity with abundant fatty tissue filling the majority of the intramedullary space. We were able to recover red blood cell counts and hemoglobin to some degree upon transfusion of non-humanized mouse blood, however, the effect lasted only 7 days. CFSE labeling of anemic blood allowed us to detect a decreased half-life of red blood cells of 20 days compared to 30 days for humanized NSG mice. Treatment of mice with Campath, a CD52 antibody, completely reversed the phenotype at the expense of the entire human graft, demonstrating clearly that this was a graft-mediated process. However, specific ablation of B and T cells with rituximab and OKT3 did not alter the phenotype, even when administered soon after CD34+ cell transplant to suppress development of lymphocytes. Additionally, dexamethasone and IV/IG were ineffective in reversing the disease. These data, coupled with negative findings from tests to detect mouse reactive human antibodies, point towards myeloid cell involvement. Indeed, further experiments revealed that specific ablation of human myeloid cells effectively reversed the anemia. In summary, these data highlight a potential pitfall for effective myelopoiesis in the xenograft setting. More broadly, these findings demonstrate the possibility for myeloid driven bone marrow dysfunction and may warrant investigation of myeloid cells in cases of idiopathic aplastic anemia that fail to respond to therapies directed at autoimmune lymphocyte activity.

Disclosures:

No relevant conflicts of interest to declare.
PU.1 and Spi-B Oppose Transformation Of Pre-B Cells Through Activation Of Key Genes Involved In B Cell Receptor Signalling
Authors: Christie Darah A. , Turkistany Shereen A. , Xu Li S. , Li Stephen K. H. , Welch Ian , Broughton Heather C. , Bell Gillian I. , Hess David A. , DeKoter Rodney P.
Published: October 2019

B cell development is controlled by stage-specific expression of transcription factors. Aberrant expression of such factors can lead to B cell acute lymphoblastic leukemia (B-ALL). Deletion of genes encoding the E26 transformation-specific (ETS) transcription factors, PU.1 and Spi-B, in B cells (CD19+/CreSfpi1lox/loxSpib-/- mice, abbreviated to CD19-CreΔPB) leads to B-ALL at 100% incidence and with a median survival of 21 weeks. However, little is known about the target genes of PU.1 and Spi-B that explain leukemic transformation in these mice. In the current study, we investigated the developmental origins and mechanisms of leukemogenesis in CD19-CreΔPB mice. We found that B-ALL cells in CD19-CreΔPB mice had frequently rearranged both their heavy and light chain genes, but retained cell surface expression of interleukin-7 receptor (IL-7R), suggesting aberrant pre-B cell differentiation. Preleukemic CD19-CreΔPB mice had increased frequencies of pre-B cells compared to wild type mice. Pre-B cells, but not mature B cells, purified from the bone marrow of preleukemic CD19-CreΔPB mice could rapidly transfer disease to transplanted recipient mice. B-ALL cells from established tumors had uniform expression of markers indicating a pre-B cell phenotype and contained a high-frequency of leukemia-initiating cells as measured by transplantation assays. Genome-wide analysis of gene expression showed that B cell receptor signalling was the top impaired pathway in B-ALL cells from CD19-CreΔPB mice. Bone marrow cells from CD19-CreΔPB mice had increased responsiveness to IL-7R signalling and could be cultured as IL-7-dependent cell lines. Preleukemic or leukemic cells from CD19-CreΔPB mice expressed reduced levels of the gene encoding Bruton’s tyrosine kinase (Btk), which we show is a target gene of PU.1 and/or Spi-B that in combination with reduced BLNK is sufficient to explain increased IL-7R responsiveness. We conclude that mutation of PU.1 and Spi-B predispose developing B cells to leukemogenesis by impairing expression of key genes, such as Btk, that are required for BCR signalling and are involved in attenuation of IL-7 receptor signaling.

Disclosures:

No relevant conflicts of interest to declare.
NRF2 Signaling Promotes Survival and Drug Resistance Of Acute Myeloid Leukaemia Through Induction Of Mir-125b-1
Authors: Shah Niraj M , Rushworth Stuart A , Zaitseva Lyubov , Bowles Kristian M , Macewan David J
Published: October 2019

We have previously shown that Nrf2, an important regulator of several biologic processes involved in the progression of cancer, has abnormal constitutive expression in AML. As post-transcriptional regulation by micro RNAs (miRNAs) affects the malignant phenotype of a number of diseases, in this study, we aimed to identify and characterise NRF2 regulated miRNAs in AML.

Initially a qRT-PCR miRNA array was utilised to investigate miRNA expression in response to NRF2 lentiviral mediated knockdown in the AML cell line THP-1. To verify these results analysis of miRNA identified in the array were performed in response to transfection of siRNA against the NRF2 inhibitors Keap1 and Bach1. Subsequently we performed bioinformatic analysis to identify putative NRF2 binding sites upstream of the miRNA coding regions. From this we identified miR-125b as a putative NRF2 regulatory miRNA.

There are two miR125b paralogs coding for the same mature sequence in humans, miR-125b-1 on chromosome 11 in a cluster containing let-7a-2 and miR-100 and miR-125b-2 on chromosome 21 in a cluster with miR-99a and let-7c. To identify which paralog is upregulated by NRF2 qRT-PCR of the miRNA in each cluster was performed and referenced to the immature miR125b sequences which do differ between each paralog. Results show that NRF2 regulates miR-125b-1, but does not regulate miR-125b-2 or the other miRNAs within the miR125b-1 or the miR125b-2 clusters, implying that mir-125b-1 contains an alternative promoter which is regulated by NRF2.

To locate the NRF2 promoter site in miR125b-1 electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChiP) were carried out which confirmed NRF2 binding to the putative ARE site 946bp upstream of the 5’ of the miR-125b-1. To confirm NRF2 capacity to transcriptionally regulate miR-125b-1, we cloned the miR-125b-1 promoter into the PGL4 luciferase vector (PGL4/p125b) and found that NRF2 knockdown significantly inhibited PGL4/p125b luciferase activity compared to control knockdown.

Finally to identify the functional relevance of miR-125b-1 in a broad range of AMLs, miR-125b-1 mimic and antagomiRs were transfected into 3 AML cell lines and 11 primary AML samples. Results show a 10-25% increase in annexin V staining in the miR-125b-1 antagomiR experiments compared with the mimic or controls. Moreover miR-125b antagomiR increased the cytotoxicity of conventional AML therapy (daunorubicin and cytosine arabinoside). These results confirm the cytoprotective role of miR-125b-1 in AML.

In summary we find that NRF2 regulation of miR-125b-1 acts as a novel survival pathway in human AML.

Disclosures:

No relevant conflicts of interest to declare.
Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia
Authors: Kuntimaddi Aravinda , Lokken Alyson , Zhang Shubin , Thorpe Jeremy , Leach Benjamin , Lumba Bhavna , Hemenway Charles S. , Adli Mazhar , Zeleznik-Le Nancy J. , Bushweller John H.
Published: October 2019

Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis.

We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis.

1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013).

Disclosures:

No relevant conflicts of interest to declare.
Heatshockproteins-60 and 90 Are Overexpressed In Leukemia Stem Cells and Stabilize Oncogene Signaling
Authors: Kampa-Schittenhelm Kerstin M , Grandl Tanja , Akmut Figen , Illing Barbara , Schittenhelm Marcus M
Published: October 2019

Introduction

Activating mutations of the KIT class III receptor tyrosine kinase (TK) are associated with the pathophysiology of acute leukemia, especially core binding factor leukemia (CBFL), and systemic mastocytosis (SM). Despite considerable antiproliferative and proapoptotic activity of several KIT TK inhibitors in vitro, clinical efficacy in AML and SM is generally moderate. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways. Previously we reported that KIT TK inhibition results in significantly increased phosphorylation of heat shock protein (HSP) family members and that KIT is a client protein of phosphorylated (p)HSP90 putatively stabilizing KIT protein function in the presence of KIT TK-inhibitors (Kampa-Schittenhelm et al., ASH annual meeting 2010). This prompted us to further test the HSP inhibitor IPI-504 in KIT dependent CBFL, including the leukemic stem cell fraction.

Methods

Protein expression levels of (p)HSPs in leukemic blasts of high-risk and CBFL patients were studied by flow cytometry focusing on the CD34+/CD38- leukemia stem/progenitor cell fraction. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays.

Results

(p)HSP90/60 levels were preferentially upregulated in CBFL associating with KIT dysregulation. Consequently, HSP90 inhibition with IPI-504 potently degraded KIT expression causing a direct antiproliferative and antiapoptotic effect in CBFL in in vitro and ex vivo models. Efficacy of IPI-504 was potentiated when combined with TK inhibitors. Importantly, high expression of (p)HSP90 and HSP60 was particularly observed in the CD34+/CD38- putative leukemia stem cell fraction arguing for a function as protection mechanism to conserve (leukemic) progenitor cell function upon cell stress such as antileukemic treatment.

Conclusion

HSPs are upregulated in CBFL, and IPI-504 induces antiproliferative and proapoptotic effects in primary leukemia samples. Importantly, in particular the putative malignant progenitor cell pool in KIT-associated acute leukemia expresses high levels of (p)HSP90, identifying HSP90 inhibition as an attractive novel strategy to overcome the therapy-refractory behavior of malignant stem/progenitor cells. Our results provide a rationale for the evaluation of HSP90 inhibitors such as IPI-504 in CBFL.

Disclosures:

No relevant conflicts of interest to declare.
A HoxD11 Homeobox Gene Mutation Associated With Congenital Absent Radius But Without The Thrombocytopenia Of The TAR Syndrome Or a HoxA11 Mutation
Authors: Fleischman Roger A.
Published: October 2019

HoxA11 and HoxD11 are homeobox genes critical for normal development of the forearm and thus are potential candidate genes for involvement in the pathogenesis of the thrombocytopenia/absent radius (TAR) syndrome. However, we previously reported an absence of coding sequence mutations in either HoxA11 or HoxD11 in a series of 10 unrelated TAR syndrome patients (Fleischman RA et al., Br J Haematol., 116:367-75, 2002). Despite this negative finding, interest in the potential role of homeobox genes in the TAR syndrome has been supported by a report of a HoxA11 mutation occurring in two kindreds with amegakaryocytic thrombocytopenia and radio-ulnar synostosis, a less pronounced more proximal pattern of radial malformation (Thompson AA and Nguyen LT. Nat Genet., 26:397-8, 2000). Unlike HoxA11, however, no mutations in the human HoxD11 gene have been described thus far that would help elucidate the potential role of this paralogous gene in megakaryopoiesis or the TAR syndrome.

We now describe a novel mutation in human HoxD11 that results in a polyalanine sequence expansion, (GCG)6→ (GCG)8, and report that this mutation is associated with a unilateral absent radius in the affected propositus. A familial syndrome is suggested in this kindred, moreover, by the prior observation of a bilateral absent radius in a deceased maternal aunt. This mutation was not present in more than 100 unrelated normal subjects or 8 other unrelated individuals with sporadic absence of the radius. Two other living maternal relatives also carried the mutation but did not exhibit any radial defects, a finding consistent with autosomal dominance with incomplete penetrance, an inheritance pattern reported for short polyalanine expansion mutations in the related homeobox gene HoxD13 which cause synpolydactyly. In contrast to the reported HoxA11 mutation, however, neither the propositus nor the mutation carriers of this HoxD11 mutation exhibited thrombocytopenia or any other cytopenias or congenital defect. The results suggest that at least one class of mutation in human HoxD11 may be sufficient to cause an absent radius syndrome but unlike the reported HoxA11 mutation, does not adversely affect megakaryopoiesis. The findings further suggest that additional studies of the TAR syndrome may be necessary to exclude as yet undetected non-coding mutations in promoter or enhancer sequences that alter the expression of HoxA11, HoxD11 or other homeobox genes critical for radial development and/or megakaryopoiesis.

This work was supported by a VA Merit Award.

Disclosures:

No relevant conflicts of interest to declare.