bluuebunny/crossref_metadata_2025 huggingface dataset:
CXCL9 Contributes To Chemotherapy-Induced Acute Intestinal Damage Through Proliferative Inhibition Of Epithelial Cells
Authors: Lu Huili , Liu Hongyu , Shen Jiaqing , Weng Shunyan , Qian Lan , Wu Mingyuan , Zhu Shunying , Yu Yan , Han Wei , Zhu Jianwei , Moldenhauer Anja
Published: October 2019

Apart from angiostasis and chemoattraction, CXCL9 can derange hematopoiesis by its influence on mesenchymal stroma cells. The receptor of CXCL9, CXCR3, is abundantly expressed intracellularly in epithelial cells, even though it is rarely present on the surface of these cells. Here, we hypothesized that CXCL9 influences the proliferative and degenerative activity of epithelial cells in vitro and in vivo. CXCL9 inhibited the proliferation of MCF10A cells in a dose-dependent manner. In vivo, rhCXCL9 caused an intestinal weight loss of 30% in normal mice (n=6, 0.59±0.05 g of rhCXCL9 treated mice versus 0.83±0.06 g controls, P = 0.0007 determined by 2-tailed student’s t-test). Intestinal epithelial cells of 5-Fluorouracil (5-FU) treated mice developed a 2.55 fold higher level of cxcl9 expression, indicating that CXCL9 may participate in a chemotherapy-induced damage of the intestinal epithelium. Neutralization of the up-regulated endogenous CXCL9 by anti-CXCL9 monoclonal antibodies accelerated epithelial regeneration determined by villi length (317.5±19.9 μm versus no-antibody control 283.7±17.1 μm, P < 0.001) and crypt depth (78.0±8 μm versus control 67.9±10.9 μm, P = 0.326). CXCL9 function was highly associated with p70 ribosomal S6 kinase (p70S6K) activation (50.0±2.2 MFI versus control 27.9±1.4 MFI, P = 0.007), which was reversed by anti-CXCR3 (31.4±5.7 MFI, n=4). CXCL9 downstreamingly stimulated TGF-β secretion of epithelial cells through the mTOR/p70S6K pathway (66.3±17.1 pg/mL versus on-treated control 39.8±12.2 pg/mL, P <0.05), which was reversed by anti-CXCR3 (46.8±21.6 pg/mL, n=8). That explains the anti-proliferative effect of CXCL9 on these cells. Our results strongly suggest that anti-CXCL9 may help to mitigate a chemotherapy-induced intestinal damage. The work was supported by the National Science Foundation China (81273576, 30801419, 30901873), and the German Academic Exchange Service (A/09/90104).

Disclosures:

No relevant conflicts of interest to declare.
TPO plus Tanshinone IIA has neural protective effect and this effect may be mediated via its receptor c-mpl and PI3K/Akt signaling
Authors: Yang Mo , Ye Jie yu , Li Su yi , Liang En yu , Meng Fan yi , Lian Qizhou
Published: October 2019

Thrombopoietin (TPO) is a growth factor for platelet/megakaryocytic lineage. Our previous study showed TPO has cardiac protective effect(Li et al, Circulation, 2006). TPO may also has a protective effect in neural cells. Tanshinone IIA (TIIA), isolated from Danshen (Radix Salviae Miltiorrhiza Bge), is a derivative of phenanthrenequinone, which has been widely used as anti-platelet and neural protection drug for stroke for many years in China. In this study, a hypoxic–ischemic encephalopathy model was established, followed by 2 hrs of hypoxia in 7-day-old neonatal rat pups. TPO (12.5 ug/kg) or/and TIIA (10 mg/kg) were administrated daily (IP) for two weeks. The severity of brain injury was estimated by the weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significant less atrophy in TPO alone or TPO plus TIIA treated animals compared with that in controls at 7 and 21 days (P<0.05, n=8). The loss of neurons in the forelimb area of the cortex was also reduced after TPO or TPO plus TIIA treatment. NSE positive cells in the forelimb area of the cortex in the right hemisphere was significantly higher in the treatment group than that of the saline group (P<0.05, n=8). An improvement in sensory motor functions was also demonstrated after TPO or TPO plus TIIA treatment. Furthermore, TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres and cerebellum, and C17.2 cell line using RT-PCR. The expression of c-mpl protein was further confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunocytochemical staining. Its ligang TPO was also detected in human cerebrospinal fluids (n=10) by ELISA, and TPO mRNA was expressed in human cerebral hemispheres, cerebellum, and mouse neural stem cell line C17.2 by RT-PCR methods. TPO showed an anti-apoptotic effect on C17.2 cells by the AnnexinV/PI assay. Moreover, TPO activated PI3K/Akt signaling pathway which was demonstrated by Western blot. The Akt activation by TPO was inhibited by the PI3-kinase inhibitor LY294002. This study provided the evidences showing that TPO or TPO plus TIIA has neural protective effect and this effect may be mediated via its receptor c-mpl, then activated PI3K/Akt signaling.

Disclosures:

No relevant conflicts of interest to declare.
Imatinib mesylate has an inhibitory effect on hematopoiesis in a mice model
Authors: Ye Jie yu , Li Su yi , Liang En yu , Meng Fan yi , Chong Beng , Yang Mo
Published: October 2019

Imatinib mesylate (Gleevec or STI-571), a tyrosine kinase inhibitor, can potently block both platelet-derived growth factor (PDGF) receptor-α and β by inhibiting the receptor tyrosine kinase activity. Imatinib mesylate has been successfully used to treat patients with chronic myeloid leukemia (CML) and Ph positive acute lymphoid leukemia (ALL). However, a significant number of patients with imatinib mesylate treatment have developed neutropenia, thrombocytopenia and anemia (van Deventer, Hall et al. 2002). Our previous studies have demonstrated that PDGF/PDGFR plays an important role on thrombopoiesis, and blockage of PDGFR by imatinib inhibited the downstream signal pathway, which led to reduce of megakaryocyte production (Ye, et al. Haematologica, 2010). This may partially explain the imatinib-related thrombocytopenia. However, the role of imatinib on normal hematopoiesis remains to be investigated. In the present study, we identified the expression of PDGFR-β on the human megakaryoblastic cells: Meg-01, CHRF-288-11, Dami and M-07e, human erythromyeloblastoid leukemia cells: K-562 and human myeloblastic cells: HL-60 by RT-PCR, western blot and flow cytometry. We then demonstrated the effect of imatinib on normal hematopoiesis in the 8 wk old C57BL/6 mice model. Gleevec (25 mg/kg/day) dissolved in distilled water or water only were given orally for up to 2 wks. 15ul whole blood was taken from postorbital weekly to perform blood cell counts. The Gleevec -treated group showed a reduction of platelet count as compared to the control at day 14 (543.3±71.95×109/L vs. 330.0±62.23×109/L, n=5, P<0.05). Similarly, Gleevec caused a significant decrease in the number of white blood cells on day 14 (9.667×±0.8511×109/L vs. 6.080±0.8015×109/L, n=5, P<0.05). While no significant differences were found on red blood cell count between Gleevec and control groups. The mice bone marrow was harvested from each group to perform CFU assays. Gleevec significantly inhibited the formation of bone marrow CFU-MK (15.33±1.764 vs 3.333±0.6667, n=3), and CFU-GM(13.50±2.784 vs 6.333±0.7265, n=3) compared with the control. There were no significant differences found in CFU-GEMM and CFU/BFU-E formation between the Gleevec and control treatment. Our studies suggested that PDGFR were expressed on hematopoietic cells. Imatinib mesylate, an inhibitor of PDGFR, not only played an important role in malignant hematopoiesis, but also in normal hematopoiesis. Administration of Gleevec reduced white blood cells and megakaryocytes/platelets production, which implied its potential therapeutic role in treating myeloproliferative diseases, such as Essential Thrombocytosis.

Disclosures:

No relevant conflicts of interest to declare.
The Impact Of The Establishment Of a Hematology Telemedicine Consult Service In a Referral Center On Patients’ Travel Distance, Travel Cost and Satisfaction Scores
Authors: Yellapragada Sarvari Venkata , Hermosillo-Rodriguez Jesus , Sada Yvonne , Walder Annette , Sam Kathy , Mulchandani Alka , Hayes Teresa G , Jessica Davila A
Published: October 2019

Introduction

Travel cost and travel distance can decrease access to care. Telemedicine services can reduce patient travel distance and cost in subspecialty consult services. The impact of telemedicine services on travel distance and travel cost in hematology consult services has not been widely studied. We analyzed the impact of a hematology telemedicine consult service in the Michael E. Debakey VA Medical center (MEDVAMC) in Houston, Texas. The MEDVAMC provides subspecialty consult services for Community-based Outpatient Clinics (CBOC). Our hypothesis in this study was that the establishment of a hematology telemedicine clinic would reduce travel distance for the patient and travel pay made by the MEDVAMC for this specialty with high level of satisfaction among veterans.

Methods

We conducted a prospective cohort study of veterans enrolled in the hematology telemedicine clinic at the MEDVAMC from September, 2011 to June, 2013.  Any veteran referred to or established in the hematology clinic was eligible to be enrolled in the telemedicine service if they lived closer to a CBOC than to the MEDVAMC. The telemedicine encounter included meeting with a hematologist, a social worker and/or nutritionist.  Patients went to their assigned community clinic to connect with the hematology telemedicine service. Data on demographics and diagnosis (benign or malignant hematology condition) were collected. Outcome variables included travel distance, estimated travel cost and patient satisfaction scores with the telemedicine service. Cost of travel was defined as the estimated amount that the MEDVAMC reimburses eligible patients for their travel costs.

Results

The hematology telemedicine clinic enrolled 255 veterans during the study period. Among these patients, 195 (76%) had benign hematology conditions and 60 (24%) had malignant hematology conditions. 237 (93%) of patients were male, and 156 (61%) lived in a rural area. The mean travel distance saved was 64 miles (SD 11.5). This translated into an estimated average of 21 dollars of travel pay saved per patient. Out of all telemedicine encounters, there was a 42% survey return rate. In 94% of these surveys veterans reported they were satisfied with the visit and that they would recommend the hematology telemedicine program.

Conclusion

The establishment of a telemedicine consult service in a referral center decreased travel distance for patients and travel pay cost. Most patients that answered the feedback surveys reported they were satisfied and that they would recommend the program. Other studies looking at cost-effectiveness, and patient and disease-related outcomes will help understand further the impact and benefits of such a program.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic CMR Parameters For Cardiac Complications In Large Cohort Of Well Treated Thalssemia Major Patients
Authors: Meloni Antonella , Tassi Cristina , Batzella Maria Grazia , Commendatore Francesca Valeria , Giannotti Giorgio , Serra Giuseppe , Keilberg Petra , Maggio Aurelio , Valeri Gianluca , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

Cardiac complications are the main cause of death in thalassemia major (TM) patients. Cardiovascular Magnetic Resonance (CMR) plays a key role in their management, assessing myocardial iron overload (MIO), biventricular function, atrial dimensions, and myocardial fibrosis. We evaluated the predictive value of CMR parameters for cardiac complications, including heart failure (HF), arrhythmias and pulmonary hypertension (PH).

Methods

We followed prospectively 537 white TM patients enrolled in the MIOT network. Fifty patients were excluded from the analysis because a cardiac complication was present at the time of the first CMR. All prognostic variables associated with the outcome at the univariate Cox model were placed in the multivariate model and were ruled out if they did not significantly improve the adjustment.

Results

At baseline the mean age was 29.5±9.0 years and 222 patients were males. The mean follow-up time was 58±18 months. After the first CMR only the 37.8% of the patients did not change the chelation regimen or the frequency/dosage.

Conclusions

We detected few cardiac events thanks to a MR-guided, patient-specific adjustment of the chelation therapy. Severe and homogeneous MIO, myocardial fibrosis and ventricular dysfunction identify patients at high risk of heart failure. Heart T2* doesn’t have any power in predicting arrhythmias while male sex and atrial dilation are independent prognosticators. Male sex, severe and homogeneous MIO, myocardial fibrosis and ventricular dysfunction identify patients at high risk of cardiac complications globally considered.

Disclosures:

No relevant conflicts of interest to declare.
Surveillance Of Healthcare Associated Infections In Hematology: Prospective Study About 237 Patients
Authors: Fares Salma , Lamchahab Mouna , Zerouali Khalid , Serhier Zineb , Benchekroun Said , Quessar Asmaa
Published: October 2019

Background

Immunocompromised children or adults patients are a population at high risk of infection, thus constituting a major cause of morbidity and mortality. They are 2 times more common in oncology and hematology patients (9-12%) than in not cancerous patients (6-7%).The incidence of hematology nosocomial infections (NI) is around 20%. Although data on NI are available, those about healthcare associated infections (HAI) are poorly studied.

Objective

Our aim was to define the incidence rate and risk factors of HAI of hematology patients over a period of 7 months from February to August 2012 in preliminary study to create a local register for HAI and establish strategies for hygiene and preventive policy of our unit.

Patients & methods

We took forward all aged over 18 patients hospitalized and monitored for hematologic malignancies in the adult and transplant unit at hematology and oncology center at the 20th August 1953 hospital in Casablanca. Informations were collected and validated by dealing doctors using a farm and data analysis was done by biostatisticians team using Epi Info software. Rates of HAI were calculated for patients- 1000 hospitalization day.

Results

Among this population of 237 patients, we diagnosed 183 HAI in 94 patients, witch 62.8% were followed for acute myeloid leukemia (AML). Incidence rate of HAI was 42.1% with confidence interval (CI) of 95% at [37.4-46.9%] against 29.9% for NI and incidence density of HAI by 1000 hospitalization day was 31.6 versus 24.6 for NI. Risk factors have implicated significantly neutropenia 87.4% (p<0.001), chemotherapy 83% (p<0.001) and temporary central catheter 60% (p<0.001). HAI were microbiologically documented in 41% witch bacteremia in 28%. Pneumonia was noted in the third of cases. Infection was of unknown origin in 25%. Germs isolated were mainly bacteria (93%), specially Staphylococci (32%) and Enterobacteriaceae (29%). Majority of our isolates have one or many antibiotic resistance.

Conclusion

Incidence rate of HAI was high in our study relative to NI, so they should be preferred because they’re covering a broader definition and therefore can be used as a quality indicator for prevention programs. Our study presented a preliminary step in the creation of a HAI register and the initiation of a prevention policy adjusted on ecological profile of our unit.

Disclosures:

No relevant conflicts of interest to declare.
Gvhd-Associated Early Impairment of Marrow Function Is Frequent after Allogeneic Transplantation and Is a Sensitive Biomarker for Prediction of Treatment Related Mortality and of Overall Survival
Authors: Milone Giuseppe , Avola Giuseppe , Camuglia Maria Grazia , Leotta Salvatore , Cupri Alessandra , Spadaro Andrea , Tripepi Giovanni
Published: October 2019

INTRODUCTION: Aim of our study was to determine the role of acute-GVHD in influencing early marrow function and whether assessment of “GVHD-associated marrow impairment” may predict transplant outcome.

METHODS: We have prospectively studied 62 patients who received T-replete allogeneic stem cell transplantation because of various malignancies. At day +18/+19, we determined in bone marrow: CD34+ cells, frequency of clonogenic cell (CFU-GM and BFU-E) and, in 20 patients, also CD34+ cells showing apoptosis (AnnexinV/7-AAD). Results were related to acute-GVHD and to clinical outcome in terms of Treatment Related Mortality (TRM), Relapse Rate (RR) and Overall Survival (OS). To distinguish the effect of a-GVHD from that determined by corticosteroid, patients were divided in three groups according to time of presentation of a-GVHD: “Early a-GVHD, “No a-GVHD” and “Impending a-GVHD”. The latter group consisted of patients presenting a-GVHD after engraftment.

RESULTS: In univariate analysis “Febrile neutropenia” and” Acute GVHD” were important factors for a reduction of frequency of marrow clonogenic cells assessed on day +18/+19. However, in multivariate analysis only “Acute GVHD” was able to influence frequency of marrow CFU-GM and BFU-E at day +18/+19. Patients not developing “a-GVHD” until day +90, had on day+18 a median growth of CFU-GM of 202/10e5 plated cells while patients suffering “early GVHD” had a marrow CFU-GM growth significantly reduced: 82/10e5 plated cells, (p= 0.0009). Median CFU-GM was found significantly reduced also in patients defined as “impending GVHD” (onset of a-GVHD at day +20-/+90) (p=0.01). Apoptotic CD34+ cells in marrow cells at day +18/+19 were inversely correlated to frequency of marrow BFU-E (r= -0.5, p=0.04). Taking into account competing risks, Cumulative Incidence of TRM at 2 y in the group of patients having a frequency of marrow CFU-GM over median was 5% while it was 30% in the group having CFU-GM below the median (log-rank: p=0.004). Cumulative incidence of Relapse was not significantly different in these two groups (31% versus 34%). OS was significantly better in group having CFU-GM over median: 62% versus 35% (logrank: p=0.02). Frequency of CFU-GM over median at day +18/+19 was a factor important for a reduced risk of death (HR=0.358; p=0.004) also after adjusting, using multivariate Cox analysis, for Disease Status (Early versus Advanced).

CONCLUSIONS: acute-GVHD impairs early and significantly marrow function, marrow-GVHD is a sensitive biomarker for prediction of TRM and OS.

Disclosures

No relevant conflicts of interest to declare.
The SRC Kinase Family Member Lymphocyte Specific Kinase (p56Lck) Regulates Endothelial Cell Proliferation and Survival
Authors: Betapudi Venkaiah , Merkoulov Sergei M. , Kundu Suman , Ji Ou , Wu Meifang , McCrae Keith R.
Published: October 2019

Background and Objective: The Src family consists of Src, Lck (p56Lck), Fyn, Hck, Blk, Lyn, Fgr, Yes, and Yrk protein tyrosine kinases, which display cell and tissue specific expression. These non-receptor protein tyrosine kinases regulate many cellular processes including cell growth, differentiation, shape, migration, and survival. p56Lck is primarily expressed in T lymphocytes where it stimulates T cell receptor-mediated signaling to regulate thymocyte development. Similar to other SRC family members, p56Lck activity is negatively regulated by phosphorylation of Tyr505 and undergoes autophosphorylation on Tyr394 in the active state. Our previous studies showed that p56Lck activation occurred in proliferating endothelial cells exposed to cleaved high molecular weight kininogen (HKa), which induced endothelial cell apoptosis.The purpose of this study is to define the role of Lck in regulation of endothelial cell survival and angiogenesis.

Methods: Primary cultures of endothelial cells were treated with Lck siRNA or with mammalian expression vectors or lentiviral constructs containing Lck cDNA. The proliferative capacity, viability, and tube-forming ability of Lck and control endothelial cells were determined.

Results: Transfection of endothelial cells with Lck siRNA markedly changed their morphology and significantly enhanced their ability to proliferate in response to bFGF. In contrast, endothelial cells transfected with a Lck expression vector or transduced with a Lck-lentivirus not only failed to proliferate in response to growth factors, but also underwent apoptosis on several different extracellular matrix proteins. Finally, overexpression of Lck in endothelial cells led to impaired tube formation in a matrigel-based, in vitro angiogenesis assay; apoptosis of endothelial cells in this system was suggested by increased staining with Annexin V.

Conclusion: Taken together, these results suggest that regulation of Lck activation provides a key node in determination of endothelial cell proliferation and survival that is likely to be relevant to the regulation of angiogenesis. Moreover, they suggest that specific endothelial cell Src family kinase members may play contrasting roles in regulation of these processes. Activation of Lck may represent a potential mechanism for controlling aberrant angiogenesis in pathological conditions.

Figure: Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis. Figure:. Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis.

Disclosures

No relevant conflicts of interest to declare.
Calcium Calmodulin Dependent Kinase Kinase 2 Regulates Hematopoietic Stem Cell Regeneration and Quiescence
Authors: Lento William , Huang Wei , Doan Phuong , Chao Nelson J. , Racioppi Luigi
Published: October 2019

Extracellular free calcium (Ca2+) is the most abundant signaling molecule in the hematopoietic stem cell (HSC) niche, and intracellular calcium is an important second messenger controlling critical functions in HSCs. Accordingly, Ca2+ is a master regulator of HSC biology, and nodes of the Ca2+ signaling network are emerging as critical components of the molecular machinery governing HSC regeneration and quiescence. Ca2+ controls many cell functions by forming a complex with Calmodulin (CaM), which binds and activates a family of Calcium/Calmodulin dependent kinase (CaMK) that includes CaMKI, CaMKII, CaMKIV, CaMKK1 and CaMKK2. We have previously investigated the function of CaMKK2 in blood cells and established this protein as a critical component of the calcium signaling pathway that regulates granulopoiesis and macrophage activation. Here, we investigated the function of CaMKK2 in the regenerative response of HSCs. To determine the expression of CaMKK2 under homeostatic conditions, we examined transgenic reporter mice and identified robust reporter activity in primitive long-term hematopoietic stem cells, but reduced signal in the majority of lineage positive blood and bone marrow cells. To establish whether CaMKK2 has a functional role in controlling hematopoiesis and HSC self-renewal, we examined hematopoietic regeneration in mice deficient for CaMKK2. We demonstrate that Camkk2 -/- mice have accelerated bone marrow and peripheral blood regeneration following hematopoietic injury. Camkk2 -/- HSCs showed increased proliferative capacity in vivo and mobilized into the peripheral blood to establish extra-medullary hematopoiesis during regeneration. To identify the proximal target of CaMKK2, we assessed the phosphorylation status of CaMKI, CaMKIV and adenosine monophosphate activated protein kinase (AMPK), which are known targets of CaMKK2. Our results indicate Camkk2 -/- stem and progenitor cells (HSPC) fail to accumulate phospho-AMPK in response to ionizing radiation. Since AMPK is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, we also analyzed the levels of phospho-ribosomal 6 protein (rpS6), which is a distal target of the mTOR cascade that positively regulates cell size and proliferation. We found that irradiated Camkk2 -/- HSPCs accumulate higher levels of phospho-rpS6 compared to wild type cells. This finding implicates CaMKK2 as a critical component of a negative regulatory circuit that links calcium signaling to AMPK activation and cell proliferation in HSPC. Cumulatively, our data demonstrate that CaMKK2 regulates HSC function and suggests CaMKK2 inhibition is a novel approach to stem cell expansion for clinical therapy.

Disclosures

No relevant conflicts of interest to declare.
Downregulation of Dyskerin Impairs Expansion of Normal Hematopoietic Progenitors and Erythroid Colony Formation in the Absence of Apparent Telomere Shortening
Authors: Kumari Ashu , Richards Laura A , MacKenzie Karen L
Published: October 2019

Dyskerin is an RNA binding protein that functions as a core component of the telomerase holoenzyme as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. Within the telomerase holoenzyme, dyskerin stabilizes the telomerase RNA component (hTR) that functions as a template for synthesis of telomeric DNA at chromosome ends. Telomerase is active in malignant cells as well as normal hematopoietic stem and progenitor cells where it enables long-term cell replication.

Mutation of the gene encoding dyskerin (DKC1) is the underlying cause of the inherited disease dyskeratosis congenita (DC), which is characterized by low levels of telomerase activity and shortened telomeres. Bone marrow failure is the most common cause of mortality in DC, however since dyskerin has telomerase-independent roles in fundamental cellular processes, it is not clear whether telomerase insufficiency is the sole cause of hematopoietic dysfunction in this disorder.

To gain further insight to the consequences of deregulated expression of dyskerin in haematopoietic cells, we transduced normal human cord blood CD34+ progenitor cells with retroviral vectors encoding two different shRNAs targeting dyskerin. Transduced cells from seven independent experiments were FACS sorted then assayed for expansion and differentiation in vitro, clonogenic potential, telomerase activity and telomere length maintenance. Results from the functional assays showed that depletion of dyskerin impaired cell expansion and compromised the formation of BFU-E colonies. In contrast to BFU-E, CFU-GM colonies did not appear to be altered by dyskerin depletion. Analysis of non-transuduced cells subject to ex vivo expansion along erythroid and myelomonocytic lineages revealed that erythroid precursor cells expressed disproportionately high levels of dyskerin, and correspondingly high telomerase enzyme activity. shRNA-mediated downregulation of dyskerin lowered hTR levels and quenched telomerase activity in the erythroid precursors without altering expression of the telomerase catalytic component, hTERT. These results suggest that upregulation of dyskerin bolsters telomerase activity and renders erythroid precursors vulnerable to dyskerin depletion. However in spite of the reduction in telomerase activity induced by dyskerin shRNA, Southern blot analysis showed that the rate of telomere length shortening was not significantly altered by dyskerin depletion over the time course of these experiments. Together the data show that dyskerin is upregulated during erythroid differentiation, and has an essential function in the erythroid lineage that appears to be independent of its role in telomere length maintenance.

Disclosures

No relevant conflicts of interest to declare.
Silencing Mutations in NOTCH1 Activate Calcium Signaling in B Cells
Authors: Love Cassandra L , Healy Jane , Greenough Adrienne , Dave Sandeep S. , Li Guojie
Published: October 2019

The NOTCH1 pathway is known to be up-regulated in many cancer types including non Hodgkin lymphomas (NHLs), colorectal, breast and pancreatic cancers. The precise downstream effects of NOTCH1 mutations in lymphoma are not known.

We applied high throughput sequencing in three different non Hodgkin lymphomas including diffuse large B cell lymphoma (DLBCL, N=94), mantle cell lymphoma (MCL, N=58) and Burkitt lymphoma (BL, N=59). We also sequenced mutations from 208 patients with immune deficiency. In all, we identified 31 mutations in NOTCH1. Five of these mutations were either nonsense or frameshift mutations. Silencing mutations in the PEST domain have been reported in MCLs, and many other silencing mutations were observed in the other lymphoma cohorts as well as immune deficiency cases. The finding of silencing mutations was unexpected, given that in most cancers, NOTCH1 and its pathway are often constitutively active.

Calcium signaling is an important mechanism for activating B cells. Given the shared mutations between NHLs and immune deficiencies, we investigated the role of silencing NOTCH1 mutations in altering calcium signaling by engineering lymphoma cells with a deletion of the transmembrane region resulting in silencing of NOTCH1 using the CRISPR system.

We designed guide RNAs that spanned a 57 base pair region from exon 28 that encodes the transmembrane domain of the NOTCH1 gene. Each guide RNA was ligated into a pX458 Cas9 GFP expressing plasmid. Namalwa NHL cells were transfected, FACS-sorted for GFP positivity and subcloned. Successful deletion of the target region in Cas9 NOTCH1 cells was confirmed by Sanger sequencing and Western blotting.

Cells that were wild type, Cas9 control, or Cas9 NOTCH1 were incubated in a calcium free buffer containing Fluo-4 for detection of intracellular free calcium. Cells were washed and subjected to flow sorting where baseline calcium was recorded. The cells were stimulated with anti-IgM (Fab’)2 and recorded again for response. To test extracellular calcium influx, 4mM CaCl2 was added to the calcium free buffer containing cells and recorded again for response.Upon stimulation with anti-IgM (Fab’)2, we observed a significant increase (p<10-6) of free cytosolic Calcium in the Cas9 NOTCH1 cells compared to both wild type and Cas9 control. Similar results were obtained when measuring extracellular Calcium influx upon addition of CaCl2to the buffered solution of cells.

These findings indicate that silencing of NOTCH1 has an activating role in calcium signaling that could contribute to B cell survival and function in NHLs and immune deficiencies. Similar roles have been reported for activating mutations in PIK3CD and STAT3 that contribute to both immune deficiency and cancer. Our study is the first to identify the role of silencing mutations of NOTCH1 in NHLs and immune deficiencies.

Disclosures

No relevant conflicts of interest to declare.
Specific NK Cell Activation to Treat Relapsed/Refractory (r/r) Hodgkin Lymphoma (HL) Patients – Final, Updated Data on Clinical Outcome, Pharmacokinetics and Pharmacodynamics of a Phase 1 Study Investigating AFM13, a Bispecific Anti-CD30/CD16A Tandab
Authors: Rothe Achim , Sasse Stephanie , Topp Max S. , Eichenauer Dennis A. , von Tresckow Bastian , Hummel Horst-Dieter , Ravic Miroslav , Marschner Jens-Peter , Engert Andreas
Published: October 2019

Introduction: AFM13 is a bispecific, tetravalent TandAb antibody that targets CD16A and CD30. Preclinical data demonstrated that AFM13 specifically harnessed NK cells to exert potent cytotoxic effects on tumor cells expressing CD30. Furthermore, AFM13 showed a favorable toxicity profile in cynomolgus monkeys. HL is characterized by the presence of CD30+ Reed-Sternberg cells and by a high medical need in the r/r setting, especially since the response to brentuximab vedotin in this patient group is mostly short. Here, we present the final results of a phase 1 clinical study in r/r HL including a focus on the pre-treatment with brentuximab vedotin.

Methods: Heavily pretreated patients (pts.) with r/r HL received stepwise escalated doses of intravenous AFM13 (0.01 to 7.0 mg/kg) weekly or 4.5 mg/kg twice weekly over 4 weeks. Primary objectives were safety and tolerability, secondary objectives included pharmacokinetics (PK), pharmacodynamics (PD), and clinical efficacy measured using Cheson criteria.

Results: 28 pts. were recruited, 24 pts. received AFM13 weekly and 4 pts. received AFM13 twice weekly. AFM13 was well tolerated with mainly mild to moderate adverse events. The maximum tolerated dose was not reached. AFM13 demonstrated activity, which was consistently more pronounced for all assessed parameters at doses ≥1.5 mg/kg (n=13). 3 of 13 pts. (23%) exhibited partial responses and disease control was reached in 10 of 13 pats. (77%). Importantly, AFM13 was also active in pts. refractory to brentuximab vedotin as most recent treatment. PK data revealed an AFM13 half-life of up to 19 hours. In peripheral blood, the portion of activated NK cells (CD69+), relative to total number of NK cells, increased up to 3-fold 24 hours after infusion compared to baseline. Kinetics of NK cell activation corresponded to kinetics of AFM13 serum levels. Soluble CD30 declined to zero at the end of treatment in almost all pts.

Conclusion: Phase 1 data indicate that AFM13 is active and well tolerated in heavily pretreated r/r HL pts. including brentuximab vedotin failures. To further enhance efficacy of AFM13 both, the dose regimen (based on PK/PD profile) and the duration of treatment, will be optimized in a phase 2 study. This study is currently in preparation by the German Hodgkin Study Group.

Disclosures

Topp: Affimed Therapeutics AG: Consultancy. von Tresckow:Takeda: Honoraria, Other; Novartis: Honoraria, Research Funding. Marschner:Affimed Therapeutics AG: Employment. Engert:Millenium Takeda: Consultancy; Affimed Therapeutics AG: Consultancy.
Presence of Calreticulin Mutations in JAK2-Negative Polycythemia Vera
Authors: Girodon Francois , Broseus Julien , Park-Alexandre Ji-Hye , Hermouet Sylvie , Carillo Serge
Published: October 2019

Calreticulin (CALR) mutations have recently been reported in JAK2- and MPL-negative Myeloproliferative Neoplasms (MPN), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF).The clinical course of sporadic CALR-mutated patients seems to be more indolent than that of JAK2-mutated patients. In contrast, no CALR mutation has been found in the 647 published cases of Polycythemia Vera (PV) patients tested. Consequently, CALR mutations were considered exclusive to JAK2 and MPL mutations. Since 98% of PV patients harbor a JAK2 mutation (mostly the V617F mutation in exon 14 and more rarely, in exon 12), the absence of CALR mutations in PV seemed logical. Here, we describe two JAK2V617F-negative PV patients who presented with a CALR mutation at the time of diagnosis.

Patient # 1 had hemoglobin at 168 g/L, hematocrit at 51.3%, and increased red cell mass (RCM) at 128% associated with a normal erythropoietin level. The bone marrow biopsy showed hypercellularity for age, panmyelosis associated with normal megakaryocytes and rare isolated abnormal enlarged forms. Using reticulin stain, no myelofibrosis was noted. Patient # 2 had hemoglobin at 194 g/L, hematocrit at 53% and low erythropoietin level without any dehydration. Both had moderately elevated platelet counts (658 and 575 x109/L respectively) with normal leukocyte counts. They were negative for BCR-ABL. No mutation was found in JAK2 exons 12, 13 and 14 by HRM and allele-specific real-time PCR or in MPL exon 10. Using HRM analysis, CALR mutations were suspected in both patients and confirmed using Sanger sequencing and product sizing analyses: CALR mutations were in both patients type 1 deletions (52-bp deletion; c.1092_1143del). To complete genomic tests made on peripheral blood granulocytes, we performed colony assays in methylcellulose and in collagen, picked single BFU-E colonies grown after 14 days in the presence of erythropoietin, and genotyped each colony individually for CALR. Of the 27 colonies genotyped, 6 had no PCR amplification and 21 harboured the same CALR mutation observed in peripheral blood granulocytes, i.e 52-bp deletion; c.1092_1143del. BFU-E were found heterozygous for CALR, with a mean allele burden of 49%. To our knowledge, these patients are the first cases of CALR-mutated PV to be reported. However, since a biclonalJAK2V617F and CALR MPN case recently reported, we cannot rule out the possibility of a biclonal disease involving a yet unknown mutation associated with a CALR mutation. On the other hand, the presence of a CALR mutation both in peripheral granulocytes and in BFU-E suggests that the CALR mutation plays a role in the polycythemia phenotype. Our observations highlight the fact that in the absence of JAK2 mutation, CALR mutations can also be associated with PV.

In conclusion, our data indicate that testing JAK2-negative PV patients for CALR mutations may be useful.

Disclosures

No relevant conflicts of interest to declare.
Increased PKR Expression Promotes MDS Evolution to Acute Leukemia in the NHD13 Mouse Model
Authors: Byrne Michael T. , Bennett Richard L , Cheng Xiaodong , Carter Christopher M , May W. Stratford
Published: October 2019

Protein kinase R (PKR) is an interferon-inducible serine-threonine kinase that is activated in response to inflammatory cytokines and cellular stressors. When over-expressed in murine hematopoietic tissues, PKR results in peripheral blood (PB) and bone marrow (BM) features consistent with myelodysplastic syndrome (MDS). We hypothesized that PKR can cooperate with MDS driver mutations to promote MDS and acute leukemia. To test this, we crossed the Nup98-HoxD13 (NHD13) transgenic model of MDS with our TgPKR and PKR-KO mice to generate NHD13-TgPKR and NHD13-KO mice. These mice were aged alongside NHD13 and wild type (WT) controls. Using RT-PCR, we evaluated PKR expression in NHD13 mice. 9/13 NHD13 mice developed acute leukemia with leukocytosis and/or increased BM blasts whereas 4/13 had MDS only without PB or BM evidence of acute leukemia. Non-leukemic NHD13 mice had PKR expression levels below WT controls (RQ: 0.412 to 0.744). Alternatively, PKR expression in the leukemic group was significantly higher than WT controls and non-leukemic mice (RQ: 3.3 vs. 1.0 and 0.5, respectively) and was associated with shortened survival (mean: 304 vs. 345 days; p < 0.05). We then compared NHD13-TgPKR vs. NHD13-KO mice and discovered significantly lower white blood cells (WBC) counts in the NHD13-TgPKR group (2.7 vs. 5.6 x 106 cells/ml, p <0.05; n=8/group) with significant differences in lymphocytes and neutrophils. With aging, NHD13-KO mice lived significantly longer than NHD13 mice (352.1 vs. 314.3 days, p <0.05) and NHD13-TgPKR mice (273.9 days, p <0.05). There were minor differences in the frequency of acute leukemia between the groups (NHD13-TgPKR: 58.3% vs. NHD13-KO: 41.6%). Next, we evaluated the frequency of somatic mutations in PB reticulocytes using the PIG-A assay that we have previously validated for measuring genomic instability of BM derived cells. We discovered an increased frequency of somatic mutations, indicative of greater genomic instability of the MDS HSPC, in 9-month-old NHD13-TgPKR (3.64%) vs. WT (<0.01%), NHD13 (0.26%), and NHD13-KO (0.18%) mice (p <0.05). Finally, the pharmacologic inhibition of PKR in NHD13 mice improved the WBC count (1.6 vs. 3.6 x 106 cells/ml, p <0.05) and significantly reduced genomic instability (0.11 vs. 0.39%, p <0.05). Collectively, these data indicate that increased PKR expression is associated with the development of acute leukemia and shortened survival in the NHD13 mouse model. Further, NHD13-TgPKR mice, when compared to NHD13-KO mice, have a more aggressive MDS phenotype with deeper PB cytopenias, increased BM blasts, and shortened survival resulting from increased genomic instability. These data indicate that PKR can cooperate with MDS driver mutations and suggests that PKR may be a promising target for future MDS therapies, particularly in patients with high-level PKR expression.

Disclosures

No relevant conflicts of interest to declare.
Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia
Authors: Berg Valeska , Rusch Marion , Vartak Nachiket , Juengst Christian , Schauss Astrid , Waldmann Herbert , Hedberg Christian , Pallasch Christian P. , Bastiaens Philippe , Hallek Michael , Wendtner Clemens-Martin , Frenzel Lukas P.
Published: October 2019

Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined.

Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis.

Conclusion: The identification of the de-palmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation.

Disclosures

No relevant conflicts of interest to declare.
A Novel in Vitro Three Organ System Assay Identifies a Small Molecule Ubiquitin Analog with in Vivo Activity Against Myeloma
Authors: Vatolin Sergei , Phillips James G , Govindgari Shravya , Grabowski Dale , Parker Yvonne , Lindner Daniel , Cotta Claudiu , Reu Frederic J.
Published: October 2019

Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one sandwich setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our sandwich assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation.

Figure 1: Sandwich Three Organ System Assay Figure 1:. Sandwich Three Organ System Assay

Disclosures

No relevant conflicts of interest to declare.