bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a New Subclass of ALK Negative Anaplastic Large Cell Lymphoma Expressing Aberrant Levels of ERBB4 Transcripts
Authors: Scarfò Irene , Pellegrino Elisa , Kwee Ivo , Agnelli Luca , Mereu Elisabetta , Bergaggio Elisa , Abate Francesco , Machiorlatti Rodolfo , Messana Katia , Rinaldi Andrea , Tiacci Enrico , Serra Sara , Deaglio Silvia , Neri Antonino , Falini Brunangelo , Rabadan Raul , Bertoni Francesco , Inghirami Giorgio , Piva Roberto
Published: October 2019

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.

To discover biomarkers and/or genes potentially involved in ALK- ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a large gene expression profiling (GEP) data set including 249 cases of T-NHLs and normal T-cells. Among the top outliers, ERBB4 and COL29A1 genes were exclusively expressed in a subset of ALK- ALCL cases, and resulted highly correlated. Differential analysis comparing the expression profiles of ERBB4/COL29A1+ to ALK+ ALCL patients identified 48 genes up- and 37 down-regulated (qval < 0.0076). The most significantly over-represented functional category included transcriptional regulators. Gene-set-enrichment-analysis (GSEA) indicated that ERBB4/COL29A1+ samples shared activation of specific pathways such as inflammatory response, TNF-NF-κB, JAK-STAT, cytokine, and angiogenesis. RNA sequencing and 5’RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two ERBB4 truncated transcripts (referred as I20ΔERBB4 and I12ΔERBB4), displaying Transcription Starting Sites (TSS) in intron 12 and 20, respectively. On the contrary, full length COL29A1 mRNA was expressed in ERBB4/COL29A1+ samples. RT-qPCR analysis performed on 170 T-NHL samples (51 PCTL-NOS, 44 ALK+ ALCL, and 75 ALK- ALCL) highlighted specific expression of ERBB4 aberrant transcripts in 25% of ALK- ALCL (18 out of 75). ERBB4+ patients showed higher expression of I20ΔERBB4 as compared to I12ΔERBB4 transcript. ERBB4 expression at protein level was confirmed by immunohistochemistry and Western Blotting on selected cases. Inspection of intronic regions spanning two hypothetical TSS revealed the presence of Human Endogenous Retrovirus (HERV) Long Terminal Repeats (LTR) displaying promoter activity, as demonstrated by luciferase assays.

In conclusion, we defined a new subclass of ALK- ALCL characterized by ectopic expression of ERBB4 and COL29A1 genes. To the best of our knowledge, ERBB4 truncated transcripts carrying intronic 5’UTR have never been described before. Further studies are required to address whether the expression of ERBB4 aberrant transcripts may contribute to the ALCL transformation and lymphoma maintenance. This information might lead to more rational therapeutic approaches for ERBB4+ ALCL patients.

Disclosures

No relevant conflicts of interest to declare.
Activation-Induced Release of the TNF-Family Member BAFF By NK Cells Facilitates Resistance of Chronic Lymphoid Leukemia Cells to Direct and Rituximab-Induced NK Lysis
Authors: Wild Julia S , Raab Stefanie , Schmiedel Benjamin J , Maurer Andreas , Schneider Pascal , Kanz Lothar , Salih Helmut R
Published: October 2019

NK cells are cytotoxic lymphocytes that play an important role in the immunosurveillance of leukemia and, due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab. Available data indicate that the ability of NK cells to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear. The TNF family member B cell activating factor (BAFF) was described to be aberrantly produced in mature B cell malignancies and contributes to disease pathophysiology e.g. by acting as a growth and survival signal for CLL cells. Here we report that NK cells express and release BAFF, and NK cell activation, notably including FcγRIIIa stimulation by Rituximab, results in increased secretion of BAFF (but not its close relative APRIL). Expression on the cell surface was neither detectable in resting nor in activated state. NK cell-derived BAFF enhanced the metabolic activity of primary CLL cells and protected CLL cells from chemotherapy-induced cell death. Moreover, exposure to BAFF profoundly diminished direct and Rituximab-induced lysis of primary CLL cells by allogeneic and autologous NK cells, while NK activation and degranulation per se remained unaffected. Notably, sensitivity of CLL cells to both chemotherapeutic treatment as well as direct lysis and Rituximab-induced ADCC of NK cells could be restored by the anti-BAFF antibody Belimumab (Benlysta®), which is approved for the treatment of systemic lupus erythematosus. Thus, our data provide evidence for the involvement of BAFF in the resistance of CLL cells to chemotherapy. Moreover, our results offer a functional explanation for the reportedly compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias as well as their impaired ability to mediate ADCC upon Rituximab treatment in CLL patients. Our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for chemo-immunochemotherapy of B cell malignancies.

Disclosures

No relevant conflicts of interest to declare.
[68Ga]Pentixafor: A Novel PET Tracer for Imaging CXCR4 Status in Patients with Multiple Myeloma
Authors: Philipp-Abbrederis Kathrin , Herrmann Ken , Schottelius Margret , Eiber Matthias , Gerngroß Carlos , Pietschmann Elke , Lückerath Katharina , Franke Katharina , Rudelius Martina , Knop Stefan , Götze Katharina , Schwamborn Kristina , Steidle Sabine , Beer Ambros , Peschel Christian , Einsele Hermann , Buck Andreas , Schwaiger Markus , Wester Hans-Jürgen , Keller Ulrich
Published: October 2019

Chemokine receptors form a large family of G-protein coupled receptors that mediate chemotaxis of cells towards a gradient of chemokines. The C-X-C chemokine receptor-4 (CXCR4, also known as fusin, CD184) exerts its biological effects by binding its ligand CXCL12 (or Stromal-cell derived factor-1, SDF-1) which activates downstream pathways, ultimately resulting in altered cell adhesion and cell homing. Physiologically, the CXCR4/CXCL12 interaction plays a pivotal role in the recruitment and homing of hematopoietic stem/ progenitor cells (HSPC) and immune cells. In cancer, CXCR4 expression is associated with tumor dissemination and prognosis. Using multiple myeloma (MM) as an exemplary CXCR4-expressing cancer entity, we report the evaluation of [68Ga]Pentixafor-PET as a method for in vivo mapping of CXCR4 expression density by means of Positron Emission Tomography. [68Ga]Pentixafor-PET provides images with excellent specificity and contrast when assessed in mice xenografted with human CXCR4-positive MM. We next analyzed CXCR4 expression using [68Ga]Pentixafor-PET in a cohort of fourteen patients with advanced MM. These patients also underwent standard [18F]Fluoro-Deoxyglucose-PET (FDG-PET). Nine of fourteen FDG-PET scans were rated visually positive, whereas ten of fourteen Pentixafor-PET scans revealed MM manifestations. Assessment of blood counts and standard CD34+ flow cytometry did not reveal significant blood count changes or alterations in HSPC frequency associated with tracer application. These data document the first methodology for clinical PET imaging of CXCR4 in a cohort of MM patients. [68Ga]Pentixafor-PET opens a broad field of clinical investigations on CXCR4 expression and regulation in the cancer field and beyond.

Disclosures

Wester: SCINTOMICS, > Radiopharmaceutical Technology: Employment.
Effect of Young Age on Outcomes in Pediatric Acute Promyelocytic Leukemia (APL): North American Intergroup Study CALGB 9710 (Alliance)
Authors: Kutny Matthew A. , Geyer Susan , Laumann Kristina M , Gregory John , Willman Cheryl L. , Stock Wendy , Larson Richard A. , Powell Bayard L. , Feusner James H.
Published: October 2019

Purpose: Clinical trials in APL have generally reported similar outcomes for children and adults. However, sub-group analysis of pediatric patients treated on 2 consecutive trials of the European APL group demonstrated that children <5 years (yr) had an increased risk of relapse (Bally et al., JCO, 2012). Here we report the effect of age < 5 yr on outcome for the North American Intergroup Study C9710.

Methods: Outcomes were compared among young children (age <5 yr, n=16) to those of older age (5-12 yr, n=25 and 13-18 yr, n=45) enrolled on C9710. Treatment on C9710 included induction with daunorubicin, cytarabine and ATRA followed by two consolidation cycles with daunorubicin and ATRA and maintenance for one year with ATRA +/- mercaptopurine and methotrexate. Patients of age ≥15 yr were randomized to receive or not receive two 5-week cycles of arsenic trioxide (ATO) before the chemotherapy consolidation. However, analysis in this study of young children compared to those of older age was restricted to patients who did not receive ATO.

Results: Clinical remission (CR) rates were similar between the 3 age groups (age <5 yr= 88%, age 5-12 yr= 80%, age 13-18 yr= 82%; P=0.93). Overall survival (OS) Kaplan-Meier estimates were not significantly different (log rank P=0.84), and 5 yr OS rates were 77% (age <5 yr), 80% (age 5-12 yr), and 84% (age 13-18 yr). Similarly, event-free survival (EFS) Kaplan-Meier estimates were not significantly different (log rank P=0.48), and 5 yr EFS rates were 56% (age <5 yr), 47% (age 5-12 yr), and 59% (age 13-18 yr). Young children did not have a higher relapse rate (5-yr cumulative incidence of relapse by age: <5 yr=29%, 5-12 yr=49%, 13-18 yr=29%).

Conclusions: Results of this intergroup trial demonstrate that ATRA administration during induction, consolidation and maintenance leads to CR rates >80% and 5 yr OS rates >75%. These results are superior to recently published results of pediatric non-APL acute myeloid leukemia and confirm results from the prior APL intergroup trial (INT0129) that demonstrated that ATRA in induction and/or maintenance significantly improved outcomes for pediatric APL (Gregory et al., Ped Blood and Cancer, 2009). Our sub-group analysis showed no association of age with outcomes in children treated with this regimen.

Disclosures

Off Label Use: Daunorubicin- labeled for use in AML for adults and for pediatric ALL (not pediatric AML) Arsenic Trioxide- labeled for use in relapsed/refractory APL (not de novo APL) Mercaptopurine and Methotrexate- labeled for use in pediatric ALL (not APL).
Molecular Mechanisms Underlying the Synergism of Erib and Hht in Treating t(8;21) AML with C-KIT mutations
Authors: Chen Bing , Chen Xin-Jie , Chen Sai-Juan
Published: October 2019

AML1-ETO oncoprotein (AE) resulting from t(8;21)(q22;q22) translocation is known to play a pivotal role in leukemogenesis of t(8;21) AML, yet the activating mutations or over-expression of C-KIT occur in half of the AML patients with t(8;21). Animal models have proven that activating C-KIT mutations cooperate with AE in causing overt AML. The aberrant C-KIT expression was thus thought to be the major “second hit” in stepwise leukemogenesis of t(8;21) AML.

Previous studies have demonstrated that Eriocalyxin B (EriB), a diterpenoid extracted from Isodon eriocalyx, induced apoptosis in t(8;21) leukemia cells through the NF-κB and MAPK signaling pathways and triggered degradation of AE dependent on caspase-3. By targeting AML1-ETO oncoprotein and activating apoptosis pathways simultaneously, EriB has potential to treat the t(8;21) leukemia.

In this study, we observed the synergistic effect of EriB and the chemotherapy drug Homoharringtonine (HHT) on Kasumi-1, an AML cell line harboring t(8;21) and C-KIT mutations. Compared with using EriB or HHT alone, the combination of EriB and HHT (E+H) exhibited a higher efficacy to induce apoptosis and inhibit proliferation of Kasumi-1 cells. Data further showed that HHT enhanced the AE degradation caused by EriB in a dose/time-dependent manner. In addition, HHT significantly decreased the expression of C-KIT in Kasumi-1 cells, and the combination with EriB markedly enhanced this effect. Mechanism study revealed that E+H combination down-regulated the expression of transcriptional factor Sp1 through up-regulating miR-29b, separated Sp1 protein from Sp1/NF-κB complex, and released the Sp1/NF-κB complex from the C-KIT promoter. These data point to a mechanism involving Sp1/NF-κB/miR-29b regulatory network whereby E+H combination down-regulate C-KIT mRNA expression. In addition, HHT induced a proteasome-mediated degradation of C-KIT protein, which was further enhanced by EriB.

From these observations, we conclude that the combined use of EriB and HHT might offer a potential therapeutic avenue for t(8;21) AML with C-KIT mutations.

Disclosures

No relevant conflicts of interest to declare.
JQ 1, a Selective Bromodomain Inhibitor, Decreased the Expression of the Tolerogenic Molecule PDL1 in Antigen-Presenting Cells (APCs) and Restores the Responsiveness of Anergic CD4+ T Cells
Authors: Wang Hongwei , Cheng Fengdong , Xing Limin , Zhao Xiaohong , Villagra Alejandro , Pinilla-Ibarz Javier , Tao Jianguo
Published: October 2019

Bromodomain and extraterminal (BET) is a protein domain that recognizes acetylated lysine residues such as those on the N-terminal tails of histones. This recognition is often a prerequisite for protein-histone association, chromatin remodeling and gene transcription. The role of BET proteins in regulating the response of inflammatory cytokine genes through translation of histone marks is poorly understood.

Given that the inflammatory status of the APC is critical in determining T-cell activation versus T-cell tolerance and that epigenetic modifications of specific genes in the APC play a key role in this process, we recently determined the functional consequences of inhibiting BET in APCs.

First, we evaluated the effects of JQ 1, a selective small-molecule BET bromodomain inhibitor on APC’s function and its regulation of antigen-specific CD4+ T-cells response. In vitro treatment of peritoneal elicited macrophages (PEM) or bone marrow derived dendritic cells (DCs) with increasing concentrations of JQ 1 resulted in decreased expression and protein production of the anti-inflammatory cytokine IL-10 and IL-6 in response to LPS stimulation. At the concentration used, JQ 1 did not affect the viability of treated APCs. Second, analysis of the expression of MHC class molecules and co-stimulatory molecules revealed a decreased expression of the tolerogenic PDL1 molecule in JQ 1- treated APCs as compared to untreated APCs. Third, we evaluated the ability of JQ 1 treated APCs to present cognate antigen to naïve or tolerant antigen-specific CD4+ T-cells. We found that treatment of either PEM or DC with JQ 1 enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, JQ 1- treated APCs were able to restore the responsiveness of tolerant CD4+ T-cells isolated from lymphoma bearing hosts.

Taken together, we have found that APCs treated with the Bromodomain specific inhibitor JQ 1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and more importantly, are capable of restoring the responsiveness of tolerant T-cells. Our studies therefore have unveiled a previously unknown immunological effect of BET inhibitors and have broadened their clinical scope as promising adjuvants in cancer immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
Modeling Warfarin Associated Intracerebral Hemorrhage (ICH) in Cynomolgus Monkeys
Authors: Liu June , Li Zhiqun , Ni Jia , Deng Jilin , Cui Guoqiang , Wang Song , Feng Jianming , Li Hanjian , Tu Rong , Zhang Kevin
Published: October 2019

Intracerebral hemorrhage (ICH) accounts for about 15% of strokes and leads to higher mortality. However, its underlying pathophysiologic mechanism remains gloomy, partially due to the lack of suitable animal models in research. Approximately 20% ICH cases are associated with the use of oral anticoagulants, warfarin for the most instances in clinic, especially following the long-term treatment. Even though the coumarin drugs has been used in rodents for comparison of standard-of-care effects in ICH studies, the elucidation of results must bear the significant differences of gyrencephalic brain structure, brain vasculature and various biological interactions affecting the hemorrhage and hemostasis between rodents and human being. Quite a few works have been reported in study of strokes, particularly, the cerebral ischemic stroke in cynomolgus monkeys (Macaca fascicularis) for its high homology to human. In order to establish a more clinical relevant model for ICH and saver anticoagulant research, we studied ICH in cynomolgus monkeys by injection of collagenase in brain and characterized the effect of warfarin treatment by MRI measurement. Following injection of type IV collagenase into internal capsule, distinct acute, sub-acute, and chronic phases of ICH were observed, with the highest level of cerebral hematoma volume at 3 days after ICH induction measured by a GE signa excite 1.5T MRI scanner. Pretreatment with 0.3 mg/kg of warfarin daily for 6 days significantly increased the hematoma volumes to proximal 10% of cerebral hemisphere compared to the 4% with saline treatment. In addition, the AUC of hematoma volume changes was also remarkably enlarged with the warfarin treatment as measured at different time points following ICH. Transfusion of 30 ml of autologous fresh frozen plasma (FFP) and injection of 1 mg Vitamin K reduced the effects of warfarin treatment in hematoma and AUC enlargement, and ameliorated ICH stroke associated symptoms. The results of coagulation tests were all in agreement with the warfarin associated effects on cerebral hematoma in cynomolgus monkeys. Our data demonstrated the successful establishment of ICH model in cynomolgus monkeys and the clinical relevant treatments, such as warfarin and FFP, can be incorporated as the controls for proper researches in ICH and its therapeutics. With the high similarity in brain and high cross reactivity in target to human, the cynomolugs monkey ICH model adds a valuable tool to evaluate new therapeutic approaches and the safety of novel anticoagulants, especially for the human target-specific interventions, such as plasma products, biologics, oligonucleotides, and peptides.

Disclosures

No relevant conflicts of interest to declare.
Efficacy of a Transdermal Granisetron Patch in Controlling Chemotherapy-Induced Nausea and Vomiting (CINV) in Hematologic Cancer Patients
Authors: Niesvizky Ruben , Fernández Velasco Ana , Wong Doris S , Braccia Deborah
Published: October 2019

Introduction: Multiday, highly emetogenic chemotherapy regimens are commonly used in the management of hematologic malignancies. Despite progress in prophylaxis, CINV, especially during the delayed phase, can remain a significant barrier to attaining planned chemotherapy dose on time for some patients. A granisetron transdermal system (GTS) has been shown to be as effective as oral granisetron in controlling CINV across multiple tumor types. This post-hoc analysis specifically examined the efficacy and safety of GTS in patients with hematologic malignancies.

Methods: A randomized, phase 3 study has been published comparing GTS (7 day application) to oral granisetron (2 mg/day) in patients receiving either moderately or highly emetogenic chemotherapy for 3-5 days. Data for this analysis were limited to patients with a primary tumor diagnosis of lymphoma (n=51), leukemia (n=27), or multiple myeloma (n=5). The rates of complete control (CC; no vomiting, mild nausea, no rescue medication) and complete response (CR; no vomiting, no rescue medication) using either GTS or oral granisetron were compared during both the acute (first 24 hours) and delayed phase (days 2 to 5) following chemotherapy. Need for rescue medication and patient assessment of response to therapy with GTS were also compared.

Results: 83 hematologic cancer patients (31 GTS, 52 oral granisetron) were included. The majority of patients received a non-cisplatin regimen with corticosteroids (59 patients; 71%). Patients received chemotherapy for 3 days (37; 45%) or 4 to 5 days (46 patients; 55%). During the acute phase of CINV, the CC rate of 94% and CR rate of 94% in the GTS group were similar to rates in the overall population (94% and 95%, respectively). There was no difference in CC and CR between GTS and oral granisetron in the acute phase (P=0.90 and 0.59, respectively). In the delayed phase, GTS resulted in CC in 87% and CR in 90%; compared with CC in 77% and CR in 81% of patients given oral granisetron (P=0.26 and 0.25, respectively). The use of rescue medication over the entire study period and patient assessment of overall response to therapy were not different between arms (P=0.60 and 0.92, respectively). GTS was well tolerated; the only treatment related adverse event was one case of mild constipation.

Conclusion: This retrospective analysis suggests GTS may be an appropriate option for prevention of CINV in hematologic cancer patients. A trend toward improved control in the delayed phase was observed and further investigation of a benefit in delayed CINV for hematologic malignancies may be warranted.

Disclosures

Fernández Velasco: ProStrakan: Employment. Wong:ProStrakan: Employment. Braccia:ProStrakan: Employment.
Pomalidomide Induces Mir-125b with Subsequent Downregulation of P53 in CD34+cells Conferring Decreased Apoptosis: A Potential Mechanism of Secondary Primary Malignancies
Authors: Zheng Yijie , Li Shirong , Fu Jing , Mapara Markus , Lentzsch Suzanne
Published: October 2019

Introduction:IMiD® compounds such as lenalidomide (LEN) and pomalidomide (POM) represent a novel class of agents in the treatment of multiple myeloma. Increasing concerns about its potential toxicity on stem cells resulting in an increase of secondary malignancies primarily affecting the myeloid cells have been reported. However, the exact mechanism is unclear. MicroRNAs (miRNAs) are emerging as important regulators in stem cell development. Therefore, the present study investigated the effect of IMiD® compounds on stem cell renewal/proliferation and the role of miRNAs involved in this process.

Methods and Results: When human CD34+ cells were cultured with POM or DMSO under conditions favoring myeloid development, POM significantly increased the percentage of CD34+cells in S phase in cell cycle analysis with concomitant dramatic decrease of apoptotic cells. This resulted in a strong and significant proliferation of CD34+cells as well as induction of colony formation. Systematic screening of miRNAs expression profile by microarray showed that POM significantly enhanced the expression of miR-125b. Upregulation of miR-125b was further confirmed by realtime PCR. Interestingly, recently miR-125b was shown to function as an oncomir by inducing tumorigenesis in myeloid cells by So et al., Blood 2014. In accordance with that we found that overexpression of miR-125b in CD34+ cells exhibited similar effects as POM on CD34+ cells, such as increased cell number in S-phase, decreased cell apoptosis resulting in significant induction of colony formation and the absolute number of hematopoietic progenitors. Bioinformatics prediction models revealed a potential binding site of miR-125b on 3'UTR of P53. Indeed subsequent experiments showed that P53 was downregulated when miR-125b was overexpressed in CD34+cells or when treated with POM. Further analysis of the P53 down stream targets revealed that POM decreased the expression of PUMA and BAK1 all critical for control of cell proliferation and apoptosis. The POM-induced upregulation of mir-125b was independent of the CRBN/IKZF1 induced protein degradation.

Conclusion: Based on our results we postulate that POM upregulates the oncomir miR-125b in CD34+cells. Mir-125b controls proliferation and apoptosis by downregulating P53 and genes involved in the P53 pathway including BAK1 and PUMA. Thus, our findings on the effects of POM on the miR-125b/P53 axle provide for the first time a potential mechanism for the development of secondary hematologic malignancies in patients treated with IMiD® compounds.

Disclosures

Lentzsch: Celgene: Consultancy, Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy.
The Fifth Region of Epidermal Growth Factor like-Domain of Thrombomodulin Is Essential to Produce Cytoprotection
Authors: Ikezoe Takayuki , Yang Jing , Nishioka Chie , Nakamura Keigo , Yurimoto Hiroya , Sakai Yasuyoshi , Honda Goichi , Yokoyama Akihito
Published: October 2019

We previously found that the fourth and fifth regions of epidermal growth factor-like domain of thrombomodulin (TME45) protects vascular endothelial cells from calcineurin inhibitor-induced cell damage in association with activation of extracellular signal-regulated kinase (ERK)/myeloid cell leukemia-1 (Mcl-1) signal pathways via activated protein C (APC) independent manner (Arterioscler Thromb Vasc Biol. 2012;32:2259-70). The present study attempted to identify the minimum region of TME45 that is essential to mediate the cytoprotective function of thrombomodulin. The blocking antibody for TME5 but not for TME4 hampered the ability of TME45 to increase levels of Mcl-1 in human umbilical vein endothelial cells (HUVECs), indicating the important roles of TME5 in cytoprotection. We next divided TME5 into three parts and synthesized oligonucleotides for each region. None of the fragment was as potent as TME5 to induce Mcl-1 in HUVECs, suggesting that TME5 was essential for induction of Mcl-1. Importantly, TME5 was not able to produce APC. Furthermore, TME5 counteracted calcineurin inhibitor-induced vascular permeability and stimulated angiogenesis in vitro as well as in vivo. Taken together, TME5 may be useful to prevent or treat endothelial cell damage associated lethal complications developing after hematopoietic stem cell transplantation such as sinusoidal obstruction syndrome and thrombotic microangiopathy.

Disclosures

Honda: Asahi Kasei Pharma: Employment.
Gas6 Is Required for Thrombin-Induced Expression of VCAM-1 through Foxo-1 in Endothelial Cells
Authors: Bertin Francois-Rene , Robins Richard , Lemarie Catherine A , Blostein Mark D
Published: October 2019

Venous thromboembolism (VTE) is a leading cause of morbidity and mortality. Gas6-/- mice are protected against VTE suggesting an important role for Gas6 in the pathophysiology of this disorder. VCAM-1 is a key adhesion molecule, expressed on the endothelium, implicated in the pathophysiology of venous thrombosis. We previously demonstrated that Gas6 induced the phosphorylation of Forkhead box O1 (FoxO-1) through the activation of the PI3K/Akt signaling pathway. Thus, we hypothesize that Gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO-1 in endothelial cells. Thrombin induces VCAM-1 mRNA and protein expression in WT but not in Gas6-/- endothelial cells. Silencing FoxO-1, using siRNA, enhances VCAM-1 expression both in WT and in Gas6-/- endothelial cells. Thrombin induces FoxO-1 phosphorylation and nuclear exclusion in WT but not in Gas6-/- endothelial cells. Inhibition of FoxO-1 phosphorylation by overexpression of a phosphorylation resistant FoxO-1 mutant (Tm-FoxO-1) decreases VCAM-1 expression, both in WT and Gas6-/- endothelial cells. Inhibition of Akt by LY294002 prevents FoxO-1 phosphorylation and VCAM-1 expression. Finally, thrombin-induced VCAM-1 expression in WT endothelial cells was associated with an increase in bone marrow mononuclear cell (BM-MC) adhesion. BM-MC adhesion was further increased when endothelial cells were transfected with a FoxO-1 siRNA and decreased when transfected with the Tm-FoxO-1 plasmid. Thus, we demonstrate that Gas6 regulates VCAM-1 expression through phosphorylation and nuclear exclusion of FoxO-1, thereby promoting BM-MC adhesion. These data suggest that the Gas6/FoxO-1 signaling axis plays an important role in VCAM-1 expression during thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Characterization of Antithrombin-Specific RNA Aptamers for Use in Anticoagulant Therapy
Authors: Fortenberry Yolanda
Published: October 2019

Historically, unfractionated heparin (a complex polysaccharide classified as a glycosaminoglycan) has been used in the treatment and prevention of thromboembolic disorders. However, due to complications associated with heparin (such as bleeding and heparin-induced thrombocytopenia), low molecular weight heparins (LMWHs) have been developed and are being used in place of unfractionated heparin. LMWHs have proven effective as antithrombotic agents; however, the effects of LMWHs cannot be readily reversed, while unfractionated heparin can be reversed by protamine sulfate. The overall goal of this project was to characterize an antithrombin (AT)-specific RNA aptamer that will combine the safety and efficacy of LMWH with the reversibility of unfractionated heparin. I have developed an RNA molecule (aptamer) that binds to AT. Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) we created a combinatorial library consisting of single stranded RNA molecules with 20-40 randomized positions resulting in approximately 1014 different sequences. The RNA libraries after round 12 consisted of several different aptamers, and there was a progressive decrease in library complexity. We isolated four individual RNA molecules that bind in the nanomolar range to AT. Each displayed the same general properties, they accelerated factor Xa inhibition by AT in a dose-dependent manner. It should be noted that as anticipated, the aptamers did not promote the enhanced inhibition of thrombin by AT. Overall, our results show that these molecules are able to enhance the inhibition of factor Xa by AT. We suspect that these aptamers mimic the action of LMWH by binding to the D-helix of AT. Our previous AT specific RNA aptamer, Aptamer 7-4.16 was shown to prolonged bleeding in vivo in a vascular injury model. These data showed that Aptamer 7-4.16 is as effective as heparin in preventing clotting after vascular injury. We suspect that these new aptamers will have a similar effect in vivo. Proof of the concept was established by the work of Rusconi et al. (Nature, 2002), who developed an RNA aptamer and RNA aptamer antidote directed against coagulation factor IXa. The factor IXa aptamer completely inhibits the activation of factor X. Its anticoagulant action is controlled by the antidote, which is able to reverse the effects of the factor IXa aptamer within seconds. This aptamer/antidote pair is currently being tested clinically. Our data suggesting that antidotes to our AT aptamers have the ability to reverse its activity. If successful, this aptamer/antidote pair will have advantages over LMWHs because not only will it be able to control thrombosis in a fashion similar to LMWHs, but the availability of an antidote will allow for better therapeutic regulation and intervention.

Disclosures

No relevant conflicts of interest to declare.
PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis
Authors: LIU Fan , Cheng Guoyan , Perna Fabiana , Haiming Xu , Hamard Pierre-Jacques , Luciani Luisa , Nimer Stephen
Published: October 2019

Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology.

Disclosures

No relevant conflicts of interest to declare.
An Erythroid-Specific Intron Retention Program Regulates Expression of Selected Genes during Terminal Erythropoiesis
Authors: Pimentel Harold , Parra Marilyn , Gee Sherry , Mohandas Narla , Pachter Lior , Conboy John G.
Published: October 2019

Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proerythroblasts differed substantially from that in orthochromatic erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation.

Disclosures

No relevant conflicts of interest to declare.
Activation Status of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation
Authors: Yamanouchi Jun , Hato Takaaki , Matsubara Etsuko , Azuma Taichi , Nakanishi Hideyuki , Fujiwara Hiroshi , Yakushijin Yoshihiro , Yasukawa Masaki
Published: October 2019

Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations. It has been reported that CALR is a potential regulatory protein of integrin activation based on the interaction between CALR and a conserved sequence of GFFKR in the integrin α cytoplasmic tails. Recent studies suggest that calreticulin activates β1 integrin and modulates integrin-associated signaling. In this study, we examined the effect of CALR mutations on integrin αIIbβ3 activation. We first identified mutations of JAK2, MPL, and CALR genes in 37 patients with WHO defined ET and explored clinical characteristics of patients with CALR mutation. The patients with JAK2 V617F were 22 (59%), MPL W515L was 1 (3%), and CALR mutations were 10 (27%). The two types of CALR mutations were found; deletion (52-bp deletion; c.1092_1143del) and insertion (5-bp insertion; c.1154_1155insTTGTC) mutations. The patients with CALR mutations had lower hemoglobin and leukocyte count compared with JAK2 V617F patients, but platelet count did not have a difference between the CALR and JAK2 mutation groups. Nine (41%) of 22 patients with JAK2V617F had a thrombotic event while 1 (10%) of 10 patients with CALR mutation did (p<0.05), suggesting that patients with CALR mutation had a lower risk of thrombosis than JAK2 V617F patients. Two patients with CALR mutations developed myelofibrosis while no patient with JAK2V617F did. One patient with CALR mutation developed acute myeloid leukemia, with persistence of the CALR mutation in his leukemic cells.To see if the CALR mutation affects activation status of αIIbβ3, we examined the binding of PAC1, a monoclonal antibody recognizing the active conformation of αIIbβ3, to platelets from 5 patients with CALR mutation and 12 patients with JAK2V617F in the presence or absence of ADP. Platelets from all the 5 patients with CALR mutation showed the same level of PAC1 binding as platelets from healthy subjects. Overexpression of recombinant CALR proteins in CHO cells expressing αIIbβ3 by transfection of a protein-expression vector containing wild-type, deletion, or insertion mutant CALR had no effect on PAC1 binding. On the other hand, platelets from 4 of 12 patients with JAK2V617F had an increase in PAC1 binding in the presence and absence of ADP compared with platelets from healthy subjects. All 4 (100%) of 4 patients with increased PAC1 binding had a thrombotic event while 4 (30%) of 13 patients with normal PAC1 binding did. Our study suggests no activation of integrin αIIbβ3 by CALR mutation, which may explain a clinical finding of a low risk for thrombosis in patients with CALR mutation.

Disclosures

No relevant conflicts of interest to declare.
Ultra-Deep Sequencing Defines Stem Cell-Specific Diversity Patterns in Acute Myelogenous Leukemia
Authors: Mencia-Trinchant Nuria , Roboz Gail J. , Carroll Martin P. , Guzman Monica L. , Hassane Duane C
Published: October 2019

Acute myelogenous leukemia (AML) is a fatal disease with dismal outcomes. Despite aggressive chemotherapy regimens, most patients relapse and ultimately succumb to their disease. Relapse is thought to be driven by a surviving residual population of leukemia stem cells (LSCs) that remain quiescent and survive chemotherapy. Moreover, relapsed disease has been shown to be frequently more genetically complex, acquiring new mutations not seen in de novo disease. Since the acquisition of new mutations and survival of LSCs are both evident with relapse, we hypothesized that genetic diversity of LSCs is a likely contributor to disease outcomes. To assess diversity, we performed ultra-deep targeted next generation sequencing at up to 15000x non-duplicate depth of coverage, constructed local haplotypes, and performed subclonal variant analysis on AML samples across varying molecular and cytogenetic risk strata. The resulting high resolution molecular portrait enabled us to examine the clonal composition of both lymphocytes and leukemia, including LSCs.

Consistent with previous reports, we detected established pre-leukemic mutations in DNMT3A(R882H) evident in both patient-matched sort-purified lymphocytes and leukemic cells. In this patient, the pre-leukemic acquisition of DNMT3A(R882H) observed in lymphocytes was followed by additional acquisition of NRAS(G13D) seen exclusively in the LSC and non-LSC compartments, but not the sorted lymphocyte compartment. Interestingly, we found that LSC populations frequently exhibited more complex clonal architecture and nucleotide diversity (p < 10-18) compared to non-stem matched patient counterparts. Because LSCs inherently resist chemotherapy, genetic lesions occurring in the surviving LSC pool are capable of supporting sustainable outgrowths of new drug resistant clones. Indeed, within this complex stem cell architecture, we identified deeply subclonal lesions in clinically relevant AML genes including FLT3. For example, in one high risk patient, the drug-resistant tyrosine kinase domain mutant FLT3(D835Y) was evident at below 0.25% allele frequency in the LSC compartment. This FLT3 allele demonstrated penetrance into the bulk tumor, but not into normal lymphocytes, indicating the AML-specific context of this subclonal variant.

The data suggest that, in addition to surviving chemotherapy, LSCs represent a diverse reservoir of surviving residual cells that can support emergence of drug resistant mutants and refractory relapse. Moreover, ultra-deep sequencing of LSCs and bulk disease will enable improved precision medicine approaches to enhance AML outcomes via early detection of emergent and clinically impactful mutant alleles.

Disclosures

No relevant conflicts of interest to declare.
Identification of a Novel and Efficacious Oral Proteasome Inhibitor As a Potential Clinical Development Candidate
Authors: Won Kwang-Ai , Kim Byung Gyu , Jung Cheol-Kyu , Park Eok , Baek Eunbok , Park Jonghoon , Kang Seung-Wan , Kim Tae Hun , Jung Youngjoo
Published: October 2019

The proteasome is a validated anti-cancer target and various small-molecule inhibitors are currently in clinical development or on the market. However, adverse events and resistance associated with those proteasome inhibitors call for a new generation of drugs.

LC53-0110 and its analogues were identified as novel, reversible, selective, potent, and orally bioavailable proteasome inhibitors which have physicochemical profiles that are distinct from bortezomib, carfilzomib or ixazomib. As compared to those inhibitors, LC53-0110 is far more selective for the chymotrypsin-like proteolytic (β5) site of the 20S proteasome, with an IC50 value of less than 10 nM over the trypsin-like site and the caspase-like site. LC53-0110 treatment showed accumulation of ubiquitinated proteins and inhibited cell viability with a low nM range potency in various hematologic and solid tumor cell lines including multiple myeloma (MM), lymphoma, colon tumor, breast tumor, and lung tumor.

When a single dose was administered orally to tumor-bearing mice, LC53-0110 showed both greater maximum and sustained tumor proteasome inhibition as compared with ixazomib in MM and solid tumor xenograft models. The robust pharmacodynamic responses in tumor translated to the efficacy studies. In the RPMI8226 (MM) efficacy model, oral administration twice a week at 50 mg/kg inhibited tumor growth 68% by three weeks. In the MM.1S (MM) efficacy model, oral administration twice a week at 100 mg/kg completely abolished measurable tumor mass by one week and prevented any tumor re-growth during the three-week study period.

In the initial studies, LC53-0110 also showed potent activity on freshly isolated CD138+ cells from MM patients (pts) who are resistant/refractory (r/r) to current FDA-approved drug treatment. Further studies, including comparison of the naïve pts vs. thalidomide r/r pts and bortezomib r/r pts as well as their potential clinical implications will be discussed.

Disclosures

Won: LG Life Sciences: Employment. Kim:LG Life Sciences: Employment. Jung:LG Life Sciences: Employment. Park:LG Life Sciences: Employment. Baek:LG Life Sciences: Employment. Park:LG Life Sciences: Employment. Kang:LG Life Sciences: Employment. Kim:LG Life Sciences: Employment. Jung:LG Life Sciences: Employment.
Risk of Wernicke’s Encephalopathy in Patients with Myeloproliferative Neoplasm
Authors: Wu Jasmanda , Mirza Nadeem , Partisano Angela M. , Gallagher Christina , Vaze Anjali , Zhang Ling , Gallagher Meghan , Lin Stephen , Juhaeri Juhaeri
Published: October 2019

Background :Wernicke’s encephalopathy (WE) is an acute neuropsychiatric syndrome resulting from thiamine deﬁciency. This neurological deterioration is potentially dangerous because it predisposes the patient to hypothermia and decreased immunity. Cancer patients are at high risk of WE due to chronic malnutrition, chemotherapy-induced nausea and vomiting, and consumption of thiamine by rapidly growing tumors. Very little published research addresses the risk of WE in patients with myeloproliferative neoplasm (MPN).

Objectives: To estimate the incidence of WE among patients with MPN, and compare it with those without MPN.

Methods: Patients with a diagnosis of MPN, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) were identified from 1/1/2003 to 03/31/2013 using the US MarketScan database. The control cohort consisted of a random sample matched to the MPN cohort by age and gender in a 1:1 ratio. Diagnosis of WE was ascertained using ICD-9-CM 265.0, 265.1, 291.1, 294.0 or J code J3411. The two study cohorts were followed for WE from the index date. Cox proportional hazards modeling was used to compare the rates between two cohorts and also control for confounding variables.

Results: A total of 39,761 MPN patients, including PV (51%), ET (42%), post-PV MF (0.2%), post-ET MF (1%), and PMF (2.8%) were identified. Approximately 27% of them were > 65 years of age, and 51% were male. Patients with MPN had higher rates of WE, compared to those without MPN (MPN vs. non-MPN: 1.09 vs. 0.39/1000 person-year, hazard ratio = 2.19, 95% confidence interval 1.43-3.34). The incidence rate of WE was higher in males (male vs. female: 0.93 vs. 0.55/1000 person-year). No specific pattern was observed in age subgroups for both cohorts (e.g. MPN: age <18 vs. 18-39 vs. 40-49 vs. 50-59 vs. 60-69 vs. >70 = 0 vs. 0.85 vs. 1.09 vs. 1.16 vs. 0.72 vs. 0.86/1000 person-year).

Conclusions: Patients with MPN had higher incidence rates of WE, compared to those without MPN. Given the potentially dangerous outcomes associated with WE, physicians who care for patients with MPN should be aware of the risk of WE in this population.

Disclosures

Wu: Sanofi: Employment. Mirza:Sanofi: Employment. Partisano:Sanofi: Employment. Gallagher:Sanofi: Postdoctoral Fellow Other. Vaze:Sanofi: Employment. Zhang:Sanofi: Employment. Gallagher:Sanofi: Employment. Lin:Sanofi: Employment. Juhaeri:Sanofi: Employment.