bluuebunny/crossref_metadata_2025 huggingface dataset:
The Crosstalk Between Leptin Receptor Activation and iNKT Mediated Anti-Tumor Immunity in Multiple Myeloma
Authors: Favreau Mérédis , Menu Eline , Vanderkerken Karin , Faict Sylvia , Maes Ken , Van Valckenborgh Els , De Bruyne Elke , Govindarajan Srinath , Drennan Michael , Leleu Xavier , Zabeau Lennart , Tavernier Jan , Venken Koen , Elewaut Dirk
Published: October 2019

In recent years, researchers have showed a growing interest in the mechanistic relationship between bone marrow adipose tissue and adjacent tumors. However, the impact of bone marrow adipocytes on development of hematological malignancies, particularly multiple myeloma is unknown. With aging, bone marrow changes occur and fatty deposits can occupy up to 70% of the BM cavity. Interactions of bone marrow adipose tissue with bone cells and other immune cells, possibly suggest indirect ways in which bone marrow adipocytes may affect MM disease progression. Leptin, an adipokine released by adipocytes and crucial in energy homeostasis, displays immune modulatory properties but its role in anti-tumor immunity remains unclear. In this study we aimed to investigate the intriguing relationship between leptin receptor activation and invariant natural killer T (iNKT) cell mediated anti-tumor immunity in multiple myeloma. The murine immunocompetent 5T33MM model, mimicking the human disease closely, and human samples of newly diagnosed patients were used to investigate this research topic.

A marked increase in serum leptin levels and an upregulation of the leptin receptor expression on iNKT cells was observed in multiple myeloma, in both mice and newly diagnosed patients. In vitro functional analysis demonstrated a direct role for leptin in the downmodulation of the iNKT cell function and an indirect role by acting on the MM cells itself, potentiating the immunosuppressive effect on iNKT cells. We next evaluated the in vivo effects of blocking the leptin receptor together with activating iNKT cells with the prototypic glycolipid a-galactosylceramide (a-GalCer), in the 5T33 myeloma model. Remarkably, strong protection was seen in the combined regimen (a-GalCer and leptin receptor blockade), which was found to be linked to an amplified and sustained activation of iNKT cells, preventing them to become hypoactive. These findings suggest that leptin has a crucial immune suppressive role in myeloma development. Overall, our data reveal the leptin receptor axis as a novel target to treat multiple myeloma.

Disclosures

Leleu: TEVA: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.
ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma
Authors: Kumar Subodh , Nanjappa Purushothama , Talluri Srikanth , Shammas Masood A , Munshi Nikhil C
Published: October 2019

Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated.

In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application.

Disclosures

No relevant conflicts of interest to declare.
Bortezomib-Containing Regimens for the Treatment of Newly Diagnosed AL Amyloidosis: Experience of Two Centers in Canada
Authors: Alahwal Hatem , Song Kevin W. , Duggan Peter , Sutherland Heather J. , Neri Paola , Broady Raewyn , Bahlis Nizar J. , Jimenez-Zepeda Victor
Published: October 2019

Introduction Bortezomib is a novel, dipeptide boronic acid molecule that has shown anti-tumor activity against Multiple Myeloma (MM) and more recently has been demonstrated to be efficacious in the treatment of Light-chain Amyloidosis (AL). Bortezomib-based regimens have changed the landscape of the treatment of AL Amyloidosis due to high levels of response as well as the adequate tolerance. Based on the above mentioned, we aimed to assess the role of Bortezomib-containing regimens (BCR) for the treatment of newly diagnosed AL amyloidosis. Methods 80 consecutive patients have been treated with bortezomib-containing regimens at BCCA and TBCC in Calgary, AB and Vancouver, BC, Canada from 01/2010 to 01/2016. Among these cases, 51 (63.8%) have received CyBorD, 19 (23.7%) bortezomib (1.3 mg/m2) and dexamethasone and the rest Bortezomib in different combinations (Table 1). The primary objective of the study was to assess hematological and organ response. Results Clinical characteristics are shown in Table 1. Median age at diagnosis was 66 years and 51.1% of patients were male. At the time of analysis, 56 patients are still alive and 14 patients have already progressed. A HR was seen in 65/72 evaluable patients (ORR, 90%) after a median of 6 cycles (range, 1-24). Fourteen patients (17.5%) did not have measurable disease based on light chain levels. CR (34.7%) and VGPR (26.4%) were seen in 61% of cases. Organ response was achieved in 49% of cases. Cardiac response was observed in 40% of cases. Median OS and PFS have not been reached (Estimate, 67 and 41 months, respectively). (Fig 1a) However, OS was shorter in the group with severe heart involvement (NT-pro-BNP>5000 or BNP>1100) (p=0.03) (Fig 1b). In conclusion, BCR are efficacious for the treatment of newly diagnosed AL Amyloidosis, providing with a high degree of organ and hematological responses. The impact of BCR on advanced heart dysfunction cases remains unclear but it seems that these patients still require a different approach maintaining a dismal prognosis.

Overall survival and advanced cardiac disease Overall survival and advanced cardiac disease Figure 1 PFS for the whole AL group treated with BCR Figure 1. PFS for the whole AL group treated with BCR

Disclosures

Song: Janssen: Honoraria; Otsuka: Honoraria; Celgene: Honoraria, Research Funding. Neri:Celgene and Jannsen: Consultancy, Honoraria. Bahlis:Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria; BMS: Honoraria. Jimenez-Zepeda:Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene, Janssen, Amgen, Onyx: Honoraria.
C75 Fatty Acid Synthesis (FAS) Inhibitor Has Potent Immunosuppressive Activity
Authors: Senyuk Vitalyi , Mahmud Dolores , Oh Annie L. , Patel Pritesh R. , Rondelli Damiano
Published: October 2019

Fatty acid synthesis (FAS) or oxidation (FAO) are important regulatory pathways in immune response. In fact, FAS plays a pivotal role in antigen presentation and T cells activation and FAO leads to fatty acid degradation which has been previously shown to regulate hematopoietic stem cell maintenance. Here we hypothesized that FAS can be a new target to suppress T cell alloimmune responses in solid organ or stem cell transplantations. Therefore, we tested if the FAS inhibitor C75 could suppress T cell alloreactivity without impairing normal hematopoiesis.

The immuno-suppressive (IS) effect of moderate FAS inhibition was demonstrated in mixed leukocyte cultures (MLC) where C75 at 10 mkg/ml significantly reduced T cell proliferation and prevented the expansion of CD3+CD25+ and CD3+CD69+ T cells. In T cells stimulated by alloantigen, C75 also induced the downregulation of NF-kB gene expression and the upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene involved in ubiquitination and degradation of NF-kB protein.

When compared to other standard IS agents, such as anti-thymocyte globulin (ATG), Cyclosporine A, Rapamycin or inhibitor of FAO Etomoxir, C75 showed similar anti-T cell activity.

The same dose of C75 (10 mkg/ml) did not cause apoptotic death of human CD34+ cells in vitro, nor affected CD34+ cell clonogenicity in vitro. In fact, C75 increased the number of BFU-E and CFU-GM colonies (P < 0.05). We observed that the expression of de novo DNA methyltrasferases DNMT3A and DNMT3B, which are important regulators of stem cell renewal, was strongly reduced in CD34+ cells co-cultured for 3 days with allogeneic T cells. On the contrary, in the presence of C75 the expression of DNMT3A and DNMT3B was not different from baseline control. To test the in-vivo effect of C75 we utilized a xenograft model of stem cell rejection where 2 x 105 human CD34+ cells and HLA incompatible T lymphocytes were injected in immunodeficient nonobese diabetic/ltsz-scid/scid - IL2 receptor gamma chain knockout (NSG) mice at 1:1 ratio. Four weeks after transplantation, control NSG mice showed complete rejection of huCD45+CD34+ cells and the expansion of T cells in the marrow and spleen. NSG mice treated with intra-peritoneum injections of C75 every 3 days for 2 weeks, instead, showed 10-15% human CD45+ myeloid cells in the marrow and spleen at week 4 after transplant, suggesting at least a partial effect on preventing rejection of incompatible stem cells.

We showed here that moderate FAS inhibition may represent a novel immunosuppressive strategy and our findings will prompt preclinical investigations exploiting the effect of FAS inhibitors alone or in combination with standard IS agents in models of allogeneic transplantation or bone marrow failure.

Disclosures

No relevant conflicts of interest to declare.
Patients with Hematologic Malignancies Can Benefit More from Allele-Level HLA Matching Than Antigen-Level after Single Unit Umbilical Cord Blood transplantation: a Retrospective Single Center Clinical Analysis
Authors: Guangyu Sun , Zimin Sun , Xiaoyu Zhu
Published: October 2019

Introduction:The increasing number of unrelated umbilical cord blood (UCB) has been used as an alternative graft because greater degrees of donor-recipient HLA mismatch are tolerated. Generally, lower resolution

HLA typing (antigen-level) for HLA-A and -B and at the allele level for HLA-DRB1 is used to select UCB units; HLA-C and -DQB1 are not typically considered. A few studies have suggested that HLA-matching based upon allelic typing for 8 HLA loci(HLA-A, -B, -C and -DRB1 )should be taken into account when searching UCB units; and the NMDP also encourages extended high-resolution typing of umbilical cord blood units to facilitate further study of the impact of HLA. Nevertheless, few studies have taken into consideration that if HLA-matching based upon allelic typing for 10 loci (HLA-A, -B, -C,-DRB1 and -DQB1) can benefit more patients than traditional HLA typing method (antigen-level for HLA-A,-B and allele-level for HLA-DRB1). In this study, we aim to compare the clinical outcomes between patients using allele-level HLA typing and those using traditional HLA typing method, such as engraftment, relapse and survival after unrelated cord blood transplantation (UCBT).

Patients and methods: 309 patients with hematologic malignancies who underwent UCBT in Anhui Provincial Hospital from May 2008 to December 2015 were analyzed. All patients received a single UCB unit after intensified myeloablative conditioning regimens and a combination of cyclosporine A (CsA) and mycophenolate mofetil (MMF) was given for graft-versus-host disease (GVHD) prophylaxis. Of 309 patients at enrollment, 95 patients used traditional HLA typing method (antigen level for HLA-A,-B and allele level for HLA-DRB1, at least 4/6 HLA loci matching must be met) and 214 patients used allele-level HLA typing method (allele-level for HLA-A,-B,-C,-DRB1 and -DQB1, at least 5/10 HLA loci matching must be met). The follow-up assessment was conducted before May, 31, 2016.

Results: Patients using allele-level HLA typing method had a statistically significant higher cumulative incidence of platelet engraftment (P=0.039), and the risk of grades 2-4 acute GVHD was statistically significant lower than patients using traditional HLA typing method (P=0.01). The risk of transplant-related mortality (TRM) by 1 year was also lower than patients using traditional HLA typing method (P=0.043), while there was no increase in the risk of relapse (P=0.338). Conclusion: Using allele-level HLA typing method when searching UCB units (at least 5/10 HLA loci matching must be met) can improve the engraftment of platelet, reduce the risk of grades 2-4 acute GVHD and TRM, which can take the place of traditional HLA typing method to search UCB units.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell Transplantation for Thalassemia Major from Alternative Donors: A Single Center Experience from India
Authors: Philip Chepsy C , Mathew Amrith , Kakkar Naveen , Sobti Praveen , John M Joseph
Published: October 2019

Allogeneic stem cell transplant (aSCT) which remains the only curative option for patients with β thalassemia major (TM) is often limited by a lack of ideal donors. The role of alternative (related phenotypic donor- RD and Unrelated donor -UD) donor hematopoietic cell transplantation in thalassemia is not well established. Use of conventional myeloablation with busulphan and cyclophosphamide in alternative RDs (either phenotypically identical or 1-antigen-mismatched donors) has been associated with higher graft failure and acute graft versus host disease (aGvHD) prompting novel approaches (Gaziev et al., 2000, 2013). We undertook a retrospective analysis of patients who underwent aSCT from alternative donors in TM.

From February, 2009 to June, 2016, 51 HLA matched transplants for TM was done at our center. Among them 19 (37.3%) used alternate donors. In this cohort, the median age was 9 years (range: 2-18) and there were 13 (68.4%) males. Thirteen (68.4%) were Pesaro Class III and 8 (42.1%) were in the Class III higher risk group as defined earlier (Mathews et al., 2007). All 19 received a treosulfan based regimen. PBSC (Peripheral Blood Stem Cell) was the source of stem cells in 78% and bone marrow in the rest. All patients received a cyclosporin (CSA) plus short course methotrexate GvHD prophylaxis regimen. There were 4 (12%) deaths. The major cause of death was fungal infection (3 had invasive fungal infection of whom 1 developed candidemia following a very late UD deferral).

Of the alternative grafts 7 (36.8%) received RD grafts and 13 (68.4%) from UD. We compared the results of RD with HLA-matched sibling (matched sibling donors [MSDs]) aSCT in 32 patients and UD in 12 patients (Table 1). One (14.3%) patient developed aGvHD (Grade 2-4). The entire RD group had sustained engraftment. Rejection incidence was 0% in the RD, 8.3% in UD and 3.4% in MSD groups, with respective thalassemia-free survival probabilities at one year of 68.6% ±18.6%, 83.3% ±15.2%, 90.0% ±5.5% (P = .549) Fig 1.

In conclusion, the present data shows that a treosulfan based reduced toxicity myeloablative regimen with a PBSC graft has the potential to allow patients to safely undergo aSCT from phenotypically identical RD.

Disclosures

No relevant conflicts of interest to declare.
Nrf2 Regulates Sensitivity of Leukemias to Artemisinins
Authors: Bai Jennifer , Chen Xiaochun , Civin Curt I.
Published: October 2019

The artemisinins are a class of drugs widely used to treat severe malaria due to their potency and few adverse effects. In addition to antimalarial activity, artemisinins have been shown to have potent anti-cancer activity, specifically in leukemia. Therefore, artesunate (AS), the WHO-recommended treatment for severe malaria, and a new dimeric artemisinin derivative (ART-838) are promising agents to repurpose for leukemia treatment. Reactive oxygen species (ROS) have been implicated in the mechanism of artemisinin activity in both malaria and cancer; however, the full mechanisms of artemisinin activity in cancer have not yet been elucidated. In 2014, Ariey et al, published a paper on malaria identifying the Kelch (K13) propeller domain as a molecular marker for artemisinin resistance. Since Keap1 ̶ the human homolog of K13 ̶ is involved in ROS regulation, it may also play a role in artemisinin action in cancer. To investigate the role of Keap1-Nrf2 signaling cascades in mediating artemisinin action and resistance in leukemia, qPCRs and Western blots were performed to quantify endogenous levels of selected Keap1 pathway molecules (Keap1, Nrf2, Nqo1, and Gsta1) in 23 human acute leukemia cell lines. However no significant correlation was observed between the levels of these molecules (mRNA or protein) and AS/ART-838 sensitivity. Using an antioxidant response element (ARE) luciferase reporter that can be activated by Nrf2, a significant increase in ARE activity was observed in K562 cells upon AS or ART-838 treatment. In addition to increased transcription upon drug treatment, the ARE downstream proteins Nqo1 and Gsta1 were upregulated upon AS or ART-838 treatment. K562 cell lines with Keap1 and Nrf2 stably knocked down (KD) were generated. The Nrf2 KD K562 cell line had increased sensitivity to AS and ART-838. In contrast, KD of Keap1 did not result in a substantial change in drug sensitivity to either artemisinin. In conclusion, these studies suggest involvement of Nrf2 in human leukemia cell resistance to artemisinins. Further studies on Nrf2 may help us better understand the molecular actions of the artemisinins in order to optimize their efficacy as single agents or in drug combinations for leukemia treatment.

Disclosures

No relevant conflicts of interest to declare.
Burden of Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation on Health Care Resource Utilization in the Management of Acute Leukemia and Myelodysplastic Syndrome
Authors: Lachance Silvy , Bibeau Joelle , Lachaine Jean
Published: October 2019

Background: Allogeneic Hematopoietic Stem Cell Transplantation (aHSCT) represents the only curative modality for unfavorable acute leukemia (AL) and myelodysplastic syndrome (MDS). Despite its curative intent, a significant number of recipients relapse. There is no standardized approach for the management of relapse following transplants and therapeutic options vary among centers, which represent a major challenge. Post transplant relapse is usually associated with a poor outcome while the impact of the treatment choice on health care resource utilization and survival is unknown. The objective of this study was to measure the health care resource utilization for the management of relapse following AHSCT and how the treatment choice impacted on survival.

Methods: A retrospective medical chart review was conducted at H™pital Maisonneuve-Rosemont (HMR) after research and ethic committee approval. Patients were selected using the Hematopoietic Stem Cell Transplant (HSCT) program database. Eligible patients were diagnosed with AL or MDS and relapsed following a HLA identical aHSCT between January 1st 2011 and December 31st 2014. Patients' and disease characteristics as well as relapse-related health care resource utilization were collected from the date of transplant relapse diagnosis until death or last follow-up.

Results: During the study period, of the 645 HSCT performed at HMR, 303 were allogeneic. A total of 36 patients who relapsed met the inclusion criteria and were included in the survival analysis. Healthcare resource utilization analysis was conducted on the 25 patients for whom complete records were available. Patients' characteristics at relapse, mean health care resource utilization per patient and survival by treatment choice are presented in tables 1, 2 and 3 respectively. The mean time from relapse to death was 10.6 months (SD=13.2). Relapse-related hospitalization duration represented on average 20.3% of patients' follow-up period (SD=26.0). For a mean follow-up time of 9.5 months (ranged from 6 days to 4.8 years), the mean number of relapse-related hospitalization was 2.2 per patient (SD=2.6). The mean length of stay was 40.4 days per patient (SD=54.5). The mean hematologist consultation number was 32.4 per patient (SD=37.3

Conclusion: Relapse following AHSCT is associated with a poor prognosis and survival and significant use of health care resources. Aggressive treatment rarely leads to a second transplant. Innovative approaches should be developed to address this unmet medical need. Healthcare resources devoted to the care of patients in relapse post AHSCT provide a comparative basis for the development of cellular therapy.

Disclosures

No relevant conflicts of interest to declare.
Decentralized Data Storage to Promote Epidemiological Analysis for Subtypes Non Hodgkin Lymphoma
Authors: Bellesso Marcelo , Centrone Renato , Dias Daniela Ferreira , Santucci Rodrigo
Published: October 2019

Advanced disease means a poor prognosis factor in patients with non-Hodgkin Lymphoma (NHL). There are few Brazilian data about epidemiologic incidence of NHL, however it was observed that high grade B cell Lymphoma is more frequent than in North America, Argentina and Chile, besides that we have deficient information about Brazilian private service care. Such knowledge is essential to plan economic resources to promote the best treatment and diagnosis. Moreover, is fundamental to elaborate electronic data storage to promote a continuous supply flow of information during clinical practice. We have designed an accessible and automated data collection model built into the electronic medical records for patients with NHL. Here, we show some epidemiological parameters and classic risk factors about NHL in a private care institute stocked by continuous electronic data storage.

We evaluated data from patients with NHL during November 2015 to May 2016. Information was obtained by decentralized automated model after each outpatient care. We studied 229 patients with NHL. It was observed 112(48.9%) patients with Diffuse Large B cell Lymphoma not otherwise specified (DLBCL), 65(28.3%) Follicular Lymphoma (FL), 21(9.1%) Mantle Cell Lymphoma (MCL), 12(5.2%) Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue gastric lymphoma (GMALT), 6(2.9%) Extranodal marginal zone lymphoma of nonGastric MALT lymphoma (non-GMALT), 4(1.7%) Splenic B-cell marginal zone lymphoma (SL), 1(0.4%) nodal marginal zone lymphoma (NMZL), 1(0.4%) Sporadic Burkitt Lymphoma (BL), 2(0.8%) peripheral T cell lymphoma not otherwise specified (PTCL), 2(0.8%) anaplastic large T Cell lymphoma, ALK negative (ALTCL), 2(0.8%) Extranodal NK/T-cell lymphoma, basal type and 1(0.4%) mycosis fungoides (MF).

It was evidenced advanced disease stage III/IV in DLBCL, FL and MCL: 70(62.5%), 54 (87.1%), 18 (85.7%); Extranodal infiltration sites in DLBCL, FL, MCL: 35/94 (37.2%), 11/50 (22%) and 5/18( 27.7%); Elevated serum LDH in DLBCL, FL, MCL: 31/74( 43.6%), 10/38 (26.3%) and 4/14 (28.5%), Performance status ≥ 2 in DLBCL, FL, MCL: 18/75(24%), 2/28 (4.1) 12/20 (60%), respectively.

In this study, we have demonstrated that decentralized electronic data storage was useful and could appear an attractive model for clinical practice; moreover, it was possible observed high incidence of DLCBL and FL with advanced disease.

Disclosures

No relevant conflicts of interest to declare.
TCIRG1 Mutations As a Cause for Chronic Neutropenia
Authors: Makaryan Vahagn , Fletcher Breanna , Kelley Merideth L. , Bolyard Audrey Anna , Link Daniel C. , Duncavage Eric J. , Qualls David Augustus , Dale David C.
Published: October 2019

Background: Heterozygous mutations in TCIRG1 are recently associated with congenital neutropenia. [Makaryan et al, Human Mutation, 2014] Recent large scale population study revealed statistically significant association between absolute neutrophil count and variations in TCIRG1 gene. [Rosenthal et al, Genetic Epidemiology, 2016]

Aims: To investigate frequency and reveal possible asymptomatic cases of TCIRG1 associated neutropenia.

Methods: The Severe Chronic Neutropenia International Registry (SCNIR) enrolled and followed 2 multigenerational families with genetic neutropenia of unknown etiology with a clear autosome dominant inheritance. Exome sequencing of selected affected members from these families revealed two previously reported heterozygous single point mutations in TCIRG1. Confirmatory Sanger sequencing ratifies exome sequencing results.

Results:

Family 1: P161L (c.482C>T) single point substitution detected in 3 members of this family. This variant is very rare in population and has relatively low minor allele frequency (MAF) score: 0.08. Proline residue at this position is highly conserved and has a high genomic evolutionary rate profiling (GERP) score: 3.46. PolyPhen-2 analysis suggests this substitution as possibly damaging.

Family 2: I721N (c.2162T>A) single point substitution detected in 3 members of this family. This variant is also very rare in population and has MAF score: 0.07. Isoleucine at this position is relatively conserved and has GERP score: 2.73. PolyPhen-2 analysis suggests this substitution as probably damaging.

Summary/Conclusion: Understanding the genetic, epidemiological and biological basis of TCIRG1 associated neutropenia is critical for improving the diagnosis and treatment of this form of hereditary neutropenia. The pathological mechanisms for TCIRG1-associated neutropenia are not yet known. In our original report [Makaryan et al, Human Mutation, 2014] of the large family with TCIRG1-associated neutropenia, all carriers of the pathogenic variant had neutropenia, with variable severity. Some members have severe neutropenia and associated infections whereas others are mildly affected. The report of these two new variants in these families suggests that there may be other patients and families with neutropenia caused by variants in TCIRG1.

Disclosures

Dale: Amgen: Consultancy, Honoraria, Research Funding.
Platelet Factor 4 Induces Leukocyte Responses through Integrin Mac-1 (CD11b/CD18)
Authors: Podolnikova Nataly P. , Yakubenko Valentin P. , Ugarova Tatiana P.
Published: October 2019

The proteins/peptides secreted from α-granules of activated platelets not only aid in thrombus formation and blood coagulation, but also exert various immune-modulating effects. Among many secreted products, Platelet Factor 4 (PF4), a chemokine that belongs to the CXC family, is one of the most abundant platelet proteins. While PF4 assignment to the chemokine family is based on its structural similarity with other CXC chemokines and chemotactic activity, to date no receptor for PF4 on leukocytes has been identified. Our recent elucidation of the recognition specificity of a major leukocyte integrin αMβ2 (Mac-1) allowed the prediction that PF4 contains several putative Mac-1 recognition motifs and thus could potentially interact with this receptor. Using a peptide library spanning the sequence of PF4, we showed that the αMI-domain of Mac-1 bound several overlapping PF4-derived peptides. The biolayer interferometry analyses demonstrated that PF4 bound recombinant active αMI-domain of Mac-1 in a concentration-dependent manner with a KD of 1.3 ± 0.2x10-6 M. No interaction of PF4 with the inactive αMI-domain (α7 helix extended) or the αLI-domain of a homologous integrin αLβ2 was detected. The full-length recombinant PF4 and the αMI-domain-binding peptide (residues 58-70) identified in the peptide library supported strong adhesion and spreading of Mac-1-expressing cells, including neutrophils, U937 monocytic and Mac-1-transfected HEK293 cells. The cell adhesion to PF4 was partially inhibited by anti-Mac-1 mAbs and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface proteoglycans act cooperatively with integrin Mac-1. PF4 induced a potent migratory response of wild-type, but not Mac-1-deficient, macrophages in a Transwell system. PF4 also enhanced phagocytosis: coating of E. coli bacteria or latex beads with PF4 enhanced ~ 4-fold their phagocytosis by macrophages, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by wild-type, but not Mac-1-deficient, macrophages. These results identify PF4 as a ligand for integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated via interaction with Mac-1.

Disclosures

No relevant conflicts of interest to declare.
Identifying Functional Differences Between Protein S and Gas-6 in Pancreatic Cancer
Authors: Zacarias Cosette , Pilli Vijaya Satish Sekhar , Plautz William E. , Wells A'drianne , Majumder Rinku
Published: October 2019

Introduction: Procoagulants such as Factor IX and thrombin play major roles in cancer cell proliferation and migration; however, a role for anticoagulant proteins in cancer biology has not been elucidated. The anticoagulant Protein S (PS), its homologous protein Growth Arrest Specific protein-6 (GAS-6), and the receptors for these proteins, Tyro-3, Axl and Mertk (TAM), are over expressed in many cancer cells. TAM family receptors regulate functions such as cell survival, proliferation, migration, and apoptosis. The consequences of activation of each of these receptors varies, although the mechanism that leads to different outcomes is unknown. We hypothesized that the PS and GAS-6 ligands are responsible for the variations in the functions of these signaling cascades.

Methods: We used qPCR to analyze the pancreatic cancer cell lines Miapaca-2 and Panc-1 for variations in the expression of GAS-6 and PS. We sequestered PS and GAS-6 with antibodies and used FACS analysis to detect effects on the cell cycle and on cell cycle regulators.

Results: GAS-6 was observed to be highly expressed in proliferating Miapaca-2 cells compared with Panc-1 cells, whereas there was no significant difference in PS mRNA levels between these cell lines. For the cell line Miapaca-2, antibody sequestration of GAS-6 arrested the cell cycle in S-phase and increased p53 phosphorylation; conversely, inhibition of PS reduced p53 phosphorylation.

Conclusion: Our results indicate that PS and GAS-6 act antagonistically in controlling pancreatic cancer cell proliferation, and we hypothesize that the ratio of GAS-6 to PS expression is key to this regulation. We will further confirm our hypothesis by overexpressing and knocking down PS and GAS-6 in the pancreatic cancer cell lines.

Disclosures

No relevant conflicts of interest to declare.
Bleeding and Thrombotic Outcomes Following Perioperative Interruption of Direct Oral Anticoagulants in Patients with Prior Venous Thromboembolic Disease
Authors: Shaw Joseph , de Wit Carine , Le Gal Gregoire , Carrier Marc
Published: October 2019

Introduction: The use of Direct Oral Anticoagulants (DOACs) for the prevention and treatment of Venous Thromboembolic Disease (VTE) is increasingly common. Patients on longstanding anticoagulation for the prevention of VTE frequently undergo invasive procedures that necessitate interruption of anticoagulation in order to avoid excessive bleeding during the procedure. There is little evidence surrounding the safety of perioperative DOAC interruption in patients with VTE.

Methods: This study represents a retrospective analysis of adult patients on DOAC therapy for prior VTE, who underwent temporary interruption of anticoagulation therapy for inpatient or outpatient invasive procedures. The timing to hold and resume DOAC anticoagulation was based on the estimated half-life of the DOAC, as well as the bleeding risk of the procedure (Standard vs High). Our primary outcomes included the 30-day thromboembolic complication rate, as well as the 30-day major bleeding rate (ISTH non-surgical and surgical major bleeding criteria). Secondary outcomes included clinically relevant non-major bleeding (CRNMB) and overall mortality.

Results: To date, a total of 87 patients have been included in the analysis, 68% of which were male. The mean age of the cohort was 58.3 years. All patients were on DOAC anticoagulation for acute treatment or secondary prevention of recurrent VTE. A large majority of patients (94%) were anticoagulated with rivaroxaban. Procedures were performed on an inpatient or outpatient basis in 24 and 63 patients, respectively. Forty six patients underwent procedures with standard bleeding risk. Mean time to anticoagulation discontinuation for standard and high bleeding risk procedures was 41.3 (SD = 20.8) and 49.3 (SD = 17.7) hours, respectively. The 30-day thromboembolic complication rate was 1.2% (95% CI: 0.2 to 6.2%), whereas the 30-day major bleeding rate was 0% (95% CI: 0 to 4.2%). The rate of CRNMB was 3.5% (95% CI: 1.2 to 9.7%). Overall mortality was 0% (95% CI: 0 to 4.2%).

Conclusion: The perioperative interruption of direct oral anticoagulation for invasive procedures in patients with prior VTE appears to be associated with a relatively low risk of major bleeding, as well as recurrent VTE. Prospective studies are needed to evaluate the benefits and risks of perioperative interruption of direct oral anticoagulation in patients with prior VTE.

Disclosures

Carrier: BMS: Research Funding; Leo Pharma: Research Funding.
The ALK-2 Inhibitor, TP-0184, Demonstrates High Distribution to the Liver Contributing to Significant Preclinical Efficacy in Mouse Models of Anemia of Chronic Disease
Authors: Peterson Peter , Kim Wontak , Haws Hillary , Whatcott Clifford J. , Siddiqui-Jain Adam , Bearss David J. , Warner Steven L.
Published: October 2019

In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD.

Disclosures

Peterson: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.
Identification of New BMP6 Pro-Peptide Mutations in Patients with Unexplained Iron-Overload
Authors: Piubelli Chiara , Castagna Annalisa , Marchi Giacomo , Rizzi Monica , Busti Fabiana , Marchetti Monia , De Gobbi Marco , Roetto Antonella , Xumerle Luciano , Giorgetti Alejandro , Delledonne Massimo , Olivieri Oliviero , Girelli Domenico
Published: October 2019

Background: Hereditary Hemochromatosis (HH) is a genetically heterogeneous disorder caused by mutations in at least 5 different genes (HFE, HJV, TFR2, SLC40A1, and HAMP) involved in the production and function of the liver hormone hepcidin, a key regulator of iron metabolism. Nevertheless, patients with a HH-like phenotype that remains unexplained, despite extensive sequencing of the known genes, are not infrequently seen at referral centres, implicating the role of still unknown genetic factors. A compelling candidate is Bone Morphogenetic Protein 6 (BMP6), a member of TGFb superfamily, whose expression is stimulated by increased iron stores in the liver. BMP6 acts as a major activator of the BMP-SMAD signalling pathway, ultimately leading to the upregulation of hepcidin gene transcription. Indeed, early this year French Authors have described 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with mild to moderate, late onset, unexplained iron overload (Daher R, Gastroenterology 2016).

Methods: we recently updated our next generation sequencing (NGS)-based second level genetic test for the molecular diagnosis of non-HFE HH (Badar S, Am J Hematol 2016), by adding a number of novel potential candidate genes, including BMP6, to the panel of the 5 known HH genes. This test was applied to 38 patients evaluated at our tertiary referral centre for iron disorders, because of an unexplained iron overload phenotype.

Results: we found 3 heterozygous missense mutations in BMP6 gene in 4 patients with unexplained, late-onset, iron overload, from 3 different families. Their relevant clinical data are summarized into Table 1. Of note, 1 mutation (p.Leu96Pro) was the same recently described by Daher et al. and proven to be functional. The other two mutations (p.Glu112Gln, p.Arg257His) were novel, predicted damaging by bioinformatic tools, and both located in the pro-peptide domain, known to be crucial for appropriate BMP6 processing and secretion. They were further studied by in silico modelling, based on the available 3D structure of the TGFb, which also resulted to be consistent with their pathogenetic role.

Conclusions: to the best of our knowledge, our results provide the first independent confirmation of the likely causal role of BMP6 mutations in late onset, moderate iron overload phenotype, unrelated to mutations in the established 5 HH genes.

Disclosures

No relevant conflicts of interest to declare.
Extramedullary Erythropoiesis in Spleen of HIF Prolyl 4-Hydroxylase-2 Deficient Mice Is Mediated By Notch Signaling Downregulation
Authors: Myllymäki Mikko , Määttä Jenni , Dimova Elitsa , Izzi Valerio , Väisänen Timo , Myllyharju Johanna , Koivunen Peppi , Serpi Raisa
Published: October 2019

Erythrocytosis, an increase in absolute red cell mass, is mainly driven by erythropoietin, while hypoxia-inducible factor (HIF) regulates the expression of a number of genes involved in it, including erythropoietin. Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), the major regulator of the stability of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis and premature death. Although bone marrow is the primary site for erythropoiesis, spleen retains a capability for extramedullary erythropoiesis. We studied HIF-P4H-2 hypomorphic mice (Hif-p4h-2gt/gt) which show slightly induced erythropoiesis only upon aging despite no increased erythropoietin levels. Spleen was identified as the site of extramedullary erythropoiesis in these mice. Hematopoietic stem cells (HSCs) from spleens of the Hif-p4h-2gt/gt mice showed increased growth of BFU-Es and the mice were protected against anemia by induced extramedullary erythropoiesis. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligands and target Jag1, Jag2, Dll1 and Hes1 became downregulated upon aging dependent on HIF-2α. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased growth of BFU-Es in the Hif-p4h-2gt/gt mice. We conclude that HIFα stabilization can mediate non-erythropoietin-driven extramedullary erythropoiesis in the spleen via altered Notch signaling.

Disclosures

Myllyharju: FibroGen Inc.: Equity Ownership, Research Funding.
Distinct Roles of CHK2 and Cell Death in Suppressing Oncogenic Potential of Tandem DNA Breaks in Human Hematopoietic Stem Cells
Authors: Biechonski Shahar , Yassin Muhammad , Aqaqe Nasma , Olender Leonid , Rall Melanie , Wiesmüller Lisa , Raz Yael , Beider Katia , Nagler Arnon , Milyavsky Michael
Published: October 2019

DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells.

Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing.

Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC.

To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress.

Disclosures

No relevant conflicts of interest to declare.
Ifnα Mediated Remodeling of the Bone Marrow Stem Cell Vascular Niche
Authors: Prendergast Áine M. , Kuck Andrea , van Essen Mieke , Haas Simon , Blaszkiewicz Sandra , Essers Marieke A. G.
Published: October 2019

In the bone marrow (BM), endothelial cells (ECs) are a major component of the hematopoietic stem cell (HSC) vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha (IFNα). In the BM, IFNα induces rapid cell cycle entry of HSCs in vivo. However, the effect of acute IFNα treatment on BM ECs has not been described. Here, we demonstrate that IFNα leads to rapid stimulation of BM ECs in vivo, resulting in increased BM vascularity and vascular leakage. We find that stimulation of BM ECs involves the expression of key inflammatory and EC-stimulatory markers, including ESAM, VE-Cadherin and Laminin. Using the anti-VEGF antibody, Avastin, we could confirm that IFNα-mediated activation of BM ECs is dependent in part on VEGF signaling in BM hematopoietic cell types, including HSCs. Thus, this implies a role for HSCs in remodeling of the BM niche in vivo followinginflammatory stress. Taken together, these data increase our current understanding of the relationship between HSCs and the BM niche under inflammatory stress. In addition, we have clarified the response of BM niche ECs to acute IFNα treatment in vivo. IFNα treatment has been associated with anti-angiogenic effects in patients and therefore our described acute response of the BM vasculature may additionally inform future regimes of combination therapy.

Disclosures

No relevant conflicts of interest to declare.