bluuebunny/crossref_metadata_2025 huggingface dataset:
CRISPR/Cas9 Unsettle PIP4K2A and α, β, and γ Globin Genes Expression
Authors: Malimpensa Danaê , Faber Eliel , Corat Marcus A.F. , Lana Marlous V.G. , Strauss Bryan E. , Costa Fernando F. , Jorge Susan Elisabeth , Sonati Maria de Fátima
Published: October 2019

Phosphatidylinositol-phosphate kinases (PIPKins) belong to a family of lipid kinase enzymes that generate lipid messengers derivate from inositol, including the second messenger phosphatidylinositol-4,5-biphosphate [PI(4,5)P2], which participates in several cell regulation processes including gene expression. Previous studies developed in our laboratory suggested that the enzyme phosphatidylinositol-4-phosphate kinase-II-alpha (PIP4KIIα) may be related with the expression of the globin genes. Thus, the aim of this work was to evaluate the expression levels of the α, β and γ globin genes after disruption and overexpression of the PIP4KIIα gene (PIP4KIIA) in KU812 erythroleukemic cell line. The PIP4KIIα gene disruption was performed by using CRISPR/Cas9 and CRISPR/dCas9 techniques: KU812 cells were electro-transfected (Lonza 4D Nucleofector) with CRISPR/Cas9 lentiviral plasmid construct containing 3 gRNA targets to exon 8 of PIP4KIIA to knockout the gene; RNA samples from the pool of cells were collected and quantified by qPCR after 9 (t1) and 14 days (t2) of transfection. To overexpress PIP4K2A, CRISPR/dCas9 system (five lentiviral plasmids - 3 gRNAs targeting 214 nt upstream the transcriptional start site of PIP4K2A and 2 SAM accessorial factors) was transfected (Lipofectamine 3000) and RNA quantified by qPCR 24 (h1), 48 (h2) and 72 (h3) hours after the procedure. β-ACTIN and GAPDH genes were used as endogenous controls for qPCR. Our results with transfected cells without selection revealed, as expected, a reduction of PIP4KIIA compared to the control (no transfected cells - t1 and t2). The α globin genes presented a variation of 42% (increase in t1) and a 60% reduction in t2 (RQ = 1.42; RQ = 0.40, respectively), while β globin genes presented a 123% increase in t1 and remained similar to the control at t2 (RQ = 2.23; RQ = 1.01). The γ globin genes presented variation of 18% and 59% reduction in t1 and t2, respectively (RQ = 0.82; RQ = 0.41). The overexpression results suggested that PIP4KIIA leads to a higher γ globin expression in h2 and h3 (RQ = 3.26; RQ = 2.01) as well as a lower β globin expression in h1, h2 and h3 (RQ = 0.54; RQ = 0.002; RQ = 0.18), while a globin remained similar to the control at all analysed times (RQ variation of 1.26; 0.82 and 1.25, respectively). These results, although preliminary, suggest a possible relationship between PIP4KIIα and the regulation of expression of the globin genes. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp.

Disclosures

No relevant conflicts of interest to declare.
Chemically Controlled, Immunosuppression-Resistant, Anti-Bcma CAR-T Cells for Treatment of Antibody-Mediated Autoimmunity
Authors: Rajavel Sowndharya , Ito Cade E. , Abe Keith , Guerrero Valerie , Uenishi Gene I. , Scharenberg Andrew M. , Cost Gregory J.
Published: October 2019

Auto-reactive antibody production by plasma cells is the direct cause of many auto-immune diseases. In such cases elimination of plasma cells would ameliorate the disease. Chimeric antigen receptor T (CAR-T) cells with cytotoxicity toward cells expressing B-cell maturation antigen (BCMA) have shown remarkable promise for the treatment of multiple myeloma, a plasma cell neoplasm. Elimination of non-malignant plasma cells is a side-effect of anti-BCMA CAR-T treatment of multiple myeloma, suggesting the use of these anti-BCMA CAR T cells for auto-immune indications. Unfortunately, CAR-T administration requires use of lymphodepletion to achieve efficient cell engraftment, and is often accompanied by cytokine release syndrome (CRS), a potentially life-threatening side-effect.

As lymphodepletion and CRS pose morbidity/mortality risks that are unacceptable for therapy of many auto-immune diseases, we have utilized CRISPR-Cas9 gene editing to develop a controllable CAR-T cell platform that provides for (1) engraftment with non-cytotoxic transient immunosuppression; and (2) small-molecule dependent CAR T-cell expansion. We have implemented this platform using a unique dual targeting approach in which a BCMA CAR transgene is integrated into the TRAC locus, and additional payloads are integrated into a second locus, thus also enabling an allogeneic manufacturing process. Transgene integration occurred in >50% of cells individually with several percent of cells targeted at two loci. TRAC-targeted, anti-BCMA CAR T cells demonstrated CAR-dependent, target-cell-BCMA-dependent cytotoxicity towards both high-BCMA- and low-BCMA-expressing cell lines and in multiple myeloma cells xenografted into NSG mice. Drug-regulation properties and immunosuppression resistance are the subject of ongoing experiments. Anti-BCMA CAR T cells that are chemically controlled, incapable of graft-versus-host disease, and insensitive to immunosuppression may be an attractive treatment option a variety of antibody-mediated auto-immune conditions.

Disclosures

Rajavel: Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Guerrero:Casebia Therapeutics: Employment. Uenishi:Casebia Therapeutics: Employment. Scharenberg:Casebia Therapeutics: Employment; Generation Bio: Equity Ownership; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.
Secdrug: A Computational Method for Discovering Novel Secondary Drug Combinations Using Large-Scale Pharmacogenomics Databases
Authors: Chen Li , Mukherjee Ujjal Kumar , Rankine Emily , Van Ness Brian G. , Mitra Amit Kumar
Published: October 2019

Drug resistance is a major obstacle in achieving complete and sustained therapeutic effect in cancer chemotherapy. Chemo-resistance may also lead to over-dosing and unwanted exposure to ineffective anti-tumor agents thereby increasing the risk of negative side-effects and the cost of drug development. Therefore, our goal is to utilize a large-scale pharmacogenomics database (Genomics of Drug Sensitivity in Cancer or GDSC, the largest public resource of drug-sensitivity data on over 250 drugs in more than 1000 human cancer cell lines of common and rare types of adult and childhood cancers of diverse origin) and develop a prediction method to identify novel secondary drug combination regimens that may effectively reverse drug resistance.

We utilized a greedy algorithm-based set-covering computational optimization method followed by a regularization technique to seek all secondary drugs that could kill maximum number of cell lines of the test disease (B-Cell cancers) resistant to the test drug (the Proteasome inhibitor/PI drug Bortezomib/Bz/Velcade) in a sequential manner ordered by the number of cell lines killed. The predicted top secondary drug combinations in PI-resistant B-cell cancers are shown in Figure 1.

To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib with the predicted best secondary drugs. Figure 2 depicts In vitro chemo-sensitivity profiles of PI-resistant HMCLs and other B-cell cancer cell lines showing percent survival compared to untreated control at increasing concentrations of secondary drugs, as single agents or in combination.

Furthermore, for each drug in the predicted drug combination, we identified differentially expressed (DE) genes by comparing the expression profiles between extraordinary- sensitive and resistant cell lines. These significantly regulated DE genes were used to identify pathways associated with the successful drug combinations.

Finally, we developed an R software package secDrug based on this computational pipeline for predicting novel secondary therapies in chemotherapy-resistant cancers. secDrug takes a query of any cancer type and any test drug, and outputs a list of the top secondary drug combinations with confidence score and biological pathway visualization. Thus, secDrug has potential application in clinical decision-making for discovering resistance-reversing cancer chemotherapy regimens.

Disclosures

No relevant conflicts of interest to declare.
Potential Long-Term Treatment of Hemophilia a By Early Postnatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stem Cells
Authors: Gao Kewa , Kumar Priyadarsini , Hao Dake , Reynaga Lizette , Farmer Diana Lee , Rose Melanie , Nolta Jan A. , Zhou Jianda , Zhou Ping , Wang Aijun
Published: October 2019

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Both mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been used independently to correct the HA phenotype, but poor survival and insufficient retention of transplanted cells in the long term limit their application. In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) with placental mesenchymal stem cells (PMSCs) can achieve stable, long-term engraftment. To investigate stem cell engraftment and therapeutic potential, ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector. To distinguish between PMSCs and ECFCs in the co-transplantation group, a Td-Tomato containing lentiviral vector was used to label PMSCs while a green fluorescent protein (GFP) labeling vector was used to label ECFCs. They were then transplanted intramuscularly either as ECFC only group, PMSC only group or co-transplantation of ECFCs and PMSCs group into neonatal (3 days) or adult (12 weeks) immune deficient NOD scid gamma (NSG) mice. Using an in vivo bioluminescence imaging system, we investigated cell engraftment for up to 26 weeks after transplantation. ECFC only and the co-transplantation groups achieved better engraftment than PMSCs only group in both neonatal and adult mice during the entire study period. The co-transplantation group showed higher engraftment rate than the ECFC only group at 16 weeks and 20 weeks post-transplantation. In addition, transplantation of ECFCs only or in combination with PMSCs in neonatal period achieved higher engraftment than transplantation in adult period. Immunohistochemical analyses were further employed to determine the fate and functionality of the engrafted cells. We found that engrafted ECFCs maintained endothelial phenotype and expressed high level of FVIII at 26 weeks post-transplantation. The presence of ECFCs in the co-transplantation group also promoted formation of functional vasculature after transplantation. To study the functional reversal of HA phenotype we co-transplanted ECFCs and PMSCs into F8 knock-out HA mice. The transplanted cells reduced the blood loss volume from 562.13±19.84µl of blood to 155.78±44.93µl in a tail clip assay in these HA mice. This work demonstrates that co-transplantation of ECFCs with PMSCs at the neonatal period is a promising strategy to achieve stable, long-term engraftment of transplanted cells, and thus holds great promise for the treatment of HA and other genetic diseases and tissue regeneration applications.

Disclosures

No relevant conflicts of interest to declare.
PIM Inhibitor SMI-4a Induces Cell Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphocytic Leukemia Cells Via the JAK2/STAT3 Pathway
Authors: Kuang Xingyi , Xiong Jie , Wang Weili , Li Xinyao , Lu Tingting , Wang Jishi
Published: October 2019

The serine/threonine PIM protein kinases are critical regulators of turmorigenesis in mutiple hematologic malignancies and solid cancers. We used real-time PCR to detect the expression of PIM in B-cell acute lymphocytic leukemia (B-ALL) patients, and found the expression of PIM in B-ALL patients was significantly higher than that in normal controls. SMI-4a is a pan-PIM small molecule inhibitor, and this agent exhibits demonstrable preclinical antitumour activity in a wide range of hematologic malignant cell lines. To further explore the effect of SMI-4a on B-ALL cells, B-ALL cell lines CCRF-SB and Sup-B15 were treated with this small molecule inhibitor, and the results showed that SMI-4a inhibited B-ALL cell proliferation in a dose- and time-dependent manner. Moreover, SMI-4a significantly promoted B-ALL cell apoptosis and caused cell cycle arrest in the G0/G1 phase. The results of Western blot showed that SMI-4a increased the expression of Caspase-3, Caspase-9, Bax and P21, and decreased the expression of Bcl-2 and CDK4. Furthermore, we found that SMI-4a significantly inhibits the activation of the JAK2/STAT3 pathway and HO-1 interferes with the JAK2/STAT3 pathway to inhibit SMI-4a-induced ALL cell apoptosis. Finally, xenograft experiments in NOD/SCID mice were operated to investigate the potential role of SMI-4a in B-ALL tumorigenesis in vivo. To observe the effect of SMI-4a on tumor growth in vivo, NOD/SCID mice were transplanted with B-ALL devied cells, and the tumor-bearing mice were intraperitoneally injected with saline and SMI-4a, respectively. As a result, tretment with SMI-4a resulted in a significant inhibition on tumor growth. In addition, PIM inhibtor obviously reduced the volume and weight of B-ALL cell-derived tumors. TUNEL assay revealed the proportion of apoptotic cells was higher in the SMI-4a-treated group than in the control group. Taken together, our data showed PIM inhibitor (SMI-4a) significantly inhibits the growth of B-ALL cells in vitro and in vivo and promotes apoptosis and cell cycle arrest. This suppressive effect is mediated partly through inhibiting the JAK2/STAT3 pathway activation.

Disclosures

No relevant conflicts of interest to declare.
Downstream Pathway of AML1-ETO Targeted ARHGEF12 Might be Targetable for Rock Inhibitor to Improve Therapy Outcome
Authors: Xie Yangyang , Wu Ruichi , Chen Xiaoxiao , Sun Xiao-Jian , Shen Shuhong
Published: October 2019

As a transcription factor AML1-ETO modulate gene expression of its targets. Scanning our ChIP-seq data we found AML1-ETO bind to an AML1 binding motif in the promotor region of ARHGEF12. Exploring acute myeloid leukemia (AML) microarray databases we confirmed that the expression of ARHGEF12 was up-regulated consistently in AML1-ETO-positive AML patient samples. To confirmed this finding, we quantitated expression of ARHGEF12 in pediatric AML samples from Shanghai children's medical center. Not out of expectation, all AML1-ETO-positive cases have higher ARHGEF12. Moreover, the ARHGEF12 expression was an independent poor prognostic factor for overall survival comparing between patients with higher and lower expressed ARHGEF12 (P=0.0258) (Figure A).

To understand the role of ARHGEF12 in leukemogenic function of AML1-ETO fusion gene, we knocked down ARHGEF12 expression in kasumi-1 cells by shRNA. Growth of kasumi-1 cells in vitro was significantly inhibited with cell cycle exit and more apoptosis. Further, we transformed fetal liver cells of Arhgef12 conditional knockout mice to leukemia model by retrovirus conducted AML1-ETO9a(AE9a) expression. Induced Arhgef12 deletion delayed full blown of leukemia in vivo. The overall survival prolonged 15.5 days (median 83.5 vs. 68 days, P=0.0072). (Figure B) ARHGEF12 is a well-known guanine nucleotide exchange factor for RhoA GTPase. Arhgef12 deletion decreased activated GTP-RhoA and MYPT1 phosphorylation, which is one of ROCK kinase substrates. Spleen cells from leukemic mice were cultured in vitro, the ROCK inhibitor Y-27632 significantly decreased the cell proliferation, which mimic the tamoxifen-induced Arhgef12 knockout phenotype. Y-27632 combined with tamoxifen treated cells almost eradicate alive leukemia cells on the ninth day in vitro (Figure C) and delay leukemogenesis in vivo (Figure D).

These findings suggest that ARHGEF12 as a transcriptional target of the AML1-ETO fusion protein and plays an essential role in leukemogenesis. The ARHGEF12-RHOA-ROCK pathway may serve as a new therapeutic target for AML1-ETO+ AML

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Good Care and Long-Term Follow-up in Young Hemophiliacs
Authors: Iscan Zeynep , Koc Basak , Kilicoglu Onder , Atalar Ata , Taser Omer , Cetik Gulten , Zulfikar Bulent
Published: October 2019

Introduction: Hemophilia is a hereditary bleeding disorder that presents with primarily hemarthrosis and hematoma. The most common and frequent complication, especially in severe hemophilia, where factor VIII or IX is not administered prophylactically, is the hemophilic arthropathy that develops as a result of recurrent bleeding. In this study, the relationship between arthropathy development and timing of the prohylaxis and orthopedic interventions in hemophiliacs was presented.

Materials and Methods: The data of 99 haemophilia (HA) and hemophilia B (HB) patients under the age of 25 years at our center were reviewed retrospectively. Age, type of disease, factor level, follow-up duration, prophylaxis dose and frequency, annual bleeding rate (ABR), target joints, arthropathic joints, applied radioactive synovectomy and orthopedic interventions were recorded.

Results: Seventy-six HA and 23 HB were included. The median age was 14 (range 1-25) years old. Ten of them (10.1%) were mild, 15 (15.2%) were moderate and 74 (74.7%) were severe. Severe hemophiliacs were most of the patients in both types. Eighty-nine (89.9%) patients including 17 of mild-to-moderate type with target joint and / or surgical intervention were under prophylaxis and 10 (10.1%) were receiving on-demand therapy. The annual bleeding rate was 1.38. Twelve (12%) patients (8 HA, 4 HB) with degenerative hemophilic arthropathy had a mean age of 22 years old and a mean age of application was 12.3 years, a mean annual bleeding rate was 1.54 and a mean follow up and prophylaxis time was 9.5 years. Six patients (4 HA, 2 HB) with median age 21.8 years (range: 18-25) had 9 major orthopedic surgeries (arthrodesis (2), radial head resection (2), TEP, contracture releasing, acyloplasty, deformity correction, arthroscopic sinovectomy) and 22 patients with median age of 17.8 years had 29 minor orthopedic interventions (radiosynovectomy (28), closed reduction to femur fracture). Orthopedic interventions were performed at an advanced age, often who met with prophylaxis too late.

Discussion: In our study, it has been shown that who had prophylaxis at an early age and prophylaxis for a longer period had less arthropathies which is the most important complication and additionally less arthroplasties. The fact that, the prevalence of permanent musclosceletal deformity was 59% before year 2000 (historical data) which reduced to 12% nowadays shows the sensitivity of hemophilia care in our center. Early and regular prophylaxis remains the most current treatment modality for the prevention of hemophilia complications.

Disclosures

No relevant conflicts of interest to declare.
MiR-221 Sensitizes Chronic Myeloid Leukemia Cells to Imatinib By Targeting STAT5
Authors: Xu Xiucai , Jiang Xiaoxiao , Xia Lijun
Published: October 2019

Imatinib, a tyrosine-kinase inhibitors (TKIs), is the first-line treatment in the early chronic phase of chronic myeloid leukemia (CML). In the last decade, the survival of patients with CML has significantly increased with the use of TKIs. However, the drug resistance is one major barrier in CML treatment. At present, although the research on CML TKIs resistance mechanism has made great progress, the underlying mechanism is complex and still not fully elucidated, especially the lack of understanding of the upstream common mechanism. MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this work, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to K562 cells and patients with optimal responses respectively. We also observed the expression of STAT5 expression inversely correlated with miR-221 expression in CML cells. Additionally, STAT5 was validated in this study as a direct target of miR-221 in dual-luciferase reporter vector assays. MiR-221 restoration and STAT5 knockdown in K562/G cells increased the sensitivity of CML cells to imatinib by reducing the Bcl2: Bax ratio. Collectively, our data suggest that miR-221-STAT5 axis played crucial roles in controlling the sensitivity of CML cells to imatinib.

Disclosures

No relevant conflicts of interest to declare.
Increasing CD28-FOXP3+CD8+ Treg and Senescent CD8+NK2GA+Eomes+ NK-like T Cells in Peripheral Blood of Patients with Multiple Myeloma
Authors: Kasakovski Dimitri , Zeng Xiangbo , Xu Ling , Li Yangqiu
Published: October 2019

Immune dysfunction in patients with multiple myeloma (MM) includes TGF-β induced dendritic cell dysfunction, regulatory T cell (Tregs)/Th17 cell imbalance, accumulation of Tregs and myeloid derived suppressor cells. These tumor-induced dysfunctions may contribute to immune escape and even suppress immune cells introduced in adoptive cellular immunotherapy. CD28 independent T cells of the effector memory (TEM) and CD45RA+ effector memory (TEMRA) population were shown to accumulate with age and contribute to the immunosuppressive tumor microenvironment in several solid tumors and hematological cancers. Especially in the CD8+ population, the loss of CD28 is associated with high cytotoxicity and regulatory function while showing high diversity and defective antigen-induced proliferation. In the CD4 subset, regulatory and senescent T cells were studied extensively, while in the CD8 positive subset their heterogeneity is still not clearly defined. Their potential immunosuppressive role and distribution in healthy individuals (HI) as well as patients with multiple myeloma (MM) remains to be observed. Furthermore, a recently characterized CD8+CD28- NK-like T cell subset showing expression of NK-related inhibitory receptors and TCR independent effector function is potentially of interest in the progression of MM. In the present study, we compared the changes of distribution of CD8+CD25+ and CD8+FOXP3+ regulatory T cells (Treg), CD28-CD57+ senescent T cells (Tsen), and CD8+KIR/NK2GA+EOMES+ NK-like T in peripheral blood (PB) between HI and patients with MM by multicolour flow cytometry (Gating strategy shown in Figure 1A). When comparing 35 HIs (Median age is 54) and 14 MM patients (Median age is 52), it was shown that there is no significant change in the proportion of senescent T cells in CD8 (P = 0.2452), TEM/CD8 (P = 0.1686) and TEMRA/CD8 (P = 0.4861) between HIs and the MM group, while both the CD25+FOXP3+ regulatory T cells of the CD4 population (P = 0.0031) and CD28-FOXP3+ regulatory T cells of the CD8 population (P = 0.0014) were shown to increase. There is no significant difference in the percentages of KIR/NK2GA+EOMES+ in the CD8 T cell and TEMRA/CD8 T cell population between HIs and the MM group. Remarkably, although there was no overall increase in senescent T cell in MM patients, senescent CD8+NK2GA+EOMES+ NK-like T cells increased in MM patients in comparison to HIs (P = 0.0068) (Figure 1B). In conclusion, the increase of regulatory T cells of both the CD4 and CD8 population as well as the increase of senescent NK-like T cells in the CD8 population potentially contributes to cancer progression through creation of suppressive microenvironments. Moreover, we found that regulatory CD8 T cells and CD8 NK-like T cells only contribute to a small part of the overall CD28- senescent T cell pool.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
UBR5 Mutations in Mantle Cell Lymphoma Lead to Increased Proliferation through a Cyclin D1-Dependent Mechanism
Authors: Kutovaya Olga , Hung Stacy , Viganò Elena , Telenius Adele , Woolcock Bruce W , Meissner Barbara , Colborne Shane , Gascoyne Randy D. , Villa Diego , Morin Ryan D. , Scott David W. , Morin Gregg B , Steidl Christian
Published: October 2019

Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma, with patient outcomes inferior to most other lymphoma subtypes. Recent progress in describing recurrent somatic gene mutations has led to a better understanding of MCL pathogenesis. However, the functional and clinical implications of many alterations remain to be elucidated. Here, to uncover the role of recurrent UBR5 gene mutations in lymphomagenesis, we studied a cohort of 248 MCL patients by targeted sequencing and performed genome editing of MCL-derived cell lines to investigate UBR5-mutation associated phenotypes in vitro. We identified deleterious UBR5 exon 58 hotspot mutations in 8% of MCL patients, all of which were mutually exclusive with CCND1 mutations. Proteomics analysis of Granta-519 and Jeko-1 cell lines with engineered UBR5 exon 58 indel mutations showed differential expression of genes involved in cell cycle and ubiquitination, and led to the discovery of decreased phosphorylation of CCND1 in the UBR5-mutated lines. Accordingly, in vitro studies of engineered genome-edited Granta-519, Jeko-1 and Mino cells revealed accumulation of cells in the S phase of the cell cycle, increased phosphorylation of retinoblastoma protein (Rb), and increased lymphoma cell proliferation. Our results demonstrate that UBR5 mutations, in addition to the hallmark t(11;14) translocation drive proliferation of MCL cells, potentially rendering mutation-carrying cells more sensitive to targeted therapies.

Disclosures

Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Tioma: Research Funding.
Comparing Randomized Controlled Data with Grouped Data from the Literature to Inform Decision Making When Standard of Care Has Low Level of Evidence Available
Authors: Andreozzi Valeska , Vandewalle Björn , Parreira Jorge
Published: October 2019

Objectives: To apply indirect comparison methods to inform decision-making when standard of care has low level of evidence available.

Methods: Simulated treatment comparison is one of the methods available to generate indirect comparison of data in different formats and levels of evidence. It is particularly helpful when patient level data exists for one set of data and there is the need to compare it with single-arm studies. In theses cases evidence is deemed ''unanchored'' due to a lack of a common comparator. We use the case of second line treatment of chronic immune thrombocytopenic purpura (ITP). Splenectomy has been historically recommended in guidelines mainly due to the lack of evidence for other second-line therapies for ITP. We use individual patient data from eltrombopag clinical trials and one of the only studies published in the literature reporting long term outcomes for splenectomy to generate an indirect comparison of eltrombopag and splenectomy in second line treatment of ITP.

Results: A subset of patients (n=84) treated with eltrombopag but not subject to splenectomy in the randomised, phase 3 study RAISE (PMID:20739054) were indirectly compared with a large retrospective cohort of patients (n=233) who underwent splenectomy for ITP in the study by Vianelli et al (PMID:23144195). After controlling for gender, age, baseline platelet count and number of previous therapies, we estimate that the proportion of patients with ITP responding to second line therapy is higher with eltrombopag in comparison to splenectomy: complete response (platelet count >100x109/L) 68.6% vs 54.0%; partial response 18.6% vs 24.6%; no response (platelet count <30x109/L)) 12.8% vs 21.4%.

Conclusions: Indirect comparison with simulated treatment comparison is useful for decision making when no head to head data exists between new treatment options and standard of care and also when standard of care has low level of evidence available.

Disclosures

No relevant conflicts of interest to declare.
Long-Term Effect of Mesenchymal Stromal Cell Derived Extracellular Vesicles on the Restoration of Engraftment of Stem Cells in Radiation Exposed Mice
Authors: Wen Sicheng , Goldberg Laura R , Dooner Mark S , Pereira Mandy , Del Tatto Michael , Papa Elaine , Cheng Yan , Quesenberry Peter J.
Published: October 2019

Extracellular vesicles (EVs) including exosomes and microvesicles, have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that pretreated irradiated murine bone marrow stem cells with human or murine MSC-EV in vitro, could significantly improve the engraftment capacity of radiation-damaged stem cells up to 9 months post-transplantation. Interestingly, the restoration of engraftment was not significantly observed within the first month of post-transplant, the predominant reversal effect occurred on later period of post-transplant from 3 months up to 9 months. This is indicating a long-term effect of MSC-EVs on reversal of radiation damage of stem cell engraftment capacity. To confirm this hypothesis, in our current study, the effect of human MSC-EVs on reversal of engraftment capacity of bone marrow stem cells post-radiation was investigated by an in vivo study. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-EVs or vehicle were then injected intravenously 24, 48 and 72 hours after irradiation. The whole bone marrow were harvested at 6, 12, 26 and 53 weeks post EV-injection and then transplanted into 950 cGy exposed B6.SJL mice and engraftment evaluated at 1 and 3 months post-transplantation. In those transplanted mice at 6 weeks post-EV injection, there was slight increase in the restoration of engraftment rate (the percent of irradiated mice with EV/Vehicle treatment engraftment rate compared to healthy non-irradiated mice engraftment rate) in EV treated mice (17.58±2.32% compared to untreated mice (13.80±1.41%) after 1 month post-transplantation. However, for those mice transplanted at 12, 26, and 53 weeks post-EV injection, there were the significant higher restorations of engraftment rate in EV treated mice (40.48±6.03%, 33.93±3.76%, and 56.62±3.635) compared to untreated mice (12.39±1.30%, 15.14±2.21%, 36.21±3.63%) after 1 month transplantation respectively. The similar restorations of engraftment were also seen in 3 months post-transplantation. Our study also showed that there was a significant inhibition of stem cell engraftment at 53 weeks post 500cGy whole body radiation mice which was 36.21±3.63% of engraftment rate from healthy mice. Thus our data suggest that there is a long-term effect of MSC-EVs on the restoration of engraftment of stem cells in radiation-exposed mice.

Disclosures

No relevant conflicts of interest to declare.
Favorable Outcome of Sequential HLA-Haploidentical Transplantation Using Ptcy As GvHD Prophylaxis in High Risk AML and MDS of the Elderly
Authors: Fraccaroli Alessia , Prevalsek Dusan , Häbe Sarah Elena , Bücklein Veit , Schulz Christoph , Drolle Heidrun , Fritsch Susanne , Ledderose Georg , von Bergwelt Michael , Hiddemann Wolfgang , Tischer Johanna
Published: October 2019

Sequential conditioning regimens, comprising cytoreductive chemotherapy shortly applied prior to reduced intensity conditioning are successfully used for high-risk (HR) AML/MDS in matched related and unrelated donor hematopoietic stem cell transplantation. However, few data are available for sequential conditioning in the context of HLA-haploidentical transplantation (haplo-HSCT), especially in the elderly. To investigate the relative merits of sequential haplo-HSCT in the elderly we retrospectively analyzed the outcome of thirty-five patients (pts) with advanced AML/MDS (>=50 years old).

Thirty-three pts suffering from HR AML, defined by refractory, relapsed or secondary leukemia, or complete remission with adverse-risk genetics according to ELN criteria and two pts with HR MDS according to IPSS-R, who underwent T-cell-replete haplo-HSCT at our institution between January 2009 and November 2016 were included. Disease was active in 29 pts while 6 had achieved CR. Pre-transplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) which was ≥3 in 13 pts (median HCT-CI:2, range 0-8). A sequential therapeutic concept using either FLAMSA (n=26) or clofarabine (n=9) for cytoreduction was used prior to RIC in all pts. Bone marrow (54%) and peripheral blood stem cells (46%) were both used as graft source. Post-grafting immunosuppression consisted of high-dose cyclophosphamide, tacrolimus and MMF in all pts.

Median age was 60 years (50-70). One graft rejection occurred. Three pts died early in aplasia. Neutrophil and platelet engraftment was achieved in 95% and 77% of evaluable pts, respectively at a median of 16,5 (13-89) and 31,5 (11-103) days.Acute GvHD grade I-III occurred in 25/32 of the pts (grade III n=2); no patient developed grade IV aGvHD. Chronic GvHD was observed in 13/29 pts and was most frequently assessed as mild (n=6) or moderate (n=5) while 2 pts developed severe cGvHD. No GvHD related death was observed. CI of NRM at day 100, 1-year and 3-years was 11%, 23% and 23%, respectively. CI of relapse at 1- and 3-years was 15% and 27%, with a median time to relapse of 152 days (20-413). At a median follow up of 27 months (4-74), estimated one- and three-year overall survival (OS) was 62% and 52% respectively. One- and three-year leukemia-free survival (LFS) was 59% and 52%.

Our results suggest that using a sequential therapeutic concept in PTCY-based haplo-HSCT is safe and properly tolerated while it provides a favorable disease control when treating elderly HR MDS/AML pts. Thus, sequential haplo-HSCT seems to be a valuable alternative in pts who lack a conventional donor or are in urgent need for prompt transplantation.

Disclosures

Tischer: Jazz Pharmaceuticals: Other: Jazz Advisory Board.
Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses
Authors: Podolnikova Nataly , Balabiyev Arnat , Ugarova Tatiana P.
Published: October 2019

CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress.

Disclosures

No relevant conflicts of interest to declare.
Adoptive Therapy with Cord Blood Regulatory T Cells Does Not Impair Anti-Leukemia Activity of Infused Donor Cells
Authors: Ma Hongbing , Nishimoto Mitsutaka , Zeng Ke , Nguyen Tuongvan D , Schuler William , Kellner Joshua , Peedikayil Jaimol , Lyu Mi-Ae , Thangavelu-Devaraj Santhana , Parmar Simrit
Published: October 2019

Introduction

Cord blood derived ex vivo expanded regulatory T cells(CB-Tregs) have been verified that they can effectively prevent xenogenic graft-versus-host disease(GVHD) in NSG mice, which provide evidence to further investigate the effect of CB-Tregs in clinical setting. However, due to the potent suppression function of CB-Tregs, it needs to elucidate the concerning about whether CB-Tregs worsen leukemia or dampen anti-leukemia activity of donor cells. Beatrice et al. report donor derived in vitro expanded Treg can also prevent GVHD successfully while allowing a powerful graft-versus-leukemia(GVL) in murine models. We hypothesize cord blood derived ex vivo Treg are effective in preventing GVHD without compromising GVL.

Methods

Tregs are isolated from cord blood and expanded with CD3CD28 beads and IL-2 in SCGM medium. Then the cells are harvested on day14 after culturing. We use CD4+CD25+CD127low or - Foxp3+ to confirm Tregs, viabilty and purification on flow cytometry. The suppresive assay of CB-Tregs are based on CFSE. 3 or 10 million HL-60 cells are used to build AML model in NSG mouse. AML VS AML+Tregs or AML+PBMC/Tcons VS AML+PBMC/Tcons+Tregs are set up in each experiments. The primary end point is survival, secondary point is AML percentage and weight loss as well as the cytokines in the serum and tissue pathology.

Results

62-135 folds of CB-Tregs compared original number are harvested on day14, >90% cells are CD4+CD25+CD127low or - Foxp3+, There is no any difference in weight loss, AML percentage of peripheral blood and survival between AML+PBMC and AML+PBMC+Treg (Figure1 a, b,c and d,e,f). There is no significant difference in AML percentage between AML+PBMC/Tcons +Treg and AML+PBMC/Tcons, but mice in AML+Tcons+Treg survived longer than mice in AML+Tcons (Figure 2a,b,c,d).

Conclusion

CB-Tregs do not deteriorate the development of AML, they also do not impair anti-leukemia activity of PBMC or Tcons while they provide protection from GVHD. Our findings lay down the basis for testing impact CB-Tregs on CAR T cell activity in Vivo.

Disclosures

No relevant conflicts of interest to declare.
Aged Neutrophils in Sickle Cell Disease Impede Ischemic Repair
Authors: Okwan-Duodu Derick , Archer David R. , Taylor W Robert
Published: October 2019

INTRODUCTION: Repeated bout of tissue ischemia is a hallmark of sickle cell disease (SCD). Neutrophils are critical first responder cells that initiate host response to sterile injury, including ischemia. Of the numerous neutrophils in circulation, a small proportion undergo physiologic changes (termed aging) which directs their homing to the bone marrow for clearance. How neutrophil homeostasis is regulated in sickle cell disease (SCD) to influence ischemic repair is unknown.

HYPOTHESIS: We hypothesized that SCD may alter the homeostasis and function of aged neutrophil.

METHODS: Aged neutrophils from the Townes humanized sickle cell (SS) mice were compared to aged neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI.

RESULTS: Compared to wild-type mice (7 ± 1.2%), the proportion of CXCR4HiCD62Llo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%). However, in contrast with aged neutrophils from MerKO mice, those from SS mice showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, perfusion recovery in the MerKO and wild type mice were similar, but this was dramatically impaired in SS mice. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), but NAC therapy had no impact on MerKO mice.

CONCLUSIONS: Although SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that oxidant stress causes aged CXCR4HiCD62Llo neutrophils in SS to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health in SCD.

Disclosures

No relevant conflicts of interest to declare.
Adamts-13 Inhibitor Testing Is Often Negative on Initial Testing
Authors: Chaudhry Hina , Sholzberg Michelle , Pavenski Katerina
Published: October 2019

Background:

Thrombotic thrombocytopenic purpura (TTP) presents with microangiopathic hemolytic anemia and thrombocytopenia and is caused by severe ADAMTS13 deficiency. TTP can be the result of autoantibodies to ADAMTS13 or genetic defect in the ADAMTS13 gene. ADAMTS-13 is an enzyme that specifically cleaves unusually large von Willebrand Factor (VWF) multimers which mediate platelet thrombus formation under high shear. When ADAMTS13 is deficient, unusually large VWF multimers accumulate causing excessive platelet aggregation and thrombosis in the microvasculature.

Methods:

St. Michael's Hospital, Toronto is home to a large reference laboratory for special coagulation. We use a commercial ELISA Technoclone Technozym ADAMTS-13 activity assay for the determination of ADAMTS-13 activity and Technoclone Technozym ADAMTS-13 inhibitor assay to identify anti-ADAMTS-13 antibodies. We send samples to another Canadian laboratory for validation of our results as they use an in-house ELISA assay for ADAMTS13 activity and anti-ADAMTS13 antibody.

Results:

We performed a retrospective review of all ADAMTS13 activity tests performed by our laboratory between January 1, 2013 and June 30, 2018. The total number of tests was 466 from 203 unique patients. 24% had an ADAMTS-13 activity under 10% (N = 144) which is consistent with the diagnosis of TTP.

When specimens with severe ADAMTS-13 deficiency were tested for presence of anti-ADAMTS13 antibody, 46% were negative. Four of these specimens were sent to the other laboratory and all had detectable, albeit very low titre, inhibitors. Furthermore, on repeated testing over the study period, the vast majority of patients who presented with low ADAMTS13 activity and no detectable antibody subsequently became antibody positive. Fifty-two patients remained antibody negative by our internal and send-out testing. Five of them were known to have or were subsequently diagnosed with hereditary TTP (hTTP). Only one patient continues to have negative antibody but whose clinical course is not consistent with hTTP.

Conclusions:

We found that a commercial ADAMTS13 (Technoclone Technozym) antibody assay is falsely negative in a substantial proportion of patients with autoimmune TTP, the majority of which likely had a low titer inhibitor, below the threshold of test detection. More sensitive assays and/or repeated testing, presumably as inhibitor titre increases during the course of the disease, may detect antibody presence in the majority of samples of patients with autoimmune TTP. This is an important finding as this could impact the types of therapies offered to patients with negative antibody screens and may also avoid unnecessary, expensive genetic testing .

Disclosures

No relevant conflicts of interest to declare.
Single Center Real-World Experience of Daratumumab and Extra-Medullary Myeloma
Authors: Bishnoi Rohit , Shah Chintan , Moreb Jan , Murthy Hemant S.
Published: October 2019

Background: Daratumumab (Dara) was recently FDA approved for the treatment of relapsed/refractory multiple myeloma (RRMM). We evaluated a real-world single-center experience of Dara salvage therapy for RRMM.

Methods: Patients treated at the University of Florida with Dara for RRMM between January 2016 and February 2018 were included. Data was collected by chart review and analyzed for patient demographics, disease characteristics, treatment, and outcomes.

Results: 25 patients (13 as monotherapy and 12 in combination) were included, with a median age of 63 (range, 42-86) years. Median time from MM diagnosis to Dara initiation was 51.3 (range, 8.4 -162.3) months. The median duration of Dara therapy was 72 days (range, 7 - 622). The median number of Dara doses was 7 (range, 2- 49). The best response to DARA was Very Good Partial Response (VGPR) in 1 (4%), Partial Response (PR) in 17 (68%), while 7 (28%) had Progressive Disease (PD). Overall Response Rate (ORR) to Dara was 72%. By the end of the study period, another 11 (44%) patients progressed while on DARA with a total of 18 (72%) patients having PD at the time of analysis. Estimated median progression free survival (PFS) after starting Dara was 155 days (95% CI: 21 - 289) and estimated median overall survival (OS) in the entire cohort was 11.7 months (95% CI: 4.2 - 19.2). Post DARA, 1 (4%) patient with VGPR underwent ASCT, while 9 (36%) went on to have other therapies which included HyperCVAD, Melphalan, and clinical trials, and 8 (32%) were treated with only best supportive care.

Of 18 patients who had PD, 10 (40%) patients had a progressive extramedullary disease (EMD), of whom 6 (24%) had EMD prior to starting Dara, while another 4 (16%) patients developed new EMD while on Dara. PFS in the EMD cohort was 63 days (95% CI: 23 - 103), which was shorter than patients with no EMD (333 days; 95% CI: 42 - 624) (P= 0.08). Estimated median OS in patients with EMD was 5.4 months while in patients with no EMD was 16.1 months (p =0.16).

Conclusions: DARA therapy does have good ORR (72%) in RRMM, however, there was an unusually high incidence of progressive or newly diagnosed EMD in RRMM patients treated with Dara/Dara-containing regimens, suggesting that Dara may not be effective in treating or preventing EMD. Moreover, we noticed patients who have EMD or develop new EMD tends to have worse outcomes. The underlying escape mechanism for EMD remains unknown and warrants further investigation.

Table. Table.

Disclosures

No relevant conflicts of interest to declare.