bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Expression of NEK2 Induces Drug Resistance by Inhibiting Premature Senescence Through Activation of the PI3K/Akt Pathway in Myeloma and Other Cancers,
Authors: Zhan Fenghuang , Xiao Fang , Zangari Maurizio , Xu Hongwei , Tricot Guido J. 
Published: October 2019

Abstract
Abstract 3923

Background:
Autologous transplantation in combination with the newer drugs has significantly prolonged survival of patients with myeloma (MM). However, the majority of patients still relapses. Our previous study has shown that NEK2, a chromosomal instability (CIN) gene, is up-regulated in many types of cancer cells and is highly related to drug resistance. Recently, premature senescence has been determined to be an indicator of the efficacy of anti-cancer drugs. In this study, we investigate the correlation between the inhibition of premature senescence and NEK2 induced drug resistance.

Materials and Methods:
Four MM cell lines (Δ47, KMS28PE, ARP1 and U266) and 2 other cancer cell lines (MCF7, H1299) over- and under-expressing NEK2 using a lentiviral delivery system were studied. NEK2 induced drug resistance was evaluated by colonogenic and standard apoptotic assays. The phenotypic changes of premature senescence were determined by using SA β-galactosidase (SA β-gal) staining. The mechanisms of NEK2-induced anti-senescence activity were studied in vitro and in the 5TGM1 murine MM mouse model.

Results:
Over-expression of NEK2 stimulated cancer cells into increased S and G2/M phase, resulting in polyploidy. All NEK2 over-expressing cancer cells were able to override doxorubicin-induced premature senescence and to maintain their proliferative potential. Mechanistic studies revealed that NEK2 activated the PI3K/Akt pathway. This was confirmed by the use of a specific PI3K/Akt inhibitor, KP372-1, which abrogated NEK2-induced drug resistance by decreasing clonogenicity of NEK2 over-expressing cells and by decreasing tumor burden and extending mouse survival in the 5TGM1 myeloma mouse model even better than bortezomib. In contrast, knockdown NEK2 by shRNA also induced premature senescence. We determined that NEK2 activates PI3K/Akt pathway by directly interacting with pp1. And the Akt down-stream targets GSK-3, MDM2, NF-kB and the tumor suppressor genes p53, p21, p27, the pro-apoptotic and pro-survival genes of the Bcl-2 family as well as the ABC transporters that related to drug resistance were identified to contribute to the inhibition of premature senescence resulting in drug resistance.

Conclusion:
NEK2 inhibits doxorubicin-induced premature senescence, resulting in increased drug resistance by activating the PI3K/Akt pathway. Alternatively, down regulation of NEK2 by shRNA inhibits cell proliferation and decreases drug resistance. Thus, targeting PI3K/Akt pathway should eliminate NEK2 induced drug resistance by re-establishing drug-induced premature senescence.

Disclosures:
No relevant conflicts of interest to declare.
TNF-α/Fas-RIP-1-Induced Cell Death Signaling Separates Hematopoietic Stem Cells/Progenitors Into Two Distinct Populations
Authors: Xiao Yechen , Volk Andrew , Zhang Shubin , Wei Wei , Breslin Peter , Zhang Jiwang 
Published: October 2019

Abstract
Abstract 393
Tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) have been found to induce a negative regulatory effects on hematopoiesis and have been implicated in the pathogenesis of human bone marrow failure (BMF) syndromes. However, the molecular mechanism by which these factors inhibit hematopoiesis is still not completely known. We previously reported that Tak1-knockout mice (Tak1−/−) develop BMF due to the mass apoptosis of hematopoietic cells, including hematopoietic stem cells and progenitors (HSC/Ps). Taking advantage of this mouse model, we studied the effects of TNF-α and Fas-induced death signaling on HSC/Ps by examining their contributions to the development of BMF syndromes in Tak1−/− mice. To do so, TNF-α and Fas-induced signaling were genetically inactivated in Tak1−/− HSC/Ps in order to examine to what degree both the apoptosis of HSC/Ps and BMF in vivo can be prevented. We found that complete inactivation of TNF-α signaling by the deletion of both Tnfr1 and Tnfr2 (TNF receptors 1 and 2) is able to protect up to 30–40% of Tak1−/− HSC/Ps from apoptosis. In vitro studies suggested that Fas signaling also contributes to less than 10% of Tak−/− HSC/P death. However, since Fas works on the same population of cells as TNF-α, and because TNF-α signaling is dominant in vivo, inactivation of Fas signaling failed to inhibit the apoptosis of HSC/Ps and BM damage in Tak1−/− mice. In addition, inhibition of RIP-1 (Receptor-Interacting Protein-1) activity by the specific inhibitor Nec-1 (Necrostatin-1) but not inhibition of FADD/caspase-8 signaling was able to protect the same percentage of the Tak−/− HSC/Ps from death as complete inactivation of TNF-α signaling did, but was unable to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSC/Ps (Tak1, Tnfr1 and r2 compound mutant). This suggests that TNF-α, acting through RIP-1, induces death in 30 to 40% of HSC/Ps. To investigate the causes of apoptosis in the remainder of cells, we looked for factors which either protect Tak1−/−Tnfr1−/−r2−/− HSC/Ps from death or further induce such death. We found that the expression of major pro-survival genes is significantly down-regulated in Tak1−/− HSC/Ps. The survival of the Tak1−/−Tnfr1−/−r2−/− HSC/Ps can be further improved by transducing the over-expression of dominant negative (DN)-caspase-9, as well as by Bcl-xl. Our studies suggest that there is heterogeneity in BM HSC/Ps. Only a portion of HSC/Ps is responsive to TNFα/Fas-RIP-1-induced cell death, whereas the death of the remaining HSC/Ps is induced by an intrinsic apoptotic mechanism. Tak1 is involved in mediating hematopoietic cytokine- and pro-inflammatory factor-induced survival signaling, protecting against both the TNF-α/Fas-RIP-1-dependent and independent death of HSC/Ps.

Disclosures:
No relevant conflicts of interest to declare.
Pomalidomide and Dexamethasone in Relapsed Myeloma: Results of 225 Patients Treated in Five Cohorts Over Three Years,
Authors: Lacy Martha Q , LaPlant Betsy R. , Laumann Kristina , Gertz Morie A , Hayman Suzanne R , Buadi Francis K , Dispenzieri Angela , Kumar Shaji , Lust John A. , Russell Stephen , Dingli David , Zeldenrust Steven R , Greipp Philip R. , Fonseca Rafael , Bergsagel P.Leif , Roy Vivek , Stewart Keith , Reeder Craig B. , Hall Robert L , Rajkumar S. Vincent , Mikhael Joseph R. 
Published: October 2019

Abstract
Abstract 3963

Background:
Pomalidomide at doses of 2 or 4 mg/d has demonstrated excellent activity in patients with relapsed multiple myeloma (MM). Between November 2007 and November 2010, we opened 5 sequential phase 2 trials using the pomalidomide at differing doses with weekly dexamethasone (Pom/dex) regimen to study the efficacy of this regimen.

Methods:
The five cohorts consisted of: Cohort 1 (N=60): relapsed MM with 1–3 prior regimens, 2 mg dose; Cohort 2 (N=34): lenalidomide refractory, 2 mg dose; Cohort 3 (N=35): bortezomib/lenalidomide refractory, 2 mg dose; Cohort 4 (N=35): bortezomib/lenalidomide refractory, 4 mg dose; and Cohort 5 (N=60) lenalidomide refractory, 1–3 prior regimens, 4 mg dose. Pomalidomide was given orally 2 mg daily or 4mg daily on days 1–28 of a 28-day cycle with oral dexamethasone given 40 mg daily on days 1, 8, 15 and 22. Response was assessed by the International Myeloma Working Group Uniform Response criteria. All patients received aspirin 325 mg daily for DVT prophylaxis or full dose anticoagulation.

Results:
A total of 225 patients were enrolled across all 5 cohorts. One patient was ineligible and excluded from analysis. The median age was 63 years (32.0–88.0). The median time since diagnosis was 53 months. Forty percent had high-risk molecular markers. Eighty-nine percent had received previous IMIDs including thalidomide (53%) and lenalidomide (81%). Sixty-two percent had previous bortezomib and 73% had prior transplant. The median follow-up is 12.6 months, but varies from 9.4 months for the most recent cohort to 30 months for the first cohort. Sixty-nine percent are alive and 30% remain progression free. Toxicities ≥ grade 3 are shown in table 1 and patient outcomes are shown in Table 2.

Conclusions:
Pom/dex is remarkably active and well tolerated even in heavily pretreated patients. Responses are durable. Response rates and toxicity are similar between the 2 mg and 4 mg doses.

Disclosures:
Lacy: Celgene: Research Funding. Fonseca:Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.
Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,
Authors: Shima Haruko , Shino Mika , Yamagata Kazutsune , Aikawa Yukiko , Koseki Haruhiko , Kitabayashi Issay 
Published: October 2019

Abstract
Abstract 3998
Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells.
Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency.
To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B.
Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells.

Disclosures:
No relevant conflicts of interest to declare.
Lung Endothelial Injury Induced by HNA-3a Antibodies in TRALI
Authors: Bayat Behnaz , Tjahjono Yudy , Akylbek Sydykov , Hippenstiel Stephan , Weissmann Nobert , Sachs Ulrich J , Santoso Sentot 
Published: October 2019

Abstract
Abstract 40
Transfusion related acute lung injury (TRALI) is a severe side-effect of blood transfusion characterized by the acute onset of non-cardiogenic pulmonary edema. Meanwhile, TRALI turned out to be the leading cause of transfusion related fatalities. Accumulated evidence demonstrates that antibodies against human neutrophil antigens (HNAs) play a major role in the pathomechanism of TRALI. Recent studies show that antibodies against the allelic isoform of choline transporter like protein 2 (CTL-2 Arg154 isoform; also known as HNA-3a) are associated with high TRALI mortality. The mechanism underlying this fatal reaction, however, is not clear. In this study, we aimed to identify the mechanism of fatal TRALI induced by HNA-3 antibody under in vitro as well as in vivo conditions.
Analysis of mRNA by real-time PCR in different blood cells and endothelial cells (EC) revealed abundant copies of CTL-2 transcripts in ECs in comparison to neutrophils and platelets. This result was confirmed by immunochemical analysis using rabbit antibody specific for CTL-2 as well human antibodies against HNA-3a. In contrast to treatment of neutrophils, treatment of EC with HNA-3a antibodies, but not with antibodies against the antithetical allelic CTL-2 isoform (Gln154 isoform; HNA-3b), leads to significant production of reactive oxygen species (ROS). When HNA-3aa EC monolayers were treated with human anti-HNA-3a antibodies, a significant increase in albumin-FITC influx in transwell system was observed when compared to controls. In line with this observation, a strong reduction of transendothelial electrical resistance was measured. Microscopically, drastic stress fiber formation and gap formation was visible. Immunoblotting analysis of HNA-3a treated ECs showed a significant degradation of VE-cadherin. This observation indicates that anti-HNA-3a antibodies induce EC barrier disturbance via ROS-mediated destabilization of VE-cadherin in cell junctions. Accordingly, this antibody-mediated barrier leakage can be ameliorated by the vasoactive peptide adrenomedulin (ADM), which prevents cell destruction in response to oxidative stress.
In an in vivo murine model of TRALI, with lipopolysaccharide primed C57BL/6 mice upon injection of HNA-3a antibodies a significant increase in lung weight and elevated concentration of albumin and number of neutrophils in the bronchoalveolar lavage was observed, indicating the formation of lung edema in these mice. Neutrophil depletion mitigated this effect in mice, but failed to prevent TRALI.
Our data demonstrate the direct influence of transfused HNA-3a antibodies on endothelial barrier integrity in vitro as well as in vivo. This novel mechanism of TRALI may be helpful in defining prevention and treatment strategies in order to decrease transfusion mortality.

Disclosures:
No relevant conflicts of interest to declare.
Excess Heparan Sulphate Inhibits CXCL12-Mediated Hematopoietic Cell Migration and Engraftment After Bone Marrow Transplant in Mice with Mucopolysaccharidosis Type I,
Authors: Watson H. Angharad , Holley Rebecca J , Langford-Smith Kia J , Wilkinson Fiona L , van Kuppevelt Toin H , Wynn Robert , Wraith J. Ed , Merry Catherine L R , Bigger Brian W 
Published: October 2019

Abstract
Abstract 4010
The primary axis of migration for transplanted hematopoietic stem and progenitor cells (HSPC) is CXCL12/CXCR4. Heparan sulphate (HS) is required for CXCL12 presentation and receptor binding, but the functional role of HS is poorly defined. The alpha-L-iduronidase knockout mouse (Idua−/−) accumulates HS and dermatan sulphate, recapitulating the neurodegenerative lysosomal storage disease Mucopolysaccharidosis I Hurler (MPSIH). MPSIH is primarily treated with HSPC transplant, but clinical experience suggests a historical engraftment defect in patients.
We show significantly reduced HSPC migration in Idua−/− recipients and under limiting engraftment conditions we show a significant haematopoietic engraftment defect in Idua−/− recipients. No significant donor cell effect was observed. Bone marrow but not peripheral blood CXCL12 levels are slightly elevated in Idua−/− mice. CFU frequency in BM is unchanged between genotypes but reduced significantly in peripheral blood of Idua−/− mice. In whole bone marrow, and on mesenchymal stem cells from Idua−/− mice, HS is present in significant excess, particularly in extracellular matrix, and cell surface locations, with significant increases in all sulphation modifications, especially 2-O-sulphation. Finally we show that excess HS, and particularly HS with increased 2-O -sulphation, functionally inhibit haematopoietic progenitor cell migration in vitro. These data provide novel insight into the influence of highly sulphated HS in CXCL12 mediated haematopoietic progenitor cell migration and help to explain why HSCT engraftment has been historically low in MPSIH.

Disclosures:
No relevant conflicts of interest to declare.
MyD88-Dependent Expansion of An Immature GR-1+CD11b+ Population From Donor Bone Marrow Reduces Early Mortality After Allogeneic Hematopoietic Stem Cell Transplantation,
Authors: Lim Ji-Young , Park Gyeongsin , Youn Hyewon , Choi Eun-Young , Jeong Dae-Chul , Min Chang-Ki 
Published: October 2019

Abstract
Abstract 4011
Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity. MyD88 is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the toll-like receptor (TLR) family. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs.
We therefore investigated how the MyD88-dependent expansion of MDSCs from donor bone marrow (BM) contributes to protection of acute GVHD. To test this, we employed an intestinal GVHD murine model, C57BL/6 (H-2b) → B6D2F1 (H-2b/d), which differs at major and minor histocompatibility loci. Lethally irradiated recipient mice were transplanted with wild-type (WT) or MyD88 knock out (KO) mice T cell-depleted (TCD)-BM together with WT spleen T cells. Morbidity and mortality of GVHD was significantly worse in recipients of MyD88 KO TCD-BM with higher intestinal pathologic grading. Animals that underwent syngeneic HSCT did not show early mortality regardless of presence of MyD88 in BM, which ruled out myelosuppression-associated toxicity. The expression of Gr-1+CD11b+ in blood, mesenteric lymph nodes and liver on day 13 was significantly reduced in the recipients of MyD88 KO TCD-BM compared with those of WT TCD-BM while the percentage of donor T cells infiltrating colon and liver was significantly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of MyD88 KO TCD-BM. Injection of MDSCs from BM of non-tumor bearing donor markedly inhibited GVHD lethality in recipients of MyD88 KO TCD-BM. Moreover, in vivo administration of lipopolysaccharide (LPS), a TLR ligand, to donor mice expanded GR-1+CD11b+ in BM with enhanced expression of MyD88 mRNA. Recipients of TCD-BM from WT mice injected LPS showed attenuated GVHD severity as measured by weight loss and survival compared to those of TCD-BM from WT mice injected diluent.
In summary, MyD88-dependent expansion of GR-1+CD11b+ population from donor TCD-BM appears to be critical for survival after allo-HSCT. Incomplete expansion of GR-1+CD11b+ population in target organs correlates with decreased apoptosis and increased infiltration of donor T cells into the target organs.

Disclosures:
No relevant conflicts of interest to declare.
Impaired Donor T Cell Egress From Recipient Lymphoid Tissue Profoundly Diminishes Acute GvHD,
Authors: Fulton LeShara M , Coghill James , West Michelle L. , Foger Niko , Bear James , Serody Jonathan S 
Published: October 2019

Abstract
Abstract 4022
The migration of donor T cells into and out of host lymphoid tissue is critical for the induction of acute Graft-versus-Host Disease (GvHD). A number of groups including ours have found that the lack of lymphoid tissue or impaired migration into that tissue prevented acute GvHD. However, it is not clear if preventing egress of effector T cells from lymphoid tissue can inhibit GvHD. Coronin 1A (Coro 1A) is an actin binding protein involved in cell migration and motility, and has previously been shown to contribute to the ability of T cells to migrate within lymph nodes. To elucidate a role for Coro 1A in GvHD pathogenesis we used mice deficient in Coro 1A (Coro 1A−/−). Methods : A haplotype-matched Hematopoietic Stem Cell Transplant (HSCT) model was used. C57BL/6 (H-2b, “B6”) mice were used as donors while C57BL/6 x DBA2 F1 (H-2bxd, “B6D2”) functioned as recipients. Na•ve conventional T cells (Tns) were isolated from the spleens of wild type B6 (WT), enhanced green fluorescent protein expressing B6 (WT eGFP), Coro 1A−/−B6, and Coro 1A−/−eGFP mice. B6D2 mice were lethally irradiated to 900 rads on day −1 and injected with 4 × 106 Tns from WT, WT eGFP, Coro 1A−/−, or Coro 1A−/−eGFP mice supplemented with 3 × 106 WT B6 T cell depleted bone marrow cells on day 0. Recipients were followed for survival and assessed for GVHD twice weekly using a validated scoring system. In those mice given WT eGFP or Coro 1A−/− eGFP Tns, recipient organs were imaged by stereofluorescence microscopy on days 3 and 14 post transplantation. Results : Mice that received Coro 1A−/− Tns displayed significantly attenuated GvHD with greater than 90% surviving to transplant day 60. In contrast, those animals receiving WT Tns demonstrated a 100% mortality rate by the end of the study period (Fig 1A, p<0.05 by Fisher's exact test). Stereomicroscopy performed 3 days after transplantation demonstrated a lower eGFP signal within the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) of those mice given Coro 1A−/−eGFP T cells versus those receiving WT eGFP cells. By 14 days post transplantation, however, the converse was observed, with more cells found within the MLNs and PPs of recipient mice receiving Coro 1A−/−eGFP cells (Fig 1B). Total T cells numbers in the liver and colon, however, were decreased at this time point in Coro 1A−/−eGFP recipients, indicating an impaired ability for these cells to exit host lymphatic tissue and traffic to GvHD target organs. Conclusion : These data indicate that impaired egress from lymphoid tissue, by blocking the function of Coro 1A, leads to a profound decrease in GvHD pathology and donor T cells in GvHD target organs. These data suggest that blocking the migration of donor T cells out of lymphoid tissue is a new viable approach to prevent acute GvHD.

Disclosures:
No relevant conflicts of interest to declare.