bluuebunny/crossref_metadata_2025 huggingface dataset:
Contrasting Effects of Enhanced Sirt1 Activity in Normal and BCR-ABL1-Depedent Haematopoiesis
Authors: Hu Ming , Khorashad Jamshid S , Chaidos Aristeidis , Nteliopoulos Georgios , Khongkow Mattaka , Roberts Irene , Goldman John , Reid Alistair , Lam Eric W-.F. , Apperley Jane , Karadimitris Anastasios
Published: October 2019

Abstract 1215

Sirt1 is a NAD+-dependent class III histone deacetylase that regulates various cellular processes including cell growth, apoptosis, senescence and stress responses. Variations in Sirt1 expression and activity are associated with pleiotropic and profound biological effects that are often cell context-dependent. Here we tested the impact of enhanced Sirt1 activity in normal and BCR-ABL1-dependent leukemic haematopoiesis in vitro.

Using 5-FU enriched murine primary hematopoietic stem and progenitor cells (HSPC) as a model, we report that: 1. Sirt1 over-expression induces 2–3 fold increased, both in frequency and absolute number, of HSPC cells (Lin-Sca1+ckit+) in ‘stem cell-like’ culture condition and enhanced clonogenicity in serial transplantation assay. qRT-PCR analysis showed significantly higher expression of GATA-2 gene in HSPC cells suggesting that Sirt1 promotes GATA-2 mediated HSPC cell proliferation. 2. When HSPC cells were induced to differentiation in the presence of lineage specific growth factor EPO, G-CSF and IL-7, respectively, Sirt1 expression significantly increased the frequency and absolute number of terminal differentiated erythroid (ter119+, 2–3 folds), granulocytic (Gr-1+Cd11b+, 2–3 folds) and B (B220+CD19+, 3–4 folds) cells. Thus, enhanced Sirt1 activity promotes HSPC self-renewal and multiple lineage differentiation.

We next tested the effects of Sirt1 activity in Chronic Myeloid Leukaemia (CML). By transducing Sirt1 retrovirus into IL3-dependent BaF3, IL3-independent BCR-ABLp210-expressing BaF3 and primary HSPC cell, we found that Sirt1 over-expression significantly inhibited BCR-ABL1-driven cell proliferation and clonogenicity in cytokine depleted media. In addition, Sirt1 over-expression further reduced the growth rate of BCR-ABLp210 expressing BaF3 cells when treated with TGF-b1, suggesting that Sirt1 might play as a downstream effector of TGF-b signal. Finally, Resveratrol (RSV), a Sirt1 activator, alone and in co-operation with Imatinib, induced cell death and inhibited clonogenicity of CD34+ cells from CML patients with chronic phase.

Taken together, our data demonstrate that Sirt1 exhibits contrasting effects in normal and BCR-ABL1-dependent haematopoiesis and suggest that manipulating Sirt1 activity may be a useful strategy to differentially promote normal and inhibit leukemic haematopoiesis in CML.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stem Cells Engineered to Inhibit Complement-Mediated Damage
Authors: Soland Melisa , Bego Mariana , Colletti Evan , Porada Christopher D , Zanjani Esmail , St. Jeor Stephen , Almeida-Porada Graca
Published: October 2019

Abstract 1253

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express low levels of molecules, such as CD59, which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. It has been demonstrated that transduction of MSC with a retrovirus encoding HCMV US proteins resulted in higher levels of MSC engraftment and diminished recognition by the immune system, due to a decrease in HLA-I expression. Here we investigate whether engineering MSC to express US2, US3, US6, or US11 HCMV proteins can alter complement recognition, and thereby protect MSC from complement attack and lysis. US HCMV proteins increased MSC CD59 expression to differing degrees, as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI) (n=3). CD59 MFI on untransduced MSC was 128±33, and this value remained largely unchanged on MSC transduced with an empty retroviral vector (MSC-E) and on MSC transduced with US11 (MSC-11). In contrast, a significant increase in CD59 MFI was seen in MSC transduced with US2 (MSC-2), US3 (MSC-3), and US6 (MSC-6), with MFIs of 273±35 (p<0.05), 319±64 (p<0.05), and 265±16 (p<0.05), respectively. Although overexpression of HCMV proteins on MSC did not change the MFI for membrane cofactor protein (CD46) and complement decay accelerating factor (CD55), it significantly altered the percentage of MSC that expressed these two complement-protective proteins (n=3). Specifically, while no statistically significant difference was seen in the percentage of MSC-E, MSC-6, or MSC-11 expressing CD46 (17.9±1%, 19±0.1%, 21.3±1.4%, respectively), 27.7±0.7% of MSC-3 (p<0.05) and 27.7±1.6% of MSC-2 (p<0.05) expressed CD46. Similarly, while comparable percentages of MSC, MSC-E, and MSC-3 expressed CD55 (37.8±3%, 42.8±1%, and 45.2±1, respectively), the overexpression of HCMV US2, US6, and US11 each led to a significant increase in the percentage of MSC expressing CD55 (MSC-2: 49±1% (p<0.05); MSC-6: 56±0.6% (p<0.05); MSC-11: 60±1.3% (p<0.05)). Because the HCMV US2 protein was the most efficient at up-regulating all three complement regulatory proteins, we used two different complement-mediated cytotoxicity assays to investigate whether MSC-2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis of MSC-2 by 59.10±12.89 % when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV US proteins in complement evasion, and our results demonstrate that over-expression of HCMV US proteins on MSC could serve as a strategy to generate cells protected from complement lysis.

Disclosures:

No relevant conflicts of interest to declare.
Reactivation of Telomerase in Late Generation Tert−/− Mice Results in Reversal of Anemia
Authors: Raval Aparna , Kusler Brenda , Garbuzov Alina , Artandi Steven , Mitchell Beverly S.
Published: October 2019

Abstract 1258

We previously reported that telomere shortening in a late generation telomerase K/O mouse model results in significantly reduced RBC and erythroblast (CD71+Ter119+) numbers in the peripheral blood and bone marrow, respectively. We also observed significant decreases in erythroid progenitor (Lin-cKit+Sca1-CD34-CD16/CD32-) and HSC (Lin-cKit+Sca1+ CD34-CD150+) numbers in G5 Tert−/− mice. We now report the mechanism underlying the specific defect in the erythroid lineage in these mice. First, there is a higher number of CD34-KLS CD150lo and CD150neg cells in the mutant mice relative to age matched G0 +/− mice. Methyl cellulose colony formation assay showed that CD34-KLS CD150hi cells give rise to a greater number of CFU-GEMM colonies while CD34-KLS CD150lo and CD34-KLS CD150neg cells both had a reduction in the numbers of GEMM colonies, suggesting that the loss of erythroid differentiation is associated with loss of CD150hi expression within the CD34-KLS population. Thus, a significant reduction in CD150hi HSC (p value < 0.001) and an increase in CD150lo (p value < 0.001) and CD150neg (p value < 0.04) HSC in G5 Tert−/− mice underlies the decreased number of erythroblasts in these mice. Secondly, we found that the reduction in telomere length as measured by quantitative PCR is greater in sorted erythroblasts (CD71+Ter119+) than in similarly purified myeloid progenitors (Gr1+Mac1+) in G5 Tert −/− mice. This finding correlates with the presence of increased DNA damage and apoptosis in erythroid progenitors and erythroblasts as compared to myeloid progenitors. In order to establish a cause and effect relationship between telomere shortening and defective erythropoiesis, we reactivated telomerase activity using a tamoxifen-inducible Cre recombinase to excise a Lox-Stop-Lox cassette located in intron 2 of telomerase gene in G5 Tert−/− mice. Reactivation of telomerase activity resulted in increased CD150hi HSC, erythroid progenitors and erythroblasts numbers in the bone marrow of these mice 3 months after tamoxifen treatment. In addition, RBC numbers and Hb levels in the peripheral blood improved in the treated mice, whereas neither improved in control G5 Tert−/− mice treated with sunflower oil. These data show for the first time a direct link between the loss of telomerase function, telomere shortening and a selective defect in erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments underway to define the intracellular signaling pathways that are affected in the HSC and erythroid precursors of G5 Tert−/− mice, demonstrate the lineage-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Suppression of the Hematopoietic Defect in TF-1 Cells Depleted of Shwachman-Diamond Syndrome Protein: Correlation with Decreased eIF6 Levels
Authors: Singh Sharon , Blanc Lionel , Henson Adrianna , Sezgin Gulay , Ellis Steven R , Liu Johnson M.
Published: October 2019

Abstract 1270

Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6.

Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p<0.01 135+/−6.8 p=0.64 Erythroid 89+/−8.4 48+/−4 p<0.001 89+/− 4.7 p=0.94

Disclosures:

No relevant conflicts of interest to declare.
A Homozygous Telomerase Reverse Transcriptase T Motif Variant Resulting in Markedly Reduced Telomerase Repeat Addition Processivity in Siblings with Hoyeraal Hreidarsson Syndrome
Authors: Gramatges M. Monica , Qi Xiaodong , Sasa Ghadir S. , Chen Julian J-L , Bertuch Alison A.
Published: October 2019

Abstract 1272

Hoyeraal Hreidarsson syndrome (HHS) is a severe form of dyskeratosis congenita (DC) characterized by bone marrow failure, intrauterine growth retardation, developmental delay, microcephaly, cerebellar hypoplasia, immunodeficiency, and extremely short telomeres. As with DC, mutations in genes that encode factors required for telomere maintenance, such as the telomerase reverse transcriptase (TERT), have been found in patients with HHS. We describe two sibling cases of HHS caused by a homozygous mutation (p.T567M) within the TERT T motif, which is a highly conserved motif specific to the telomerase-class of reverse transcriptases. This mutation resulted in a marked reduction in the capacity of telomerase to processively synthesize telomeric repeats, indicating for the first time a role for the T motif in this unique aspect of telomerase function. The consanguineous parents, heterozygous for this mutation, exhibited telomere lengths around the first percentile and no evidence of a DC phenotype. Although heterozygous processivity defects have been associated with familial adult-onset pulmonary fibrosis, these cases demonstrate the severe clinical and functional impact of biallelic processivity mutations. Thus, despite retaining the capacity to catalyze the addition of short stretches of telomeric repeats onto the shortest telomeres, the sole expression of telomerase repeat addition processivity mutants leads to a profound failure of telomere maintenance and early onset disease. In the course of our investigation of this mutant, several commonly used algorithms predicted no effect of TERT p.567M upon protein function. To examine this further, we compiled missense changes that are associated with a clinical phenotype and correlated each with its reported telomerase activity and presence of the phenotype in more than one family member. We then summarized the predicted functional impact for each of these mutations based upon four commonly used algorithms. Our results demonstrate these algorithms to be limited in their capacity for predicting the effect of missense changes upon telomerase function, emphasizing the importance of in vitro functional analyses including processivity assays for TERT variants of unknown significance.

Disclosures:

No relevant conflicts of interest to declare.
Adaptor Protein Lnk Binds to and Inhibits Normal and Leukemic FLT3
Authors: Lin Dechen , Yin Tong , koren-Michowitz Maya , Ding Ling-Wen , Gueller Saskia , Gery Sigal , Tabayashi Takayuki , Bergholz Ulla , Kazi Julhash , Rönnstrand Lars , Stocking Carol , Koeffler Phillip
Published: October 2019

Abstract 1312

Background

The production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML.

FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3.

Methods and Results

Co-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL.

Conclusions

This study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases.

Disclosures:

No relevant conflicts of interest to declare.
Plasma Membrane Proteomics Identifies Biomarkers Associated with T(4;14) Multiple Myeloma
Authors: Xie Zhigang , Gunaratne Jayantha , Cheong Lip Lee , Liu Shaw Cheng , Koh Tze Loong , Huang Gaofeng , Blackstock Walter , Chng Wee-Joo
Published: October 2019

Abstract 1314

Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. Patients with t(4;14) have very poor prognosis. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry based on stable isotope labelling by amino acids in cell culture (SILAC) to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 55 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was universally over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. Quantitative RT-PCR (qPCR) analysis indicated MMSET shRNAs resulted in significant reduction of SLAMF7 mRNA, suggesting MMSET might regulate the transcription level of SLAMF7. ChIP followed by qPCR analysis indicated MMSET protein binding was concentrated in an upstream region (near −1,500 bp) of SLAMF7 transcript start site. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. SLAMF7 is a self-ligand receptor, and we found that SLAMF7 extracellular domain (ECD) could specifically recognize t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM, and SLAMF7 ECD might be an alternative approach for targeting SLAMF7 on t(4;14) MM.

Disclosures:

No relevant conflicts of interest to declare.
Geminin Is a Newly Identified Cks1 Interaction Partner and Overexpressed in a Murine Model During Lymphomagenesis
Authors: Hoellein Alexander , Bassermann Florian , Schoeffmann Stephanie , Graf Steffi , Peschel Christian , Gloeckner Johannes , Ueffing Marius , Keller Ulrich
Published: October 2019

Abstract 1328

Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells.

We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation.

Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Combination Therapy Based On the Genetics of Follicular Lymphoma
Authors: Wendel Hans-Guido
Published: October 2019

Abstract 1330

Follicular lymphoma (FL) is a common and incurable form of Non-Hodgkin's lymphoma characterized by the translocation t(14;18) that results in increased expression of the anti-apoptotic BCL2 protein. Exactly what drives expansion of the malignant B-cells in FL is not clear. Here we report recurrent genetic lesions targeting cell cycle controls in FL. These include mutations in the CYCLIN D3 gene (6%), chromosomal gains of CDK4 (31%), and losses of CDKN2a/b (9%), and RB1 (10%). Consistent with the genetic data, 46% of FLs show abundant RB phosphorylation, and together these lesions identify patients with advanced tumours who fare badly with standard therapies. In a murine FL model loss of cell cycle control acts as an oncogenic driver and cooperates with BCL2. Moreover, xenografted FLs respond to CDK4/6 inhibition and combinations with BH3 mimetic drugs cause extensive apoptosis and consolidate the therapeutic effect. Hence, loss of cell cycle control is an oncogenic event in ∼50% of FL and the simultaneous blockade of CDK4 and BCL2 is an effective combination therapy for FL.

Disclosures:

No relevant conflicts of interest to declare.
Tregs Specific for Minor Histocompatibility Antigens
Authors: Veerapathran Anandharaman , Pidala Joseph , Beato Francisca , Janssen William E. , Yu Xue-Zhong , Anasetti Claudio
Published: October 2019

Abstract 1891

Background:

The risk of acute GVHD after HSCT is increased in male recipients of female grafts. Disparities for the male-associated H-Y and other minor histocompatibility antigens (mHAs) have the capacity to sensitize alloreactive donor T cells and cause GVHD in HLA-matched recipients. These mHAs are polymorphic proteins that differ between donor and recipient and are presented as peptides by HLA molecules on recipient or donor antigen-presenting cells to donor immune cells. Currently, there is no evidence that minor histocompatibility antigen specific Tregs exist. Earlier in our laboratory, we have measured the frequency, growth requirements, and function of human blood Tregs specific for allo-MHC. In the present study, we sought to detect the frequency, expansion kinetics and characteristics of the minor antigens specific Tregs in the blood of HLA-matched sibling pair.

Methods:

CD4+CD25+CD127− Tregs were isolated by immunoabsorption from sibling donors, and cultured with HLA-matched sibling recipient antigen-presenting cells in the presence of IL-2, IL-15 and rapamycin. We detected 30–50 fold increase in H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical sibling compared to self DC. The precursor frequency of mHA-specific Tregs are between 7 and 43 (median - 13) cells per one million blood Tregs. The frequency of mHA-specific conventional CD4 T cells among total blood CD4 T cells is similar in HLA-matched sibling donors. Ex vivo expanded mHA-specific Tregs maintained higher levels of Foxp3 expression, retained the lymphoid homing receptor CD62L and a chemokine receptor, CCR7, suggesting that they are functional and are able to migrate to lymphoid tissue in vivo. Split well assay on day 12 demonstrated the mHA specificity, since Treg responded to restimulation with DC from the original HLA-identical sibling, but not self DC. The mHA-specific Tregs expanded to more than 100 fold in vitro, and exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 6 mHA-specific Treg clones that retained TGF-beta secretion in response to the sibling's mHA-disparate DC but not self DC.

Conclusion:

We demonstrated for the first time that it is possible to detect and expand mHA specific Tregs from HLA-matched sibling pairs, immunotherapy with mHA-specific Tregs may prevent GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Ascertainment of Iron Depletion and Deficiency in Blood Donors Through Screening Questions for Pica and Restless Legs Syndrome.
Authors: Bryant Barbara J. , Yau Yu Ying , Arceo Sarah M. , Hopkins Julie A. , Leitman Susan F.
Published: October 2019

Abstract 2283

Background:

Pica and restless legs syndrome (RLS) are associated with iron depletion and deficiency. Pica refers to the compulsive craving for and persistent consumption of non-nutritive substances. Pagophagia, the pathological consumption of ice, is a specific pica strongly associated with iron deficiency. RLS is a neurological movement disorder characterized by uncomfortable sensations in the lower extremities, with a compelling urge to move the extremities to relieve the discomfort. Primary RLS is a central nervous system disorder; however, secondary RLS may be caused or exacerbated by iron deficiency. The purpose of this study was to prospectively assess the prevalence of pica and RLS in blood donors presenting to a hospital-based donor center, to correlate the findings with donor hemoglobin and iron levels, and to study the effects of oral iron replacement on the resolution of symptoms.

Methods:

During a 39-month period, 1236 blood donors deferred for fingerstick hemoglobin <12.5 g/dL and 400 non-deferred “control” donors underwent health screening and laboratory testing (CBC, ferritin, iron, transferrin). Iron deficiency was defined as a ferritin level below the institutional reference range of 9 mcg/L in females and 18 mcg/L in males. Iron depletion was defined as a ferritin of 9 – 19 mcg/L in females and 18 – 29 mcg/L in males. Pica and RLS were assessed by direct questioning. Deferred donors and iron-deficient control donors were given ferrous sulfate 325 mg daily for 60 days. Reassessments were performed and additional iron tablets dispensed at subsequent visits.

Results:

Pica was reported in 11% of donors with iron depletion/deficiency, compared with 4% of iron-replete donors (p<0.0001). The prevalence of pica rose to 21% in females with ferritin <9 mcg/L. Pagophagia (ice pica) was most common and was often of extraordinary intensity, with characteristics of an addiction. Donors with pagophagia given iron reported a marked reduction in the desire to eat ice by day 5–8 of therapy, with disappearance of symptoms by day 10–14. RLS was reported in 16% of subjects with iron depletion/deficiency compared with 11% of iron-replete donors (p=0.012). Iron replacement generally resulted in improvement of RLS symptoms, however, at least 4–6 weeks of iron therapy was necessary. The positive predictive values of pica and RLS in iron deficient/depleted blood donors were 73% and 58%, respectively.

Conclusion:

The presence of pica is highly predictive of iron depletion/deficiency in blood donors; however, RLS lacks a strong correlation in this population. Screening questions for pica, in particular pagophagia, may be useful in the ascertainment of iron depletion and deficiency in blood donors and may identify those who would benefit from oral iron therapy.

Disclosures:

No relevant conflicts of interest to declare.
Phase II Study of Pomalidomide (Pom) in Genomically Defined High Risk Relapsed and Refractory Multiple Myeloma (RRMM)
Authors: Usmani Saad Z , Hansen Emily , Steward Doug , Waheed Sarah , Panozzo Susan B. , Petty Nathan M , Atrash Shebli , Abdallah Al-Ola , Ahmad Nisar , Hoering Antje , Crowley John , Barlogie Bart
Published: October 2019

Abstract 4083

Background:

Pom is a third generation immunomodulatory drug (IMiD) which has demonstrated safety and efficacy in RRMM with prior exposure/resistant to other IMiDs and bortezomib. This is the first report on a phase II clinical trial employing Pomalidomide (Pom) in genomically defined high risk RRMM.

Methods:

First cycle Pom was given at 4mg orally Days 1–21 every 28 days; dexamethasone (DEX) or bortezomib (BOZ) could be given in absence of PR or better from Cycle #3. In the absence of at least PR, Pom dose was escalated to 5mg. Cox regression modeling was employed for univariate and multivariate analyses, whereas Kaplan-Meier curves were used for overall survival (OS) and progression free survival (PFS).

Results:

30 patients with RRMM were enrolled. Baseline characteristics included age >=65yr in 47%, ISS stage >=II was seen in 74% of patients, cytogenetic abnormalities (CA) within 6 months in 79%, and GEP-defined high risk in 66% by GEP70-gene model, 97% by GEP80-gene model, 13% had extramedullary disease (EMD), and 7% had secondary plasma cell leukemia (SPCL). 26/30 patients (87%) had prior autologous stem cell transplant, 23/30 patients (77%) had had at least 2 transplants. 22 patients (73%) had disease progression while on lenalidomide, bortezomib or both within 60 days of enrollment. At least 2 cycle of treatment were administered to 28 (93%) patients enrolled, 20 (67%) patients received >4 cycle of treatment and 16 (53%) received > 6 cycles. 15 patients (50%) discontinued therapy primarily due to progression or death. 5 (17%) patients achieved PR or better as best response. PFS benefit was observed in older patients (HR=0.29, p=0.008), with high LDH presenting an increased risk for OS (HR=14.64, p=0.013.) Most common toxicities, counting all toxicities (>=grade 3) were: thrombocytopenia (63%), leukopenia (63%), anemia (40%), hypophosphatemia (20%) and hypocalcemia (23%). Overall and progression- free survival at 12 months were 64% and 20%, respectively.

Conclusions:

The present trial is the first investigation evaluating Pom's clinical activity in high risk RRMM and demonstrates good anti-myeloma activity in this heavily pre-treated patient population. Given the activity in poor prognostic clinical features such as EMD and SPCL, there is precedence to evaluate Pom as an IMiD of choice in combination with other novel agents for this subset of RRMM.

Disclosures:

No relevant conflicts of interest to declare.
Abrogation of Myeloid Derived Suppressor Cell-Like Inhibitory Activity of K562 Restores Antigen Presenting Cell Functions
Authors: Tanimoto Kazushi , Muranski Pawel , Hensel Nancy F. , Keyvanfar Keyvan , Fujiwara Hiroshi , Barrett A. John , Melenhorst J. Joseph
Published: October 2019

Abstract 4118

The human leukemia cell line K562 represents an attractive platform for creating an artificial antigen presenting cell (AAPC): it is readily expandable, does not express HLA class I and II and can be stably transduced with various genes. To generate an AAPC to expand CMV antigen-specific T cells for adoptive immunotherapy, we stably transduced K562 with HLA-A2 in combination with 4-1BB ligand, or CD64 or HLA-DR15. In preliminary experiments, irradiated K562 cells expressing HLA-A2 and 4-1BB ligand pulsed with CMV pp65 and IE-1 HLA-A2 specific peptides failed to elicit antigen-specific CD8+ T cells in HLA-A2+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Since CMV peptides added directly to the PBMC readily expanded antigen-specific CD8+ T cells, we concluded that K562 AAPC inhibited the T cell response. We found that both parental K562 cells and AAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcus enterotoxin B (SEB), anti-CD3 stimulation with OKT3, and in MLR. The inhibitory effect of K562 appeared to be T cell specific since K562 cells did not suppress EBV-transformed B cells.

Transwell experiments demonstrated preservation of the inhibition, suggesting that suppression was mediated by a soluble factor. MLR inhibition was not reversed by neutralizing anti-TGFβ antibody or PGE2 inhibitors. Finally, the full abrogation of the suppressive activity of K562 was achieved by a brief fixation of cells with formaldehyde at concentrations as low as 0.1%: The MLR was restored when K562 was fixed, and donor T cell response to SEB- and OKT3-loaded K562 AAPC was significantly higher when using fixed K562 cells compared to unfixed cells. Moreover, fixed pp65 and IE-1 peptide-loaded HLA A2+ AAPC expressing 4-1BB ligand induced robust (3–5 fold improved) expansion of CMV-specific T cells from all tested HLA-A2+ donors when compared with irradiated AAPC control. Thus, fixed K562 cell constructs efficiently presented antigen and stimulated T cells.

Overall, we demonstrate that K562 line can serve as a source of AAPC for cell therapy approaches after abrogation of their suppressive activity using formaldehyde. However, our results also revealed a previously unappreciated feature of K562 biology, clearly indicating that these commonly used cells are potent inhibitors of peptide antigen-, superantigen-, and OKT3- driven T cell proliferation. Thus K562 line displays a myeloid-derived suppressor cell-like functionality. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemias and other tumors.

Disclosures:

No relevant conflicts of interest to declare.
Azacytidine Compromises NK-Cell Activity in AML and MDS Patients Undergoing MRD-Based Pre-Emptive Treatment After Allogeneic Stem Cell Transplantation
Authors: Sockel Katja , Schönefeldt Claudia , Sopper Sieghart , Wermke Martin , Schmitz Marc , Ehninger Gerhard , Bornhäuser Martin , Wolf Dominik , Platzbecker Uwe
Published: October 2019

Abstract 4122

The hypomethylating agent azacytidine (AZA) represents the standard treatment for many high-risk MDS and AML patients. While the clinical efficacy has been confirmed in several studies, the precise molecular mechanism of action has not been fully understood yet.

Human NK-cells play an important role in the regulation of immune responses against malignant cells. Their function is controlled by a complex interplay of activating and inhibitory receptors - some of them being regulated by methylation of the respective genes. We, therefore explored, whether AZA modulates in vitro NK-cell function as well as in vivo during minimal-residual disease (MRD)-guided treatment of imminent relapse in MDS and AML patients treated within the prospective RELAZA trial (NCT00422890).

Methods:

After purifying NK-cells of healthy donors by MACS (magnetic cell sorting), NK-cells were exposed in vitro to different concentrations of AZA (100nM, 1μM, 3μM) with or without IL-2. In parallel, the NK-cell phenotype of patients (n=12) with AML or MDS, undergoing MRD-guided treatment with AZA after stem cell transplantation was monitored by FACS from peripheral blood samples on day 1, 5 and 7 of the first and second AZA cycle. All patients were still in complete haematological remission at the time of therapy.

Results:

In vitro, we observed a significant reduction (3,1% to 1,8% p=0.028) of the immature and cytokine-regulating CD56bright NK-cell subpopulation with increasing concentrations of AZA. There was a trend towards a reduced expression of the death-ligand TRAIL, the activating receptors NKG2D and NKp46 and for an increased expression of the inhibitory KIR CD158b1/b2, whereas we could not detect any changes in the expression of FAS-L, Perforin, Granzyme B, NKp30, NKp44, CD69, CD57, DNAM-1, CD16, and NKG2A-CD94. Confirmatory, we observed a significant decrease in the expression of TRAIL (p=0.003), NKG2D (p=0.03) and NKp46 (p=0.006) during AZA treatment in-vivo. Interestingly, these changes appeared to be reversible.

The observed reduction of NK-cell activating receptors and TRAIL during AZA treatment correlated with a reduction or stable course of MRD in all analyzed patients.

Conclusion:

In summary these data suggest that the clinical effects of AZA are not mediated by enhancing NK-cell activity. In fact, the drug may have inhibitory effects on NK-cell function which should be considered when applying AZA in the post-transplant setting.

Disclosures:

Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Why Is Cancer Care So Expensive: Potential Implications of Fraud and Abuse
Authors: Lu Zhiqiang , Qureshi Zaina Parvez , Chen Brian , Xirasagar Sudha , Ray Paul , Rao Gowtham , Lopez Isaac , Felder Tisha , Ablin Richard , Sartor Oliver , Bennett Charles
Published: October 2019

Abstract 4275

Introduction:

Annually, $82 billion to $272 billion is reportedly lost to federal health care fraud. Between 1996 and 2005, 379 federal health care fraud cases initiated by qui tam relators (“whistle blowers”) concluded, resulting in $9.3 billion in recoveries. Of these, pharmaceutical companies accounted for 13 cases (False Claims Act (FCA) cases, the primary statute invoked in health care fraud and abuse), but $3.9 billion of recoveries (4% of the cases and 39% of the financial recoveries). We report concluded FCA cases involving pharmaceutical manufacturers between 2006 and 2011. Oncology accounts for the largest per cent of total pharmaceutical expenditures. Over 90% of all new cancer pharmaceuticals cost > $20,000 for 12-weeks of treatment.

Methods:

Websites for the Department of Justice (DOJ), Taxpayers Against Fraud, Health and Human Services Inspector General's Office, Health Care Fraud and Abuse Control Project, and Lexis/Nexis were queried for pharmaceutical FCA cases (2006 to 2011).

Results:

Between 2006 and 2011, the DOJ closed 54 cases with pharmaceutical FCA violations, 38 with and 16 without qui tam relators, accounting for recoveries of $11.3 billion (mean $296 million) and $2.6 billion (mean, $165 million), respectively. Illegal marketing is the most common fraud allegations invoked against pharmaceutical manufacturers (19 cases). Pharmaceutical manufacturers accounted for 31% of total FCA cases, and 71.5% of total FCA recoveries (Table 1).

Conclusion:

Since the DOJ's shift of focus to pharmaceutical corporations in 2001, the trend has intensified, with virtually every large pharmaceutical corporation settling at least one FCA case. Pharmaceutical cases now account for 31% of the federal fraud cases and 71% of the financial recoveries. Fraud and abuse may be an important component of the high costs of cancer care in the United States. Moreover, unless fundamental changes occur, the pharmaceutical industry will continue to be the main FCA investigative target as this sector has the deepest pockets and is the health care sector most resistant to deterrence.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of Blood Graft Cellular Content in Myeloma (MM) Patients and Non-Hodgkin Lymphoma (NHL) Patients Mobilized with Chemotherapy Plus G-CSF with or without Plerixafor
Authors: Jantunen Esa , Varmavuo Ville , Silvennoinen Raija , Valonen Piia , Kuittinen Taru , Nousiainen Tapio , Mäntymaa Pentti
Published: October 2019

Abstract 4414

Background:

Cellular composition of blood graft collected to support high-dose therapy may have important implications in regard to hematopoietic and immune reconstitution after high-dose therapy. Mobilization method used may have effect on graft composition as well as previous therapy given to the patients.

Methods:

We have retrospective analyzed by flow cytometry cellular composition of freezed blood grafts in 34 patients with NHL (chemotherapy + G-CSF mobilization 15, add-on plerixafor 19 patients) and compared the observations with those of 17 MM patients mobilized either with low-dose cyclophosphamide + G-CSF (n=12) or with add-on plerixafor (n=5). The analyses were performed from the first graft collected from the chemomobilized patients or from the first draft collected after the plerixafor injection, respectively. Antibodies used included CD34, CD38, CD133 in regard to CD34+ subclasses and CD3, CD4, CD8, CD19 and CD3CD16/CD56 to analyze lymphocyte subsets. In addition, 7-aminoactinomycin D staining was used to assess the amount of nonviable CD34+ and lymphoid cells.

Results:

In regard to the patients mobilized without plerixafor, MM patients had higher content of CD34+ cells (4.9 vs. 2.0 × 106/kg, p=0.009) as well as lower proportion of more primitive stem cells (0.8 vs. 1.9 % of all CD34+ cells, p=0.075) when compared to NHL patients. The amount of B-lymphocytes was also significantly higher in MM patients (0.5 × 106/kg vs. 0.0, p< 0.001). No differences were observed in the total amount of T (CD3+) cells, NK cells or in the proportion of nonviable CD34+ or lymphoid cells. Of the plerixafor treated patients, the amount of B-lymphocytes was higher in MM patients (1.8 × 106/kg vs. 0.0, p<0.001) as well as CD4/CD8 ratio (2.56 vs. 1.0, p=0.004) when compared to NHL patients.

Conclusion:

As there appears to be differences between MM and NHL patients mobilized with or without plerixafor, it might be advisable to analyze separately effects of mobilization therapy to graft content and post-transplant outcomes in these diseases. Apparently the type and intensity of previous therapy given to the patients (e.g. rituximab) has important effects on graft cellular composition.

Disclosures:

Jantunen: Genzyme: Has participated in EU Leadership meeting organized by Genzyme as well as Medical Advisory Board meeting organized by Genzyme Other, Honoraria. Silvennoinen:Genzyme: Consultancy.
Double and Triple Hit Diffuse Large B Cell Lymphomas and First Line Therapy
Authors: Jaglal Michael V. , Peker Deniz , Tao Jianguo , Cultrera Jennifer L.
Published: October 2019

Abstract 4885

Background:

B cell lymphoma, unclassifiable, with features intermediate between diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL) or Burkitt like lymphoma is a high grade lymphoma, with a high proliferation index and a complex karyotype mostly involving MYC and BCL-2 genes, so called “double hit” as well as BCL-6 genes, so called “triple hit”. This high grade lymphoma shows an aggressive behavior for which the most appropriate therapeutic approach is not established.

Methods:

This was a single center retrospective review of patients with a confirmed diagnosis of B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL from 2006 to 2012. The definitive diagnosis in all cases was based on morphological and phenotypic features, and genetic abnormalities involving MYC, BCL-2, and BCL-6. Clinicopathological data was extracted including age, gender, primary disease location, phenotypic subtype, genetic abnormalities by FISH, IPI scores, treatment regimens and survival data. Descriptive statistical analyses were utilized. Kaplan-Meier method was used to estimate OS and log rank test was used to compare the groups. All data were analyzed using SPSS version 19.0 statistical software.

Results:

31 patients with “Double or Triple Hit” lymphomas were identified between 2006 and 2012. The age range at diagnosis was 33–87 years with median age of 71. 23 of 31 patients (74 %) were ≥ 60 years old. Male to female ratio was 1.58:1 (19:12). The median ECOG PS was 1. No patients had HIV. A majority of patients presented with extranodal disease 21 out of 31 patients and only 10 patients presented with nodal disease. The mean overall survival of the entire cohort was 28 months with ranges 3 to 40 months. 20 of 31 patients (65%) are currently alive. RCHOP was the most utilized treatment regimen in the cohort of alive patients 13 out of 20 patients (65%). Patients who presented with CNS disease had a worse prognosis when compared to the entire cohort with a mean overall survival of 13 months versus non CNS disease overall survival was 32 months (p=0.014). In patients treated with RCHOP for first line therapy the overall survival was 33 months versus 17 months for non RCHOP regimens first line (p=.048). The non RCHOP regimens included Hyper CVAD, ESHAP, and RICE.

Conclusions:

RCHOP was the most utilized treatment regimen in the cohort of alive patients. The treatment regimen with the best efficacy in this retrospective study was RCHOP in first line treatment of “Double or Triple Hit” lymphomas. CNS disease is associated with worse prognosis in patients with “Double or Triple Hit” lymphomas in this retrospective study.

Disclosures:

No relevant conflicts of interest to declare.
Cytotoxic Effect of Bendamustine Combined with Kinase Inhibitors in Hematologic Cell Lines
Authors: Hagiwara Kazumi , Kojima Yuki , Miyata Yasuhiko , Nagai Hirokazu
Published: October 2019

Abstract 4912

Bendamustine, a kind of alkylating agent, has demonstrated a favorable anti-tumor activity both alone and in combination with rituximab in low grade B cell lymphoma. Because the kinase cascades, including B-cell receptor signaling, are necessary for the proliferation and survival of the lymphoma cells, the kinase inhibitors have been developed as molecular target agents of malignant lymphomas. We investigated the cytotoxic effect of bendamustine, both as a single agent and in combination with these new kinase inhibitors in human lymphoid cell lines. We used three B-cell lines, BALL-1 (acute lymphoblastic leukemia), SKLY16 (B-cell lymphoma) and DHL4 (diffuse large B cell lymphoma), and T-cell line THP-6 in this study. These cells were treated with bendamustine alone or combined with enzastaurin (a PKC-β inhibitor), CAL-101 (a p110δ PI3K inhibitor), PCI-32765 (a Btk inhibitor) or R406 (a Syk inhibitor) for 48 hours. Drug-induced cytotoxicity was evaluated using the MTT assay. Cell viability in each experiment was normalized using untreated controls. The treatment with bendamustine alone decreased cell viability in a dose-dependent manner on these three B-cell lines. In contrast, T-cell line THP-6 showed the resistance to bendamusitine. For the combination study, cells were exposed to bendamustine (10μM) and one of these kinase inhibitors at various concentrations simultaneously for 48 hours. CAL-101 and PCI-32765 did not enhance the cytotoxic effect on all of B-cell lines. In BALL-1 and SKLY16 cells, the combination with enzastaurin or R406 resulted in a higher cytotoxic activity than that induced by bendamustine alone. In DHL4 cells, the treatment combined with R406 inhibited cell growth effectively, but not with enzastaurin. To evaluate whether these drug combinations are synergistic, a combination index (CI) was calculated and normalized isobolograms were constructed from non-constant ratio drug combinations using Calcusyn software. The CI values were less than 0. 7 in SKLY16 cells treated with bendamustine and enzastaurin, indicating that these produced synergistic cytotoxic effects in cell line-dependent manner. Our results show that enzastaurin might potentiate the cytotoxic activity of bendamustine in vitro and be a good candidate for the combination with bendamustine.

Disclosures:

No relevant conflicts of interest to declare.