Blood

Abstract Bone marrow sinusoidal endothelial cells (ECs) are an integral component of the hematopoietic stromal microenvironment and have been implicated in hematopoietic stem and progenitor cell (HSPC) homeostasis, though their role in regulation of hematopoietic trafficking remains uncertain. In this study, we demonstrate that bone marrow (BM) sinusoidal ECs actively regulate HSPC retention and enforced egress out of BM quite surprisingly by serving as “gatekeepers” of HSPC within the marrow niche, a non-paradigmatic mechanism independent of traditional adhesive/chemotactic mechanisms. Treatment with G-CSF (100 µg/kg, 4 days) resulted in down-regulated expression of VE-cadherin (50±3.3% reduction) and CD31 (35±2.1%) along the gap junctions connecting ECs (CD45neg Ter119- VEGFR3+) forming BM sinuosoids, reducing cell-cell contact and allowing for increased HSPC trafficking to the peripheral blood. To validate the functional contribution of ECs in HSPC trafficking, mouse (C166 cells) or human (HUVEC) endothelial monolayers were treated with G-CSF for 24 hours in vitro. Treatment of EC monolayers with G-CSF increased dextran-FITC permeability across EC monolayers and increased migration of HSPCs across endothelium, suggesting that a component of G-CSF mobilization activity is the “opening” of EC gap junctions to allow successful HSPC migration through the EC layer. To confirm this increase in endothelial monolayer permeability in response to G-CSF treatment, we measured EC cell-cell contacts both in vitro and in vivo. Immunofluorescence analysis of HUVECs demonstrated significant VE-cadherin+ cell-cell contact in our cultured HUVEC monolayers with a significant loss of cell-cell contact after G-CSF exposure. Histological examination of femur sections from G-CSF treated mice showed a similar disruption of cell-cell contact of VEGFR+and Dil-Ac-LDL sinusoids. Previous reports have demonstrated that pharmacologic antagonism of the dipeptidyl peptidase CD26, or its gene deletion, results in reduced HSPC egress in response to G-CSF. As CD26 is highly expressed on BM ECs, and expression dynamically changes in response to G-CSF treatment, we hypothesized that sinusoidal ECs expressing CD26 may regulate sinusoidal integrity and HSPC egress. Blockade of CD26 activity, or gene deletion of CD26 in ECs, completely reversed the G-CSF-mediated reduction in EC cell-cell junctions accompanied by reduced trans-endothelial migration of HSPC. Given that CD26 is an enzyme that exerts physiologic effects by N-terminal cleavage of effector proteins, we performed a systematic protein sequence search for proteins containing putative CD26 recognition sites. Intriguingly, the neurotransmitter neuropeptide Y (NPY) can be cleaved by CD26, and the full length and cleaved versions have differing functions on NPY receptors expressed on EC. While HSPC mobilization in response to G-CSF was reduced in the absence of CD26 activity, mobilization was restored by treating mice with the cleaved NPY (NPY3-36) and was inhibited by antagonizing NPY2 and Y5 receptors on ECs, suggesting that reduced mobilization in CD26 knockout mice is caused by a lack of cleaved NPY, preventing an opening of EC cell-cell junctions and allowing for HSPC egress. If ECs are acting as active “gatekeepers” of HSPC within the marrow space, regulated by CD26 and NPY, we hypothesized HSPC egress in response to agents other than G-CSF would also depend on EC cell-cell junction opening. Interestingly, LPS administration, a mimic of bacterial infection, and AMD3100, a CXCR4 antagonist, also reduced VE-cadherin and CD31 expression along BM sinusoidal EC junctions coincident with enhanced HSPC mobilization. Our results identify an unappreciated active role for bone marrow niche ECs in maintaining HSPC within the marrow space, and describe a heretofore unknown CD26-mediated EC-neuropeptide Y axis regulating optimal HSPC trafficking. These results further refine our knowledge of hematopoietic trafficking mechanisms and identify potential new pharmaceutic targets to regulate trafficking in homeostatic and stress conditions. Disclosures: Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.

Abstract Using the spleen colony-forming technique to assay hemopoietic stem cells, (CFUs) experiments have been carried out to investigate the role of thymus cells in hemopoiesis. The experiments were carried out by injecting live or killed thymus cells into lethally-irradiated mice together with hemopoietic tissue containing CFUs. The effect of thymus cells on endogenous colony formation in sublethally-irradiated mice was also investigated. Colony formation by normal bone marrow or spleen is unaffected by the addition of thymus cells, but cell populations damaged by radiation all demonstrate increased colony numbers when live thymus cells are injected within 48 hr of initiating colony formation. Endogenous colony formation resulting from an x-ray dose of 475 red is increased 2.9 times: exogenous colony formation from bone marrow or spleen populations whose CFUs content has been reduced to approximately 20% of normal with 180-rad γ-rays is increased 2.1 times. Studies investigating this enhancement demonstrate that at least some of the colonyforming cells require the cooperation of live thymus cells. Under the conditions of these experiments, at least 106 thymus cells must be injected to produce maximum enhancement.

Abstract Background: Diffuse Large B cell lymphoma, the most commonly diagnosed type of non-Hodgkin lymphoma is curable in many cases, despite this, up to 30% of patients will relapse after initial therapy, necessitating salvage chemotherapy and transplantation if feasible. There is a pressing need for novel treatment strategies in highly chemo refractory cases. ONC201 is a small molecule that induces p53-independent cell death in tumor cells while sparing normal cells through a number of putative mechanisms, including inactivation of the pro-survival kinases Akt and ERK. ONC201 is currently entering Phase I/II clinical trials as a monoagent in adult advanced cancers. ONC201 promotes up-regulation of TRAIL gene transcription by inactivating AKT and ERK kinases which leads to translocation of transcription factor Foxo3a into the nucleus. It appears to act on a p53 independent pathway. ABT-199 is a selective, potent and orally bioavailable small molecule that selectively inhibits BCL-2 and triggers apoptosis. The Bcl-2 family of proteins is key regulator of the apoptotic process, comprising proapoptotic and prosurvival proteins. BH3-only proteins (such as Bim) bind to pro-survival proteins and cause increased permeability of mitochondrial membrane, release of cytochrome c and activation of caspases through release of Bax and others. Methods: Cell lines Pfeiffer and Toledo were purchased from ATCC and one patient DLBCL cell specimen from the ascitic fluid was cultured for use in experiments. ABT-199 was purchased from MedKoo Biosciences and ONC201 was provided from Oncoceutics. Cytotoxicity was evaluated by using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturerÕs instructions. Cell viability was measured over time in response to treatment with ONC201(1-64μM) and ABT-199(128 nM-4μM). Western Blotting was performed on treated cells with well-established methodologies, with antibodies to c-Myc, Bcl-2, pAKT, pERK. Results: Immunohistochemical Staining Pattern of Cell Lines Table 1. Patient Toledo Pfeiffer Bcl-2 +++ ++ + Bcl-6 + ++ ++ c-Myc +++ ++ + p-ERK + + ++ p-AKT + ++ + Bax ++ NA NA Bim ++ NA NA Mean IC50 Calculated for Cell Lines Table 2. Cell Line Therapeutic Agent ABT199 ONC201 Patient Sample 8 μM 5 nM Toledo 9 μM 28 nM Pfeiffer 6 μM 2 μM SHAPE Conclusion/Discussion: The patient cell line, an ascitic fluid sample of DLBCL was sensitive to both ONC201 and ABT-199 and manifested bright Bcl-2 expression, the target of ABT-199. In this series there was a higher sensitivity to ABT199 in DLBCL cells with higher Bcl-2 expression. ONC201 down regulated pAKT expression, as seen in Western Blots in treated cells, consistent with prior investigation with the agent. We further found that ONC201 synergizes to potentiate cytotoxicity with ABT199, as demonstrated in the cell viability assay for Toledo cell lines (at the 24 hour time point), which were the least sensitive to ONC201 (highest IC50) when given as a single agent. Yet, when combined with increasing doses of ABT199, there was synergistic lymphoma cell kill with a fixed dose of ONC201. Together these results suggest that ONC201 has potential as an antitumor agent in NHL as monoagent and in combination with ABT-199, which may be further explored in phase Ib/II trials. Further analysis in larger patient sample series may elucidate the biomarkers that predict for greater therapeutic sensitivity to these highly potent lymphoma agents. Figure 1. Figure 1. Disclosures Allen: Oncoceutics, Inc: Employment, Equity Ownership. Eldeiry:Oncoceutics, Inc: Equity Ownership.

Abstract Mice homozygous for the gusmps allele lack beta-glucuronidase activity and provide a useful model for human Mucopolysaccharidosis type VII (MPS VII), also known as Sly syndrome. Bone marrow (BM) transplantation was shown to correct the metabolic defect and to increase the life span of diseased animals. We have used this murine model in a preclinical study aimed at evaluating whether the techniques currently available for gene transfer into large mammalian and human BM cells will provide efficient enzyme replacement therapy in MPS patients. Autologous BM was transplanted into deficient mice after retrovirus-mediated transfer of the human beta-glucuronidase cDNA. Conditioning of recipients was performed by a single sublethal irradiation of 4.5 Gy, giving rise to low donor engraftment. In recipient mice analyzed until 145 days after gene transfer, the percentage of genetically modified hematopoietic cells was less than 5%. Nevertheless, beta-glucuronidase enzyme activity was detectable in various organs, including the brain, and disappearance of lysosomal storage was obvious in the liver and spleen. These results show that the autologous transplantation of genetically engineered BM cells could be beneficial in MPS patients.

Abstract Introduction: Mutations (mut) in the Calreticulin gene (CALR) were recently described in BCR-ABL1-negative myeloproliferative neoplasms (MPN). They occur frequently in essential thrombocythemia (ET; 15-30%) and primary myelofibrosis (PMF; 25-35%), but not in cases with polycythemia vera (PV). Other well-known mutations in ET and PMF are JAK2V617F (50-60%) and MPLW515 (5-10%). These three mutations are nearly mutually exclusive of each other. Furthermore, also chromosomal aberrations are frequently detected in PMF (40%), whereas they are rare in ET (3%). However, no association of CALRmut with any cytogenetic aberration has been reported yet. Aims: Investigate CALRmut and JAK2V617F in cytogenetic subgroups of BCR-ABL1-negative MPN. Patients: We studied 220 patients with cytomorphological confirmed BCR-ABL1-negative MPN excluding PV and with following chromosomal aberrations: del(20q) (n=64), trisomy 8 (+8; n=57), +1q (n=30), del(5q) (n=25), del(13q) (n=23), monosomy 7/del(7q) (-7/del(7q); n=21). Of these 220 cases, 25 and 12 patients were in accelerated and blastic phase, respectively. The cohort comprised 85 females (38.6%) and 135 males (61.4%). Median hemoglobin (Hb) level was 12.3 g/dl (range: 6.0 - 17.8 g/dl, n=167), platelet count 277,500x109/L (range: 16,000 –1,877,000x109/L; n=170) and white blood cell (WBC) count 16,000 x109/L (range: 1,600 -305,000 x109/L, n=181). Methods: Chromosome banding analysis was performed using standard G-banding. Screening for CALRmut was done by fragment analysis and subsequent Sanger sequencing of positive cases. JAK2V617F and MPLW515 were analyzed by melting curve analysis. MPLW515 was only analyzed in CALR/JAK2V617F-negative patients. Results: All 220 patients were screened for CALRmut and JAK2V617F. The frequency of CALRmut was 16.8% (37/220) and of JAK2V617F 58.2% (128/220). Mutations in these two genes were mutually exclusive (p<0.001). MPLW515 occurred in 3/55 (5.5%) of CALR/JAK2V617F-negative cases. CALR mutations presented as type 1 (p.Leu367Thrfs*46) in 56.8% (21/37) and as type 2 (p.Lys385Asnfs*47) in 27.0% (10/37) according to the nomenclature of Klampfl et al. (NEJM, 2013). The remaining 6 cases represented different mutation types all resulting in the same C-terminus of the mutated CALR protein. Analysis of gene mutations and cytogenetic aberrations showed that CALRmut associated significantly with del(13q) (with vs. without: 10/23, 43.5% vs. 27/197, 13.7%, p=0.001), whereas they were rare in +8 patients (2/57, 3.5% vs. 35/163, 21.5%, p=0.001; Figure 1). Additionally, no CALRmut was detected in patients with -7/del(7q) (0/21, 0% vs. 37/199, 18.6%, p=0.029). For JAK2V617F an association with del(20q) was detected (44/64, 68.8% vs. 84/156, 53.8%, p=0.050). Exclusion of MPN in accelerated or blastic phase from analyses resulted in the same associations between distinct cytogenetic abnormalities and CALRmut. Only the negative correlation to chromosome 7 aberrations lost its significance, probably due to low case numbers (0/9, 0% vs. 28/174, 16.1%, n.s.). For JAK2V617F the association with del(20q) was still present, even though the statistical significance was lost (37/55, 67.3% vs. 77/132, 58.3%, n.s.). Furthermore, we analyzed the distribution between type 1 and type 2 CALR mutations (n=31) in cytogenetic subgroups. Type 1 mutations were more frequent in cases with del(13q) (9/9, 100.0% vs. 12/22, 54.5%, p=0.030), whereas the frequency of type 2 mutations was higher in del(20q) (6/10, 60.0% vs. 4/21, 19.0%, p=0.040). Analysis of clinical data showed that CALRmut vs. wild-type patients had lower Hb levels (mean: 10.9 vs. 12.1 g/dl, p=0.019) and JAK2V617F cases had lower WBC counts vs. JAK2V617F-negative patients (19,308 vs. 30,786 x109/L, p=0.041). Additionally, Hb levels were higher in JAK2V617F patients compared to cases with CALRmut (12.2 vs. 10.9 g/dl, p=0.017). Conclusions: The highest CALR mutation frequency was observed in del(13q) cases (43.5%) and nearly all of them were type 1 mutations (90.0%). In contrast, CALRmut were rare in the cytogenetic subgroups with +8 and -7/del(7q). The highest JAK2V617F frequency was detected in patients with del(20q) (68.8%). Thus, in PMF and ET specific patterns are detectable based on cytogenetic and molecular data. Figure 1: Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns. Figure 1:. Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.

Abstract Abstract 5299 Background: Thalassemia is one of the most common genetic blood disorders worldwide. With recent improvements in medical therapy, patients with transfusion-dependent thalassemia, i.e., thalassemia major, are living longer. As a result, there is a greater need to address endocrine complications related to chronic iron overload. Adrenal insufficiency (AI), in particular, is important to identify because therapies are available and can be life-saving. Objectives: The objectives of this study are to determine the prevalence of AI in our population of subjects with thalassemia major; to identify risk factors that predict AI in these individuals; and to localize the origin of the AI within the hypothalamic-pituitary-adrenal (HPA) axis. Methods: This is a prospective study of individuals with thalassemia major with an enrollment goal of 30 subjects. All subjects enrolled were initially tested for AI using a glucagon stimulation test. Those found to have AI (stimulated cortisol <18 mcg/dL) subsequently underwent an ovine corticotrophin-releasing hormone (oCRH) stimulation test for confirmatory purposes and to define the physiological basis for the AI. Results: Eleven subjects (8 - 29 years old, 6 female) have been enrolled to date. In our population of patients with TM, the prevalence of AI was 55%. There was no correlation between age, number of years transfused, or ferritin levels and AI. All male patients failed the glucagon stimulation test, whereas 5 of 6 females passed the glucagon stimulation test, p = 0.0024. There was no correlation between 8 AM ACTH levels and 8 AM cortisol levels. There was a significant correlation (p = 0.025) between 8 AM cortisol level and peak cortisol level following glucagon stimulation testing. Of the six subjects with AI, two subjects subsequently failed the oCRH stimulation test (peak cortisol < 21.9 mcg/dL). In these two subjects, peak oCRH ACTH levels were elevated, 144 and 164 pg/mL, respectively, suggesting primary adrenal insufficiency. Conclusions: We conclude that 8 AM cortisol level is a good predictor of adrenal insufficiency in our population, and can potentially be used as a simple screening test for AI with a strong negative predictive value. There appears to be a male predominance of AI in our population. This may indicate a protective role of female sex in this population. Two subjects had classic primary AI with robust ACTH levels in the face of inadequate cortisol production following oCRH testing. Four subjects (all males) who failed the glucagon stimulation test subsequently demonstrated normal ACTH and cortisol response to oCRH, indicating a possible hypothalamic origin to their AI. This dysfunction is likely independent of iron overload and warrants further investigation. Alternatively, these subjects may have impaired sympathetic nervous system function leading to hypoglycemic unawareness. Both outcomes are novel to the field and of medical significance. Disclosures: Geffner: Daiichi- Sankyo: Steering Committee for Clinical Trial; Eli Lilly, Inc.: Research Contract; Endo Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Genentech, Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ipsen: Data Safety Monitoring Board and Research Contract; Novo Nordisk: Research Funding; Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Abstract 3312 The European Network of Rare Bleeding Disorders (EN-RBD) was established to bridge the gap between knowledge and practice in the care of patients with RBDs (rare bleeding disorders frequency <1/0.5-2M). The aim of this first report was to explore the relationship between the coagulation factor activity levels and clinical bleeding severity in patients with RBDs. A total of 592 records on patients with RBDs were cross-sectionally collected over a period of three years. Data on clinical bleeding episodes were available for 495 patients and were classified into four categories according to severity. The mean age of patients was 31 years (range, 7 months-95 years), with 51% being females. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F)FX, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. It is of clinical relevance to identify levels that are not associated with spontaneous major bleeding, which was done by constructing receiver operating characteristic curves. These levels were highest for FXIII deficiency (25 U/dl), followed by FV+VII (15 U/dl), FVII (15 U/dl), FX (5 U/dl), and FV (1 U/dl) deficiencies. For fibrinogen deficiency, a factor activity level of 20 mg/dl is needed to ensure absence of major spontaneous bleeding. There is a clear relation between coagulation factor activity level and clinical bleeding severity in RBDs, which is different for the missing clotting factor. Disclosures: Peyvandi: NovoNordisk, CSL Behring: Honoraria. Bidlingmaier:CSL Behring, Bayer Schering: Research Funding; CSL Behring, NovoNordisk, Pfizer, Bayer Schering: Membership on an entity's Board of Directors or advisory committees; Baxter, Biotest, CSL Behring, NovoNordisk, Pfizer, Bayer Schering: Honoraria. Schved:LFB, CSL Behring, Novonordisk, Bayer Schering: Research Funding.