ALBERT

Description: Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Description: We show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α5β1, α3β1, α6β1, and α1β1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Description: Caspases play a critical role in regulation of apoptosis, cell differentiation, inflammation, and innate immunity, and several are mutated or have altered expression in non-Hodgkin lymphoma (NHL). To study the impact of genetic variation in caspases on NHL risk, we analyzed tag single nucleotide polymorphisms (SNPs) in 12 caspase and related genes in 3 population-based case-control studies (1946 cases and 1808 controls). Gene-based analysis, adjusting for the number of tagSNPs genotyped in each gene, showed significant associations for CASP8, CASP9, and CASP1. SNP-based analysis showed that CASP8 rs6736233 (odds ratio (OR) CG = 1.21; ORCC = 2.13; P trend = .011); CASP9 rs4661636 (ORCT = 0.89; ORTT = 0.77; P trend = .011); and CASP1 rs1785882 (ORAT = 1.12; ORAA = 1.30; P trend = .0054) were significantly associated with NHL risk and consistent across studies. It is noteworthy that genetic variants in CASP8 were associated with risk of all major NHL subtypes. Our findings suggest that genetic variation in caspases may play an important role in lymphomagenesis.

Description: Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC50 (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Eμ-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Description: Romiplostim is a thrombopoietin receptor agonist that increases platelet counts in patients with chronic immune thrombocytopenia (ITP). Thrombopoietin receptor agonists are reported to increase the risk for reticulin fiber deposition within bone marrow. This report describes bone marrow findings from romiplostim-treated rats, a retrospective analysis of reticulin observed in romiplostim ITP clinical trials, and a prospective clinical study of the effects of romiplostim on bone marrow morphology. In rats, romiplostim produced a dose-dependent increase in bone marrow fibrosis that resolved after treatment withdrawal. Of 271 ITP patients in romiplostim clinical trials, 10 were reported to have reticulin deposition; reticulin grade was increased in 4 of 5 patients with both pretreatment and on-treatment bone marrow results. Reticulin grade often decreased soon after romiplostim discontinuation. In the prospective study, reticulin grade during romiplostim treatment remained within the normal range for all patients and was increased in only 1 of 6 patients with pretreatment and on-treatment bone marrow results. This report suggests that romiplostim produces reversible, dose-dependent bone marrow changes in rats and produces modest increases in bone marrow reticulin in some ITP patients that decrease when therapy is discontinued. These studies were registered at www.clinicaltrials.gov as #NCT00102323, #NCT00102336, #NCT00861224, and #NCT00116688.

Description: Abstract 3430 Poster Board III-318 Background and Significance Chronic lymphocytic leukemia (CLL) is a malignant B cell lymphoproliferative disorder characterized by the clonal expansion and accumulation of CD5+ B cells. CLL is quite heterogeneous in clinical behavior, where some patients remain asymptomatic and require no therapy for many years, whereas other patients follow an aggressive clinical course with early need for therapy and short overall survival. Those patients with somatic deletions and / or mutations of p53, a central mediator of cell cycle control, apoptosis, and a potent tumor suppressor, follow the most aggressive clinical courses of all patients with CLL. Furthermore, these patients tend to be refractory to conventional CLL chemotherapy regimens, and therefore novel therapeutic approaches are needed. Cenersen (Eleos Inc.) is a 20-mer antisense molecule and binds to a coding region of exon 10 in p53 mRNA and has been shown in vitro to increase apoptosis in lymphoma cell lines. We hypothesize that the addition of cenersen to chemo-immunotherapy will enhance leukemic cell apoptosis and thus increase chemotherapy sensitivity in vivo for patients with aggressive CLL. Here we report early clinical responses to cenersen in combination with FCR chemo-immunotherapy at a planned interim analysis for efficacy and safety after enrollment of 17 patients. Methods We have undertaken a clinical trial at Duke University Medical Center of cenersen in combination with chemo-immunotherapy for patients with CLL or SLL (ClinicalTrials.gov identifier: NCT00636155). Patients with relapsed CLL or previously untreated patients with a deletion or mutation of p53 requiring therapy were enrolled. Cenersen was administered at 2.4 mg/kg/day as a continuous infusion over 24 hours starting on day 1 and ending on day 4. Fludarabine was administered daily at 25 mg/m2 on days 2 through 4, cyclophosphamide was administered daily at 250 mg/m2 on days 2 through 4, and rituximab was administered at 375 mg/m2 on day 2. Cycles were repeated every 28 days for a maximum of 6 cycles. The primary outcome measure was the overall response rate (complete and partial responses by iwCLL response criteria), and secondary outcome measures included progression free survival, event free survival and overall survival. All patients received growth factor support as well as prophylaxis with acyclovir and trimethoprim / sulfamethoxazole. Interphase cytogenetics and sequencing analysis for somatic mutations of p53 was performed in all patients. Results To date, 17 patients have been enrolled to a protocol-defined interim analysis of efficacy and safety. Of the enrolled patients, 1 was previously untreated, and the remaining 16 were relapsed (median number of prior therapies 4, range 1 - 8). Among previously treated patients, the mean response duration of the most recent prior regimen was 2 months. 15 of 16 had previously received fludarabine and 14 of 16 patients had previously received rituximab. 10 of 16 patients tested showed 17p deletions by FISH. Among the 14 patients tested to date, 8 showed somatic mutations in p53 by sequence analysis, of these, 6 showed both deletion of 17p as well as somatic mutation of p53. Of the 17 patients enrolled, 14 are evaluable for response: 1 patient has not yet received 3 cycles of therapy and 2 patients voluntarily discontinued trial participation. To date, 5 patients show a partial response by iwCLL criteria, including 2 patients who have concluded all therapy and show a partial response with incomplete hematologic recovery (PRi) with no detectable CLL B cells by flow cytometry analysis of bone marrow aspirate. One of these patients has subsequently undergone allogeneic stem cell transplantation. The remainder of the patients had stable disease (SD), though 3 were removed from study prior to the completion of 3 cycles for inadequate response as determined by the treating physician. There have been 4 episodes of febrile neutropenia, and there have been no toxicities specifically attributed to cenersen. Conclusions We have observed encouraging clinical responses to the combination of cenersen with FCR chemo-immunotherapy among a group of extensively pretreated patients with high risk genetic features. There does not appear to be any excess toxicity associated with the investigational agent. The results to date suggest that there may be therapeutic benefit to p53-targeted therapies in high risk CLL. This study will be continued to full accrual. Disclosures Lanasa: Genentech: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Rituximab, in combination with fludarabine and cyclophosphamide, for the treatment of CLL. Cook:Eleos: Employment. Rizzieri:Eleos: Research Funding.

Description: Abstract 212 Epigenetic gene regulation is emerging as a major mechanism of regulating genetic programs and pathways involved in various forms of cancer. Specifically, recently published data and preliminary evidence suggests that histone modifications directly and indirectly affected by the Mixed Lineage Leukemia (MLL) protein may play a fundamental role in the pathogenesis of certain leukemias. Rearrangements of the MLL gene are found in a subset of pediatric and adult acute lymphoid and myeloid leukemia (ALL and AML). Leukemias with MLL-rearrangements tend to have a poor prognosis, particularly infant-ALL and treatment associated AML. MLL is a histone modifying enzyme, methylating histone 3 at lysine 4 (H3K4). In most MLL fusions, the domain harboring the methyl transferase activity (Set-domain) is lost. However, several fusion partners of MLL, such as AF4, AF9, AF10 and ENL, have been shown to bind and potentially recruit another histone methyl transferase, DOT1L, that methylates histone 3 at lysine 79 (H3K79). Chromatin immunoprecipitation studies in MLL-rearranged leukemia cells indeed revealed elevated H3K79 methylation at MLL-fusion target loci. These results were consistent in leukemic cells from Mll-AF4 conditional knock in mice, MLL-AF4 expressing cell lines and primary human t(4;11) (MLL-AF4) leukemia cells. H3K79 is a chromatin modification associated with actively transcribed genes, and H3K79 methylation profiles correspond well to expression profiles in MLL-rearranged cells. This prompted the hypothesis that certain MLL-fusions transform cells in part by mis-targeting DOT1L, and promoting inappropriate histone methylation. We decided to test this hypothesis using an RNAi approach. Transduction of human leukemia cell lines carrying a t(4;11) translocation (MLL-AF4) with 2 different lentiviral shRNA constructs directed against DOT1L show a 60-80% reduction in global H3K79 methylation. This reduction was also observed on known MLL target loci such as the 5' HoxA cluster genes, which are central to MLL-mediated leukemogenesis. HoxA5 and HoxA9 expression levels from hypomethylated loci were greatly reduced in cell expressing the DOT1L shRNAs. Phenotypically, DOT1L knockdown adversely affected in vitro viability and proliferation of 2 ALL cell lines expressing the MLL-AF4 fusion gene, SEM-K2 and RS4;11. Nalm-6 and Jurkat ALL control cells were unaffected by DOT1L suppression. We developed a xenograft mouse model using SEM-K2 and Jurkat cells which stably express luciferase, thus allowing monitoring of leukemia development in live mice using bioluminescence imaging. In this in vivo model, DOT1L suppression led to a significant reduction in the time to onset of leukemia in t(4;11) SEM-K2 cells, but not Jurkat control cells. We are currently extending our studies to include a larger panel of human leukemia cells with different cytogenetic abnormalities, including AML cell lines. The results presented here, particularly if confirmed in a larger panel of cell lines and primary patient cells, should establish DOT1L as a highly promising therapeutic target for MLL-rearranged leukemias. Disclosures: Armstrong: Epizyme Pharmaceuticals: Consultancy.

Description: Abstract 2840 Poster Board II-816 Multiple myeloma (MM) remains incurable with a median survival of 3–4 years. Despite high dose therapy and autologous stem cell transplant (ASCT) most patients relapse with median progression-free survival (PFS) of 2.5–4 years and overall survival (OS) of 4–5 years. Although allogeneic SCT (allo-SCT) is potentially curative due to a graft-versus-myeloma effect, its applicability is significantly limited by high transplant related mortality (TRM). Therefore, the identification of additional independent biological predictors of outcome is required in order to tailor therapy to disease. Natural killer (NK) cells provide first line defence against tumors. NK cells have been shown to recognize and kill myeloma cells both in the allogeneic and autologous settings and donor NK genotype has been shown to influence leukemia free survival following allo-SCT. The aim of this study was to investigate the impact of KIR genotype on event-free (EFS) and OS following ASCT for MM. We performed KIR genotyping on 190 patients with MM receiving a first autologous transplant. KIR genotype and haplotype frequencies were comparable to those published for normal controls. Factors found on univariate analysis to be associated with a shorter EFS included haplotype Bx (containing at least 1 of the KIR B haplotype-defining loci- KIR2DL5, 2DS1, 2DS2, 2DS3, 2DS5, or 3DS1) (median 547 vs 656 days, P = 0.036), ≥3 activating KIR genes (median 547 vs 615 days, P = 0.046), the presence of activating KIR genes KIR2DS1 and KIR3DS1 (median 456 vs 589 days, and 464 vs 619 days, P=0.045 and 0.01 respectively). Disease status at ASCT was the most highly predictive factor for EFS. In patients with good risk disease (CR or PR at ASCT) KIR3DS1 status was highly predictive for EFS 464 days (341–586) vs 731 days (599–862) (P = 0.003) and OS 807days (713-901) vs 967 (925-1009) (P=0.023). KIR3DS1 was not predictive in patients with poor risk disease (P=0.36). Of note KIR3DS1+ve patients were equally represented in good risk (CR and PR) and poor risk (refractory or relapsed) groups at ASCT (around 30% in both groups). Notably the median EFS for KIR3DS1+ good risk patients was not significantly different to poor risk disease patients (P = 0.061). ASCT outcome was then determined according to 3 main groups based on disease status and KIR3DS1 status; A: Good Risk, KIR3DS1-ve; B: Good Risk, KIRDS1+ ve; and C Poor risk (KIR3DS1+ve or -ve). The RR of relapse or death was 1.0, 1.9 (P=0.002, 95% CI 1.3-3.1), and 3.0 (P=0.0001, 95% CI 1.9-4.8) respectively. By multivariate analysis, after adjusting for the presence of adverse cytogenetics and serum albumin and β2m, the KIR3DS1 status and grouping remained highly predictive of poor EFS, RR of 1.0, 2.7 (P= 0.021, 95%CI 1.2-6.2) and 5.3 (P= 〈 0.0001, 95%CI 2.4-11.7) respectively. The prognostic value of KIR3DS1 however, was greatest in patients in whom the ligand for the corresponding inhibitory KIR3DL1, Bw4 was missing. KIR3DS1+ KIR3DL1+ HLA-Bw4 negative patients had significantly reduced median EFS of 400d (315-495) vs 615 (545-684) for all other patients (P=0.048). Again this was most striking in good risk patients. Patients who had the genotype KIR3DS1+ KIR3DL1+ HLA-Bw4 –ve had a significantly shorter EFS survival of 372 days compared to 509 days in KIR3DS1+KIR3DL1+HLA-Bw4+ patients and 793 days for KIR3DS1 negative individuals (P=0.004). In conclusion: Our data from 190 patients with MM suggests that KIR3DS1, a gene previously linked to an increase risk of progression to invasive cervical carcinoma, independently predicts for poor EFS and OS following ASCT. A significant proportion (30%) of patients who are defined as good risk at ASCT (CR and PR) are KIR3DS1+ve and have an EFS which is not significantly different from patients who have refractory/relapsed disease at ASCT. This effect of KIR3DS1 is more significant in the absence of HLA-Bw4, the ligand for the inhibitory receptor KIR3DL1. The mechanism for this is effect is unclear and we are currently performing functional studies to further understand these findings. Disclosures: Apperley: Novartis: Consultancy, Honoraria. Marin:Novartis: Consultancy, Research Funding.

Description: Abstract 1177 Poster Board I-199 BACKGROUND: High dose melphalan followed by an autologous peripheral blood stem cell transplant (ASCT) is standard initial therapy for multiple myeloma (MM). However, toxicity and efficacy of this treatment are variable, with the sources of variability being poorly understood. We hypothesized that obesity and renal insufficiency modulate the pharmacokinetics of melphalan and therefore impact treatment toxicity. METHODS: We evaluated 39 patients with MM undergoing high-dose melphalan followed by ASCT. Patients received melphalan on day -2 at a dose of 200 mg/m2 (one patient with poor renal function received 180 mg/m2) and ASCT on day 0. We assessed body composition using dual energy x-ray absorptiometry (DEXA) and renal function using 24 hour urine creatinine clearance. We assessed toxicity on day +7 using the Oral Mucositis Assessment Scale (OMAS), a physician evaluated measurement of erythema and ulceration (on a scale of 0-5, with higher scores indicating worse mucositis). We also assessed patient reported symptom scores for mouth and throat soreness using the Mucositis Daily Questionnaire (MDQ) periodically during the first month (on a scale of 0-10, with higher scores indicating worse soreness). RESULTS: The median age was 55 years (range 37-70). 59% were male. The median weight was 83 kg (48-128), with median percentage body fat as measured by DEXA of 31% (range 15–53), and the median glomerular filtration rate (GFR) was 105 ml/min (range 28-194). Results from univariate and multivariate analyses are shown in the table below. In univariate analyses, we observed a weak and statistically insignificant direct correlation between percent body fat and toxicity (as measured by OMAS score or peak soreness), and a weak and statistically significant indirect correlation between GFR and OMAS score. In a multi-variable linear regression model including percent body fat, weight, GFR, and actual melphalan dose, we observed a correlation between OMAS score and percent body fat, with an increase in body fat percentage of 0.10 associated with a 0.65 unit increase in OMAS score (p=0.01). In a similar multi-variable model, we observed a correlation between MDQ soreness score and percent body fat, with an increase in body fat percentage of 0.10 associated with a 2.07 increase in MDQ soreness score (p=0.05). CONCLUSION: More obese patients, as measured by percent body fat, have more severe oral mucositis after high dose melphalan, independent of melphalan dose, weight, and renal function. We are measuring melphalan pharmacokinetics in this group of patients, and further research should help guide rational chemotherapy dosing for this highly effective treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Cyclic adenosine monophosphate response element binding (CREB)–binding protein (CBP) and p300 are multidomain transcriptional coactivators that help assemble large regulatory complexes at sites of active transcription. Nullizygosity of CBP or p300 results in pervasive defects in hematopoiesis. To systematically assess the structural domains of p300 required for normal hematopoiesis, we used recombinase-mediated cassette exchange to create an allelic series of coisogenic embryonic stem cells, each expressing a different mutant of p300 from the endogenous locus. We found that deletion of either the KIX or CH1 domain caused profound and pervasive defects in hematopoiesis, whereas the loss of most other domains had only lineage-restricted effects. When expressed from the p300 locus, an extra copy of CBP largely compensated for a lack of p300. Surprisingly, mutation of the p300 histone acetyltransferase (HAT) domain had minimal effects on hematopoiesis, and actually increased progenitor and stem cell numbers and proliferative potential. Our results suggest that, in distinct contrast to other organ systems, HAT activity does not provide a critical function for hematopoietic development and emphasizes the importance of enzyme-independent functions of p300.