bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Allosteric Inhibitors of the Interaction of AML1 and CBFβ for the Treatment of Leukemia.
Authors: Douvas Michael G. , Roudaiya L. , Grembecka J. , Speck N. , Bushweller J.H.
Published: October 2019

The protein-protein interaction between the subunits of the nuclear transcription factor core binding factor, RUNX1 (CBFα) and CBFβ, plays a critical role in hematopoiesis. Chromosomal rearrangements that target CBF genes are among the most common mutations in leukemia, including t(8;21) found in approximately 12% of cases of AML and resulting in the protein AML1-ETO. We describe the development of small molecule inhibitors of the interaction between RUNX1 and CBFβ. We used virtual screening to identify lead compounds. These leads were used to generate a library of compounds to explore the structure-activity relationships and optimize activity, resulting in the identification of low micromolar IC50 inhibitors of this protein-protein interaction. We confirmed by FACS FRET that inhibition also occurs in mammalian cells. HEK-293 cells were transfected with Cerulean-Runt domain and Venus-CBFβ. FRET emission from cells was monitored on a flow cytometer to assess binding of CBFβ and the Runt domain. Results with drug were compared to FRET between Runt and wild-type CBFβ and FRET between Runt and a mutant CBFβ with 2 point mutations abrogating binding to Runt. The small molecule inhibitor KG-3-275 inhibited CBFβ - Runt domain binding in a dose dependent manner (Table 1). Treatment of the t(8;21) leukemia cell lines Kasumi-1 and SKNO-1 results in inhibition of proliferation. SKNO-1 cells are inhibited in a dose-dependent manner by KG-3-275 but not by the weak inhibitor KG-1-253 (Fig 1). KG-3-275 does not inhibit the growth of renal tubular and hepatocellular cell lines (Fig 1). Kasumi-1 and SKNO-1 undergo apoptosis in a dose-dependent manner upon treatment with KG-3-275 (Table 2). Finally, KG-3-275 shows synergy with ATRA in increasing differentiation of Kasumi-1 cells as measured by CD11b expression, consistent with recent published results establishing a link between AML1-ETO and repression of RAR signaling (Fig 2). These data indicate drugs inhibiting CBF interactions hold promise as targeted agents in the treatment of CBF leukemias.

Fluorescence Resonance Energy Transfer Geometric Mean in HEK-293 Cells Cerulean-Runt domain + Venus-CBFb 4.07 +/− 0.1 Cerulean-RD + Venus-CBFb61/104 1.85 +/− .09 C-RD + V-CBFb + KG-3-275 200 mcm 3.05 +/− .15 C-RD + V-CBFb + KG-3-275 100 mcm 3.25 +/− 0.1 C-RD + V-CBFb + KG-3-275 50 mcm 3.45 +/− .25 C-RD + V-CBFb + KG-3-275 25 mcm 3.6 +/− 0.2 % Cells Annexin V/PI Negative at 72 Hrs Kasumi-1 0.25% DMSO 91.2 +/− 0.9 KG-3-275 25 mcm 91.2 +/− 1.2 KG-3-275 50 mcm 85.9 +/− 2.4 KG-3-275 100 mcm 66.7 +/− 6.7 SKNO-1 0.25% DMSO 77.6 +/− 3.4 KG-3-275 25 mcm 77.6 +/− 2.3 KG-3-275 50 mcm 63.7 +/− 3.6 KG-3-275 100 mcm 13.7 +/− 6.8 Cellular Proliferation as measured by MTTAssay at 72 Hrs Cellular Proliferation as measured by MTTAssay at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs
Expression Pattern and Prognostic Impact of Vascular Endothelial Growth Factors VEGF and VEGF-C and Their Receptors Flt-1, KDR and Flt-4 in Peripheral T-Cell Lymphomas.
Authors: Jørgensen Judit M. , Sørensen Flemming B. , Bendix Knud , Nielsen Johan L. , Funder Anette D. , Tainola Tapio , Karkkainen Marika , Sørensen Annette B. , Pedersen Finn S. , Kühle Astrid V. , d’Amore Francesco
Published: October 2019

The vascular endothelial growth factor family (VEGF, VEGF-B, -C and -D) is the main regulator of angiogenesis and lymphangiogenesis via their receptors, Flt-1, KDR and Flt-4. The aim of the present study was to estimate the microvascular density and investigate the expression of VEGF, VEGF-C and its receptors Flt-1, KDR and Flt-4 in peripheral T-cell lymphomas (PTCL).

Material and methods: Microvessel density (MVD) was determined after CD34 immunohistochemical (IH) staining of endothelial cells in pre-therapeutic lymph-node biopsies from 107 cases of PTCL (64 unspecified, 1 angiocentric, 10 angioimmunoblastic and 32 anaplastic large cell T/0). 44 of these cases (28 unspecified, 1 angiocentric, 4 angioimmunoblatic and 11 anaplastic large cell T/0) were investigated for expression of angiogenic molecules, VEGF, VEGF-C and their receptors by IH at protein level. Furthermore, VEGF and VEGF-C mRNA expression was detected by non-isotopic ′in situ′ hybridization. The angiogenic scores were correlated to pre-therapeutic clinical parameters, treatment response and survival.

Results: Tumoral expression of VEGF, VEGF-C and of their receptors was detected in the majority of the PTCL biopsies investigated (VEGF mRNA: 73%, VEGF protein: 95%; VEGF-C mRNA: 79%; VEGF-C protein: 79%; Flt-1 and KDR: 100%; Flt-4: 26%). All biopsies contained VEGF, Flt-1 and KDR positive vessels and VEGF-C was also widely expressed by the endothelial cells (3 negative cases), while endothelial Flt-4 expression was detected only in 43% of cases. High MVD scores correlated with an advanced clinical stadium (Ann Arbor) (p=0.047). A strong diffuse VEGF mRNA expression significantly correlated with a poorer overall survival (p=0.0015) as compared to focal or negative ISH signal. The predictive value of this parameter persisted at multivariate level independently of the International Prognostic Index (HR: 3.95, p=0.012, Cox regression).

Conclusion: VEGF, VEGF-C and their receptors are expressed by both lymphoma and endothelial cells in the majority of PTCL cases. VEGF expression seems to have an adverse impact on survival. Larger studies of angiogenesis/lymphangiogenesis in PTCL are needed to clarify the biological role of these molecules and identify possible therapeutic targets.
Hfe Acts in Hepatocytes To Prevent Hemochromatosis.
Authors: Muckenthaler Martina U. , Vujic-Spasic Maja , Kiss Judit , Herrmann Thomas , Galy Bruno , Gröne Herrmann-Josef , Stremmel Wolfgang , Hentze Matthias W.
Published: October 2019

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder hallmarked by intestinal iron hyperabsorption, hyperferremia and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the ubiquitously expressed HFE/Hfe gene. To understand the molecular mechanism(s) underlying HH and to identify the cell type in which Hfe acts to prevent HH we generated mice with tissue-specific Hfe ablations. We demonstrate that hepatocyte-specific Hfe deficiency fully recapitulates the phenotype observed in Hfe−/− mice with severe liver iron accumulation, reduced splenic iron content, increased Tf saturation, hyperferremia and reduced hepcidin expression. These findings unambiguously and directly show that Hfe acts in hepatocytes to prevent hemochromatosis.
MicroRNA-155 Promotes Myeloid Proliferation and Is Overexpressed in Acute Myeloid Leukemia.
Authors: Rao Dinesh S. , O’Connell Ryan M. , Chaudhuri Aadel A. , Boldin Mark , Taganov Konstantin , Nicoll John , Paquette Ronald , Baltimore David
Published: October 2019

Recent discoveries have implicated microRNAs, which are small 22–24 nucleotide long RNA molecules, as important regulators of many cellular processes, including differentiation and development. The microRNA-155 (miR-155) is known to be overexpressed in subsets of B-cell neoplasms and is thought to be important in the activation and function of B-lymphocytes. Here, we show that miR-155 is signficantly overexpressed in human acute myeloid leukemia and that its overexpression is most consistently seen in acute myelomonocytic leukemia. These findings led us to investigate the effects of overexpression of miR-155 in hematopoietic cells. By utilizing retroviral infection and transfer of murine bone marrow, we introduced miR-155 overexpressing hematopoietic stem cells into lethally irradiated recipient mice. Following reconstitution of hematopoietic organs, the mice demonstrated a profound myeloproliferative condition in the bone marrow characterized by replacement of the marrow by proliferating and dysplastic myelomonocytic cells. In addition, extramedullary hematopoiesis was observed in the spleen and examination of the peripheral blood revealed anemia and thrombocytopenia. To begin to explore the mechanisms whereby miR-155 overexpression results in this myeloproliferative phenotype, we utilized computational methods to identify targets predicted to be regulated by miR-155. This revealed several genes that have previously been implicated in myeloid development and neoplasia, which were confirmed to be targets of miR-155 by reverse-transcription/quantitative polymerase chain reaction (RT/QPCR) and by downregulation of luciferase protein upon fusion of the luc gene with the respective 3′ untranslated regions. These studies show a hitherto uncharacterized role of miR-155 in myeloid development and proliferation. Importantly, these findings lay the groundwork for understanding the complex regulatory networks underlying myeloid development in the context of microRNAs, and may point to new therapeutic opportunities in the treatment of myeloid malignancies.
Nilotinib Is Highly Active and Safe in Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Patients with Imatinib-Resistance or Intolerance.
Authors: Kantarjian Hagop M. , Hochhaus Andreas , Cortes Jorge , Martinelli Giovanni , Bhalla Kapil N. , Giles Francis J. , Ossenkoppele Gert , Gattermann Norbert , Haque Ariful , Gallagher Neil , Baccarani Michele , le Coutre Philipp
Published: October 2019

Background: Nilotinib is a novel, selective BCR-ABL inhibitor, designed to bind the ATP-binding site of BCR-ABL protein with higher affinity than imatinib. It is more potent than imatinib (IC50 <30 nM) against wild-type BCR-ABL and active against 32/33 imatinib-resistant BCR-ABL mutants.

Methods: This open-label study was designed to evaluate the safety and efficacy of nilotinib in patients (pts) with Philadelphia (Ph+) imatinib-resistant or -intolerant CML-CP. Planned nilotinib dose was 400 mg twice daily (BID) with escalation to 600 mg BID for inadequate responses. The primary endpoint was the rate of major cytogenetic response (MCyR) determined on the conventional intent-to-treat patient population. Cytogenetic response (CyR) was assessed by bone marrow karyotyping; peripheral blood FISH was used if a marrow sample cannot be obtained.

Results: This analysis includes data from 320 pts who received at least 6 months of nilotinib therapy (70.6% imatinib-resistant; 29.4% imatinib-intolerant). The median age was 58 years, the median duration of CML was 57.3 months, and 50.3% were males. Treatment with nilotinib is ongoing in 188 pts (58.8%) and 132 pts (41.3%) discontinued the treatment [51 (15.9%) for disease progression, 51 (15.9%) for adverse events (AE)]. The median duration of nilotinib exposure was 341 (1–624) days and the median average dose intensity was 792.1 mg/d (47.9–1111.6 mg/d). The dose was escalated to 600 mg BID in only 51 pts (15.9%). Complete hematological remission (CHR) at baseline was reported in 114 pts. Of the remaining 206 pts, 157 (76.2%) achieved CHR in 1 month. MCyR was observed in 180 pts (56.3%): 128/320 pts (40.0%) had complete cytogenetic response (CCyR). The median time to CHR and to first MCyR was 1.0 and 2.8 months, respectively. The median duration of MCyR has not been reached. Minor and Minimal CyR was seen in 22 (6.9%) and 42 (13.1%) patients respectively. The most frequent Grade 3/4 hematologic laboratory abnormalities included thrombocytopenia (27%), neutropenia (30%), anemia (9%), and asymptomatic serum lipase elevation (15%).

Conclusion: The present study confirms nilotinib is an effective therapeutic option in CML-CP pts with imatinib-resistance or -intolerance, with an acceptable safety and tolerability profile. With longer follow up, cytogenetic response continues to increase and no change in safety profile has been observed on nilotinib therapy.
MMY-3002: A Phase 3 Study Comparing Bortezomib-Melphalan-Prednisone (VMP) with Melphalan-Prednisone (MP) in Newly Diagnosed Multiple Myeloma.
Authors: Miguel J.F. San , Schlag R. , Khuageva N. , Shpilberg O. , Dimopoulos M. , Kropff M. , Spicka I. , Petrucci M. , Samoilova O. , Dmoszynska A. , Abdulkadyrov K. , Schots R. , Jiang B. , Palumbo A. , Mateos M. , Liu K. , Cakana A. , Van de Velde H. , Richardson P.
Published: October 2019

Background: In a phase 1/2 trial of VMP in 60 newly diagnosed MM patients (median age 75 years), the CR/nCR rate was 43%, with 32% CR; the 3-year survival was 85% (Mateos et al, Blood 2006; Mateos et al, EHA/IMW 2007). MMY-3002 is a phase 3 study comparing VMP with standard MP in patients with previously untreated MM who are not candidates for high-dose chemotherapy/stem-cell transplant.

Methods: Approximately 680 patients were randomized to VMP or MP and stratified according to baseline β2-microglobulin and albumin, and geographic region. Eligibility criteria required the presence of measurable disease, KPS ≥60% and hematology/chemistry laboratory values meeting predefined criteria. Patients in the VMP arm received intravenous bortezomib 1.3mg/m2 twice weekly (weeks 1, 2, 4, 5) for four 6-week cycles (8 doses per cycle), followed by once weekly (weeks 1, 2, 4, 5) for five 6-week cycles (4 doses per cycle) in combination with oral melphalan 9mg/m2 and prednisone 60mg/m2 once daily on days 1–4 of each cycle. Patients in the MP arm received 9 6-week cycles of MP once daily on days 1–4. For both groups, treatment continued for a maximum of 9 cycles (54 weeks) unless disease progression or unacceptable treatment-related toxicity occurred. The primary endpoint was time to progression (TTP), and secondary endpoints included progression-free survival (PFS), overall survival (OS), overall response rate, time to and duration of response, and safety.

Results: Between December 2004 and September 2006, 682 patients from 151 centers in 22 countries across Europe, North and South America, and Asia were randomized. The median age was 71 years with 30% of patients aged ≥75 years. Fifty percent were male and 88% were of Caucasian origin. Median KPS was 80%, and 34% of patients had KPS ≤70%. Sixty-three percent had IgG myeloma, 25% had IgA myeloma, and 8% had light-chain disease. Bone involvement with >10 lytic bone lesions was reported in 27% of patients, 33% had β2-microglobulin >5.5mg/L, and 60% had albumin <35g/L. The study population therefore included a high proportion of patients with advanced disease and high-risk factors. The Independent Data Monitoring Committee is scheduled to review data from a pre-planned interim analysis to determine whether TTP was significantly longer with VMP vs MP and therefore whether the protocol-specified statistical boundary for the primary endpoint has been reached.

Conclusion: Efficacy and safety analyses will be reported at the meeting.
Small-Molecule XIAP Antagonist Restores Caspase-9-Mediated Apoptosis in XIAP-Positive Diffuse Large B-Cell Lymphoma Cells.
Authors: Cillessen Saskia A.G.M. , Reed John C. , Pinilla Clemencia , Meijer Chris J.L.M. , Hooijberg Erik , Hess Corine J. , Castricum Kitty C.M. , Kortman Pim , Ossenkoppele Gert J. , Oudejans Joost J.
Published: October 2019

Clinical outcome in patients with diffuse large B-cell lymphomas (DLBCL) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including XIAP. XIAP suppresses apoptosis through inhibiting active caspases-3, -7 and -9. In this study we investigated if the small-molecule XIAP antagonist 1396–12 induces cell death in cultured lymphoma cells of DLBCL patients and whether it is possible to predict whether a DLBCL will be sensitive to the XIAP antagonist. Treatment with this XIAP antagonist resulted in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy refractory and responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal center B-cells from healthy donors. XIAP antagonist-sensitive cases were characterized by high expression levels of XIAP and relatively low expression levels of Bcl-2. In addition, we found that XIAP antagonist sensitive lymphomas are characterized by constitutive caspase-9 activation and that the apoptosis inducing effect of the XIAP antagonist depends on this constitutive caspase 9 activity. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in DLBCL cells by restoring caspase 9 mediated apoptosis and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers suggesting the possibility of pre-defining patients most likely to benefit from XIAP antagonist therapy.
Genetic Polymorphisms Identify the Likelihood of Bone Disease in Myeloma: Correlations with Myeloma Cell DKK1 Expression and High Risk Gene Signatures.
Authors: Durie Brian G.M. , Van Ness Brian , Ramos Christine , Stephens Owen , Haznadar Majda , Hoering Antje , Haessler Jeff , Katz Michael S. , Mundy Gregory R. , Kyle Robert A. , Morgan Gareth J. , Crowley John , Barlogie Bart , Shaughnessy John
Published: October 2019

Background: Bone disease in myeloma occurs as a result of complex interactions between myeloma cells and the bone marrow microenvironment. To date, no studies have evaluated the potential impact of genetic polymorphisms upon and/or within this microenvironment. Patients and

Methods: Peripheral blood DNA from 282 patients enrolled in the Total Therapy 2 (TT2) protocol was studied using the previously reported Affymetrix 3k BOAC custom chip to evaluate relevant genetic polymorphisms. DKK1 gene expression and high risk GEP gene signatures were assessed as previously reported (Blood109:4470–4477 and 109:2276–2284 2007). Patients were classified using both full skeletal x-rays and MRI findings. The lower cut-off used was absence of focal abnormalities on x-ray and/or <7 focal lesions on MRI (see JCO25:1121–1128 2007).

Results: The top 200 SNPs were first evaluated based upon univariate P values linked to limited or extensive bone disease. The top 50 SNPs with the smallest P values were then selected (eliminating closely linked genes) for recursive partitioning analysis. The recursive partitioning was conducted both with and without the insertion of known biologically relevant SNPs. The pruned tree developed with recursive partitioning proved to be quite stable and incorporated 4 dominant SNPs: rs 3766934 Epoxide hydrolase (EPHX1); sr3783408 MAP kinase; sr 1062637 RNA helicase DDX18; and sr3181366 TNFSF8-TNF-α. No alternate SNP substituted for EPHX1 in the recursive model. The 4 SNP signature of EPHX1 GG, MAP4K5 AG/AA, DDX18 GG/CC plus TNFSF8 CT/TT was highly correlated with the number of MRI lesions: mean 8.66 lesions versus 3.33 for alternate SNPs (P<.001). In the univariate association of standard prognostic factors, 4 SNP signature and 70 and 17 gene models for high risk, the best correlation with bone disease was with the 4 SNPs (P<.0001) followed by the 17 gene GEP model (P=0.05). Using stepwise multivariate regression analysis the best correlation with bone disease was Log2DKK-1 expression (P=.0001) but closely followed by SNPs EPHX1 (P=.0009); MAP4K5 (P=.0071) plus TNFSF8 and serum LDH (P=.02). The various analyses training (2/3) and validation sets were used with assessment of sensitivity and specificity. The microenvironmental 4 SNP signature combined with GEP DKK1 provided the best prediction of myeloma bone disease and overall outcome.

Conclusions: This first assessment of genetic polymorphisms linked to myeloma bone disease in myeloma has led to the identification of polymorphisms with both potential biologic relevance and utility in prognostic models of myeloma bone disease including risk stratification for bisphosphonate use. Validation of these findings may allow a search for potential therapeutic targets.
Treatment with hERG1 K+ Channel Inhibitors Reduces Acute Myeloid Leukemia Cell Lines Engraftment into Nonobese Diabetic/Severe Combined Immunodeficient Mice and Prolongs Survival of Injected Mice.
Authors: Pillozzi Serena , De Lorenzo Emanuele , Masselli Marika , Crociani Olivia , Cilia Emanuele , Torre Eugenio , Mugnai Antonio , Becchetti Andrea , Arcangeli Annarosa
Published: October 2019

hERG1 K+ channels are expressed in a broad range of human leukemic cell lines and primary acute myeloid and lymphoid leukemias (Pillozzi S, et al Leukemia16:1791–1798, 2002; Smith GA, et al, JBC227:18528–18534, 2002). hERG1 activity is necessary for leukemia cell proliferation as well as migration in response to angiogenic factors (VEGF). This effect can be traced back to the assembly of a macromolecular signaling complex on the plasma membrane of leukemia cells. Such complex comprises hERG1 channels, the beta1 subunit of integrin adhesion receptors and the VEGF receptor 1, FLT-1. In vivo experiments in NOD/SCID mice injected with AML cells showed that herg1 over-expression confers a greater malignancy, witnessed by a higher bone marrow (BM) angiogenesis, and a stronger leukemia blast exit into the peripheral blood (PB) and into extramedullary organs. What is more, herg1 expression in AML patients correlates with a higher probability of relapse and shorter survival periods (Pillozzi S et al, Blood110:1238–1250, 2007). On the whole we can conclude that hERG1 channel expression in AML represent a progression factor and can be envisaged as a novel target for therapy. We tested this latter hypothesis in NOD/SCID mice injected with AML cell lines expressing hERG1 channels at various extent, and treating injected mice with a selective hERG1 inhibitor, E4031. E4031 was administered i.p. starting one week after inoculum, at the dose of 20 mg/kg, daily for two weeks. In a first set of experiments mice were sacrificed after a total 3 weeks after inoculum. It emerged that E4031 reduced both BM engraftment and PB invasion of AML cells. In particular, BM angiogenesis was significantly lower in mice treated with E4031. In a second set of experiments, mice were treated as above and the survival time was measured. It emerged that E4031 treatment prolonged survival compared with the control-treated group. We have developed a monoclonal antibody against an extracellular loop of hERG1 channels. The immunoreactivity and activity on hERG1 channels of such antibody was first tested in leukaemia cells. Treatment of NOD/SCID mice injected with AML cell lines with this antibody is ongoing.
Phase I Study of Sapacitabine, an Oral Nucleoside Analogue, in Patients with Advanced Leukemias or Myelodysplastic Syndromes (MDS).
Authors: Kantarjian Hagop , Garcia-Manero Guillermo , Faderl Stefan , Cortes Jorge , Estrov Zeev , Boone Patricia , Stuart Iain , Westwood Robert , Green Simon , Chiao Judy H. , Plunkett Willilam
Published: October 2019

Background: Sapacitabine, a 2′-deoxycytidine nucleoside analogue with unique ability to cause irreparable single-strand DNA breaks and as a result induce G2 cell cycle arrest, is undergoing clinical evaluation for the treatment of cancer. The recommended Phase II dose (RD) in patients with advanced solid tumors was 75 mg twice daily (b.i.d.) x 7 days orally every 21 days. The major DLT was myelosuppression. Here, we present the initial results of a Phase I study of sapacitabine in patients with advanced leukemias or MDS. The primary objective was to define the MTDs of two dosing schedules, b.i.d. x 7 days orally every 21 days or b.i.d. x 3 days per week for 2 weeks every 21 days. The secondary objectives were to characterize the PK/PD effects of sapacitabine and its major metabolite CNDAC.

Methods: Eligible patients had relapsed/refractory leukemias or MDS, or untreated disease if not willing to proceed with conventional systemic chemotherapy, adequate organ functions and performance status of 0–2. At least 3 patients were enrolled at each dose level. The MTD was the highest dose level at which ≤2/6 patients experienced a DLT during the first treatment cycle.

Results: Forty-seven patients received sapacitabine, including 35 treated with the 7-day schedule and 12 treated with the 3-day per week schedule. Median age was 65 (range: 36 – 91). The majority of patients had AML (n=36) or MDS (n=4). Median number of prior chemotherapies was 2 (range: 0 – 6). Cytogenetic abnormalities were present in 27; 30 had relapsed disease or were refractory to cytarabine or high-dose cytarabine regimens. MTD was reached at 375 mg b.i.d. on the 7-day schedule, and 475 mg b.i.d. on the 3-day per week x 2 schedule. DLTs consist of abdominal pain/small bowel obstruction (n=1), neutropenic colitis (n=2) and diarrhea (n=3). One patient died from complications of neutropenic colitis. Common non-hematologic adverse events (all grades, regardless of causality) included fatigue, nausea, vomiting, diarrhea, anorexia, cough, dyspnea, and abdominal pain, most of which were mild to moderate in intensity. PK and PD data are being analyzed. To date, 11 patients of 42 evaluable (9 AML, 2 MDS) had a reduction in bone marrow blast counts to ≤ 5% including 2 CRs, 2CRs with incomplete recovery of platelets, 1 CR of extramedullary disease, and 1 PR of extramedullary disease. 5/11 had relapse-resistance disease on prior cytarabine.

Conclusion: The MTD of sapacitabine is 375 mg b.i.d. by the 7-day schedule and 475 mg b.i.d. by the 3-day per week schedule. The predominant DLT is gastrointestinal toxicity. Sapacitabine is well tolerated and has promising antileukemic activity in patients with relapsed or refractory AML and MDS.
SGN-33, Lintuzumab, Demonstrates Anti-Leukemic Activity in Preclinical Models of AML.
Authors: Sutherland May S.K. , Yu Changpu , Morris-Tilden Carol , Lewis Timothy S. , Miyamoto Jamie B. , McEarchern Julie A. , Oflazoglu Ezogelin , Gerber Hanspeter , Sievers Eric L. , Grewal Iqbal S. , Law Che-Leung
Published: October 2019

SGN-33 is a humanized IgG1 antibody with signaling properties that also mediates effector functions including Antibody-Dependent Cellular Cytotoxicity (ADCC). SGN-33 targets CD33, a sialoadhesion family member expressed on acute myeloid leukemia (AML) cells, eliciting complete remissions in some AML patients. In the laboratory, SGN-33 mediates natural killer (NK) cell lysis of both multi-drug resistant (MDR- positive) and MDR-negative AML cell lines. Additionally, SGN-33 significantly increases survival in SCID mouse disseminated models of MDR-negative and MDR- positive AML. Immunomodulatory compounds such as thalidomide and its analog, lenalidomide (Revlimid®), have been shown to enhance NK cell function and anti-cancer activity. In particular, Revlimid in combination with rituximab demonstrated greater tumor cell killing compared to the antibody alone. Given these reports, we investigated whether thalidomide or lenalidomide would enhance the anti-tumor effects of SGN-33. In vitro, lenalidomide augmented NK function in a dose-dependent fashion, manifested as significant increases in perforin expression. Thalidomide and lenalidomide enhanced ADCC activity (% maximal specific lysis) while having no significant effects on tumor cell levels of CD33. In vivo, treatment of SCID mice with lenalidomide resulted in a 2-fold increase in the absolute numbers of lymphocytes (including NK cells). In vivo studies evaluating the efficacy of the combination of SGN-33 and lenalidomide in SCID mouse models of AML are in progress. In summary, our studies demonstrate the anti-leukemic activity of SGN-33 against MDR-positive AML cell lines, suggesting that this unconjugated anti-CD33 antibody may yield benefit in clinical settings where other therapies fail due to the MDR phenotype of the tumor cells. Additionally, agents augmenting immunologic effector function might provide clinical benefit in combination with SGN-33. The combination of SGN-33 and lenalidomide is now being evaluated in a clinical trial.
Factor Xa Inhibitors Can Be Differentiated in Xa, IIa and Microparticle Generation and Other Whole Blood Based Assays.
Authors: Iqbal Omer , Adiguzel Cafer , Hoppensteadt Debra , Cunannan Josephine , Fareed Jawed
Published: October 2019

Currently used oral anticoagulants such as Vitamin K antagonists have drawbacks, which reportedly limit their safety and efficacy. Oral Factor Xa and IIa inhibitors are claimed to overcome these limitations. Factor Xa inhibitors provide more complete suppression of thrombin generation than Factor IIa inhibitors. Four Factor Xa anticoagulant agents, both direct and indirect, namely A,B,C and D with Ki values 6–200 nM, were studied in various assays at equigravimetric final concentration of 10 ug/ml to determine their relative potencies. Apparent differences in their biochemical profiles were noted in thrombin generation, Factor Xa generation and microparticle generation inhibition assays. Furthermore, the anticoagulant potential of these Xa inhibitors was studied in celite activated clotting time (ACT) and modified celite ACT system using different concentrations of tissue factor. Microparticle generation was performed using agent- supplemented whole blood that was incubated for three minutes with varying concentrations of tissue factor. Following three minutes the reaction was stopped using EDTA stop solution. The functional assay for the determination of microparticle procoagulant activity in the plasma was performed using Zymuphen MP-Activity assay kit from Hyphen BioMed (Neuville-sur-Oise, France). While three of the Xa inhibitors studied showed microparticle generation inhibition levels of 31.37 nM, one agent provided a level of 17.87 nM compared to a saline control value of 39.28nM. Supplementation studies were also carried out in whole blood PT, APTT and Heptest assays in the concentration range of 0 to 10 ug/ml. The whole blood prothrombin time assay showed a maximum of 208.8, 61.3, 77.8 and 15.9 seconds at a final concentration of 10 ug/ml for the respective anticoagulant agents when compared to a normal value of 12.3 seconds. Similarly the whole blood APTT assay showed a maximum of 267.9, 161.8, >300 and 71.9 seconds respectively when compared to a control value of 46.8 seconds. The whole blood Heptest assay showed a varying maximum anticoagulant effect from >300, 42.7, >300 and >300 seconds respectively when compared to a control value of 13.8 seconds. In each of these assays Factor Xa inhibitors showed concentration-dependent effects and had varying potencies. In TF-mediated platelet activation assays the different Xa agents produced varying effects which were not proportionate to their Ki values. Differentiation of Factor Xa inhibitors have a clinical impact in dosage selection, dosage adjustment and their monitoring when given in large dosages. Synthetic factor Xa agents exhibit Ki values of 20–200 nM, while pentasaccharide -AT complex has a Ki value of 60 nM. However, these results do not translate into proportionate anticoagulant effects to their Ki values. Large-scale clinical studies are necessary to validate these preliminary results.
Fibrinolytic Agents for Restoration of Central Venous Catheter Patency: A Systematic Review.
Authors: Wang Myra , Kagoma Yoan , Lim Wendy , Clase Catherine M. , Crowther Mark A.
Published: October 2019

Introduction. Thrombolytic agents are frequently used as an aid to restore catheter patency despite limited evidence supporting their use. We therefore conducted a systematic review to evaluate the efficacy of tPA (alteplase) and reteplase in restoring catheter patency.

Methods. We searched MEDLINE (1999 to May Week 3 2007) and the Cochrane Controlled Clinical Trials Register (Second Quarter 2007) and performed a manual search for further studies. Studies published before 1999 were excluded since they have been previously reviewed; furthermore, most such studies used urokinase, which is no longer used to restore catheter patency in North America. Studies were included if they described the efficacy of alteplase or reteplase in restoring patency of central venous catheters (either for hemodialysis or infusional treatment) and where more than 5 thrombotic episodes were reported. This latter exclusion criteria was used to reduce the likelihood of bias likely to be found in very small case series. Two independent researchers abstracted total thrombolytic dose administered, dwell times, catheter patency rates and adverse event (including bleeding rates). For purposes of comparison, dwell times were stratified as ≤ 30 minutes and 30 to 120 minutes.

Results. 25 articles were identified in the initial search, of which 17 met one or more exclusion criteria. Of the remaining 8 studies, 7 evaluated central venous catheters used for intravenous infusions and 1 study evaluated occluded hemodialysis catheters. Except for 1 randomized trial comparing thrombolytic agents to placebo in non-hemodialysis patients, all studies described uncontrolled cohorts or case series. The mean study size was 214 patients (range 6–995). At doses of 0.5–2.5 mg of alteplase, the range of catheter patency rates at dwell times of ≤ 30 minutes and 30 to 120 minutes were 52–54% (1301 total uses) and 69–91% (1466 total uses) respectively. In comparison when used with a ≤ 30 minute dwell time, reteplase was reported to restore catheter patency in 67–73% cases (154 total uses). In all reports bleeding complications attributed to thrombolytic therapy were uncommon.

Conclusion. Our review suggests that the installation of alteplase or reteplase will restore catheter patency in more than 50% of cases when used with a dwell time of at least 30 minutes. Longer dwell times appear to increase patency rates. Reteplase may be more effective than alteplase, although this observation should be validated in an adequately powered clinical trial. Overall, although modern thrombolytic agents appear to provide catheter patency in the majority of patients, the evidence supporting their use is of low quality and is not based on the results of randomized trials.
A Novel HIFalpha Mutation Associated with Familial Erythrocytosis Supports This Isoform Being the Major Regulator of Erythropoietin in Humans.
Authors: Percy Melanie J. , Furlow Paul W. , Lucas Guy S. , Li Xiping , Lappin Terry R. , McMullin Mary F. , Lee Frank S.
Published: October 2019

Red cell homeostasis is crucial to maintain a sustained tissue oxygen supply. Oxygen is sensed by the kidneys and hypoxia induces renal erythropoietin (Epo) to stimulate red cell production. At the molecular level this process is controlled by the hypoxia inducible factor (HIF) complex, comprised of a labile a-subunit and a constitutively expressed b subunit. Key prolines present in the oxygen-dependent degradation (ODD) domain of the a subunit are hydroxylated by prolyl hydroxylase domain proteins (PHD1-3). The von Hippel Lindau (VHL) protein then binds and targets HIFa to the proteasome. There are 3 isoforms of the a subunit, each of which shows differential tissue distribution. The HIF-1, HIF-2 and HIF-3a subunit, in association with the b subunit, exhibit diverse affinities for the hypoxia response elements (HREs) located in the promoters and enhancers of HIF target genes. It is unclear whether Epo synthesis is under the control of HIF-1 or HIF-2 in humans although recent studies in mice support HIF-2 as the critical regulator. Dysregulation of Epo production can give rise to the red cell disorder of erythrocytosis. Defects in the oxygen sensing pathway are commonly associated with erythrocytosis and mutations in both the VHL and PHD2 genes have been described. Previously, screening the ODD domain of HIF-1a detected a polymorphism, Pro582Ser, that did not impair the function of HIF-1a. Subsequently, when sequencing HIF-2a, a novel heterozygous c.G1609T mutation was uncovered in 3 generations of one family with erythrocytosis. All affected individuals exhibited greatly elevated serum Epo levels. The c.G1609T mutation was absent in other unaffected family members as well as a cohort of 200 normal control samples. The mutation predicts the replacement of Gly by Trp at amino acid 537, which is close to the main site of proline hydroxylation at 531. Functional assays confirmed the Gly537Trp variant was less hydroxylated and exhibited reduced affinity for PHD2. Furthermore, the increased stability in normoxia of the Gly537Trp variant would result in elevated transcription of HIF-2 target genes. Consequently, these findings support a critical role for HIF-2a in control of Epo production in humans.
Diabetes Impairs Bone Marrow Microenvironment Affecting Progenitor Cell Number, Mobilization and Engraftment in Humans.
Authors: Rizzoli Vittorio , Masselli Elena , Prezioso Lucia , Ferraro Francesca , Lodi-Rizzini Elisa , Mangoni Marcellina , Graiani Gallia , Frati Caterina , Spinetti Gaia , Reni Carlotta , Emanueli Costanza , Emanueli Costanza , Madeddu Paolo , Quaini Federico
Published: October 2019

Abstract 1482

Poster Board I-505

Alterations in cell trafficking and chemoattractive properties may represent basic mechanisms responsible for organ pathology in diabetes mellitus (DM). These functional events involving both inflammatory and progenitor cells may be translated in the proneness to major septic events and effaced reparative response characterizing the clinical course of the disease. To determine whether DM affects BM progenitor cell number and function, tissue resident and GSF mobilized disease-free BM CD34pos cells were evaluated in 19 DM and 38 non-diabetic control (CTR) biopsies and in 18 DM and 48 CTR patients undergoing autologous BM transplantation. DM was documented by the presence of glucose intolerance, HbA1C > 6 g% and need of high dose insulin treatment. CTR were selected on the basis of normal glucose tolerance and the absence of neoplastic infiltration and/or morphologic changes in cellular hematopoietic compartments compatible with hematologic disorders. Reduction in trabecular bone, fat accumulation, rearrangement of microvascular structures and marked DNA oxidative damage characterized BM structural and cellular remodelling in DM. Immunohistochemical and FACS analysis documented that DM produced a 50% reduction in the number of both BM resident and mobilized CD34pos Linneg cells. In addition only 16% of diabetic patients were able to mobilize >10 × 106 CD34pos cells/kg compared to 62% of CTR. After infusion of mobilized CD34pos progenitors, BM engraftment was partially prolonged in diabetic patients. Severe impairment of SC engraftment by DM was striking by the comparative analysis of the infusion of similar number of mononuclear cells (nearly 180 ×106/Kg) directly harvested from the bone marrow of DM and CTR patients. In this setting, PMN and Plts recovery was delayed by nearly 2 and 4 weeks, respectively. Thus, DM severely affects human progenitor cell function and BM microenvronment in vivo inevitably impairing the outcome of BM transplantation. Whether all these changes are due to specific perturbation of the GSF-dependent SDF-1/CXCR4 axis and whether drugs interfering with ROS mediated damage may reverse this phenomenon is under intense investigation.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide Shuts Down the Translational Machinery in Multiple Myeloma Cells Resulting in Down-Regulation of Critical Transcription Factors Such as C/EBPβ and IRF4.
Authors: Li Shirong , Pal Rekha , Galson Deborah L , Schafer Peter H , Ouyang Hongjiao , Mapara Markus Y , Lentzsch Suzanne
Published: October 2019

Abstract 1844

Poster Board I-870

Background: Lenalidomide is an immunomodulatory drug that has been shown to be highly active in the treatment of multiple myeloma (MM). In combination with dexamethasone, remission rates of up to 61% are achieved in newly diagnosed MM patients. Nevertheless, the mechanisms of how immunomodulatory compounds induce high remission rates in MM remains elusive. Methods and Results: Here we investigate the underlying anti-MM mechanism of lenalidomide. Lenalidomide significantly (p<0.01) inhibited DNA synthesis at concentrations as low as 0.01 μM in MM.1S or OPM2, as shown by thymidine uptake analysis. Inhibition of proliferation was accompanied by cell cycle arrest at G0/G1 in MM cell lines. Even at high concentrations up to 100 uM lenalidomide did not induce the expected level of apoptosis in MM cell lines. Previously we have demonstrated that C/EBPβ is an important transcription factor which controls growth and proliferation of MM cells and also regulates the transcription of interferon regulatory factor-4 (IRF4). Our present data show that lenalidomide significantly inhibited the protein expression of C/EBPβ in MM cell line and primary MM cells. Furthermore, we found that lenalidomide does not decrease the mRNA level of C/EBPβ, nor induce the protein degradation of C/EBPβ, suggesting that the protein translation of C/EBPβ was inhibited. Consistent with this notion, analysis of the effects of the compounds on the translational machinery in MM cells revealed that the protein eukaryote initiation factor 4E (eIF4E) was significantly down-regulated in both lenalidomide-treated MM cell lines and primary MM cells. The resulting decreased C/EBPβ protein expression impaired the transcription of downstream IRF4, BLIMP1 and XBP1, which are all critical for MM survival. Conclusions: Our studies, for the first time, provide evidence that lenalidomide inhibits MM cell proliferation and survival by likely inhibiting the protein translation of C/EBPβ and consequently repressing gene expression of multiple downstream transcription factors of C/EBPβ including IRF4, BLIMP1 and XBP1.

Disclosures:

Schafer: Celgene Corporation: Employment. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.
Cell Specific Consequences of Notch Signaling: PARP1/HES1 Interaction Reveals a Novel Tumor Suppressor Mechanism.
Authors: Kannan Sankaranarayanan , Zweidler-McKay Patrick A.
Published: October 2019

Abstract 2388

Poster Board II-365

The Notch signaling pathway is a critical regulator of cell fate determination and differentiation during development, which is highly cell type specific. Similarly, Notch signaling plays both oncogenic and tumor suppressor roles in a wide variety of malignancies, depending on cell type. In contrast to T cell acute lymphoblastic leukemia (T-ALL) where Notch activation promotes leukemogenesis, induction of Notch signaling in B-ALL leads to growth arrest and apoptosis. The Notch target gene Hairy/Enhancer of Split1 (HES1) is sufficient to reproduce this tumor suppressor phenotype in B-ALL, however the mechanism is not yet known.

Here we report the novel finding that HES1 forms distinct complexes in B-ALL versus T-ALL. This suggests that HES1 interacting proteins may contribute to the cell-type specific consequences of Notch/HES1 signaling. During characterization of these complexes, we identified the novel interaction between HES1 and PARP1 through immunoprecipitation and MALDI-TOF protein sequencing. This interaction was dependent on the HES1 bHLH and Orange domains and PARP1 and HES1 co-localize to a genomic HES1 binding site by ChIP. This interaction both inhibits HES1 repressor function and induces PARP1 activation in B-ALL. HES1-induced PARP1 cleavage leads to enhanced poly ADP ribosylation of PARP1, consumption of NAD+, diminished ATP levels, and translocation of the Apoptosis Inducing Factor (AIF) from mitochondria to the nucleus, resulting in apoptosis in B-ALL, but not T-ALL. Importantly the potential therapeutic Notch agonist peptide “DSL” also induces cell-specific growth arrest and apoptosis (A+B), followed by poly-ADP ribosylation of PARP1 (C), and nuclear translocation of AIF in B-ALL but not T-ALL cells (D).

These data reveal a novel interaction of HES1 and PARP1 in B-ALL which modulates the function of the HES1 transcriptional complex and signals through PARP1 to induce apoptosis. This novel tumor suppressor mechanism involving a Notch driven, cell-type specific pro-apoptotic pathway may lead to the development of Notch agonist-based cancer therapeutics.

Disclosures:

No relevant conflicts of interest to declare.
Glanzmann's Thrombasthenia Mutation of the Genes ITGA2B and ITGB3.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: October 2019

Abstract 2411

Poster Board II-389

Glanzmann Thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by a life-long mucocutaneous bleeding tenedency and absence or severely reduced platelet aggregation in response to ADP, epinephrine, and collagen, but relatively normal in response to ristocetin, where the glycoprotein (GP) IIb/IIIa a calcium dependent heterodimer complex is deficient or present but dysfunctional. GPIIb 172kbp is composed of 30 exons and GPIIIa 65Kbp is composed of 15 exons, have their own separate genes on the long arm of chomosome 17 (17q21-32), specific genetic abnormalities of each GP include missense, non sense, splite site mutation, deletions and point mutation. Mutation abrogates ligand binding to the activate integrin to the adhesive protein: fibrinogen, vWF, fibronectin, vitronectin, CD40L and the platelet are unable to mediate outside-inside signaling promoting actin plymerization and cytoskeletal reorganization such as clot retraction, talin and kindlin protein activation. We have studied four patients with mutation of the gene encoding platelet GPIIb (ITGAIIB) exon 17 mutation 1750 C (cystein)-T (threonin) phenotype non sense R584X,the proband showed a platelet with 3%-10% a fibrinogen binding and GPIIb/IIIa receptors; exon 23 mutation 2333 A (alanin)-C (cystein) phenotype missense Q778P, produced truncated protein, cystein residue is ipermethylated with a reduction of adhesion <8% cystein is postulated to be critical for post translational processing of GPIIb; and the gene encoding platelet GPIIIa (ITGB3) exon 11 mutation 1813 G (glycin)-A (alanin) phenotype missense H306P,ifluencing the Ca2+ dependent stabilty of the GPIIb/IIIa complex to divalent cation chelation; exon 3 mutation 355 C (cystein)- T (threonin) phenotype missense R119W escluding at codon 355 leader sequence, producing a frame-shift and protein termination and a stop codon with a great abnormalities of GPIIb/IIIa heterodimer inter subunit surface interaction, and intracellular trafficking. The patients have all severe bleeding included epistaxis, petechiae, gum bleeding, and a high grade of relapsing, refractory bleeding often requiring the transfusion with HLA compatible platelet concentrats and/or administration of recombinant FVIIa. This study confirmed that the genetic mutation can be significatly associated with the frequency and severity of bleeding and these approach may be the future of the management of GT.

Disclosures:

No relevant conflicts of interest to declare.
Radioprotection by Mesenchymal Stem Cells Overexpressing Extracellular Superoxide Dismutase.
Authors: Deng Weiwen , Abdel-Mageed Aly S , Senagore Anthony J , Pietryga Daniel W , Connors Robert H , Giambernardi Troy A , Hay Rick V
Published: October 2019

Abstract 4510

Oxidative stress caused by ionizing radiation is a major determinant of radiation injuries. Extracellular superoxide dismutase (ECSOD), a potent antioxidant enzyme, alleviates oxidative stress. Mesenchymal stem cells (MSCs), a subset of adult stem cells from bone marrow, have been found to migrate to radiation injured tissues after intravenous administration. Here, we show for the first time that intravenous administration of MSCs genetically modified to secrete ECSOD improves survival, extends lifespan, retards cataract formation, and prevents carcinogenesis in irradiated mice. These findings suggest that intravenous administration of MSCs overexpressing ECSOD could become a critical medical countermeasure against radiation.

Disclosures:

No relevant conflicts of interest to declare.