bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen Activator Inhibitor 1 Deficiency Reduces the Progression of Atherosclerosis in a Murine Model of Human Familial Hypercholesterolemia.
Authors: Iwaki Takayuki , Victoria Ploplis A. , Castellino Francis J.
Published: October 2019

Atherosclerosis has been described as a “self-perpetuating” inflammatory disease which progresses in discrete stages and involves a number of cell types and effector molecules. Lipid metabolic disorders are heavily involved in the genesis and progression of atherosclerosis in humans, and thrombotic complications substantially contribute to the coronary artery disease that frequently accompanies atherosclerosis. A relevant mouse model of human atherosclerosis with elevated LDL-cholesterol (C) is one in which both the Ldlr and Apobec1 genes are deleted (Ldlr−/−/Apobec1−/−), since these mice lack the ability to convert apoB-100 to apoB-48 in liver and also are defective in LDL clearance. We have generated Ldlr−/−/Apobec1−/−/PAI-1−/−and characterized atherosclerosis progression in these mice. VLDL, LDL, HDL, and total C levels were identical between Ldlr−/−/Apobec1−/−and Ldlr−/−/Apobec1−/−/PAI-1−/−mice (VLDL-C; 30.3±1.8 vs 26.0±1.7 (mg/dl), LDL-C; 268.7±6.3 vs 274.1±13.1 (mg/dl), HDL-C; Total-C; 353.8±8.1 vs 356.8±14.5 (mg/dl), respectively). However, both the plaque size in the aortic sinus and the extent of plaque area in aorta were significantly smaller in Ldlr−/−/Apobec1−/−/PAI-1−/−mice relative to Ldlr−/−/Apobec1−/−mice (plaque in aortic sinus; 158792.9±11958.2 vs 227243.5±15291.1 (μm2), plaque area in aorta; 3.90±0.34 vs 5.10±0.27 (%), respectively). To elucidate the cellular functions, especially macrophages, in the plaque, bone marrow-derived macrophages (BMMs) were isolated and utilized for Dil-oxidized (Ox) LDL uptake assays and migration assays using 8 μm pore culture inserts. There were no significant differences in Dil-OxLDL uptake by BMMs between the two genotypes. The kinetics of migration of BMMs was more rapid in Ldlr−/−/Apobec1−/−/PAI-1−/−cells than Ldlr−/−/Apobec1−/−cells. Furthermore, thioglycollate-induced intraperitoneal migration of macrophages was increased in Ldlr−/−/Apobec1−/−/PAI-1−/−mice vs Ldlr−/−/Apobec1−/−mice. Results from these studies indicate that cell migratory differences may contribute to attenuating atheroma formation in a PAI-1 deficient state.
Calcineurin/NFAT Pathway Is a Novel Target of Therapeutic Interest in Lymphoid Malignancies.
Authors: Medyouf Hind , Alcade Helene , Berthier Caroline , Janin Anne , de Thé Hugues , Ghysdael Jacques
Published: October 2019

Calcineurin (PP2B) is an ubiquitously expressed serine/threonine protein phosphatase dependent on calcium calmodulin. Calcineurin plays a pivotal role in many important biological processes, including the development and function of the immune system. Sustained signalling through calcium/calcineurin results in the activation of transcription factors of the NFAT family (Nuclear Factor of Activated T cells). Mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFAT activation and function in many developmental processes. In lymphoid cells, the calcineurin/NFAT signalling pathway is essential to specific aspects of T cell development and plays a central role in the activation of the immune response and in its homeostatic control.

Despite their central role in T cell development, immune function and homeostasis, deregulation of calcineurin/NFAT pathway have not been shown so far to have a pro-oncogenic role in lymphomagenesis. Using our TEL-JAK2 transgenic mouse model of T-ALL, we investigated the activation status and importance of the calcineurin/NFAT signalling pathway in leukemogenesis. We found that the calcineurin/NFAT pathway is activated in leukemic cells of TEL-JAK2 transgenic mice. Western blot and EMSA experiments showed that all NFAT members expressed in the lymphoid lineage (NFAT1, 2 and 4) are found in an active hypophosphorylated form, they are located in the nucleus, and exhibit an increase in their DNA binding activity. In order to investigate whether constitutive calcineurin/NFAT activity was specific to TEL-JAK2 leukemia or whether it is a more general property of leukemic cells, we analyzed NFAT activation in leukemic cells of transgenic mice aberrantly expressing oncoproteins involved in lymphoma in human. This showed that calcineurin/NFATpathway is not only aberantly active in leukemic cells of TEL-JAK2 transgenic animals, but also in other mouse models of human leukemia. We also found that the activation of the calcineurin/NFAT pathway is only observed when cells are rapidly analyzed following their explantation and dissociation from diseased animals. Indeed, if cells are incubated in tissue culture for a short period of time (60–120min.), essentially stochiometric rephosphorylation, meaning inactivation, of NFAT is observed. This indicates that activation of the calcineurin/NFAT pathway depends upon signals generated in the in vivo tumor cell environment and is not -or not solely- under the control of the initiating oncogene. Using these mouse models we conducted pre-clinical studies that indicated that calcineurin/NFAT pathway is an attractive novel target of therapeutic interest in lymphoid malignancies.
Cerebrospinal Fluid (CSF) Proteomics in Children with Acute Lymphoblastic Leukemia (ALL).
Authors: Trueworthy Robert C. , Stork Linda , Zhong Yanping , Pine Sharon , Matloub Yousif , Laderas Ted , McWeeney Shannon , Fan Guang
Published: October 2019

Relapse within the central nervous system (CNS) remains a major challenge for the treatment and cure of childhood ALL. With current intensive treatment, at least one-third of relapses occur within the CNS. Evaluation of CSF is essential to the management of childhood ALL and relies on morphologic examination of cytospin smears. CSF has been re-discovered in the post genomic era as a source of potential protein biomarkers for various diseases. Such proteins may help establish a diagnosis, provide insight into pathogenesis, or identify therapeutic toxicity. In the present study, acellular CSF supernatants from newly diagnosed children with standard risk ALL treated on CCG-1991 were used for protein profiling.

The morphologically negative CSF samples had previously been classified as minimal residual disease (MRD) negative (−) or positive (+) using real-time PCR techniques on centrifuged cell lysates. Three MRD −, 3 MRD +, and 2 morphologically + CSF samples were concentrated and desalted using Microcon filter device. Albumin was removed using a Human Albumin Kit, Albuminomics™. The MRD − and MRD + samples, each containing 15 mcg of protein, were pooled separately and processed for protein identification and quantitation using a multidimensional LC/MS/MS method. Custom software was developed (TandTRAQ) for database searching and peptide quantitation. Protein annotation was primarily derived from ENSEMBL and NCBI sources. Rank order summarization was used to identify putative candidates. In the comparison between MRD + and MRD− samples, we identified 226 unique genome identifiers, with 89 having functional annotation. Of these 89, 49 have high quality protein-level annotation, 35 have mRNA-level annotation, and 5 are predicted (have functional protein evidence). Using a 1.5 fold cut-off, there were 18 up-regulated and 12 down-regulated proteins among the 89 proteins in MRD + compared to MRD − supernatants. Focusing on the 49 with high quality protein-level annotation, 8 proteins show higher-level expression and 2 show lower level expression in MRD + CSF samples (Table). Eight highly expressed proteins are involved in either DNA binding interactions, cell migration, apoptosis, or immune response. Two under-expressed proteins are involved in cell adhesion. The 2 morphologically + CSF samples confirmed 4 over expressed and 1 under-expressed proteins.

This research is the first attempt at protein profiling of CSF from patients with ALL. The proteins identified in this study may further our insight into the pathogenesis of CNS leukemia. Furthermore, future research may find that protein profiling of supernatants is a sensitive way to detect leukemia in CSF and to monitor treatment-related CNS toxicities.
Immunogenicity of Childhood T-ALL.
Authors: Panzer-Grümayer Renate , Inthal Andrea , Krapf Gerd , Felzmann Thomas , Gadner Helmut , Dohnal Alexander
Published: October 2019

Background: Acute lymphoblastic leukemia (ALL) with a T cell immunophenotype accounts for about 15% of childhood ALL. While contemporary treatment protocols have improved the relapse free survival for children with T-ALL, it has for a long time been considered a malignancy with a dismal prognosis. In fact, this type of leukemia is still at a higher risk for treatment failure and early relapses than B cell precursor ALLs. Further intensification of conventional treatment or stem cell transplantation may increase the cure rate for only some subtypes of ALL but is also associated with considerable treatment-related mortality and morbidity. Thus, new treatment strategies that are less toxic and, at the same time, more effective for patients with a resistant disease, are needed. The aim of the study was to evaluate whether a specific immune reaction is triggered in children with T-ALL and to identify the respective targets. We chose SEREX (serological identification of leukemia derived antigens by recombinant expression cloning) to screen a T-ALL expression library with plasma from 18 children with T-ALL.

Results: Thirteen antigens with homology to known genes that are involved in critical cellular processes were detected. Some of them have already been implicated in the induction of an immune response in a variety of cancer types. From four of these genes novel isoforms were identified and further analyzed. mRNA expression of three isoforms (HECTD1Δ, CX-ORF-15Δ and hCAP-EΔ) was restricted to more than 70% of T-ALLs (n=22) and specific antibodies against these isoforms were detected in up to 30% of patients (n=16) with the highest frequency for HECTD1Δ. The alternative splicing leads to the deletion of one glutamic acid located in a putative PEST domain thereby decreasing the PEST score suggesting a higher stability of the protein. Interestingly, the HECTD1 protein was present at high abundance in T-ALLs while is was not detectable in normal hematopoietic tissues. Since the leukemia-associated antigens detected in this study have an intracellular localization, a feature that is shared by the majority of SEREX defined antigens, the generation of immune effector responses most likely requires antigen presentation. To test this assumption, dendritic cells were loaded with HECTD1Δ protein and used for T cell stimulation. A specific T cell response was induced in vitro in all three donors, including a former T-ALL patient.

Conclusion: Leukemia-associated antigens, identified by SEREX, appear to be capable of inducing both a humoral and cellular immune response in children with T-ALL. Thus, these data support further studies to establish new approaches for immunotherapy.
Cellular Cholesterol Modulates VEGFR-1 Function on Acute Leukemia Cells.
Authors: Fragoso Rita , Casalou Cristina , Dias Sergio
Published: October 2019

Vascular endothelial growth factor (VEGF) and its receptors play a crucial role in malignancy and in disease, regulating the survival, proliferation, and migration of several cell types, such as endothelium and also leukemia cells. Following our recent report on the role of VEGFR-1 (FLT-1) in ALL (Fragoso R et al, 2006), in the present study we analyzed the molecular mechanisms whereby it modulates acute leukemia cell migration in response to VEGF/Placental Growth Factor (PLGF). First, we observed the formation of cell protrusions on ALL cells after VEGF/PLGF stimulation, with evidence for polymerized actin and FLT-1 co-localization (as determined by phalloidin, immunofluorescence staining, and confocal microscopy). Western blot analysis revealed that PLGF/VEGF stimulation resulted in increased RhoA and Rac1 GTPases expression. Co-treatment with LY200942 significantly decreased RhoA and Rac1 induction and cell migration by PLGF/VEGF, demonstrating this effect is modulated via Pi3 kinase. Next, we investigated the mechanisms whereby FLT-1 and actin co-localize at the cell “leading edge” (protrusions), after VEGF/PLGF stimulation, and the relevance of such co-localization for cell migration. We addressed this question by impairing the formation of lipid rafts/caveolae using drugs that either sequester (nystatin) or deplete (methyl-β-ciclodextrin) total cholesterol. Accordingly, co-treatment of leukemia cells with nystatin or MβCD and PLGF/VEGF blocked cell migration, an effect that was associated with a decrease in FLT-1 polarization and co-localization with actin filaments. Instead, FLT-1 was now found mostly in the cell cytosol. Given that leukemia cells have an increased rate of cholesterol up-take we sought to understand if increased cholesterol levels affected FLT-1 function in leukemia cells. Cholesterol repletion in leukemia cells enhanced leukemia cells migration in response to VEGF/PlGF (about 3 folds). This significant increase was associated with an increase in FLT-1 protein expression that, very interestingly, was particularly concentrated intracellulary in the cytoplasm. At this time we are trying to understand if this increase in FLT-1 expression after cholesterol repletion is associated with increase protein translation or impairment in proteasome activity. Finally, our preliminary in vivo experiments using Nod-Scid mice subjected (n=3) or not (n=3) to high fat diet (that results in increased cholesterol levels in the BM and in the spleen), showed this metabolic condition worsens disease symptoms and significantly decreases mouse survival. These results reveal for the first time some of the molecular mechanisms involved in FLT-1-mediated leukemia migration, namely the involvement of cholesterol metabolism, which may be crucial for new therapeutics delineation.
SBDS Protein Plays a Role in Ribosome Biogenesis.
Authors: Ganapathi Karthik A. , Austin Karyn M. , Malsch Maggie , Shimamura Akiko
Published: October 2019

Shwachman-Diamond syndrome is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow failure, and leukemia predisposition. The majority of patients with Shwachman-Diamond syndrome harbor mutations in the SBDS gene. SBDS is a novel gene of unknown function and is highly conserved throughout evolution. Studies of the yeast orthologue, YLR022c/SDO1, suggest that SBDS may play a role in ribosome biogenesis. In support of this hypothesis, we have found that the SBDS protein shuttles in and out of the nucleolus. Previously we have shown that SBDS nucleolar localization is regulated in a cell cycle-dependant manner. We now find that SBDS nucleolar localization is also lost following exposure to actinomycin D, suggesting that SBDS nucleolar localization is dependent on active ribosomal RNA (rRNA) transcription. In cell survival assays, SBDS−/− patient-derived cells are sensitive to actinomycin D treatment relative to normal control cells. Introduction of the wild-type SBDS cDNA into SBDS−/− cells corrects their actinomycin D sensitivity, confirming that the observed sensitivity is SBDS-dependent. In contrast, SBDS−/− cells do not exhibit increased sensitivity to cyclohexamide, a protein translation inhibitor. Consistent with this result, SBDS protein co-localizes with ribosomal precursor subunits but not with mature polysomes upon sucrose gradient sedimentation. No differences in polysome profiles are observed between SBDS−/− cells and wild type control cells. Gel filtration studies suggest that SBDS associates into a complex with other proteins. SBDS co-immunoprecipitates with other nucleolar proteins involved in rRNA biogenesis. RNA immunoprecipitation studies reveal that SBDS also associates with the 28S rRNA but not the 18S rRNA. These findings support the hypothesis that SBDS plays a role in ribosome biogenesis
Forodesine IV (Bcx-1777) Is Clinically Active in Relapsed/Refractory T-Cell Leukemia: Results of a Phase II Study (Interim Report).
Authors: Furman Richard R. , Gore Lia , Ravandi Farhad , Hoelzer Dieter
Published: October 2019

Background: Forodesine, a potent, rationally designed purine nucleoside phosphorylase inhibitor, has shown clinical activity in T-cell leukemia following IV administration. This phase II study was undertaken to evaluate further the efficacy and safety of IV forodesine in patients with relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL).

Methods: In this open-label, single-arm, multicenter, ongoing study, patients received forodesine, 40 mg/m2 IV, 5 days each week (1 cycle) for a total of 6 cycles. The dose was escalated to 90 mg/m2 in patients who were not responding after cycle 2. The primary end point was objective response (OR), which included complete response (CR), CR with incomplete recovery of peripheral blood cell counts (CRi), and partial response (PR).

Results: In total, 34 T-ALL patients (median age, 31 yr; range, 3–76 yr) with relapsed/refractory T-ALL (median, 3 prior treatments) received forodesine IV. The OR rate was 32.4% (11 patients; 7 [20.6%] CR, 0 CRi [0%] and 4 [11.8%] PR). Patient demographics and response for the 7 CR patients are shown in the Table. One CR patient has continued to receive treatment for almost 1 year. Two patients with CR proceeded to hematopoietic stem cell transplantation following remission. The restoration of normal hematopoiesis observed in patients while receiving therapy indicates a specificity of forodesine for leukemic cell populations. A total of 18 patients were dose escalated to 90 mg/m2 of forodesine after cycle 2 due to a lack of response. None of these patients demonstrated an improved response with dose escalation. The most frequently reported possibly drug-related AEs without regard to grade were nausea (22%), headache (16%), thrombocytopenia (14%), leukopenia (14%), asthenia (14%), and anemia (11%). Possibly drug-related AEs of grade 3 and higher occurring in ≥2 patients were thrombocytopenia (3 patients; 8%) and leukopenia and lymphopenia (2 patients each; 5%).

Conclusions: IV forodesine is effective as a single agent in the treatment of patients with relapsed or refractory T-ALL and is safe and well tolerated. Additionally, 40 mg/m2 has been confirmed as the dose for subsequent phase II development in T-ALL.

T-ALL Patients With Complete Response Patient No. No. of Previous Treatments Age/Gender Relapsed/Refractory Weeks of Tx Time to CR Time to Progression(Days) Overall Survival (Days) #Patient died in CR due to a cardiovascular event *Patient discontinued treatment and then progressed †Patient underwent transplantation following remission 1# 9 23/M Relapsed 9 2 weeks 77 77 2 1 60/M Relapsed 31 6 weeks 238 238+ 3* 4 28/F Relapsed 10 2 weeks 119 459+ 4 2 3/F Relapsed 51 2 weeks 398+ 398+ 5† 2 20/F Refractory 7 6 weeks 207 224 6 2 76/M Refractory 12 2 weeks 91 91+ 7† 3 27/M Refractory 6 2 weeks 180+ 180+
Phase II Study of Forodesine, a PNP Inhibitor, in Patients with Relapsed or Refractory B-Lineage Acute Lymphoblastic Leukemia.
Authors: Ritchie Ellen , Gore Lia , Roboz Gail J. , Feldman Eric , Ravandi Farhad , Furman Richard
Published: October 2019

Background: Forodesine is a potent, rationally designed purine nucleoside phosphorylase (PNP) inhibitor that has shown activity in acute lymphoblastic leukemia (ALL). Although the drug was thought to be most active in T-cell malignancies, preclinical data and results of a phase I/II trial showed clinical activity in B-lineage ALL (B-ALL).

Methods: Twelve patients with relapsed/refractory B-ALL were enrolled (median age of 10 adults, 48.5 yr; median age of 2 children, 10 yr). Three patients had relapsed after allogeneic bone marrow transplantation (BMT), and 9 patients relapsed after a median of two prior regimens. The patients were treated with a 30-minute infusion of forodesine, 80 mg/m2 for 5 days/wk for a 4-week period. If the patient showed clinical improvement at 4 weeks, the drug was continued for additional cycles.

Results: Two of 12 patients achieved a complete response. One of these patients had relapsed after allogeneic BMT and restored complete donor chimerism after 16 weeks of forodesine treatment. As restoration of donor chimerism was achieved, the patient developed hepatic graft-versus-host disease (GVHD) and was treated simultaneously with dexamethasone beginning at 4 weeks; this was tapered off and restarted when GVHD again worsened. The second patient failed one prior regimen and had no leukemic blasts in the bone marrow after 4 weeks of treatment. Both these patients continue to receive therapy (5+ and 2+ months respectively). There were no patients with a partial response. Four patients presented with high white blood cell (WBC) counts; each demonstrated an initial decrease in WBCs without an increase in neutrophils while they were receiving forodesine but then developed persistent pancytopenia with residual bone marrow disease at 4 weeks. Dexamethasone was added to the treatment of 3 nonresponders, with no clinical benefit. Three patients with Ph+ B-ALL were treated on the study. Two rapidly progressed on the medication and were taken off the study before the end of 2 weeks; one had persistent pancytopenia and bone pain with residual marrow disease at 4 weeks and was taken off the study. Adverse events included increases in liver transaminase (grade ≥ 2, 2 patients) and alkaline phosphatase levels (grade 2,3, 2 patients), hyperkalemia (grade 2,3, 2 patients), and hypomagnesemia (grade 1, 1 patient). Two patients with increased liver transaminase levels underwent liver biopsies: 1 showed leukemic infiltration and the other GVHD. Patient accrual continues for this study.

Conclusions: Forodesine is a safe, well-tolerated drug with preliminary evidence of activity as a single agent in B-ALL.
PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation.
Authors: Ozpolat Bulent , Akar Ugur , Barria Magaly , Lopez-Berestein Gabriel
Published: October 2019

Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.
Development of a Novel Xenotransplantation System for Studying Human Acute Myeloid Leukemia.
Authors: Park Christopher Y. , Majeti Ravindra , Tan Brenton T. , Weissman Irving L.
Published: October 2019

The formal demonstration of leukemia stem cells (LSC) relies on the ability of primary human leukemias to engraft in transplanted xenohosts with the subsequent demonstration of serially transplantable tumors to establish the presence of a self-renewing population. Several immunodeficient and transgenic mouse strains have been utilized as xenohosts for human AML (e.g. NOD, NOD/SCID, NOD/SCID,β2m); however, robust engraftment frequently requires that the mouse recipients be pre-treated with antibodies against mouse immune cells or that leukemic blasts and/or mouse hosts are treated with exogenous hematopoietic growth factors before and/or during transplantation. While such treatments result in a higher percentage of engrafting AML’s, the overall effect on leukemic blasts, particularly the leukemic stem cell population, is unknown. In order to avoid these confounding factors as well as to potentially develop a more robust model of xenotransplantation, we have evaluated the use of the RAG2/gamma common chain double knockout mouse model to xenotransplant human AML’s. Using primary human samples (n=32, patient age range 1–86, median 55), we have determined that newborn pups intravenously injected via the facial vein efficiently engraft human AML’s without the use of additional growth factors or depleting antibody therapy. Among the AML’s tested, 65.6% (21/32) engrafted in mice as assessed by >5% human chimerism in either the peripheral blood, spleen, or bone marrow at 12 weeks post-transplant. In contrast to other xenotransplantation mouse models, circulating leukemic cells were typically identified in the circulation by 4 weeks post-transplant and were present in stable or increasing numbers in the circulation until engraftment was assessed (12 weeks) or until recipient mouse demise. Engrafting AML’s were composed of both CD34+ and CD34- AML’s and included all WHO/FAB subtypes except for acute promyelocytic leukemia. These AML’s included de novo and relapsed AML, as well as therapy-related AML and AML arising from pre-existing MDS or CMPD. While the number of samples tested was limited, there was no statistically significant association between engraftment and WBC at diagnosis, cytogenetic risk group, MLL or FLT3 status, age, or achievement of CR following induction therapy; however, among CD34+ AML’s the percentage of putative leukemic stem cells (defined as CD34+CD38- leukemic blasts), showed a statistically significant correlation with engraftment (student T-test p < 0.05). All CD34+AML’s containing >18% CD34+/CD38− leukemic blasts engrafted in the transplanted hosts. Overall, these findings demonstrate that the RAG2/gamma common chain double knockout mouse is a robust system for studying human AML and will be a useful model for future functional studies of human AML LSC’s.
Effect of Failure To Respond to Targeted Therapy on Response to Cytotoxic Therapy in Pts Age ≥ 60 with Newly-Diagnosed AML.
Authors: Estey Elihu H. , Garcia-Manero Guillermo , Giles Francis , Kantarjian Hagop M.
Published: October 2019

An increasing number of studies are emphasizing use of less toxic, often called “targeted”, therapies (TT) in pts age ≥ 60 with untreated AML. A significant proportion of TTs are unlikely to produce even “hematologic improvement”, raising the question whether response to subsequent cytotoxic therapy (CT) is adversely affected in pts who fail to respond to TT. From 11/00–7/06, 25 pts received CT (containing ara-C in 20 cases) as 1st salvage therapy after failing to respond to a 1st course of TT (23 pts) or relapsing after brief CRs with such therapy. The TTs were decitabine or 5azacitidine +/− valproic acid 9 pts, PKC412 5 pts, tipifarnib 3 pts, HDAC inhibitors 3 pts, others 3 pts. The median age of the 25 pts was 71 years; 12 of the 24 whose cytogenetic status was determined had a normal karyotype (“diploid”), 7 had chromosome 5 and/or 7 abnormalities (-5/7), and 5 various other poor prognosis abnormalities. The CR rate after CT salvage in the 12 diploid pts was 8/12; their median survival was 44 weeks. Among 114 diploid pts who, from 11/00–7/06, met eligibility criteria for TT studies (WBC < 20,000, performance status < 3) but were given ara-C-containing CT as 1st therapy CR rate was 68% and median survival 53 weeks. Only 1/12 pts with abnormal karyotypes who received CT as 1st salvage achieved CR; median survival was 13 weeks. In contrast, in the 161 pts with an abnormal karyotype (50% -5/-7) who were given CT as 1st therapy, CR rate was 37% (p = 0.06) and median survival 19 weeks. Subject to various potential selection biases our conclusions are: (1) in diploid pts failure to respond to TT does not materially affect outcome with CT and (2) failure to respond to TT may identify pts with abnormal cytogenetics who are unlikely to respond to CT; in any event, there is no reason to give such pts CT salvage.
Azacitidine Plus Gemtuzumab Ozogamicin (GO): A Novel Combination in the Treatment of Acute Myeloid Leukemia (AML) and High-Risk Myelodysplastic Syndromes (MDS) in the Elderly.
Authors: Nand Sucha , Godwin John , Smith Scott , Barton Kevin , Germano Eliza , Stiff Patrick
Published: October 2019

AML and high-risk MDS in the elderly carry a poor prognosis. Only 46% of AML patients receiving standard chemotherapy achieve complete remission (CR) and treatment-related mortality approaches 30% above age 60. In 2005, we initiated a Phase II trial for elderly patients with newly diagnosed AML or MDS, using the following outpatient treatment schema: If white blood cell (WBC) count at presentation was >10,000/ul, pt was started on hydroxyurea 1500 mg twice daily by mouth. Leukapheresis was performed if WBC >100,000/ul. Once WBC count was <10,000/ul, the patient received azacitidine 75 mg/m2 s/cu D1–7 and GO 3 mg/m2 on D8. A bone marrow was performed on D14 and induction therapy repeated for residual disease. Those who achieved CR were given one consolidation treatment with azacitidine+GO in same doses after recovery of blood counts. A total of 13 pts have been treated to date. Eleven had AML by WHO classification and 2 MDS (both RAEB). The median age was 77 (62–83) and 7 were male. Ten patients required retreatment on D14. Ten patients (76%) achieved CR. Six patients developed grade 3 toxicities: 5 neutropenic fever and 1 typhlitis, all requiring hospitalization. There were no treatment-related deaths. Median follow up is 7 months (2–13 months) and eleven patients remain alive. Two patients have died from relapsed or refractory disease. Nine patients remain in CR with a median duration of remission of 7 months (2–13 months). The trial is ongoing with an accrual goal of 20. Our early experience with this novel combination is quite encouraging. Cytoreduction with hydroxyurea and leukapheresis followed by azacitidine and GO appears to be a safe and effective regimen for elderly patients with AML and the therapy can be given in the outpatient setting. These preliminary results need to be confirmed in a larger cohort of patients.
SGN-33 Modulates Cytokine and Chemokine Production by Activated Monocytes and Macrophages.
Authors: Sutherland May S.K. , Lewis Timothy S. , Yu Changpu , McEarchern Julie A. , Drachman Jonathan G. , Sievers Eric L. , Grewal Iqbal S. , Law Che-Leung
Published: October 2019

SGN-33, a humanized IgG1 anti-CD33 antibody that targets CD33, a sialoadhesion family member expressed on myeloid precursor cells, macrophages, and monocytes, is currently in Phase I clinical trials for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). To further characterize and define the mechanism of action of SGN-33, we undertook a series of experiments in AML cell lines and in cultures of primary normal human monocytes and macrophages. SGN-33 has previously been shown to eliminate tumor cells through antibody-mediated effector functions including Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC). We have now demonstrated that SGN-33 binding also initiates Antibody-Dependent Cellular Phagocytosis (ADCP). Because CD33 has been hypothesized to mediate signal transduction, we explored the biochemical effects of SGN-33 binding on AML cell lines and normal monocytes and macrophages. SGN-33 binding stimulated tyrosine phosphorylation of CD33 and recruitment of SHP-1, with maximal effects between 5 to 30 minutes. Initiation of signaling by SGN-33 was not dependent upon antibody crosslinking. Furthermore, SGN-33 exposure significantly reduced the syntheses of pro-inflammatory cytokines (TNF-a, IL-6, IL-1b) and chemokines (RANTES, MCP-1, IL-8) by macrophages cultured in the presence of tumor cell conditioned media. Production of these cytokines by monocytes and macrophages activated in vitro by IFN-g and TGF-b were also blocked by SGN-33 binding (up to 100% reduction in TNF-a levels in vitro). These data demonstrate that the mechanisms of action of anti-CD33 immunotherapy may be broader than previously known. Because pro-inflammatory cytokines have been implicated in the growth and survival of tumor cells in patients, the capacity of SGN-33 to block these factors may yield clinical benefit. This hypothesis is currently being evaluated in clinical studies of SGN-33.
Farnesyl Transferase Inhibitor (Tipifarnib, Zarnestra; Z) in Combination with Standard Chemotherapy with Idarubicin (Ida) and Cytarabine (ara-C) for Patients (pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS).
Authors: Alvarez Ricardo H. , Kantarjian Hagop , Garcia-Manero Guillermo , Estrov Zeev , Ravandi-Kashani Farhad , Verstovsek Srdan , Giles Francis , O’Brien Susan , Asa Koller Charles , Faderl Stefan , Thomas Deborah , Wright John J. , Cortes Jorge
Published: October 2019

Tipifarnib is a non-peptidomimetic farnesyl transferase inhibitor (FTI) with clinical activity in hematologic malignancies, including AML, MDS and CML. Pre-clinical data suggests that tipifarnib may be synergistic with some chemotherapeutic agents. We designed a phase I/II study where pts age 15 to 70 years with previously untreated AML or high-risk MDS (blasts >10%) received induction with Ida 12 mg/m2/d on days 1–3, ara-C 1.5 g/m2 IV over 24 hours daily on days 1–4 (days 1–3 only if age ≥ 60 years) and Z with first cohort (6 pts) receiving 200mg orally twice daily (BID), and all others 300 mg BID x 21 days every 28 days. Pts achieving CR received consolidation (5 courses) with Ida 8 mg/m2/d, days 1–2, ara-C 0.75 g/m2/d, days 1–3, and Z 300 mg BID x14 days every 4–6 weeks. Maintenance was with Z 300 mg BID x 21 d every 4–6 wk for 6 months. We have treated 74 pts, median age 50 yrs (17–61 yrs). All pts are evaluable for response. Fifty seven pts (77%) responded: 48 (65%) achieved CR and 9 (12%) CRp. Overall response rate (CR+CRp) in a similar historical population treated with the same chemotherapy regimen (IA) without Z was 68% (p=0.29). Response by cytogenetics was: 26/30 (86%) for diploid, 12/16 (76%) with -5/-7, 1/2 with t(8;21), and 17/25 (68%) with other abnormalities. Response by Flt3 was 11/15 (73%) for unmutated Flt3, 39/46 (85%) for mutated Flt3. Response by age was 26/34 (76%) if ≤50yrs, 9/13 (69%) if >50yrs with diploid karyotype and 13/27 (48%) if >50yrs with abnormal karyotype. With median follow-up of 22 weeks, 34 pts are still on study. Of the 48 pts that achieved CR, 2 died in CR of unrelated causes and 9 pts have relapsed after 5, 11 13, 14, 16, 26, 29, 34, and 39 weeks, respectively. The most common grade 3 adverse events include diarrhea in 31 (42%) and hyperbilirrubinemia in 10 (14%), both transient. Thirty nine (53%) pts have required treatment interruptions/dose reductions during induction, 17 (16%), and 2 (7%) during consolidation and maintenance respectively. We conclude that Z combined with Ida and ara-C induces a high rate of CR in AML, with increased incidence of diarrhea and hyperbilirrrubinemia. Direct comparison with chemotherapy alone is warranted in a randomized trial.
Improved Survival in Acute Myeloid Leukaemia Patients Aged over 40 Given Cladribine in Combination with Standard Remission Induction (DA 3+7) and Consolidation Treatment (HD AraC). Seven Year Follow-Up of Prospective, Cooperative PALG Study.
Authors: Holowiecki Jerzy , Grosicki Sebastian , Hellmann Andrzej , Skotnicki Aleksander , Robak Tadeusz , Jedrzejczak Wieslaw W. , Konopka Lech , Kuliczkowski Kazimierz , Kyrcz-Krzemien Slawomira , Stella-Holowiecka Beata
Published: October 2019

The aim of this study was to evaluate long-term outcome of acute myeloid leukemia (AML) patients treated within the PALG 1999 DAC vs. DA Study. [b][Within 3 years (1999–2002) 445 patients, aged 18–60, were randomized 1:1 to the induction treatment DAC-7: daunorubicin 60 mg/m2/d iv 1–3; cytarabine 200 mg/m2/d ci d 1–7; cladribine (2-CdA) 5 mg/m2 2h inf. iv d 1–5 and standard DA-7 regimen (the same therapy excluding cladribine). Patients achieving CR received two courses of subsequent intensive consolidation:

HAM (HD AraC, mitoxantrone) HD AraC with or without cladribine in the DAC-7 or DA-7 arm, respectively.

In case of PR after the first induction course the same regimen was repeated, Post-consolidation therapy was comparable in both arms with following proportions of autoHCT, alloHCT and maintenance: DAC-7 17%, 14%, 69%; DA-7 21%, 14%, 65%, respectively.

As previously reported, a single course of DAC-7 induction resulted in 17% higher CR rate compared to the DA-7 treatment (p=0,0008). The difference was particularly pronounced in patients: aged >40 years and with initial WBC >100x109/L. In the latter subgroup also the overall CR rate (achieved after entire induction program) was higher in the DAC-7 arm (71% vs. 43%). [Leukemia.2004;18:989–97]

In the present report we analyzed seven-year long-term outcome (median follow-up 5 yrs) in the whole study population and in subgroups stratified according to age, initial WBC, cytogenetics, sex, FAB subtype, and preceding myelodysplasia. In the whole group the overall survival (OS) rate equaled 29,5% for DAC-7 and 24% for DA-7 arm (p=NS) and leukemia free survivall (LFS) 30% vs. 28% (p=NS), respectively.

Of note, in patients aged >40 years, the therapy containing cladribine was associated with improved OS (26% vs. 14,5%, p=0.03), and LFS (28% for DAC-7 vs. 18,5% for DA-7, p=0.02).

Other subgroup analyses revealed higher probability of the OS in patients with initial WBC ≥ 50 G/l assigned to DAC-7 compared to DA-7 arm (32% vs. 20,5%, p=0.04). The LFS rate equaled 35% and 27% (p=NS), respectively.

In women receiving DAC-7 induction therapy in comparison with those treated in arm without 2-CdA reached higher OS: 29% vs. 19,5%, p=0,03, respectively. LFS in these subgroups was comparable: 25% vs. 22%, p=NS, respectively.

We conclude that addition of cladribine to induction and consolidation therapy of AML improves long-term outcome in patients: older than 40 y, as well as in those with high tumour burden. The better outcome in older patients results mainly from reduced risk of relapse, whether that in cases with high WBC seems to be linked to a higher CR rate.
In Vivo Analysis of the Anti-Leukemic Activity of Alpha-Tocopherol in Acute Promyelocytic Leukemia.
Authors: Abreu e Lima Rodrigo S. , Silva Ana Silvia G. , Scheucher Priscila S. , Panepucci Rodrigo A. , Proto-Siqueira Rodrigo , Santos Guilherme Augusto F. , Teixeira Hamilton Luiz G. , Saggioro Fabiano P. , Falcao Roberto P. , Rego Eduardo M.
Published: October 2019

As2O3 has been successfully been employed in the treatment of patients with acute promyelocytic leukemia (APL), however it is unavailable to most APL patients in Latin America, and the lack of alternative treatments lead to a very poor prognosis of relapsed/ATRA resistant patients. Here we investigate the anti-leukemic effect of alpha tocopherol succinate (VES) (a vitamin E analog with antiproliferative activity against several cancer cell types) which was shown to activate the intrinsic cell death mediators of caspase-9 and -3 by both its strong redox activity and inhibition of protein kinase C (PKC) and target Akt and JNK pathways. We used a syngenic transplantation model of APL: blasts from hCG-PML/RARalpha transgenic mice (TM) were IV injected in irradiated non-transgenic littermates. Massive infiltration of bone marrow (BM), spleen and liver was detected by 21st day post transplant. Recipient mice were treated with: VES (50UI/g/d) (n=15), As2O3 (2.5μg/g/d) (n=15), VES + As2O3 (n= 15) at the same doses, or vehicle (Control; n=15) starting from Day 4 for 21 days. Molecular remission was determined by RT-PCR for PML/RARalpha. The mean survival time in the Control group was of only 25,6 days (95% C.I. = 20,9 – 30,3), whereas in the VES it was 160,40 (95%CI = 134,2 – 185,6); in the As2O3 of 162,1 (95% CI = 137,97 −186,3) and in the VES+ As2O3 of 163 (95% CI = 139,93 – 186,1). Compared to controls, all treatments significantly prolonged survival. Molecular remission was attained in 86,5% of mice in the VES arm, 80% in the As2O3, and 86,5% in VES + As2O3. No significant organ toxicity was found by histopathological analyses. Another group of twelve recipients was treated with VES or DMSO for 6 days, and the percentage of apoptotic CD117+ leukemic cells in spleen and liver was determined by flow cytometry. Differentiation was evaluated morphologically on Leishman stained BM cytospin preparations after 72h of treatment with VES. The mean percentage of CD117+ apoptotic cells in VES treated mice was significantly higher (41,17 ± 5 versus 62,11 ± 4%, in spleen p<0,03; and 94,65 ± 1% versus 12,94 ± 11,95%, in liver, p<0,01). No significant difference in the number of mature granulocytic cells was detected. Furthermore, we compared the gene expression profile of BM cells obtained from leukemic mice treated with VES or DMSO (n=6, per group) using nylon microarrays representing 598 genes. Data mining from our microarray results revealed the higher expression of Mitogen Activated Protein Kinase 3, Protein Kinase C delta and BCL2 antagonist Killer-1 genes in VES treated samples. These were confirmed by Real Time-PCR. In conclusion, our results demonstrate that VES induces prolonged remissions and that it activates of MAPK and PKC signaling pathways leading to apoptosis.
Loss of Major Histocompatibility Class II (MHCII) Gene Expression in Diffuse Large B Cell Lymphoma (DLBCL) Is Not Generally Due to Genetic Mutations in the Coding Regions of CIITA or RFX.
Authors: Roberts Robin A. , Miller Thomas P. , Rimsza Lisa M.
Published: October 2019

Loss of expression of the MHCII in a subset of DLBCL cases studied by the Leukemia and Lymphoma Molecular Profiling Project (LLMPP) has been previously associated with extremely poor prognosis. Large genetic deletions of the MHC II loci were not seen in these cases. Furthermore, gene expression profiling analysis demonstrated that the MHCII gene expression was coordinated and most likely suppressed through altered transcription. We therefore investigated the possibility of small deletions or genetic mutations of 2 key transcriptional regulators of MHCII, CIITA and RFX, as possible causes of decreased MHCII expression in DLBCL. These transcription factors were chosen because mutations in the coding region of these proteins have been shown to cause the rare genetic disease, bare lymphocyte syndrome, in which MHCII expression is lost. We designed primers to amplify all coding exons of CIITA and RFX (a multimer containing RFX5, RFXB, and RFXAP), including internal splicing regions, in a minimal number of amplifications. DNA samples were amplified by 6 multiplex PCRs of genomic DNA, then the products sequenced with separate sequencing primers and compared to NCBI curated sequences. DLBCL DNA samples for which gene expression profiling data on MHCII expression was available, were obtained from the LLMPP research group. 23 of these samples were from the lowest 10% de novo untreated average MHCII expressers, 4 from non-de novo samples expressing MHCII in the same range, 4 from low MHCII expressers in the lowest 10–25% range, and 15 were primary mediastinal B cell lymphoma (PMBL) samples. The PMBL subset of DLBCL expresses MHCII at a lower range than other DLBCL. A number of other MHCII positive and negative lymphoma samples and cell lines were also sequenced. Although various SNPs and silent changes were noted, there were few small point mutations, deletions, or splicing mutations in the low MHCII DLBCL expressers that would explain loss of MHCII expression. In RJ2.2.5, an MHCII negative Burkitts lymphoma cell line derived from Raji, which is known to have only one CIITA allele expressing RNA lacking exons 11 and 12, the genomic deletion was sequenced. One of the lowest 10% MHCII expressing LLMPP samples had an insertional duplication that caused a frameshift in the C-terminus of all copies of the CIITA gene. Another tumor sample showing functional mutations was an MHCII negative T cell lymphoma (non-LLMPP), which had nonsense mutations in both RFXAP and RFX5, all copies. In conclusion, critical deletions or mutations were not common in the studied samples. These results confirmed previous data implying loss of MHCII expression in DLBCL was most likely due to altered transcriptional regulation, and indicate that this unfortunate circumstance may frequently be amenable to therapeutic intervention to upregulate the MHCII pathway.