bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies to TRAIL Receptors R1 and R2 Induce Apoptosis in Non-Hodgkin’s Lymphoma Cells.
Authors: Smith Mitchell Reed , Jin Fang , Joshi Indira
Published: October 2019

Monoclonal antibodies and chemotherapy can be effective, but not curative, therapy for non-Hodgkin s lymphoma (NHL). Rituximab acts by several mechanisms, including directly signaling apoptosis of CD20+ cells via the mitochondrial pathway involving caspase 9. Chemotherapy also activates this apoptotic pathway. TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. Thus, we investigated the effects of agonistic monoclonal antibodies to TRAIL-R1 and -R2 on NHL cell lines (DoHH2, WSU-FSCCL and FC-TxFL2, each t(14;18)+, EBV-). TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) antibodies were from Human Genome Sciences (Rockville, MD). FC-TxFL2 expressed the highest cell surface levels of TRAIL-R1 (DR4, target of HGS-ETR1) and TRAIL-R2 (DR5, target of HGS-ETR2). WSU-FSCCL expressed lower, but significant, levels of each. DoHH2 expressed dim TRAIL-R1 and the lowest levels of TRAIL-R2 of the 3 cell lines. IC50 (MTT assay after 72 hr incubation with antibody) was 0.25mcg/ml for WSU-FSCCL and FC-TxFL2 with either HGS-ETR1 or HGS-ETR2, while DoHH2 was minimally inhibited by either antibody. Both HGS-ETR1 and HGS-ETR2 antibodies induced dose dependent increases in apoptosis (annexin V assay) of WSU-FSCCL and FC-TxFL-2, but not DoHH2. Caspase activation (flow cytometry using fluorescent substrates; Western analysis) was seen 4–6 hr after antibody addition. As expected, caspases 3 and 8 were activated by both antibodies in the two sensitive cell lines. Caspase 9, however, was also activated. Thus, TRAIL-R1 and TRAIL-R2 antibody binding inhibits growth, induces apoptosis and activates caspases 3, 8 and 9 in NHL cells expressing the targets. The induction of caspase 9 suggests cross-talk between the extrinsic and intrinsic pathways, in which activation of caspase 8 leads to cleavage and translocation of bid, which in turn leads to activation of caspase 9, and ultimately caspase 3. We have confirmed that bid cleavage does occur in these NHL cells after HGS-ETR1 and HGS-ETR2 binding. We have further shown that both the pan-caspase inhibitor ZVAD and a specific caspase 8 inhibitor block HGS-ETR1 and HGS-ETR2 induced apoptosis. As predicted, a specific inhibitor of caspase 9 only partially blocks apoptosis. This suggests that combination of the agonist HGS-ETR1 and HGS-ETR2 antibodies with agents that act via the caspase 9 pathway would be rational combinations to test for therapeutic potential in NHL.
Oral Melphalan, Cyclophosfamide and Prednisone (MCP) in 72 Newly Diagnosed Waldenström’s Macroglobulinemia Patients: Results and Costs Analysis.
Authors: Petrucci Maria Teresa , Annibali Ombretta , Levi Anna , Tirindelli Maria C. , Fossati Carolina , Gallucci Cristiano , Del Bianco Patrizia , Mandelli Franco , Avvisati Giuseppe
Published: October 2019

Treatment of Waldenström’s macroglobulinemia (WM) is based on the use of oral alkylating agents, even though recently, more expensive drugs have been proposed for its treatment.The aim of this study was to report the results obtained combining oral melphalan, cyclophosphamide and prednisone (MCP) in 72 WM patients, and to compare the results and related costs with those observed using more aggressive and expensive protocols. Between July 1973 and April 2002, we have observed 100 newly diagnosed WM, of whom 72 were symptomatic and therefore requiring a treatment. The antineoplastic treatment consisted of melphalan (6 mg/m2, days 1–7), cyclofosfamide (125 mg/m2, days 1–7) and prednisone (40 mg/m2, days 1–7) administered orally for a total of 12 courses. Thereafter, responding and stable patients were maintained with chlorambucil (3 mg/m2) and prednisone ( 6 mg/m2) until progression. Levels ≥1.5 g/dL of IgM immunoglobulin associated to 30% of lymphoplasmocytic cells in the bone marrow aspirate were required for diagnosing WM. Among these patients, 46 (63%) were men and 26 (47%) were women. Median age, IgM Immunoglobulin and Hb levels were at diagnosis: 61 years (range 42–87 years), 3.8 gr/dl ( range 1.5–6.5) and 12 gr/dl ( range 6.5 – 16) respectively.

Of these patients, we evaluated response rate, overall survival (OS), response duration (RD), freedom from progression (FFP), event free survival (EFS) duration, toxicities and costs/course in € of the drugs utilized.Of the 72 treated patients,71 (98.6%) were evaluable and 55 (77.5%) achieved a response, 7 (9.9%) showed a disease stabilization and 9 (12.6%) disease progression. After a median follow-up of 72 months (range 3–195 months), median OS, RD, FFP and EFS were: 66, 64, 55 and 47 months, respectively. No grade III or IV WHO toxicities were observed and toxicity was limited to transient nausea, vomiting and mild neutropenia. The cost/course for MCP was € 13, similar to that of standard protocols based on chlorambucil, and much less expensive compared to the costs of more aggressive protocols proposed for the treatment WM, ranging from € 74 to € 3260.90.In conclusion,the MCP is, as chlorambucil based protocols, a cost effective and safe option for treating patients with WM. Moreover, the obtained results do not appear inferior to those so far reported with more expensive, aggressive and toxic intravenous protocols.
Role of Anemia in Survival of Elderly Aggressive Non-Hodgkin’s Lymphoma Patients Following Chemotherapy.
Authors: Zinzani Pier Luigi , Tani Monica , Molinari Anna Lia , Visani Giuseppe , Gentilini Patrizia , Guardigni Luciano , Stefoni Vitorio , Alinari Lapo , Marchi Enrica , Gabriele Annalisa , Musuraca Gerardo , Fina Maria Paola , Baccarani Michele
Published: October 2019

Recent literature data suggest that baseline anemia is an important poor prognostic factor for non-Hodgkin’s lymphoma (NHL) outcome. We evaluated the prevalence, risk factors, and prognostic value of anemia following chemotherapy in elderly patients with aggressive NHL treated with conventional front-line chemotherapy. Patients and Methods. The retyrospective sample included 168 patients with diagnosis of aggressive NHL treated between March 1993 and February 2001 with VNCOP-B regimen in five italian institutions. The model included sex, age, hemoglobin level following chemotherapy, and response to chemotherapy. Tumor remission at the end of chemotherapy was associated to a highly significant reduction in risk (RR, 0.215; p=0.0001); advanced staging before chemotherapy independently predicted a poorer survival (II-IV vs. I-II; RR, 1.55; p=0.0023). The prognostic value of hemoglobin level at chemotherapy end, adjusted for all above factors, was statistically significant (RR for 1-g/dL increment, 0.76; p= 0.0041). Our preliminary results suggest the prognostic role of hemoglobin value after chemotherapy in terms of overall survival. This concept introduces the potential role of anemia correction in improving the survival rate in elderly aggressive NHL.
Acceptable Outcomes for Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation (AlloSCT) for Relapsed Aggressive Non-Hodgkin’s Lymphoma (NHL).
Authors: Doocey Richard T. , Song Kevin W. , Toze Cynthia L. , Connors Joseph M. , Nevill Thomas J. , Gascoyne Randy D. , Barnett Michael J. , Forrest Donna L. , Hogge Donna E. , Lavoie Julye C. , Nantel Stephen H. , Shepherd John D. , Sutherland Heather J. , Voss Nicholas , Smith Clayton A.
Published: October 2019

Background: AlloSCT for the treatment of aggressive histology NHL is appealing on the grounds of providing both a tumour free graft and a graft versus lymphoma mechanism for disease clearance. Currently only modest experience has been acquired concerning this treatment modality because of the fear of increased treatment related mortality (TRM).

Methods: Retrospective review of 47 consecutive patients with aggressive histology NHL who proceeded to AlloSCT between 1987–2003 in Vancouver. Patients with Burkitt’s lymphoma and lymphoblastic lymphoma were excluded.

Characteristics: WHO classification histology: 24, diffuse large B cell (including 5 T cell Rich B cell and 3 primary mediastinal); 16, transformed indolent to diffuse large B cell; 7, peripheral T cell unspecified. Gender M:F 27:20. Median age at AlloSCT 40 years (range 19–55 years). Disease status at AlloSCT: 33 (71%) chemosensitive relapse; 12 (25%) primary resistant /resistant relapse; 2 (4%) first complete remission. 39 (83%) had advanced stage disease at SCT. Stem cell source: 33 (71%) HLA-identical sibling bone marrow; 3 (6%) HLA-identical sibling peripheral blood; 8 (17%) unrelated donor bone marrow; 3 (6%) unrelated donor peripheral blood. Cyclophosphamide /TBI conditioning was used in 81% of patients.

Results: 22 patients remain alive post AlloSCT with a median follow-up of 40 months (3–135 months). The 5 year overall survival and event free survival (EFS) for the 47 patients was 47% (95% CI 32% – 63%) and 43% (95% CI 27% – 59%) respectively. The 5 year EFS for the individual disease groups was diffuse large B Cell 37%; transformed indolent B Cell 12%; peripheral T cell - unspecified 86%. Chemosensitivity at SCT was associated with a 5 year EFS of 41% (95% CI 23% – 60%) as compared to 37% (95% CI 8% – 67%) for those with chemorefractory disease (p = 0.61). The cumulative incidence of TRM at 3 months and relapse at 3 years were 17% (95% CI 8% – 29%) and 32% (95% CI 18% – 47%) respectively for the entire 47 patients. Cumulative incidence of relapse at 3 years was 54% for patients with chemoresistant disease versus 25% for those with chemosensitive disease (p=0.049). The incidence of acute and chronic graft versus host disease (GVHD) was 57% and 51% respectively.

Conclusion: AlloSCT for aggressive NHL results in an acceptable TRM and favourable long term EFS and OS for patients with advanced stage disease having relapsed after primary treatment. Patient with chemoresistant disease can have a durable remission post AlloSCT although they are at increased risk of relapse.
A Defined Donor Activating Natural Killer Cell Receptor Phenotype Protects Against Leukemic Relapse after HLA-Identical Hematopoietic Stem Cell Transplantation.
Authors: Demanet Christian J. , Verheyden Sonja J. , Schots Rik J.
Published: October 2019

Objective: Killer immunoglobulin-like receptors (KIRs) on Natural Killer (NK) cells recognize groups of HLA class I alleles. Failure to recognize the appropriate KIR ligand on a HLA class I-mismatched cell triggers NK cytotoxicity. Because KIR and HLA genes are located on different chromosomes matching for HLA does mostly not result in matched KIR genes in related HLA-identical hematopoietic stem cell transplantation (HSCT). Moreover, KIR genotypes are very diverse in the population and therefore we explored whether this KIR diversity could have any Graft-versus-Leukemia (GvL) effect in related, HLA-identical HSCT.

Methods: A total of 65 consecutive patients with leukemia (AML, CML, ALL) who received a first transplant at our institution, were included in the analysis. The median age of the patients was 34 years. The conditioning regimen included TBI + cytosine arabinoside + cyclophosphamide in the majority of the patients (85%). The source of stem cells was bone marrow (72%) or peripheral blood (28%). The NK KIR genotype (6 inhibiting and 5 activating KIRs) and molecular HLA compatibility (locus A, B, C, DRB1, DQB1, DPB1) were studied in all patients and their sibling donors.

Results and conclusion: Our analysis showed that the combined presence of the activating KIR receptors 2DS1 and 2DS2 on the donor NK cells is of major importance in protection against leukemic relapse in HLA-identical HSCT. In the patient group with a KIR2DS1(+)2DS2(+) donor, relapse was significantly lower (2/15; 13%) compared to all other donors (23/50; 46%) (P=0.01). Additionally, we found a direct relationship between the number of HLA class I - activating KIR interactions and relapse. The more activating KIR receptor-HLA-C ligand interactions possible, the less relapse observed (P=0.05). However, we cannot exclude at this moment that a hitherto unknown ligand (such as a viral protein or a tumor associated or -specific protein) could substitute for a HLA class I antigen. HLA class I down-regulation is frequently observed on leukemic cells and decreases the interaction with inhibitory NK receptors. The presence of sufficient donor activating NK receptors to overcome the remaining inhibition is likely the mechanism that mediates the elimination of residual leukemic cells.
Tumor Antigen Immunization of Sibling Stem Cell Transplant Donors in Multiple Myeloma.
Authors: Neelapu Sattva S. , Munshi Nikhil C. , Jagannath Sundar , Watson Thelma M. , Pennington Robin , Barlogie Bart , Kwak Larry W.
Published: October 2019

Strategies to enhance the specific anti-tumor effect of the graft without increasing the risk of graft-versus-host disease (GVHD) are needed to improve the outcome in allotransplant recipients. In this pilot study, we vaccinated sibling donors against a defined, patient tumor-derived antigen prior to bone marrow harvest. The objective of this strategy was to induce tumor antigen-specific immunity in healthy donors and to transfer this immunity to the recipients by bone marrow transplantation (BMT). For this purpose, the unique antigenic determinants (Idiotype [Id]) of the immunoglobulin secreted by the myeloma tumor can serve as a safe, tumor-specific antigen for active immunotherapy. The vaccine formulation consisted of Id isolated from the plasma of myeloma patients chemically conjugated to a carrier molecule keyhole limpet-hemocyanin (KLH) and administered together with GM-CSF as an adjuvant. Immunization of the HLA-matched sibling donors (n = 5) prior to bone marrow harvest, induced specific cellular and/or humoral immune responses against Id and KLH in all five donors. No short-term or long-term toxicities were observed in any of the donors following vaccination. The recipients received priming vaccination pretransplant and booster immunizations starting approximately 3 months after the myeloablative transplantation. Following BMT, two patients died within 30 days due to transplant related complications. Id-specific and KLH-specific T cell responses were detected in all three surviving patients post-, but not pre-BMT. In at least one patient, Id-specific T cell immunity persisted beyond 18 months after completion of the vaccination schedule. All three surviving patients converted from a partial response to a complete response following BMT. Two of the three patients (both recipients of immune BMT from syngeneic twins) remain in complete remission 8 years and 7 years after the transplant, and the third patient developed chronic steroid-dependent GVHD and died of renal failure after 5.5 years while in complete remission from the disease. Our results suggest that Id vaccination induces specific T cell immunity in the donors that is transferable by BMT, is associated with a prolonged disease-free survival in recipients and may represent a novel strategy to enhance the graft versus myeloma effect. These encouraging results have prompted us to initiate a clinical trial to evaluate this approach of donor immunization in the context of non-myeloablative allogeneic peripheral blood stem cell transplantation in myeloma patients.
CD40 Ligand Stimulation of Multiple Myeloma Cells Results in Upregulation of Surface Expressed Heat Shock Proteins and Increased Antigenicity.
Authors: Gullo Charles A. , Hwang William , Au Melvin , Greenfield Edward A. , Anderson Kenneth C. , Teoh Gerrard
Published: October 2019

Effective immune-based therapies against the plasma cell malignancy, multiple myeloma (MM), are currently lacking. Identification of novel antigens (Ag) on the surface of MM cells to use as cellular targets for the destruction of cancer cells by the body’s immune system has been of great interest. We and others have demonstrated that CD40 stimulation of MM cells results in marked upregulation of membrane bound proteins such as Ku86. Using CD40 triggered MM cells as immunogens and hybridoma technology; we generated a monoclonal antibody (mAb), 6D11, that recognizes a CD40 induced cell membrane Ag on MM cells. This Ag is detectable on the surface of MM cells using indirect immunofluorescence flow cytometric analysis. Moreover, in Western immunoblotting assays, 6D11 mAb reacts with a 94 kDa protein, which is strongly associated with a 78 kDa protein. Using high performance liquid chromatography and protein microsequencing, we confirm that these proteins are the heat shock proteins (HSP), glucose-regulated peptide 94 (GRP94) and GRP78, respectively. These data were confirmed using co-immunprecipitation experiments. Furthermore, we demonstrate through indirect immunofluorescence flow cytometric analysis and quantitative real time reverse transcription polymerase chain reaction (RQ-PCR) that CD40 ligand (CD40L) stimulation of MM cells results in rapid upregulation of both GRP94 and GRP78. Since HSPs have been shown to play a role in both Ag presentation, as well as the intracellular transport of cellular Ags, it is tempting to speculate that cell membrane expression of tumor-specific peptides could also be induced via CD40 triggering. Accordingly, CD40 induced cell membrane HSP expression resulted in increased antigenicity as determined by increased co-stimulatory molecule expression on Ag presenting cells (APC) and by increased immunoreactivity in mixed lymphocyte reactions (MLR). This suggests that CD40 induced HSP expression may indeed result in increased recognition of MM cancer by the immune system. Our study therefore supports the development of CD40-based targeted cell therapies against MM.
In Vitro Activity of a Novel Small Molecule Cyclin Dependent Kinase Inhibitor, CYC202 (seliciclib or R-Roscovitine), in Multiple Myeloma (MM).
Authors: Raje Noopur , Kumar Shaji , Hideshima Teru , Ishitsuka Kenji , Roccaro Aldo , Yasui Hiroshi , Hamasaki Makoto , Shiraishi Norihiko , Chauhan Dharminder , Munshi Nikhil C. , Green Simon R. , Anderson Kenneth C.
Published: October 2019

Perturbation of cell cycle is central to tumorigenesis, resulting in unrestricted cell proliferation. Cyclin dependent kinase (CDK) inhibitors have the potential to induce cell cycle arrest, followed by apoptosis, in cancer cells. CYC202 (seliciclib or R-roscovitine; Cyclacel, Dundee, UK) is a potent inhibitor of CDKs currently undergoing phase II clinical testing. In vitro data has shown this tri-substituted purine to have greatest potency against CDK2/cyclin E, although it also inhibits CDK2/cyclin A, CDK7/ cyclin H, and CDK9/cyclin T. It has been tested against a broad range of tumor cell lines, ultimately inducing cell cycle changes and apoptosis. This orally bio-available compound has also induced regression of human tumor xenografts in nude mice, prompting its clinical testing. Phase I studies have demonstrated favorable pharmacokinetics and toxicity profiles, and phase II trials are currently ongoing in combination with gemcitabine /cisplatin for non-small cell lung cancer and as a monotherapy in B-cell malignancies. We have previously shown that MM cell lines demonstrate relatively low levels of p21WAF1, suggesting that CDKs are constitutively activated, thereby promoting uncontrolled cell cycle regulation, growth, and proliferation. Conversely, inhibiting CDK activity may therefore trigger MM cell growth inhibition. Here we studied the in vitro activity of CYC202 in MM cells. Our data demonstrates that CYC202 has potent cytotoxic effects against MM cells that are both sensitive (MM1.S, RPMI 8226, U266, H929) and resistant (MM1.R, Dox-40, LR5, MR 20) to conventional chemotherapy. MM cell line cytotoxicity, as evidenced by MTT assays, is noted at 24 hours, with IC50 ranging from 25–50 mmol. In contrast, this dose was not toxic to normal peripheral blood mononuclear cells. Cell cycle analysis demonstrated an increase (35–50%) in MM cells in sub-G1 phase at 24 hrs induced by CYC202 (25 mmol), suggesting that CYC202 triggers apoptosis. Caspase-8 and poly ADP-ribose polymerase (PARP) cleavage, evidenced by western blot analysis further confirmed apoptosis of MM cells. Importantly, CYC202 triggered a rapid down-regulation of MCL-1, a known anti-apoptotic protein in MM. Treatment of MM cells with CYC202 also resulted in decreased phosphorylation of retinoblastoma protein. Protein expression of certain CDKs, specifically CDC2, CDK4, and CDK6, were also down-regulated after treatment. Ongoing studies are delineating the specific signaling cascades affected by CYC202 treatment. These studies provide the pre-clinical basis for a clinical trial of CYC202, either alone or in combination with other agents to improve patient outcome in MM.
Zoledronic Acid Inhibits Protein Prenylation in Plasmacytoma Tumors In Vivo and Enhances Survival in the INA-6 SCID Mouse Model.
Authors: Guenther Andreas , Gordon Sharon , Bakker Frank , Burger Renate , Green Jonathan R. , Rogers Michael , Gramatzki Martin
Published: October 2019

Bisphosphonates such as zoledronic acid (ZOL) are effective at preventing osteolytic bone disease in patients with multiple myeloma. ZOL inhibits bone resorption by inhibiting FPP synthase and preventing the prenylation of small GTPases in osteoclasts. In vitro studies have demonstrated previously that ZOL can also directly affect the growth and viability of myeloma cells by inhibiting protein prenylation and therefore could, potentially, have a direct anti-tumour effect in vivo in addition to effects on osteoclasts. To examine this further, the effect of ZOL on six myeloma cell lines, including the IL-6 dependent INA-6 line, was investigated. ZOL caused cell cycle arrest and concentration-dependent growth inhibition in all six cell lines, with varying sensitivity (IC50= 30–285 μM).

The potential anti-tumour effect of ZOL on INA-6 cells in vivo was studied in a SCID mouse xenograft model, in which mice injected intraperitoneally with INA-6 cells develop plasmacytomas but do not develop significant osteolytic lesions. In several experiments involving more than 50 mice, ZOL was administered subcutaneously (sc) or intravenously (iv). ZOL, at a dose of 8 μg or 2 μg three times per week for two weeks after inoculation of INA-6 cells, significantly reduced tumour burden and increased survival of the mice (p= 0.002). The effect of ZOL on protein prenylation in plasmacytomas dissected from the mice was measured by western blotting to specifically detect the unprenylated form of the small GTPase Rap1A. Unprenylated Rap1A was virtually absent from tumour samples of untreated animals, while a single iv injection of 8 μg ZOL induced a marked accumulation of unprenylated Rap1A in the tumours after 24–72 hours.

These studies are the first to demonstrate that ZOL can inhibit protein prenylation in plasmacytomas in vivo. Together with the decreased tumour burden and increased survival, the data suggest that ZOL may have direct anti-tumour effects in this animal model. Thus, nitrogen-containing bisphosphonates such as ZOL may have therapeutic potential in multiple myeloma beyond the prevention of osteolytic lesions.
High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.
Authors: Kuchenbauer Florian C. , Kern Wolfgang , Schoch Claudia , Kohlmann Alexander , Hiddemann Wolfgang , Haferlach Torsten , Schnittger Susanne
Published: October 2019

Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.
Phase 3 Randomized Trial of Fludarabine/Cyclophosphamide Chemotherapy with or without Oblimersen Sodium (Bcl-2 Antisense; Genasense; G3139) for Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL).
Authors: Rai Kanti R. , Moore Joseph O. , Boyd Thomas E. , Larratt Loree M. , Golenkov Anatoliy K. , Koziner Benjamin , Chanan-Khan Asher , Bociek R. G. , Itri Loretta M.
Published: October 2019

Bcl-2 is an anti-apoptotic protein that has been closely linked to chemotherapy resistance and inferior survival in patients (pts) with CLL. Preliminary studies showed that oblimersen, an antisense oligonucleotide that targets Bcl-2, enhanced apoptosis induced by fludarabine (Flu), dexamethasone, and rituximab in cultured CLL cells. In a Phase 1 study, oblimersen displayed modest single-agent activity in CLL pts who had received extensive prior therapy. We conducted a randomized, multinational, Phase 3 trial of chemotherapy given with or without oblimersen in pts with advanced CLL. Eligibility included: symptomatic disease; intermediate or high-risk Rai stage; ≥ 1 Flu-containing regimen; ECOG PS ≤ 2; adequate organ function. Exclusions: stem cell/allogeneic transplant; autoimmune anemia/thrombocytopenia. Pre-randomization strata: prior treatment (1 or 2 vs. ≥ 3 regimens); response vs. refractory to prior Flu; and response duration to last therapy (> or ≤ 6 mos). All pts received Flu 25 mg/m2/d, plus cyclophosphamide (Cy) 250 mg/m2/d, daily for 3 days. Pts randomized to oblimersen received 3 mg/kg/d x 7 days by IV infusion, beginning 4 days before Flu/Cy and on each day of Flu/Cy treatment. Subsequent cycles were repeated every 28 days, for a maximum of 6 cycles, or to the time of CR, intolerable toxicity, or progression. All pts received prophylactic filgrastim, TMP/SMZ, and acyclovir. Response assessment was conducted at every cycle during treatment, and every 2 months thereafter for up to 36 months. The primary endpoint of the study was to compare the proportion of pts who achieved complete (CR) and nodular partial (nPR) responses. Secondary endpoints included: overall response rates (CR/nPR/PR); durable responses; response duration; time-to-progression; clinical benefit; and safety. Bone marrows in responding pts underwent central blinded review. A total of 241 pts were randomized; the last patient was enrolled in the 2nd quarter 2003. All pts have completed ≥ 1 year of follow-up, and results of this trial will be presented.
Perturbed Endocytosis by CALM-Containing Fusion Proteins: A Leukemogenic Mechanism in AML.
Authors: Chao Mwe Mwe , Walker Ann C. , Pendergast Molly B. , Bohlander Stefan K. , Wechsler Daniel S.
Published: October 2019

Background: We recently identified a novel fusion protein, MLL-CALM, in an infant with fatal AML. Although disruption of normal MLL function clearly contributes to leukemogenesis, perturbed CALM function may also be important. The native CALM protein plays a key role in clathrin-dependent endocytosis (CDE) and intracellular vesicle transport. Interestingly, CALM was first described as a partner for the AF10 gene - itself an MLL partner - in aggressive leukemias and lymphomas. Recently, it was shown that mutations in calm are responsible for hematopoietic abnormalities in inbred fit1 mice. Taken together, these observations suggest an important role for CALM in both normal and malignant hematopoiesis. We hypothesize that disrupted CDE resulting from the presence of CALM-containing fusion proteins interferes with downregulation of growth factor signaling, thereby contributing to leukemogenesis.

Objective: 1) To determine whether expression of MLL-CALM or CALM-AF10 interferes with endocytosis of transferrin (TF), 2) To analyze the importance of distinct CALM domains which affect endocytosis, and 3) To determine whether MLL-CALM expression reduces the IL-3 growth factor dependence of FL5.12 cells.

Design/Methods: The relative ability of leukemia cell lines that harbor MLL and CALM translocations to endocytose AlexaFluor633-conjugated TF was assessed by flow cytometry. The degree of Texas Red (TR)-TF endocytosis in COS7 cells expressing GFP-tagged MLL-CALM and CALM-AF10 was quantitated using densitometry. CALM deletion constructs were prepared and endocytosis of Texas Red-TF was examined in COS-7 cells transfected with these plasmids. Finally, IL-3-dependent FL5.12 cells engineered to stably express MLL-CALM were grown in decreasing amounts of IL-3-containing WEHI conditioned medium and survival was determined.

Results: U937 and P31/Fujioka cells (that harbor the CALM-AF10 translocation) showed reduced endocytosis of AF633-TF, but cells that lack CALM translocations (THP-1, K562, MonoMac6, HL-60) showed robust AF633-TF uptake. COS7 cells expressing MLL-CALM or CALM-AF10 showed 40–50% less endocytosis of TR-TF compared with untransfected cells or cells transfected with empty vector. CALM aa 484–620 appear to be important in inhibiting endocytosis. IL-3-dependent FL5.12 cells transfected with an MLL-CALM expression vector grew better in low concentrations of IL-3-containing WEHI conditioned media in comparison with FL5.12 cells transfected with empty vector.

Conclusions: Expression of MLL-CALM or CALM-AF10 interferes with endocytosis, and MLL-CALM expression renders FL5.12 cells less dependent on exogenous IL-3. The ability of MLL-CALM and CALM-AF10 to interfere with CDE may result in sustained growth factor signaling and contribute to the development of aggressive hematopoietic malignancies.
Identification of Genomic Imbalances in AML with Complex Karyotype Using Matrix-Based Comparative Genomic Hybridization.
Authors: Rucker Frank G. , Schwanen Carsten , Lipka Daniel , Wessendorf Swen , Frohling Stefan , Kestler Hans , Lichter Peter , Bentz Martin , Dohner Konstanze , Dohner Hartmut
Published: October 2019

Approximately 10 to 15 % of acute myeloid leukemia (AML) cases exhibit complex karyotypes, i.e., three or more chromosome abnormalities without presence of a specific fusion transcript. Using chromosome banding analysis, the majority of such cases cannot be completely described due to the low resolution of this method. Comparative genomic hybridization to microarrays (matrix-CGH) is a novel technique that allows genome-wide screening for genomic imbalances at high resolution and thus may facilitate the identification of novel regions harboring potential disease-related genes. We constructed a microarray consisting of 2799 different human genomic DNA fragments cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors. A set of 1502 of these clones covers the whole human genome with a physical distance of approximately 2 Mb. The remaining 1297 clones either contiguously span genomic regions known to be frequently involved in hematologic malignancies (e.g., 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q) (n=610) or contain oncogenes or tumor suppressor genes (n=687). In this study 45 AML cases with complex karyotypes were analyzed. Matrix-CGH detected genomic aberrations in all cases and genomic losses were found more frequently than gains. The most frequent aberration was deletion 5q in 38 of 45 cases. Three consensus regions were delineated mapping to chromosomal bands 5q11 (23 cases), 5q34 (22 cases), and 5q35 (21 cases). Further frequent deletions affected 17p (60%); two consensus regions were identified mapping to chromosomal bands 17p13 (60%), and 17p11-q11 (33%). Other chromosomal losses affected 7q (51%), 16p (31%), 16q, 18q (29% each), 15q (24%), 10p (22%), 14q, 4p (20% each), 11q, 20q (18% each), 3p, and 12p (15% each). The most frequent genomic gain was identified at 8q (40%). Further frequent gains were detected on 11q (38%), 21q (27%), 1p, 2q (24% each), 9p (22%), 6p, 19p (20% each), 3q (18%), 7p, 20p (15% each), 13q, and 20q (13% each). Genomic amplifications were identified on chromosomal band 8q24 including the MYC gene (2 cases), 9p24 (JAK2, 2 cases), 10p15 (PRKCO, 1 case), 11q23 (MLL, 2 cases), 11q23.3 (ETS1 and FLI1, 2 cases each), 12p13 (CCND2 and FGF6, 2 cases), 13q12 (CDX2, 3 cases), 20q11.1 (BCL2L, 1 case), 21p (1 case), 21q (2 cases), and 22q (1 case). These data demonstrate the potential of matrix-CGH with regard to spatial resolution and sensitive detection of genomic imbalances. Analysis of a large series of AML cases with complex karyotype may lead to the identification of novel critical regions and pathogenetically relevant genes.
Triptolide-Induced Apoptosis Is Dependent on Caspase Activation and NFκB Signaling and Mediated by XIAP and Survivin Downregulation through the Mitochondrial Pathway in Leukemic Cells.
Authors: Carter Bing Z. , Schober Wendy D. , McQueen Teresa , Evans Randall L. , Andreeff Michael
Published: October 2019

Triptolide, an immunosuppressor isolated from the Chinese herb, Tripterygium wilfordii Hook. F, has recently shown anti-tumor activities in a broad range of solid tumors. We examined its effects on leukemic cells and investigated mechanisms of apoptosis. Triptolide, at less than 100 nM, arrested cell growth and potently induced cell death in myeloid and lymphoid leukemic cells tested, including OCI-AML3, U937, Jurkat, KBM5, and K562 cells. In OCI-AML3 cells, triptolide induced caspase 3 activation, PARP cleavage and annexin V positivity with an IC50 of about 30 nM, at 24 hrs, all of which were inhibited by a general caspase inhibitor suggesting caspase dependent cell death. However, Triptolide-induced cell growth arrest was not affected by caspase inhibition. Treatment of OCI-AML3 cells with triptolide decreased XIAP and survivin expression, but did not affect Bcl2 and BclXL levels. Forced overexpression of XIAP attenuated Triptolide-induced cell death. Triptolide induced Bid cleavage, but Jurkat cells deficient in caspase 8 were only slightly less sensitive to triptolide than the wild-type counterpart indicating that Triptolide-induced cell death is caspase 8 independent. Jurkat cells deficient in receptor interacting protein (RIP) and therefore deficient in NFκB activation were resistant to Triptolide demonstrating that NFκB signaling is essential for Triptolide-induced cell death. Triptolide treatment induced cytosolic release of cytochrome C and loss of mitochondrial membrane potential, overexpression of Bcl2 effectively suppressed apoptosis induced by Triptolide, and caspase 9 knockout MEF cells were resistant to Triptolide suggesting criticality of the mitochondrial pathway. The antioxidants GSH (5 mM) and vitamin C (150 μM) did not protect from apoptotic cell death induced by Triptolide. In addition, Triptolide-induced apoptosis of blast crisis CML KBM5 cells was independent of their sensitivity or resistance to Imatinib: Triptolide killed Imatinib resistant KBMSTI cells as effectively as Imatinib sensitive KBM5 cells. Ex vivo studies showed that Triptolide also induced cell death in primary AML blasts. Collectively, our studies demonstrate that Triptolide potently induces caspase-dependent apoptosis and arrests cell growth in leukemic cells. Triptolide-induced cell death is dependent on NFκB signaling, and mediated by downregulation of XIAP and survivin through the mitochondrial pathway. The potent anti-leukemic activity of Triptolide in vitro warrants further investigation of this compound for the treatment of leukemia and other malignancies. This drug may also be potentially useful in overcoming Imatinib resistance in CML and Philadelphia chromosome positive ALL.
Reoxygenation Induces Premature Senescence of FA Hematopoietic Progenitor/Stem Cells through a Pathway Involving ATM, P53, and P21CIP1/WAF1.
Authors: Zhang Xiaoling , Li June , Sejas Daniel P. , Pang Qishen
Published: October 2019

Hematopoietic cells are often exposed to transient hypoxia as they develop and migrate between blood and tissues. We sought to test the role of continuous cycles of hypoxia and reoxygenation in the pathogenesis of bone marrow failure in Fanconi anemia (FA). We isolated Lin− BM cells from wild-type (WT) and Fancc−/− mice, and subjected them to two cycles of hypoxic (1% O2) then reoxygenation (20% O2). Expansion of Fancc−/− Lin− BM cells was significantly decreased in response to the oxidative stress of reoxygenation, as compared with WT Lin− BM cells (1.3 vs. 4.9-fold by day 6, respectively). This inhibition was attributable to a marked decrease of Fancc−/− progenitor (Lin− ScaI− c-kit+) cells, as well as a slower expansion of Fancc−/− stem (Lin− ScaI+ c-kit+) cells than the WT stem cells following reoxygenation. Fancc−/− Lin− BM cells transduced with FANCC retroviruses exhibited a significant growth advantage to untransduced cells, particularly in hypoxia and reoxygenation conditions where the predominance of FANCC/GFP+ progenitors was observed in both the short-term liquid culture and clonogenic assays. There was no evidence of increased apoptotic death in these reoxygenated Fancc−/− BM progenitor and stem cells compared to their WT counterparts, as assessed by a flow cytometric method for caspase 3 activation (early apoptosis). Interestingly, evaluation of the apoptotic profile and cell cycle of stroma-supported long-term BM culture (LTBMC; two weeks after second reoxygenation) revealed that reoxygenated Fancc−/− LTBMC cells had reduced apoptosis compared to reoxygenated short-term culture. However, a vast majority (70.6%) of reoxygenated Fancc−/− LTBMC cells was residing in the G0 + G1 phases compared with 55.8% in WT LTBMC cells. Fancc−/− LTBMC cells stained intensely for SA-b-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21CIP1/WAF1 and to a lesser extent, p16INK4A. Moreover, reoxygenation-induced phosphorylation of p53ser15 was dependent on the ATM kinase but not ATR, as inhibition of ATM signaling by the kinase inhibitor 2-aminopurine or siRNA knockdown of ATM decreased p53ser15 phosphorylation and reduced cell senescence. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc−/− BM hematopoietic progenitor and stem cells by signaling through the ATM to p53, upregulating p21 and causing senescent cell cycle arrest. In addition, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic stem cell depletion and BM failure in FA. <B style=%
Investigations on the Mechanisms of Resistance to Pixantrone in Tumor Cells.
Authors: Hazlehurst Lori A. , Alsina Melissa , Hacker Miles P.
Published: October 2019

Pixantrone is an aza-anthracenedione currently undergoing Phase 3 clinical trials and has shown significant activity in non-Hodgkin’s lymphoma. Preclinical studies with the aza-anthracenediones demonstrated a remarkable structure activity requirement, i.e., the ring nitrogen must be in the 2 position for anti-tumor activity. More importantly, Pixantrone which lacks hydroxyl groups at either the 1 or 4 positions of the chromophore, lacked any cardiotoxicity in pre-clinical trials. These pre-clinical findings have been substantiated during clinical development of Pixantrone. Given that there is a very real possibility that Pixantrone will progress to broader clinical use, we reasoned that understanding potential mechanisms by which tumor cells may become resistant to the drug were essential. By continuous in vitro exposure of MCF-7 cells to increasing concentrations of Pixantrone, we have developed a cell line (MCF7/aza) 20 fold resistant to the drug. These cells are cross resistant with mitoxantrone, surprisingly to a much great degree of resistance to mitoxantrone than to Pixantrone. Two compounds BBR 3438 and BBR 3576, represent a new class of anticancer drugs- the aza-anthrapyrazoles- have entered clinical trials and both were cross resistant in the MCF 7/aza cells. Using western blotting techniques we have demonstrated that the MCF 7/aza cells express elevated levels of BCRP but not Pgp. Further, resistance to Pixantrone was reversed with fumitremorgin C, a classic BCRP inhibitor that reverses mitoxantrone resistance in BCRP expressing cells. Resistance to Pixantrone was not reversed by verapamil, the classic Pgp inhibitor that reverses mitoxantrone resistance in MDR cells. Microscope evaluation of cells treated with Pixantrone demonstrated a unique intracellular distribution of the drug in MCF 7/aza cells in that the drug was sequestered in cytoplasmic vesicles rather than in the nucleus. More recently wehave completed a microarray analysis of MCF 7/Aza compared to MCF 7 cells and not surprisingly found a large number of genetic alterations in the resistant phenotype. One of the most striking alterations was a greater than 30 fold increase in BCRP. The results of our studies indicate that Pixantrone, a non-cardiotoxic mitoxantrone analogue, induced a resistant phenotype more closely related to that seen with mitoxantrone than the multi-drug resistance seen with many other anti-cancer drugs. It is of interest that an aza-anthracenedione, BBR 2378, was not cross resistant in MCF 7/aza cells. The unique structural feature of BBR 2378 is the presence of tertiary amines at the terminus of both sides arms of the compound in contrast to Pixantrone, BBR 3438, BBR 3576 or mitoxantrone all of which lack tertiary amine side arms. BBR 2378 has been previously been shown to be active in MDR cell linesas well. These results suggest that side arms play a pivotal role in determining activity or resistance in a variety of drug resistance tumor phenotypes.
Anemia and Ineffective Erythropoiesis Caused by Somatic RAS Mutation.
Authors: Braun Benjamin S. , van Ziffle Jessica , Gavin Joehleen , Tuveson David A. , Jacks Tyler , Shannon Kevin
Published: October 2019

Ras proteins modulate cell fates by integrating extracellular stimuli and transducing signals that regulate proliferation, survival, and differentiation via downstream effector cascades. Somatic activating mutations in RAS oncogenes are found in approximately one third of cases of myelodysplastic syndrome (MDS), and are common in myeloproliferative disorders (MPDs) such as CMML and JMML that frequently have a dysplastic component. We have modeled these human diseases in the mouse using a conditional allele of oncogenic Kras, which is activated in mice bearing the inducible Mx1-Cre transgene by injection with polyinosinic-polycytidilic acid (pI-pC). Mice treated with pI-pC to induce K-rasG12D expression at 21 days of life develop not only myeloid proliferation, but also progressive anemia which is the likely cause of death with median survival 84 days after injection. Erythroid precursors are abundant in spleens of anemic mice, suggesting ineffective erythropoiesis. These features are highly reminiscent of human MDS, and suggest that anemia in MDS is a direct consequence of hyperactive RAS signaling. Further analysis reveals that although erythroblasts are absent from the bone marrow, earlier erythroid progenitors such as BFU-E and CFU-E are abundant in both bone marrow and spleens of anemic mice. However, despite hyperproliferation of primitive erythroid cells, there is a block in terminal erythroid differentiation. This is also seen in primary E14.5 fetal liver cells acutely induced to express K-rasG12Din vitro, and in wild type recipients of Mx1-Cre, KrasG12D bone marrow, demonstrating this to be a cell-intrinsic property of erythroid progenitors expressing oncogenic Ras. Together, these findings support a model in which RAS mutations in MDS both confer a proliferative advantage to a neoplastic clone as well as directly impede differentiation, resulting in ineffective erythropoiesis and anemia. The improved efficiency of differentiation observed in spleen relative to bone marrow also suggests that microenvironmental influences modify the cell-intrinsic effects of oncogenic Ras.
Inhibition of p38 MAPK by SCIO-469 Suppresses TNF Generation and Promotes CD34+ Cell Survival in an In Vitro MDS Cell Culture Model.
Authors: Navas Tony A. , Nguyen Aaron N. , Ma Jing Y. , Stebbins Elizabeth G. , Haghnazari Edwin , Heaton Ruth , List Alan F. , Higgins Linda S.
Published: October 2019

Progress in the development of more effective therapeutics for myelodysplastic syndrome (MDS) has been limited by the lack of targets critical to the pathobiology of the disease. Ineffective hematopoiesis in MDS is characterized by accelerated proliferation and premature apoptotic death of progenitors and their progeny that is potentiated by the local generation of inhibitory molecules, including TNFa, TGFß, FasL, and VEGF. To identify upstream regulatory signals that may coordinate activation of inhibitory molecules, we used an in vitro cell culture model incorporating a CD34+ MDS cell line isolated from a RAEB-t patient, normal bone marrow stromal cells (BMSC), and/ or bone marrow mononuclear cells (BMMNC) to determine effects of cell-cell interactions on secretion of inhibitory hematopoietic cytokines. The role of p38 MAP kinase, a regulatory kinase involved in the convergence of inhibitory cytokine activation and signaling, was evaluated in this interaction. We found that p38 MAPK is induced under basal culture conditions in the MDS cell line and is further activated by TNFa or TGFß. In all cases, p38 activation was reduced by SCIO-469, a potent and specific inhibitor of p38a activity. SCIO-469 does not directly block p38 activation, suggesting a feedback loop is interrupted when p38 kinase activity is inhibited in MDS cells. To determine the effects of cellular interactions, the MDS cell line was co-cultured with either BMSC, BMMNCs or both from normal donors, and TNFa and FasL secretion were measured after 3 days incubation. TNFa and FasL were detected in culture supernatants when the MDS cell line was co-cultured with BMMNC but not when co-cultured with BMSC. TNFa secretion by BMMNCs was dependent on MDS cell contact and was significantly inhibited by SCIO-469. The addition of BMSC to the MDS and BMMNC co-culture prevented TNFa elevation, suggesting BMSCs as a dominant source for anti-inflammatory signal(s). VEGF, FGF-ß, TGFß2, BDNF, TIMP-1, TIMP-2 and IL-6 secretion by BMSC was induced by MDS co-culture, whereas SCIO-469 blocked cytokine induction. To determine the effects of SCIO-469 and MDS clone-induced BM cytokine secretion on normal CD34+ proliferation, we co-cultured BMMNCs and BMSC in transwell inserts in the presence or absence of the MDS cell line with or without SCIO-469. CD34+ proliferation was assessed in cells cultured in outer wells. CD34+ progenitors proliferated in culture at the same rate as those co-cultured with BMSC, BMMNC and MDS for 6 days. At longer intervals, viability of progenitors cultured with the MDS line declined, whereas treatment with SCIO-469 abrogated the decrease in CD34+ viability. These results implicate p38a as a critical target in the induction of pro-apoptotic cytokines in MDS, and that selective inhibition of p38 by SCIO-469 may provide a novel therapeutic strategy for MDS.