bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Multiple Myeloma Cell Adhesion to Fibronectin by Ephrin Ligation.
Authors: Ratner Stuart , Schiffer Charles A. , Zonder Jeffrey A.
Published: October 2019

Multiple myeloma (MM) cell adhesion to fibronectin (FN), mediated via VLA-4 and VLA-5, has been shown to induce resistance to several chemotherapeutic drugs. Disruption of MM cell adhesion to FN and other marrow microenvironment elements might therefore enhance the effects of therapy. We now present the first evidence that Eph-ephrin signaling may be exploited to inhibit MM cell binding to fibronectin. Ephs are transmembrane tyrosine kinases and ephrins are their cell-surface ligands. There are two classes of Ephs and ephrins, A and B. Both Ephs and ephrins can transduce repulsive signals that cause interacting cells to lose contact with each other and with extracellular matrix. We are not aware of any previous systematic study of Eph and ephrin expression or function in MM cells. We have found MM cell lines H929, U266, and RPMI 8226 express members of the A classes of both Ephs and ephrins, but not the B classes. First, we demonstrated ligation with commercially available anti-ephrin A3 antibody was followed by ephrin capping and shedding from the cell surface. We next explored whether ephrin ligation affects MM cell adhesiveness in culture. Whereas H929, U266, and RPMI 8226 cells adhered rapidly to fibronectin-coated plastic surfaces, all three cell lines failed completely to adhere to a mixed coating of FN and rabbit anti-ephrin A3 antibody for a period of 2 hrs. This effect was not seen with FN + normal rabbit Ig. This suggests binding of ephrin A3 (or another cross-reacting A-class ephrin) by solid-state antibody triggers intracellular signals that interfere with initial steps of integrin-mediated adhesion. After 2 hr, spontaneous partial recovery of adhesion occurred, reaching a plateau of approximately 30% of control values by 24 hr. We postulate this recovery occurs via clipping of the extracellular ephrin domain by transmembrane metalloproteases, since recovery of FN adhesion was partially prevented by the metalloprotease inhibitor GM6001 (25 uM). Also consistent with this theory, we found in a separate experiment that GM6001 reduced the shedding of cross-linked A-class ephrins from MM cell lines. In summary, we have demonstrated that manipulation of EPH-ephrin signaling can impair MM-cell adhesion to FN, and that this effect is enhanced by simultaneous inhibition of metalloprotease activity. We are currently studying the effect of A-class ephrin ligation on adhesion-mediated drug resistance in MM cell lines. We also intend to evaluate EPH-ephrin expression in marrow specimens from patients with MM.
Increased Peripheral Platelet Destruction and Caspase-3-Independent Programmed Cell Death of Bone Marrow Megakaryocytes in Myelodysplastic Patients.
Authors: Houwerzijl Ewout J. , Blom Nel R. , van der Want Johannes J.L. , Louwes Henk , Esselink Mariet T. , Smit Jan W. , Vellenga Edo , de Wolf Joost Th.M.
Published: October 2019

To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelets <100 x 109/L. Furthermore, plasma thrombopoietin and glycocalicin-index (a parameter of platelet or megakaryocyte destruction) were determined. Mean platelet life (MPL), corrected for the degree of thrombocytopenia, was reduced in 50% of the patients (4.3 ± 0.9 days (mean ± SD) vs 6.0 ± 1.3, p=0.0003). Platelet production rate (PPR, i.e. the number of platelets entering the circulation to maintain the platelet count) was reduced in 83% (68 ± 34 x 109/d vs 220 ± 65, p<0.0001), although 80% of these patients had normal or increased numbers of bone marrow megakaryocytes. Thrombopoietin levels (patients 223 ± 194 vs controls 118 ± 33 pg/ml, p=0.04) were not significantly correlated with the PPR. However, the glycocalicin-index was significantly higher compared to controls (15 ± 16 vs 0.7 ± 0.2, p=0.001) and significantly and inversely correlated with the PPR (p=0.02, r=−0.5), but not with the MPL (p=1.8). These findings indicate to ineffective thrombopoiesis as a result of increased destruction of platelets and megakaryocytes in the bone marrow leading to a diminished release of platelets to the peripheral blood (i.e. PPR). To investigate whether programmed cell death (PCD) of bone marrow megakaryocytes might be responsible for this ineffective thrombopoiesis ultrastructural studies were performed in 5 patients MDS patients. In the majority of megakaryocytes (66 ± 15%) ultrastructural features of necrosis-like PCD were found, without signs of apoptotic or apoptotic-like PCD, especially in mature megakaryocytes. The data correspond with a negative immunohistochemical staining for activated caspase-3 of bone marrow megakaryocytes. These results indicate that the main cause of thrombocytopenia in MDS might be caspase-3-independent necrosis-like PCD resulting in a decreased PPR in conjunction with an increased glycocalicin-index.
A Phase I/II Multicenter, Safety and Efficacy Study of Combination Treatment with Melphalan, Arsenic Trioxide and Vitamin C (MAC) in Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Berenson James R.
Published: October 2019

Background: An urgent need exists for new treatments to overcome chemoresistance in MM patients. Recent in vitro and In vivo studies in our laboratory show that arsenic trioxide (ATO) can sensitize chemoresistant MM cells to melphalan-induced cytotoxic effects. Pre-clinical studies also show the most profound anti-MM effects when ATO, ascorbic acid and melphalan are used in combination compared with the effects observed when the drugs are used alone or combinations of any two of these agents. Based on encouraging results from a pilot study1, a larger, multicenter trial was recently started.

Methods: MM patients who showed relapse after responding to first-line chemotherapy and/or having proved to be refractory to chemotherapy are enrolled. Patients received a loading dose of ATO at 0.25 mg/kg IV followed by ascorbic acid 1 g IV days 1–4 of week 1 of each six-week cycle. ATO/ascorbic acid was given twice-weekly for the next 4 weeks of each cycle. Low-dose melphalan (0.10 mg/kg) was administered orally for the first 4 days of each cycle. Patients received a maximum of 6 cycles followed by weekly maintenance treatment with weekly ATO followed by ascorbic acid. The primary objectives of this study are to determine response rate and safety and tolerability of MAC therapy.

Results: Twenty patients have received at least one treatment cycle. Preliminary data show that eight (4 PR, 4 MR) of the 14 evaluable patients (57%) had an objective response, an additional three patients achieved stable disease, resulting in a total of 11 patients (79%) with disease control. Since responses were seen after 2 to 5 treatment cycles, it is possible that some patients with stable disease may experience additional disease response. Seven of the eight responding patients had failed two or more treatments: five patients had received prior thalidomide therapy, two had received melphalan and bortezomib, and two patients had undergone autologous peripheral stem cell transplantation. Of the six patients who have now completed the maximal numbers of cycles, four achieved PR, one MR, and one SD. The regimen was well tolerated with few significant side effects reported; mild cytopenias were reported as reversible.

Conclusions: These preliminary results in this treatment group of heavily pre-treated MM patients who had either relapsed or were refractory to standard and/or investigational multiple myeloma treatments suggests that the MAC treatment regimen (1) shows efficacy using a low dose of melphalan supporting the preclinical evidence that ATO can sensitize tumors to chemotherapy; (2) is well tolerated; (3) may require multiple cycles before response.
A Monoclonal Antibody Specific for Free Human Kappa Light Chains Induces Apoptosis of Multiple Myeloma Cells and Exhibits Anti-Tumor Activity In Vivo.
Authors: Asvadi Parisa , Jones Darren R. , Dunn Rosanne D. , Choo Andre B.H. , Raison Matthew J. , Raison Robert L.
Published: October 2019

Despite high dose chemotherapy and autologous stem cell transplant, multiple myeloma (MM) remains an incurable malignancy, with a median 5 year survival of less than 20%. With the exception of idiotype, few antigen targets have been identified that would facilitate specific immunotherapy of MM. We have previously described a murine monoclonal antibody that recognizes a conformation-dependent epitope on free human kappa light chains and a cell surface antigen, KMA, expressed on kappa MM plasma (MMκ) cells (Raison, RL and Boux, HA Mol Immunol 1985 22:1393). Here we show that the murine antibody, designated mKap, bound specifically to a range of MMκ cell lines and inhibited the in vitro growth of these cells. Flow cytometric analysis (Annexin-V and PI staining) of MMκcell lines incubated with mKap demonstrated a dose dependent induction of apoptosis. Furthermore, the presence of activated caspases in mKap treated cells was detected using the CaspACE™ FITC-VAD-FMK reagent. The induction, by mKap, of apoptosis in MMκ cells occured in the absence of cross-linking second antibody or effector cells. In vivo, anti-tumor activity by mKap was demonstrated in a SCID mouse tumor xenograft model. Tumor growth was measured by quantitation of secreted myeloma Ig over a period of 6 weeks. From week 4 onwards, significantly lower serum concentrations of myeloma Ig were detected in animal groups receiving 3.0, 1.5, 0.3 and 0.15 mg total mKap compared to the untreated control (P<0.005 at week 4; P<0.0001 at week 5). The tumor-restricted specificity of mKap, coupled with its ability to inhibit MMκ cell growth in vitro and in vivo, suggests the potential use of either a chimeric or humanised version of this antibody, alone or in combination with chemotherapy, for the treatment of kappa MM.
Genes Regulated by NPM-ALK Fusion Kinase Play a Key Role in the Activation of AP-1 Transcription Factors.
Authors: Staber Philipp B. , Linkesch Werner , Schauer Silvia , Moser Gerit , Kadin Marshall E. , Dirks Willi , Hoefler Gerald
Published: October 2019

Background: About half of nodal anaplastic large cell lymphomas (ALCL) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is the product of a t(2;5)(p23;q35) chromosomal translocation. Expression of this protein has been shown to result in neoplastic change. Combining suppression subtractive hybridization (SSH) and cDNA microarray analysis we aimed at elucidating the consequences of NPM-ALK expression.

Methods: SSH cDNA libraries were constructed using mRNA from human embryonic kidney (293) cells transfected with active or kinase-dead NPM-ALK constructs, as well as pools of NPM-ALK positive and negative ALCL cell lines. The resulting cDNA clones and genes relevant for cancer pathogenesis were spotted, generating specific cDNA microarrays comprising 4992 genes. mRNA expression patterns were analyzed in individual cell lines. Real time quantitative RT-PCR of 20 selected genes validated the mRNA expression data of the microarrays.

Results: Expression of a set of 102 genes distinguishes NPM-ALK-negative (FE-PD, MAC-2A) from NPM-ALK-positive ALCL cell lines (SU-DHL-1, JB-6, SUP-M2, SR-786, DEL and Karpas 299). The majority are involved in regulation of cell cycle and apoptosis. 38 of these genes also discriminate 293 cells with respect to their NPM-ALK expression. Interestingly, AP-1 target genes, such as GM-CSFRA, GM-CSFRB, ARF5, FAS, FASL, and BCL3, are increased in NPM-ALK expressing cell lines. Electrophoretic mobility shift assay (EMSA) verifies NPM-ALK dependent AP-1 DNA binding activity.

Conclusion: This study reveals genes specifically regulated by NPM-ALK lymphoma kinase. Further, we demonstrate that AP-1 activation is a critical target of NPM-ALK signalling.
PKC412 Inhibits the ZNF198-FGFR1 Fusion Tyrosine Kinase and Is Efficacious in Treatment of t(8;13)(p11;q12) Associated Stem Cell Myeloproliferative Disease.
Authors: Chen Jing , DeAngelo Daniel J. , Kutok Jeffery L. , Williams Ifor R. , Lee Benjamin H. , Wadleigh Martha , Duclos Nicole , Cohen Sarah , Adelsperger Jennifer , Okabe Rachel , Coburn Allison , Galinsky Ilene , Huntly Brian J.P. , Cohen Pamela , Meyer Thomas , Fabbro Doriano , Banerji Lolita , Griffin James D. , Xiao Sheng , Fletcher Jonathan A. , Stone Richard M. , Gilliland D. Gary
Published: October 2019

Human stem cell leukemia-lymphoma syndrome usually presents as a myeloproliferative disease (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-FGFR1 fusion tyrosine kinase. Current empirically-derived cytotoxic chemotherapy is inadequate treatment of this disease. We hypothesized that small molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo. Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLCγ, STAT5 and PI3K/AKT, required the proline-rich, but not the zinc-finger domains of ZNF198. A small molecule tyrosine kinase inhibitor, PKC412 (N-benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1 induced myeloproliferative disease. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disease with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.
FLT3 Expression Is Increased by MEIS1 and Collaborates with NUP98-HOX Fusion Genes in the Induction of Acute Myeloid Leukemia.
Authors: Palmqvist Lars , Pineault Nicolas , Argiropoulos Bob , Wan Adrian , Humphries Keith R.
Published: October 2019

The TALE family member and HOX cofactor MEIS1 is important in leukemic transformation. MEIS1 has, although non-leukemogenic on its own, been shown to strongly collaborate with several HOX genes and NUP98-HOX fusions to induce acute myeloid leukemia (AML). We have recently described a novel in vitro culture system of cell lines established from murine primary bone marrow cells transduced with the AML-associated fusion gene NUP98-HOXD13 or an engineered NUP98-HOXA10 fusion. These pre-leukemic NUP98-HOX cell lines are transplantable and can efficiently be converted into AML-inducing cells upon MEIS1 transduction. Conveniently, the MEIS1 transduced cells can be purified and preserve their leukemogenic potential even after extensive in vitro expansion. Thus, the availability of the NUP98-HOX cell lines system provided the opportunity to investigate and characterize the mechanism of MEIS1-mediated AML-conversion. Potentially interesting target or candidate genes were screened for expression changes between the parental pre-leukemic lines and AML-inducing MEIS1 transduced cell lines with quantitative RT-PCR and Western blotting. Aberrant expression or mutations of the receptor tyrosine kinase FLT3 gene is a common finding in human AML. Interestingly, Flt3 was found induced 5 to 10 fold in MEIS1 transduced cell lines compared to the parental cell lines. The observed increase in Flt3 expression provided the MEIS1 transduced cells with Flt3 ligand driven growth. This was not seen in the parental cell lines, which could not proliferate with Flt3 ligand as single cytokine or with a MEIS1-homeodomain mutant expressing cell line. Importantly, the Flt3 inhibitor AG1295 could block the proliferative effect of the Flt3 ligand in the MEIS1 transduced cell lines. To test whether Flt3 could substitute for MEIS1-mediated induction of AML in NUP98-HOX pre-leukemic cells, a NUP98-HOXA10 cell line was transduced with an MSCV-Flt3-IRES-YFP construct. The resulting Flt3-transduced cells were shown to express Flt3 at levels similar to that of MEIS1 transduced cells without any significant increase in endogenous Meis1 expression. Transplantation of these cells into mice led to lethal and transplantable AML with a median disease onset of 116 days (n=8) compared to 59 days for MEIS1 (n=4), whereas control transplants remained healthy (n=2). In conclusion, this study demonstrates that MEIS1 can induce Flt3 expression and that Flt3 can collaborate with NUP98-HOX fusion genes in the induction of acute myeloid leukemia. Furthermore, theses results suggest a model in which the leukemogenic synergism of MEIS1 on HOX-mediated leukemia might in part be mediated through FLT3-dependent pathways.
The Dimerization Domain and C-Terminal NCoR/SMRT Interacting Zinc-Finger Domain of t(8;21) Fusion Protein AML1-ETO Have Critical and Opposite Effects on Leukemogenesis.
Authors: Yan Ming , Kanbe Eiki , Peterson Luke F. , Boyapati Anita , Hines Robert , Zhang Dong-Er
Published: October 2019

Previous analyses demonstrated that full length AML1-ETO requires additional mutagenic events to promote leukemia. Here we describe a novel shorter form of the AML1-ETO fusion protein (AML1-ETOtr), which lacks C-terminal NHR3 and NHR4 [NCoR/SMRT interacting zinc-finger (ZnF)] domains. Unlike full length AML1-ETO, expression of AML1-ETOtr results in rapid development of acute myeloid leukemia (AML) in mice. Interestingly, naturally occurring alternatively spliced AML1-ETO variants exist in t(8;21) AML patient samples. AML1-ETOexon9a that includes one additional exon Exon 9a and AML1-ETOintron5 that uses a polyadenylation signal in Intron 5 are two such variants. AML1-ETOexon9a is structurally similar to AML1-ETOtr. AML1-ETOintron5 lacks the NHR2 heterodimerization domain as well as missing the NHR3 and NHR4 domains. To elucidate their putative role in leukemia development, we performed retroviral transduction and transplantation experiments with AML1-ETOexon9a, AML1-ETOintron5, and two zinc-finger domain (NHR4) mutants [AML1-ETOdZnF (deleted ZnF) and AML1-ETOmZnF (mutated ZnF)]. All of the mice transplanted with AML1-ETOexon9a (n = 8), AML1-ETOdZnF (n = 7), and AML1-ETOmZnF (n = 12) expressing cells rapidly developed AML, displaying a similar phenotype to AML1-ETOtr. None of AML1-ETOintron5 (n = 8) mice showed leukemia. These results suggest that the NHR2 domain of AML1-ETO is required whereas the NHR4 NCoR/SMRT interacting zinc finger domain is inhibitory for leukemia development. These observations suggest a novel mechanism of leukemogenesis for the AML1-ETO gene that can transcribe both tumor suppressive and/or oncogenic chimeric protein(s) by alternative RNA splicing.
Vaccination with the PR1 Leukemia-Associated Antigen Can Induce Complete Remission in Patients with Myeloid Leukemia.
Authors: Qazilbash Muzaffar H. , Wieder Eric , Rios Rosa , Lu Sijie , Kant Shreya , Giralt Sergio , Estey Elihu H. , Thall Peter , de Lima Marcos , Couriel Daniel , Champlin Richard E. , Komanduri Krishna , Molldrem Jeffrey J.
Published: October 2019

BACKGROUND: Several studies have established the PR1 peptide (VLQELNVTV) as a human leukemia-associated antigen. PR1 is derived from proteinase 3, an aberrantly expressed protein in myeloid leukemia cells, and can be presented on HLA-A2 to cytotoxic T lymphocytes (CTL) that preferentially kill leukemia over normal hematopoietic progenitors.

METHODS: Thirty-five HLA-A2+ patients with AML, MDS and CML were vaccinated with the PR1 peptide in an incomplete Freund’s adjuvant (Montanide ISA-51) and 75 mg of GM-CSF. Eligibility included AML with smoldering disease or ≥ 2nd CR, CML not responding to upfront treatment or relapsed disease, and MDS with ≥ 5% blasts. Patients were assigned at random to receive 0.25 mg, 0.50 mg, or 1.0 mg PR1 SQ every 3 weeks for 3 total injections. Immune responses (IR) were assessed by PR1/HLA-A2 tetramer staining and intracellular IFN-γ production by CTL, and clinical responses were assessed by bone marrow biopsy before study entry and 3 weeks after the 3rd vaccination.

RESULTS: Thirty-five patients with a median age of 50 (26–82) were treated at a median of 26 months from time of diagnosis, and follow up was 1 to 4 years. Toxicity was limited to grade 1 and 2 injection site reactions. Overall survival was 33% at 4 years. Immune responses (IR) were elicited in 20 of 33 evaluable patients (60%). Of 16 patients with relapsed or refractory AML, there were 4 (25%) clinical responses (3 CR, 1 PR). All 4 AML patients treated during CR remain in CR. Of 10 CML patients, there was 1 cytogentic CR. Three CML patients refractory to allogeneic transplant, interferon and imatinib had stable disease with some hematological improvement since these patients were able to discontinue hydroxyurea and anagrelide. Of 5 MDS patients, there was 1 PR (17% blasts reduced to 4%). Overall survival at 4 years was 14 of 20 patients with IR vs. 0 of 13 without IR (p < 0.0001). Progression-free survival for patients with or without IR was 6.4 months vs. 2.4 months, respectively (p = 0.003).

CONCLUSION: PR1 peptide vaccination is safe and can elicit both immunological and clinical responses in patients with refractory and relapsed myeloid leukemia, which improves progression-free survival of patients with IR to PR1. This is the first study to show complete molecular remission induced by peptide vaccination. These results warrant further study of immunization strategies in the treatment of leukemia.
Inhibition of Breast Cancer and Leukemia Cell-Induced Endothelial Angiogenesis by Low Molecular Weight Heparins (LMWH).
Authors: Marchetti Marina , Vignoli Alfonso , Pagnoncelli Marcella , Falanga Anna
Published: October 2019

Background: Clinical studies have suggested a survival advantage associated with LMWH administration in cancer patients. Angiogenesis is an important step in tumor progression and it has been demonstrated that LMWH can affect in-vitro vessel growth induced by purified proangiogenic factors.

Objective: In this study we investigated whether two different LMWHs, enoxaparin (ENX) and dalteparin (DLT), and unfractionated heparin (UFH), were able to counteract the tube formation capacity of human microvascular endothelial cells (HMEC-1) induced by tumor cell conditioned media (TCM) or by purified factors (i.e. VEGF, bFGF, TNF-alpha and IL-1beta).

Methods: TCM were obtained after a 24h growth from three breast cancer cell lines, i.e. MCF-7, ZR75.1 and MDA.MB.231, and one human promyelocytic leukemia cell line, i.e. NB4 cells. HMEC-1 cells were resuspended in TCM or purified cytokine solutions in the presence or absence of 10 IU/ml heparins (DLT, ENX or UFH) and then seeded in a 96-well plate pre-coated with growth factor-reduced Matrigel. After 24h, tube formation was examined under phase-contrast microscopy and tube length determined by image analysis software. To characterize TCM cytokine contents, TCM were first screened for their VEGF, bFGF, TNF-alpha and IL-1beta levels by ELISA, and then inhibition studies were performed by pre-incubating TCM with specific neutralizing antibodies directed against each of these factors.

Results: TCM from all 4 tumor cell lines induced a significant (p<0.05) increase in total tube length (60 to 95% mean increase) compared with control TCM. Purified proangiogenic factors significantly (p<0.05) promoted tube formation (46 to 68% mean increase), although to a lesser extent than TCM. Angiogenesis induced by each TCM was significantly affected (76 to 100% mean inhibition) by ENX and DLT, but was less sensitive to UFH. A similar pattern of inhibition was observed with tube formation induced by purified angiogenic factors, particularly the angiogenesis induced by VEGF, bFGF, TNF-alpha or IL-1b was 100% inhibited by ENX and 65–75% by DLT, whereas minor or no inhibition was observed with UFH (VEGF= 27%; bFGF = 0%; TNF-alpha= 53%; IL-1beta= 53%). Inhibition studies with neutralizing MoAbs against proangiogenic factors demonstrated that anti-VEGF MoAb was the most effective in inhibiting tube formation induced by both breast cancer and leukemic cells (77 to 84% mean inhibition).

Conclusion: Our data indicate that heparins can inhibit the proangiogenic stimulus of both tumor cells and purified angiogenic factors on microvascular endothelium, and that the LMWHs are more effective than UFH in this experimental system.
Reduced Plasma Fibronectin Leads to Delayed Thrombus Growth in the Injured Arterioles in Vivo.
Authors: Matuskova Jana , Cambien Beatrice , Piffath Crystal , Wagner Denisa D.
Published: October 2019

In addition to von Willebrand factor and fibrinogen, plasma fibronectin (pFN) contributes significantly to thrombus development in arteries. Complete deficiency of plasma fibronectin (in pFN conditional knockout mice) affects thrombus formation and growth and subsequent occlusion of injured arterioles in vivo. We wanted to study a more physiological decrease of plasma fibronectin without having to induce interferon production in the mice to excise the FN gene. Reduced pFN levels are common in patients with liver disease, sepsis and following trauma or surgery. To evaluate the effect of a reduced amount of plasma fibronectin on thrombus formation at arterial shear rate, we used the ferric chloride model of arterial injury in fibronectin heterozygote mice which have 50% of the normal plasma level of FN. Two groups of mice were observed by intravital microscopy - FN heterozygote (FN+/−) and corresponding age matched wild-type controls (FN +/+). The reduced level of pFN did not affect the early platelet interaction with subendothelium but caused a delay of several minutes in appearance of the first thrombus in the injured arteriole (15 min in FN +/− vs 6 min in FN+/+, p<0.001). Although the thrombi were stably anchored to the vessel wall, the growth of the thrombus was slowed down because of the constant platelet shedding in fibronectin heterozygote mice. Consequently, this lack of firm adhesion of the platelets led to occlusion delay (36 min in FN +/− vs 28 min in FN+/+, p<0.01) with the majority of arterioles in the FN+/− mice not occluding by 40 minutes after injury. In conlusion, the phenotype of thrombosis in the mice with 50% of normal pFN level strikingly resembles the phenotype observed in mice with induced complete deficiency of pFN. This further emphasizes the fundamental role of this plasma protein in thrombosis in vivo in the arterial system.
Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) Induces Long-Term Responses in Patients with Relapsed or Refractory Follicular Lymphoma (FL).
Authors: Schilder Russell J. , Witzig Thomas E. , Flinn Ian , Gordon Leo I. , Emmanouilides Christos , Wang Hua , Vo Katie , Molina Arturo
Published: October 2019

Background: Radioimmunotherapy (RIT) is an emerging clinical treatment option for patients with non-Hodgkin’s lymphoma (NHL). Yttrium 90 (90Y) ibritumomab tiuxetan (Zevalin®) RIT was approved by the FDA in February 2002 for the treatment of patients with relapsed or refractory low-grade, follicular, or transformed B-cell NHL, including patients with rituximab-refractory follicular NHL. In 4 registrational trials of 90Y ibritumomab tiuxetan conducted between 1996 and 1999, 211 patients with B-cell NHL were treated. Of these 211 patients, 153 patients (73%) had follicular lymphoma (FL). With ongoing follow-up, long-term durable responses have been observed, but until now have not been more fully characterized.

Methods: Responding patients with time to progression (TTP) of ≥12 months were identified and characterized as long-term responding (LTR) patients.

Results: In the 4 registrational trials, 78 of the 211 patients (37%) were identified as LTR patients. Characteristics of these LTR patients were as follows: median age 58 years (range, 24 to 80), 44% >60 years old, 55% male, 76% with follicular lymphoma, and 41% with lymphomatous marrow involvement. LTR patients had a median of 2 prior regimens (range, 1–9): 59% had ≥2 prior therapies, 33% had ≥3 prior therapies, and 37% had no response to their last prior therapy. Thirty percent of LTR patients had bulky disease (tumor size >5 cm) and 83% had stage III/IV disease. At the time of this analysis, the median duration of response (DR) and TTP for LTR patients were 28 months (range 11–80) and 29 months (range 12–82), respectively, with a median follow-up of 50 months (range 13–82). The median DR to the last prior therapy for LTR patients was 12 months. The complete response rate (confirmed [CR] and unconfirmed [CRu]) among LTR patients was 65%, and the median DR and TTP were 29 and 31 months, respectively, for CR/CRu patients. In ongoing responders the median DR is 52 months (range 48–80). Among the 153 patients with FL, 59 (39%) were identified as LTR patients. Compared to the overall LTR patients, LTR patients with FL had similar disease characteristics, DR, TTP, and CR/CRu rates.

Conclusions: Ongoing follow-up indicates that 90Y ibritumomab tiuxetan frequently produces durable long-term responses (TTP ≥12 months) in patients with relapsed or refractory B-cell NHL. Failure to respond to the therapy immediately prior to treatment with 90Y ibritumomab tiuxetan does not appear to affect the ability to achieve long-term responses with 90Y ibritumomab tiuxetan. Durable long-term responses were achieved in 37% of all patients and 39% of patients with FL treated in 4 registrational trials of 90Y ibritumomab tiuxetan at 30 centers in the US. Of these LTR patients, a high proportion were >60 years old and had received ≥3 prior therapies.
The Anti-CD30 Monoclonal Antibody SGN-30 Inhibits Hodgkins Disease Growth and Sensitizes Cells to Established Chemotherapeutics.
Authors: Cerveny Charles G. , Law Che-Leung , McCormick Renee S. , Lenox Joel S. , Hamblett Kevin J. , Westendorf Lori E. , Yamane Andrew K. , Petroziello Joseph M. , Francisco Joseph A. , Wahl Alan F.
Published: October 2019

SGN-30, a chimeric monoclonal antibody with apoptotic-signaling activity against CD30-positive malignancies, is currently in Phase II clinical evaluation against anaplastic large cell lymphoma and Hodgkin’s disease (HD). Mechanisms underlying SGN-30’s anti-tumor activity were evaluated using cDNA array analysis of SGN-30-treated L540 HD cells. Changes in expression of growth and apoptotic related genes including cyclin D2 and p21CIP1/WAF1 followed treatment of L540 and L540cy cells with SGN-30. Intra-cellular staining showed increases in both activated caspase-3 and p21CIP1/WAF1 in L540cy. These alterations coincided with increased Annexin V/PI staining. In addition to directly inducing apoptosis, SGN-30 mediated alterations in gene expression and could sensitize cells to standard chemotherapeutics. To evaluate this potential, L540cy cells were treated for 24 h with SGN-30 followed by exposure to a panel of chemotherapeutics commonly used in HD therapy and the combinations quantified by the Combination Effects method. Most of the chemotherapies examined were at least additive or better in combination with SGN-30, with bleomycin producing the strongest synergistic response. In vitro data were confirmed by the significantly increased efficacy of SGN-30 and bleomycin against established L540cy HD tumor xenografts in SCID mice. Results suggest that in addition to direct cell killing, SGN-30 binding to CD30 on HD tumor cells effects both growth arrest and drug sensitization by altering gene expression of cell cycle and apoptotic-related genes. Importantly, tumor cell sensitization to established chemotherapies, in particular DNA damaging agents used to treat HD, suggests that SGN-30 could improve the outcome of current drug-based therapies for treating HD.
CXCR4-Dependent Repulsion of Mature Single-Positive CD4 Cells Mediates Emigration from Thymus.
Authors: Vianello Fabrizio , Kraft Paul , Hart William , Mok Ying Ting , Poznansky Mark C.
Published: October 2019

Developing thymocytes undergo maturation during their passage through the cortex to the medulla of the thymus before emigrating to peripheral lymphoid organs. Chemokines control T cell migration within and away from the thymus. We have previously shown that the chemokine SDF-1, produced by thymic stroma, repels mature thymocytes away from thymus via a CXCR4 mediated mechanism. We proposed that CXCR4 plays a specific role in thymic emigration. We evaluated the emigration of mature thymocytes away from thymus in a modified fetal thymic organ culture (FTOC) system in fetal thymi from CXCR4+/− (Control) and CXCR4 −/− (KO) mice at day 15.5. Fetal thymic emigrants were collected using a transwell system with a polycarbonate membrane (3 μm pore size). The immunophenotype of thymic emigrants was distinct from that of thymocytes resident in the thymus in both control and CXCR4-KO thymi. From day 9 of culture onwards, only mature SP cells were able to move away from thymus into the lower chamber of the transwell. Only SP CD4 thymocytes expressing CD62Lhigh were found among emigrants, whereas two distinct populations of SP CD8 cells expressing CD62Lhigh and CD62Llow were present in the emigrant fraction. Incubation of FTOC with pertussis toxin significantly inhibited emigration of mature SP thymocytes out of the thymus in control CXCR4 mice (p=0.002). The total number of emigrants collected from CXCR4-KO thymi was always significantly reduced compared to emigrants from control thymi. In addition, the total number of SP CD4 emigrants from CXCR4-KO thymi were strikingly reduced in comparison to control SP CD4 emigrants (p=0.035). There was no statistically significant difference in the maturation of intrathymic thymocytes from CXCR4-KO as compared to control FTOC. Analysis of CD62L expression in CXCR4-KO thymocytes showed retention of SP CD4 cells in the thymus with the immunophenotypic characteristic normally associated with recent thymic emigrants (p<0.05). Treatment of FTOC with the specific CXCR4 antagonist, AMD3100, significantly increased the total number of SP CD4 cells retained in the thymus compared to untreated FTOC (p=0.002). Treatment with AMD3100 significantly reduced the absolute number of emigrants compared to untreated FTOC (p=0.015) and again SP CD4 cells were most affected in their ability to migrate away from the thymus. Treatment of FTOC with sphingosine-1-phosphate (S1P) resulted in an increase in the emigration of SP CD4 cells from CXCR4-KO thymi (p=0.02). In conclusion these data support the view that CXCR4-mediated repulsion of mature thymocytes away from the medullary chemokine SDF-1, termed fugetaxis, is a key mechanism controlling T cell egress from the thymus. These data support the existence of a CXCR4 dependent mechanism governing thymic emigration which may itself have S1P dependent and/or independent components.
The Role of the bZip Protein Bach2 in Umbilical Cord Blood (UCB) T Lymphocytes.
Authors: Huang Y. , Fanning L. , Masri O. , Kaminski B. , Kadereit S. , Laughlin M. J.
Published: October 2019

Graft versus host disease (GVHD) occurring after allogeneic stem cell transplantation is mediated by donor T cell alloreactivity against major and minor histocompatibility antigens presented by recipient antigen presenting cells (APC). Clinical reports identify that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD due to reduced cytokine production. Nuclear factor of activated T cells (NFAT1) plays a pivotal role in the transcription of cytokine genes and other immune responses. NFAT1 regulates the expression of cytokine genes through direct interaction with AP-1 (Fos/Jun, basic-lucine zipper bZip proteins). NFAT1 and AP-1 cooperatively bind to a 15-bp DNA that contains both NFAT1 and AP-1 sites. We used Affymetrix oligonucleotide microarrays to compare gene expression of primary purified CD4+ UCB T-cells to adult peripheral blood CD4+ T-cells (AB) at baseline, 6, and 16 hours after primary stimulation with anti-CD3/anti-CD28. NFAT-regulated genes exhibited lower expression in UCB CD4+ T-cells including: GM-CSF, IFN- g, TNF-a, IL-3, IL-4, IL-5, IL-13, IL- 2R (CD 25). In addition, AP-1 (Fos/Jun) did not show a significant change. However, Bach2, an 841- amino-acid-residue containing bZip protein, did show a significant increase in UCB CD4+ T-cells. Previous reports identify from the crystal structure of the quaternary complex of NFAT1/AP-1/DNA (Chen et al., Nature 1998) that NFAT1 is in contact with the lucine zipper region of AP-1 while the basic region of AP-1 binds to the DNA site at TGTTTCA. The amino-acid residues that make contact with DNA in the bZip regions of Jun and Fos are highly conserved in the Jun and Fos families. These amino acid residues are also 100% conserved in the human and mouse Bach2. The high similarity of DNA-contacting amino-acid residues between Bach2 and AP-1 strongly suggests that Bach2 and AP-1 may bind to the same DNA site. We hypothesize that Bach2 may act as a transcriptional repressor of cytokine genes due to the fact that increased Bach2 in UCB may compete with AP-1 for same DNA binding site, which in turn may partly block the interaction of NFAT1 with AP-1 and the expression of cytokine genes in UCB. We are currently pursuing this hypothesis. Ongoing studies are to confirm whether the protein levels of Bach2 and AP-1 are different in UCB compared to with AB CD4+ T-cells. This includes using gel-shift assays in vitro to test Bach2 binding to the DNA site of AP-1 as well as to test potential competitive binding of Bach2 and AP-1 to the same DNA site.
Identification of Immunodominant Alloreactive T-Cell Epitopes on the Jka Red Blood Cell Protein Inducing Either Th1 or Th2 Cytokine Expression: Role of DRB1*01 Molecules in Jka Peptide Presentation.
Authors: Ansart-Pirenne Helene , Zeliszewski Dominique , Lee Ketty , Martin-Blanc Stephanie , Rouger Philippe , Noizat-Pirenne France
Published: October 2019

Background: although antibodies implicated in red blood cell (RBC) blood group antigen alloimmunization are studied for many years, little is known about helper T cell responses that drive their production. The aim of this work was to determine T cell antigenic determinants involved in T cell responses against RBC antigens, and the subsequent patterns of stimulatory cytokine production.

Study Design: the Kidd blood group antigen (Jka), was selected as a model. We investigated cytokine expression by helper T cells after stimulation by Jka peptides. Peripheral mononuclear cells were isolated from Jka primed donors producing anti-Jka antibodies and from non anti-Jka alloimmunized donors as negative controls. Four 16-mer peptides mimicking the Jka sequence, 266–293, including the Asp280 polymorphism, and overlapping each over by 12 amino acids were tested to map alloreactive T cell epitopes. A sensitive method, the real time RT-PCR, was chosen to quantify Th1- and Th2-type cytokines (respectively IL-2 and IL-4) after Jka peptide stimulations. DRB1* and DQB1* low resolution typing, and DRB1* DNA subtyping were performed.

Results: lymphocytes from non anti-Jka alloimmunized donors produced low level of IL-2 or IL-4 (<10copies), while lymphocytes from all anti-Jka alloimmunized donors responded in a much higher level to at least one of the four Jka peptides. A clear Th1/Th2 dichotomy in the cytokine response was observed in this population. Indeed, among the 10 anti-Jka alloimmunized donors tested, 4 produced IL-2 alone and 6 produced IL-4 alone. Sequences among two peptides (Jka1and Jka2) were together able to elicit a response (IL-2 or IL-4) in 8 among 10 of these anti-Jka alloimmunized donors and represent a pool of immunodominant peptides. This Th1/Th2 dichotomy was not due to delays in kinetics of IL-2 and IL-4 productions. It was neither related to particular donor DRB1* or DQB1* molecules. The DRB1*01 phenotype frequency was increased (82%) in the anti-Jka alloimmunized donors as compared to the expected frequency (18%) in the caucasian population. This observation raised the question as to whether these molecules are associated to genetic susceptibility to anti-Jka alloimmunization.

Conclusion: These data indicate that Th1 and Th2 subsets are associated to the specific memory humoral immune response against Jka RBCs. The cytokine pattern (IL-2 or IL-4) is characteristic of the donor, whatever the peptide tested. DRB1*01 seems to be implicated in the peptide presentation to the T lymphocyte.

A detailed understanding of peptides and cytokines involved in T cell responses to Jka protein and the HLA class II molecules implicated in the peptide presentation constitutes a first step toward evaluation of peptide immunotherapy to prevent or treat anti-RBC alloimmunization.
Update of the Hyper-CVAD and Imatinib Mesylate Regimen in Philadelphia (Ph) Positive Acute Lymphocytic Leukemia (ALL).
Authors: Thomas Deborah A. , Faderl Stefan , Cortes Jorge , O’Brien Susan , Giles Francis , Garcia Guillermo , Kornblau Steven , Andreeff Michael , Beran Miloslav , Verstovsek Srdan , Pierce Sherry , Letvak Laurie , Salvado August , Champlin Richard , Kantarjian Hagop
Published: October 2019

The intensive combination chemotherapy program hyper-CVAD in newly diagnosed Ph+ ALL yields complete remission (CR) rates of 90%, but remissions are brief with median CR duration of 16 months [Kantarjian et al, JCO 18:547, 2000]. The activity of the tyrosine kinase inhibitor imatinib given as a single agent in relapsed or refractory Ph+ ALL or chronic myelogenous leukemia in lymphoid blast phase was 20% [Druker et al, NEJM 344:1084, 2001]. A phase II trial of imatinib and hyper-CVAD was conducted in newly diagnosed Ph+ ALL. Imatinib was given 400 mg days 1–14 of each course of therapy (fractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone alternating with high dose methotrexate and ara-C). Preliminary results were published in the first 20 patients (pts) treated, with CR rates of 100% in the de novo group, and improvement of disease-free survival (DFS) compared with hyper-CVAD alone [Thomas et al, Blood 103:4396, 2004]. To date, 32 pts with Ph+ ALL have been treated from April 2001 to February 2004. Twenty-six patients had active disease, either untreated (n=21) or refractory (n=5) to one induction course without imatinib. Six pts were in CR at study entry after one induction course without imatinib mesylate. Median age was 48 years (range, 17–75); 59% were male. Five had CNS disease (16%). Twenty-five of 26 pts (96%) with active disease at study entry achieved CR (1 failed to meet platelet criteria for CR). Median days to response was 21 days. Two of 26 pts (8%) required 2 courses to achieve CR. Allogeneic stem cell transplant (SCT) was performed in 13 pts in CR within a median of 3 months from start of therapy (range, 1–12). After a median follow-up of 2 years (range, 4–36 months), 1 primary refractory pt relapsed at 12 mos (bcr-abl/abl RT-PCR ratio <.05 at 9 mos), 1 pt relapsed day 149 after matched related SCT despite negative nested PCR for bcr-abl, and 2 pts changed therapy after 5 mos for persistent marrow Ph+ metaphases without overt leukemia relapse. Five pts died in CR, 3 older pts related to comorbid conditions (2 were negative for bcr-abl by nested PCR) and 2 related to complications of allogeneic SCT. Molecular response rate (negative bone marrow RT-PCR for bcr-abl confirmed by nested PCR) was approximately 50% in 19 pts who did not undergo allogeneic SCT. Outcome appears better with the hyper-CVAD and imatinib regimen with 2-year DFS rates of 87% (all pts) compared with 28% for hyper-CVAD alone or 12% for VAD (p<.001). Unexpected toxicities related to the addition of imatinib mesylate were not observed. The hyper-CVAD and imatinib regimen with or without allogeneic SCT continues to appear promising with additional accrual and longer follow-up.
Development of Lymphohematopoietic Progenitors during Human Embryonic Stem (hES) Cell Differentiation on OP9 Stromal Cells.
Authors: Slukvin Igor I. , Vodyanik Maxim A. , Bork Jack A. , Thomson James A.
Published: October 2019

hES cells provide an unique opportunity to study the earliest stages of hematopoietic commitment which are not easily accessible in the human embryo. To model early hematopoietic development, we cultured H1 and H9 hES cell lines on OP9 stromal cells without the addition of cytokines. On day 2 of co-culture, hES cells up-regulated brachyury expression and began to form mesodermal-like colonies. A transient population of blast colony-forming cells (CFCs) with the potential to differentiate into blood and endothelial cells was detected on days 3–6 of co-culture. CD34+ cells first appeared on day 3–4 of co-culture, which was coincident with induction of the transcription factors GATA-1, GATA-2 and SCL. CD43+ and CD41a+ cells along with CFCs emerged 2 days later within CD34+ population; 3–4 days before the appearance of CD45+ cells. We were able to obtain up to 20% of CD34+ cells from hES/OP9 co-culture and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8–9 days of culture. The hES cell-derived CD34+ cells were highly enriched in CFCs, displayed CD90+CD117+CD164+CD38- phenotype of primitive hematopoietic progenitors, and contained ALDHhigh cells as well cells with verapamil-sensitive ability to efflux rhodamine 123. Isolated CD34+ cells differentiated into lymphoid (NK cells) as well as myeloid (neutrophils and macrophages) lineages when cultured on MS-5 stromal cells in the presence of SCF, Flt3-L, IL7 and IL3. These data indicate that hES cell/OP9 co-culture reproduces the major events that are observed during embryonal hematopoietic development, including the formation of lympho-myeloid progenitors. We employed OP9 system for identification of the phenotype of early hematopoietic progenitors in humans and to directly differentiate hES cells into different blood lineages.