bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Invariant NKT Cell-Dependent Alloreactivity by Administration of G-CSF after Bone Marrow Transplantation
Authors: Morris Edward S , MacDonald Kelli P A , Kuns Rachel D , Morris Helen M , Banovic Tatjana , Don Alistair LJ , Rowe Vanessa , Burman Angela C , Markey Kate A , Godfrey Dale I , Smyth Mark J , Hill Geoff
Published: October 2019

G-CSF is often used to hasten neutrophil recovery following allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences invoked remain unclear. We examined this in murine models and found that administration of both standard G-CSF and pegylated G-CSF early after BMT significantly increased graft-versus-host disease (GVHD). This effect was seen in the B6 → B6D2F1, BALB/c → B6 and C3H.SW → B6 systems of GVHD to either MHC or multiple minor histocompatibility antigens. This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DC) responsive to G-CSF by up-regulating their expression of the G-CSF receptor as determined by real-time PCR. This induction of G-CSFR expression was not seen following busulfan (Bu), cyclophosphamide (Cy) or fludarabine. The enhanced GVHD was present when G-CSF was administered to both WT and G-CSFR−/− donors but not G-CSFR−/− recipients, confirming that host signalling was critical for this effect. G-CSF administration after BMT had no effect on inflammatory cytokine generation but enhanced in vivo CTL activity after BMT when administered to WT but not G-CSFR−/−, CD1d−/−, IFNgR−/− or CD40−/− recipients. Furthermore, donor iNKT cell activation was absent in CD11c Diptheria Toxin Receptor recipient transgenic mice depleted of dendritic cells (DC) by diphtheria toxin and treated with G-CSF. Thus, stimulation of host DC by G-CSF subsequently unleashed a cascade of events characterized by CD1d dependent donor iNKT cell activation, IFNg secretion and CD40-dependent amplification of donor CTL function during the effector phase of GVHD. Critically, the detrimental effects of G-CSF on GVHD were present when administered early following TBI conditioning and at a time when residual host APC were still present (day +1), but had no effect when administered at day +8 when host DC were not detectable by phenotypic or functional analysis. This is consistent with the inefficient cross presentation of host Ag within MHC class I by donor DC after BMT. In addition, the administration of G-CSF after Bu/Cy conditioning had no effect, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries since TBI conditioning predominated only in the positive European study. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have important effects on outcome and suggest a guide to the safe use of G-CSF after allogeneic BMT.
Soluble Lymphotoxin Plays a Critical Role in Acute Graft-Versus-Host Disease
Authors: Markey Kate A , Burman Angela C , Kuns Rachel D , Morris Edward S , Banovic Tatjana , Raffelt Neil C , Olver Stuart D , Engwerda Christian R , Korner Heinrich , MacDonald Kelli P A , Hill Geoff R
Published: October 2019

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies.

We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P<0.0002). Having observed a potentially TNF-independent but TNFR dependent pathway in the generation of GVHD we next examined mRNA expression by real-time PCR. This demonstrated that the majority of LTα mRNA was within donor CD4 T cells and that expression was unbalanced (> 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P<0.01) in both systems while LTβ−/− grafts induced GVHD that was equivalent to, or more severe than in recipients of WT grafts. This data confirms soluble LTα3 but not membrane bound LTα1β2 as an additional pathogenic molecule in GVHD. Using a luciferase-expressing, host-type leukemia (P815) in the B6 → B6D2F1 system, relapse occurred at an identical rate in recipients of WT and LTα−/− grafts confirming that GVL is maintained in the absence of donor derived soluble LTα3. Thus the combined neutralization of both LTα3 and TNF represents an important and logical therapeutic intervention in transplant medicine.
Mechanisms of Synergy Between Toll-Like Receptor 4 and Triggering Receptor Expressed on Myeloid Cells-1 in Human Neutrophils
Authors: Radsak Markus P , Schild Hansjörg , Haselmayer Philipp , Salih Helmut R
Published: October 2019

The triggering receptor expressed on myeloid cells 1 (TREM-1) is an important player in the innate inflammatory response to microbial infections. Activation and expression of TREM-1 by polymorphonuclear neutrophils (PMN) occurs in concert with Toll-like receptors (TLR) such as TLR4 for bacterial lipopolysaccharide. However, it is currently unclear how this is mediated on a molecular level. Using pharmacologic inhibitors and western blot analysis we demonstrate that phosphatidyl inositide 3-kinase, phospholipase C and the mitogen activated kinase p38 are essential for the TREM-1 and TLR4 mediated respiratory burst of human PMN. The down stream phosphorylation of protein kinase B and extracellular signal related kinase show characteristic phosphorylation patterns upon single or co-ligation indicating individual activation pathways of both receptors. TREM-1, but not TLR4 mediated respiratory burst depended on calcium flow via store operated calcium entry channels, while transient receptor potential channels were important for TLR and TREM-1. Taken together, we provide new insights on the mechanisms how TREM-1 and TLR interact creating synergistic activation in PMN. These results shed a new light on our understanding how the innate inflammatory responses are regulated and might contribute to the development of future concepts for the treatment of severe inflammatory conditions such as sepsis.
Early Diagnosis and Treatment of the Patients with Acquired Hemophagocytic Lymphohistiocytosis
Authors: Wang Zhao , Wang Yini , Feng Cuicui , Tian Liping
Published: October 2019

Acquired hemophagocytic lymphohistiocytosis (HLH) is a life threatening condition characterized by uncontroling hyperinflammation on the basis of various infection, tumor and inherited immune deficiency. Awareness of the clinical symptoms and of the diagnostic criteria of HLH is crucial in order not to overlook HLH and to start life-saving therapy in time. In this study, we reviewed 57 suspected HLH patients from March 2006 to June 2008. 25 healthy subjects were enrolled in the study as control. NK cell activity in peripheral blood was tested by a released LDH assay. Meanwhile, solution interleukin-2 receptor (sCD25) was examined with ELISA double antibody sandwich assay. The level of glycosylated ferritin was also detected and the ratio of glycosylated ferritin to ferritin was determined. 41 out of 57 patients were definitely diagnosed according to HLH-2004 diagnostic criteria in this study and 16 patients were excluded. We found that the level of NK cell activity and the ratio of glycosylated ferritin in the all 41 final diagnozed HLH patients were significantly lower than those in the 16 excluded patients and 25 healthy control subjects (p<0.01). Meanwhile, the level of sCD25 in peripheral blood was much higher in all the 41 HLH patients than that in the excluded and healthy people (p<0.05). We compared the coincidence of each diagnostic index in the 41 HLH patients before and after final diagnosis. It was found that 100% patients had abnormal expression on NK cell activity, sCD25 and glycosylated ferritin in the early disease. The three diagnostic indexes were more sensitive and specific than other indexes, such as fever, hepatosplenomegaly, cytopenia, hyper-triglyceridemia, hypo-fibrinogenemia. 41 diagnosed patients received the regimen containing methylprednisolone and immunoglobulin, with or without fludarabine, 26 out of 41 were markedly improved after treatment, 10 out of 41 were exacerbated, and other 5 patients gave up treatment. It is concluded that detection of NK cell activity, sCD25 and glycosylated ferritin may play a very important role in the early diagnosis of HLH. Our data also suggest that fludarabine combined with methylprednisolone and immunoglobulin (FDIg) may provide a new viewpoint for HLH therapy.
PU.1 Plays a Critical Role in Maintenance of Mature Dendritic Cell Pool
Authors: Iino Tadafumi , Chong Yong , Mizuno Shin-ichi , Ito Kyoko , Tenen Daniel G. , Reizis Boris , Akashi Koichi
Published: October 2019

PU.1, a hematopoietic transcription factor, is absolutely required for development of myelo-lymphoid cells from hematopoietic stem cells (HSC). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. In this study, by disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool. By crossing PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC, we disrupted PU.1 at the CD11c+ DC stage. In these mice, development of DC precursors such as Lin−c-KitloFLT3+MCSFR+ DC progenitors, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <30%, <10%, <10% and <5% of DC numbers in control mice, respectively. In contrast, mice possessed normal numbers of granulocytes/monocytes, B cells, and naïve, effector or regulatory T cells. These mice have not developed any significant hematological or immune disorders at least until 6 months after birth. These results clearly show that PU.1 is required not only for DC development but also for maintenance of the peripheral DC pool. We are currently trying to elucidate the underlying mechanism for PU.1 to maintain mature DC numbers in peripheral organs.
Spatial Analysis of Hematopoietic Stem and Progenitor Cells in the Bone Marrow
Authors: Nombela-Arrieta Cesar , Harley Brendan , Levantini Elena , Mahoney John E , Pivarnik Gregory , Protopopov Alexei , Tenen Daniel G. , Silberstein Leslie E.
Published: October 2019

Sustained production of all mature blood cell types relies on the continuous proliferation and differentiation of a rare population of self-renewing, multipotent hematopoietic stem cells (HSCs). HSC maintenance and lineage differentiation are strictly regulated by distinct microenvironments, termed niches, defined by cellular components, soluble regulators, and by the extracellular matrix. Definitive identification of the location as well as cellular and extracellular characteristics of HSC niches in the bone marrow (BM) has not been completed due to limitations of conventional imaging techniques. We have employed a novel imaging technology, Laser Scanning Cytometry (LSC) to define the localization of hematopoietic stem and progenitor cells (HSPCs) within different regions of the BM. LSC allows imaging and objective quantitative analysis of the anatomical position(s), number, and frequency of specific cell populations within the native tissue microenvironment. Analysis of whole femoral longitudinal sections of Bmi-GFP mice, in which GFP is expressed at its highest levels in HSPCs, revealed that within the bone diaphysis, HSPCs (Bmi-GFPhi c-kit+) cells were highly enriched in endosteal regions (within 100nm away from inner bone surface) compared to the central medullary region. Importantly, our data show that HSPCs are found at highest frequencies in the metaphysis of long bones, suggesting that these areas, which display characteristic morphological features, are functionally distinct from the diaphyseal region and a preferential location for HSPC-specific niches. We are currently employing LSC to identify HSPC niche cellular constituents by quantifying the relative frequency at which these cells are found in association with previously proposed niche-components such as osteoblasts, BM endothelial sinusoidal cells and CXCL12-abundant reticular cells. A detailed understanding of niche-derived signals regulating unique properties of HSCs will certainly prove relevant in human HSPC transplantation and cell therapy.
High-Dose Chemotherapy and Autologous Stem-Cell Transplantation for Primary CNS Lymphoma: Updated Results from a Pilot and Phase II Study
Authors: Illerhaus Gerald , Müller Fabian , Feuerhake Friedrich , Finke J.ürgen
Published: October 2019

Introduction: High-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) demonstrated high efficacy in the treatment of newly-diagnosed primary CNS lymphoma (PCNSL) in younger patients (pts.). A 5-year overall survival probability (OS) of 69% could be demonstrated in 30 pts within a phase-II trial on HDT and ASCT with consolidating whole-brain-irradiation (WBRT) (Illerhaus et al. JCO 2006). A subsequent pilot trial on HDT and ASCT without WBRT showed a 5-year OS of 77% (Illerhaus et al. Haematologica 2008). Here we give an update of our two different treatment regimens and future perspectives.

Patients and Methods: Thirty pts. ≤65 years were treated within the phase II trial, chemotherapy (CHT) consisted of 3 cycles of high-dose methotrexate (MTX, 8 g/m2), 1 cycle of AraC (2× 3 g/m2) plus thiotepa (TT, 40 mg/m2) followed by rG-CSF and stem-cell-mobilization. Conditioning regimen included BCNU (400 mg/m2) and TT (2×5 mg/kgBW) followed by ASCT. Hyperfractionated WBRT (45 Gy, 2×1Gy/d) was administered as consolidation. In our subsequent pilot trial 13 pts. (age 38–67 years) were treated without consolidating WBRT; CHT was intensified with 4 cycles MTX 8g/m2, 2 cycles AraC (2× 3 g/m2) and TT (40 mg/m2). Dose escalated HDT included BCNU (400 mg/m2) and TT (4×5 mg/kgBW) followed by ASCT. WBRT was reserved for pts. not responding to CHT.

Results: Median follow-up of the 30 pts. treated within our phase II trial was extended to 95 months (mo), the updated 5-year OS of all pts. is 66.6% and 82,3% of the subgroup of pts. who underwent HDT and ASCT (n=23), respectively. Three additional deaths occurred due to relapse (n=2) after 45 and 71 mo and due to comorbidity (n=1) after 103 mo. Five of 30 pts. developed severe leukoencephalopathy during follow-up. With a median follow-up of 35 mo in the 13 pts. treated within the pilot-phase without consolidating WBRT 3-year OS of all pts. is 77%. No further relapse or non-relapse mortality occurred in this pilot-group during. Most recent follow-up data will be presented in detail.

Conclusion: Sequential systemic application of high-dose cytostatic agents followed by HDT+ASCT is highly effective as initial therapy for pts. with PCNSL. The restriction of WBRT to refractory disease shows similar OS rates and a decrease in neurotoxicity. In an ongoing multicenter phase-II trial immunotherapy with rituximab is combined with HDT and ASCT to further increase remission rates. A future randomized trial should be focused on the efficacy of consolidation with HDT supported by ASCT.
Gem-(R)CHOP Versus (R)CHOP: A Randomized Phase II Study of Gemcitabine Combined with (R)CHOP in Untreated Aggressive Non- Hodgkin’s Lymphoma - EORTC Lymphoma Group Protocol 20021
Authors: Aurer Igor , Eghbali Houchingue , Raemaekers John M. , Khaled Hussein M , Van Glabbeke Martine , Baila Liliana , van der Maazen Richard
Published: October 2019

Despite recent advances many patients with aggressive NHL succumb to their disease and improvements in front-line chemotherapy are still needed. Gemcitabine is active in lymphoma. We embarked on a randomized phase II trial to determine the efficacy, feasibility and toxicity of the addition of gemcitabine to standard front-line chemotherapy. The trial consisted of two parts. The first part was designed to determine the maximal tolerated dose of gemcitabine and the second to determine the response rate, feasibility and toxicity of the treatment. Patients, 18–70 years old, with stage II–IV previously untreated B-large cell, follicular grade 3, anaplastic large cell or peripheral T cell lymphoma were eligible. They were randomized to receive either 8 cycles of standard CHOP given every 3 weeks or the same treatment combined with gemcitabine (Gem-CHOP). In the second part of the trial patients with B-NHL also received 8 cycles of rituximab (R). The starting dose of gemcitabine was 800 mg/m2 on days 1 and 8. Dose escalation to 1000 and 1250 mg/m2 and reduction to 800 mg/m2 on day 1 only were foreseen. Twenty-five patients, 7 with T and 18 with B-NHL, were enrolled in the trial; 15 in the dose-finding part and 10 in the second part before early closure due to low accrual. Twelve received Gem-(R) CHOP and 13 (R)CHOP. Maximal tolerated dose of gemcitabine was 800 mg/m2 given on days 1 and 8; dose limiting toxicity was hematologic. Five patients (42%) treated with Gem-(R)CHOP achieved CR in comparison to 10 (77%) treated with (R)CHOP. Three out of 4 patients with T-NHL responded to Gem-CHOP and 2 out of 3 to CHOP. Median time to treatment failure was 1.49 years for the Gem-(R)CHOP arm and 3.13 years for the (R)CHOP arm. Four patients receiving Gem-(R)CHOP had serious pulmonary toxicity, as compared to none receiving (R)CHOP. One patient died of severe pneumonitis. The apparently immunologically mediated pulmonary toxicity prompted us to add in the second part of the trial a three-day course of prednisone starting on day 8, together with the second gemcitabine dose. After this change, no additional cases of pulmonary toxicity were seen. The combination of gemcitabine with standard (R)CHOP results in significant but acceptable hematologic toxicity. Maximal tolerated dose of gemcitabine is 800 mg/m2 given on days 1 and 8 every 3 weeks. In this small group of patients, addition of gemcitabine did not seem to improve outcomes. The use of gemcitabine in previously untreated patients with aggressive NHL results occasionally in severe, potentially fatal, pulmonary toxicity. Therefore, further exploration of this combination does not seem to be warranted.
Combination Therapy with Histone Deacetylase Inhibitor Vorinostat Plus Aurora Kinase Inhibitor MK-0457 Leads to Enhanced Lymphoma Cell Killing with Stabilization of p53 and Repression of C-Myc, hTERT, and Myc-Responsive miRNAs
Authors: Kretzner Leo , Scuto Anna , Jove Richard , Forman Stephen J. , Yen Yun , Kirschbaum Mark
Published: October 2019

Background: Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few therapeutic options after salvage therapy and transplant, and new agents are thus needed. MK-0457 is a novel aurora kinase inhibitor (AKI) which leads to polyploidy and G2 arrest by disregulating mitosis. We demonstrate that the addition of vorinostat, a broad spectrum HDACi, leads to the reactivation of proapoptotic and DNA damage response elements leading to enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination using MTS, Annexin V, and flow cytometry studies.

Results: Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by at least 50% in all lines tested. For example, 1.5 mM vorinostat results in averages of 50% growth reduction and 30% apoptotic cells at 48 hours in the L540 HL cell line. MK-0457 alone at 100 nM – 500 nM results in up to a 50% reduction in cell growth and 20% apoptosis in the tested cell lines. However, the combination of 1.5 mM vorinostat and 100 nM MK-0457 results in 50 – 70% apoptosis of the L540 and KM-H2 Hodgkin cell lines at 48 hours. Cell cycle analyses by FACS show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat there is a significant increase in the sub G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination suggest an epigenetic as well as a protein acetylation mechanism for this activity. Prosurvival genes such as bcl-XL and hTERT are downregulated five-fold by combination drug treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. The p53 tumor suppressor is stabilized both by increased acetylation in response to vorinostat as well as by reduced inhibitory phosphorylation at Ser315 by MK-0457-inhibited aurora kinase, leading to a corresponding increase in the p53 target genes p21 and Noxa. Protein and mRNA levels of c-myc are reduced. Repression of microRNAs (miRNAs) in the myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, is demonstrated with vorinostat and TSA.

Conclusions: The HDACi vorinostat leads to both transcriptional and post-transcriptional changes which create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the AKI, MK-0457.

[We acknowledge Merck Inc for providing Vorinostat, MK-0457, and research support.]<
Cytogenetic Analysis and FISH in the Evaluation of Patients with MDS: A Retrospective Analysis of Concordance Rate, Utility and Benefit of Performing Both Studies Simultaneously
Authors: le Shammo Jami , Gimelfarb Alla , Hsu Wei-Tong , Paz Irene Dehghan- , Venugopal Parameswaran , Larson Melissa L. , Fung Henry C. , Farhat Mohamed , Rizman Amy , Loew Jerome , Gregory Stephanie A.
Published: October 2019

Background: The Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders, characterized by cytopenia, and risk of progression to acute leukemia. Chromosomal abnormalities are detected in 40–60%of patients with MDS. Cytogenetic findings are critical for diagnosis, prognosis and monitoring therapy. FISH has been reported to detect occult clonal abnormalities in 15–7.8% of karotypically normal patients in some studies, but found to be of limited value in others. We sought to evaluate whether FISH can be a valuable diagnostic/prognostic adjunct to conventional cytogenetic (CC) analysis.

Methods: Since the implementation of FISH panel at our institution, we have identified 62 MDS patients who had both CC and FISH panel tests performed simultaneously. Our FISH panel was designed specifically to detect abnormalities in chromosomes 5, 7, 8, 11 and 20.

Results: In these patients, concordance between the 2 methods was noted in 43 patients (69.3%). Clonal chromosomal abnormalities were detected by CC in 36 patients (58 %). FISH defined additional clones in 7/26 patients with normal and 3/36 patients with an abnormal karyotype increasing the detection rate by 16%. Importantly, 9/36 patients with abnormal CC had clonal abnormalities in chromosome regions not analyzed, thus not detected, by our FISH panel.

To further evaluate the prognostic significance of the additional clones detected by FISH, we calculated and compared the IPSS risk category for the 10 patients identified with discordant results utilizing data derived from CC and FISH. 3 patients had no change in their IPSS scores, 7 patients had an upgrade in their IPSS scores as follows: from low to Int-1: 4, Int-1 to Int-2: 2, and Int-2 to High: 1. If one were to apply NCCN guidelines with regard to treatment options according to IPSS risk categories, FISH upgraded scores could potentially result in change of therapy in 2/10 patients with discrepancy, and in 2/62 (3%) patients altogether.

Conclusion: Our results suggest that FISH has the potential of increasing the rate of detecting chromosomal abnormalities in complete CC studies that have failed to reveal clonal abnormalities. However, the practical impact of finding such clones seems to be very low resulting in potential change of clinical management in only 3% of patients. Furthermore, in cases with identified cytogenetic abnormalities, FISH was of limited benefit. Our series is small and retrospective in nature; prospective studies addressing the utility, benefit, and cost justification of this method are needed.
The Immunotoxin HM1.24-ETA′ Is a Potent Inducer of Apoptosis in Malignant Plasma Cells
Authors: Staudinger Matthias , Muskulus Anja , Burger Renate , Guenther Andreas , Repp Roland , Gramatzki Martin , Peipp Matthias
Published: October 2019

Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma (MM) still needs improvement. Antibody-based targeted therapies are increasingly used for tumor therapy, and may represent interesting options for MM patients. HM1.24 is a surface molecule that is over expressed on malignant plasma cells and efficiently internalized from the cell surface. It may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the development and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of an HM1.24-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and purified to homogeneity by affinity chromatography. Specific binding to HM1.24 was demonstrated by immunofluorescence staining and flow cytometry using antigen positive and negative cells. HM1.24-ETA′ efficiently inhibited growth of IL-6 dependent and IL-6 independent myeloma cell lines (INA-6, RPMI8226, U266). Half maximal growth inhibition was observed at low nanomolar concentrations. Further analyses demonstrated that target cell killing occurred via induction of apoptosis, as evidenced by Annexin V/propidium iodide staining and detection of PARP cleavage. The cytotoxic effect was completely blocked by adding excess of unconjugated parental antibody, demonstrating that the effect was antigen-specific and not mediated by non-specific uptake of the immunotoxin. Importantly, HM1.24-ETA′ efficiently triggered apoptosis (>80% Annexin V positive cells) of freshly isolated plasma cell leukemia cells within 48h. In conclusion, HM1.24-ETA′ efficiently triggered apoptosis of multiple myeloma cell lines as well as freshly isolated tumor cells. These results indicate that HM1.24 may represent a promising target structure for efficient antigen-specific delivery of cytotoxic compounds to plasma cell tumors.
An Animal Model to Study Bisphosphonates Associated Osteonecrosis of the Jaw
Authors: Pozzato Gabriele , Chiandussi Silvia , Zacchigna Serena , Dore Franca , Moimas Silvia , Zanconati Fabrizio , Cavalli Fabio , Mecchia Arturo , Faraon Michela , Giacca Mauro , Di Lenarda Roberto , Giacca Mauro , Biasotto Matteo
Published: October 2019

The pathogenesis of osteonecrosis of the jaws (ONJ) in patients treated with intravenous bisphosphonates is still not completely understood, and likely involves both reduction in blood supply and the activity of oral bacteria. We present an animal model of ONJ, which will significant contribute to the understanding of the aetiopathogenesis of this condition. Five skeletally-mature female Wistar rats (weight 450 g) were given intravenous zoledronic acid 0.04 mg in saline solution (0.2 mg/ml) once a week for 5 weeks. After 2 weeks, the animals underwent the extraction of the upper right first molar, followed by the creation of a 4 mm-diameter bone defect on the same site under general anaesthesia. After 7 weeks from the extraction, the animals were clinically examined and a static bone-scintigraphy using 99mTc-MDP methylene diphosphonate was performed with a gamma camera equipped with pinhole collimator (Siemens-Ecam). After an additional week, the rats were sacrificed and a Computed Tomography (CT) was carried out. Samples obtained from the bone defect were decalcified and prepared for histological assessment. Five rats, not treated with zoledronic acid and exposed to the same surgical treatment, were used as controls. Histological and imaging features were assessed blindly. At 7 weeks after the extraction, all the rats treated with zoledronic acid showed impair healing, expansion of the defect and bone exposure. These features were confirmed by the scintigraphy, which showed abnormal localized activity in comparison with the surrounding tissues. On clinical examination, the rats of the control group demonstrated epithelialization of the bone defect and a normal uptake of the contrast medium during the scan. The CT scan disclosed irregularity of the cortical margin and destruction of the cortical bone, which were not evident in the control group. The histology showed sheets of necrotic bone, with loss of osteocytes from their lacunae and peripheral resorption. No inflammatory infiltrate was observed. The control group instead demonstrated normal bone healing. All animals were treated according with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Based on this study, the rat treated with zoledronic acid can be considered a novel, reliable and reproducible model to better understand the pathophysiology of ONJ and to develop a therapeutic approach.
Sp1 Transcription Factor as a Novel Therapeutic Target in Multiple Myeloma (MM)
Authors: Fulciniti Mariateresa , Tassone Pierfrancesco , Hideshima Teru , Nanjappa Puru , Podar Klaus , Adamia Sophia , Amin Samir B , Prabhala Rao , Lopez Rocio , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Sp1 transcription factor has an important role in regulating expression of cell differentiation, cell cycle and apoptosis related genes. Increased Sp1 activity has been shown to augment growth and metastatic potential of tumor cells. In multiple myeloma (MM), modulation of autocrine IL-6 secretion by Sp1 has been described. Using genome expression profiling analysis we observed high expression of Sp1 in primary myeloma cells obtained from bone marrow aspirates of MM patients. This observation was confirmed by western blot analysis. Therefore, we evaluated Sp1 as a novel therapeutic target in MM, analyzing both in vitro and in vivo anti-MM activity of a Sp1 inhibitor (Terameprocol, EM-1421). Terameprocol significantly (≥80% inhibition, P≤0.001) inhibited DNA synthesis in 12 MM cells lines tested in a dose-dependent fashion at 24 hour with an IC50 of 1–10 uM. Longer exposure of MM cells to Terameprocol resulted also in cytotoxity, as assessed by MTT assay, in all cell lines tested (IC50 of 20–40 uM at 72 hours). Terameprocol induced apoptosis in a time- and dose-dependent manner in all MM cell lines examined, as assessed by Annexin V+/PI staining, and triggered caspase-9/3 and PARP cleavage. Since Sp1 modulates transcription of survivin and survivin is an anti-apoptotic protein overexpressed in most human cancers that inhibits caspases-3 and -9 activity, we next examined effect of Terameprocol on survivin protein expression. Consistent with induction of caspase-3 and apoptosis, Terameprocol significantly reduced survivin protein in a time-dependent manner. To identify additional potential downstream targets of Sp1 inhibition in MM, we evaluated change in expression profiling in MM cells following Terameprocol treatment (10 uM for 12 hours). We observed alteration (>2-fold changes) in 68 genes. In particular, we recognized significant down-regulation of c-Jun and up-regulation of p53 and p21 following exposure to Terameprocol. Changes in protein expression of these genes were also confirmed by western blot analysis. Since bone marrow stromal cells (BMSCs) trigger growth, survival, and drug resistance of MM cells we next confirmed the ability of Terameprocol to overcome protective effects of BMSCs on MM cell growth and survival. Finally, we have confirmed in vivo activity of Terameprocol on MM cell growth in a xenograft murine model of human MM. In conclusion, our results demonstrate that Sp1 may be an attractive target for MM therapy and provide the rationale for clinical evaluation of Terameprocol in MM.
Validation of a Novel Clonality Assay in Patients with Clonal Hematologic Disorders
Authors: Agarwal Neeraj , Swierczek Sabina , Nussenzveig Roberto , Samuelson Scott James , Parker Charles J. , Prchal Josef T.
Published: October 2019

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. They are useful in the diagnosis and assessment of therapeutic response in clonal hematologic disorders, especially in absence of an identifiable somatic mutations. Skewing of X-chromosome allelic-usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in ~30% elderly women, precluding the use of this assay in elderly patient (>65 years of age). Using a quantitative, transcriptionally-based clonality assay, we did not detect clonal hematopoiesis in >200 healthy non-elderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in forty elderly women (age 65–92, mean 81.3 years), using a novel, quantitative qPCR transcriptional clonality assay. In this assay, mRNA transcribed from five X-chromosome polymorphic genes expressed in peripheral blood neutrophils is quantified by real time, allele specific RT-PCR. We did not detect clonal hematopoiesis in any of the elderly women. However, using HUMARA assay, 30% of these elderly women were detected to have monoallelic methylation of the HUMARA gene locus, consistent with previously reported literature. We concluded that our novel transcriptional clonality assay is suitable for evaluation of clonal hematopoiesis in all women including elderly women (Swierczek S, Agarwal N et al. Blood 2008, July 18 epub). Using this novel assay, we detected clonal hematopoiesis in 31 out of 32 well characterized patients with myeloproliferative disorders:

polycythemia vera (all fourteen patients were clonal by our assay and all were JAK2V617F positive), essential thrombocytosis (nine out of ten patients were clonal by our assay, one out of ten patients was cMPLW515L positive, seven out of ten were JAK2 V617F positive; however one subject with low JAK2 V617F allelic burden was polyclonal by our assay), and primary myelofibrosis (all eight patients were clonal by our assay and two of them were positive for JAK2V617F).

In addition, we detected clonal hematopoiesis in 4 patients with unexplained anemia (two eventually evolved in to myelodysplastic syndrome), and in one patient with persistent leukocytosis (eventually found to be cMPLW515L positive). Using our assay we did not detect clonal hematopoiesis in 10 patients with reactive or secondary erythrocytosis, thrombocytosis or leukocytosis. We conclude that our novel transcriptional clonality assay is suitable for detection of clonal hematopoiesis in patients with clonal hematologic disorders, especially in patients lacking known somatic mutation. Studies to detect an emerging clone in milieu of polyclonal hematopoiesis (such as seen in PNH or early stages of clonal hematological disorders) by comparison of X-chromosome allelic usage ratio in myeloid cells and in T lymphocytes are underway.
FGFR3 Associates with and Tyrosine-Phosphorylates p90RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation
Authors: Kang Sumin , Elf Shannon , Dong Shaozhong , Hitosugi Taro , Guo Ailan , Ruan Hong , Lonial Sagar , Khoury Hanna , Williams Ifor , Karsenty Gerard , Hanauer André , Gu Ting-Lei , Chen Jing
Published: October 2019

Dysregulation of receptor tyrosine kinase FGFR3 has been implicated to play a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates p90 Ribosomal S6 Kinase2(RSK2) at Y529, which consequently regulates RSK2 activation [Kang et al, Cancer Cell 2007 Sep;12(3):201–14]. Here we identified Y707 as an additional tyrosine site of RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory αL-helix on the C-terminus of RSK2, unlike Y529 phosphorylation that facilitates ERK binding. To elucidate the role of tyrosine phosphorylation at Y707 induced by FGFR3 in RSK2 activation, we characterized the RSK2 mutants with single Y→A and Y→F substitutions at Y707. RSK2 Y707F demonstrated decreased kinase activity, suggesting substitution of Y707 attenuates activation of RSK2 induced by FGFR3. Tyrosine phosphorylation at Y529 by FGFR3 regulates RSK2 activation by facilitating inactive ERK binding, whereas substitution of Y707 in RSK2 does not similarly attenuate inactive ERK binding to RSK2. Phosphorylation at Y707 may regulate RSK2 activation by affecting the structure of the autoinhibitory C-terminal domain of RSK2 since the Y707 is localized at the C-terminal tail region which represents a conserved putative auto-inhibitory alpha helix. Since other tyrosine kinases including FGFR1 and Src also phosphorylate RSK2 at Y529 and Y707, tyrosine phosphorylation may be a general requirement for RSK2 activation through the ERK/MAPK pathway. Together, our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2, and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, and subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation.
Expression of Leukemogenic Ptpn11 Causes a Fatal Myeloproliferative Disorder by Affecting Multiple Stages of Hematopoiesis
Authors: Chan Gordon , Kalaitzidis Demetrios , Usenko Tatiana , Kutok Jeffery , Yang Wentian , Mohi Golam , Neel Benjamin
Published: October 2019

The Src homology-2 (SH2) domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is an ubiquitously expressed non-receptor protein tyrosine phosphatase (PTP) that positively regulates Ras/Erk activation in many receptor protein tyrosine kinases (RTK) and cytokine receptors. Mutations in PTPN11 are found in ~35% of patients with juvenile myelomonocytic leukemia (JMML) and at lower incidence in other neoplasms. To model JMML pathogenesis, we generated knock-in mice that conditionally express the leukemia-associated mutant Ptpn11D61Y. Ptpn11D61Y expression in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly with extramedullary hematopoiesis, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin−Sca1+cKit+ (LSK) compartment is expanded and “right-shifted”, accompanied by increased stem cell factor (SCF)-evoked colony formation and Erk and Akt activation. However, stem cell activity is decreased in diseased mice, and mice engrafted with Ptpn11D16Y stem cells fail to develop MPD. Ptpn11D16Y common myeloid (CMP) and granulocyte-monocyte (GMP) progenitors produce cytokine-independent colonies in a cell-autonomous manner, and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-SCF) stimulation. Ptpn11D61Y megakaryocyte-erythrocyte progenitors (MEP) yield increased numbers of erythrocyte burst-forming units (BFU-E), but MEP and erythrocyte-committed progenitors (EP) produce fewer erythrocyte colonyforming units (CFU-E), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cellautonomous, and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.
Bone Marrow Stromal Cells Reverse Proapoptotic Effects of JAK2 Inhibitor Atiprimod in Cells Carrying the JAK2V617F mutation
Authors: Manshouri Taghi , Estrov Zeev , Burger Jan , Livun Ana , Zhang Ying , Ma Shuhuang , Gaikwad Amos , Harris David , Kantarjian Hagop M , Verstovsek Srdan
Published: October 2019

Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The recent reports of an activating somatic mutation in codon 617 of the JAK2 gene (JAK2V617F mutation) in patients with myeloproliferative disorders (MPDs), has opened new avenues for the development of targeted therapies for these malignancies and clinical trials with JAK2 inhibitors are underway. We report here the activity of Atiprimod (N,N-diethyl-8-dipropyl-2-azaspiro[4,5]decane-2-propanamine), a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2V617F mutant-expressing murine FDCP-EpoR cells, set-2 cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2WT or mutant JAK2V617F, and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2V617F cells (IC50 0.42 μM) and set-2 cells (IC50 0.53 μM) than FDCP wildtype JAK2WT cells (IC50 0.69 μM). Atiprimod inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose- and time-dependent manner. It induced apoptosis, as evidenced by increase in mitochondrial membrane potential, caspase3 activity, and cleavage of PARP protein. The anti-proliferative effect on expanded PV patient progenitor’s cells was paralleled by a decrease in JAK2V617F mutant allele frequency in BFU-E or CFU-GM clones in clonogenic assay. However, co-culturing of JAK2V617F mutant cells with three different bone marrow stromal cell lines (Hs5, ABM-MSC, NK-Tert) either directly (cell on cell) or indirectly (separated by 0.4 μm micropore membranes) for 48 hours resulted in a significant protection of mutant cells from the effect of Atiprimod. Co-culturing of bone marrow stromal cells prevented Atiprimod (0.4 and 0.8 μM) induced apoptosis, and reversed the inhibition of phosphorylation of STAT proteins. Our results suggest that cytokines secreted by stromal cells might play an important role in protecting the hematopoietic cells from a JAK2 inhibitor. Further dissection of the nature of interactions between JAK2V617F mutant cells and marrow stromal cells may lead to new therapeutic avenues for patients with MPD.
Role of Human Endogenous Retroviruses in Lymphoma Pathogenesis and a Possible Biomarker of Disease
Authors: Gitlin Scott D. , Galindo Rafael Contreras- , Kaplan Mark H. , Markovitz David M.
Published: October 2019

Actively replicating retroviruses entered hominid species millions of years ago and through mutations preventing replication now exist as 8% of the human genome. Active retroviral particles and antigens from the supposedly dormant human endogenous retrovirus, HERV-K (HML2), have been identified in several cancer cell lines. We have recently demonstrated very high RNA titers of HERV-K (HML2) in the plasma of HIV positive individuals by nucleic acid sequence-based amplification (NASBA) and RT-PCR. We now demonstrate very high HERV-K (HML2) RNA titers in the plasma of patients with HIV positive and HIV negative non-Hodgkin lymphoma (NHL) and in Hodgkin Disease (HD), but not in normal individuals. Different copies of HERV-K (HML-2) present throughout the human genome exist as Type 1 viruses which encode a new oncoprotein, NP9, or as Type 2 viruses which encode a functional envelope (env) and express the Rec oncoprotein. Both Types 1 and 2 viruses appear in NHLs but only Type 1 appears in the plasma of those with HD. HERV-K (HML2) Env and Gag proteins, Env and Gag RNA, and Reverse Transcriptase (RT) activity are isolated from patients with a variety of NHLs, but not in normal controls or in patients with non-malignant diseases. Viral titers dramatically decrease, up to an approximately 7.5 log drop, when patients with NHL or HD go into remission following treatment. To further establish the presence of functional viruses in NHL and HD, immuno-gold electron microscopy allowed demonstration of HERV-K (HML2) particles in the plasma of lymphoma patients. Preliminary analysis of the effect of antiretroviral agents on cell lines infected with HERV-K (HML2) demonstrate a drug class-specific reduction in viral expression at drug concentration levels that range from 0.125 – 1 mcg/mL. In conclusion, we have demonstrated evidence that human endogenous retroviruses are found in the plasma of patients with NHL and HD, suggesting that these viruses, previously presumed to be inactive, may play a role in lymphoma pathogenesis. The observation that viral expression parallels declines in disease activity with treatment of disease may allow use of HERV-K (HML2) expression as a biomarker of lymphoma activity. The role of the HERV-K (HML2)-encoded oncoproteins in disease pathogenesis is under study, as is the potential role of antiretroviral therapy for these malignancies.