bluuebunny/crossref_metadata_2025 huggingface dataset:
A Human Antibody, Cloned from a Patient with Heparin-Induced Thrombocytopenia, That Binds Heparin/Platelet Factor 4 Complexes.
Authors: Dessain Scott K. , Rybinski Katherine E. , Adekar Sharad P. , Rauova Lubica , Sachais Bruce S. , McKenzie Steven E. , Reilly Michael P.
Published: April 2020

In heparin-induced thrombocytopenia (HIT), patients receiving heparin develop IgG antibodies that bind complexes formed between heparin and platelet factor 4 (PF4). Most patients are asymptomatic, but some develop life- and limb-threatening arterial or venous thrombosis. We previously described a transgenic mouse model of HIT that demonstrated four factors are necessary and sufficient to recapitulate the clinical manifestations of HIT with thrombosis: heparin, human PF4, a heparin/PF4 antibody, and platelet activation via FcγRIIA. We hypothesize that specific quantitative and qualitative characteristics of heparin/PF4 antibodies determine which patients with HIT will develop thrombosis. Because heparin/PF4 antibodies isolated from patients with HIT are generally polyclonal and heterogeneous, it has been impossible to directly test this hypothesis. We recently developed a novel means of cloning human antibodies, in which we fuse primary human B-cells to a murine cell line that ectopically expresses human telomerase (hTERT) and murine interleukin-6 (mIL-6). This method readily generates heterohybridoma cells that stably secrete human antibodies. We have used this method to clone human heparin/PF4 antibodies from patients with HIT and thrombosis. We identified a patient with clinical HIT, exhibiting a heparin-dependent drop in platelet counts, plasma heparin/PF4 IgG antibodies by ELISA, and a deep venous thrombosis. We fused peripheral blood lymphocytes from this patient to our novel fusion partner cell line, and isolated 2 independent hybridomas that express IgM antibodies that strongly bind heparin/PF4 complexes. We assayed the specificity of the cloned antibodies by testing serial dilutions for heparin/PF4 binding activity by ELISA. Each cloned antibody bound heparin/PF4 with an absorbance >2X background at dilutions that ranged from 1:32–1:64, whereas three negative control antibodies gave no detectable binding at dilutions of 1:2 or greater. cDNA sequencing indicated that both antibodies have the same heavy chain sequences, including a novel CDR3 region, suggesting that the antibodies were derived from post-germinal center memory B-cells rather than from naive B-cells. Using recombinant DNA techniques and ectopic expression in CHO cells, we produced an IgG1 version of one of these antibodies (8E5). By ELISA, the 8E5 IgG1 antibody binds human PF4 at optimal ratio of 10 μg/ml to 0.4 U/ml of heparin (see Figure). We are currently exploring the nature of the 8E5 IgG/heparin/PF4 complex and its platelet activating/thrombotic potential.

Figure Figure
Prevention of Acute Graft-Versus-Host Disease Despite Compensatory Function of Lymphoid Organs In Vivo.
Authors: Beilhack Andreas , Schulz Stephan , Baker Jeanette , Beilhack Georg F. , Nishimura Ryosei , Landan Gilad , Baker Enosh M. , Herman Edward I. , Butcher Eugene C. , Contag Christopher H. , Negrin Robert S.
Published: April 2020

Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (>120 d, p<0.0001). Our study shows that multiple priming sites are involved in GVHD initiation, the spleen compensating for the lack of PP and mesenteric LN, and vice versa. In contrast, splenectomy and antibody blocking resulted in a clear survival benefit for all recipients.
The Role of Annexin II in Stem Cell Homing to Endosteal Osteoblasts - Molecular Dissection of the Hematopoietic Stem Cell Niche.
Authors: Taichman Russell S. , Jung Younghun , Wang Jingcheng , Song Junhui , Wang Jianhua H. , Havens Aaron , Sun Yanxi X. , Krebsbach Paul H.
Published: April 2020

HSC differentiation occurs in direct proximity to osteoblasts (OBs) with in the bone marrow cavity. Using a novel cell blotting system in which OB membranes were run on non-denaturing discontinuous gels and blotted with labeled HSC cell lines, two major adhesive bands were identified. The two proteins were identified as annexin II monomers and multimers.

Immunohistochemistry and laser capture and microarray studies demonstrated that annexin II is preferentially expressed at endosteal surfaces and endothelial cells. Antibody studies against annexin II blocked the adhesion of HSC to OB in vitro. siRNA knockdown and over-expression studies of annexin II resulted in either reduced or enhanced binding characteristics, respectively, as expected. These findings were verified using primary human CD34+ bone marrow cells and human OBs where the binding of the progenitor cells and CFU-activities were linked with annexin II. Peptide mapping demonstrated that the majority of the HSC binding activity resides in the N-terminal aspect of the peptide.

To assess the role of annexin II in hematopoietic reconstitution, lethally irradiated mice were transplanted with bone marrow cells in the presence or absence of antibody to annexin II, antibody to the p11 annexin II binding partner, or isotype matched controls. Additional groups included use of the N-terminal p1-12 annexin II synthetic peptide or control. Animals that did not receive any marrow cells died within 14 days. Of the mice transplanted with 2x10^5 bone marrow cells, 1/8 animals died at day 11, representing an 80% survival rate. In contrast, when mice were transfused with the same number of marrow cells together with antibody to annexin II, none of the mice survived; the control treated animals survived (n = 8). Equivalent antibody doses against the annexin II binding partner p11 decreased the survival rate of the treated animals compared to IgG controls. However this treatment was less effective in inhibiting survival compared to the antibody to annexin II. Like wise, the survival of mice treated with two different doses of the p1-12 peptides at two different cell concentrations was significantly relative to the groups treated with a scrambled peptide control. These data suggest that the N-terminal twelve amino acids of annexin II facilitate stem cell homing to the marrow. Together these in vitro and in vivo data suggest a novel role for annexin II in the marrow microenvironment.
Bortezomib Induces Selective Depletion of Activated T Lymphocytes and Modifies the Cytokine’s Production Pattern.
Authors: Blanco Belen , Perez-Simon Jose A. , Sanchez-Abarca Luis I. , Carvajal-Vergara Xonia , Mateos Juan , Vidriales Belen , Lopez-Holgado Natalia , Alberca Mercedes , Villaron Eva , Schenkein D. , Canizo Consuelo , Pandiella Atanasio , San Miguel Jesus F.
Published: April 2020

The NF-kB family has emerged as a key transducer of inflammatory signals involved in dendritic cell maturation and T lymphocyte activation. Accordingly, the diverse signaling pathways downstream of TCR/CD3 converge on several transcription factors including NFAT, AP-1 and NF-kB. We explored the ability of the proteosome inhibitor bortezomib, which prevents Nuclear Factor kB (NF-kB) activation, to block T-cell activation, proliferation and survival within alloreactive as compared to resting T-cells. For this purpose, T-cells were stimulated with PHA, aCD3/aCD28, allogeneic dendritic cells (APC) or through mixed lymphocyte cultures. NF-kB expression increased in activated T-lymphocytes as compared to resting T-cells. Interestingly, the higher the NF-kB expression, the more intense the proliferative blockade induced by bortezomib. This effect on proliferation was due to a selective induction of apoptosis among activated T-cells. Thus, after mixed lymphocyte reaction (MLR) cultures, alloreactive T-cells were 2 logs more sensitive to bortezomib-induced apoptosis than the resting T-cell counterpart as assessed by annexin / 7AAD staining. This effect of bortezomib was partially blocked by adding the caspase inhibitor Z-VAD to the culture. The addition of bortezomib not only decreased the proliferation and viability of activated T-lymphocytes but also the levels of IFN-g and IL-2, which were significantly decreased among activated T-cells cultured with bortezomib at doses ranging from 10 to 100 nM. Secondary mixed lymphocyte cultures demonstrated a selective loss of alloreactive T-cells while activation and proliferation against third party donor was partially preserved.

In conclusion, at concentrations reached in the clinical setting, bortezomib induces selective apoptosis and decreases Th1 response among alloreactive T-lymphocytes while it barely affects unstimulated T-cells. These results establish the basis for the clinical use of bortezomib in the management of GVHD
Enhanced Immune Reconstitution and Function by CD4+CD25+ Regulatory T Cells Following Allogeneic Hematopoietic Cell Transplantation.
Authors: Nguyen Vu H. , Shashidhar Sumana , Zeiser Robert , Ho Lena , Brown Janice M.Y. , Negrin Robert S.
Published: April 2020

Regulatory T cells (Treg), defined phenotypically by CD4+CD25+ expression, reduce the incidence and severity of acute graft-versus-host disease (GvHD) in murine models of major-MHC mismatched hematopoietic cell transplantation (HCT), presumably by dampening the proliferation and activation of mature effector T cells. It is unclear whether the effect of Treg on effector T cells is a selective or nonselective process or if Treg regulate the process of intrathymic and peripheral T cell maturation and selection following HCT, particularly given the intrinsic link of GvHD and immune reconstitution. The current study assessed the impact of Treg on the quantitative and functional lymphoid reconstitution in a murine model of major-MHC mismatched HCT. Treg (5x105) from the spleen and lymph nodes of FVB/N (H2q) mice were co-transplanted into lethally irradiated Balb/c (H2d) host along with wild-type FVB/N T-cell depleted bone marrow (TCD-BM) cells (5x106) and splenocytes (1.25x106) (Tcon), the latter to induce GvHD. Chimerism studies were performed on day 14 and 40 post-transplantation to measure the level of donor immune reconstitution. At both time points, total lymphoid reconstitution was delayed in the GvHD control group and enhanced in the recipients transplanted with Treg (p-values=0.0005 on day 14, <0.0001 on day 40). T cell reconstitution, particularly CD4+ cells, was enhanced in the Treg group at both time points and reached statistical significance on day 40 (p-value=0.003). The number of donor natural killer cells was particularly enhanced on day 14 in the Treg recipients (p-value=0.0003). B-cell reconstitution, as measured by percentage of CD19+ cells, was minimal for all groups at day 14, but was enhanced at day 40 in recipient animals that received Treg (p-value=0.006). The T cell repertoire assessed by V-beta TCR screening with FACS analysis showed a polyclonal distribution. To determine if the improved and diverse lymphoid reconstitution is associated with increased immune function, mice were challenged with murine CMV (5x10e5 pfu/mouse) intraperitoneally at day 14 post-transplantation. Two weeks after infection, 66% of animals which received Treg in addition to Tcon, and 11% of animals which received Tcon alone were alive (p-value=0.05). Uninfected mice in the respective groups served as controls to separate the effect of CMV infection and GvHD on survival. Compared to infected animals, no deaths were observed in the respective uninfected groups at this time point (Tcon alone, p-value=0.0004; Treg+Tcon, p-value=0.21). In both infected and control uninfected animals, Treg treated animals had no evidence of significant clinical GvHD while animals that received Tcon alone had severe GvHD. These findings indicate that Treg enhance both the quantitative and functional recovery of the lymphoid cell populations while providing protection against GvHD.
Complete Absence of the Lineage-Determining Transcription Factor C/EBPα Results in Loss of Myeloid Identity in Bcr/abl Induced Malignancy.
Authors: Wagner Katharina , Zhang Pu , Rosenbauer Frank , Drescher Bettina , Kobayashi Susumu , Radomska Hanna S. , Kutok Jeffery L. , Krauter Jürgen , Tenen Daniel G.
Published: April 2020

The lineage-determining transcription factor C/EBPα is required for myeloid differentiation. Decreased function or expression of C/EBPα is often found in human acute myeloid leukemia. However, the precise impact of C/EBPα deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl induced myeloproliferative disease. The expression of bcr/abl in C/EBPα+/+ and C/EBPα+/− fetal liver cells lead to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl expressing C/EBPα−/− fetal liver cells fail to induce a myeloid disease in transplanted mice, but instead cause a fatal, transplantable erythroleukemia. Accordingly, increased expression of SCL and GATA-1 in hematopoietic precursor cells of C/EBPα−/− fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl positive cell line. Consistent with findings of the transplant model, expression of C/EBPα and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was upregulated upon C/EBPα expression. Chromatin immunoprecipitation was done and C/EBPα binding to a 3 prime enhancer of the Id1 gene was observed. Downregulation of Id1 by RNA interference impaired C/EBPα induced granulocytic differentiation. Thus, Id1 is a direct and critical target of C/EBPα. Taken together, our study provides the first evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPα.
Telomerase Promotes the Self-Renewal of Human Hematopoietic Progenitor Cells but Does Not Maintain Telomere Length.
Authors: Wei Junping , Wunderlich Mark , Mulloy James C.
Published: April 2020

Telomerase is a reverse transcriptase that adds telomeric sequences onto chromosome ends. During hematopoiesis, telomerase activity is high in proliferating hematopoietic stem and progenitor cells, but decreases sharply as cells differentiate. Approximately 90% of all human cancers are telomerase positive, including samples from AML patients. To understand the role of telomerase in human hematopoietic stem and progenitor cell proliferation and differentiation, and in the development of leukemia, we established stable long-term cultures expressing high levels of telomerase by retrovirus-mediated transduction of the h-TERT (catalytic subunit of telomerase) gene into AML1-ETO-expressing CD34+ (AE) cells. We have previously shown that these AE cells maintain CD34 expression and proliferate for 7 months in culture but gradually lose telomere length and are not immortal, providing a good model for studying the molecular mechanisms involved in telomere shortening. Strikingly, telomerase expression enhanced the self-renewal ability of the human hematopoietic progenitor cells, as shown by continuous replating ability in methylcellulose assays. We also detected a pronounced delay in the differentiation of the progenitor cells upon telomerase expression, and an expansion of the progenitor pool. Telomerase expression promoted proliferation due to increased cell cycle progression as well as a survival advantage. At the same time, these cells demonstrated a progressive decline in telomere length, despite telomerase activity equivalent to that detected in leukemia cell lines. We conclude that expression of hTERT expands the pool of hematopoietic progenitors but does not maintain long telomeres in human CD34+ cells. hTERT plays a critical role not only in telomere homeostasis for genetic stability but also in cellular proliferation, differentiation, and self-renewal, functions that may be part of hTERT involvement in leukemogenesis. These data open an important debate regarding the specific contribution of telomerase expression to the leukemic phenotype and the potential timing of this essential hit in the progression of the disease.
Strong Mutagenic Potential of Temozolomide in Bone Marrow Cells In Vivo.
Authors: Weiss Brian , Schleimer David , Geiger Hartmut
Published: April 2020

Therapy-related myeloid leukemias (t-ML) have emerged as severe late complications of cancer treatment in as many as 5–10% of patients who are otherwise cured of a primary neoplasm with aggressive multi-modal regimens. A new alkylating agent, temozolomide (TMZ), has shown efficacy in the treatment of adult high-grade glioma and refractory leukemia. Due to this early success, TMZ is now being incorporated as upfront therapy into more than 40 studies for a range of cancers including glioblastoma and melanoma. TMZ’s method of action depends on the methylation of guanine bases at the O6 position, resulting in O6-methylguanine and G:C‡A:T transitions. While some early in vivo studies of TMZ demonstrated tumorigenicity after contact with normal tissues, until recently no cases of secondary malignancies from TMZ exposure had been reported in the literature. However, two groups have now reported secondary myeloid malignancies after TMZ treatment (De Vita, De Matteis et al. 2005; Su, Chang et al. 2005). As TMZ moves into the frontline of our chemotherapeutic armamentarium, further investigation of its in vivo mutagenic potential are warranted.

We have employed a transgenic genomic mutation indicator mouse strain (small blue mouse) to determine the in vivo mutagenic potential of TMZ on bone marrow (BM) cells in comparison to cyclophosphamide (CP). In this mouse model, the mutational target is the non-transcriptionally active lacZ portion of the plasmid pUR288. The plasmid is stably integrated in multiple copies into the genome, and the mutation frequency in the lacZ gene is determined with a plasmid rescue procedure applied to genomic DNA derived from BM (or any other tissue) and a subsequent selection for lac-Z negative clones. The type of mutation can then be further determined by PCR amplification and restriction digestion.

For this study, animals were treated with TMZ (175 mg/kg/day i.p for 5 days), CP, or PBS, and BM was harvested 10 days after the last treatment. CP treatments were 200 mg/kg i.p. either once or weekly for 6 weeks. Determination of the mutation frequency revealed that the 1-day CP treatment increased the mutational load in BM approximately 2–3 fold over control, whereas the TMZ regimen resulted in an almost 40-fold increase over control. BM cells in animals treated 6 times with CP did not show an increase in the mutation frequency over animals treated only one time with CP. As expected, over 80% of all mutations in response to TMZ treatment were point mutations. BM cells from animals treated with CP showed less than 30% point mutations, with the remaining mutations being either translocations or deletions. This data emphasizes the strong mutagenic potential of TMZ on BM cells in vivo in our mouse model system. Further investigation is needed to determine whether the increased mutation rate seen with TMZ exposure is indicative of an elevated risk of therapy-induced leukemia.
Interplay between Tumor Cell-Associated and Circulating Coagulation Factors in Establishing Metastatic Potential.
Authors: Palumbo Joseph S. , Massari Jessica V. , La Jeunesse Christine M. , Flick Matthew J. , Hu Zhiwei , Talmage Kathryn E. , Degen Jay L.
Published: April 2020

Tumor cell-associated tissue factor (TF) is an established determinant of metastatic potential. In order to explore the interplay between tumor cell-associated TF and circulating hemostatic system components in malignancy, we established a line of hRas-transformed tumor cells from C57Bl/6-derived TF−/− embryonic fibroblasts. These cells were subsequently transfected with expression vectors encoding wildtype murine TF (TFWT), a mutant form of murine TF lacking the cytoplasmic tail (TFΔTail), or no transgene product (TFO). Stable transfectants expressing TFWT and TFΔTail were selected by fluorescence-activated cell sorting using ICON protein (a chimeric derivative of murine fVIIa linked to the Fc portion of human IgG). All three tumor cell lines supported similar and robust tumor growth following subcutaneous transplantation into immunocompetent C57Bl/6-inbred mice. However, consistent with previous reports, TFWT cells were dramatically more metastatic than TFO cells. Loss of the cytoplasmic tail significantly reduced metastases relative to TFWT cells, but TFΔTail cells were far more metastatic than TFO cells, indicating that the TF cytoplasmic tail is important, but not strictly-required to support metastasis. Imposing genetic deficits in either platelet function (i.e., Gαq deficiency) or coagulation function (i.e., fibrinogen deficiency) significantly diminished the metastatic potential of TF-expressing tumor cells. The low metastatic potential of TFΔTail cells was further diminished in mice with impaired platelet activation, indicating that loss of the cytoplasmic tail does not uncouple metastatic potential from circulating hemostatic system components. Studies of the early fate/survival of [125I]-iododeoxyuridine radiolabeled TFWT and TFO cells revealed that neither fibrinogen nor tumor-associated tissue factor were required for initial tumor cell adhesion within the pulmonary vasculature. However, the combination of tumor cell TF and circulating fibrinogen was shown to enhance the longer-term survival of tumor cells within the lungs. Based on the earlier finding that both platelets and fibrinogen support metastasis in part by impeding natural killer cell mediated clearance of embolic tumor cells, a favored working hypothesis is that circulating hemostatic factors couple tumor cell-associated TF to evasion of innate anti-tumor immune surveillance mechanisms.
Jak3 Kinase Domain Crystal Structures and Implications for Jak-Specific Drug Design.
Authors: Boggon Titus J.
Published: April 2020

Janus (Jak) family non-receptor tyrosine kinases are critical for appropriate signaling of many growth factors and cytokines. The four vertebrate Jak kinase family members demonstrate differential receptor cytoplasmic tail binding associations and transduce discrete signals on extracellular binding of ligand to the transmembrane cytokine or growth factor receptor. On ligand binding, a rapid tyrosine phosphorylation mediated signaling cascade is initiated, culminating in translocation of cytosolic latent transcription factors, signal transducer and activator of transcription (Stat) proteins, to the nucleus and targeted activation of transcription. Dysregulation of this Jak-mediated signaling pathway is documented in a number of hematological diseases: Improper upregulation of Jak activity is seen in certain hematological malignancies [1] and inability to appropriately transduce signals from the common gamma chain, γc, through Jak3 is responsible for approximately 60% of human severe combined immunodeficiency cases [2]. In addition, Jak2 point mutation V617F is frequently documented in myeloproliferative disorders including polycythemia vera; this activating mutation may disrupt an autoinhibited conformation [3]. Therapies targeting restraint of Jak family tyrosine kinase activity may be useful for treating inappropriate activation of Jak signaling cascades or for suppressing the immune response.

Advances towards structure-directed drug design of Jak-specific inhibitors were made recently with solution of the Jak3 kinase domain X-ray crystal structure [4], representing the first three-dimensional structural data for any portion of the Jak family of tyrosine kinases. Here three further crystal structures of the kinase domain of Jak3 are presented: an improved resolution co-crystal structure with staurosporine analog AFN-941 and two crystal forms of Jak3 kinase domain in complex with the kinase inhibitor small molecule compound QAD-409. Comparisons between these three solved Jak3 kinase domain crystal structures illustrate conformational flexibility between the kinase domain lobes and in the area of the catalytic cleft. Further structure analysis is also presented documenting in silico modeling of the binding of small molecule CP-690,550 [5] to the different Jak3 kinase domain crystal forms. Potential binding conformations of this inhibitor to the Jak3 kinase domain are suggested with one highly scored binding conformation predicted for all crystal forms. The crystal structures and modeling studies presented further define the extent of the Jak kinase catalytic cleft, demonstrate conformational plasticity in the active conformation Jak3 kinase domain and will aid the design of higher specificity Jak inhibitors.
Soluble Syndecan-1 (sCD138) Is an Independent Prognostic Factor in Patients with Chronic Lymphocytic Leukemia.
Authors: Jilani I. , Keating M. , William W. , Ferrajoli A. , Kantarjian H. , O’Brien S. M. , Giles F. J. , Albitar M.
Published: April 2020

Syndecan-1 (sCD138) is a transmembrane heparan sulfate-bearing proteoglycan expressed in epithelial cells as well as hematopoitic cells that demonstrate plasmacytioid differentiation. CD138 is believed to play a role in cell-cell and cel-matrix interaction. A soluble form of CD138 (sCD138) has been reported to be elevated in multiple myeloma. Higher levels of sCD138 have been reported to correlate with poor outcome in myeloma. While some cells in patients with chronic lymphocytic leukemia (CLL) can demonstrate plasmacytoid differentiation, CD138 is usually not expressed in B-cell CLL. We investigated the levels of circulating sCD138 in the plasma of 104 patients with CLL and correlated these levels with clinical behavior. sCD138 levels were elevated in patients with CLL as compared with normal control subjects (median, 52.8, range: 13.4-252.7 ng/mL) (P<0.01). Patients with levels of sCD138 higher than the median (53 ng/mL) had significantly shorter survival (Figure; P=0.0002). More importantly, this association was independent of both the IgVH mutation status and beta2- microglobulin levels. The same was true whether patients were previously treated (40 patients, P=0.004) or not (64 patients, P=0.01). Patients who had mutated IgVH but high sCD138 levels (>53 ng/mL) had significantly shorter survival than those with mutated IgVH and lower levels of sCD138. Similarly, patients with unmutated IgVH but high levels of sCD138 (>53 ng/mL) had significantly shorter survival than those with lower levels of sCD138 and unmutated IgVH (P=0.007). When CLL patients were dichotomized into 2 groups according to the median level of sCD138, there was significant positive correlation with age, platelet count, male sex, white cell count, and sCD23 level (all P<0.05). In contrast, no significant correlation with these prognostic factors or survival was found when sCD138 was considered as a continuous variable. This suggests that the role of sCD138 is more related to its presence or absence. The data presented here suggest that sCD138 is a powerful independent prognostic factor in CLL, and further studies are needed to explore its biological role and the potential of targeting this pathway as a therapeutic approach.

Figure Figure
Combination Chemotherapy with Fludarabine, Cyclophosphamide and Mitoxantrone (FCM) Induces a High Response Rate in Previously Untreated CLL.
Authors: Bosch Francesc , Ferrer A. , Villamor N. , Gine E. , Abella M.A. , Gardella S. , Besalduch J. , Gonzalez M. , Altes A. , Escoda L. , Ferrer S. , Gonzalez-Barca E. , Perez-Ceballos E. , Asensi A. , Montserrat E.
Published: April 2020

Treatment with FCM results in responses rates of 60% in relapsed or refractory CLL patients. Against this background, we started a trial of FCM in untreated patients diagnosed with CLL younger than 65 yrs. FCM consisted of fludarabine 25 mg/m2 i.v. on days 1 to 3, cyclophosphamide 200 mg/m2 on days 1 to 3, and mitoxantrone 6 mg/m2 i.v. on day 1, given at a 4-week intervals up to six courses. Patients received support with G-CSF and prophylaxis with cotrimoxazole. Response was assessed two months after treatment and included bone marrow and minimal residual disease (MRD) analysis by four-color flow cytometry and PCR. Out the 64 evaluable patients (74% male, median age 58 years), 83% were in advanced (B and C) Binet’s clinical stage and 62% had increased (>20%) ZAP-70 expression. FISH analysis disclosed del(13q) in 25%, +12 in 22%, del(17p) in 10% of cases and del(11q) in 23%. Eighty-three per cent of the patients received the entire planed treatment. Overall response rate was of 88%. MRD-negative CR was obtained in 24%, MRD-positive CR in 23%, nPR 22% and PR 11%. Two out of 14 nPR cases were MRD-negative. Duration of response was 55% at 36 months. Initial parameters associated with CR achievement were the presence of del(17p) (p=0.003), increased serum LDH (P=0.014), and splenomegaly (p=0.04). Hematological toxicity was mild, with grade III-IV neutropenia in 8% of the cases, and moderate infections in 12%. Two patients developed fulminant B hepatitis, one of them dying as a direct consequence of it. In conclusion, in untreated CLL patients, FCM induces a high CR rate, including an important number of MRD negative CR. This places FCM among the most effective regimens for CLL and serves to build up a new immunochemotherapy regimen for CLL (R-FCM) currently under investigation.
Drug-Dependent Murine Monoclonal Antibodies (mAbs) Mimic Antibodies Found in Patients with Drug-Induced Immune Thrombocytopenia (TP).
Authors: Bougie Daniel W. , Birenbaum Jessica , Ahl Scott , Aster Richard H.
Published: April 2020

Drug-induced immune thrombocytopenia (DITP) is a serious and sometimes life-threatening complication of treatment with many drugs. In most instances (excluding heparin-induced TP), platelet (plt) destruction is caused by antibodies (abs) that recognize distinct epitopes on platelet membrane glycoproteins (GP) only when the sensitizing drug is present in soluble form. How drug at pharmacological levels promotes tight binding of antibody to a specific target is unknown, in part because only polyclonal human abs have been available for study. We sought to produce monoclonal abs (mAbs) that mimic the behavior of human drug-dependent abs to create tools that can be used to study the molecular basis for this interaction. Mice were immunized with GPIIb/IIIa isolated from human platelets together with soluble quinine (Qn), tirofiban (Tf), or eptifibatide (Ef), three drugs that commonly cause DITP. Hybridomas were prepared from splenic B cells using a standard protocol and approximately 550 supernatants from each cultured hybrid line were screened for DDAbs by flow cytometry using normal platelets as targets. To date, 11 Abs that react with GPIIb/IIIa only in the presence of the immunizing drug (2 Qn-, 3 Tf- and 6 Ef-specific) have been identified. Preliminary studies show that the Qn-specific abs bind reversibly to GPIIb/IIIa at 50 nM Qn, a concentration much lower than is achieved pharmacologically, and are not inhibited by Qn at 5 mM, the limit of solubility. Quinidine (Qd) the diastereoisomer of Qn, supports only weak ab binding at a concentration of 50 uM. The tirofiban and eptifibatide-dependent abs recognize GPIIb/IIIa occupied by these RGD ligand-mimetic GPIIb/IIIa inhibitors. In each of these respects, reaction patterns of the three groups of mAbs closely resemble those of abs from patients experiencing TP after treatment with one of these drugs. These findings show that mAbs mimicking the behavior of human drug-dependent abs can be produced by immunizing mice with GP and soluble drug to produce probes suitable for characterizing the molecular basis of ab-drug-target interactions leading to platelet destruction in DITP. It is noteworthy that these mAbs were induced using soluble drug and protein for immunization because this suggests that the immune response leading to DITP does not require the sensitizing drug to be covalently linked to a protein, i.e, does not require the drug to act as a classical hapten.
Thrombocytopenia and Cardiomyopathy (trac): A New Single Gene Mutation Resulting in Severe Platelet and Heart Abnormalities in Mice.
Authors: Shultz Leonard D. , Lyons Bonnie L. , Bronson Roderick T. , Donahue Leah Rae , Burzenski Lisa M. , Gott Bruce , Harris Belinda S. , Italiano Joseph E. , Johnson Kenneth R.
Published: April 2020

We have discovered a new spontaneous mouse mutation, named “ thrombocytopenia and cardiomyopathy” (trac), in a colony of A/J mice at The Jackson Laboratory. Homozygosity for this recessive mutation results in severe pathologic changes in platelets, dilated cardiomyopathy, and infertility. Although platelet counts in A/J-trac/trac mice are normal at 3 weeks of age, there is a precipitous drop in platelet numbers and increases in platelet volumes by 4 weeks of age. Quantitative analyses of peripheral blood in A/J-trac/trac mice at 10 weeks of age showed a 20-fold reduction in platelet numbers accompanied by a 3-fold increase in platelet volumes compared with non-mutant (+/?) sex-matched controls. Other abnormalities in trac/trac platelets include greatly increased size of the microtubule coil and increased CD41 expression. Thrombocytopenia in trac/trac mice was associated with greatly increased bleeding times. Homozygotes (trac/trac) showed other hematological changes that included mild microcytic anemia accompanied by a doubling of reticulocyte numbers and two-fold decrease in WBC counts. Histological examination of blood smears confirmed abnormally enlarged platelets as well as the presence of megakaryocytoid cells. Increased numbers of megakaryocytes were present in BM, spleen, and lungs of trac/trac mice. Ultrastructural studies showed a lack of well-defined demarcation channels in trac/trac megakaryocytes. In order to determine whether the trac/trac platelet abnormality is intrinsic to defects at the level of BM progenitor cells, reciprocal BM chimeras were examined. Lethally irradiated +/+ recipients of 5 x 106 trac/trac BM cells were reconstituted with normal platelet numbers and volumes. In contrast, lethally irradiated trac/trac recipients of 5 x 106 +/+ BM cells remained thrombocytopenic. Backcrossing of the trac mutation onto the BALB/cBy strain background facilitated the identification of homozygotes prior to weaning and indicated the presence of modifying genes. Our preliminary data has produced a fine structure map of a 5 megabase interval containing the trac locus on mouse Chromosome 17. The human synteneic region is Chr 2p21-p22. We hypothesize that identification of the molecular basis of this mutation will reveal a novel gene that plays a critical role in the homeostasis of platelets and cardiomyocytes.
RanBP10 Is a Cytoplasmic GDP/GTP Exchange Factor That Modulates Microtubule Dynamics in Late Megakaryocytes.
Authors: Schulze Harald , Dose Marei , Korpal Manav , Italiano Joseph E. , Shivdasani Ramesh A.
Published: April 2020

Megakaryocytes are large cells within the bone marrow that undergo complex fragmentation to release up to thousands of virtually identical blood platelets into the periphery. Each platelet contains a characterisitic microtubule (MT) marginal band that is derived from MT filaments present in long protrusion-like intermediate structures, designated proplatelets, that are immediate precursors of platelets. These MT filaments are generated in the MK periphery, where they require massive mobilization that is supposed to be different from either normal interphase MT nucleation that commonly depends on γ-tubulin in the MT-organizing center. or from MTs in the mitotic spindle that require Ran·GTP, which is generated along condensed chromosomes by the chromatin-asociated guanine nucleotide exchange factor (GEF) RCC1. We first demonstrated that γ-tubulin is absent in most of the mature or proplatelet-forming MKs, where it is therefore unlikely to attribute to the total MT nucleation. MTs are tubular cytoskeletal structures that contain polymerized α- or β-tubulin subunits. Mammalian genomes share 5–6 β-tubulin isotypes of which β1-tubulin is the most divergent, especially in its C-terminal domain. β1-tubulin expression is restricted to late MKs and platelets, where it accounts for most of the β-tubulin in MT filaments. Its ablation in the mouse results in thrombocytopenia, spherocytosis and attenuated platelet function. We therefore sought to identify proteins that bind to β1-tubulin and performed a yeast two-hybrid screen using a MK-derived cDNA library. We identified a cytoplasmic Ran-binding protein, RanBP10, as a factor that associates with cellular MTs and unexpectedly harbors GEF activity toward Ran. Loss of RanBP10 in cultured MKs disrupts MT organization and its overexpression drives accumulation of extranuclear Ran and assembly of thick and abnormally long MTs. RanBP10 thus functions as a localized β-tubulin binding protein that harbors GEF activity toward Ran in the cytoplasm, much like RCC1 in the nucleus. Our results suggest that spatiotemporally restricted generation of Ran·GTP in the cytoplasm organizes specialized MTs required for thrombopoiesis and that RanBP10 provides a molecular link between Ran and non-centrosomal MTs.
Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).
Authors: Kalra Kavita B. , Cheng Xiangfei , Womak Marion , Gocke Christopher , Patel Jyoti B. , Njar Vincent C.O. , Takebe Naoko
Published: April 2020

All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.
Development of a Measure of Health-Related Quality of Life for Non-Hodgkin’s Lymphoma Clinical Research: The Functional Assessment of Cancer Therapy - Lymphoma (FACT-Lym).
Authors: Cella David , Webster Kimberly , Cashy John , Kutikova Lucie , Burgess Michael F. , Lin Boris K. , Bowman Lee , Liepa Astra M. , Gauthier Janine E. , Gregory Stephanie A. , Johnson Stephen A. , Cheson Bruce D. ,   on Behalf of the FACT-Lym Development Group
Published: April 2020

Background: The FACT-Lym was developed to address the disease-specific quality-of-life (QOL) issues for patients with non-Hodgkin’s lymphoma (NHL).

Methods: Items for inclusion in the lymphoma subscale for the FACT instrument were generated from interviews with health care providers and published literature on NHL. Items were selected based on symptom relevance, disease-specificity and clinical relevance. Validation testing was conducted with NHL patients. Patients completed the FACT-Lym and other measures at three timepoints: baseline (T1), 3–7 days (T2), and 8–12 weeks (T3). These patients were also interviewed about item content.

Results: From a pool of 69 items, 22 items were selected for testing based on relevance ratings of 17 experts. Items included B symptoms, sleep disturbance, anorexia, pruritus, pain, swelling, dyspnea, fatigue, emotional lability, worries, and uncertainty about the future. 75 NHL patients participated in validation testing (55% female, 59% with indolent disease, and 84% currently receiving treatment). Patient interviews confirmed item relevance and comprehensiveness. Item correlations, expert relevancy ratings, and patient input further reduced the subscale to 15 items. Internal consistency coefficients (alpha) for the 15–item subscale (.80, .86, .83) and test-retest stability (.85) suggest very good reliability. Correlations between the subscale and the SF-36 physical (r=.62) and mental (r=.47) summary scores reflect concurrent validity. Responsiveness to ECOG performance status rating and treatment status (on vs. off) equaled or exceeded that of the more established FACT subscale scores. From T1 to T3, subscale scores declined (−6.8, effect size= −0.86) in 14 patients who reported themselves as worsened, increased (3.9, effect size= 0.49) in 28 patients who reported themselves as improved, and did not change (−0.2, effect size= −0.02) in 18 patients who reported themselves as unchanged.

Conclusions: The FACT-Lym addresses QOL concerns that are relevant to NHL patients and is reliable and valid for use in clinical research.
Prognostic Gene-Expression Signatures in Adult Acute Myeloid Leukemia with Normal Karyotype.
Authors: Bullinger Lars , Bair Eric , Kranz Raphael , Dohner Konstanze , Frohling Stefan , Schlenk Richard F. , Tibshirani Robert , Dohner Hartmut , Pollack Jonathan R.
Published: April 2020

Acute myeloid leukemia (AML) encompasses a large number of morphologically similar but molecularly distinct variants. Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value. However, despite recent successes in detecting novel molecular markers like FLT3 (fms-related tyrosine kinase 3) mutation, treatment stratification is still difficult, especially for the 40–45% of patients with intermediate-risk, normal karyotype disease. To better characterize AML at the molecular level, and to address the need for improved risk stratification, we recently profiled gene expression in a large series of adult AML patients (Bullinger et al., N Engl J Med350:1605, 2004). By unsupervised analysis we identified new prognostically-relevant AML subgroups, and using a supervised learning algorithm we constructed a gene-expression based outcome predictor, which accurately predicted overall survival across all patients, including for the subset of AML cases normal karyotype. Having demonstrated the presence at diagnosis of normal karyotype signatures correlating with clinical outcome, we have now sought to refine a prognostic signature specific for normal karyotype disease. Towards this goal, we have now profiled 119 samples of adult AML patients with normal karyotype using 42k cDNA microarrays from the Stanford Functional Genomics Facility. By semi-supervised analysis using the supervised principal component method (Bair et al., PLoS Biology2:511, 2004), we built a cross-validated gene-expression based outcome predictor in a randomly partitioned training set (n=60 samples). This outcome signature, comprising only 16 genes, significantly predicted outcome class for normal karyotype samples in the independent test set (n=59 samples; P=0.001). In multivariate analysis, the 16-gene signature was a strong [odds ratio=0.35 (0.13 to 0.91); P=0.01] factor in predicting overall survival, independent of known prognostic factors including FLT3 mutations and preceeding malignancy. Our findings support the utility of expression profiling for improved risk stratification and clinical management of the clinically important subclass of AML patients with normal karyotype disease.