bluuebunny/crossref_metadata_2025 huggingface dataset:
IRF-4 Functions as a Tumor Suppressor in Early Stages of B Cell Development.
Authors: Acquaviva Jaime , Ren Ruibao
Published: April 2020

Interferon regulatory factor 4 (IRF-4) is a hematopoietic cell restricted transcription factor important for hematopoietic development and immune response regulation. IRF-4 is essential for mature B and T lymphocyte development, dendritic cell differentiation and some macrophage functions. It was also originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. Recent studies show that expression of IRF-4 is essential for the maintenance of multiple myeloma cells, making IRF-4 an attractive target for the development of cancer therapies. In addition to its role in late stages of lymphocyte development, IRF-4 functions redundantly with its closely related family member, IRF-8, to promote differentiation at the pre-B to immature B transition, apparently by repressing the pre-B cell receptor signaling and promoting light chain gene rearrangement. In contrast to its oncogenic properties, expression of IRF-4 is downregulated in BCR/ABL positive acute B-lymphoblastic leukemia (B-ALL) and is increased in response to imatinib, a selective BCR/ABL tyrosine kinase inhibitor. To assess the role of IRF-4 in the pathogenesis of BCR/ABL-positive B-ALL, we examined the effect of both loss of and forced expression of IRF-4 in BCR/ABL leukemogenesis using a mouse bone marrow transduction and transplantation model system. We found that BCR/ABL induced B-ALL significantly faster in IRF-4-deficient cells than control cells. On the other hand, forced expression of IRF-4 potently suppressed BCR/ABL transformation of bone marrow derived B lymphoid precursors in vitro and BCR/ABL induced B-ALL in vivo. These results provide evidence that IRF-4 functions as tumor suppressor in early B cell development and may allow elucidation of new molecular pathways significant to the pathogenesis of BCR/ABL positive B-ALL. In a parallel study, we have shown that IRF-4 functions as a tumor suppressor in myeloid lineage. The context dependent roles of IRF-4 in oncogenesis should raise caution in the development of cancer therapies targeting IRF-4. Further dissecting the mechanisms by which IRF-4 functions as an oncogene or a tumor suppressor gene is crucial for developing therapeutic strategies precisely targeting the IRF-4 oncogenic pathway as well as for developing therapeutic strategies taking advantage of the IRF-4 tumor suppressor pathway.
NF-E2 Overexpression Delays Erythroid Differentiation and Increases Erythrocyte Production
Authors: Mutschler Manuel , Magin Angela S. , Buerge Martina , Will Britta , Pilz Ingo H. , Migliaccio Anna Rita , Pahl Heike L.
Published: April 2020

The transcription factor Nuclear-Factor-Erythroid2 (NF-E2) is overexpressed in the vast majority of patients with polycythemia vera (PV). PV affects precisely those lineages expressing NF-E2: hematopoietic precursors, erythroid, megakaryocytic and granulocytic cells. In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of EPO. EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic symptom of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression lead to a delay in erythroid differentiation, manifested by a belated appearance of GlycophorinA-positive mature erythroid cells. Differentiation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted differentiation lead to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in BFU-E formation. These data argue that NF-E2 overexpression delays the early phase of erythroid differentiation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV.
Peripheral Blood Cytogenetic Studies in Myelofibrosis: Overall Yield and Comparison with Bone Marrow Cytogenetic Studies
Authors: Hussein Kebede , Ketterling Rhett P. , Dewald Gordon W. , Van Dyke Daniel L. , Mesa Ruben , Tefferi Ayalew
Published: April 2020

Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies in either primary (PMF) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis. In a retrospective study, we examined overall yield from PB cytogenetic studies in MF and where possible compared the results with those obtained by BM cytogenetic analysis.

Methods: Standard laboratory techniques were used to perform cytogenetic studies in both the PB and BM. A successful study constituted the acquisition of at least two analyzable metaphases and an abnormal clone required the presence of a specific cytogenetic abnormality in at least two metaphases. Clinical and laboratory correlations were made using variables collected at the time of cytogenetic studies.

Results I: A total of 242 PB cytogenetic studies were reviewed to identify 59 MF cases; 39 were PMF and 20 post-PV/ET MF. Median age was 60 years (range 36–77) and 37 were male patients. The median duration of the disease at the time of PB cytogenetic studies was 3.5 years (range 0.2–35). At least two analyzable metaphases (median 20, range 2–31) were obtained in 49 (81%) of the 59 study patients; in univariate analysis, this was predicted by presence and degree of circulating myeloid progenitor cells (p < 0.0001), higher PB CD34 count (p < 0.0001), higher leukocyte count (p = 0.0008), and absence of myeloid growth factor therapy (p = 0.005). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance.

Results II: Twenty two cases had concomitant PB and BM cytogenetic studies performed. Of these, 9 patients had abnormal BM cytogenetic findings; the concomitant PB cytogenetic studies were also abnormal in 5 (56%) patients but normal in the other 4. Conversely, PB cytogenetic studies were abnormal in 12 patients; the concomitant BM cytogenetic studies were abnormal in 5 (41%) and normal in the remaining 7. PB cytogenetic studies were not successful in 4 of the 22 total cases; BM studies were successful in 3 of these 4 cases and the results were normal in all.

Conclusion: In the presence of circulating myeloid progenitor cells, PB can be considered as an alternative to BM for cytogenetic studies in MF patients, especially in view of the difficulty getting BM aspirates in this disease. However, considering the prognostic value of specific cytogenetic abnormalities in MF, the non-trivial discordance between the BM and PB cytogenetic findings, observed in the current study, warrants a prospective evaluation for further clarification.
Platelet vWF, ADAMTS-13, and Neutrophil Activation in Patients with Essential Thrombocythemia (ET)
Authors: Falanga Anna , Marchetti Marina , Russo Laura , Barbui Tiziano
Published: April 2020

ET is a chronic myeloproliferative disorder, the benign clinical course of which can be complicated by both thromboses and hemorrhages. The levels of the largest multimers of plasma vWF, are relevant for the hemostatic balance in this disease. vWF is stored in the cellular granules of both endothelial cells and platelets, from which is released upon activation. Neutrophils and platelets circulate in an activation status in ET patients, leading to an increased formation of platelet/neutrophil aggregates. In this study we evaluated in ET patients, the platelet vWF content and its relation to neutrophil activation. In addition, the activity of ADAMTS13, the enzyme responsible for maintaining the normal size distribution of vWF multimers, was studied. A group of 61 consecutive ET patients (50% carrying the JAK2 V617F mutation) and 30 healthy controls were included into the study. Total vWF antigen (vWF:Ag) and activity (vWF:Act) levels were determined in both plasma and in isolated washed platelets by ELISA and collagen binding assay (CBA), respectively. ADAMTS13 activity was measured in plasma according to Gerritsen’s method. CD11b and leukocyte alkaline phosphatase (LAP) were measured on neutrophil membrane as activation markers. Plasma vWF:Ag levels resulted significantly increased in ET patients compared to controls (p<0.05), the subgroup of patients with the JAK2 mutation showing the highest levels; whereas vWF:Act was not statistically different. The vWF Act/Ag ratio was significantly lower in ET compared to controls (0.85±0.15 vs 0.95±0.23; p<0.01). A significant inverse relation between platelet count and plasma vWF:Act existed. On the other hand, platelets from these patients had a decreased content of both vWF:Ag and vWF:Act than platelets form controls (p<0.01), suggesting an increased platelet release of vWF. In addition, platelets from ET JAK2 mutation carriers had significantly lower vWF:Act compared to wild-type subjects. Accordingly, the platelet vWF Act/Ag ratio of JAK2 mutation carriers was significantly (p<0.01) lower vs wild-type patients and controls. Neutrophil confirmed to be activated in these patients, as previously described. Indeed, surface activation markers were increased in ET patients compared to controls, and, in particular, CD11b levels were inversely related to platelet vWF:Act (p<0.01). The measurement of plasma ADAMTS13 showed a reduction in the activity of this protease in ET patients (68±16%) compared to normal control mean (p<0.01). In conclusion, in ET patients and particularly in JAK2 mutation carriers, a decrease in platelet vWF:Ag and Act is found and correlates with neutrophil activation, suggesting a role of neutrophil in platelet degranulation. Increased vWF release and reduced ADAMTS13 activity can represent a mechanism of hypercoagulation in these patients.
High Skp2 Expression Is an Independent Predictor of Unfavorable Outcome in 333 Patients with Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Seki Ritsuko , Ohshima Koichi , Kawano Fumio , Murayama Toshihiko , Moriuchi Yukiyoshi , Ogawa Ryosuke , Shimoda Kazuya , Suzushima Hitoshi , Tsuda Hiroyuki , Arima Nobuyuki , Tsukazaki Kunihiro , Suzuki Keiko , Iwahashi Masahito , Utsunomiya Atae , Matsumoto Tadashi , Okamura Takashi
Published: April 2020

The addition of rituximab to CHOP (CHOP-R) chemotherapy has resulted in an improved outcome for patients with DLBCL and has recently been shown to diminish the prognostic impact of several recognized biomarkers. S-phase kinase-associated protein 2 (Skp2) is a proto-oncogene that has been shown to be expressed in a number of tumors. We have reported that Skp2 expression in tumor cells is an unfavorable prognostic factor in DLBCL. In the present study, we investigated the significance of Skp2 expression in the patients with DLBCL treated with CHOP or CHOP-R. DLBCL patients (333 cases) were entered into this study, based on the availability of paraffin blocks for interpretable immunohistochemistry for all antigens (CD10, Bcl-6, MUM1, Bcl-2, Skp2). All patients were treated with either CHOP (201) or CHOP-R (132) from 1996 to 2005, and were diagnosed as having DLBCL at the twenty different hospitals. All specimens were histopathologically reviewed before entering into this study. Their clinical characteristics, including either the IPI or R-IPI factors, were evenly matched. The median follow-up of living patients was 3.7 and 2.1 y for CHOP vs CHOP-R, respectively. DLBCL were assigned to GCB subtype (40.8%: 136/333) or non-GCB subtype (59.2%: 197/333) based on the method of Hans et al., Blood 103: 275–82 (2004), with similar distribution in both treatment groups. Expression of bcl-6 (p<0.05) or GCB subtypes (p<0.05) was associated with better overall survival (OS), whereas expression of bcl-2 (p<0.05) was associated with worse OS in CHOP treatment group. The addition of R was associated with an improved survival in the non-GCB subtype and resulted in same as that of GCB subtype. The survival benefit of both low Bcl-2 and high Bcl-6 expressions diminished in combined treatment with R to CHOP. There were 97 patients with high Skp2 expression (>60% positive cells) (97/333: 29.1%). High Skp2 expression was found in both GCB subtype (28.6%) and non-GCB subtype (30.3%). In advanced clinical stage or extranodal involvement (>2), the patients with high Skp2 expression had worse survival than those with low Skp2 expression (p<0.05). Interestingly, in CHOP-R group, high Skp2 expression was the strong biomarker of worse prognosis (p<0.05). DLBCL patients with high Skp2 expression did not benefit from the addition of R to CHOP. Therefore, Skp2 may be a useful prognostic marker in recent rituximab era. The new treatment strategy is necessary for the DLBCL patients with high Skp2 expression.

Figure Figure
Serum B-Cell Activating Factor (BAFF) in Diffuse Large B-Cell Lymphoma: Its Prognostic Significance in the Rituximab Era.
Authors: Kim Seok Jin , Sung Hwa Jung , Choi Chul Won , Kim In Sun , Kim Byung Soo , Yoon Soo-Young , Won Jong-Ho , Kim Jun Suk
Published: April 2020

The introduction of rituximab, a chimeric monoclonal antibody against the CD20 antigen has significantly improved the treatment outcome of diffuse large B-cell lymphoma (DLBCL). However, not all patients with DLBCL respond to rituximab plus CHOP (R-CHOP) chemotherapy. Thus, resistant clones of DLBCL remain a significant clinical problem. Although the underlying mechanisms of this resistance are still not clear, considering that the dysregulation of the balance between cell survival and programmed cell death is a central feature of lymphomagenesis, a factor promoting survival of lymphoma cells may be associated with resistance of DLBCL to rituximab. B-cell activating factor (BAFF), a member of the tumor necrosis factor (TNF) superfamily has shown to be a key mediator in the formation and regulation of normal B-cell response. BAFF is usually expressed by monocytes and macropahges, and is promoting cell survival and preventing apoptosis. Therefore, we performed this study to determine the clinical impact of serum BAFF on the treatment outcome of DLBCL treated with R-CHOP. 48 patients diagnosed as DLBCL were enrolled, and all the patients were treated with R-CHOP every 3 weeks. Before treatment and every two cycles of R-CHOP, their serum was taken and cryopreserved. The level of BAFF was measured by ELSA method (Human BAFF immunoassay, R&D systems, USA). We also analyzed the expression of BAFF receptor in tumor sections by immunohistochemistry (Polyclonal antibody to BAFF receptor, Abcam, UK). Median age of the patients was 56 years old (range 21–79). 20 patients were stage I/II, and 28 were stage III/IV. Rituximab was administered at the dosage of 375mg/m2 on day1, and CHOP chemotherapy was combined with rituximab until 6 – 8 cycles. The mean level of BAFF at the time of diagnosis was 2284.54pg/mL, and the median was 1326.60pg/mL (138.80–13537.92pg/mL). When the patients were dichotomized into high and low BAFF group based on the median value, 21 patients showed complete response in low BAFF group, however, only 3 patients showed complete response in high BAFF group (p=0.011). Among 16 patients showing relapse, 11 patients belonged to high BAFF group. The expression of BAFF receptor was also increased in relapsed patients compared to non-relapse group (p < 0.05). In the analysis of relapse-free survival, low BAFF group did not reach the median value as yet while high BAFF group showed 379 days (p=0.046). Thus, baseline serum BAFF may be associated with response of DLBCL to R-CHOP. When we serially checked the level of serum BAFF, the level of BAFF was increased during treatment (After the 2nd cycle 6448.56pg/mL, and after 4th cycle 7421.28pg/mL). In conclusion, serum B-cell activating factor may be a useful indicator predicting response to R-CHOP and prognosis in patients with DLBCL.

Figure Figure
Signaling Pathways of All-Trans Retinoic Acid (ATRA)-Induced Apoptosis in Mantle Cell Lymphoma (MCL).
Authors: Singh Amareshwar T.K. , Bhalla Savita , Ryan Robert O. , Nguyen Thanh-Son , Singh Karthik T.K. , Evens Andrew M. , Gordon Leo I.
Published: April 2020

MCL, characterized by the t(11;14), results in the overexpression of cyclin D1, and typically has an aggressive clinical course. The disease is not curable by conventional therapy, and new modalities are needed. A MCL cell line, Granta, has been studied and has a complex karyotype and genetic alterations of cell cycle regulatory genes. ATRA is a metabolic derivative of retinoic acid (RA). Upon binding with ATRA, its receptors become transcriptionally activated and transactivate their target genes leading to cell growth arrest or apoptosis. We hypothesized that ATRA would lead to apoptosis or cell growth arrest in Granta MCL cells. We used ATRA nanodisks for increased solubilization and in vivo delivery. Reactive oxygen species (ROS) were measured with fluorescence-activated cell sorting (FACS) following incubation at 6 hours (h) and 24 h with ATRA (12.5–50nM). At 6 h, ATRA induced dose-dependent generation of ROS, which returned to baseline at 24 h. The levels of ROS in untreated controls were unchanged over all the incubation time points. To determine the effects of ATRA on apoptosis, Granta cells were treated with various concentrations for 6 h, 24 h, and 48 h; apoptosis was measured with annexinV/propidium iodide (annexin/PI) binding and FACS analysis. We found increasing apoptosis in Granta at 24 h, reaching significant levels at 48 h. At 48 h, the lowest doses of ATRA (12.5nM) resulted in >70% increase (as a percentage of control) in annexin/PI. To determine the biologic consequences of ATRA exposure in Granta, RARα protein levels were measured in control and ATRA treated cells at 2 h, 6 h, and 24 h. RARα protein was marginally increased at 6 h and significantly downregulated following 24 h ATRA exposure (50nM). Furthermore, ATRA significantly increased p21 protein expression at 24 h as assessed by immunoblotting. RT-PCR data showed that SESN1(T2) (an oxidant gene) transcript was virtually absent in Granta, and that SESN2 and CDKN1A transcripts were not affected by ATRA exposure. In summary, these data demonstrate that ATRA, complexed with nanodisk delivery particles, stimulates ROS generation as early as 6 h in Granta cells. In addition, ATRA induced significant apoptosis at 48 h, downregulated RARα protein, and increased p21 protein. Further studies investigating the role of ATRA in MCL are warranted.
Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.
Authors: Klier Margit , Anastasov Natasa , Angermeier Daniela , Raffeld Mark , Fend Falko , Quintanilla-Martinez Leticia
Published: April 2020

Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system.

Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.

Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2.

Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.
Synergistic Induction of Apoptosis by Simultaneous Disruption of the Bcl-2 and mTOR/Akt Pathways in Acute Myeloid Leukemia.
Authors: Zeng Zhihong , Samudio Ismael , Andreeff Michael , Konopleva Marina
Published: April 2020

Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway provides survival signals for leukemic cells and blockade of this pathway may facilitate cell death. Akt/mTOR activation is a frequent event in AML that translates into significantly shorter survival of AML patients (S.M. Kornblau, ASH 2007). Bcl-2 family proteins are key regulators of apoptosis that are known to promote tumorigenesis and chemoresistance. We have recently reported that ABT-737, a small molecule BH3 mimetic, effectively kills AML progenitor cells via the disruption of Bcl-2:Bax, but its activity is largely diminished in cells overexpressing Mcl-1 (M. Konopleva, Cancer Cell 2006). We have now investigated anti-leukemia efficacy of concomitant blockade of mTOR/Akt, known to regulate Mcl-1 levels, and of Bcl-2 signaling. Combined use of rapamycin derivative temsirolimus and ABT-737 induced striking apoptotic responses in AML cell lines OCI-AML3 and MOLM13 leukemic cells. Mechanistically, temsirolimus downregulated expression of the anti-apoptotic protein Mcl-1 and induced expression levels of the proapoptotic BH3-only protein Noxa which specifically neutralizes Mcl-1. These effects were seen upon prolonged temsirolimus treatment that inhibited mTORC2 complex (Zeng, Blood 2007) and downregulated Akt phosphorylation. In primary AML samples co-cultured with bone marrow-derived stromal cells, ABT-737 at 25nM induced apoptotic cell death in CD34+ AML progenitor cells from 13 of the 16 samples tested. Temsirolimus/ABT-737 combination markedly enhanced apoptosis induction in 8/9 primary samples that were sensitive to temsirolimus alone (specific apoptosis, temsirolimus 15±5%; ABT-737, 28±5.9%; temsirolimus+ABT, 45±5.9%, p<0.001) and in 3/7 samples resistant to temsirolimus (p=0.7). Further, combined blockade of mTOR/AKT and Bcl-2 pathway effectively induced apoptosis in 3 AML samples resistant to ABT-737 alone. Taken together, these results indicate that temsirolimus can directly alter the Bcl-2 apoptotic rheostat via induction of Noxa and decreased Mcl-1 expression sensitizing leukemia cells to apoptosis induced by BH3 mimetics. The combined use of agents interfering with AKT/mTOR and Bcl-2 pathways may be a highly effective anti-leukemia strategy that induces apoptosis in AML progenitor cells in the context of bone marrow microenvironment.
Phase I Study of S-trans, Trans-farnesylthiosalicylic Acid (FTS), a Novel Oral Ras Inhibitor, in Patients with Refractory Hematologic Malignancies.
Authors: Borthakur Gautam , Ravandi Farhad , O’Brien Susan , Beran Miloslav , Williams Betsy , Goldsweig Howard , Giles Francis
Published: April 2020

Background: Ras activation (mutational or non-mutational) is a key pathway for survival and proliferative advantage of leukemic cells. Farnesyl transferase inhibitors (FTI) that are thought to alter lipid modification of Ras and thereby alter its membrane targeting, are in clinical trial in hematologic malignancies but their target may not be specific for Ras. Toxicities of FTIs include diarrhea, skin rash and hepatic toxicity. FTS is an oral Ras inhibitor that causes dislocation of Ras from its membrane location by competing directly with farnesylated Ras to bind to its putative membrane binding proteins. FTS does not inhibit farnesyl transferase enzyme.

Methods: We report on an ongoing phase I study of FTS in patients with relapsed/refractory hematologic malignancies in which FTS is administered orally twice daily on days 1–21 of a 28 day cycle in a “3+3” dose escalation design.

Results: To date, 18 patients (pts) have been enrolled, with 17 pts treated (1 cycle=2, 2 cycles=8, 4 cycles=2, 5 cycles=1, 6 cycles=2, 8 cycles=1 and 9 cycles=1). Four pts were enrolled at dose level (DL) of 100 mg, 3 pts at each DL of 200, 400, 600 and 800 mg and 2 pts at DL of 900 mg. One pt enrolled at 100 mg DL was never treated. Median age was 72 years (range, 35 to 85 years), median ECOG status 1 (range 0–2) and median no. of prior therapies 2 (range, 0–7). Diagnoses included: Acute myelogenous leukemia (AML) = 9 pts, myelodysplastic syndrome (MDS) = 5 pts, chronic myelomonocytic leukemia (CMML) = 2 pts, chronic myelogenous leukemia (CML) = 1 pt. and Myeloproliferative Disorder = 1 pt. Preliminary analysis of pharmacokinetic data indicates that both Cmax and AUC increase with dose, but less than dose proportionately. Detailed PK data will be available for presentation at the American Society of Hematology 2007 Annual Meeting. Of 17 pts evaluable for response, 7 pts (41%) (4 MDS, 1 CMML, 1 CML, 1 AML) had hematological improvement (HI) (1 in three lineages, 6 in one lineage) without any complete remission. These responses lasted for a median of 9 weeks (range, 4 to 26 weeks). Currently, 2 patients are continuing on study with improvement in cytopenia and stable disease for over 20 weeks each. Grade 1–2 diarrhea has been the only non-hematologic toxicity seen, observed in 14 of 17 (82%) pts evaluable for toxicity and is completely correctable with oral antidiarrheals.

Conclusion: FTS shows promising activity in refractory hematologic malignancies and to date has minimal toxicity.
Expressing N-RasG12D from the Endogenous Promoter Induces Myeloproliferative Disease (MPD) and Cooperates with Retroviral Insertional Mutagenesis (RIM) To Generate Acute Myeloid Leukemia (AML).
Authors: Li Qing , Haigis Kevin , McDaniel Andrew , Huang Benjamin , Miller Michelle , Shieh Angell , Kogan Scott , Wolff Lynda , Jacks Tyler , Shannon Kevin
Published: April 2020

Somatic NRAS and KRAS mutations are common in myeloid malignancies, particularly juvenile and adult chronic myelomonocytic leukemia (JMML and CMML) and AML. Use of the interferon-inducible Mx1-Cre transgenic strain to activate oncogenic KrasG12D expression from the endogenous Kras promoter induces an aggressive MPD with 100% penetrance that is fatal by ∼100 days of age. These Mx1-Cre, KrasG12D mice do not develop AML, which suggests that additional mutations are required. To identify genes that might cooperate with KrasG12D in leukemogenesis, we performed RIM with MOL4070LTR virus. Mx1-Cre, KrasG12D pups were injected with this virus at birth and with pIpC at 3 weeks of age to induce Cre recombinase expression. Approximately 12% of these mice developed either AML or a transplantable MPD-like leukemia. In parallel studies, we intercrossed Mx1-Cre mice with a novel strain of loxP-STOP-loxP (LSL) NrasG12D “knock in” mice to investigate the effects of expressing NrasG12D in the same experimental system. Although Mx1-Cre, NrasG12D mice develop MPD, this disease is remarkably less aggressive with a much later onset of disease and longer survival compared to Kras mutant mice. Consistent with the attenuated disease phenotype, myeloid progenitors from Mx1-Cre, NrasG12D mice form fewer cytokine independent colonies in methylcellulose cultures and are less hypersensitive to growth factors than KrasG12D progenitors. Importantly, 90% of Mx1-Cre, NrasG12D mice that were injected with MOL4070LTR at birth and with pIpC at 3 weeks old developed AML or MPD-like leukemia, most of which demonstrate myelomonocytic features that are reminiscent of human M4 AML. We are cloning retroviral integrations from these leukemias and have identified Evi1 and other common insertions. Although both NRAS and KRAS are commonly mutated in myeloid malignancies, our data indicate that expressing these oncoproteins at endogenous levels in primary stem/progenitor cells results in distinct phenotypes. We recently showed that fetal liver cells infected with MSCV viruses expressing NrasG12D, Kras4aG12D, or Kras4bG12D demonstrate similar patterns of cytokine-independent colony formation and growth factor hypersensitivity. These data raise the possibility that variable levels of expression of Ras isoforms in different hematopoietic compartments may underlie some of our results. These models provide tractable systems for identifying cooperating genes in leukemogenesis and platforms for testing and designing new therapeutic agents.
Outcomes for Patients Who Fail High Dose Chemoradiotherapy and Autologous Stem Cell Rescue for Relapsed and Primary Refractory Hodgkin Lymphoma.
Authors: Moskowitz Alison , Yahalom Joachim , Kewalramani Tarun , Tejerina Manfred , Zelenetz Andrew D. , Moskowitz Craig H.
Published: April 2020

Background: Limited data exists regarding the outcome for patients (pts) with relapsed and primary refractory Hodgkin Lymphoma (HL) who fail high dose chemoradiotherapy (HDT) and autologous stem cell rescue (ASCR). The aim of this study is to determine survival time and evaluate the role of second transplantation in pts who have HL-related ASCR failure.

Results: At Memorial Sloan Kettering Cancer Center, from 1995–2005, two hundred and twelve pts with relapsed or primary refractory HL underwent HDT/ASCR after responding to ICE-based salvage chemotherapy on one of four consecutive clinical trials. Eighty-one patients failed HDT, including 10 treatment related mortality (4 deaths within 100 days from ASCR, 5 deaths due to secondary malignancies, and 1 death, 10 years after ASCR due to cardiomyopathy); leaving 71 (33%) pts with biopsy-confirmed progression; median time to progression was 5.7 months. There was 43 males (60%), median age at the time of ASCR was 29 (range 17–65). At a median follow-up of 2 years for surviving patients, the median overall survival from ASCR failure was 25 months. Only seventeen of the 71 pts are currently alive (24%), nine (12.6%) of whom are in remission. Remission duration from ASCR of < 3 months predicted for OS; 1 of 17 pts who relapsed within 3 months are currently alive vs 16 of 54 pts with remission duration of > 3 months; median OS are 13.5 and 27.3 months respectively (p=0.02). Treatment regimens following ASCR failure varied and included gemcitabine-based chemotherapy, MOPP, investigational therapies, radiotherapy and second transplantation. The impact of the various treatment regimens on OS following HL-related ASCR failure was difficult to assess due to the heterogeneity of regimens administered. Treatment with a second transplant had a favorable impact on OS, however only two of 17 pts with remission duration < 3 months were able to receive a second transplant because of rapid disease progression. Twenty-three patients underwent a second transplant of which seven are failure free (1/4 autologous, 6/19 allogeneic). The median OS was 40 months for patients transplanted vs. 19.3 months (p=0.05) for patients ineligible for a second transplant.

Conclusion: Patients who fail HDT/ASCR due to HL progression have a median OS of two years. Relapse within 3 months of ASCR is associated with a poor OS and investigational or novel therapy should be explored in this cohort of pts. An allogeneic transplant following HL-related ASCR failure is associated with a 30% chance of long term survival and should be strongly considered in pts whose remission duration from their initial ASCR is >3 months.
FANCD2 Purification and Identification of a Functionally Important Phosphorylation Event at Serine 331.
Authors: Zhi Gang , Tomashevski Andrei , Ward Michael , Newcomb William , Brown Jay , Semmes Oliver J. , Kupfer Gary M.
Published: April 2020

Fanconi anemia (FA) is an inherited hematological disorder characterized by birth defects, bone marrow failure, and cancer susceptibility. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to cross linking reagents such as mitomycin C (MMC). Cloned FA genes cooperate in a signaling pathway activated by DNA damage during replication, and monoubiquitination of FANCD2 is the critical downstream event. The check point kinase ATR is required for the efficient monoubiquitination of FANCD2. Inducible ATR-dependent phosphorylation of threonine 691 and serine 717 are reported to enhance the monoubiquitination of FANCD2 and resistance to MMC. In the present study, we have devised means to purify the FANCD2 protein into multiple subcomplexes. In doing so, we have identified at least 3 distinct subcomplexes: a pure multimeric precursor form comprised of FANCD2 polypeptides, a 1 MD form that is chromatin bound, and a 2 MD form that is in the nucleosol. Electron microscopic analysis of the precursor form reveals homogenous appearing complexes. Mass spectroscopic analysis of the 1MD FANCD2 complex has identified the FANCI protein, among others, which confirms recent reports of FANCD2-FANCI interaction. Further mass spectroscopic analysis of FANCD2 has identified a novel phosphorylation site on FANCD2, serine 331, which is further confirmed by immunoblot using phosphospecific antibody against phospho-serine 331. This phosphorylation site is conserved in other species, including mouse, rat, xenopus, and tetraodon nigroviridis. Single amino acid mutation by alanine at this site results in a complete loss of monoubquitination of FANCD2 and resistance to MMC, suggesting that the phosphorylation of S331 is a functionally significant event. Moreover, this site fits the consensus sequence for a CHK1 substrate, which in turn is a substrate of target for CHK1, a substrate of ATR, which is also required for monoubiquitination of FANCD2. These findings further support the correlation between phosphorylation and monoubiquitination of FANCD2 in the DNA damage response with the intervention of CHK1 as a mediator of ATR and suggest dynamic regulation of FANCD2 subcomplexes within the FA pathway.
Targeted Disruption of GAS6-Mertk Pathway Leads to Defects in Physiological Clearance of Expelled Nuclei from Erythroblasts by Bone Marrow Macrophages.
Authors: Kadi Linda , Burnier Laurent , Sugamele Rocco , Carmeliet Peter , Lemke Greg , Earp H. Shelton , Matsushima Glenn K. , Schapira Marc , Angelillo-Scherrer Anne
Published: April 2020

Late in erythropoiesis, nuclei are expelled from erythroblasts and engulfed by macrophages located in the blood island. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as a signal for their engulfment by macrophages. PS opsonins milk-fat-globule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors avb5 (and avb3) and Axl/Mertk/Tyro3, are involved in the phagocytosis of apoptotic cells. Because fetal liver and bone marrow macrophages do not express MFG-E8, GAS6-Mertk pathway might constitute a major pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from the spleens of phlebotomized mice (500 μl), and tested the capacity of bone marrow-derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), Axl (Axl−/−), Mertk (mertkkd) or Tyro3 (Tyro3−/−) to internalize these nuclei. Spleen erythroblasts were isolated 4 days after phlebotomy and cultured for 5 hours. Nuclei were obtained after disconnection from reticulocytes by mechanical shaking. Released nuclei were then identified by flow cytometry according to their size and their positive staining for the erythroid lineage marker Ter119 and Annexin V for PS labelling. Purity of the preparation was double-checked by morphological examination of cytospin preparations. Primary BMDM isolated from wild-type (WT) controls and GAS6−/−, Axl−/−, Mertkkd, Tyro3−/−, Axl/Tyro3−/−, Axl−/−/Mertkkd mice were incubated with nuclear preparation for 3 hours. BMDM were then washed to remove un-engulfed nuclei, analyzed in bright field and stained with May-Grünwald-Giemsa. Phagocytosis was determined by counting the number of BMDM with ingested nuclei and the phagocytosis index indicated the number of engulfed nuclei per macrophage. We found that GAS6−/− BMDM cleared 30% less nuclei than WT BMDM (p<0.01). We observed a slight decrease of internalization capacity for Axl−/− BMDM, whereas Tyro3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, Mertk deficiency nearly abolished the nuclei phagocytosis (p<0.001). Axl/Tyro3−/− and Axl−/−/Mertkkd BMDM were tested in comparison with WT BMDM and single knockouts, and did not show any cumulative effects when compared to single knockouts. Thus, Mertk was critical for the phagocytosis of nuclei from erythroblasts whereas the role of Axl and Tyro3 appeared to be negligible. In conclusion, we have shown that GAS6 and its receptor Mertk were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island. Indeed, GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and Mertk receptor on BMDM, allowing their efficient clearance.
A Case of HPP with a Novel Combination of α and β Spectrin Mutations.
Authors: Natarajan Kavita , Kutlar Ferdane , Li Tao , Gallagher Patrick , Kutlar Abdullah
Published: April 2020

Hereditary pyropoikilocytosis (HPP) is a red cell membrane disorder classified under the broad umbrella of hereditary elliptocytosis (HE). It is usually the most severe form of HE and is inherited in an autosomal recessive manner. HE results from alterations in RBC membrane cytoskeleton proteins: spectrin, ankyrin, pallidin, band 4.1, and band 3. Quantitative or qualitative deficiencies result in varying clinical syndromes. In general, mutations in the α gene are closer to the amino terminal, and those in the β spectrin gene are nearer the carboxyl terminal and affect the formation of tetramers. We describe a patient associated with a novel mutation in the β and α spectrin genes. A 19 year old black female with a lifelong history of anemia and jaundice that worsened since age 15 presented with history of early satiety of a few months duration. Neither parent had a history of a blood disorder; a maternal uncle had history of splenectomy in his thirties. Her pre-splenectomy labs showed a Hb of 4.8g/dl with a MCV of 62, RDW of 38.8, and chemistries consistent with non-immune hemolytic anemia. Imaging and clinical exam revealed a spleen of 19cms and bizarre red cell morphology including RBC fragmentation and microcytosis. Post splenectomy the patient is entirely asymptomatic with a Hb of 13.2 with pathologic exam of the spleen showing benign, enlarged, red pulp consistent with hypersplenism. Genomic DNA was extracted from peripheral blood. Sequencing of the β-spectrin exon 30 was undertaken at the MCG Sickle Cell Center that revealed a GCT → CCT mutation (Ala → Pro) in codon 2053. This mutation has been previously reported in the homozygous state in a young boy from Mali with HPP (β spectrin Kayes). Since we could not explain the relatively severe phenotype in this patient with heterozygous β spectrin Kayes, further molecular analyses was performed at Yale. This revealed a mutation in the α-spectrin exon 5; L207P, α spectrin St.Louis. This is the 2nd reported case of HPP due to compound heterozygosity for an α and β spectrin mutation and the first combination of α spectrin St. Louis and β spectrin Kayes.

African American female, 19 years old Diagnoses: “Pyropoikilocytosis” Sequence data: Spectrin bate-Kayes African American female, 19 years old Diagnoses: “Pyropoikilocytosis” Sequence data: Spectrin bate-Kayes
Polyphosphate Shortens the Clotting Time of Hemophilic and Anticoagulated Plasma.
Authors: Smith Stephanie A. , Morrissey James H.
Published: April 2020

Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain abundant polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway, promoting factor V activation, and abrogating tissue factor pathway inhibitor function. We now report that polyP shortens the clot time of hemophilic plasmas and plasmas containing a variety of anticoagulant drugs.

Methods: Citrated plasmas were obtained from normal donors, patients receiving coumadin, and individuals with severe hemophilia A or B. In some experiments, clinically relevant concentrations of unfractionated heparin, enoxaparin, or argatroban were added to normal plasma. In other experiments, therapeutic concentrations of FIX, FVIII, or FVIIa were added to hemophilic plasma. Clotting was initiated by dilute thromboplastin.

Results: Adding 33 uM polyP to normal plasma shortened clot times at all doses of heparin (by 37–55%), enoxaparin (by 35–57%), and argatroban (by 28–59%; see Figure). PolyP shortened clot times of plasmas from coumadin patients (by 29–39%) with INR values ranging from 1.7 to 4.8. PolyP also shortened the clot times of plasma from patients with hemophilia A (by 54–75%) and B (by 51–79%). PolyP alone shortened clot times of hemophilia A or B plasma equivalent to adding 6 to 15 nM recombinant FVIIa, and the polyP effect was additive to that of FVIIa, FVIII or FIX.

Conclusions: PolyP shortened the in vitro clot time for all plasmas tested and was additive with replacement of missing clotting factors or recombinant FVIIa for hemophilic plasmas. These results suggest that treatment with polyP may be useful for reversal of anticoagulation in patients treated for thrombophilia, or for treatment of bleeding episodes in patients with hemophilia. Furthermore, polyP could potentially reduce the dose of replacement clotting factors or recombinant FVIIa needed to treat bleeding episodes in hemophilia.

Figure Figure
EKLF Is Recruited to the γ-Globin Gene Promoter as a Co-Activator and Is Required for γ-Globin Gene Induction by Short-Chain Fatty Acids.
Authors: Perrine Susan P. , Mankidy Rishikesh , Boosalis Michael S. , Bieker James J. , Faller Douglas V.
Published: April 2020

The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.
Identification of a Shared CD4+ and CD8+ T Cell Epitope within Human Neutrophil Elastase for Peptide Vaccination in HLA-A02 Negative Leukemia Patients.
Authors: Le Quan , Melenhorst J. Joseph , Hewitt Rachel E. , Hensel Nancy , Barrett A. John
Published: April 2020

Human Neutrophil Elastase (HNE) is aberrantly expressed by myeloid leukemia cells and contains the antigenic PR1 peptide sequence inducing HLA-A*0201 restricted cytotoxic T cells (CTL) against leukemic cells. Our previous studies demonstrated that full length HNE protein can induce both CD4+ and CD8+ T cell responses in healthy individuals including HLA- A*0201 negative individuals, indicating an immunogenic potential of HNE beyond the PR1 peptide (Fujiwara H et al Blood 2004). To identify new HNE epitopes, a peptide matrix consisting of 15mers overlapping by 11 amino acids spanning HNE was screened using peripheral blood mononuclear cells (PBMC) from patients with acute myeloid leukemias (AML). Peptide candidates predicted using syfpeithi predictive algorithms were evaluated by measuring responses to candidate epitopes. We found a novel HNE peptide E17 (HNE: 65–80), recognized by both CD4+ and CD8+ T cells in an HLA-A (11, 31) patient with AML. E17 induced significant proliferation of CD4+ T cells 3.2X and CD8+ T cells 4.6X above background, as measured by CFSE flow cytometry-based proliferation assay. E17 induced IFN-g expression in CD4+ T cells 7.4X and in CD8+ T cells 2.5X above background in flow cytometric assay (see figure). E17 (HNE: 65–80) peptide induced IFN-g mRNA expression 3 hours after peptide treatment of PBMC, as measured by quantitative Real-Time RT-PCR assay. Finally, we showed by flow cytometry that E17 induced cell surface expression of CD137 in CD4+ T cells 2.4X and CD8+ T cells 1.7X above background 8 hours after peptide treatment of the cells. Antigen-driven surface expression of CD137 was used to isolate peptide E17 antigen-specific T cells, for further in vitro expansion and characterization of the T cell response. Thus we have identified a shared CD4+ and CD8+ T cell epitope in HNE, inducing the proliferation and effecter functions in CD4+ and CD8+ T cells. Since this epitope (HNE: 65–80) recruits both helper and cytotoxic T cells, it is well suited for peptide vaccine applications. This study provides the rationale for using the novel E17 (HNE: 65–80) peptide to vaccinate patients with myeloid leukemias.

Figure Figure