bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Gammopathy of Undetermined Significance with an IgE Paraprotein.
Authors: Ayto Robert M. , Lambert Christopher , Lampert Irvin , Salooja Nina
Published: October 2019

Monoclonal Gammopathy of Unknown Significance (MGUS) is a non-malignant condition characterized by a serum paraprotein, low numbers of plasma cells on bone marrow examination(<10%) and the absence of end organ damage. The transformation rate of MGUS to multiple myeloma is frequently quoted as about 1% per year. IgG, IgM and IgA paraproteins account for virtually all cases of MGUS with only the rare literature report of IgD or IgE isotypes. To our knowledge there is only one published case of IgE MGUS (Ludwig and Vormittag 1980; BMJ, 281:6239, 539–540). IgE Myeloma is an extremely rare entity and reported to have a comparatively poorer prognosis, but the incidence of pre-existing IgE MGUS is unknown. Here we report the case of a 74 year old gentleman with a stable low level IgE paraprotein. The patient presented in March 2003 for investigation of a persistent borderline normocytic anaemia recorded as 13.3g/dl at initial review (lower range of normal 13.5g/dl) with an ESR of 27mm/h. Co-morbidity included hypertension, ischaemic heart disease and hypercholesterolemia but general examination was unremarkable. Screening investigations revealed normal haematinics, renal function and corrected calcium. Analysis of immunoglobulins revealed mild immunoparesis of his IgM titre only (0.33g/l; Reference range 0.5–2.0g/l) and the presence of both a blood and urinary paraprotein. The blood paraprotein was quantified as 3g/l with immunofixation identifying kappa light chain restriction without the presence of one of the commonly encountered heavy chain isotypes (IgG, IgM or IgA). Further probing of the abnormal gamma globulin region revealed the paraprotein to be IgE in isotype. Immunofixation of a spot urine sample revealed kappa light chain restriction with a protein concentration of 0.03g/l. Bone marrow examination demonstrated 1% plasma cells on the aspirate with mild dysplastic change within the erythroid series. Cytogenetics were not performed. The trephine specimen revealed 10% plasma cells with strong immunostaining for IgE, dysplastic features within the megakaryocytic and erythroid lineages together with confirmation of kappa light chain restriction. Interestingly, recent further immunostaining of the original specimen revealed the plasma cells to be CD56-ve but cyclin D1 positive. Skeletal survey was normal. His investigations fulfil the WHO diagnostic criteria for MGUS, although it could be argued that with 10% plasma cells in the trephine biopsy, he could be classified as a borderline smouldering myeloma. Four years later, however, this gentleman remains well on routine follow-up with no evidence of myeloma related organ or tissue impairment. His most recent paraprotein from August 2007 is stable at 4g/l. The reporting of plasma cell dyscrasias with rare immunoglobulin isotypes should be encouraged to contribute to the bank of knowledge of these infrequently encountered entities.
The BCL9 Oncogene Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic and Angiogenic Properties to Myeloma Cells
Authors: Mani Mala , Dutta Jui , Zhang Yunyu , Carrasco Daniel E , Zhou Yiming , Gatt Moshe , Barlogie Bart , Shaughnessy John D. , Anderson Kenneth C , Carrasco Daniel Ruben
Published: October 2019

Wnt signaling plays an important role in tissue development and maintenance during embryogenesis, cell differentiation, and stem cell growth. Several components of the Wnt signaling cascade have been shown to function as either tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis. Deregulation of the canonical Wnt/b-catenin pathway has been implicated in numerous human epithelial malignancies as well as hematologic malignancies including multiple myeloma (MM), generating immense interest in these molecules as targets for cancer therapy. Activation of Wnt/b-catenin in cancer has been associated with mutations that enable b-catenin to escape degradation by the proteasome, thereby allowing its accumulation in the nucleus where it functions as a transcriptional regulator in conjunction with coactivators by constitutively activating target genes such as c-Myc and Cyclin D1. To date, however, no mutations in Wnt pathway have been documented in MM, suggesting that mechanisms other than gene mutation may contribute to Wnt pathway deregulation. BCL9, a key component of the Wnt pathway, is required for b-catenin transcriptional activity and resides on chromosome 1q21, a region frequently involved in secondary chromosomal aberrations associated with MM tumor progression. Here we provide evidence that dysregulation of BCL9 expression is a novel oncogenic mechanism of Wnt pathway activation in MM. Using in vitro and in vivo functional analyses, we demonstrate that BCL9 is a bonafide oncogene that is aberrantly expressed in MM and associated with survival. Using the TCF- specific luciferase reporter, we show that enforced expression of BCL9 in MM cells enhanced b-catenin mediated transcription by >12 fold, suggesting a possible role of BCL9 overexpression in the pathogenesis of MM. BCL9 enhanced proliferation (1.5 fold, P<0.02), migration (3.5 fold, P<0.0001) and the metastatic potential of MM cells. We also showed that BCL9 plays an important role in tumor progression by regulating Cyclin D1 and c-Myc mediated cell proliferation, CD44 mediated tumor metastasis, as well as VEGF mediated host angiogenesis. Importantly, BCL9 knockdown significantly increased the survival in a xenograft mouse model of human MM (P=0.001), associated with decreased tumor burden and host angiogenesis. In summary, we have demonstrated that BCL9 is a novel and potent oncogene of the Wnt pathway in MM, playing fundamental roles in tumor progression by regulating proliferation, migration, invasion, angiogenesis and the metastatic potential of tumor cells. The pleiotropic roles of BCL9 and its aberrant expression highlight its importance as an attractive and novel therapeutic target in the treatment of MM.
MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia
Authors: Park Christopher Y. , Han Yoon-Chi , Bhagat Govind , Fan Jian-Bing , Weissman Irving L , Gu Hua
Published: October 2019

microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.
The Mir-17-19b Microrna Cluster Quantitatively Regulates the Self- Renewing Leukemia Stem Cell Compartment in MLL Leukemia
Authors: Wong Piu , Iwasaki Masayuki , Chen Chang-Zheng , Cleary Michael
Published: October 2019

The critical molecular determinants for leukemia stem cell (LSC) generation and maintenance are largely unknown. While the majority of studies focus on investigating key transcription factors and cytokines involved in hematological malignancies, very little is known about the roles of recently discovered microRNAs in leukemogenesis. Bioinformatic analysis of the Mouse Retroviral Tagged Cancer Gene Database (Neal Copeland et al.) revealed the 5′ regulatory region of the miR-142 gene as a recurrent target for retroviral integrations in a variety of mouse leukemia and lymphoma cancer models, including MLL-induced leukemia, thereby implicating miR-142 as a potential collaborating oncomir in hematologic malignancies. Subsequent expression analyses of differentially expressed microRNAs in LSC-enriched cell fractions (c-kit+) from MLL leukemias in comparison with various stages of normal hematopoietic progenitors indicated that the miR-17-5p family of microRNAs is highly expressed in LSC-enriched fractions and their possible normal counterparts, granulocyte-macrophage progenitors. Moreover, the expression of miR-142 and miR-17-5p family microRNAs was substantially reduced upon differentiation and loss of self-renewal in cells transformed by the conditional MLL-ENL-ER oncogene following withdrawal of tamoxifen. Forced expression of the miR-17-19b polycistron or miR-142 substantially shortened the latency for MLL leukemia development. In particular, leukemias expressing high levels of the miR-17-19b polycistron displayed a higher frequency of LSCs due to an efficient block of differentiation and enhanced proliferation associated with reduced expression of p21, a cyclin dependent kinase inhibitor previously implicated as a direct target of miR-17-19b microRNAs. Knock down of p21 in MLL oncogene transduced cells recapitulates the MLL-leukemic phenotype induced by over-expression of the miR-17-19b cluster including a significant decrease in MLL leukemia latency. This observation validates p21 as a biologically relevant in vivo target of the miR-17-19b polycistron in MLL leukemia. Thus, microRNAs are pathogenically important for quantitatively modulating the self-renewing LSC compartment in a murine model of MLL leukemia. Potential requirements of the miR-17-19b microRNA cluster for induction and/or maintenance of MLL leukemia are currently under study.
TRAF-1 Deficient Mice Show Impaired Monocyte Recruitment and Decreased Atherogenesis
Authors: Missiou Anna , Köstlin Natascha , Münkel Christian , Pfeifer Dietmar , Zirlik Katja , Bode Christoph , Libby Peter , Zirlik Andreas
Published: October 2019

Members of the tumor necrosis factor (TNF) interleukin/toll-like receptor superfamily such as CD40L, TNFa, and IL-1b potently promote atherogenesis in mice and likely also in humans. TNF receptor associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines. We recently reported over-expression of TRAFs in murine and human atheromata and demonstrated dependency of classic inflammatory functions on TRAFs in endothelial cells and macrophages. Here we test the hypothesis that TRAF-1 modulates atherogenesis in vivo. TRAF-1--/LDLR--mice fed a high cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions compared with LDLR--controls. Intimal lesion size decreased by up to 56±6% and 33±5% in sections of the aortic arch and aortic root, respectively (n>10 per group, P<0.01 each). Plaques of TRAF-1-deficient animals contained up to 46±9% and 55±4% fewer macrophages while smooth muscle cell content increased by up to 32±6 and 36±7%, characteristics associated with non disrupted plaques in humans. Lipid content, collagen content, and lymphocyte content remained unchanged. In vitro, gene expression profiling revealed reduced expression of adhesion molecules (VCAM-1, ICAM-1), chemokines (CCL2, CXCL2), and growth factors (M-CSF) in TRAF-1-deficient endothelial cells as well as of integrins (CD29, CD11b) and chemokines/chemokine receptors (CXCL2, CCR1) in TRAF-1-deficient macrophages, verified by siRNA studies in human cells. Finally, both deficiency of TRAF-1 in endothelial cells and neutrophils/monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic adhesion assays. We present the novel finding that TRAF 1 deficiency attenuates atherogenesis in mice, an effect most likely mediated by impaired monocyte recruitment to the vessel wall. These data identify TRAF-1 as potential treatment target for chronic inflammatory diseases such as atherosclerosis.
ZKSCAN3, a Novel Zinc Finger Transcription Factor, Regulates Cyclin D2 Expression in Multiple Myeloma (MM) Cells
Authors: Yang Lin , Orlowski Robert Z.
Published: October 2019

Background: Dysregulation of cyclin D has been proposed to represent an early oncogenic event in MM, and often occurs as a result of chromosomal translocations. Cyclin D2 can be induced as a result of translocations involving c-maf, mafB, and FGFR3/MMSET, but its expression is also increased in hyperdiploid and nonhyperdiploid cells without these translocations through unknown mechanisms. We previously identified a novel zinc finger transcription factor, ZKSCAN3, as a new “driver” of colon cancer progression, and found that the cyclin D2 promoter harbored several ZKSCAN3 binding sites, prompting us to investigate a possible role of ZKSCAN3 in MM pathogenesis.

Methods: ZKSCAN3 expression was studied in patient-derived primary tumors and normal cells, as well as in human MM cell lines.

Results: Tissue microarray studies showed that ZKSCAN3 was strongly expressed in the neoplastic cells of 7/10 MM patients by immunohistochemistry, while in all samples the surrounding normal bone marrow cells showed only background staining. Real-time polymerase chain reaction (qPCR) analysis revealed some level of ZKSCAN mRNA expression in 5/5 MM cell lines studied (RPMI 8226, U266, KAS-6/1, INA-6, ANBL-6), but not in pooled CD19+ normal B-cells. By Western blotting, ZKSCAN3 was most highly expressed in RPMI 8226 and KAS-6/1 cells, which notably do not bear cyclin D dysregulating translocations, while U266 and ANBL-6, which have such translocations, showed the lowest levels of ZKSCAN3 expression. In RPMI 8226 cells, but not the other MM lines, six copies of the ZKSCAN3 gene were detected, suggesting the presence of a previous amplification event. cisRED analysis predicted that ZKSCAN3 may be transcriptionally regulated by paired box gene 5 (Pax5), an essential factor for maintaining the commitment of the B cell lineage which is inactivated during plasma cell differentiation. Consistent with a role for Pax5 in controlling ZKSCAN3 level, a reporter assay showed that Pax5 inhibited ZKSCAN3 promoter activity, and Pax5 expression negatively correlated with ZKSCAN3 expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed direct binding of ZKSCAN3 to the endogenous cyclin D2 promoter in these MM cell lines. Knockdown of ZKSCAN3 using short hairpin RNAs reduced Cyclin D2 mRNA and protein expression in RPMI 8226 and KAS-6/1 cells, and also decreased their proliferative rates.

Conclusions: These findings support the possibility that ZKSCAN3 contributes to myeloma pathogenesis by dysregulation of cyclin D2, and that events impacting upon ZKSCAN3 may provide MM cells with an alternative pathway for induction of Cyclin D2 in the absence of an activating translocation. ZKSCAN3 may therefore be an attractive candidate for MM therapy.
Autologous Stem Cell Transplantation after Myeloablative Therapy in First Remission May Be Beneficial in Patients with Advanced Stage Follicular Lymphoma after Front-Line Therapy with R-CHOP. An Analysis of Two Consecutive Studies of the German Low Grade Lymphoma Study Group (GLSG)
Authors: Hiddemann Wolfgang , Buske Christian , Kneba Michael , Dreyling Martin , Schmitz Norbert , Lengfelder Eva , Schmits Rudolf , Reiser Marcel , Metzner Bernd , Unterhalt Michael
Published: October 2019

The addition of Rituximab (R) to front-line combination chemotherapy has substantially improved the long term perspectives of patients with advanced stage follicular lymphoma (FL) resulting in median progression free survival (PFS) times of more than 36 months. In this situation the potential role of myeloablative herapy with subsequent autologous stem cell transplantation (ASCT) in 1st remission needs to be critically reassessed. For this purpose two consecutive studies of the GLSG were analyzed both randomizing in 1st remission for Interferon alpha (IFN) maintenance therapy versus ASCT. In the GLSG trial ‘96 initial therapy comprised a randomized comparison of CHOP versus Mitoxantrone, Chlorambucil and Prednisone (MCP) (total patient number 312) while in GLSG study ‘00 CHOP was randomly compared to R-CHOP (total patient number 268). Hence, both studies had an identical treatment arm of CHOP followed by either IFN maintenance or ASCT. In both studies, the identical treatments revealed superimposable results with a 5 years PFS of 27% after CHOP and IFN maintenance and 66% after CHOP followed by ASCT. Taking these data as internal control R-CHOP followed by IFN maintenance achieved a 5 year PFS of 67% which is hence comparable to CHOP followed by ASCT. R-CHOP followed by ASCT, however, revealed a 5 year PFS of 79% with only one relapse after 24 months.

Figure Figure

Although the difference between R-CHOP followed by IFN maintenance or ASCT is currently not yet significant it strongly suggests a beneficial effect of ASCT in the era of R-CHOP front-line therapy particularly for intermediate of high risk patients with advanced stage FL.
Generation of vFLIP Transgenic Mice: A Model to Study KSHV-Associated Lymphomagenesis
Authors: Ballon Gianna , Chadburn Amy , Perez Rocio , Liu Yi-Fang , Sasaki Yoshiteru , Cesarman Ethel
Published: October 2019

Primary effusion lymphoma (PEL) is a distinct subtype of aggressive non-Hodgkin’s lymphoma (NHL), specifically associated with infection by Kaposi’s sarcoma-associated herpesvirus (KSHV) and occurring more frequently in HIV-infected individuals. Several in vitro observations suggest that vFLIP, a viral protein expressed during latency, is an important viral oncogene. It is essential for the survival of KSHV-infected PEL cells, mainly by constitutively activating the NF-kB pathway. In order to assess the role of vFLIP in the pathogenesis of PEL, we knocked a cDNA encoding vFLIP, preceded by a loxP-flanked neoR-Stop cassette and followed by Frt-flanked IRES-eGFP sequences, into the ubiquitously expressed ROSA26 locus. A specifically restricted expression of the transgene in CD19+ B-cells has been achieved by crossing the ROSA26. vFLIP knock-in mice with mice expressing cre recombinase under the control of the CD19 promoter. These mice have also been crossed with transgenic mice expressing KSHV LANA, which is considered to also be a viral oncogene, to assess a potential synergistic effect of these two KSHV latent proteins in the lymphomagenic process of PEL. vFLIP expression in the CD19+ B-cells results in splenomegaly, with an increase in both T and B-cells, and with a relative increase of the T versus B-cell ratio. Although primary follicles were enlarged, the expression of vFLIP in the CD19+ B-cells results in lack of germinal center formation in the spleen, lymph nodes and intestine, and in partially impaired class-switching recombination. vFLIP transgenic mice had an increased number of plasmablast-like cells expressing lambda light chain, reminiscent of a phenomenon seen in KSHV-associated multicentric Castleman’s disease (MCD). These results indicate that by constitutively activating the NF-κB pathway in pre-germinal center B-cells expressing CD19, the normal B-cell differentiation is impaired, and provide clues about possible aberrant differentiation in PEL and MCD.
Rituximab (R) + Hypercvad Alternating with R-Methotrexate/Cytarabine after 9 Years: Continued High Rate of Failure-Free Survival in Untreated Mantle Cell Lymphoma (MCL)
Authors: Romaguera Jorge , Fayad Luis , Rodriguez Alma , Hagemeister Fb , Pro Barbara , McLaughlin Peter , Younes Anas , Samaniego Felipe , Shah Jatin , Weaver Pamela , Hartig Kim , Cabanillas Fernando , Kwak Larry , Kantarjian Hagop , Wang Michael
Published: October 2019

MCL has a poor outcome with current non-intense therapies. We present an update of our results utilizing an intense chemotherapy regimen comprising fractionated cyclophosphamide, doxorubicin, vincristine and dexamethasone to which rituximab has been added (R-HyperCVAD, one cycle), alternating with rituximab and high dose methotrexate/cytarabine (R-MA, one cycle) for a total of 6–8 cycles (J Clin Oncol 2006 Feb 1;24(4):724). Treatment was started at most within 2 months from the initial evaluation. Overall survival (OS) was defined as the time from start of treatment until recurrence or death from any cause. Failure-free survival (FFS) was defined as the time from start of treatment until recurrence or death from toxicity or death from treatment-related malignancy. Of 97 consecutive evaluable patients, 87% achieved a complete response (CR) or unconfirmed CR after 6 cycles and did not receive additional therapy. With a median follow up on 84 months (7 years), the overall survival and failure-free survival at 7 years was 60% and 43%, respectively. Among patients 65 years of age or younger, the 7-year OS and FFS was 68% and 52%, respectively. Important prognostic variables included age (65 yrs or less vs > 65 yrs; initial serum B2 microglobulin 3 or less mg/dL vs > 3); and initial serum lactate dehydrogenase (normal vs > normal). Further subset analysis will be presented at the meeting.

Figure Figure
Rituximab Maintenance Treatment of Relapsed/Resistant Follicular Non- Hodgkin’s Lymphoma: Long-Term Outcome of the EORTC 20981 Phase III Randomized Intergroup Study
Authors: Van Oers Marinus H.J. , Van Glabbeke Martine , Baila Liliana , Giurgia Livia , Klasa Richard , Marcus Robert E , Wolf Max , Kimby Eva , Hagenbeek Anton
Published: October 2019

Background. In 2005 we analyzed the results of a prospective randomized phase III intergroup trial evaluating the role of rituximab (R) both in remission induction and maintenance treatment of 465 relapsed/resistant follicular lymphoma (FL) patients. Major conclusions were that addition of R to CHOP induction yielded an increased ORR and CR rate, and that R maintenance strongly improved median progression free survival (PFS), both after induction with CHOP and R-CHOP, and overall survival (OS (van Oers et al Blood2006;108:3295). At that time the median follow for the maintenance phase was 33 months. Now we report the long-term outcome of maintenance treatment, with a median follow up of 6 years from start of maintenance.

Study design. Patients with stages III or IV FL at initial diagnosis and relapsed after or resistant to a maximum of two non-anthracycline containing systemic chemotherapy regimens, were randomized to remission induction with either 6 cycles of standard CHOP (once every 3 weeks) or CHOP + R (375 mg/m2 at day 1 of each cycle of CHOP). Those with a complete or partial remission after 6 cycles of therapy underwent a second randomization to no further treatment (observation) or maintenance treatment with R (375 mg/m2 once every 3 months) until relapse or for a maximum period of two years.

Results. 465 patients were randomized to induction with either CHOP (231) or R-CHOP (234). As reported, CHOP and R-CHOP induction yielded similar partial response rates (57% vs.56%), but significantly different CR rates (16% and 29%; p=0.0001). 334 patients were randomized to either R maintenance treatment (167) or observation (167). R maintenance resulted in a highly significant improvement of PFS: median 3.7 years versus 1.3 years in the observation arm (p<0.0001; hazard ratio 0.55). The advantage of R maintenance was observed both after CHOP induction (p< 0001: HR 0.37) and R-CHOP induction (p= 0.003; HR 0.69). The 5 years OS was 74% in the R maintenance arm and 64 % in the observation arm (p=0.07). That the highly improved PFS after R maintenance did not translate into a significant OS advantage might partially be explained by the fact that 41% of progressing patients received R as salvage therapy. This varied according to treatment arm: from 59% after CHOP followed by observation to 26% after R-CHOP followed by R maintenance. R maintenance was associated with a significant increase in grade 3/4 infections: 9.7% vs.2.4% (p= 0.01). 7 of the 167 patients had to discontinue R maintenance because of toxicity, mostly recurrent infection.

Conclusion. With long term follow-up we confirm the superior PFS with R maintenance. The improvement of OS did not reach statistical significance, possibly due to the abundant use of R in post-protocol salvage treatment.
Bortezomib Induces Thrombocytopenia by Inhibiting Proplatelet Formation but Not Proliferation and Endomitosis in Human Primary Megakaryocytes
Authors: Nogami Wataru , Yamane Akiko , Nakamura Takanori , Matsuki Eri , Ikeda Yasuo , Miyakawa Yoshitaka
Published: October 2019

The proteasome inhibitor bortezomib has therapeutic activity in patients with multiple myeloma. The most common adverse event from its application is thrombocytopenia, which has kinetics that differ from those induced by other cytotoxic agents. After treatment with bortezomib, platelet counts usually decrease within a couple of days but rapidly recover toward baseline during the rest periods between each cycle. The lowest count of platelets in each cycle does not worsen during the 8 courses of bortezomib treatment. Furthermore, bortezomib does not induce any cytotoxic injury in megakaryocyte in the murine model. Therefore, we postulated that bortezomib-induced thrombocytopenia is caused by inhibition of the platelet releasing process without megakaryocyte toxicity. In vitro assays using human bone marrow-derived CD34-positive hematopoietic stem cells revealed that bortezomib did not inhibit colony formation and endomitosis of human primary megakaryocytes in the presence of recombinant human thrombopoietin (rhTPO). As proplatelet formation (PPF) is often used as the indicator of the platelet releasing process in vitro, we evaluated the inhibitory effects of bortezomib for PPF. Seven days after culture of human CD34-postive cells with 10 ng/ml rhTPO, mature megakaryocytes were enriched by discontinuous bovine serum albumin gradients (purity>90%). The enriched mature megakaryocytes were treated with various concentrations of bortezomib for a further 4 days and the percentage of megakaryocytes bearing PPF was calculated under a microscope. Bortezomib dose-dependently inhibited PPF from mature megakaryocytes. Other proteasome inhibitors such as lactacystin and MG132 also demonstrated inhibitory effects on PPF without inhibiting colony formation of megakaryocytes. Since the inhibition of transcriptional factor NF-kB activity is one of the major pathways of proteasome inhibitors, we evaluated the effects of NF-kB inhibitors such as (−)-DHMEQ and Bay11-7082. Both of these inhibitors also demonstrated inhibitory effects on PPF but did not inhibit the colony formation of megakaryocytes. To exclude the direct effects of bortezomib on human platelets, we analyzed the effects of bortezomib for the activation of caspase-3 and mitochondrial potential in human platelets. We found that bortezomib did not directly induce apoptosis in human platelets. Our results demonstrate that bortezomib induces thrombocytopenia by inhibiting PPF but does not affect proliferation of megakaryocytes, endomitosis and platelet apoptosis. We believe this is the first report using human primary megakaryocytes to clarify the pathogenesis of thrombocytopenia caused by bortezomib therapy.
BMP4 and TGFbeta Differentially Regulate CD34+ Progenitor Development in Human Embryonic Stem Cells through SMAD-Dependent Pathway
Authors: Wang ZacK Z. , Bai Hao , Arzigian Melanie , Gao Yong-Xing , Wu Wen-Shu
Published: October 2019

Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (Nat Biotech.25:317, 2007). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.
Rare Polymorphisms Associated with Neonatal Alloimmune Thrombocytopenia (NATP) and New Mutations Identified in Platelet Glycoproteins (GP)
Authors: Peterson Julie A. , Gitter Maria , Kanack Adam , Curtis Brian , McFarland Janice , Aster Richard H.
Published: October 2019

Neonatal Alloimmune Thrombocytopenia (NATP) is caused by maternal immunization against alloantigens carried on paternal platelet glycoproteins (GP). Although common antigens and their corresponding antibodies can be reliably detected, about two-thirds of suspected cases of NATP go unresolved. We are investigating the extent to which maternal immunization against low frequency antigens not usually tested for contributes to the relatively low diagnostic yield in NATP evaluations. In the course of this work, we have identified eight previously undescribed amino acid substitutions located in four different platelet GPs (Table 1).

Table 1. Eight new mutations identified in platelet glycoproteins

Name Sta Kno Sol Brc Bec Aus Rou2 Rou1 GP IIIa IIb IIIa Ia IIIa Ibb IIb IIb Mutation K137Q T619M E628K E505V K646E D13H G790S T9A

The incidence of each mutation in the general population is less than 1.0%. Antigens Sta and Kno were each identified in fathers of suspected NATP cases and in the affected infants. In addition, maternal antibodies reacted with recombinant GPs modified to contain the relevant mutations, making it very likely that the antibodies were the cause of NATP in these instances. Antigen Sol was identified in the father and maternal antibody reacted strongly with paternal GPIIb/IIIa. However, the infant has not yet been typed. It is likely, but not certain that immunization against Sol caused NATP in this case. Mutation Brc was found in the father of an infant with possible NATP. However maternal serum failed to react with GPIa from father’s platelets and it was not possible to type the affected infant. Mutations Bec, Aus and Rou2 were identified in paternal DNA but the affected infants were negative for these mutations. Mutation Rou1 was identified in maternal DNA and could not have been an immunogen. It is highly likely that three of the eight new mutations (Sta, Kno, and Sol) caused maternal immunization leading to NATP. However, the other five (Bec, Aus, Brc, Rou1 and Rou2) appear to have been identified incidentally in the course of the NATP evaluations. Before concluding that maternal-fetal incompatibility for a rare antigen identified in the father is the immunogen responsible for triggering NATP these findings emphasize the importance of typing an affected infant to determine whether a low frequency antigen identified in the father was inherited. In addition it is necessary to confirm that maternal serum reacts with the suspected paternal antigen incorporated into a recombinant protein or carried on platelets from an unrelated individual.
Protein Synthesis Escapes mTORC1 Control and Constitutes a Promising Therapeutic Target in Acute Myeloid Leukemia.
Authors: Tamburini Jerome , Green Alexa Samantha , Bardet Valérie , Chapuis Nicolas , Willems Lise , Park Sophie , Uzunov Madalina , Ifrah Norbert , Dreyfus Francois , Mayeux Patrick , Lacombe Catherine , Bouscary Didier
Published: October 2019

The deregulation of translation, leading to a preferential translation of oncogene-related mRNAs, markedly contributes to the malignant phenotype in human cancers. The assembly of the eukaryotic translation Initiation Factor 4F (eIF4F) complex is limiting for malignancy-related translation events and is hence an attractive anticancer therapeutic target. The Mammalian Target Of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through eIF4E Binding Protein 1 (4E-BP1) phosphorylation. The mTORC1 inhibitor rapamycin has been developed as an anti-cancer agent but is of limited efficacy in the treatment of Acute Myeloid Leukemia (AML). We show in our study that in primary AML cells, 4E-BP1 phosphorylation is mTORC1-independent and is in fact controlled by the Pim- 2 serine/threonine kinase: neither the rapamycin derivate RAD001 nor Raptor siRNA inhibited 4E-BP1 phosphorylation contrary to siRNA-mediated Pim-2 knock-down. We utilized the 4E-BP1 mimetic 4EGI-1, as a direct inhibitor of translation in AML. In contrast to RAD001, 4EGI-1

totally inhibited eIF4F assembly; markedly decreased the proportion of polysome-bound c-Myc mRNA; and dramatically decreased the accumulation of oncogenic proteins that are regulated at the translational level.

Moreover, 4EGI-1 demonstrated potent anti-leukemic activity ex vivo in primary AML blast cells through a selective suppression of proteins implicated in proliferation and in anti-apoptotic mechanisms. Significantly, these effects occurred with a minimal degree of toxicity against normal hematopoietic processes. Our present results therefore strongly implicate an mTORC1-independent mechanism of deregulated synthesis of oncogenic proteins in human myeloid leukemogenesis. The direct inhibition of the eIF4F translation initiating complex, by 4EGI-1, by the recently developed eIF4E antisense oligonucleotides or by specific Pim-2 inhibitors when available, thus represents an attractive option for the development of new therapies for AML.
mTORC1 Activity Is Independent of PI3K/Akt but Controlled by AMPK in Acute Myeloid Leukemia.
Authors: Green Alexa Samantha , Tamburini Jerome , Willems Lise , Chapuis Nicolas , Park Sophie , Bardet Valérie , Foretz Marc , Viollet Benoit , Ifrah Norbert , Dreyfus Francois , Mayeux Patrick , Lacombe Catherine , Bouscary Didier
Published: October 2019

In most cancer models, the PI3K/Akt pathway controls the activity of the mammalian Target of Rapamycin Complex 1 (mTORC1) that regulates cell growth through the phosphorylation of P70S6K and 4E-BP1. In Acute Myeloid Leukemia (AML), both constitutive activation of PI3K (PI3K+) and of mTORC1 are detected. We previously reported in AML that PI3K activity is due to the expression of the p110d isoform of class Ia PI3K and is constitutive in 50% of samples at diagnosis. We show here in primary AML samples that the constitutive activity of mTORC1, detected by the phosphorylation of P70S6K on Thr389, is not due to PI3K/Akt activation. In PI3K+ samples, the specific inhibition of p110d with IC87114 does not inhibit mTORC1. Furthermore, a constitutive activity of mTORC1 is detected in almost all AML samples regardless of their PI3K/ Akt activation status. Similarly, the inhibition of ERK/MAPK activity with UO126 does not result in mTORC1 inhibition. Interestingly, in primary AML cells, we shows that mTORC1 is fully inhibited following leucine starvation as observed in non-transformed cells, suggesting that the kinase activity of mTOR is not deregulated and remains accessible to target inhibition. We thus focused on the links between the AMP kinase (AMPK) and mTORC1. AMPK is a physiological cellular energy sensor and becomes phosphorylated at Thr172 downstream of LKB1 in response to a decrease in cellular ATP levels. AMPK then controls multiple metabolic processes such as fatty acid oxidation, glucose production and protein synthesis. Interestingly, AMPK is usually unphosphorylated in cancer cells including AML and thereby represents a potential therapeutic target. We thus investigated the effects of metformin, a potent AMPK activator developed as an oral anti-diabetic drug, in primary AML cells. This compound induces a dose-dependent activation of AMPK that correlates with a decrease of mTORC1 activity. Primary AML blast cells cultured with metformin show a significant inhibition of proliferation, of clonogenic growth and apoptosis suggesting that AMPK may be an attractive anticancer therapeutic target in AML. However, as metformin may exerts AMPK-independent effects, we are producing a lentiviral construct expressing a constitutively activated AMPK mutant to more specifically determine the consequences of AMPK activation in mTORC1 signaling and blast cells survival.
Tailored Temozolomide Therapy for Elderly Patients with Acute Myeloid Leukemia.
Authors: Medeiros Bruno C. , Gotlib Jason R. , Coutre Steven E. , Mignea Angela , Zehnder James
Published: October 2019

Alternative treatment modalities are greatly needed for elderly patients with acute myeloid leukemia (AML). Previous preclinical data and clinical observations show that only patients lacking expression of O6-alkylguanine-DNA alkyltransferase (AGAT) in leukemic blasts are sensitive to temozolomide. Furthermore, AGAT enzymatic activity can be significantly decreased by protracted exposure to low doses of temozolomide. Based on these data, we have designed a phase II clinical trial tailoring temozolomide therapy to elderly patients with AML according to AGAT status. AGAT promoter methylation status was determined in pretreatment leukemic samples. Patients demonstrating evidence of AGAT promoter methylation were stratified to conventional doses of temozolomide at 200mg/m2 orally x 7 days. Patients demonstrating lack of AGAT promoter methylation (unmethylated) were stratified to receive protracted doses of temozolomide (100mg/m2 orally x 14 days) followed by conventional doses of temozolomide. Those patients who achieved CR were given up to 5 similar consolidation treatments. Eleven patients have been treated to date. The median age was 78 (71–83) and 6 were male. Seven patients were newly diagnosed and 6 patients had a normal karyotype. Nine patients had an unmethylated AGAT promoter in their leukemic blasts. Correlative AGAT protein expression in leukemic blasts will be presented at the meeting. All patients had an intact mismatch repair pathway. Median HCT-CI score was 1 (0–5). Four patients (4/10) achieved a complete remission after 1 cycle of therapy (1/2 for patients with methylated AGAT promoter and 3/8 for patients with unmethylated AGAT promoter). Nonhematologic toxicities attributed were minimal. Drug-related hematologic toxicities were difficult to distinguish from disease-related cytopenias. Six patients developed neutropenic fever (four patients developed neutropenic fever before the start of therapy). Three (3/10) patients remain alive with a median duration of remission of 4.5 months (4–5 months). Four patients have died from disease progression. And two patients died of neutropenic sepsis. One patient who achieved a CR, experienced prolonged marrow aplasia (47 days) following consolidation cycle #1 and withdrew from the study. The trial is ongoing with an accrual goal of 36 patients. These preliminary results suggest that temozolomide therapy may be individually tailored to elderly patients with AML according to AGAT promoter status.
Allogeneic Stem Cell Transplantation Results in a Low Relapse Rate in Patients with Peripheral T-Cell Lymphoma.
Authors: Nickelsen Maike , Canals Carmen , Kyriakou Charalampia , Schmitz Norbert , Buzyn Agnes , Engelhardt Monika , Glass Bertram , Conde Eulogio , Lopez-Guillermo Armando , Socie Gerard , Sureda Anna
Published: October 2019

Patients (pts) suffering from mature (peripheral) T-cell lymphoma are known to have a poor outcome compared to pts with aggressive B-cell lymphoma when receiving conventional therapies. However, case reports and single centre experiences show promising results of allogeneic stem cell transplantation (allo SCT) for these pts. We here present a retrospective analysis of 103 pts with mature T-cell lymphoma who received an HLA-identical allo SCT in EBMT centres between 2000 and 2005. Histological subtypes of the patients were anaplastic large cell lymphoma (n=25; 4 anaplastic lymphoma kinase [ALK] positive, 11 ALK negative, 10 ALK unknown), peripheral T-cell lymphoma unspecified (n=51), angioimmunoblastic T-cell lymphoma (n=18) and aggressive T-cell lymphoma unspecified (n=9). Patients with primary cutaneous T-cell lymphoma or T-lymphoblastic lymphoma were excluded. Median follow up for surviving patients was 40 months (6–98), median age at allo SCT 42 years (18–68) and median time to allo SCT 17 months (3–233). 39 pts had failed a prior autologous SCT; 68 pts had chemosensitive disease at allo SCT (of these 15 in CR1) and 30 pts had chemorefractory or untested relapsed or progressive disease. Donors were HLA identical siblings for 73 and matched unrelated donors for 30 pts; 82 pts received peripheral blood stem cells. 54 pts were treated with reduced intensity conditioning before SCT. The cumulative incidence (CI) of acute graft versus host disease (GvHD) 100 days after SCT was 52% and had no effect on non-relapse mortality (NRM) or relapse rate (RR). 34 of 74 evaluable pts experienced chronic GvHD (14 limited, 20 extensive), CI 46% at 2 years. Introducing chronic GvHD as a time dependent covariate, this factor was associated with a higher NRM (p<0.001) and a lower RR (statistically not significant), resulting in a lower progression free survival (PFS) (p=0.001). For all patients CI of NRM and RR at 3 years were 36.7% and 24.9%, respectively, resulting in 41.4% PFS and 46.9% OS. Median time to relapse / progression was 4 (1–18) months. In multivariate COX analysis for PFS refractory disease (relative risk 3.4, p=0.001), prior failed auto SCT (relative risk 2.6, p=0.001) and poor performance status at SCT (relative risk 2.5, p=0.04) were significant adverse factors. Additionally, there were trends for T-cell depleted grafts (p=0.1), CMV status other than negative-negative (p=0.1) and reduced intensity conditioning (p=0.2) to be adverse factors. A subgroup of 39 good risk pts (good performance status, chemosensitive disease, no prior autologous SCT) had a PFS of 59%.

This retrospective analysis on allo SCT in peripheral T-cell lymphoma shows an encouraging low RR but efforts have to be made to reduce NRM. Prospective studies are warranted to further define pts who will profit from an early allo SCT.
An Open-Label Study of CEP-701 in Patients with JAK2 V617F-Positive Polycythemia Vera and Essential Thrombocytosis
Authors: Moliterno Alison R , Roboz Gail J. , Carroll Martin , Luger Selina , Hexner Elizabeth , Bensen-Kennedy Debra M.
Published: October 2019

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal hematopoietic stem cell disorders characterized by the over production of phenotypically normal circulating blood cells. Most PV and approximately half of ET patients harbor the activating mutation JAK2 V617F. CEP-701 is an orally available, potent low nanomolar inhibitor of both the wild-type and mutated JAK2 tyrosine kinase, with its inhibitory effect demonstrated both in enzymatic and cellular assays and in vivo, where CEP-701 significantly inhibited the growth of JAK2 V617F-positive HEL.92 xenografts in mice. These findings suggest that CEP-701 is an attractive candidate for clinical evaluation in JAK2 V617F-positive myeloproliferative disorders. The purpose of this study is to test the safety and efficacy of CEP-701 administration in JAK2 V617F positive ET and PV patients. The primary endpoint is reduction in JAK2 V617F neutrophil allele burden; secondary endpoints are reduction in phlebotomy rates, improvement in hemoglobin, white cell and platelet counts, reduction in hydroxyurea (HU) dose, and reduction in spleen size. The secondary endpoints include the pharmacokinetics and pharmacodynamics of CEP-701 and the safety of CEP-701 treatment in patients with JAK2 V617F-positive PV and ET. This is a multicenter study with an anticipated enrollment of 40 PV and ET patients. Inclusion criteria include JAK2 V617F-positive PV and ET patients; patients with PV either have a neutrophil count greater than 7,000/ul or are receiving HU, while ET patients are receiving concomitant HU. Other inclusion criteria include an ECOG performance status of 0, 1 or 2, and 18 years of age or older. Exclusion criteria include the active use of anegrelide or interferon, or a recent history of venous or arterial thrombosis. This is an 18 week trial with an optional 1 year extension period; doses will escalate from 80 mg twice daily to a maximum of 120 mg twice daily. To date, 20 subjects, 11 PV and 9 ET, comprised of 11 females and 9 males, ages 34 to 74, have enrolled. Approximately 27% of the PV patients were taking HU. The most common adverse events have been gastrointestinal (GI) and constitutional in nature. No related serious adverse events have been observed. Five patients have discontinued study participation, all for adverse events: 1 due to disease progression, 1 leg cramps, and 3 GI. To date, 7 patients have completed 18 weeks of therapy and 6 of these patients will continue to receive CEP-701 on the extension phase of the trial. Five of 8 subjects with splenomegaly have responded with reductions in spleen size evident within 6 weeks of therapy initiation. Updated results on current and future patients will be presented at the meeting.