bluuebunny/crossref_metadata_2025 huggingface dataset:
HCV and HIV Envelope Proteins Co-Operatively Induce Fas-Mediated Apoptosis Via a Novel Stat1 Signaling Pathway.
Authors: Balasubramanian Anuradha , Ganju Ramesh K. , Groopman Jerome E.
Published: October 2019

Hepatitis C virus (HCV) co-infects approximately 40% of patients with human immunodeficiency virus (HIV). HCV/HIV co-infected patients often have progressive liver disease that can lead to cirrhosis and death. We observed that hepatocytes exposed to HCV and HIV envelope proteins undergo apoptosis via an ‘innocent bystander’ mechanism due to cell surface binding of viral proteins independent of direct viral infection. HCV envelope protein E2 (1.5 nM) and HIV envelope protein gp120 (0.8 nM) derived from M-tropic and T-tropic viruses induce significant apoptosis in both hepatocytic cell lines and primary hepatocytes, while either of these viral proteins alone does not. Now, we have elucidated the signaling mechanisms that mediate this effect. HCV-E2 and HIV-gp120 were found to significantly upregulate Fas ligand (FasL). We then examined the Stat family of proteins known to participate in FasL and apoptotic pathways. We observed an increased DNA binding activity of Stat1 upon HCV-E2 and HIV-gp120 stimulation. Furthermore, overexpression of wild type Stat1αincreased apoptosis and FasL expression in HepG2 cells, whereas a C-terminal domain deleted mutant, Stat1β, decreased HCV-E2 and HIV-gp120 mediated apoptosis and FasL upregulation. Overexpression of Stat1αand Stat1β in primary hepatocytes confirmed that Stat1αenhanced apoptosis upon HCV-E2 and HIV-gp120 treatment. We observed a tyrosine dependent activation of Stat1 and a subsequent serine phosphorylation of Stat1. TYK2, lyn kinase, RAFTK and MAP kinases were activated upstream of Stat1. In addition, Stat1 associated with the death domain-containing adapter protein TRADD. TRADD is known to induce inflammatory signaling through the NFκB pathway. Here, the association of Stat1 with TRADD would reduce the availability of TRADD to induce NFκB. Thus, Stat1 sequestration of downstream apoptotic signaling molecules would block the host inflammatory response. Further characterization of Fas-mediated apoptosis revealed that caspase 3 and caspase 7 were activated following HCV-E2 and HIV-gp120 stimulation. However, we were not able to detect significant activity of either caspase 8 or caspase 9. We also found a loss in mitochondrial membrane potential upon HCV-E2 and HIV-gp120 stimulation, which leads to the release of cytochrome C and AIF into the cytosol. Taken together, these studies indicate that the viral proteins of HCV and HIV co-operate in causing the apoptosis of hepatocytes, independent of direct infection, by induction of novel Stat1 downstream signaling pathways at the expense of a normal host inflammatory response.
Human Herpesvirus-8 (HHV-8/KSHV) Induces Reactive Oxygen Species in Endothelial Cells That Facilitate Virus Infection.
Authors: Wang Jian Feng , Zhang Xuefeng , Chandran Bala , Groopman Jerome E.
Published: October 2019

Reactive oxygen species (ROS) can activate replication of certain viruses, induce the production of various inflammatory mediators and play a critical role in carcinogenesis and tumor development. Kaposi’s sarcoma (KS) is the most prevalent HIV-associated cancer and is caused by infection with human herpesvirus-8 (HHV-8/KSHV). KS tissue has been reported to possess increased levels of ROS. We studied if ROS generation is related to HHV-8 infection and its role in virus entry into endothelial cells. Incubation of dermal microvascular endothelial cells (DMECs) with highly purified HHV-8 induced rapid increases in the production of intracellular hydrogen peroxide (H2O2), one of the major forms of ROS. Intracellular H2O2 was also induced by the treatment of DMECs with the HHV-8 envelope glycoprotein B (gB). The gB protein possesses an RGD motif, binds to the integrin molecules, alpha3 and beta1, and is a major mediator of virus entry into target cells. To test if it was integrin ligation that induces the production of ROS, we treated DMECs with fibronectin or laminin, the respective natural ligands for alpha3 and beta1 integrins. We observed a similar induction of intracellular ROS in DMECs by either matrix protein. These results indicated that the HHV-8-induced production of ROS was, at least in part, mediated by stimulation of integrins through the RGD-containing viral gB protein. ROS have recently been shown to function as second messengers in cellular signaling. To assess at which steps of cell signaling ROS may be functioning, we studied the signaling cascade in DMECs activated by the HHV-8 gB protein. Previous studies have shown that HHV-8, through gB/integrin interaction, induces cytoskeletal rearrangement and activates focal adhesion kinase (FAK), Src kinase and Akt, which are critical for virus entry into the target cells. We found that the activation of FAK, c-Src or Akt by this viral protein was inhibited by pretreatment with N-acetyl-L-cysteine (NAC), a potent thiol antioxidant. These results suggested that generation of ROS was involved in HHV-8-triggered signaling. We next examined if a change in ROS production modulated HHV-8 virus entry. We used green fluorescent protein (GFP)-labeled HHV-8 at a multiplicity of infection of 5–6, and quantitated the infection by fluorescence analysis of the DMECs. Short term exposure to low concentrations of H2O2 enhanced HHV-8 infection in DMECs, while treatment with NAC significantly decreased infection. These data indicated that ROS generation participated in HHV-8-mediated signaling and entry into target cells. Our study demonstrates a novel role of ROS in virus pathogenesis and provides a framework for the development of novel antioxidant strategies in AIDS-KS treatment.
Phase II Trial of Bortezomib in Mantle Cell Lymphoma.
Authors: Belch Andrew , Kouroukis C. Tom , Crump Michael , Sehn Laurie , Gascoyne Randy , Klasa Richard , Jean Powers , Eisenhauer Elizabeth
Published: October 2019

We report the results of a phase II trial of an investigational new drug bortezomib (PS-341, Velcade) for the treatment of mantle cell lymphoma. Advanced stage previously untreated patients or those given up to two prior chemotherapy regimens were eligible. Additional entry criteria included measurable nodal disease, minimum laboratory requirements, and written informed consent. Central pathology review, to confirm eligibility and the presence of cyclin D1, was required after study entry. Accrual was closed July 2004 having reached the preplanned target of 30 patients. Bortezomib 1.3 mg/m2 IV bolus was given days 1, 4, 8 and 11 in 3-week cycles. Patients were assessed by CT scanning after every two cycles of therapy and response assessed according to International Working Group recommendations (Cheson B, J Clin Oncol 1999). Non progressors were to receive a maximum of 4 cycles. Responding patients were to receive bortezomib for 2 cycles following documentation of maximal partial response (PR) or complete response (CR). To date demographic data are available on 26 patients. The median age was 67 (48–78 yrs), all had stage 3 or 4 disease (55% bone marrow involvement), 7 were female and 10 had received no prior chemotherapy. A median of 4 treatment cycles has been given (range 1–7) and 25 patients are evaluable for toxicity. Grade ≥ 2 adverse effects thought to be related to study drug occurred as follows: anorexia 8%, nausea 16%, vomiting 4%, diarrhea 20%, fatigue 60%, dizziness 4%, sensory neuropathy 12%, edema 8%, hypotension 4%, vascular leak syndrome 4%, arthralgia 12%, myalgia 12%, neuropathic pain 12%, dyspnea 12%, rash 12%. Nine patients discontinued therapy because of toxic effects (6 of whom had neuropathy or myalgia). During accrual of the first 14 patients, 5 serious adverse events occurred in patients with pre-existing edema, dyspnea, and/or effusion. Therefore, the eligibility criteria were amended to exclude such patients thereafter and no further serious adverse events have occurred. To date, 24 patients are evaluable for response. Of the 10 patients having no prior chemotherapy there were 3 PR (range 2.5–14.8m) 6 SD (1.3–13.6m) and 1 PD for a response of 30%. Of the 14 previously treated patients we obtained 1 CRu (14.1m), 4 PR (2.4–13.1m), 7 SD (1.2–14.3m) and 2 PD. The overall response rate is 33% (95% C.I. 16–55%) but interestingly it is similar in both previously untreated and treated groups. We conclude that bortezomib is an active agent in mantle cell lymphoma, but at this dose and schedule complete remission is rare. Since higher doses will not be possible given the frequency of neuropathy and myalgia, alternative schedules or novel combinations with other active agents will be of interest to pursue.
The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.
Authors: Gould Weston R. , Baxi Sangita , Perrin Lisa A. , Leadley Robert J.
Published: October 2019

At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.
The Ubiquitin Ligase Triad1 Is Involved in Myelopoiesis and Interacts with the Transcriptional Repressors Gfi1 and Gfi1b.
Authors: Van der Reijden Bert A. , Marteijn Jurgen A.F. , Van Emst Liesbeth , De Witte Theo , Jansen Joop H.
Published: October 2019

We identified Triad1 as a gene that is upregulated by retinoic acid during the granulocytic differentiation of acute promyelocytic leukemia cells. In normal hematopoiesis, we show that Triad1 is weakly expressed in immature CD34+ bone marrow cells, and highly expressed in mature monocytes and granulocytes. Together, this suggests that Triad1 plays a role in the differentiation of hematopoietic cells. Triad1 contains a tripartite domain including two RING fingers, indicating that this protein might function as a ubiquitin E3 ligase, catalyzing the the conjugation of ubiquitin to substrate proteins thereby marking them for targeted degradation by the 26S proteasome.

Using GST pull down experiments, we show that Triad1 binds to the ubiquitin conjugating (E2) enzymes UbcH6 and 7. In addition, immunoprecipitation of Triad1 in cells that were transfected with FLAG-tagged ubiquitin shows that Triad1 binds to ubiquitinated proteins, and that Triad1 is capable of self-ubiquitination, further corroborating the assumption that Triad1 acts as a E3 ubiquitin ligating enzyme.

To study the role of Triad1 in hematopoiesis we overexpressed the gene in primary murine bone marrow cells using a retroviral vector that contains Triad1 in front of an IRES-GFP cassette. GFP positive cells were FACS sorted and used in colony assays (CFU-GM). Compared to empty vector controls (GFP alone), Triad1 expression resulted in more than 80% inhibition of clonogenic growth. Importantly, addition of the proteasome inhibitor MG132 (10E-8 M) reversed the Triad1-induced suppression of colony formation. Furthermore, three Triad1 expression constructs in which one of the conserved cys/his residues of the TRIAD domain (essential for function) were mutated did not show the suppressive effect on colony formation. Together, these data show that Triad1 is involved in myelopoiesis and acts through the ubiquitination of specific substrate proteins.

To identify these substrates, a yeast-two-hybrid screen of a human bone marrow cDNA library was performed using the Triad1 protein as a bait. Interestingly, the transcriptional repressor Gfi1b was found to bind to Triad1. The interaction was confirmed by immunoprecipitation using GFP-Triad1 and FLAG-tagged Gfi1b transfections in mammalian cells. We show that Triad1 binds to the zinc finger region of Gfi1b. This region is very (>98%) homologous to the paralogue Gfi1. Further immunoprecipitation analyses showed that Triad1 also binds to the zinc finger region of Gfi1. Gfi1 plays an essential role in neutrophil development and Gfi1 pointmutations result in neutropenia in man. Currently, we are studying the direct ubiquitination of Gfi and Gfi1b by Triad1 in in vitro ubiquitination assays. In addition, we are studying the effect of Triad1 on the transcriptional repression of the ELA2 and other promoters by Gfi1.
The Transcription Factor Nf-E2 Is Overexpressed in Patients with Polycythemia Vera.
Authors: Goerttler Philipp S. , Maerz Edith , Steimle Cordula , Will Britta , Schmitt-Graeff Annette , Pahl Heike L.
Published: October 2019

Despite recent advances in characterizing molecular markers for the diagnosis of polcycythemia vera (PV), the aberrations leading to disease development remain unknown. We therefore used expression profiling to identify candidate genes involved in the pathophysiology of PV. RNA from purified granulocytes of 40 PV patients was analyzed by hybridizing individual samples to a pool of 50 healthy controls. Of the 7,496 genes represented in the cDNA array, 253 were upregulated more than 1.5 fold in PV compared to healthy controls (p< 0.01, FDR corrected). Promoters for 26 of the 253 genes overexpressed in PV are regulated by members of the Sp1 family of transcription factors. We have therefore hypothesized that altered activity of one or several Sp1-like transcription factors may contribute to the molecular etiology of PV. Here we report that one of the Sp1 target genes identified, the transcription factor NF-E2, is overexpressed in 37 of the 40 PV patients (92.5%) assayed by microarray. NF-E2 overexpression was confirmed by Northern Blot and quantitative RT-PCR analysis. Transcription factor overexpression varies from 2.3 to 40 fold, with a median increase of 7 fold in PV patients compared to healthy controls. The NF-E2 protein is readily detected in PV granulocytes by Western Blot whereas it is undetectable in healthy control cells. Immunohistochemistry revealed that in PV bone marrow, NF-E2 is overexpressed in megakaryocytes as well as erythroid and granulocytic precursors. Several published observations suggest that NF-E2 is an exceptionally promising candidate in the molecular etiology of PV. Firstly, the transcription factor is expressed in hematopoietic precursors as well as in erythroid, megakaryocytic and granulocytic cells, those lineages affected in PV. Secondly, Sayer et al. have shown that overexpression of NF-E2 in fetal liver cells leads to the development of Epo-independent erythroid colonies, analogous to the endogenous erythroid colonies (EECs) observed in PV patients. Furthermore, ectopic expression of NF-E2 results in the spontaneous emergence of morphologically mature erythroid cells in the absence of Epo and can reprogram monocytic cells towards erythroid and megakaryocytic differentiation. These data support the hypothesis that the concentration of an individual transcription factor can control lineage commitment. We thus propose that in PV patients elevated concentrations of NF-E2 alter the physiological transcription factor balance leading to an overproduction of erythroid and, in select patients, megakaryocytic cells/platelets. In this model the level of NF-E2 overexpression determines both the severity of erythrocytosis and the concurrent presence or absence of thrombocytosis.
Incorporation of Bone Marrow Derived Cells in Colonic Adenoma.
Authors: Cogle Christopher R. , Theise Neil D. , Jorgensen Marda L. , Smith Doug , Thung Swan L. , Liu Chen , Wingard John R. , Scott Edward W.
Published: October 2019

Bone marrow contains stem cells that can engraft in various distant organs. These observations prompted us to investigate the cellular origin of post-transplant solid tumor neoplasias found in women having received a bone marrow transplant at our facility. In women having received bone marrow transplantation from male siblings we found colonic adenomas post-transplant. Presence of donor-derived cells were examined using a combination of immunohistochemistry for cells expressing the pan-leukocyte antigen, CD45, and the colonocyte antigen, cytokeratin 20, in conjunction with fluorescent in situ hybridization for X and Y chromosomes. Confocal microscopy was used to evaluate for evidence of cell fusion. In these cases, recipients of gender-mismatched transplants showed evidence of Y-chromosome positive, cytokeratin positive cells within the adenomas. The male cells accounted for 1 to 4% of the adenoma cells in histologic section. Confocal microscopy permitted evaluation of entire nuclei (Z-stack steps of 0.25 microns) and demonstrated no male cell with more than one X chromosome. A total of 40 male cells expressing cytokeratin were found throughout the adenomas. When considering how bone marrow derived cells incorporate into colonic adenomas, two possibilities should be regarded. First, bone marrow derived cells may migrate to neoplasias due to upregulation of tumor growth factors, which also act as inflammatory cytokines and chemokines, such as stromal derived factor 1 (SDF-1). Up- or down-regulating SDF-1 has profound effects on recruiting circulating bone marrow stem/progenitor cells for engraftment as end-organ epithelia. SDF-1 staining of the colonic adenomas found in our patients demonstrated intense, patchy SDF-1 expression compared to normal colonic epithelia. Second, roving donor-derived bone marrow cells may have engulfed the recipient adenoma cells. A donor scavenger cell could have phagocytosed an apoptotic adenoma cell. In these situations the fusion of nuclear material should result in a multiplicity of sex chromosomes (XXY or XXXY). However, as noted, the donor-derived epithelial cells within the adenomas demonstrated no extra X chromosomes. In the liver, where cell fusion has been demonstrated in severe disease stress states, it has been postulated that 28% of donor-derived hepatocytes are due to reduction division, resulting in diploid daughter cells. Based on the probability of binomial distribution, the chance that we would find 40 out of 40 diploid donor-derived epithelial cells amidst a background fusion resolution rate of 28% is one in 1x10e22. Thus, direct differentiation, rather then absolutely complete and perfect resolution of every fusion event is the most likely explanation of our current findings.
Identification of Non-Hematopoietic Stem Cells in Mouse Bone Marrow Osteoblastic Zone.
Authors: Sato Hidetaka , Arai Fumio , Shibata Mami , Hirao Atsushi , Suda Toshio , Sakurada Kazuhiro
Published: October 2019

Bone marrow contains hematopoietic and non-hematopoietic stem cells. Although the character of hematopoietic stem cells (HSCs) in vivo has been extensively studied, our understanding of non-hematopoietic stem cells is derived only from in vitro expanded culture such as mesenchymal stem cells (MSCs). Since the number of non-hematopoietic stem cells in bone marrow is small, their characteristics, anatomical location and physiological role in vivo remain unknown. We attempted to overcome this problem by introducing a novel method. We extracted bone marrow cells from osteoblastic zone by enzymatic digestion. Bone marrow extracted cells (BMECs) contained non-hematopoietic cells (CD45−/Ter-119− cells) more than flushed out bone marrow cells (BMCs). Non-hematopoietic cells in BMECs were divided into two types, CD34+ (BME 34+) cells and CD34− (BME 34−) cells.

When BME 34− cells were cultured to expand to a confluent layer in MSC culture condition, they formed fibroblastic cells, which have the ability to differentiate into osteocytes, chondrocytes and adipocytes, indicating that BME 34− cells are the in vivo origin of MSCs.

On the other hand, BME CD34+ cells contained mostly non-hematopoietic SP fraction and they were Sca-1+ cells. Immunohistochemical analyses show that CD31-BME 34+ cells localized on osteoblasts and endothelial cells in bone marrow. When BME 34+ cells were co-cultured with stromal (OP9) cells, they formed a colony of myogenic cells, endothelial cells or adipogenic cells. However, different from BME 34− cells, fibroblastic cells expanded from BME 34+ cells did not differentiate to osteocytes, chondrocytes or adipocytes in MSC culture condition. When 4,000-6,000 BME 34+ cells were transplanted into irradiated mice, these cells engrafted as non-hematopoietic Vimentin+ fibroblastic-like cells in various organs such as liver, spleen, pancreas, colon, intestine, kidney, skin, brain, lung, heart, skeletal muscle, and bone marrow. In spleen and liver, transplanted cells formed clusters of Vimentin+ fibroblastic cells indicating that BME 34+ cells have ability to expand in vivo to some extent. When BME 34+ cells were directly injected into tibial muscles which were damaged by cardiotoxin, these cells gave rise to mature skeletal muscle cells and endothelial cells.

In summary, these studies show distinguishable two types of non-hematopoietic stem cells in bone marrow. It is possible that mobilization and recruitment of these bone marrow-derived non-hematopoietic stem cells play a major role in the setting of wound healing, organ regeneration and tumor growth. In addition, non-hematopoietic two types of BMECs may contribute to clarify the debate on the stem cell plasticity of bone marrow cells.
Outcome for Adolescent and Young Adults 16–21 years of age (AYA) with Acute Lymphoblastic Leukemia (ALL) Treated on the Children’ s Cancer Group (CCG) 1961 Study.
Authors: Nachman J. , Siebel N. , Sather H. , Steinherz P. , DeLaat C. , Fryer D. , Mattano L.A. , Hastings C. , Rubin C. , Bertolone K. , Franklin J. , Mitchell T. , Ettinger L. , Pyesmany A. , Gaynon P.S. ,   for the Children’s Oncology Group (COG), Arcadia, CA
Published: October 2019

AYA Patients 16–21 years of age with ALL are treated on both pediatric and adult ALL protocols. Recent publications have suggested that AYA patients treated on pediatric protocols have a significantly better outcome than those treated on adult protocols. From November 1996 to May 2002, 262 AYA ALL patients were entered on the CCG 1961 protocol. Treatment was based on the following observations from the previous CCG 1882 Protocol (1989–1995). 1) For patients with rapid early response (< = 25% blasts in a day seven bone marrow aspirate-RER), in the context of CCG modified BFM therapy (S-BFM), pre-symptomatic central nervous system therapy with intensive intrathecal (IT) methotrexate (MTX) alone provided equivalent results to standard IT MTX and cranial radiation (CRT). 2) For patients with slow early response (> than 25 blasts on day 7-SER), the augmented BFM (A-BFM) regimen produced a significant improvement in outcome compared to S-BFM. A-BFM featured a significant increase in dose intensity for vincristine (VCR), L-asparaginase (L-ASP), and steroid, the use of intravenous MTX without rescue in conjunction with VCR and L-ASP in interim maintenance (IM), and a second IM and delayed intensification (DI) phase. RER patients on 1961 were randomized to one of four arms; S-BFM, S-BFM with a second standard IM and DI phase, A-BFM with only 1 IM/DI phase, and full A-BFM. SER patients were randomized to A-BFM or A-BFM with Idarubicin/Cytoxan pulses added to the 2 DI phases. One hundred ninety patients had < 50K WBC (73.4%) and 21% had T cell immunophenotype. Seven patients had a t (9/22) and three patients had a t (4/11). Five year EFS for AYA patients treated on CCG 1961 was 68% ± 3.7% and the five-year survival was 77% ± 3.2%. The majority of events were isolated bone marrow relapses. For RER AYA patients, five year EFS for patients treated on the augmented intensity arms (N=87) was 83.2% compared to 60.8% for patients treated on the standard intensity arms (N=76) (P = .10). Within the augmented intensity arms, there was no difference in EFS for patients randomized to one or two DI phases. There was no difference in EFS for the 2 SER treatment arms. 5 year EFS was 78% for the Idarubicin/Cytoxan arm and 74% for the ABFM arm. Five year EFS for AYA patients with < 50K WBC was 73.2% (N=190) versus 54% for AYA patients with > 50K WBC. Unlike younger patients, there was no difference in outcome for males and females. Conclusions: 1) AYA patients with ALL have a 68% 5 year EFS when treated with BFM based chemotherapy. 2) WBC < 50K predicts for a favorable outcome. 3) Early augmented intensity therapy appears to improve outcome for AYA ALL patients showing a rapid response to induction therapy.
Phase II Trials of Clofarabine in Relapsed or Refractory Pediatric Leukemia.
Authors: Jeha Sima , Razzouk Bassem I. , Rytting Michael E. , Gaynon Paul S. , Kadota Richard , Rheingold Susan , Luchtman-Jones Lori , Shen Violet , Arceci Robert J. , Fernandez Manuel , Weitman Steven , Steinherz Peter G.
Published: October 2019

Background: Clofarabine, a next generation nucleoside analogue, was well tolerated and demonstrated activity in adult and pediatric Phase I trials conducted in heavily pretreated leukemia patients. Multicenter Phase II studies in pediatric leukemia have completed accrual in the US and are reported here.

Methods: Two Phase 2, multicenter, open-label studies were conducted with clofarabine in children with refractory or relapsed ALL or AML. Clofarabine was administered intravenously over 2 hours at 52 mg/m2/day for 5 consecutive days. Cycles were repeated every 2 to 6 weeks based on response and toxicity.

Results: The studies enrolled 100 patients (60 ALL and 40 AML). Currently, data are available for 84 patients (49 ALL, 35 AML). Median age is 12 years (range 1 to 22 years) and median number of prior regimens is 3 (range 1 to 6). Thirty-nine percent had received prior bone marrow transplant (BMT). As determined by independent review, preliminary data indicate overall response rates of 31% in ALL (6 CR, 4 CRp, and 5 PR) and 26% in AML (1 CRp and 8 PR). Median duration of remission for ALL is 9.7 weeks (range 1.0 to 28.6) and for AML is 16.2 weeks (range 1.7 to 56.6+). Thirteen of 24 responding patients (54%) proceeded to BMT. Median survival was 42 weeks (range 7.0 to 63.1+) for responding ALL patients (CR+CRp+PR) and 39 weeks (range 7.7 to 93.6+) for responding AML patients (CRp+PR). Patients who failed treatment or were non-evaluable had shorter median survival; 7.4 weeks (range 0.9 to 40.1+) and 12.4 weeks (range 1.6 to 84.9+) for ALL and AML, respectively. Among the patients who were refractory to the last prior chemotherapy, 7/30 (23%) with ALL and 4/22 (18%) with AML achieved a response with clofarabine. Median duration of remission in these patients is 4.6 weeks (range 2.3 to 24.4+) for ALL and 20 weeks (range 1.7 to 56.6+) for AML. Most drug-related adverse events were transient including febrile neutropenia, diarrhea, nausea/vomiting, fever, skin rash, headache, elevation in liver enzymes and bilirubin, and infusion-related flushing and anxiety.

Conclusions: Clofarabine is active as a single agent in pediatric ALL and AML that are refractory to intensive salvage regimens. The overall safety profile is similar to that reported in other pediatric salvage studies. Clofarabine in combination with standard chemotherapy is currently under investigation in children.
In Vivo Evidence of Modulation of the Hemophilia Phenotype by the Factor V Leiden.
Authors: Schlachterman Alexander , Liu Jianhua , Liu Yi-Lin , High Katherine , Arruda Valder
Published: October 2019

The amelioration of hemophilia phenotype or delayed onset of the bleeding episodes in subjects with severe hemophilia A (FVIII deficiency) has been associated with inherited resistance to activated protein C due to factor V Leiden (R506Q), FVL. These observations were confirmed by in vitro systems in which homozygous phenotype of FVL increased thrombin formation in presence of < 1% FVIII by nearly 5-fold (Blood 90:3067). Here we provide in vivo evidence of the beneficial interaction of FVL on the hemophilia phenotype in mice. Animals with severe deficiency of factor VIII due to deletion of intron 16 of factor VIII gene (HA) or large gene deletion of factor IX (HB) were crossed with FVL homozygous mice [+/+] on C57Bl6 strain [Cui et al. (Blood 96:4222)] We used a modified activated partial thromboplastin time (aPTT) assay to compared clotting times among male HA (n=30), HA/FVL [+/+] (n=7), FVL [+/+] (n=5), and litermate WT mice (n=6) with age ranging from 6–12 weeks. Blood samples were collected by tail vein transection into 3.8% sodium citrate. Values for the aPTT in male HA were 67.3 ± 4 sec, and among WT or FVL [+/+] values were 37 ± 4 sec or 31± 2 sec, respectively. Whereas intermediate aPTT values of 53 ± 3.7 sec were determined in HA/FVL [+/+], which differs from HA mice (p<0.0001) but also from WT or FVL (p<0.0001). A similar shortening of aPTT was also determined among HB/FVL[+/+] which were compared to HB, 55 ± 7 sec vs. 64 ± 0.9. Hemostatic challenge by tail clipping assay failed to revealed differences in bleeding times/blood loss among hemophilia animals with or without FVL mutation. To test whether a more sensitive technique would provide further evidence of the improved hemostasis in HA/FVL mice, we assessed real-time in vivo thrombus formation by confocal and widefield microscopy. Mice were anesthetized and the cremaster muscle was exposed for intravital microscopy. Infusion of fluorescently labeled antibody to murine platelet glycoprotein IIb/IIIa complex via the jugular vein allowed monitoring of platelet deposition upon laser-mediated endothelial injury at several sites of the arterial vessel wall. No thrombus formation was observed in severe HA mice following successive vascular injuries, a finding also common in severe HB mice. However, infusion of factor VIII concentrated clearly induced the thrombi formation upon vascular injury. HA/FVL mice tested presented thrombus formation in a comparable fashion of HA-FVIII transfused mice. These in vivo data provide support to the hypothesis that the FVL mutation has the potential to improve the phenotype of severe hemophilia and may offer a novel therapeutic target for hemophilia.
Intermediate Dose Intravenous Melphalan and Dexamethasone Treatment in 144 Patients with Systemic AL Amyloidosis.
Authors: Goodman Hugh J.B. , Lachmann Helen J. , Bradwell Arthur R. , Hawkins Philip N.
Published: October 2019

The optimal intensity of chemotherapy in AL amyloidosis remains contentious. Traditional low dose oral melphalan and prednisolone therapy is ineffective in most patients, whereas high dose therapy with autologous stem cell rescue carries substantial procedure related mortality. We report here the outcome of intermediate dose intravenous melphalan (IDM) (25mg/m2 d1 + dexamethasone 20mg po d1-4, repeated every 21-28d) among 144 patients with amyloidosis (83M:61F, median age 62yrs, range 30–79). The median number of organs involved by amyloid was 2, including kidneys in 74% of patients, heart in 56%, neuropathy in 25% and liver in 20%. Median follow-up was 13mo (0.5–53). Patients received a median of 3 cycles of treatment (range 1–8). Dexamethasone was omitted in 51 cases, principally due to concern about fluid retention associated with nephrotic syndrome or cardiac amyloidosis. There were 52 deaths of which 3 (2%) were considered treatment-related (1 neutropenic sepsis, 2 dexamethasone-induced pulmonary oedema). Serum free light chain (sFLC) measurements were available before and after at least one cycle of IDM in 140 patients; 23% achieved complete response (CR, sustained normalization of sFLC ratio), 31% a partial response (PR, sustained >50% reduction in pre-treatment clonal isotype) and 46% did not respond (NR). Rates of sFLC CR+PR were higher among patients who also received dexamethasone at 64% vs 37% (p<0.0004). Paraproteins were detectable by electrophoresis and immunofixation of serum and/or urine in 115pts (80%); 85 of these pts had post-treatment data, showing CR (complete resolution of serum and urine paraprotein) in 17 (20%), PR (50% reduction) in 24 (28%) and NR in 44 (52%). Cycle by cycle assays of sFLC allowed rapid assessment of early clonal response, which was evident within 2 cycles in almost all responders. Maximal response was usually achieved by the fourth cycle. Median survival was 44 months among patients with CR or PR, and 18 months among non-responders (p<0.003). Amyloidotic organ dysfunction improved in 14%, remained stable in 51%, and deteriorated in 19% of evaluable cases. The findings support the use of IDM coupled with cycle by cycle sFLC measurements as a valid treatment strategy in patients with AL amyloidosis. Although clonal response rates in this series may be lower than selected patients treated with high dose therapy and autologous stem cell rescue, IDM can be delivered to a wider range of patients, in whom the rapidity of its action, relative simplicity of the regimen, and much lower treatment related mortality favor its continued use and further investigation.
Ectopic Fibroblast Growth Factor Receptor 3 (FGFR3) Expression Mediated by Isotype Switch Recombination.
Authors: Affer Maurizio , Robbiani Davide , Chesi Marta , Bergsagel Peter L.
Published: October 2019

FGFR3 is disregulated in approximately 15% of cases of multiple myeloma (MM) occurring by chromosomal translocation in the Igh switch region which results in the juxtaposition of FGFR3 next to the regulatory elements of the IgH locus, causing its ectopic expression in plasma cells. The acquisition of FGFR3 activating mutations in some tumors indicate a role for FGFR3 in tumor progression. In order to study its role in MM progression we developed a strain of transgenic mice in which the expression of the activated form of FGFR3 is mediated by isotype switch recombination by replacing the IgH gamma1 costant region with an FGFR3-IRES-EGFP cassette in a BAC that covers the entire murine IgH locus. We predict that in the transgenic mice B cells that undergo switch recombination to g1 on the productive allele will also undergo switch recombination to g1 at the transgenic locus and express FGFR3 and EGFP. To evaluate FGFR3 expression in vitro, we collected splenocytes from our transgenic mice and induced their proliferation and differentiation to plasma cells (mainly IgG1) using LPS and IL4. After four days of stimulation, the average number of EGFP positive cells went from 0.2% to 80%. We then analyzed FGFR3 expression by RT PCR, Northern and Western blot and detected LPS/IL4 inducible FGFR3 expression in plasma cells mediated mainly by the Igamma1 promoter and in a subset of them by the Vh promoter. We then tested if forced FGFR3 expression in B cells would affect their proliferation rate and ability to differentiate in vitro by MTT assay, cell cycle analysis and PI staining on LPS/IL4 stimulated splenocytes at different time points. With this system, we did not observe any FGFR3 mediated alteration of splenocytes growth parameters when compared to wild type controls. Similarly, in vivo no monoclonal gammopathy has been observed by serum protein electrophoresis and all the transgenic mice remaine tumor free at 1.5 years. Although we have not been able to demonstrate a tumorigenic role for FGFR3 in switched plasmacells we were able to obtaine regulated FGFR3 expression in them. It is possible that FGFR3 expression alone is not sufficient to induce plasma cells transformation and that FGFR3 has a dispensable role in tumor initiation, but that it could still play a role in tumor progression. Consistently, FGFR3 expression is lost in about 25% of multiple myeloma wih a t(4;14). To address this hypotesis we are currently crossing these mice with MMSET (the other gene disregulated in the t(4;14)) transgenic mice to investigate a possible collaborative role between these two genes in the development of MM.
Sequencing the Multiple Myeloma Kinome: Absence of Mutation in Known Malignancy-Associated Kinases.
Authors: Claudio Jaime O. , Khaja Razi , Zhuang Lihua , Bali Meenakshi , Sivananthan Kamalanayani , Christopoulos Constantine C. , Fonseca Rafael , Bergsagel P. Leif , Scherer Stephen W. , Stewart A. Keith
Published: October 2019

In approximately 50% of Multiple Myeloma (MM), translocation of non random genes into the IgH locus is believed to be the seminal event in the pathogenesis of the disease. Another 50% of cases are hyperdiploid and trisomic to certain autosomes, but do not harbor any translocation and thus are believed to have genetic alterations in unidentified loci. These observations, together with the finding of somatic mutations in FGFR3, N- and K-RAS, MYC, TP53 and CDKN2C/p18INK4C during the later stages of MM indicate that defective signaling pathways likely play a role in the progression of this malignancy. Of relevance then, in recent years recurrent mutations in kinases have frequently been implicated in malignancies including notably colon cancer and melanoma. We have therefore begun a comprehensive effort to sequence the tyrosine kinome for mutations and genetic polymorphisms in MM. Of particular interest are 90 receptor tyrosine kinases, 43 receptor tyrosine kinase-like, 5 receptor guanylate cyclase, and a lipid kinase. We report here results from our pilot high throughput exon scanning in 32 human MM cell lines which initially focused on 13 kinases known to be somatically mutated in human cancers. To date we have expanded this effort to assess 30 genes with sequence obtained which currently spans 80% coverage of the kinase domains of these genes. A total of 1.9 million bp have been sequenced across 235 exons. No recurrent mutations have been identified in the kinase domains of the cancer-associated genes: SRC, ILK1, KIT, GUCY2F, PDGFRA; in the genetic disorder-associated kinases: BTK, EPHA4, LAMA2, EPHB6, ACVR2; and in the mutation hot spots of frequently mutated cancer gene PIK3CA. A novel missense mutation is however identified upstream of the kinase domain of FGFR3 changing a Ser residue to Arg at codon 433. This residue, which is conserved across species and in FGFR1 and FGFR2, has not been reported in myeloma and in thanatophoric dysplasia, but the biological significance of this mutation is unknown. Several single nucleotide polymorphisms were identified in the coding regions of some of these kinases. Notably, synonymous polymorphisms in the kinase domains of EPHA4, PDGFRA3, KIT, MLK1, ILK1, NTRK3, FLT3, ABL1, FES, MLK4, and EGFR1 were identified that changed a codon but not the amino acid. More importantly, we identified non-synonymous amino acid variations in the kinase domains of EPHA4, GUCY2F, PTK2, and PIK3CA genes that are more likely to effect variability in the activity of these kinases. In summary, no recurrent kinase mutations of significance in Myeloma development or progression have yet been identified. Sequencing of the known cancer associated kinases in MGUS and hyperdiploid MM patients is now underway and our data set is being expanded to include all 139 kinases.
Comprehensive Genome-Wide Profile of Regional Gains and Losses in Multiple Myeloma Using Array-CGH: The 1q21 Amplification and Potential Role of the BCL-9 Gene in Multiple Myeloma Pathogenesis.
Authors: Carrasco Ruben , Tonon Giovanni , Brennan Cameron , Protopopov Alexei , Sinha Raktim , Feng Bin , Leo Christopher , Zhang Yunyu , Zhang Jean , Zhan Fenghuang , Protopopova Marina , Mitsiades Constantine , Munshi Nikhil , Chin Lynda , Bergsagel Peter , Kuehl Michael , Shaughnessy John , Anderson Kenneth , DePinho Ronald
Published: October 2019

Multiple Myeloma (MM) is characterized by a clonal proliferation of abnormal plasma cells in the bone marrow and is among the most frequent and lethal hematological diseases. In spite of significant effort towards the identification of the molecular events leading to this malignancy, the genetic alterations responsible for the pathogenesis of this disease remain poorly understood. Regional copy number alterations (CNAs) in cancer genomes have been among the most informative structural changes in cancer and have led to the discovery of many oncogenes and tumor supressor genes. Using array comparative genomic hybridization (array-CGH) and expression microarray technologies we have analyzed a large collection of cell lines and clinically annotated primary tumors. This high-resolution genomic analysis has identified all previously reported regional gains and losses as well as many novel highly recurrent genetic loci with potential biological and clinical relevance. In particular, we have identified an amplification at chromosome 1q21 as one of the most recurrent genetic changes in cell lines and in a subgroup of primary tumors. This chromosomal change has been previously implicated with disease progression. Analysis across several cell lines has allowed the identification of a Minimal Common Region (MCRs) of amplification at 1q21. Correlation between DNA copy number changes and expression profiling data has identified a limited set of candidate genes within this MCR that are amplified and overexpressed. Using shRNAi technology we have identified BCL-9 as a candidate gene residing at the 1q21 MCR. In vitro and in vivo functional data about the role of BL-9 will be presented. These data will provide critical understanding on the diverse pathways leading to Multiple Myeloma progression.
Nordic Mantle Cell Lymphoma (MCL) Project: Prolonged Follow-Up of 86 Patients Treated with BEAM/BEAC + PBSCT Confirms That Addition of High-Dose Ara-C and Rituximab to CHOP Induction + In-Vivo Purging with Rituximab Increases Clinical and Molecular Response Rates, PCR-Neg. Grafts, Failure-Free, Relapse-Free and Overall Survival.
Authors: Geisler Christian H. , Elonen Erkki , Kolstad Arne , Laurell Anna , Andersen Niels S. , Pedersen Lone B. , Jerkeman Mats , Nordstroem Marie , Lauritzen Grete F. , Ralfkiaer Elisabeth , Aakerman Maans , Sundstroem Christer , Langholm Ruth , Karjalainen-Lindsberg Marja-Liisa
Published: October 2019

Methods: In the Nordic Lymphoma Group MCL Project newly diagnosed stage II-IV MCL patients < 66 years receive induction treatment and peripheral-blood stem-cell (PBSC) harvest, followed by BEAM/BEAC with PBSC support. In the MCL1 Protocol, the induction was maxi-CHOP x 3 (CTX 1200 mg/m2, doxorubicin 75 mg/m2, VCR 2mg D1, prednisolone 100 mg D1-5). Stem-cells were mobilised by maxi-CHOP + G-CSF. Because of the high failure rate in MCL1 (Figure 1) the MCL2 protocol was activated adding 2 series of high-dose Ara-C (3g/m2 BID D1-2) and 2 standard doses of rituximab (R) (375 mg/m2) to the induction program. Stem cells were mobilised by Ara-C + G-CSF + rituximab D1 + D9 for in-vivo purging. In both protocols patient-specific molecular markers were sought established before treatment start, and stem-cells and follow-up blood and bone-marrow samples assessed for MCL by PCR or flow cytometry.

Results: Table 1

Compared Results of the Nordic Lymphoma Group MCL1 and MCL2 Protocols

NORDIC MCL PROTOCOL # MCL1 (1997–2000) MCL2 (2000-) P value Supported by the Nordic Cancer Union No. of Pts included/eval. for response 42/42 120/88 Response pretransplant 31/42 86/88 0.002 CR/resp pretransplant 11/31 51/86 0.04 No. transplanted 27 86 Eval. for response posttransplant 27 82 CR/Response posttransplant 25/27 76/82 NS Molecular response posttransplant 5/13 38/42 0.0003 PCR-neg. stem-cell products (of tested) 2/16 22/25 0.00005 3-year failure-free survival (104 pts eval.) 24% 68% 0.0001 3-year relapse-free survival (RFS) 45% 76% 0.04 3-year molecular RFS survival ND 67% 3-year overall survival 60% 85% 0.02

Posttransplant maintenance treatment: In MCL2, patients in clinical stable response but molecular relapse are offered R 4 std. doses. So far, isolated molecular relapse occurred in 11 pts. of whom 10 received R: Four became PCR neg., one did not respond and later relapsed, five are not yet evaluable. Conclusion: By comparing MCL1 results with preliminary results of the still ongoing MCL2 study we conclude that the addition of HD Ara-C and R to the induction treatment significantly increases the

• clinical response rate pretransplant

• molecular response rate

• No. of tumor-cell free grafts

• failure-free, relapse-free and overall survival

Rituximab maintenance treatment can induce 2nd molecular remission, the clinical significance hereof still unknown.

Figure Figure