bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allogeneic Stem Cell Transplantation for CML in Accelerate or Blastic Phase
Authors: Morozova Elena V , Zabelina Tatjana , Ayuk Francis , Wolschke Christine , Stübig Thomas , Bondarenko Sergey N , Alianskii Alexandr L. , Zander Axel R. , Afanasyev Boris , Kröger Nicolaus 
Published: October 2019

Abstract
Patients with advanced chronic myeloid leukemia (CML) in accelerate (AC) or blastic phase (BC) have a dismal prognosis despite the use of tyrosine kinase inhibitors (TKI). Here we report our experience for allogeneic stem cell transplantation (ASCT) in advanced and phase of CML. Between July 1990 and May 2012 88 patients with a median age of 36 years (range 7-76) received allogeneic stem cell transplantation from related (n=34) or unrelated (n=54) donors , including 19 HLA-mismatched donors after myeloablative (n=54) or reduced intensity (n= 34) conditioning. Stem cell source was bone marrow (n=37) or peripheral blood stem cells (n=51). GvHD prophylaxis consisted of calcineurin inhibitor plus short course MTX or MMF. Most of the patient received additional ATG as GvHD prophylaxis (84%).The majority of patient (75%) received TKIs before ASCT. 50% received one TKI, 8 received 2 TKIs and 2 received 3 TKI before ASCT. At time of transplantation 34 patient achieved a second or subsequent chronic phase, 28 were in accelerate and 25 in blastic phase. Overall the median number of blasts at time of ASCT was 18.75% (range 5-58%) and the time from diagnosis to transplantation was 27 months (range 3-296).
No primary graft failure was observed. The incidence of acute graft versus host disease (GvHD) grade II to IV was 43% and of severe grade III/IV GvHD was 28%. Forty-two percent of the patient experienced chronic GvHD.
The non-relapse mortality (NRM) at 1 and 5 years was 22% (95% CI; 14-30%) and 23% (95%CI: 13-33%), respectively. In a multivariate analysis (MVA) only higher number of transplanted CD34+ cells were associated with a lower risk of NRM (HR 0.850, 95%CI: 0.729-0.992, p=0.04). The cumulative incidence of relapse at 5 years was 43% (95% CI: 31-55%). In a MVA age > 40 years (HR 2.272, 95%CI: 1.112-4.645, p=0.024) and Reduced intensity conditioning (HR 2.034, 95%CI: 0.998-4.144, p=0.051) were significant factors for higher risk of relapse. After a median follow-up of 91 months (r., 52-133) the estimated 5 and 10 year overall survival was 44% (95% CI: 32-56) and 40% (95% CI: 28-52%), respectively. In an univariate analysis overall survival was significantly influenced by stem cell source, gender, CD34 transplanted cell number and blastic phase at time of transplantation. However, the only significant factor for improved survival in a MVA was a higher number of transplanted CD34+ cells (HR 0.916, 95%CI: 0.844-0.916, p=0.038).

Disclosures
No relevant conflicts of interest to declare.
Discovery of Novel EPO/EPOR/JAK2 Targets and Signal Transduction Factors Via Proteomic-Based Interrogations of Post-Translational Motif Modifications
Authors: Wojchowski Don M , Su Su , Johnson Ashley , Jachimowicz Edward , Stokes Matthew P , Green Jennifer M , Silva Jeffrey C 
Published: October 2019

Abstract
Although EPO/EPOR/JAK2 actions have been intensely studied, the majority of investigations to date have relied heavily upon conventional approaches, extant reagents and murine cell models. We therefore hypothesized that important EPOR/JAK2 targets consequently remain undiscovered, and by applying (phospho)proteomic based interrogations, have uncovered intriguing sets of new candidate mediators of EPO’s effects on human erythroid progenitor cells. Specifically, following [-] vs [+] EPO challenge, total cellular tryptic or Glu-C peptides were generated, and those post-translationally modified (PTM) due to EPO at phosphotyrosine residues (p-Y), p-TPP motifs, and MAPK plus ATK signaling nodes were isolated, and identified by duplicate tandem LC-MS/MS. EPO targets furthermore were confirmed via parallel analyses using an EPOR agonist. Known EPO/EPOR/JAK2 targets first were validated (eg., JAK2, STAT5, GAB1-3, SHIP1-3, PLCgamma, p85alpha-PI3K, LNK, SHC1, SPRY1) (and further defined with regards to specific isoforms, plus known vs novel sites of EPO-induced PTM). Beyond this, newly discovered EPO/EPOR/JAK2 regulated targets included those within the following five categories: 1] Cytoskeletal targets unexpectedly as erythroid alpha and beta spectrins (pY2332, pY15) together with pY modulation of Beta-Adducin at a calmodulin binding site (plus EPO-regulated PTM of Calmodulin per se); 2] Select EPO- regulated transcription factors, including 100-fold p-Y modulation of KLF5 at a novel transcriptional activation motif; 3] EPO/EPOR/JAK2 modulation of two new protein tyrosine phosphatase targets as PTPN18 and PTPN7; 4] Rapid EPO-induction of a notable number of novel upstream molecular adaptors including C1ORF186/”RHEX”, C1ORF150, DOK1, IRS2, SH2D2A, STAM2, SPRY2,4, DLG1,3, FRS2, AMOTL1, RAI14, CTNND1, EPS15, HGS, ITSN2, and WASL – with two novel ORF factors as C1ORF186/RHEX and C1ORF150 as prime targets in human erythroid progenitors, but absent from rat, mouse, and lower vertebrate genomes. [For C1ORF186/RHEX, select cell and molecular biological properties will be summarized]. And 5] highlighted additional novel targets (eg, transporters, metabolic regulators) that, together with the above, further emphasize a previously underestimated sophistication of EPOR/JAK2 signal transduction circuits, certain of which are proposed to be central to EPO and ESA effects on anemia (and potentially, hypertensive side-effects).

Disclosures
No relevant conflicts of interest to declare.
Fas Controls Neutrophil Lifespan during Bacterial and Viral Infection
Authors: O'Donnell Joanne A. , Kennedy Catherine L , Pellegrini Marc , Nowell Cameron , Cengia Louise H. , Masters Seth L. , Hartland Elizabeth L. , Gerlic Motti , Roberts Andrew W. , Croker Ben A 
Published: October 2019

Abstract
Introduction
The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8+ T cells, which kill antigen-stimulated T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection, can also induce neutrophil death in tissues during infection. Infection of Fas- and FasL-deficient mice with the enteropathogenic-like mouse pathogen Citrobacter rodentium (C.rodentium) is associated with neutrophil infiltration and severe diarrhoea. We hypothesized that a deficiency of Fas in neutrophils prolongs neutrophil lifespan and contributes to the accumulation of neutrophils in C.rodentium-infected mice.
Methods
Fas signaling can drive IL-1β and IL-18 production and thereby affect neutrophil accumulation in tissues, so mixed bone marrow chimeras were generated to compare the accumulation of WT and Fas-deficient neutrophils in vivo. For mixed bone-marrow chimeras, irradiated Ly5.1 mice were reconstituted with 2.5 x 106 bone marrow cells from ubiquitin-GFP or Ly5.1/5.2 mice combined with LysM-Cre Fasfl/fl at a 1:1 ratio. For infection studies, mice were infected with LCMV docile or C.rodentium. For in vitro assays, neutrophils were primed for 1 h with GM-CSF prior to treatment with Toll-like receptor (TLR) ligands or IL-18 and FcFasL. Neutrophil viability was measured by live cell imaging and automated image analysis.
Results
Using LysM-Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan in the colon during C.rodentium infection, and during LCMV infection in the lung, peripheral blood and spleen. To examine if pathogen-derived molecules can modulate Fas signaling in neutrophils, we primed neutrophils with TLR ligands, which ablated Caspase-8 processing and Fas signaling. Treatment of neutrophils with the TLR ligands Pam3CSK4, Pam2CSK4 or LPS or the MyD88-dependent cytokine IL-18 strongly protected neutrophils against Fas-induced death.
Conclusion
These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provides a mechanism to account for neutrophil resistance to Fas stimulation during infection.

Disclosures
No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia Stem Cells (LSCs) and Leukemia Progenitor Cells (LPCs) Display Overlapping and Unique Mechanisms of Genomic Instability: The Role of PI3k-AKT and PI3k-Rac2-PAK Pathways
Authors: Nieborowska-Skorska Margaret , Flis Sylwia , Skorski Tomasz 
Published: October 2019

Abstract
BCR-ABL1 fusion tyrosine kinase transforms hematopoietic stem cells (HSCs) and cause chronic myeloid leukemia in chronic phase (CML-CP), which is a stem cell (leukemia stem cell=LSC) -derived but a progenitor (leukemia progenitor cell=LPC)-driven disease. Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, nilotinib and ponatinib revolutionized the treatment of CML.
We showed that reactive oxygen species (ROS) may increase the levels oxidative DNA damage in TKI-naïve and TKI-treated Lin-CD34+CD38- LSCs, including quiescent LSCs, and in Lin-CD34+CD38+ LPCs. Since DNA repair mechanisms display reduced fidelity/activity in CML cells, overproduction of ROS may promote genomic instability (accumulation of point mutations and chromosomal aberrations) resulting in resistance to TKIs and malignant progression of the disease to accelerated (CML-AP) or blast (CML-BP) phase.
BCR-ABL1 kinase stimulates numerous signaling pathways to induce and maintain transformation of hematopoietic cells. We and others found that phosphatidylinositol-3 kinase (PI3k) plays an essential role not only in growth factor independent proliferation and protection from apoptosis of CML cells, but also in overproduction of mitochondrial ROS. Here we investigated the role of PI3k downstream effectors, AKT serine/threonine kinase and Rac GTPase in genomic instability in TKI-naïve and TKI-treated LSCs and LPCs.
Using CML-CP primary cells and an inducible model of murine CML-CP –like disease we determined that Rac2 – PAK serine/threonine pathway alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III thereby generating high levels of ROS in TKI-naïve and TKI-treated LSCs and LPCs. In addition, AKT appeared to play an essential role in generation of ROS-induced oxidative DNA damage in TKI-naïve and TKI-treated LPCs, but not in LSCs. Inhibition of AKT did not affect the activity of Rac2 and inhibition of Rac2 did not affect AKT, clearly indicating that their activation status does not depend on each other. Targeting Rac2 and AKT either by mutations/deletions/shRNAs or small molecule inhibitors/peptides reduced genomic instability resulting in diminished frequency of TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations.
Altogether TKI-naive and TKI-treated LSCs (including quiescent LSCs) display only PI3k-Rac2-PAK pathway, whereas LPCs contain PI3k-Rac2-PAK and PI3k-AKT pathways responsibble for ROS-induced oxidative DNA damage and genomic instability. Our findings may have more broad application because other oncogenic tyrosine kinases expressed in hetatopoietic malignancies induce similar mechanisms of genomic instability.

Disclosures
No relevant conflicts of interest to declare.
Phase II Study of an AKT Inhibitor MK2206 in Patients with Relapsed or Refractory Lymphoma
Authors: Oki Yasuhiro , Fanale Michelle A. , Romaguera Jorge E. , Fayad Luis , Fowler Nathan , Copeland Amanda R. M. , Samaniego Felipe , Kwak Larry W. , Neelapu Sattva S , Wang Michael , Feng Lei , Younes Anas 
Published: October 2019

Abstract
Aberrant activation of the PI3K/AKT/mTOR pathway has been implicated in promoting lymphoma cell growth and survival. Several agents targeting this pathway at the PI3K or mTOR levels have demonstrated clinical activity in a variety of lymphoma subtypes. However, there has been no clinical data with AKT inhibitors in lymphoma. MK-2206 is the first oral non-ATP competitive allosteric inhibitor of AKT 1, 2, and 3 which entered clinical development. MK-2206 demonstrated AKT inhibition and antiproliferative activity as single agent and in combination with other agents in multiple human cancer cell lines. MK-2006 synergized antitumor effects of chemotherapeutic agents in vivo and in vitro. To investigate the potential therapeutic value of targeting AKT in lymphoma, we conducted a phase II study of MK2206 in patients with relapsed or refractory lymphoma. Eligible patients were required to have lymphoma of any histology excluding Burkitt lymphoma or lymphoblastic lymphoma that have relapsed or been refractory after at least one treatment regimen, have no curative option available and have adequate organ functions. MK-2206 (200 mg) was given orally, once per week, in 28-day cycles for a maximum of 12 cycles. In the absences of limiting toxicity, MK2206 dose could be increased to 300 mg. Serum cytokines levels are measured in consenting patients on days 1, 8 and 22 of the first cycle. A total of 59 patients were enrolled including 25 patients with classical Hodgkin lymphoma. The median number of prior treatment regimen was 4 (range, 1-10). Based on intent-to-treat analysis, objective responses were observed in 8 (14%) patients, with 29 (49%) patients demonstrating reduction in their tumor measurements (Figure). The median response duration was 5.8 months. In the 25 patients with classical Hodgkin lymphoma, the response rate was 20%. Treatment was well tolerated, with skin rash being the most common toxicity (Any grade: 53%, Grade 3: 15%). Grade 3 or 4 hyperglycemia was observed in 5%, and neutropenia in 2%. There was no grade 3 or 4 thrombocytopenia. Collectively, the study showed that the AKT inhibitor MK2206 can induce responses in classical Hodgkin lymphoma and indolent lymphoma. The single agent activity, however, seems to be low against large B-cell lymphoma, T-cell lymphoma or mantle cell lymphoma. The possible mechanism of resistance to the treatment with AKT inhibitors may include suboptimal inhibition of AKT phosphorylation at low concentrations or development of alternative survival pathway. Future studies should aim at optimizing the dose and schedule of MK2206, and exploring mechanism-based combination strategies.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Surgical Control/Cure of 5TGM1 Murine Multiple Myeloma Model By Thyroparathyroidectomy
Authors: Zangari Maurizio , Yoon Donghoon , Yoo Hanna , Kim Young Sik 
Published: October 2019

Abstract
The 5TGM1 transplanted C57BL6/KaLwRij mouse is widely used to study murine multiple myeloma development. It recapitulates many features of human multiple myeloma including monoclonal paraprotein as well as bone lesions. Using this mouse model we demonstrated the crucial role of the parathyroid hormone 1 receptor (PTH1R) on the survival of mice exposed to proteasome inhibitor drugs such as bortezomib and carfilzomib; specifically the blockage of the PTH1R function by parathyroid hormone antagonist (PTH 7-34) resulted in significant abrogation of the beneficial survival effect of proteasome inhibitor drugs. To further investigate the effect of the parathyroid hormone (PTH) axis on murine myeloma development we performed thyroparathyroidectomy on the C57BL6/KaLwRij mouse before or after 5TGM1 transplantation.
Thyroparathyroidectomy was performed by trained personnel in general anesthesia with isoflurane; all surgical/postsurgical procedures and animal welfare followed an IACUC approved protocol at the University of Arkansas. To prevent hypocalcaemia at postsurgery, 1M CaCl2 solution in the drinking bottle was supplemented for a week. All tested mice received 0.5X106 5TGM1 cell infusion by intravenous injection; mice were divided into three groups; Group 1 control mice (n=10) received 5TGM1 infusion at day 0; Group 2 included 15 mice which on day 10 post 5TGM1 cell infusion underwent thyroparathyroidectomy; Group 3 included 15 mice which at 20 days post-surgery received infusion of 5TGM1 cells. 30% of animals experienced post-surgical complications and died in the first 7 days from procedure; those animals were not included in this survival analysis which is based on 10 animals for each cohort. Blood samples were collected at weekly intervals for IgG2 measurements. The survival curves of mice recovered after surgery are shown in the figure.
Figure 1 Figure 1.
All control mice (group 1) developed myeloma progression and died within 3 weeks post-transplant; 2 mice from group 2 and 5 mice of the third cohort manifested disease. All 7 animal deaths were pathologically and serologically attributed to myeloma progression. Compared to controls thyroparathyroidectomized mice (Group 2 and Group 3) showed significantly longer survival p<0.001 and p=0.003, respectively. The 13 surviving mice are still alive 6 months post-procedure and have not shown any serological or clinical evidence of disease progression. Two mice from previous experiment set are still alive and healthy after more than 2 years follow-up. In conclusion thyroparathyroidectomy achieves durable control and possibly cure of the 5TGM1 murine myeloma model suggesting an endocrinological control of myeloma progression. Currently the mechanism of action is under investigation.

Disclosures
Zangari: Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding.
Dynamin 2-Dependent Endocytosis Regulates Megakaryopoiesis
Authors: Bender Markus , Giannini Silvia , Jönsson Terese , Grozovsky Renata , Christensen Hilary , Pluthero Fred G , Kahr Walter H , Hoffmeister Karin M , Falet Hervé 
Published: October 2019

Abstract

Dynamins are large and highly conserved GTPases involved in endocytosis and vesicle trafficking. Mutations K562E/del in the ubiquitous dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in megakaryopoiesis we generated Dnm2fl/fl Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/fl Pf4-Cre mice were viable, but had severe macrothrombocytopenia with moderately accelerated platelet clearance and prolonged bleeding due to poorly functional platelets. Dnm2-null bone marrow MKs had altered demarcation membrane system, appearing at times as a compact, narrow twisting membrane system of clathrin-coated vesicles. Fetal liver cell derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2 plays a major role in MK membrane formation and thrombopoiesis. Both endogenous DNM2 and overexpressed DNM2 WT, but not DNM2 K562E/del mutants localized with the early endosome in bone marrow MKs. The endocytic pathway was disrupted in Dnm2-null MKs, as evidenced by severely reduced early endosome EEA1 and APPL1 staining and weak LysoTracker internalization. Endocytosis of the thrombopoietin (TPO) receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating TPO levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by massive MK hyperplasia and myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. However, additional Mpl genetic deletion failed to rescue the severe splenomegaly of Dnm2fl/fl Pf4-Cre mice, and Mpl-/- Dnm2fl/fl Pf4-Cre mice instead died at 4-5 weeks of age. Taken together, our data demonstrates that unrestrained MK growth and proliferation results in rapid myelofibrosis independently of Mpl expression and other bone marrow cell types, and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis and bone marrow homeostasis.

Disclosures
No relevant conflicts of interest to declare.
Bortezomib-Resistance Is Characterized By Upregulation of E3 Ligase Pirh2 Which Protects Bortezomib Induced Apoptosis in Multiple Myeloma Cells
Authors: Yang Li , He Jingsong , Li Yi , Guo Xing , Chen Jing , Chen Qingxiao , Lin Xuanru , He Donghua , Yang Yang , Zhang Enfan , Han Xiaoyan , Wu Wenjun , Zhao Yi , Zheng Gaofeng , Huang He , Cai Zhen 
Published: October 2019

Abstract
Background Proteasome inhibitor bortezomib is an effective approach to the treatment of multiple myeloma (MM), but drug resistance often emerges and limits its retreatment. However, the action mechanisms are not fully understood. Several lines of evidence link E3 ligase to myeloma and its drug resistance. Pirh2 (p53-induced RING-H2) E3 ubiquitin ligase with intrinsic ubiquitin-protein ligase activity for polyubiquitination and subsequently proteasomal degradation was predisposed for plasma cell hyperplasia and tumorigenesis as reported. In this study, we examined Pirh2 expression in plasma cells of the bone marrow (CD138+ cells) from MM patients and investigate the effect of Pirh2 on the viability, proliferation, survival and drug resistance of MM cells.
Methods Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. To identify some of the mechanisms involved, we developed myeloma cell lines OPM-2 and RPMI8226 bortezomib resistant (OPM-2-BR, RPMI8226-BR) derivative lines by continuous culture in sub-lethal concentrations of bortezomib. Clones of OPM-2-BR and RPMI8226-BR were obtained after several months, and acquired resistance to bortezomib remained stable over months with extended stimulus duration in the presence of bortezomib. Pirh2 mRNA and protein expressions were detected by real time quantitative PCR(Q-RT PCR) and Western blotting. We also generated stable Pirh2 knock down cell lines and stable Pirh2 overexpression cell lines.
Results We found differential expression of Pirh2 in bortezomib-resistant (BR) cells, compared to wild type (WT) controls by Q-RT PCR and Western blotting. We confirmed generally Pirh2 mRNA and protein expressions in MM cell lines. Besides, Pirh2 mRNA expression was also determined from bone marrow samples of MM patients. In summary, we showed that Pirh2 is more highly expressed in MM cell lines and patient MM cells (from newly diagnosed or refractory/recurrence patients with MM) than in normal bone marrow mononuclear cells from normal donors (P<.0001). Interestingly, We found that Pirh2 overexpression cell lines showed negatively effect on the response to bortezomib treatment. Consistent with this, Pirh2 knock down cell lines can overcome bortezomib resistance.
Conclusions Collectively, Our findings demonstrate a novel role for Pirh2, which suggests that Pirh2 may act by an unknown mechanism involved in bortezomib resistance of MM. An improved understanding of it can lead to better therapeutics for bortezomib retreatment which will be a rational strategy for clinical translation.

Disclosures
No relevant conflicts of interest to declare.