bluuebunny/crossref_metadata_2025 huggingface dataset:
Depletion of CD45RA+ Cells from Mobilized Peripheral Blood Stem Cell (PBSC) Collections As an Integral Part of Hematopoietic Stem Cell Transplantation (HSCT) and Cellular Therapy (CT)
Authors: Law Ping , Eldridge Paul W. , Houston James , Triplett Brandon M. , Shook David R , Leung Wing
Published: October 2019

Clinical scale depletion of CD45RA+ naïve T cells from leukapheresis collections can be achieved using CliniMACS (Bone Marrow Transplant. 2013, 1-7). In this study, PBSC collections depleted of CD45RA+ cells were infused as part of CT treatment protocols for pediatric patients with hematologic malignancies and solid tumors in 2 separate clinical trials. Between Sep 2012 and May 2014, a total of 25 CD45RA+ cell depletion procedures was performed for 25 patients. Mobilized PBSC leukapheresis units were collected (using Spectra) from HLA haplo-identical donors and held overnight in cold prior to cell selection. Characterization of the products before and after CD45RA+ cell depletion is shown in Table 1. Depletion of all CD45RA+ cell averaged to 3.39-log while depletion of CD3+CD45RA+ T cells was > 3.76-log, as this subset of cells became undetectable in 8 (out of 25) final products. CD34+ cell recovery was 65.4%, similar to that of CD3+CD45RO+ T cells (65.1%), which constituted > 99% of all T cells after depletion. From the PBSC collection, each patient received a median dose of 0.009 x 106 CD3+CD45RA+ cells/kg and 11.1 x 106 CD34+ cells/kg. The final product contained a substantial amount of CD14+ monocytes (median = 22.0%, range: 7.0 – 46.4), few CD56+ NK cells (3.1%, range: 1.3 – 8.1) and almost no CD19+ B cells (0.04%, range: 0.01 – 0.30). After processing the cells were resuspended in Plasmalyte + 5% human serum albumin and infused as soon as possible. There was no reported contamination (growth of bacteria – aerobic or anaerobic and fungus) in the final cell products and no infusion related adverse events were observed. In conclusion, we have demonstrated clinical scale depletion of naïve T cells can be achieved consistently and the final cell product can be safely infused into patients as part of HSCT and / or CT program.

Table 1: Cell Populations (Median & ranges) Before and After CD45+ Cell Depletion Before Depletion After Depletion Total Nucleated Cells 3.97 x 1010 (1.55 – 6.60) 1.47 x 1010 (0.43 – 2.92) % CD34+ Cells 0.85% (0.29 – 2.30) 1.45% (0.32 – 4.63) % CD3+ T Cells 22.8% (11.5 – 50.9) 13.1% (3.9 – 37.7) % CD45RA+ Cells 39.6% (27.2 – 64.1) 0.05% (0.01 – 0.79) % CD3+ CD45RA+ Naïve T Cells 12.3% (4.3 – 29.2) 0.01% (0 – 3.42) % CD3+ CD45RO+ Memory T Cells 6.5% (2.0 – 15.6) 13.0% (3.9 – 37.5)

Disclosures

No relevant conflicts of interest to declare.
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

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

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

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

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.
T Cell Mediated Suppression of Hematopoiesis in Myeloproliferative Neoplasm
Authors: Morse Sarah J , Luty Samuel B , Lai Hew Yeng , Druker Brian J. , Fleischman Angela G
Published: October 2019

Background:

Increased inflammation is an established feature of MPN, but its precise role in disease pathogenesis has yet to be clearly defined. T cell mediated insult on hematopoietic stem cells can drive expansion of clones that have mutated in such a way as to escape these suppressive and/or apoptotic cues (this mechanism is clearly illustrated in aplastic anemia). To investigate whether T cell mediated suppression of hematopoiesis occurs in MPN we interrogated the T lymphocyte compartment of MPN patients, guided by abnormalities known to be present in aplastic anemia.

Methods and Results:

We first asked how selective depletion of T cells from peripheral blood mononuclear cells (MNC) would affect myeloid colony formation in MPN as compared to normal controls. MNC were depleted of T cells using CD3 magnetic beads or mock depleted and plated side by side in methylcellulose (+SCF, IL-3, Epo). Removal of CD3+ T cells from bulk MNC reduced colony formation in all 29 normal control samples tested, consistent with the ability of normal lymphocytes to enhance colony formation. In contrast, removal of CD3+ T cells from MPN MNC resulted in an increase in colony formation in 20 out of 51 MPN patient samples (containing an equal distribution of polycythemia vera, essential thrombocythemia, and myelofibrosis). From JAK2V617F-positive MPN patients, colonies were plucked and genotyped for JAK2V617F mutational status. The fraction of JAK2WT colonies increased with removal of T cells, demonstrating that JAK2WT progenitors are more sensitive to T cell mediated suppression as compared to their JAK2V617F counterparts. The observation that removal of T cells enhances myeloid colony forming ability of some MPN patients suggests that T mediated suppression of hematopoiesis may be occurring in MPN.

Next, we tested whether MPN T cells have the ability to suppress colony formation of normal hematopoietic progenitors. CD3+ T cells from MPN (n=2) and normal controls (n=2) were added to normal control mobilized peripheral blood CD34+ cells (n=3). Each CD34+ sample was plated in methylcellulose by itself, with the addition of 100,000 MPN T cells/1.1ml or with the addition of 100,000 normal control T cells/1.1ml. For each CD34+ sample a unique MPN patient and normal control was used as the source of T cells. Addition of MPN T cells reduced colony formation in one of three CD34+ samples, whereas normal control T cells enhanced colony formation of all three CD34+ samples. In the remaining two CD34+ samples MPN T cells had an attenuated ability to enhance colony formation as compared to the normal control T cells.

Conclusion:

T-cell mediated suppression of myelopoiesis in vitro is demonstrable in MPN and may be a contributor to the anemia associated with myelofibrosis. Taken together, these data provide the rationale for further investigation into T mediated suppression of hematopoietic progenitors in MPN patients.

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

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

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

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

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.
Invasive Fungal Infections in Pediatric Acute Lymphoblastic Leukemia: Incidence, Characterization, Outcome and Risk Analysis of Study ALL-BFM 2000
Authors: Mann Georg , Attarbaschi Andishe , Schrappe Martin , Möricke Anja , Bleckmann Kirsten , Alten Julia , Lehrnbecher Thomas , Zimmermann Martin , Niggli Felix
Published: October 2019

With achievable survival rates of above 90%, non-leukemic events gather a high proportion of treatment failure causes with infections forming the majority. We analysed the incidence and outcome of reported invasive fungal infections as serious adverse events (SAE according to NIH criteria) in study ALL-BFM 2000 with the aim to characterize risk factors to optimize antifungal prophylaxis and treatment. A total of 4867 patients with ALL were enrolled from August 1999 to May 2010. Analysis of (mandatory) SAE reporting revealed 144 cases (3%). Fungal infections related to hematopoietic stem cell transplantation were excluded. According to the EORTC/MSG classification 65 (1.3%) were proven, 53 (1.1%) probable/possible and 26 (0.5%) not classifiable. There were 19 (0.4%) deaths and 41 (0.8%) events were classified as life threatening according to FDA definition. Positive mycological results were available in 98 cases: Aspergillus ssp. in 72, Candida ssp. in 21, Mucor in 3, Rhizopus and Fusarium in 1 case each. Fatality occurred in 16 cases with Aspergillus (22%) and in 1 each of the other species. Out of 41 life-threatening infections 30 were due to Aspergillus, 5 to Candida and 4 to the other species. Regarding all fungal infections, there was a significantly rising risk with increasing age (7.2% >15 y, 4.8% 10-<15 and 2.1% >1-< 10y; p<0.001), significance of age-dependent differences remained conserved for the life-threatening and fatal infections. Additionally, female gender appeared to impose a significant higher risk (all fungal infections, 2.4 vs. 3.7%, p=0.005). With respect to the treatment phase, the risk of fungal events was highest during induction, especially in the case of delayed treatment response, and during intensive block chemotherapy in the high-risk arm. This study on a very large population of children with ALL demonstrates that invasive fungal infections represent an important cause of morbidity and mortality in this cohort, giving support to reflect prophylactic measures for patients at high risk in the future.

Disclosures

No relevant conflicts of interest to declare.
Breast Milk from Immune Thrombocytopenic Mothers Can Contain Anti Platelet Antibodies That Are Associated with Persistent Thrombocytopenia in Neonates
Authors: Sharon Nehama , Rosenberg Nurit , Seligsohn Uri , Simmonds Aryeh , Shiff Yakov , Schechter Yaakov , Hausner Hagit
Published: October 2019

Purpose: Maternal immune thrombocytopenic purpura (ITP) accounts for 5% of all cases of pregnancy associated thrombocytopenia and is a common cause of neonatal thrombo­cytopenia. One of the common mechanism involves transfer of IgG autoantibodies against platelet receptors which are found in the blood samples of affected patients. The neonatal thrombocytopenia usually subsides within 2 months. The autoantibodies are often of the IgG type and therefore can cross the placenta and cause fetal and/or neonatal thrombocytopenia. Recently we observed persistence of neonatal ITP which disappeared following discontinuation of breast feeding. The aim of our current work was to discern whether breast milk of mothers with ITP contains anti-platelet antibodies and whether these antibodies may be the cause for persistent neonatal ITP.

Methods: Breast milk samples were collected from 12 women with ITP. Six of them were thrombocytopenic during pregnancy and their neonates also had thrombocytopenia. The remaining 6 mothers had a history of ITP but not during the current pregnancy, and neither did their neonates. As controls, breast milk from 8 healthy women was also examinied.

The presence of anti-platelet antibodies were evaluated by incubating washed platelets from healthy donors with breast milk or extracted milk – Ig. The type of immune globulin was defined by flow cytometry using fluorescence conjugated anti-human IgA, IgG or total Ig antibodies. To prove that the antibodies were against an antigen that is platelet specific and not against HLA epitopes, we also tested the samples on cultured cells expressing the fibrinogen receptor, αIIbβ3, and compared the results to mock cells (transfected with empty vectors).

Results: In the cases of women with active ITP, 3 were positive for anti-platelet antibodies, 2 were inconclusive and one was negative. In the mothers with a history of ITP, 1 was positive, 1 was inconclusive and 4 were negative. No anti-platelet antibodies were found in breast milk of healthy women. In all 6 cases of active ITP, the cell cultured assay for the fibrinogen receptor αIIbβ3 was positive, indicating that there were anti platelet specific antibodies in their milk. Of the 6 neonates of mothers with active ITP, 4 of them required treatment with oral steroids, and the average time to resolution of thrombocytopenia was 3.5 months.

Conclusion: This study demonstrates that breastmilk of women with active ITP contains anti platelet specific antibodies. The presence of these antibodies demonstrated an association with persistant and prolonged neonatal thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
Tyrosine Kinase-Directed Ubiquitin Ligases Cbl and Cbl-b Enforce Hematopoietic Stem Cell Quiescence By Negatively Regulating c-Kit and FLT3
Authors: An Wei , Nadeau Scott , Mohapatra Bhopal , Feng Dan , Zutshi Neha , Storck Matthew , Band Vimla , Band Hamid
Published: October 2019

Maintenance of quiescent hematopoietic stem cells (HSCs) is essential for life-long hematopoiesis. A prominent category of HSC quiescence regulators that determine HSC fate by directly interacting with niche-derived growth factors are receptor tyrosine kinases (RTKs). Cbl and Cbl-b are E3 ubiquitin ligases that are directed to activated tyrosine kinases and negatively regulate a number of cellular activation pathways. Previously, we established a conditional Cbl and Cbl-b double knockout (DKO) mouse strain using MMTV-Cre to delete floxed Cbl on a Cbl-b-null background, and identified that Cbl and Cbl-b function redundantly in controlling the growth factor-induced proliferation of HSCs. These DKO mice developed a rapidly fatal myeloproliferative disorder (MPD) accompanied by expansion of the HSC compartment. However, how the negative regulatory functions of Cbl-family proteins are integrated into HSC homeostatic program and the mechanistic basis for their role remain unknown.

Here, we utilize mouse models to examine the functional role of Cbl and Cbl-b in regulating HSCs and its potential mechanistic basis. Transplant analyses revealed that DKO HSCs are the disease-initiating cells. However, in vitro serial colony-forming assays showed that DKO HSCs possess a reduced colony-forming ability despite their hyper-proliferative status. Cell cycle analyses demonstrated a smaller pool of quiescent long-term HSCs in DKO mice. Non-responder assays in vivo showed a reduced frequency of functional HSCs in the DKO LSK compartment and serial HSC transplantation demonstrated compromised reconstitution ability of DKO HSCs, especially at the 2nd round of transplantation. Mechanistically, DKO HSCs exhibit sustained signaling in response to c-Kit and FLT3 ligands, especially via p-Akt but also p-Erk and p-S6. The ligand-induced cell surface c-Kit and FLT3 receptor downregulation was slower in DKO HSCs. Furthermore, while c-Kit and FLT3 ligands promote higher proliferation of DKO bone marrow cells, this hyper-proliferation leads to loss of colony-forming potential that is significantly reversed by respective kinase inhibitors.

Together, our data reveal a novel and physiologically essential role of Cbl and Cbl-b in the enforcement of HSC quiescence and protection against exhaustion by fine-tuning the signaling pathways downstream of tyrosine kinase-coupled receptors such as c-Kit and FLT3. These findings could have significant implications for hematological neoplasms associated with mutations of Cbl.

Disclosures

No relevant conflicts of interest to declare.
The P2Y6-AMPK Pathway Triggers Autophagy during CSF-1-Induced Human Monocyte Differentiation and Is a Potential Target in CMML
Authors: Obba Sandrine , Hizir Zoheir , Selimoglu-Buet Dorothée , Boyer Laurent , Pfeifer Anja , Hamouda Mohamed-Amine , Goncalves Diogo , Cerezo Michael , Marchetti Sandrine , Rocchi Stephane , Droin Nathalie , Robert Guillaume , Luciano Frederic , Robaye Bernard , Viollet Benoit , Legros Laurence , Solary Eric , Auberger Patrick , Jacquel Arnaud
Published: October 2019

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacological and genetic evidence indicate that autophagy plays pleiotropic functions in cellular homeostasis, development, survival and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by M-CSF. We have previously shown, using pharmacological inhibitors, siRNA approaches and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper CSF-1-driven differentiation of human and murine monocytes. We have also unraveled a role for autophagy in macrophage acquisition of phagocytic functions. Here, we determine that the CaMKKb-AMPKa1-ULK1 pathway is required for CSF-1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2Y6 to the induction of autophagy, and we decipher the signalling network that links the CSF-1 receptor to P2Y6-mediated autophagy and monocyte differentiation. Importantly, we show that the physiological P2Y6 ligand UDP and the specific P2Y6 agonist MRS2693 restore normal monocyte differentiation through re-induction of autophagy in primary myeloid cells from chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for P2Y6-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.

Disclosures

No relevant conflicts of interest to declare.
Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop Bone Marrow Failure
Authors: Kamio Takuya , Gu Bai-wei , Olson Timothy S. , Zhang Yanping , Mason Philip J , Bessler Monica
Published: October 2019

MDM2, an E3 ubiquitin ligase, is an important negative regulator of p53. In turn the MDM2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited by the binding of some ribosomal proteins, including RPL5 and RPL11, and that the resulting increase in p53 levels may be important in the red cell aplasia seen in DBA and in 5q- MDS. DBA and 5q- MDS are associated with inherited (DBA) and acquired (5q-MDS) haploinsufficiency of ribosomal proteins. A mutation in MDM2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation, retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis.

While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we inadvertently noticed that Mdm2 C305F/ C305F mice had perturbed hematopoiesis. These mice had a mild macrocytic anemia with reticulocytosis and high erythropoietin levels. In the bone marrow, these mice showed a significant decrease compared to wildtype (WT) littermates in Ter119+ cells, while no decrease in the number of immature erythroid cells (Ter119+CD71+) was found in the spleen, which showed significantly increased hematopoiesis. In methyl cellulose cultures BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls(p < 0.01). Further investigation revealed that there was a decrease in Lin-Sca-1+c-Kit+(LSK)cells, accompanied by significant decreases in short-term hematopoietic stem cells(ST-HSC) (p < 0.05) and multipotent progenitor(MPP) cells(p < 0.05).

These results suggest that control of the cell cycle through interactions between ribosomal proteins and MDM2 is important in the regulation of normal hematopoiesis as well as in the pathogenesis of 5q- MDS and DBA.

Disclosures

No relevant conflicts of interest to declare.
Low Toxicity Profile of the Combination of Bendamustine Plus Rituximab (BR) in Elderly FRAIL Patients with Newly Diagnosed DLBCL
Authors: Spina Michele , Luminari Stefano , Salvi Flavia , Passamonti Francesco , Fabbri Alberto , Vitolo Umberto , Cabras Maria Giuseppina , Miglino Maurizio , Merli Francesco , Fattori Pier Paolo , Liberati Anna Marina , Zaccaria Alfonso , Tosi Patrizia , Ciancia Rosanna , Pesce Emanuela Anna , Storti Sergio
Published: October 2019

Introduction: R-CHOP is the gold standard for the treatment of elderly patients with DLBCL. However, unfit and frail patients frequently do not qualify for CHOP-based chemotherapy. Alternatives are an urgent medical need. Bendamustine plus rituximab (BR) has been established as a standard treatment of indolent lymphomas and preliminary data have shown a promising activity in DLBCL, both in the relapsing and upfront setting.

Methods: Within the Fondazione Italiana Linfomi (FIL), we started a phase II study (R-BENDA frail study, EUDRACT2011-001421-24) in elderly patients (>70 years) with a newly diagnosed DLBCL not suitable for R-CHOP-based chemotherapy. All patients were evaluated according to ADL, IADL and CIRS-G and were considered FRAIL if the following criteria were meet: in patients aged 70-80 ADL<4 or IADL<5 or one grade 3 comorbidity or >8 grade 2 comorbidities; in patients older than 80 years ADL>5 or IADL>6 or 5-8 grade 2 comorbidities. Patients received bendamustine at a dose of 90 mg/m2 daily on days 1 and 2 of each 28-day cycle along with rituximab on day 1 for up to 6 cycles.

Results: From February 2012 to February 2014, 49 patients were enrolled in 24 Italian centers. The majority (57%) were male and 57% had stage III-IV with 41% elevated LDH. The median age was 82. Overall, 83% of the planned cycles were delivered without dose reduction or delay; grade 3/4 neutropenia was reported in 25% of cycles followed by anemia 21%, and thrombocytopenia 20%. One case of febrile neutropenia was observed. Grade 3-4 non-hematological toxicity was mild and reported in 6% of cycles including 3 episodes of cardiovascular events and 7 other cases of different toxicities (one creatinine increase, one fatigue, one bleeding, one peripheral neurotoxicity, one hyponatriemia, one hyperglycemia and one liver toxicity). Two deaths during treatment have been observed (cardiac failure and sudden death). At the interim analysis (23 patients) the overall response rate was 56% with a complete response rate of 39%.

Conclusions: Combination therapy with BR demonstrates low toxicity profile in this high risk population. The promising results on activity can encourage clinicians to considered BR for the treatment of FRAIL elderly patients with DLBCL not eligible for R-CHOP.

Disclosures

No relevant conflicts of interest to declare.
Initial Results of an Ex Vivo Microfluidic Cis-Coculture Csra to Predict Response for Myeloma Patients
Authors: Pak Chorom , Callander Natalie S , Young Edmond WK , Titz Benjamin , Kim KyungMann , Chng Kenny , Miyamoto Shigeki , Beebe David J
Published: October 2019

In vitro chemosensitivity and resistance assays (CSRAs) test primary tumor cells’ response to chemotherapeutic drugs. The goal of CSRAs is to potentially direct therapy based upon in vitro response. This is in contrast to empiric therapy of cancer patients where therapy is based on clinical trial evidence and the likelihood of patient response. However, most previous CSRAs have been unsuccessful; this may be because they employ tumor cells in mono-culture. increasing evidence indicates that heterogeneity in tumor cell biology and microenvironment and the interaction between the two play essential roles in regulating drug response. In addition, many of these assay and culture systems require more tumor cells per condition than can be obtained from every patient. Furthermore, coculture systems in the past often cultured tumor and non-tumor cells from different patients (trans-coculture). Therefore, a CSRA capable of being performed for all patients while incorporating both tumor cells and non-tumor microenvironmental cells from the same patient is needed.

Here, by using a microfluidic ex vivo system requiring only thousands of cells per condition, we analyzed patient CD138+ multiple myeloma (MM) cells in either microfluidic mono-culture (MicroMC) or cis-coculture (MicroC3) with their own CD138- non-tumor mononuclear cells. The MM CD138+ cells were then exposed to varying doses of bortezomib, a clinical proteasome inhibitor, for 24 hours. An in-house software program, J’experiment, was used to calculate live fractions of the tumor cells. Because plasma concentrations of bortezomib peak at 100 nM, the change in live fraction was calculated from the 0 and 100 nM dose of bortezomib. Using k-means and Gaussian mixture clustering techniques, MicroC3 responses could be grouped into two distinct data clusters which correctly identified 17/17 patients as either clinically responsive (7) or non-responsive (10). On the other hand, responses measured in MicroMC could not be unambiguously separated into clinical response groups. Thus, MicroC3 accurately identified MM patient responses as either responsive or non-responsive to bortezomib-containing therapy. Further development of MicroC3 as a CSRA with the ability to rapidly identify chemosensitive or resistant cancer patients will enable more personalized therapeutic decisions.

Disclosures

Beebe: Bellbrook Labs, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.