bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts
Authors: Tashiro Haruko , Shirasaki Ryosuke , Oka Yoko , Yamamoto Tadashi , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Abstract 4815

Background and Aims:

We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF).

Materials and Methods:

cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed.

Results and Discussion:

Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Commensal Bacteria Are Dispensable in Neutropenia-Induced G-CSF Regulation
Authors: Bugl Stefanie , Wirths Stefan , Märklin Melanie , Müller Martin R , Wiesner Tina , Frick Julia-Stefanie , Malenke Elke , Kanz Lothar , Kopp Hans-Georg
Published: October 2019

Abstract 4830

Introduction:

G-CSF is generally accepted to be the major cytokine regulating neutrophil production, but neutrophil steady-state homeostasis has still not been fully elucidated. We have previously shown in NODSCIDcγ−/− mice that feedback G-CSF expression upon antibody-based neutrophil depletion occurs independent from lymphocytes (Bugl et al., ASH 2010) and hypothesized that there may be a sensing mechanism of neutropenia relying on the presence of microbial components, similar to emergency granulopoiesis. Therefore, germ-free wildtype mice received anti-neutrophil antibodies and underwent further analysis. Moreover, passive regulation of circulating G-CSF levels by neutrophil cell mass was examined.

Methods:

Anti-Ly6-G antibody (clone 1A8) was used to induce neutropenia in germ-free C57BL/6 mice. After one week of neutrophil depletion hematopoietic tissues and peripheral blood were harvested and analyzed on cellular, protein and RNA level. Moreover, neutrophil granulocytes (granulocyte-differentiation antigen-1+ Mac-1+) purified from peripheral blood of C57BL/6 mice were transfused into neutropenic wild type mice and plasma G-CSF was monitored.

Results:

Peripheral blood neutropenia could be effectively induced in all experimental mice by anti-1A8 antibody. Transfusion of 3.5 × 106 neutrophils into neutropenic wild type mice did not significantly change plasma G-CSF levels. Filgrastim (rhG-CSF), however, caused significant downregulation of bone marrow G-CSF at the mRNA level. Germ-free C57BL/6 mice were analyzed after 1 week of antibody-induced neutropenia and showed a shift of myeloid progenitors towards granulocyte macrophage precursors (GMP) at the expense of megakaryocyte erythrocyte progenitors (MEP) as well as significantly increased numbers of hematopoietic stem cells. In addition, G-CSF, M-CSF, and CXCL12 behaved identically in both germ-free control and wild type mice under specific-pathogen free conditions.

Conclusions:

Transfusion of G-CSF-receptor (CSF3R) positive neutrophils did not significantly influence G-CSF. Moreover, exogenous G-CSF downregulated marrow G-CSF on the transcriptional level. Germ-free mice are able to mount a feedback loop including G-CSF upregulation and marrow changes in progenitor cell distribution analogous to mice carrying physiological commensal bacteria. Our data are therefore consistent with G-CSF feedback regulation occurring independent from commensal germs. Moreover, our data indicate transcriptional rather than CSF3R+ cell mass-associated passive regulation of G-CSF levels. Taken together, we propose a model of myeloid bone marrow homeostasis, where feedback loops of neutrophil production upon antibody-dependent neutropenia occur through transcriptional upregulation of G-CSF. The underlying mechanisms occur independent of lymphocytes and presence of germs.

Disclosures:

No relevant conflicts of interest to declare.
To Use CD64 Expression Level on Neutrophils As An Infection Marker in Children 10 Years Old or younger Cut-off Values Higher Than That in Adults Must Be Established
Authors: Matsushita Shoko , Inagaki Yoshiko , Nakatsuji Yukie , Endo Mikiya , Ito Shigeki
Published: October 2019

Abstract 4930

Objectives:

To develop the use of CD64 expression as an early diagnostic of adult collagen disease, we elucidated normal levels in neonates, infants and children.

Methods:

Blood samples were collected from a total of 62 symptom- and infection-free subjects recruited at our outpatient department: neonates (n=80), infants (n=41), 1- to 5-year-old children (n=77), 6- to 10-year-old children (n=58), children 11 years old or older and adults (n=95). CD64 expression per neutrophil was quantitatively determined by flow cytometry using a BD FACS Calibur HG flow cytometer (Becton Dicknson, BD) and BD CellQuest Pro analysis software (BD). Fluorescence was measured and converted to number of CD64 molecules per cell using a phycoerythrin (PE) fluorescence kit (Quantibrite PE; BD Biosciences) according to the manufacturer's instructions. This study was approved by the ethics committee of Iwate Medical University.

Results:

CD64 expression level in neonates (median, 1981 molecules per neutrophil; range, 908–9844), infants (1666.5, 572–12941), and 1- to 5-year-old children (1778, 545–11283) was significantly higher than that in children 11 years old or older and adults (1280, 533–11205) (p<0.01 or <0.05). There were no differences in expression between 6- and 10-year-old children (1529, 439–9214) and children 11 years old or older and adults.

Conclusion:

Our results suggest that the normal value for adults can not be applied for neonates, infants, and 1- to 5-year-old children. To use CD64 expression level on neutrophils as an infection marker in neonates, infants and 1- to 5-year-old children cut-off values higher than that in adults must be established.

Disclosures:

No relevant conflicts of interest to declare.
Immunologic Effects of An Oral BRAF Inhibitor in a BRAF Wild-Type Murine Model
Authors: Vosganian Gregory S. , Bos Rinke , Sherman Linda
Published: October 2019

Abstract 4935

Introduction:

Metastatic melanoma represents a clinical challenge as aggressive therapy often results in unacceptable toxicity and less intensive therapy results in suboptimal clinical outcomes. PLX-4032 is an orally available small molecule which targets constitutively activated BRAFV600E in melanoma cells. BRAF is an important regulator of cell growth, proliferation and migration. We examined the effect of PLX-4032 on the immune system in two BRAF wild type murine systems: the first to determine the effect of PLX-4032 on cytokine production and the second to determine any functional immune effect of PLX-4032 in an insulinoma model.

Methods:

Effect of PLX-4032 on cytokine production: B10.D2 BRAF WT mice were injected with CD8 lymphocytes expressing a TCR specific for HA518-526 peptide (clone 1) and CD4 lymphocytes expressing a TCR specific for HA110-119 peptide (SFE) on Day 0 and concurrently simulated with appropriate peptide, incomplete Freund's adjuvant, and poly IC. Mice received PLX-4032 200mg/kg or placebo daily for 5 days. On day 6, splenic lymphocytes were harvested and flow cytometry performed to determine CD4 and CD8 IL2, IFN gamma, and TNF alpha production. Effect of PLX-4032 on functional immune response: B10.D2 BRAF WT rat insulin promoter (RIP)-Tag2-hemagglutinin mice bearing insulinoma were injected with clone 1 and SFE lymphocytes on Day 0 and received concurrent stimulation as above. Mice received PLX-4032 200mg/kg or placebo daily for 30 days and serum glucose levels were monitored weekly.

Results:

Effect of PLX-4032 on cytokine production: We noted no significant difference in the absolute number of splenic lymphocytes recovered in the two groups. There was no significant difference in the percent of CD8+ clone 1 cells producing TNF alpha, IL2 or IFN gamma. There was similarly no significant difference in the percent of CD4+/SFE lymphocytes recovered between the treatment and control groups, nor was there a significant difference in the percent of cells producing IFN gamma. Effect of PLX-4032 on functional immune response: Treatment with PLX-4032 resulted in no significant inhibition of the immune response of transferred lymphocytes against insulinoma. Mice in both groups developed significantly elevated serum glucose levels at the same time and remained diabetic for a similar duration.

Discussion:

Our data demonstrate that PLX-4032 does not appear to exert a quantitative or functional effect on the immune system in BRAF WT mice. Melanoma therapies often include medications which augment patient immune function or specifically target immune regulation; these data suggest that PLX-4032 does not inhibit normal immune function and therefore may have a role in combination with immunologically directed therapy, possibly resulting in synergistic anti-tumor effect by targeting multiple pathways involved in tumor survival.

Disclosures:

No relevant conflicts of interest to declare.
Lymphoproliferative Disorders (LPD) in Individuals ≥80 Years of Age–Incidence, Response, and Survival
Authors: France Fallon O , Chikkappa G. , Pasquale Donald
Published: October 2019

Abstract 4945

Introduction

Clinical trials describing LPD therapy and outcomes overwhelmingly exclude individuals ≥80 years of age. Thus, ability to deliver LPD therapy to this age group is not defined, and tolerance to therapy and response rates and survival data are not available.

Methods

We retrospectively identified all individuals diagnosed with any LPD at ≥80 years of age at our institution between 1997 and 2010. Data included age at diagnosis, diagnosis, therapy for LPD, comorbidities (disease count), survival from date of diagnosis of LPD, chemotherapy dose intensity (percent of 100% dose based on standard regimen dosing), hospitalization for any reason during chemotherapy, and response. Survival was analyzed by Kaplan-Meier analysis and data sets were compared using Student's t-test or Chi-square as appropriate.

Results

Of a total of 518 individuals diagnosed with any LPD, we identified 33(6.4%) who were diagnosed ≥80 years of age. All were male consistent with our veteran population. Data is illustrated in tables 1 and 2. Fifteen (15) had Large B-cell lymphoma, 4 CLL/SLL, 2 follicular, 2 mantle zone, 1 marginal zone, 2 lymphoplasmacytic, 3 T-cell. Two (2) could not be classified despite AFIP review. Twenty-two (22) individuals were treated with 73 cycles of chemotherapy (25 CHOP, 30 R-CHOP, 3 CVP, 11 R-CVP, 4 PO cyclophosphamide). One (1) individual with LBCL had no evidence of disease following excisional biopsy and declined further therapy, and 1 received radiotherapy. A total of 27 (82%) patients died during observation. Deaths were predominantly due to LPD. Of those not treated, 5 of 9 had low-grade LPD and were among longer-term survivors. Median survival, illustrated in the figure, was 18.8 months and was not different between treated and untreated group. This compares with 72.7 months predicted survival for an average 84-year old (WWW.SSA.GOV).

Discussion

LPD is common in individuals ≥80 years of age with large B-cell lymphoma being most common in our population. Despite multiple co-morbidities, the data show that reasonable dose intensity combination chemotherapy may be delivered to treat octogenarians, however hospitalization was required for 1/3 patients during chemotherapy. While survival was not different between treated and untreated groups, the individuals who were treated likely had more severe or advanced stage LPD. Prospective studies with sufficient number of individuals within each diagnostic category should be done to clarify benefits of chemotherapy in ≥80 year old patients with LPD.

Disclosures:

No relevant conflicts of interest to declare.
Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.
Authors: Huang Jian , Klein Peter S.
Published: October 2019

Abstract 2309

Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Autologous HSCT for Ph-Positive Adult Acute Lymphoblastic Leukemia: A Curative Option in the Era of Tyrosine Kinase Inhibitors? an Analysis From the Acute Leukemia Working Party of the EBMT
Authors: Giebel Sebastian , Labopin Myriam , Gorin Norbert Claude , Caillot Denis , Leguay Thibaut , Schaap Nicolaas , Michallet Mauricette , Socie Gerard , Mohty Mohamad
Published: October 2019

Abstract 233

Outcome of patients with Ph-positive acute lymphoblastic leukemia (Ph+ ALL) improved markedly with the introduction of tyrosine kinase inhibitors (TKIs) used in combination with chemotherapy. However, despite very high rate of complete remissions the possibility of cure with conventional-dose treatment remains questionable. Hence, allogeneic hematopoietic stem cell transplantation (HSCT) is still considered a standard of care. Patients lacking appropriate donor are usually treated with TKI-based maintenance. Autologous HSCT could be an alternative approach, however, due to “negative” results of a series of prospective studies run in XX. century its use has been limited. The goal of the current analysis was to retrospectively analyze if results of autoHSCT for Ph+ ALL changed over time.

Results of 171 autologous transplantations performed in the first complete remission between 1996–2010 and reported to the EBMT registry have been analyzed. Median patient age was 48.3 (19–65) years. Conditioning regimen was based on either TBI (63%) or chemotherapy (37%). Peripheral blood was used as a source of stem cells in 84% cases.

With the median follow-up of 2 years, in the whole study group, the probability of the overall survival (OS) at 2 years was 45% (+/−4%) and leukemia-free survival (LFS) was 32% (+/−4%). The cumulative incidence of relapse (RI) and non-relapse mortality (NRM) was 54% (+/−4%) and 13% (+/−3%), respectively. LFS rates were comparable for TBI and chemotherapy-based conditioning (34% vs. 29%, p=0.53). As well, the source of stem cells had no impact on LFS (32% for PB vs. 33% for BM, p=0.91). The 2 year probability of LFS increased from 22% for transplants performed between 1996–2000 (n=70) to 32% between 2001–2006 (n=61) and 54% between 2007–2010 (n=40), p<0.001 (Fig). In respective periods the RI decreased from 65% to 47% and 46% (p=0.01), while NRM was 11%, 21% and 0% (p=0.03). In a multivariate analysis the year of transplantation (<2007 vs. 2007–2010) was the only factor independently influencing the risk of treatment failure (HR=2.98, 95%CI, 1.54–5.79; p=0.001).

We conclude that results of autologous HSCT for Ph+ ALL improved markedly in recent period with more than half of patients being alive and leukemia-free at 2 years. Therefore, it appears than in the era of TKIs autologous HSCT may be considered potentially curative option. The advantage is probably associated with more profound responses achieved with TKIs, which, however requires confirmation in a separate analysis. As well, the role of post-transplant maintenance remains to be determined.

Disclosures:

No relevant conflicts of interest to declare.
TGFβ Restores Hematopoietic Homeostasis After Chemotherapy.
Authors: Brenet Fabienne A. , Kermani Pouneh , Rafii Shahin , Scandura Joseph M.
Published: October 2019

Abstract 2344

Background:

Hematopoietic stem cells (HSCs) are required for lifelong blood cell production and, to prevent exhaustion, the majority of HSCs are deeply quiescent during steady-state hematopoiesis. Paracrine factors produced by specialized bone marrow niche cells maintain HSC quiescence. Yet, evolution demands a rapid hematopoietic response to stressors such as infection, bleeding or toxin exposure. These triggers set off a remarkable adaptation in hematopoiesis that sacrifices HSPC quiescence, and the protection it affords, to meet an urgent need for new blood cell production. A great deal is known about how the HSPCs are mobilized during these periods of stress but mechanisms driving the return to steady-state hematopoiesis after stress have not been identified.

Method/Results:

Because TGFβ is one of few negative regulators of hematopoiesis, we measured the levels of active TGFβ and assessed the effects of its downstream signaling during hematopoietic recovery after chemotherapy. During late hematopoietic regeneration, as hematopoietic homeostasis is restored, the levels of active TGFβ spike in whole bone marrow and downstream signaling (as reported by Smad2 phosphorylation) increases in hematopoietic stem and progenitor cells (HSPCs). To assess how TGFβ signaling alters hematopoietic regeneration, we administered myelosuppressive chemotherapy to mice and then, during recovery, treated cohorts with either an antibody that specifically neutralizes active TGFβ (1D11), an isotype control antibody (13C4) or nothing at all. We found that TGFβ blockade accelerated blood count and bone marrow regeneration, and promoted HSCP self-renewal/proliferation by delaying the return of HSPCs to quiescence (G0). In contrast, TGFβ blockade during homeostasis elevates neither blood counts nor bone marrow cellularity and fails to induce HSPCs to emerge from quiescence.

Conclusions:

The de facto paradigm is that homeostasis is passively reestablished as stress mediators normalize. Our data strongly suggest that this paradigm is incorrect. Rather than being a passive process, it appears that steady-state hematopoiesis is actively reimposed. TGFβ pathway activation marks regenerating HSPCs returning to quiescence and this context-depending signaling helps reestablish homeostasis during recovery from chemotherapy. Together, our data demonstrate that myelosuppression does not drive hematopoiesis using only a cytokine-fueled gas pedal but also taps an active braking mechanism once sufficient recovery has been attained. More directly, our data strongly suggests that TGFb pathway inhibitors could be used to promote multi-lineage hematopoietic reconstitution. This finding significantly extends prior work in the field by opening the door to new, potentially superior, ways to modulate the hematopoietic adaptation to stress.

Disclosures:

No relevant conflicts of interest to declare.
AML1-ETO Collaborates with the TALE Homeobox Genes MEIS1 and MEIS2 in Inducing AML.
Authors: Vegi Naidu M , Klappacher Josef , Oswald Franz , Claus Rainer , Mulaw Medhanie M , Thiel Verena , Rawat Vijay P.S , Fend Leticia Quintanilla , Plass Christoph , Döhner Konstanze , Döhner Hartmut , Hiddemann Wolfgang , Buske Michaela Feuring , Buske Christian
Published: October 2019

Abstract 2387

AML1-ETO (AE) is the most frequent fusion gene in human AML. Previously, we and others have demonstrated that the fusion is not able to cause leukemia on its own in experimental murine models, but that it needs collaborative partners. Following the ‘two-hit model’ hypothesis that class I and class II mutations have to collaborate to induce leukemia, we could previously demonstrate that AE (class II) collaborates with FLT3-length mutation (class I) to generate AML in the murine bone marrow transplantation model (BMT model). We now demonstrate that AE can collaborate with mutations of its own class in inducing AML. We focussed our analyses on the TALE homeobox gene family, namely Meis1 and Meis2, as already known in the case of Meis1 to be a potent co-factor for Hox gene associated leukemias and being classified as other homeobox genes as a class II mutation.

First, real-time RT - PCR confirmed that MEIS1 is expressed at high levels in a subgroup of AE positive AML patients. Furthermore, MEIS2 was highly and aberrantly expressed virtually in all AE patients (n=70) compared to normal bone marrow. Expression patterns of both MEIS genes correlated with their promoter methylation in the t(8;21) patients and cell lines. To test the functional relevance of MEIS expression in human AE patients, we analysed collaboration of AE with Meis genes in the BMT model: Meis1 and Meis2 were retrovirally expressed alone or in combination with AE in 5-FU treated primary murine bone marrow and injected into lethally irradiated recipients (AML1-ETO alone, n=10; EGFP control, n=7; AE+Meis1, n=14; AE+Meis2, n=4). None of the mice in the AE as well as in the control group developed disease. In contrast, mice transplanted with BM co-expressing AE+Meis1 and AE+Meis2 developed lethal disease after a median latency of 102 and 255 days respectively. AE+Meis1 induced MPS in three, AML in seven and ALL in three cases, whereas AE+Meis2 induced AML in all cases. Functional relevance of MEIS expression was further confirmed in human AML cell lines as shRNA mediated depletion of MEIS1 as well as MEIS2 impaired cell growth in AE positive AML cell lines and so far one primary AE AML sample. To understand the mechanisms of AE/MEIS collaboration co-immunoprecipitation assays were performed documenting weak binding of Meis1, but strong interaction of Meis2 with AE. ChIP-Seq is currently being performed to test whether DNA binding of MEIS genes alter in collaboration with AE. Taken together, we could demonstrate that AE collaborates with Meis genes in AML. It furthermore shows that a class II mutation can be converted into an overt oncogene by a mutation of its own class.

Disclosures:

No relevant conflicts of interest to declare.
FLT3-ITD Interacts with and Phosphorylates IL-3β, and JAK1/2 Dependent IL-3β Activation Bypasses FLT3-ITD in FLT3 Kinase Independent Inhibitor Resistance in Vitro: Evidence for the Significance of IL-3β for FLT3-ITD Dependent Oncogeneic Signaling in AML.
Authors: Rummelt Christoph , Gorantla Sivahari P. , Sigl Michael , Saenger Jana , Götze Katharina , Peschel Christian , Duyster Justus , von Bubnoff Nikolas
Published: October 2019

Abstract 2423

Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML.

Disclosures:

Duyster: Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.
A Genome-Wide Analysis of Variants Influencing Methotrexate Clearance Replicates SLCO1B1.
Authors: Ramsey Laura B. , Panetta John C , Smith Colton , Yang Wenjian , Fan Yiping , Winick Naomi , Martin Paul L. , Cheng Cheng , Devidas Meenakshi , Pui Ching-Hon , Evans William E. , Hunger Stephen P. , Loh Mignon L. , Relling Mary V.
Published: October 2019

Abstract 2466

High-dose methotrexate (HDMTX) is an important element of chemotherapy for acute lymphoblastic leukemia (ALL) and other malignancies. Methotrexate clearance influences cure and toxicity in children with acute lymphoblastic leukemia (ALL). HDMTX schedules and doses vary widely among treatment protocols. The Children's Oncology Group (COG) tested the efficacy of 6 courses of 2 g/m2 over 4 hours versus 1 g/m2 over 24 hours (P9904 and P9905 protocols). Patients were assigned to one of four arms for consolidation: A, 24-hour methotrexate infusion (1 g/m2) and no delayed intensification (DI); B, 4-hour methotrexate infusion (2 g/m2) with no DI; C, 24-hour methotrexate infusion with DI; D, 4-hour methotrexate infusion with DI. We estimated methotrexate clearance for 1279 patients treated on these protocols, with two plasma MTX concentrations per course, using a Bayesian pharmacokinetic modeling approach. Germline genetic variation was assessed using the Affymetrix 6.0 array, and other single nucleotide polymorphisms (SNPs) were imputed based on 1000 Genomes reference data, yielding 5.2 million SNP genotypes evaluable per patient.

Average MTX clearance was highly variable, with a median (range) of 164 (65–355) and 109 (49–290) ml/min/m2 for the 24-hour and 4-hour infusions, respectively. Methotrexate clearance was lower in older children (p = 7 × 10−7), girls (p = 2.7 × 10−4), and patients who received a delayed intensification phase during consolidation (p = 0.0022). Adjusting for age, gender, race, and treatment arm, a genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in SLCO1B1(p = 2.1 × 10−11), a gene that encodes for an organic anion transporter that is known to transport methotrexate. This replicates our previous findings (Trevino et al, J Clin Oncol. 2009;27(35):5972-8) that polymorphisms in SLCO1B1 influence methotrexate clearance in ALL patients treated on St. Jude protocols with three different HDMTX schedules. In a combined meta-analysis including the 1279 COG patients and 699 St. Jude patients, and adjusting for age, gender, race, and treatment arm, the association of methotrexate clearance with SLCO1B1 SNP rs4149056 yields a p-value of 3.1 × 10−19 (Figure). Even after adjustment for the rs4149056 SNP, other polymorphisms in SLCO1B1 remained significantly related to methotrexate clearance, indicating that there are multiple variants in SLCO1B1 that can influence methotrexate clearance. Validation of the association of this gene with five different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance.

Disclosures:

No relevant conflicts of interest to declare.
Whole Exome Sequencing Analysis in AML with Normal Karyotype Not Harboring FLT3/ITD Mutation Reveals Novel Genetic Alterations.
Authors: Lee Il-Kwon , Kim Namshin , Kim Yeo-Kyeoung , Kim Dennis Dong Hwan , Trinh Quang , Minden Mark D , Jung Chul Won , Jang Jun-Ho , Kim Hee-je , Moon Joon Ho , Sohn Sang Kyun , Won Jong-Ho , Kim Sung-Hyun , Huh Jungwon , Kim Nan Young , Yu Li , Lee Yun Gyeong , Chung Sung-Hoon , Ahn Jae-Sook , Yang Deok-Hwan , Lee Je-Jung , Kim Hyeoung-Joon
Published: October 2019

Abstract 2593

Background:

NK-AML represents genetically heterogeneous group of disease. However genetic lesions affecting treatment outcome in patients with NK-AML are relatively unknown.

Methods:

The discovery cohort consists of 67 NK-AML patients in complete remission (median age: 49.2, ranges: 19–70) without FLT-3 mutations. Genomic DNA was extracted from enriched AML cells at diagnosis or control specimens obtained after complete remission. Whole exomes were captured using Agilent SureSelect and sequencing were performed by HiSeq2000 with 41∼89× coverage. Bioinformatics analysis and identification of somatic mutation has been done by series of software such as BWA, Picard, GATK, VarScan 2, and custom-made scripts. All the data has been re-checked by manual inspection. Validation has been done independent set of cohort (358 NK-AML patients, median age: 51, ranges: 15–85) with Sanger sequencing on highly mutated target sites.

Results:

Filtering against dbSNP and COSMIC database generated 485 genes with somatic and structural variations. Among them, 41 genes were detected in more than two patients. In addition to well-known 28 mutations, 13 novel mutations with different frequencies were identified including genes responsible for structural maintenance of chromosome (SMC1A, 6.0%) and tumor suppressor function (FAT1, 6.0%). Most common type of mutation was missense mutation (70.8%), and substantial fraction of mutation was splicing site mutations (3.8%). The hematological system development and hematologic function were most highly enriched by the Ingenuity Pathway Analysis (IPA) as expected. CIRCOS plot analysis showed similar co-occurring pattern of recurrent mutations with previous reports. Hierarchical clustering analysis divided into four different groups according to the number of harboring mutations. In network analysis four distinct subgroups were observed ranging 21 to 3 gene network.

Conclusion:

Using whole exome sequencing approach, a catalog of recurrent mutations was successfully defined in the patients with NK-AML without FLT3/ITD mutation. This candidate list of novel mutations should be tested further for therapeutic target and prognostic marker in the patients with NK-AML.

Disclosures:

No relevant conflicts of interest to declare.
Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells
Authors: Zhao Jimmy L. , Ma Chao , O'Connell Ryan , Rao Dinesh S. , Heath James , Baltimore David
Published: October 2019

Abstract 26

During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production.

As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level.

Disclosures:

No relevant conflicts of interest to declare.
Outcome of Elderly Patients with Acute Myeloid Leukemia (AML) Post Hypomethylating Agent (HMA) Failure.
Authors: Takahashi Koichi , Kantarjian Hagop M. , Ghanem Hady , Ravandi Farhad , Cortes Jorge E. , Kadia Tapan M. , O'Brien Susan , Pierce Sherry A. , Jain Nitin , Garcia-Manero Guillermo , Jabbour Elias J.
Published: October 2019

Abstract 2627

Background and Aims:

HMA such as 5-azacitadine or decitabine have been increasingly used as induction therapy in elderly patients with AML. The prognosis of such patients who failed initial HMA and the impact of subsequent chemotherapy on outcome are unknown. Therefore the aims of the current analysis are 1) to evaluate the prognosis of elderly patients with AML post HMA failure and 2) to evaluate the efficacy of using intensive cytarabine-based salvage therapy vs. low intensity investigational therapy.

Methods:

We analyzed 54 patients older than 60 years old with AML who received induction treatment with HMA using either 5-azacitadine (n=17; 31%) or decitabine (n=37; 69%) under various clinical trials.

Result:

Median age of the group was 68 years (range; 60–84); 39% patients were female. Forty three patients (77%) had performance status (PS) ≤1 and 11 patients had PS of 2 (23%). Mean initial white blood cell count was 10.9 ± 3.2 (x103/μl), hemoglobin 9.9 ± 0.2 (g/dl), platelet count 77 ± 9 (x103/μl), and bone marrow blast count 44 ± 2.9 (%). Cytogenetic category was classified as intermediate in 30 (55%) patients and poor in 24 (45%) based on the MRC criteria. Six patients (11%) carried FLT3-ITD mutation and 1 carried FLT3 D835 mutation. Complete remission (CR) was achieved in 24 patients (44%) with median numbers of cycles required to achieve CR being 3 (range; 1–6). All patients received further salvage therapies, at the time of failure, with a median of 2.5 (range, 2–5) salvage regimens. As part of the salvage regimen, high-dose cytarabine-based regimen (HDAC) was given to 32 patients (59%). Objective response rate to HDAC was 53%, with 18 pts achieving CR. Median number of courses given was 2 (range, 1–7) with a median duration of response of 3 months. No induction death was reported. Stem cell transplant was performed in total of 9 patients (17%) as a consolidation post HDAC (n=4) or as salvage therapy (n=5). With a median follow-up of 5 months post HMA failure, 5 (9%) patients remained alive receiving therapy. The median overall survival (OS) was 5.4 months (range, 2.3–8.5). The 1-year overall survival rate was 26%. There was no difference in OS between patients who received HDAC versus those who received investigational agents (p=0.25).

Conclusion:

The outcome of untreated elderly patients with AML who failed HMA is poor with a median survival of 5 months. HDAC based regimen, although active with low treatment related morbidity, yielded no survival improvement compared to investigational agents. Such patients should benefit from more innovative approaches.

Disclosures:

Ravandi: Celgene: Honoraria, Research Funding; Eisai: Honoraria, Research Funding.
Early Response to Methotrexate, Vincristine and Procarbazine Chemotherapy-Adapted Strategy for Primary CNS Lymphoma: Omitted Radiotherapy for Patients Achieving Complete Response in Interim Analysis.
Authors: Kim Yu Ri , Kim Se Hoon , Chang Jong Hee , Suh Chang-Ok , Kim Soo Jeong , Kim yun Deok , Hwang Doh Yu , Jang Ji Eun , Hyun Shin Young , Cheong June-Won , Min Yoo-Hong , Kim Jin Seok
Published: October 2019

Abstract 2719

The aim of this study was to evaluate treatment outcomes and prognostic factors of high-dose MVP (methotrexate, vincristine and procarbazine) chemotherapy and response adapted intensification therapy in primary central nervous system lymphoma (PCNSL). We evaluated the expression of Epstein-Barr virus (EBV)-encoded RNA in tissue and pretreatment EBV-DNA in whole blood. Forty patients with PCNSL who treated with high-dose MVP chemotherapy were retrospectively reviewed. Additional radiotherapy or autologous stem cell transplantation were performed for the patients who could not achieve complete response (CR) at interim response (Figure 1). The median age was 55 years. CR rate was 50.0% and objective response rate was 80.0%. Two-year overall survival (OS) was 59.8% and 2-year progression free survival (PFS) was 47.1%. Grade 3 or 4 neutropenia was seen in 19 of 40 (47.5%) patients and thrombocytopenia was developed in 13 (32.5%) patients. Treatment-related mortality was occurred in 4 patients. Delayed neurotoxicity was occurred in 4 patients. Poor performance status, elevated cerebrospinal fluid protein level and whole blood EBV-DNA positivity showed inferior OS (P=0.036, P=0.022, P=0.013, respectively, Figure 2). We detected whole blood EBV-DNA in 10 (29.4%) of 34 patients. It was related with poor OS and PFS in high risk patients according to International Extranodal Lymphoma Study Group scores (P=0.015, P=0.027, respectively). This study suggests that high-dose MVP chemotherapy without intrathecal chemotherapy and response adapted strategy may be an effective regimen for the newly diagnosed PCNSL. Whole blood EBV-DNA positivity could predict the inferior outcome in high-risk patients.

Figure 1: Treatment scheme HD-MVP high dose methotrexate, vincristine and procarbazine, CR complete response, CRu unconfirmed complete response, PR partial response, SD stable disease, PD progressive disease, Tx treatment, NE not evaluated, ASCT autologous hematopoietic stem cell transplantation, RT radiotherapy Figure 1:. Treatment scheme HD-MVP high dose methotrexate, vincristine and procarbazine, CR complete response, CRu unconfirmed complete response, PR partial response, SD stable disease, PD progressive disease, Tx treatment, NE not evaluated, ASCT autologous hematopoietic stem cell transplantation, RT radiotherapy

Figure 2: Overall survival and progression free survival according to EBV-DNA status in high risk patients. EBV Epstein-Barr virus Figure 2:. Overall survival and progression free survival according to EBV-DNA status in high risk patients. EBV Epstein-Barr virus

Disclosures:

No relevant conflicts of interest to declare.
Inherited Biallelic Loss-Of-Function Mutations In CSF3R Define a Novel Type Of Severe Congenital Neutropenia With Full Myeloid Cell Maturation and Refractoriness To RhG-CSF
Authors: Triot Alexa , Järvinen Päivi M , Arostegui Juan I. , Racek Tomas , Puchalka Jacek , Díaz Jose Luis Dapena , Kohistani Naschla , Gertz E. Michael , Schäffer Alejandro A. , de Heredia Rubio Cristina Díaz , Ozdemir Mehmet A. , Patiroglu Turkan , Karakukcu Musa , de Toledo Codina José Sanchez , Yagüe Jordi , Unal Ekrem , Klein Christoph
Published: October 2019

Severe congenital neutropenia (SCN) is a heterogeneous group of disorders characterized by defective production and viability of neutrophil granulocytes and predisposition to life-threatening bacterial infections. Currently, OMIM lists five defined monogenic SCN: SCN1 ELANE, SCN2 GFI1, SCN3 HAX1, SCN4 G6PC3. Here, we describe a novel SCN subtype (SCN6) caused by recessively-inherited loss-of-function mutations in the gene encoding the granulocyte colony-stimulating receptor (CSF3R). We have identified four affected children in two distinct families. Family A had a homozygous missense mutation in close proximity of the highly conserved WSXWS motif (c.922T, p.Arg308Cys) and family B had two compound heterozygous small deletions provoking frameshift mutations (p.Gly316fs and p.Gly415fs). Mutated G-CSFR p.Arg308Cys protein was characterized by perturbed N-glycosylation and aberrant localization to cell surface. G-CSF induced phosphorylation of STAT3 and STAT5 was greatly diminished. In contrast to other SCN subtypes, all patients had morphological evidence of full myeloid cell maturation in bone marrow. However, none of the patients responded to granulocyte colony-stimulating growth factor (GCSF) treatment in vivo, confirming aberrant GCSF-receptor dependent signaling. Our studies highlight the genetic and morphological variability of SCN and provide evidence both for functional importance and redundancy of G-CSFR-mediated signaling in human granulopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Role Of Histone Deacetylase 6 (HDAC6) In The Modulation Of STAT Pathways and Immune Function Of APCs
Authors: Lienlaf Maritza , Perez-Villarroel Patricio , Cheng Fengdong , Lee Calvin K. , Canales Jorge , Knox Tessa , Marante Danay , Yoder Sean J , Woan Karrune V. , Pinilla-Ibarz Javier , Seto Edward , Sotomayor Eduardo M. , Villagra Alejandro
Published: October 2019

Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation.

We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6.

Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.