bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of c-SRC and FLT3 Inhibitors Has An Additive Inhibitory Effect on FLT3 ITD but Not on FLT3 TKD Positive Cells
Authors: Leischner Hannes , Grundler Rebekka , Albers Corinna , Illert Anna Lena , Götze Katharina , Peschel Christian , Duyster Justus
Published: October 2019

Abstract 2892

Activating mutations of FLT3 are frequent in patients with AML. Two types of mutations are most common: Internal tandem duplications (ITD) of the juxtamembrane domain in approximately 30% of patients and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3-ITD mutation have a significantly worse prognosis whereas FLT3-TKD mutations do not appear to influence the clinical outcome. Studies have shown that mice receiving a transplant of bone marrow expressing FLT3 ITD develop a myeloproliferative disease. In contrast, mice which were transplanted with FLT3 TKD infected bone marrow, suffer from a lymphoid disease. Thus, both FLT3 mutations seem to exert different biological functions. Interestingly, FLT3-ITD but not FLT3-TKD or FLT3-WT leads to a strong activation of the STAT5 signaling pathway.

Recently we have shown that c-SRC is the crucial signaling mediator of FLT3 ITD to activate STAT5. Based on these findings we investigated the effect of FLT3 inhibitors (Midostaurin, Sorafenib and Sunitinib) in combination with c-SRC inhibitors (Dasatinib and PD166-326) on FLT3 ITD and FLT3 TKD murine and human cell lines as well as on primary patient material.

In FLT3 ITD expressing murine myeloid 32D cells c-SRC inhibitors in combination with FLT3 inhibitors showed clear additive effects on growth inhibition, apoptosis and activation of STAT5. In contrast, c-SRC inhibitors had no additional effects in FLT3 TKD expressing cells. Accordingly, a strong additive effect of c-SRC and FLT3 inhibitors could also be demonstrated in the FLT3 ITD positive human AML cell line MV4-11. Finally FLT3 ITD and FLT3 TKD positive primary human AML cells were investigated. We were able to detect a significant additional growth inhibition of FLT3 ITD positive human cells by combining c-SRC and FLT3 inhibitors. In contrast, no further growth inhibition was observed by c-SRC inhibition in primary AML cells expressing the FLT3 TKD mutation.

Together our results confirm c-SRC as a crucial signaling mediator in FLT3-ITD but not FLT3-TKD positive AML. The combination of FLT3 and c-SRC inhibitors warrants further investigation in FLT3 ITD positive AML.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Single Nucleotide Polymorphism Interactions Associated with Survival and Risk In Multiple Myeloma Using Novel Data Mining Methods
Authors: Van Ness Brian , Haznadar Majda , Fang Gang , Wang Wen , Paunic Vanja , Steinbach Michael , Kumar Vipin , Day Patrick , David Johnson , Morgan Gareth J , Barlogie Bart , Durie Brian GM
Published: October 2019

Abstract 2973

Disease risk and therapeutic outcomes are impacted by both tumor heterogeneity as well as germline variations found in the population. Multiple myeloma (MM) shows significant heterogeneity in genetic aberrations in tumor cells, that together with inherited polymorphisms, affects disease risk and therapeutic response. In order to identify the impact of genetic variations (SNPs) on MM we have developed a Bank On A Cure platform for examining 3404 SNPs, selected in 983 genes associated with pathways affecting cellular functions important in cancer. Using SNP data sets we sought to identify genetic interactions, beyond single univariate association analysis. The challenge was to use data mining methods that take into account relatively small cohorts of patients, in which false discovery rates typically exceed the power of the study. We report results from using novel computational approaches that efficiently identify higher order SNP interactions associated with disease risk as well as survival outcomes, while minimizing the false discovery rate. The BOAC SNP panel was used to develop a data base on 143 patients selected for short (<1yr) versus long (>3yr) survival in ECOG 9486 and SWOG 9321; as well as 247 newly diagnosed patients and equal number of controls for disease risk analysis. One algorithm developed employs a discriminative pattern mining approach in which defined pathway sets of SNPs are used in combination testing. A second algorithm used identified SNPs that had some association with outcome (survival or disease status); but demonstrated a significant increase in associations when examined in combinations – we refer to this as a p-value jump association. Variations in genes associated with cell cycle, apoptosis, drug metabolism, stress response and immunity reached very low p-values, and survived multiple comparison testing when analyzed in combinations associated with both survival (PFS) predictions as well as analysis of case-control disease risk. Some of the key genetic variations identified in various combinations, included: PTRB, PTEN, CDK5, XRCC4, GSTA4, GPX, DYPD, PCNA, CYP4F2, VEGF, PON1, ALK, and BAG3. The data mining methods and algorithms used, and specific combinations associated with risk and survival, will be presented. These results are being further validated in new cohorts, and functional implications of identified genetic variants are being investigated in HapMap cell lines.

Disclosures:

No relevant conflicts of interest to declare.
An Investigational Proteasome Inhibitor MLN9708 (MLN2238) Induces Apoptosis In Human Multiple Myeloma Cells In Vitro
Authors: Masood Aisha , Chitta Kasyapa , Miles Kiersten M , Khan Nazmul H , Adelaiye Remi , Akhtar Drusilla , Sher Taimur , Chanan-Khan Asher A.
Published: October 2019

Abstract 3066

Targeting the proteasome has proven to be one of the most effective therapeutic strategies in the treatment of multiple myeloma (MM), and the proteasome inhibitor bortezomib is approved for treatment of MM. However its clinical efficacy is compromised by the acquired resistance in patients, necessitating the development of new therapeutics. Several new proteasome inhibitors are under investigation for their therapeutic efficacy in MM. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a proteasome inhibitor which shows refined pharmacokinetic and pharmacodynamic properties in preclinical studies and is currently in Phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all of the studies reported here, in which we report the efficacy of MLN2238 on three established MM cell lines-KMS11, OPM2 and U266. MLN2238 was found to inhibit the chymotrypsin-like proteasomal activity of all MM cell lines in a dose dependent manner. Investigation of the IC50 of MLN2238 on these cell lines demonstrated that KMS11 is the most sensitive (IC50 of 15.9 nM) while U266 was found to be the least sensitive cell line (IC50 of 511 nM). OPM2 cells also showed intermediate sensitivity with an IC50 of 58.6 nM. MLN2238 induced apoptosis in KMS11 cells as evidenced by annexin V staining and PARP-1 cleavage. Cleavage of caspases 9 and 3 suggested activation of the intrinsic apoptotic pathway by MLN2238. Furthermore, MLN2238 treatment was shown to increase the mitochondrial outer membrane permeability (MOMP) and decrease BCL-2 levels. Evaluation of the expression of PSMB5, the preferred proteasomal subunit target for both bortezomib and MLN2238, revealed that it is expressed at approximately 3 fold more in KMS11 cells as compared to U266, suggesting a possible mechanism for higher sensitivity of KMS11 to the proteasomal inhibitor, MLN2238. This preclinical evaluation confirms the anti-myeloma effects of MLN2238, warranting further in-depth evaluation in both in vitro and in vivo models of MM.

Disclosures:

No relevant conflicts of interest to declare.
Primary Myelofibrosis In Children
Authors: DeLario Melissa R. , Sheehan Andrea , Ataya Ramona , Bertuch Alison A. , Vega Carlos , Webb C. Renee , lopez-Terrada Dolores , Venkateswaran Lakshmi
Published: October 2019

Abstract 3079

Primary myelofibrosis is a chronic myeloproliferative disorder characterized by cytopenias, leukoerythroblastosis, extramedullary hematopoiesis, hepatosplenomegaly and bone marrow fibrosis. It is a serious medical condition in adults, often requiring major interventions such as hematopoietic stem cell transplantation (HSCT) for cure. In comparison to adults, children are rarely affected by this entity; the largest case series reports on four such patients. Most of these reports suggest that the majority of affected children have spontaneous resolution of their myelofibrosis with no long term complications. To better describe the clinical characteristics and outcomes of pediatric primary myelofibrosis, we performed a retrospective chart review of children diagnosed with myelofibrosis by bone marrow pathology at our institution from 1996 to 2009. Eighteen patients with primary myelofibrosis were identified. At presentation, all patients had one or more cytopenias and only one had leukoerythroblastosis. Three of 11 patients tested (27%) had cytogenetic abnormalities, as opposed to up to two-thirds of adults with primary myelofibrosis. Eleven of 18 patients (61%) demonstrated erythroid hypoplasia in the bone marrow, which is uncommon in adults. Based on recent molecular studies of myeloproliferative disorders in adults, the World Health Organization now includes the presence of a clonal marker such as JAK2V617F as a major criterion for diagnosis of myelofibrosis in adults. In contrast to approximately half of adults (43-63%) with primary myelofibrosis, JAK2V617F mutation in the bone marrow has not been reported in children. JAK2V617F mutation analysis was negative on 16 of 18 bone marrow specimens tested. In this series, only 5 of the 18 children (27.8%) had spontaneous recovery. No child developed malignant transformation. Eight children underwent HSCT, four of whom were cured of their myelofibrosis; the remaining four children died from infections acquired during transplantation. Four children died prior to transplantation, or were identified as having myelofibrosis at autopsy. One child transferred care to another facility and thus outcome data was not available. In all children, infection was the most common cause of death. Notably, the degree of bone marrow fibrosis did not correlate with outcome. Our series demonstrates that children with primary myelofibrosis have hematologic, bone marrow and molecular features that differ from adults. In contrast to what is found in the literature, our series of pediatric patients with primary myelofibrosis, the largest reported thus far, indicates a poor outcome for the majority of these patients.

Disclosures:

No relevant conflicts of interest to declare.
SOCS3 Phosphorylation In Myeloproliferative Neoplasms
Authors: Arnold Claire , Elliott Joanne , Johnston James A , McMullin Mary Frances
Published: October 2019

Abstract 3083

Introduction:

Suppressor of cytokine signaling protein 3 (SOCS3) is an important regulator of the erythropoietin receptor and the receptor-associated JAK2 kinase. We hypothesize that the mechanism of JAK2 mutant-mediated transformation in myeloproliferative neoplasms (MPN) involves overcoming SOCS3 inhibition. In co-expression studies SOCS3 protein is stabilized and tyrosine phosphorylated in the presence of JAK2 V617F (Hookham et al., Blood 2007) and exon 12 mutations (Elliott et al., Haematologica 2009) but not wild-type JAK2, rendering SOCS3 unable to downregulate phosphorylation of these JAK2 mutants. The purpose of this study was to determine if these observations can be reproduced in vivo, by comparing SOCS3 expression and phosphorylation in MPN patients and healthy volunteers.

Methods:

Patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) were recruited from Belfast City Hospital. The mutational status of the JAK2 gene had been previously assessed. Granulocytes were isolated from peripheral blood of patients and healthy volunteers by Ficoll density gradient centrifugation, dextran sedimentation and hypotonic red cell lysis. Estimation of the JAK2 V617F/wild type allelic ratio was performed by pyrosequencing. SOCS3 expression levels were measured by quantitative real-time reverse transcriptase-PCR. Granulocyte whole cell lysate was immunoprecipitated with anti-SOCS3 and Western blots were probed with anti-phosphotyrosine. SOCS3 phosphorylation was estimated by densitometry.

Results:

To date, 63 MPN patients and 27 healthy volunteers have been recruited. This total is comprised of 47 patients with PV (JAK2 V617F n=38, JAK2 exon 12 n=4, unknown n=5) and 16 patients with ET (JAK2 V617F n=15). Median V617F allele burden was 45% in PV (range 0–99%) and 20% in ET (range 7–84%). In 67% of patients with PV, SOCS3 expression was greater than healthy volunteers. Compared to healthy volunteers, SOCS3 phosphorylation in PV patients was higher in 67%, equivalent in 17% and lower in 17% of cases. Similarly in ET, SOCS3 phosphorylation was higher in 73%, equivalent in 18% and lower in 9% of patients compared with healthy volunteers. SOCS3 tyrosine phosphorylation was observed in association with both JAK2 V617F and exon 12 mutants.

Conclusion:

The presence of aberrant tyrosine phosphorylation of SOCS3 protein observed in some PV and ET patients supports the hypothesis that this is a potential mechanism by which mutant JAK2 escapes inhibition and leads to constitutive downstream signaling in MPN.

Disclosures:

No relevant conflicts of interest to declare.
Retrospective Study of the Utility of Follicular Lymphoma International Prognostic Index (FLIPI) and FLIPI2 In Patients with Follicular Lymphoma Uniformly Treated with Rituximab, Cyclophosphamide, Doxorubicin, Vincristin, and Prednisone
Authors: Numata Ayumi , Fujimaki Katsumichi , Tomita Naoto , Tanaka Masatsugu , Hashimoto Chizuko , Oshima Rika , Matsumoto Kenji , Matsuura Shiro , Yamamoto Wataru , Motomura Shigeki , Ishigatsubo Yoshiaki
Published: October 2019

Abstract 3100

The Follicular Lymphoma International Prognostic Index (FLIPI) is the most useful prognostic index for follicular lymphoma (FL). FLIPI was developed from retrospective data, which was based on treatment without rituximab (R) therapy and did not contain β2-microglobulin data of some cases. Recently the working group that had suggested FLIPI proposed FLIPI2, based on prospectively collected data. However, FLIPI2 contains data on several different treatment methods, such as treatment with or without R therapy. The present study aimed to retrospectively analyze the prognosis of FL uniformly treated with the combination of R, cyclophosphamide, doxorubicin, vincristin, and prednisone (R-CHOP). This study involved 114 patients consecutively diagnosed with FL who were treated with R-CHOP and were registered in the data-base of the Yokohama City University Hematology Group between January 2001 and October 2009. All the parameters required for FLIPI and FLIPI2 calculation were present in 108 patients (men, 53; women, 55, median age; 57 years [34- 78 years]). The median follow-up period was 31.7 months (0.8- 97.5 months). According to the FLIPI score, 50 patients (46.3%) were in the high-risk group (HR); 31 patients (28.7%), the intermediate-risk group (IR); and 27 patients (25.0%), the low-risk group (LR). On the basis of the FLIPI2 score, 45 patients (41.7%) were in HR; 52 patients (48.1%), IR; and 11 patients (10.2%), LR. According to FLIPI, the 5-year overall survival (5yOS) was 74.1% (95% CI, 58.1– 94.4%) for HR, 89.5% (95%, CI, 76.6– 100%) for IR, and 100% for LR (p = 0.0049); the 5-year time to failure (5yTTF) was 42.3% (95% CI, 26.0– 68.9%) for HR, 39.1% (95% CI, 22.7– 67.3%) for IR, 66.4% (95% CI, 47.2– 93.4%) for LR (p = 0.244). According to the FLIPI2 score, 5yOS was 71.3% (95% CI, 53.2– 95.6%) for HR, 85.6 % (95% CI, 72.0– 100%) for IR, and 100% for LR (p = 0.197), the 5yTTF was 35.8% (95% CI, 20.0– 64.2%) for HR, 46.7% (95% CI, 31.1– 70.3%) for IR, and 76.2% (95% CI, 52.1– 100%) for LR (p = 0.075). In conclusion, in FL patients treated with R-CHOP, FLIPI provided a more accurate OS than that provided by FLIPI2; however, FLIPI2 provided a more accurate TTF than that provided by FLIPI.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA Profiling In Activated B-Cell and Germinal Center B-Cell Diffuse Large B-Cell Lymphoma Using Formalin Fixed, Paraffin Embedded Patient Biopsies
Authors: Glinsmann-Gibson Betty J. , McMillan Dennis E , Wilkinson Sarah , Teruya-Feldstein Julie , Rimsza Lisa M.
Published: October 2019

Abstract 3111

Diffuse Large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. The Leukemia Lymphoma Molecular Profiling Project (LLMPP) collaboration has done extensive work regarding this disease including gene expression profiling to show that the cell of origin impacts the prognosis of the patient. Those cases which arise from an activated B cell (ABC) have a worse prognosis than those cases which arise from a germinal center B cell (GCB). We have used a subset of cases from our institution which have had gene expression profiling performed on frozen material and assigned ABC (n=18) or GCB (n=24) status (Rosenwald et al NEJM 2002, 346:1937). We have used matched formalin fixed paraffin embedded (FFPE) tissue block from these cases to obtain a microRNA profile utilizing the qNPA technology (High ThroughPut Genomics) as previously published (Roberts et al Lab Invest 2007, 87:979). This novel FFPE based RNAase protection assay measured 688 human microRNAs. Each microRNA was represented twice on the array and the values averaged for a signal value. The data were normalized to the total signal of the microarray. We were able to define a signature of increased microRNA for each DLBCL subtype as shown in Table 1.

ABC subtype GCB subtype hsa-miR-155 hsa-miR-28-5p hsa-miR-196a hsa-miR-138 hsa-miR-501-3p hsa-miR-151-3p hsa-miR-656 hsa-miR-151-5p hsa-miR-1247 hsa-miR-182 hsa-miR-210 hsa-miR-613

The hsa-miR-155 is of importance in lymphoma as it regulates the generation of immunoglobulin class-switch in plasma cells (Turner et al Immunity 2007, 27:847) and was previously identified as a characteristic of ABC cell lines (Lossos et al Blood 2009,113:3754). The hsa-miR-210 has been shown to modulate the MYC antagonist MNT which allows for MYC expression (Grandori et all Cell Cycle 2009, 8:2756). This microRNA profile provides an opportunity to further explore the role microRNA plays in lymphoma biology and in particular, in DLBCL subtype determination. The success of the technique in FFPE tissue holds promise for further microRNA studies on archival material.

Disclosures:

No relevant conflicts of interest to declare.
TWIST2 Was Deregulated In Human Acute Myeloid Leukemia and Might Play a Tumor Suppressor Role
Authors: Zhao Yun , Zhang Xiuyan , Zhu Yongjin , Cui Jiangxia , Shan Lidong , Yao Hong , Zhu Xiaosu , Zhu Li
Published: October 2019

Abstract 3141

TWIST encodes a typical basic helix-loop-helix transcription factor. Twist-2, as a member of this family was recently identified as a negative regulator of normal murine myelopoiesis (Sharabi AB. et al. PLoS Biol. 2008, 6:2786-2800), however the expression and functional role of TWIST2 in normal and leukemic human myeloid cells is poorly understood. To address this, we detected the transcript expression of TWIST2 from CD34+ cells of 19 AML patients compared with those of their normal counterparts from 6 healthy donors, and found that the expression of this transcript was 5-fold decreased in the patients. A lentiviral vector, which could only express YFP was used as control; and then the other lentiviral vector to express TWIST2 plus YFP was constructed and validated to efficiently infect various human AML cell lines including THP-1, Dami, HEL, HL60, NB4 and SHI-1. Using all these cell lines, we found that averagely the control cells could expand in the liquid culture 46-fold within 6 days, however the TWIST2 overexpressed cells could only increase 7-fold; moreover in the colony-forming cell (CFC) assays with both Dami and THP-1 cells, the TWIST2 overexpressed cells lost nearly 80% CFC capacities compared with those of control cells (n=3, P<0.05). Next we found that more TWIST2 overexpressed cells could reside in the G0/G1 phase compared with control cells (61% vs. 44%, n=2, P<0.05) and accordingly less TWIST2 overexpressed cells were present in the G2/S/M phase compared with control cells (39% vs. 56%, n=2, P<0.05). In addition, the control and TWIST2 viruses infected Dami (2×106 cells/mouse) and THP-1 cells (4×106 cells/mouse) were subcutaneously injected into nude mice without irradiation respectively, and the control cells could generated tumors (7/7 mice with Dami cells and 2/8 mice with THP-1 cells) but not the TWIST2 overexpressed cells (0/8 mice with Dami cells and 0/6 mice with THP-1 cells) within 4 weeks. To obtain molecular insights of how TWIST2 suppressed the tumourgenesis capacity of these cells, microarray analysis (Agilent 44K) were used to assess the differentially expressed transcripts between control and TWIST2 overexpressed THP-1 cells (from 3 independent experiments). Some examples of the differentially expressed genes included CCND1, RBBP9 and EPHB4, which were down-regulated in the TWIST2 overexpressed cells compared with control cells; in the other hand CDKN1A (p21Cip1), TP53INP1, DDI1 and AKTIP, which were up-regulated in the TWIST2 overexpressed cells compared with control cells. Taken together, these data demonstrated that the TWIST2 was deregulated in human AML patients, and suggested it might play a tumor suppressor role in these cells.

Disclosures:

No relevant conflicts of interest to declare.
Pathway Dependence on the Tyrosine Kinase TYK2 and Its Mediator STAT1 In T-Cell Acute Lymphoblastic Leukemia
Authors: Sanda Takaomi , Tyner Jeffrey W , Gutierrez Alejandro , Ngo Vu N , Moriggl Richard , Ahn Yebin , Glover Jason M , Chang Bill H , Willis Stephanie G , Staudt Louis M. , Druker Brian J , Look A. Thomas
Published: October 2019

Abstract 3155

The often aggressive and unpredictable behavior of T-cell lymphoblastic malignancies continues to pose both major clinical challenges in children and adults. To discover oncogenic pathways downstream of critical genetic abnormalities that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), and to identify novel molecular targets for anti-leukemic agents with T-cell specificity, we carried out a genome-wide functional screen in T-ALL cell lines using a retroviral library of inducible short-hairpin RNAs (shRNAs). Among the genes that are required for the growth of T-ALL cells, we found that loss of TYK2, a JAK family tyrosine kinase, was specifically lethal in each of three T-ALL cell lines that we tested in this screen. By contrast, TYK2 knock-down did not affect the growth of cell lines from diffuse large B-cell lymphoma or multiple myeloma, indicating that TYK2 is specifically required for the growth of T-ALL cells. We confirmed by knock-down with multiple independent shRNAs that the loss of TYK2 induces apoptosis in T-ALL, whereas knock-down of other JAK proteins (JAK1, JAK2 or JAK3) had no effect. We found that the TYK2 protein is constitutively phosphorylated in many T-ALL cell lines, and that these cells are sensitive to small molecule JAK/TYK2 inhibitors, including JAK inhibitor-I, AG-490 and CEP-701. To identify upstream receptors involved in TYK2 activation, we knocked down IFNAR, IFNGR, IL10R and IL12RB, and found that loss of IFNAR1 and IFNAR2 specifically inhibit the growth of T-ALL cells, as strongly as TYK2 knock-down. In addition, targeted knock-down analysis of downstream STAT proteins revealed that loss of STAT1 also inhibits the growth of T-ALL cells, indicating that this transcription factor is involved in the TYK2 pathway and required for cell survival. In fact, STAT1 protein was constitutively phosphorylated in many T-ALL cell lines and this phosphorylation was inhibited by both TYK2 knock-down and treatment with JAK/TYK2 inhibitors. Although interferon-mediated pathways are cytostatic in many cell types, our results indicate a requirement for the IFNAR-TYK2-STAT1 pathway in promoting the growth and survival of T-ALL cells. Dependence on this pathway confers unique sensitivity of T-ALL cells to TYK2 inhibition by small molecule inhibitors, thus providing a novel therapeutic target for clinical testing in patients with this disease.

Disclosures:

Druker: Molecular MD: Equity Ownership.
MicroRNA Mir-125b Causes Leukemia
Authors: Bousquet Marina , Harris Marian , Zhou Beiyan , Fleming Mark D. , Lodish Harvey
Published: October 2019

Abstract 3158

MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Symptomatic and Incidental Thromboses Are Both Associated with Mortality In Pancreatic Cancer
Authors: Menapace Laurel A. , Peterson Derick R. , Berry Andrea , Sousou Tarek , Khorana Alok A.
Published: October 2019

Abstract 3167

Background:

Incidentally diagnosed venous thromboembolism (VTE) is a growing clinical problem. Although pancreatic cancer is well-known to be associated with VTE, contemporary rates of incidental and symptomatic VTE events and their association with mortality are incompletely understood.

Methods:

We conducted a retrospective cohort study of consecutive pancreatic adenocarcinoma patients seen at the University of Rochester from 2006–2009. Radiologic reports were reviewed for presence of pulmonary embolism (PE), deep venous thrombosis (DVT), and visceral vein thrombosis. Multiple clinical variables and mortality outcomes were collected. Data were analyzed using a multivariate Cox proportional hazards model.

Results:

A total of 1151 radiologic exams for 135 patients were included. Forty-seven patients (34.8%) experienced at least one VTE event. There were 12 PEs (n=12 patients, 8.9%), 34 DVTs (n=17 patients, 12.6%), 47 visceral vein (n=31 patients, 22.9%) and 2 arterial (n=2 patients, 1.5%) events. Twenty-one patients (15.5%) experienced more than one event. Incidental events comprised 33.3% (n=4) of PEs, 17.6% (n=6) of DVTs and 100% (n=47) of visceral VTE. Median survival for the study population was 237 (95% CI 199–277) days. Patients with VTE had significantly reduced survival (73 vs. 233 days at 3 months post-diagnosis; 66 vs. 245 days at 6 months post-diagnosis). There was no significant difference between asymptomatic and symptomatic events in terms of conditional median survival at 3 months-, 6 months- or 1 year-post diagnosis. In multivariate analysis, occurrence of either DVT (HR 7.4 95% CI 3.8–14.6, P<0.0001) or visceral asymptomatic events (HR 2.5 95% CI 1.6–3.8, P=0.0001) was significantly associated with mortality along with advanced stage.

Conclusions:

VTE occurs in over one-third of pancreatic cancer patients, including a significant proportion with incidentally discovered events. Patients with visceral vein events are generally not anticoagulated but these findings suggest a similar association with mortality as symptomatic DVT. Our findings require reconsideration of prognosis and anticoagulation options in pancreatic cancer patients with both incidental and symptomatic VTE.

Disclosures:

No relevant conflicts of interest to declare.
Mapping the Interface Between Protease Activated Receptor 1 (PAR1) and PAR4 Heterodimers
Authors: Noble Daniel N. , de la Fuente Maria , Nieman Marvin T.
Published: October 2019

Abstract 3195

Antiplatelet therapy is a key component of the treatment regimen for acute coronary syndrome and stroke. A major obstacle in developing antiplatelet agents is for agents to be effective without causing bleeding complications. Protease activated receptor 4 (PAR4) is a potential attractive target for antiplatelet therapies because of its requirement for stable clot formation and limited tissue distribution. The purpose of the current study is to determine how PAR4 interacts with other proteins on the surface of platelets and how these interactions influence activation and signaling of PAR4. We have demonstrated that PAR4 forms homodimers using two techniques, bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). These interactions are specific, as PAR4 does not interact with two unrelated G-protein coupled receptors (GPCRs) rhodopsin and dopamine D2 receptor. In addition to homodimers, PAR4 forms heterodimers with PAR1. The heterodimers are also specific since PAR1 also does not interact with other GPCRs. PAR1 does not interact with rhodopsin; therefore we generated a series of rhodopsin-PAR4 chimeras to determine the region on PAR4 that interacts with PAR1. We have mapped the region on PAR4 required for heterodimerization to transmembrane helix 4 (TM4). When the rhodopsin-PAR4 junction is at the beginning of TM2, TM3 or TM4, the chimera interacts with PAR1 in a manner indistinguishable from PAR4-wt. However, when the junction is moved to the beginning of TM5, TM6 or TM7 the chimera does not interact with PAR1-wt. Molecular modeling of PAR1 and PAR4 has identified residues within TM4 that are exposed and are directed away from the other TM helices creating a potential interaction interface. Co-expression of PAR1 with PAR4 increased the efficiency of PAR4 activation by thrombin ∼10-fold indicating a functional significance for PAR1-PAR4 interactions. The results from these studies provide detailed map of the interactions between two GPCRs on the cell surface and may provide insights for developing PAR antagonists. The current strategy of developing PAR antagonists is to target the individual receptors. However our data show that the functional unit is the PAR1-4 heterodimer and, as such, this may the relevant target to disrupt thrombin activation of platelets. Antagonists to PAR1 or PAR4 need to developed and studied in the context of these interactions, not in isolation.

Disclosures:

No relevant conflicts of interest to declare.
Critical Role of CalDAG-GEFI In FCγRIIa-Dependent Platelet Activation and Thrombosis
Authors: Stolla Moritz , Stefanini Lucia , Ouellette Timothy Daniel , Ahmad Firdos , Reilly Michael P , McKenzie Steven , Bergmeier Wolfgang
Published: October 2019

Abstract 3196

Immune-mediated thrombosis is a major cause of death in autoimmune diseases and contributes to complications in drug treatments (e.g. Heparin Induced Thrombocytopenia). The major receptor on platelets for immunoglobulin-mediated activation is FcγRIIA. FcγRIIA signals through an immunoreceptor tyrosine-based activation motif (ITAM) that leads to phospholipase C activation, which induces the release of calcium and diacylglycerol (DAG). In our previous work, we identified CalDAG-GEFI (calcium and DAG regulated guanine nucleotide exchange factor I) as a key component of collagen/ITAM-mediated platelet activation. In the current study, we evaluated if CalDAG-GEFI is a potential target for the intervention with FcγRIIA receptor dependent, immune-mediated thrombosis.

Mice transgenic for the human FcγRIIA (hFCR) and deficient for CalDAG-GEFI-/- (hFCR/CDGFI-/-) were generated. In vitro, aggregation of hFCR/CDGFI-/- platelets required 50–100-fold higher concentrations of anti-CD9 antibodies than hFCR/WT controls. In comparison, inhibition of P2Y12, the target of clopidogrel, shifted the dose response curve for anti-CD9 in hFCR/WT platelets by only ∼2-fold. In addition to their aggregation defect, hFCR/CDGFI-/- platelets were characterized by markedly impaired Rap1 activation.

To assess the role of CalDAG-GEFI in FcγRIIA -mediated thrombosis in vivo, we developed a model of antibody-mediated thrombosis, were we injected mice with an Alexa-750 labeled antibody against GPIX and extracted the lungs to visualize pulmonary thrombosis on a LICOR scanner. Anti-GPIX induced pulmonary thrombosis in hFCR mice but not in WT animals. Pretreatment with clopidogrel did not provide a substantial protection from thrombosis in hFCR mice. In contrast, no pulmonary thrombi were observed in hFCR/CDGFI-/- mice.

Together, our studies are the first to highlight the importance of CalDAG-GEFI downstream of platelet Fc-receptor/ITAM signaling and suggest CalDAG-GEFI as a powerful new target in the intervention of immune-mediated thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Shortened Erythrocyte Life Span and Increased Oxidative Stress In Erythroid Precursors Are Consistent with Normocytic Anemia In Mice with Chronic Inflammation
Authors: Layman Andrew J. , Langdon Jacqueline M. , Yu Qilu , Prince Olivier D. , Walston Jeremy D. , Civin Curt I. , Roy Cindy N.
Published: October 2019

Abstract 3205

The anemia of inflammation is a common co-morbid condition associated with various infections, autoimmune disorders, and other chronic disease states. In general, hemoglobin levels are mildly to moderately decreased. In vitro studies have demonstrated that pro-inflammatory cytokines can inhibit erythropoiesis at various stages of development, but many of these studies are limited to the analysis of early developmental stages and primarily assess proliferation as opposed to maturation or survival. Sufficient data from in vivo studies and whole animal models are lacking. We characterized the anemia in 8–10 week old C57BL/6 mice with turpentine oil-induced sterile abscess. We found that following 3 weeks of sterile abscess, the mice had a normocytic, normochromic anemia.

Using in vivo biotin-labeling of peripheral blood, we found the coefficient describing erythrocyte life span significantly decreased from -0.044 ± 0.002 SE in control mice to -0.048 ± 0.002 SE in mice with sterile abscess (p = 0.04). In further support of the mechanism of increased erythrocyte turnover, we found that splenic macrophages isolated from mice with abscess significantly increased expression of Heme-regulated gene 1 (Hrg-1) 2.5 fold, (p < .001) and significantly increased expression of Ferroportin (Fpn) 2.76 fold (p = 0.003).

Oxidative stress can lead to increased erythrocyte turnover and can inhibit erythroid maturation. To determine whether erythrocytes from mice with sterile abscess had impaired capacity for survival, we assessed staining of peripheral blood and erythroid precursors with chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester (DCF). DCF fluoresces upon oxidation. We found that mean DCF fluorescence intensity (MFI) in peripheral blood increased from 3.2 ± 1.0 in control mice to 5.5 ± 0.6 in mice with abscess (p=0.004). Similarly, we found a greater percentage of bone marrow derived erythroid precursors from mice with abscess had high DCF staining (16.2 ± 3.9%) when compared to control mice (6.7 ± 1.9%, p=0.003). In conclusion, these data support the hypothesis that increased oxidative stress in erythroid precursors of mice with sterile abscess results in increased erythrocyte turnover and normocytic, normochromic anemia. Future studies will investigate whether pro-inflammatory cytokines inhibit the expression of key erythrocyte anti-oxidant enzymes or whether reactive oxygen species from neighboring granulocytes damage erythroid precursors in the bone marrow.

Disclosures:

No relevant conflicts of interest to declare.
Phase I Trial of the Oral Poly (ADP-ribose) Polymerase (PARP) Inhibitor Veliparib (ABT-888, V) Combined Wtih Topoecan (T) and Carboplatin (C) for Adults with Relapsed and Refractory Acute Leukemias
Authors: Karp Judith E. , Pratz Keith W , Litzow Mark R. , Ji Jiuping , Chen Alice , Rudek Michelle , Kaufmann Scott H.
Published: October 2019

Abstract 3276

PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7.

Acute Myelogenous Leukemia (AML, 33 patients) Acute Lymphoblastic Leukemia (ALL, 4 patients) Median Age (range) 570 (40-75) 55 (42-62) Relapsed 8 No. Prior CRs 2 (1-3) Prior Stem Cell Transplant 2 0 Refractory 25 4 No. Primary Refractory 10 1 No. Prior Regimens 3 (1-4) 3 (1-4) Prior Stem Cell Tranpslant 3 1 Secondary AML 12 Anecedent Hematologic Dx 6 Treatment-Related 6 Cytogenetics Intermediate 10 2 Adverse: Single/Complex 7/16 2 Ph+ (1 with complex) BRCA-1/BRCA-2 1 (mutation)/ 1 (13q14 deletion) 0

Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T. Of 27 pts treated at doses of V 10–20 mg orally BID Days1-8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 8 (30%) have experienced > grade 3 non-hematologic toxicity, 4 (15%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 22% (2 CR, 1 CRi, 3 PR), with 4 in relapsed AML, 1 each in refractory AML and refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V does not accumulate with multiple dose administrations in plasma but does in marrow, is not altered by T or C administration, and is detectable in marrow supernatant and cells. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to <50% of pretreatment values in 21/29 (72%) blood and 9/13 (69%) marrow samples. The MTD has not been reached and dose escalation is ongoing.

Disclosures:

No relevant conflicts of interest to declare.
Differential Dependence On Wnt Signaling Allows Chemical Mediated Eradication of Human Acute Leukemia without Affecting Normal Blood Stem Cells
Authors: Risueño Ruth M. , Szabo Eva , Dang Uyen Maria , Levadoux-Martin Marilyne , Chung Jae-Uk , Xenocostas Anargyros , Leber Brian , Bhatia Mickie
Published: October 2019

Abstract 3278

Specific targets of signaling pathways that control self-renewal and survival of acute myeloid leukemic stem cells (LSCs) vs. normal hematopoietic stem cells (HSCs) are largely unknown. Here, using a small molecule (CWP232228) derived from a parent compound that inhibits Wnt/TCF targets, we demonstrate reduction of primary human AML-blast growth and clonogenic capacity ex vivo, without effects on normal hematopoietic progenitors. Upon establishment of AML or normal hematopoiesis in immune-deficient recipients, in vivo administration of CWP232228 reduced leukemic disease and abolished LSC self-renewal, with no effect on normal HSC function. In vivo gene profiling and ex vivo molecular studies revealed that CWP232228 induces apoptosis and differentiation of AML-blasts via inhibition of Wnt/b-catenin signaling and activation of non-canonical Wnt signaling which phenocopies the effects of this small molecule. Our study reveals an in vivo differential dependence of AML on canonical vs. non-canonical Wnt signaling that allows therapeutic targeting of LSCs whilst sparing normal HSCs.

Disclosures:

Chung: Choongwae Pharma Corporation: Employment.
New Fluorescent Assay for the Direct Quantification of Heparin and Low-Molecular-Weight Heparins In Clinical Samples
Authors: Szelke Helga , Giese Christina , Krämer Roland , Harenberg Job
Published: October 2019

Abstract 3325

Heparin, a linear polydisperse polysaccharide, is used to prevent blood clotting in various diseases. It consists predominantly of a trisulfated disaccharide repeating unit and has a high negative charge density. Currently accepted methods to control blood levels of heparin or of its more commonly used low-molecular-weight (LMW) fractions include chromogenic substrate assays that analyze the biological inhibition of blood coagulation factor Xa (FXa). There are no established methods to directly determine the concentration of heparins in clinical samples.

A modified positively charged perylene diimide (Heparin Red) was developed that binds specifically to heparin and LMW-heparin (Szelke H et al., Chem. Commun. 2010;46:1667). A red fluorescence signal is dose dependently quenched in the presence of heparin and LMW-heparins. LMW-Heparin was added to human serum, plasma or urine. The chromogenic S2222 substrate assay was used to determine the anti-FXa activity of the anticoagulants. Fluorescence was recorded with a portable fluorimeter, equipped with a 550 nm excitation filter and a 610 nm emission filter.

For plasma samples spiked with 0.6–1.0 IU/mL LMW-heparin, the intraday (n=5) and interday (n=5) coefficient of variations (CVs) of the fluorimetric Heparin Red assay were in the range of 2.9–6.1% and 4.3–13.8%, respectively. The CVs of the chromogenic S2222 assay for same plasma samples were the range of 3.3–18.8% (intraday) and 6.2–13.7 (interday).

In serum and plasma samples of patients treated with LMW-heparin, correlation between the Heparin Red assay and the chromogenic anti-FXa test (S-2222) was r = 0.66. The correlation between the Heparin Red assay and the anti-FXa test in urine samples was r = 0.83. This is probably due to its lower protein concentration in urine compared to plasma and serum samples.

The fluorescent perylene diimide Heparin Red allows sensitive quantification of heparin and LMWHs in plasma, serum and urine samples. Ease of application combined with matrix-independent response make this heparin probe attractive for both routine laboratory and fast point-of-care testing as well as high-throughput pharmacokinetic studies, ideally without the need for patient-specific calibration.

Disclosures:

No relevant conflicts of interest to declare.
A Role for Splicing Factors and R-Loop Formation in the Pathophysiology of Fanconi Anemia.
Authors: Green Allison M , Collins Natalie , Longerich Simonne , Sung Patrick , Kupfer Gary M
Published: October 2019

Abstract 3371

Introduction:

Fanconi anemia(FA) is an autosomal and X-linked recessive genetic disorder characterized by congenital defects, aplastic anemia, and a predisposition to cancer. At the cellular level, patients with FA display hypersensitivity to DNA crosslinking agents and increased levels of chromosomal instability. Because of these cellular phenotypes, the FA pathway has been thought to function in DNA maintenance and repair. Our data suggest that RNA and R-loop formation may account for at least part of the genomic instability seen in FA cells.

Methods:

Nuclear extracts were incubated with ribohomopolymers to determine whether FANCD2 is able to bind RNA. In vitro binding assays were performed to determine whether FANCD2 is able to bind R-loop nucleic acid structures. siRNA transfections were used to reduce protein levels of splicing factors. Survival assays were performed to determine cell sensitivity to the interstrand crosslinking drug mitomycin C (MMC) and the topoisomerase I inhibiting drug camptothecin (CPT).

Result:

In vitro binding studies with ribohomopolymers revealed that the non-ubiquitylated form of FANCD2 is able to bind polyG RNA preferentially. Because G-rich RNA is associated with R-loop formation at IgG switch regions (Yu et al, Nat. Immunology, 2003), we wanted to determine whether FANCD2 is also able to bind R-loops. A human recombinant protein complex containing FANCD2, FANCI, and FANCE was shown to bind R-loop structures in vitro. Since depletion of the splicing factor ASF/SF2 has been demonstrated to increase cellular genomic instability through the formation of increased levels of R-loop structures (Li et al, Cell, 2005), we next wanted to determine what effect depletion of ASF/SF2 would have on the FA pathway. siRNA mediated knock down of ASF/SF2 protein levels resulted in decreased levels of FANCD2 ubiquitylation following treatment with MMC. As FANCD2 ubiquitylation is often considered a marker of activation of the FA pathway, this result suggests that depletion of ASF/SF2 inhibits activation of the FA pathway. siRNA mediated knock down of ASF/SF2 also increased cellular sensitivity to MMC and CPT. siRNA mediated knock down of the splicing factor SC35 yielded a similar result, inducing an increase in sensitivity to MMC and CPT and reducing levels of FANCD2 ubiquitylation. However, siRNA mediated knock down of a third splicing factor, SRp55, had no effect on FANCD2 ubiquitylation or cellular sensitivity to MMC and CPT.

Conclusion:

These results suggest that R-loop formation may be a source of some of the genomic instability present in FA cells. We also demonstrate that splicing factors may play a role in activation of the FA pathway. These findings may potentially provide new targets for therapeutic treatments for FA.

Disclosures:

No relevant conflicts of interest to declare.