bluuebunny/crossref_metadata_2025_split huggingface dataset:
CAL-101, a Potent Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-kinase, Attenuates Pathway Signaling, Induces Apoptosis, and Overcomes Signals From the Microenvironment In Cellular Models of Hodgkin Lymphoma
Authors: Meadows Sarah A , Kashishian Adam , Johnson Dave , Diehl Volker , Lannutti Brian 
Published: October 2019

Abstract
Abstract 3926
Phosphatidylinositide 3-kinases (PI3Ks) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The class I kinases contain 4 isoforms designated p110α, β, δ, γ, and are activated by cell surface receptors. Aberrant regulation of the PI3K signaling pathway is frequently observed in human malignancies including those of hematological origin. CAL-101 is an oral p110δ-specific inhibitor which has shown preclinical and clinical activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). This compound is a potent p110δ inhibitor (EC50 of 65 nM in a whole-blood assay) with >200-fold selectivity over the other class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. Prior in vitro NHL studies revealed that CAL-101 induces caspase-dependent apoptosis, and inhibits CD40L-, BAFF-, CXCL12- and CXCL13-derived survival signals in cellular models (Lannutti BJ, et al., Blood 2010).
To investigate the potential role of p110δ in Hodgkin lymphoma (HL) we screened a number of HL cell lines for p110δ isoform expression and constitutive PI3K pathway activation. We report high levels of p110δ protein and activated Akt in 5 of 5 HL cell lines evaluated (L428, L540, L591, L1236, KM-H2). Inhibition of p110δ with CAL-101 treatment of cell lines resulted in a reduction of Akt phosphorylation and a decrease in cellular viability. Because previous studies have established the importance of signals from the microenvironment for the survival and proliferation of malignant cells as well as for their resistance to standard therapies, we investigated the effect of p110δ inhibition by CAL-101 in HL cell line-stroma cocultures. In this setting, CAL-101 overcame tumor cell growth induced by coculture of HL cells with bone marrow stromal cells. In addition, CAL-101 induced dose-dependent apoptosis of HL cells at 48 hours. Furthermore, stromal cell coculture resulted in increased CCL5, CCL17, and CCL22 levels; productions of these chemokines by HL cells cultured in the presence of stromal cells were reduced by CAL-101 in a dose-dependent manner. These results indicate that specific inhibition of p110δ may disrupt signals between HL cells and their microenvironment, thereby providing the preclinical rationale for clinical evaluation of CAL-101 as a novel therapeutic approach in patients with Hodgkin lymphoma.

Disclosures:
Meadows: Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment.
Remarkable Efficacy of IL-1 Receptor Antagonist In Schnitzler's Syndrome: a Series of 6 Cases
Authors: Lachmann Helen J , Harding Stephen , Wechalekar Ashutosh , Gillmore Julian D , Amft E. Nicole , Hawkins Philip N 
Published: October 2019

Abstract
Abstract 3958
Schnitzler syndrome is a rare acquired autoinflammatory disease characterized by relapsing urticarial rashes, periodic fevers, arthralgias, lymphadenopathy and IgM paraproteinaemia, which can be very low level. Fewer than 100 patients have been reported and in general chemotherapy directed at the B cell clone has not been effective in controlling the inflammatory symptoms.
We describe our series of six patients, their phenotype and response to treatment. The median age at symptom onset was 55.6 years (range 52.8–78.9 years) and 4 of the patients were male. All patients presented with rash, recurrent fever, constitutional upset with appetite loss, fatigue and myalgia. The rash was urticarial in appearance but non itchy and on biopsy demonstrated a marked neutrophil infiltrate. Three patients had intermittent lymphadenopathy and one disabling bone pain. An IgM paraprotein was detected in all cases, median 7g/L (range: detected on immunofixation only – 8g/L). The IgM paraprotein was kappa in 5 cases and only one patient had abnormal serum free light chains. The Hevylite™ assay was abnormal in all 4 cases in whom the test was performed. Of note the patient with an IgM lambda paraprotein detected only on immunofixation has a clear IgM lambda excess on Hevylite™ assay. Baseline bone marrow demonstrated significant abnormalities in three patients: one has a 10% infiltrate of clonal cells consistent with lymphoplasmacytic lymphoma, one demonstrated a myeloproliferative picture with dysplastic features but no obvious plasma or lymphoid infiltrate and one was reported as reactive. Three patients had been treated with broad spectrum immunosupression without benefit and all had had corticosteroids with only partial benefit. In all cases treatment with anakinra (recombinant IL-1 receptor antagonist) as monotherapy produced a complete clinical response with normalisation of inflammatory markers. The median duration of treatment has been 9 months (range 0.1–2.7 years) and no patients have lost their response to daily ankinra injections. Median follow up from presentation has been 7.1 years with no evidence of evolution of overt lymphoproliferative disease nor change in the paraprotein concentration. In conclusion Schnitzler's syndrome is a rare acquired autoinflammatory disease which can be very effectively treated by anakinra suggesting that IL-1 is central to its pathogenesis. Treatment does not appear to have had any direct effect on clonal markers and the role of the IgM paraprotein in this disease remains unclear.

Disclosures:
Off Label Use: Anakinra - IL-1 receptor blockade, used off licence in Schniztlers syndrome.
CD40 Stimulation Sensitizes CLL Cells to CD20-Triggered Cell Death by Rituximab and GA101 Via a Different Mechanism
Authors: Jak Margot , van Bochove Gregor , Klein Christian , Umana Pablo , Eldering Eric , Van Oers Marinus H.J. 
Published: October 2019

Abstract
Abstract 3979
Although treatment results for Chronic Lymphocytic Leukemia (CLL) have improved considerably over the last decade, unfortunately a curative treatment is still not available. In part this might be due to the interaction of CLL cells with their micro-environment in lymph nodes, spleen and bone marrow. Micro-environment derived signals are not only capable of driving proliferation of CLL cells, but can also induce resistance of CLL to cytotoxic drugs. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived CLL cells can to a certain extent mimic the lymph node microenvironment and result in resistance to cytotoxic drugs (Kater AP et al. Br J Haematol 2004;127:404; Smit LA et al. Blood 2007;109:1660.; Hallaert DY et al. Blood 2008;112:5141). At present it is not known whether sensitivity of CLL cells to CD20 monoclonal antibodies (mAbs) is modulated by micro environmental stimuli. Therefore in the present study we investigated anti-CD20 mediated cell death of CD40-stimulated CLL cells from 17 CLL patients. We observed that in sharp contrast with the response towards cytotoxic drugs, CD40 stimulation sensitizes CLL cells to cell death mediated by anti-CD20 mAbs. In CD40-stimulated CLL cells, cell death induced by both Rituximab (a type I anti-CD20 mAb) and GA101 (a novel type II anti-CD20 mAb) (Moessner E et al. Blood, 2010;127:404, 115(22):4393-402) is increased. Both anti-CD20 mAbs induce a non-apoptotic, caspase- and p53-independent rapid cell death, but interestingly the mechanism of Rituximab and GA101-induced cell death appears to be different. Rituximab-induced cell death is dependent on extracellular Ca2+ and ROS production and CD40 stimulation sensitizes CLL cells by increasing basal ROS production. In contrast, GA101 induces cell death via a lysosome-dependent mechanism and CD40 stimulation sensitizes CLL cells by increasing the lysosomal volume of the cell. Moreover, in contrast to Rituximab, GA101 induces cell death in the absence of a secondary crosslinking mAb. Combination of GA101 with fludarabine, chlorambucil, bortezomib or bendamustine shows additive effects and results in strong cell death of CD40-stimulated CLL cells, even in p53 dysfunctional CLL cells. Our findings not only provide a rationale for combining cytotoxic drugs and anti-CD20 monoclonal antibodies, but also show that GA101 is a potent promising new anti-CD20 mAb for the treatment of CLL.

Disclosures:
Klein: Roche: Employment, Equity Ownership, Patents & Royalties.
Combined Low-Dose Homoharringtonine, Cytarabine and Granulocyte Colony-Stimulating Factor for Patients with Advanced Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia Transformed From MDS
Authors: Wu Lingyun , Li Xiao , Chang Chunkang , Shu Jiying , Xu Li , Zhang Zheng 
Published: October 2019

Abstract
Abstract 4004
A pilot study of the efficacy and toxicity of the low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF) (CHG protocol) in patients with advanced myelodysplastic syndromes (MDS) or MDS-transformed acute myeloid leukaemia (t-AML) was conducted. A total of 50 patients (31, advanced MDS; 19, t-AML) were enrolled in this study. All patients were administered the CHG regimen comprising low-dose cytarabine (25 mg/d, intravenous continuous infusion, days 1 C14), homoharringtonine (1 mg/d, intravenous continuous infusion, days 1 C14) and G-CSF (300 μg/d, subcutaneous injection, days 0 C14, intermitted when the peripheral WBC count reached >20 °Á 109/L). This regimen was followed by conventional chemotherapy as post-remission therapy when complete remission (CR) was achieved. The overall response rate was 68.0% after one course of the CHG regimen was applied. Of the total 50 patients, 24 (48.0%) achieved CR and 9 (18.0%) achieved partial remission (PR). The median overall survival (OS) was 14.1 months. Among the patients aged ≥ 70 years, the response rate was 81.0% (71.4% CR and 9.6% PR), while in patients aged < 70 years, the response rate was 55.2% (31.0% CR and 24.1% PR) (P < 0.01). There were no statistically significant differences for CR, PR and OS when the patients were grouped by blast percentage in bone marrow and karyotypes, respectively. 8 of the 24 CR patients who just received post-remission regimens of HA (homoharringtonine and cytarabine) and DA (daunorubicin and cytarabine) relapsed rapidly and just kept a mean CR of 4.3 months. Otherwise, the other 14 CR patients alternatively received succeeded chemotherapy, which combined mitoxantrone, idarubicin, pirarubicin or aclarubicin with cytarabine or decitabine. The mean CR duration of these 14 patients reached 15.5 months with 4 still maintaining continuous CR. No treatment-related deaths were observed. Myelosuppression was mild to moderate, and no severe non-haematological toxicity was observed. Thus, CHG priming regimen as an induction therapy is well tolerated and effective in advanced MDS or t-AML patients.

Disclosures:
No relevant conflicts of interest to declare.
MiR-15a and Mir-16-1 Affects the Angiogenesis of Multiple Myeloma by Targeting VEGF-A
Authors: Sun Chunyan , Hu Yu , She Xiaomei , Chu Zhangbo , Fang Jun , Chen Lei 
Published: October 2019

Abstract
Abstract 4039
MicroRNAs (miRNAs) are non-coding small RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of a variety of cancers. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. Here we investigated the possible role of miR-15a/miR-16 in the angiogenesis of multiple myeloma (MM). Using stem-loop quantitative reverse transcription-PCR analysis, we showed that miR-15a/miR-16 are significantly underexpressed in primary MM cells as well as MM cell lines (RPMI 8226, ARH-77, OPM-2, KM3, U266 and NIH929). The aberrant expression of miR-15a/miR-16 were detected especially in advanced stage multiple myeloma. The expression of miR-15a and miR-16-1 were remarkably lower in stage III MM patients (n=23), whereas it were significantly higher in healthy individuals (n=18) and stage 2, II MM patients (n=14) (P<0.01 and P<0.001, respectively). In human MM cell lines and normal plasma cell, expression of miR-15a/miR-16 inversely correlated with the expression of vascular endothelial growth factor (VEGF). Consistently with the proposed role of miRNAs as key regulators of angiogenesis, here we identified VEGF-A as a target for miR-15a and -16. Enforced miR-15a or miR-16 expression reduced VEGF-A protein level and the luciferase reporter assays demonstrated that miR-15a and -16 specifically suppress expression of VEGF-A by directly interacting with its 3′-untranslated region. Moreover, Ectopic overexpression of miR-15a and -16 led to decreased pro-angiogenic activity of MM cells. Conditioned medium of pri-miR-15a- and pri-miR-16- transfected RPMI 8226 cells inhibited human bone marrow microvascular endothelial cell (BMEC-1) proliferation, chemotactic motility and capillary formation in vitro as compared with conditioned medium of scramble probe-transfected RPMI 8226 cells (P<0.05). Finally, miR-15a/miR-16 was shown to greatly inhibit the process of tumor formation in an animal model. Infection of lentivirus-miR-15a or lentivirus-miR-16 can significantly inhibit the xenograft tumor growth in nude mice. The average tumor volume after 4 weeks for GFP-transfected cells was 372.5 mm3. While the average tumor volume for miR-15a-transfected cells was 47 mm3 (P<0.05) and for miR-16-transfected cells was 93.6 mm3 (P<0.05). Of interest, neoangiogenesis analysis by immunohistochemistry staining of anti-CD31 showed that there were significant reductions in microvessel density present in the miR-15a group and miR-16 group as compared to control (P<0.05). Take together, our findings suggested that miR-15a and miR-16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A. These findings have therapeutic implications and may be exploited for future treatment of MM.

Disclosures:
No relevant conflicts of interest to declare.
Expression Profile and up-Regulation of Telomere-Associated Proteins In Multiple Myeloma
Authors: de la Guardia Rafael Díaz , Elosua Carolina , Catalina Purificación , Walker Brian A , Johnson David C , Gonzalez David , Davies Faith E. , Morgan Gareth J , Leone Paola 
Published: October 2019

Abstract
Abstract 4050
The role of the telomeres in the mechanisms of ageing and carcinogenesis has generated a considerable interest as a novel approach to the treatment of many cancers. Telomeres are nucleoproteins structures that protect the ends of eukaryotic chromosomes, which are particularly vulnerable due to progressive shortening in almost all dividing cells. The telomere length was observed as a critical factor in the initiation and progression of human cancers, and it is associated to chromosomal instability.
Most immortal cells possess enzymatic activity of telomerase. This suggests that telomerase activity and telomere length maintenance may be required for unlimited cell proliferation, tumorigenesis, and protection, allowing the evasion of apoptosis in cancer development. The telomerase activity could also be regulated positively or negatively by post-trancriptional and/or post-translational modification of the enzyme without transcriptional up-regulation of human telomerase reverse transcriptase (hTERT) mRNA. In this work, we analyze the expression data of all genes involved in telomerase activity. Patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM) and plasma cell leukemia (PLC) were studied through gene expression profiling analysis (Human Genome U133 Plus 2.0 arrays, Affymetrix). We identify 21 deregulated genes, implicated directly in telomere length maintenance activity in clonal plasma cells compared with normal cells (20 up-regulated and 1 down-regulated). These genes are MYC, KRAS, HSPA9, RB1 and members of the families: Small nucleolar ribonucleoproteins (H/ACA snoRNPs), A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs), and 14-3 -3 family. In conclusion, the myeloma cells acquire the telomere maintenance capability without deregulation of the human telomerase RNA gene (hTERC) and hTERT gene expression. It is an alternative lengthening of telomeres mechanism that has effect in the regulation of the BAD activity in apoptosis. The mechanism is based on preventing the partially-denatured proteins from aggregating, telomere maintenance through the correct processing and intranuclear trafficking of hTERC, telomerase reactivation and telomere stabilization, and efficient accumulation of hTERT in the nucleus. Thus, the findings of this study may help to improve telomerase-based therapy for multiple myeloma.

Disclosures:
No relevant conflicts of interest to declare.
Activity of Dasatinib Against Novel KIT-N822I Mutation In Familial Cutaneous Mastocytosis
Authors: Wasag Bartosz , Niedoszytko Marek , Piskorz Anna , Lange Magdalena , Renke Joanna , Jassem Ewa , Debiec-Rychter Maria , Limon Janusz 
Published: October 2019

Abstract
Abstract 4088

Background:
In contrast to mastocytosis associated with somatic KIT mutations, the accounts of familial forms of mastocytosis with KIT germline mutations are extremely rare. We report the family that met the WHO criteria for cutaneous mastocytosis, which was diagnosed in the father and two children.

Patients and Methods:
The clinical follow-up of mastocytosis in the father included bone marrow histopatological/cytological examinations and flow cytometry, and histopatological examination of the skin. In children tryptase measurement and skin histopatological examination was performed. The father and children presented with urticaria pigmentosa as the only manifestation of the disease. Blood, urine and buccal swabs specimens were collected from the family members. Molecular analysis of the KIT coding sequence revealed a novel missense mutation (p.N822I) in the affected members of the family.
Ba/F3 cell lines expressing KIT-N822I, KIT-D816V and KIT-V559D mutants were treated with different concentrations of imatinib and dasatinib. The effect of treatment on proliferation, survival, and signaling was determined. In addition, Cos-7 cells were transiently transfected with plasmids expressing KIT-WT and KIT-N882I.

Results:
By in vitro assays, both imatinib and dasatinib exhibited a high efficacy toward the control, imatinib-sensitive KIT-V559D mutant. In contrast, only dasatinib potently inhibited the KIT-N822I and control, imatinib-resistant KIT-D816V isoform with an IC50 value of 68 nmol/L and 77nmol/L, respectively. Finally, experiments with Cos-7 cells showed that on the contrary to KIT-WT the KIT-N822I mutation constitutively activated KIT tyrosine phosphorylation.

Conclusion:
In summary, we proved that novel germline KIT mutation (p.N822I) has transforming potential and cause a constitutive activation of KIT. Moreover, we demonstrated that treatment with dasatinib may provide a therapeutic alternative for patients with mastocytosis whose carry imatinib-resistant KIT mutant isoforms.

Disclosures:
No relevant conflicts of interest to declare.
In Vivo Imaging of Cerebral Circulation In Mouse Models of Polycythemia Vera
Authors: Santisakultarm Thom P. , Nishimura Nozomi , Paduano Claire , Skoda Radek C. , Olbricht William L. , Schafer Andrew I. , Schaffer Chris B. , Silver Richard T. 
Published: October 2019

Abstract
Abstract 4091
Polycythemia vera (PV) is a myeloproliferative disease which is often associated with compromised cerebral microcirculation due to thrombotic and other rheological complications; this, in turn, may contribute to cognitive decline. Elevated blood viscosity may also lead to leukocyte adhesion, which further impacts cortical perfusion. An activating mutation, JAK2V617F, in the gene for JAK2, a cytoplasmic tyrosine kinase, is associated with PV in humans and transgenic mice carrying the same mutation have been developed. Our study aims to quantify the alterations in cerebral microcirculation in polycythemic mice and to determine the role of leukocyte adherence in disrupting blood flow, raising the possibility of a possible clinical therapeutic target for patients with PV. We used two models of PV: erythropoietin (EPO) injection (10-100 IU daily subcutaneous injections for 5 days) and a JAK2V617F transgenic mouse. Vascular topology and blood flow was imaged in anesthetized mice, through a craniotomy, using in vivo two-photon excited fluorescence microscopy. Texas-red dextran (0.05 mL of 2.5% w/v) and rhodamine 6G (0.05 mL of 0.1% w/v) were intravenously injected to label blood plasma and leukocytes, respectively. RBC flow speed was measured in arterioles, capillaries, and venules. Brain capillaries were classified as flowing or stalled by evaluating the motion of RBCs within individual vessels. We found a significant decrease in average capillary flow speed in EPO-injected mice, with an average hematocrit (Hct) of 60% (0.70±0.533 mm/s. 10 mice, 105 vessels, p<0.01) and JAK2V617F transgenic mice with Hct of 67% (0.55±0.362 mm/s. 3 mice, 47 vessels, p<0.01) compared to wild type controls with Hct of 48% (1.24±0.986 mm/s. 9 mice, 92 vessels), but not in surface arterioles or venules. In EPO-injected mice, 20% of the capillaries were stalled (12 mice, 6594 vessels, p<0.01), compared to only 3% in control mice (5 mice, 2431 vessels). In JAK2V617F transgenic mice, we found 25% of the capillaries were stalled (4 mice, 4574 vessels, p<0.01). Further, we observed firm leukocyte adherence in a large fraction of the stalled capillaries in the EPO-injected and JAK2V617F transgenic mice (see Figure). Our findings suggest that high Hct creates flow conditions which lead to leukocyte adherence, and may result in leukocyte activation. This work suggests that targeting leukocyte adherence and reducing the hematocrit may be clinically important in patients with PV and other diseases with high blood viscosity to ameliorate abnormal cerebral blood flow.

Disclosures:
No relevant conflicts of interest to declare.