bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells
Authors: Amit Amin Dipak , Pongtornpipat Praechompoo , Schatz Jonathan H. 
Published: October 2019

Abstract
Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase.

Disclosures:
No relevant conflicts of interest to declare.
Activities Of Commonly Used Tyrosine Kinase Inhibitors Against Wild-Type C-KIT and Potential Impact On Normal Hematopoiesis
Authors: Galanis Allison , Levis Mark 
Published: October 2019

Abstract
The receptor tyrosine kinase (RTK) c-KIT is important in early hematopoiesis, and impaired c-KIT activity is associated with abnormal erythroid and megakaryotic function in animal models. Several approved and investigational small molecule tyrosine kinase inhibitors have off-target activity against c-KIT, with resultant potential for hematopoietic toxicity. To investigate the association between c-KIT inhibition by TKIs and myelosuppression, we determined the inhibitory activity of a series of TKIs against c-KIT using immunoblot estimates of IC50 concentrations, and compared those results with the compounds’ effects on human hematopoiesis in progenitor cell assays. For this study, we used quizartinib, crenolanib, sorafenib, imatinib, pazopanib, and dasatinib. IC50s against FLT3 and c-Kit were established for each kinase with assays performed both in culture medium (RPMI/10%fetal bovine serum) and 100% human plasma. Colony formation assays with normal human bone marrow were performed in quadruplicate with clinically achievable levels of each drug, adjusting for plasma protein binding (and based on published pharmacokinetic data from plasma samples of human subjects). Out of the six TKIs studied, most showed limited to no inhibition of the formation of colony-forming unit-granulocyte macrophage (CFU-GM). Three TKIs, quizartinib, pazopanib, and dasatinib, demonstrated inhibition of burst-forming unit-erythroid (BFU-E) colonies. The highest doses of these drugs inhibited growth of BFU-E colonies by 30% versus untreated control. These same three drugs are also the most potent c-Kit inhibitors of this group, with IC50s of 35, 7.5, and 1.4 nM, respectively, in culture medium. The remaining TKIs, crenolanib, sorafenib, and imatinib, showed far less BFU-E inhibition, none inhibiting colony formation by more than 25%. These TKIs also had higher c-Kit IC50 values of 65, 395, and 122 nM, respectively. We conclude that c-KIT inhibition correlates with eythroid progenitor cell suppression in vitro, and that TKIs that achieve c-KIT inhibitory levels in vivo would be predicted to cause anemia. During clinical development of TKIs that have c-KIT inhibitory activity, attention should be paid to the therapeutic index between the targeted kinase (e.g., FLT3) and c-KIT, so as to avoid unnecessary myelosuppresive effects. Dose range determinations to spare c-Kit inhibition would lead to a decreased risk of disrupting normal hematopoiesis, making treatment far more manageable for these patients.

Disclosures:
Levis: Ambit Biosciences: Consultancy.
Oxidation Of Peroxiredoxins By Thioredoxin Inactivation Is Mediated In Arsenic Trioxide-Induced Reactive Oxygen Species Formation and Its Cytotoxicity In Acute Promyelocytic Leukemia Cells
Authors: Mun Yeung-Chul , Ahn Jee-Young , Yoo Eun-Sun , Huh Jungwon , Lee Kyoung Eun , Nam Eunmi , Lee Soon Nam , Seong Chu-Myong 
Published: October 2019

Abstract

Backgrounds
The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL). ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) and thioredoxin (TRX) remains elusive. Aim of current study is to elucidate the mechanism of redox enzymes to elevate ROS during ATO-induced apoptosis in APL-derived NB4 cells.

Methods
NB4 cell line, which is one of the human acute promyelocytic leukemia cell lines, was cultured in RPMI-1640 medium supplemented with 10% FBS in CO2 humidified atmosphere at 37°C. NB4 cells were cultured with 2 μM arsenic trioxide to induce apoptosis for 16-48 hours. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Monomer/Dimer assay for PRX I, PRX II, PRX III, and TRX I was performed by western blot using non-reducing gel.

Results
Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO but decreased after 24 hour of ATO. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Monomer, indicated active and reduced form, of peroxiredoxins was decreased and cysteine sulfinic acid (CP–SO2H) peroxiredoxins, indicated inactive and oxidized peroxiredoxins, was increased in NB4 cells after ATO treatment as time goes by. Similarily, monomer of thioredoxin-1 (active thioredoxin) was decreased and multimer of thioredoxin-1 (inactive thioredoxin) was increased in NB4 cells after ATO treatment as time elapsed.

Conclusions
Our data showed inactivation of peroxiredoxins by oxidation was developed during ATO-induced ROS generation and APL cell apoptosis. These peroxiredoxins oxidation was probably due to increment of reduced thioredoxin in NB4 cells after ATO treatment. These findings suggest ATO-induced anti-leukemic activity is more likely due to a TRX system-mediated cellular redox changes. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells.

Disclosures:
No relevant conflicts of interest to declare.
A Prospective, Multicenter Phase II Study Of Continuous Infusion Of FLAG For Patients Older Than 60 Years With Resistant Acute Myeloid Leukemia: A Comparison With Intensive Younger patients’ Trial
Authors: Kim Hawk , Lee Je-Hwan , Joo Young-Don , Bae Sung-Hwa , Lee Jung-Hee , Kim Dae-Young , Jo Jae-Cheol , Park Jae Hoo , Lee Won Sik , Ryoo Hun-Mo , Park Sang-Kyu , Lee Kyoo-Hyung 
Published: October 2019

Abstract
Relapsed/refractory acute myeloid leukemia (R/R AML) is hard to treat especially in elderly patients. We previously assessed continuous infusion (CI) of fludarabine and cytarabine plus idarubicin (CI-FLAG2) for patients under 65-years old with R/R AML. Also we started prospective phase II study of attenuated version of CI-FLAG2 for elderly patients (C-FLAG). R/R AML in elderly (¡Ã60 years old) patients were eligible. Induction chemotherapy consisted fludarabine and cytarabine (ARAC) as a 24-hr CI without idarubicin. Total 38 and 68 patients were enrolled in CI-FLAG1 and CI-FLAG2, respectively. There were no differences in terms of patients’ characteristics except for median age (p<0.001), HCT prior to salvage (p=0.002), WBC>20K/uL at salvage (p=0.004) and PB blast>40% at salvage, all which factors were unfavorable in C-FLAG. When comparing outcomes between CI-FLAG2 and C-FLAG, there were no difference in terms of CR rate (p=0.572) and objective response rate (ORR; p=0.899). Treatment failure patterns were also similar between C-FLAG and CI-FLAG2 (p=0.742). The most common treatment failure was resistant (66.7%) in C-FLAG. There were more frequent HCT in CI-FLAG2 (p<0.001) and consolidation chemotherapy in C-FALG (p=0.001). Poor predictors on ORR in C-FLAG were PB WBC>20K/uL at salvage (p=0.024) and PB blast >0% on early evaluation (p=0.013) by multivariate analysis. The overall survival of patients who achieve CR/CRp/CRi showed significantly prolonged survival compared with patients who did not in C-CLAG (p<0.001; Figure 1A). The median overall survivals were similar between CI-FLAG2 and C-FALG (p=0.427; Figure 1B).Figure 1Overall survivalFigure 1. Overall survival
Attenuated salvage regimen C-FALG in elderly patients was as effective as more intensive younger patients’ regimen CI-FALG2 in terms of response and survival although elderly patients had more unfavorable clinical characteristics.

Disclosures:
No relevant conflicts of interest to declare.
Analysis Of E2A Point Mutations In B Cell Leukemia
Authors: Soans Eroica , Choi John K 
Published: October 2019

Abstract

Introduction
TCF3 encodes for E2A protein, which belongs to the helix loop helix transcription factor family. E2A activates transcription of downstream genes by binding to E-box motifs as a homo or hetero dimer. E2A plays an important role in B lymphocyte development. Therefore deletion or mutations in TCF3 or even lowered activity of E2A are causes of B cell leukemia and lymphomas. Recently, three mutations V557E, D561E and N551K in E2A were isolated in Burkitt’s lymphoma (Schmitz, Young et al. 2012). The first two mutations are present in the homo dimerization region of E2A while N551K is present in the DNA binding region. Though the paper enumerated role of TCF3 in Burkitt’s lymphoma but the significance of these TCF3 mutations or mechanism needed further characterization. We hypothesized that these TCF3 mutations have an alternate mechanism as compared to wild type TCF3 and therefore may affect B cell development.

Methods
We characterized three TCF3 mutants by cloning them into in MIGR1 backbone using TOPO cloning. E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. DNA binding activity was measured using a DNA protein binding colorimetric assay.

Results
V557E and D561E mutants have lower activity as compared to wild type E2A as studied using E2A-specific luciferase reporter assay; while N551K showed no activity in the same assay as compared to wild type E2A activity. Similarly V557E and D561 form weaker bonds with the E box motifs while N551K showed no DNA binding activity as studied using colorimetric DNA-protein binding assay. The plasmid expressions were verified using western blot analysis.

Conclusion
Our findings suggest mutations V557E and D561E may follow a similar pathway as wild-type E2A but have lower activity. The N551K mutation has an alternate pathway to wild type TCF3 that may impact B cell proliferation, survival and development.

Disclosures:
No relevant conflicts of interest to declare.
Cardiac Derived Stromal Cells Inhibit Tumor Cell Proliferation: A Potential Role Of miR206
Authors: McNiece Ian K. , Sivajothi Santhosh 
Published: October 2019

Abstract
Stromal cells play an important role in control of proliferation and differentiation of stem cells and are a key component of the stem cell niche. Bone marrow (BM) stromal cells (also termed mesenchymal stem cells; MSC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HSCs) in part through secreted growth factors. Recent studies have demonstrated the presence of stromal cells in cardiac tissue, however the role of cardiac stromal cells (CStrC) is unclear. In this study we have compared human CStrCs to human BM-MSCs and demonstrate that CStrCs have a similar morphology and surface marker expression as BM-MSCs. To further characterize the CStrCs we performed micro array analysis of human CStrCs compared to human BM-MSCs. The CStrCs expressed a distinct cytokine and cytokine receptor profile compared to BM-MSC. In addition, a number of micro RNAs were expressed at very high levels in CStrCs compared to BM-MSC. Cardiac- associated microRNAs, including miR-1, miR-133a, and miR-206 were expressed at higher levels in CStrCs compared to BM-MSC. Given the lack of tumor development in cardiac tissue we hypothesized that CStrCs would fail to support tumor cell growth which has been described for BM-MSCs. Therefore, we cultured human tumor cell lines on CStrCs and compared the tumor cell proliferation to BM-MSCs. CStrCs inhibited the proliferation of a range of tumor cell lines including myeloid cell lines HL60, K562, myeloma cell lines U-266, RPMI 8266, ARP-1 and the B cell line Raji, while BM-MSCs supported the proliferation of tumor cells. Further we tested media conditioned by CStrCs and demonstrated inhibition of proliferation of both cell lines. Previous studies have implicated miR-206 in inhibition of proliferation of tumor cells and given the high levels of expression of miR-206 in CStrCs we hypothesize that miR-206 is a key player in the inhibitory effects of CStrCs and this effect is mediated via secreted molecules.

Disclosures:
McNiece: Proteonomix Inc: Consultancy.
A Method To Identify Copy Number Aberrations (CNAs) From Whole Exome Sequence (WES) Data and Its Application To Multiple Myeloma Cell Lines and Patient Samples
Authors: Degenhardt Jeremiah D , Hoehn Kenneth B , Kwei Kevin A , Keats Jonathan J , Kirk Christopher J. , Tuch Brian B 
Published: October 2019

Abstract
Duplications and deletions of large-scale genomic regions, including whole chromosomes, are a hallmark of many malignant tumors. Gross cytogenetic changes of this type were among the earliest genomic lesions to be observed in tumors and represent an important predictor of outcome for many tumor types. In multiple myeloma, a core set of structural variants are regularly used for disease prognosis (e.g., del17p, t(4;14), t(14,16)). The presence of one or more of these lesions is predictive of a more aggressive disease type and poor outcomes for patients. The application of next-generation sequencing (NGS) to cancer samples has enhanced our ability to detect copy number aberrations (CNAs) and identify driver mutations. The NGS approach can generate higher resolution maps of these mutations than traditional methods, which in turn enables faster identification of the functionally relevant genes they harbor.
Most NGS-based methods for the identification of CNAs in tumors require whole genome sequencing (WGS) as input. Relatively fewer methods exist for the detection of CNAs from exome sequencing and, to our knowledge, these methods all rely on having matched normal data. However, WES is still far more common than WGS and matched normal samples are frequently not available. Therefore, we have developed a novel method for the identification of CNAs from single-sample WES data. Our method combines LOWESS smoothing & discrete-wavelet-transformation to normalize the exome coverage data with an HMM for coverage segmentation. We have applied our new method to WES data from 32 myeloma cell-lines for which aCGH copy number calls were also available for training and validation. Our method shows a high correlation with the aCGH calls (mean = 95%). We are currently applying the method to a set of 102 CD138+ tumor cell samples that were collected during patient screening on three Onyx-sponsored Phase 2 clinical trials of the proteasome inhibitor carfilzomib. We will report on the frequency of known and novel lesions in these samples, comparing to what we’ve observed in cell lines and other publically available CNA datasets.

Disclosures:
Degenhardt: Onyx Pharmaceuticals: Employment, Equity Ownership. Hoehn:Onyx Pharmaceuticals: Employment. Kwei:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Tuch:Onyx Pharmaceuticals: Employment, Equity Ownership.
A Novel Mutation In Bruton Tyrosine Kinase Confers Acquired Resistance To Ibrutinib (PCI-32765) In CLL
Authors: Furman Richard R. , Cheng Shuhua , Lu Pin , Setty Menu , Perez Alexandar , Guo Ailin , Racchumi Joelle , Ma Jiao , Coleman Morton , Venkataraman Kabaleeswaran , Xu Guozhou , Chen Wei , Wu Hao , Chang Betty Y. , Leslie Christina , Wang Y. Lynn 
Published: October 2019

Abstract
The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib has produced durable remissions in chronic lymphocytic leukemia (CLL). We describe a CLL patient who progressed while receiving ibrutinib following 20 months of once daily dosing. A cysteine-to-serine amino acid replacement was identified in BTK at position 481 that disrupts the covalent, but not non-covalent, binding of ibrutinib to BTK in silico structural modeling1. The mutation was present in relapsed samples while absent in the pre-treatment and responding samples. Following the mutation, the B cell receptor (BCR) pathway was reactivated as evidenced by increased cell signaling activities and gene expression profiles. Comparing the relapsed samples with the pre-treatment and responding samples, at the cellular level, mutated CLL cells displayed higher levels of the cell proliferation marker Ki67 in vivo and higher levels of ex-vivo BrdU incorporation. Transfection of the C481S mutant construct into a sensitive lymphoma cell line rendered it much more resistant to ibrutinib treatment demonstrating the cellular impact of the mutation (see attached graph). Interestingly, the ibrutinib-resistant CLL cells remained sensitive to other BCR inhibitors such as dasatinib and SYK inhibitors. These results confirm BTK as an important pharmacologic target of ibrutinib. Further, a mechanism of resistance was revealed, and alternative therapeutic options for ibrutinib resistance were explored. (First three authors with equal contribution)

Disclosures:
Furman: Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Chang:Pharmacyclics: Employment, Equity Ownership.
Low-dose Triptolide could enhance the Idarubicin-induced apoptosis in KG1α cells through down-regulation of MDR1 and NF-κb
Authors: Xu Bing , Tang Jiahong , Chen Feili , Li Yanyan , Dong Huijuan , Liu Yuejian , Zhou Shuyun 
Published: October 2019

Abstract

Background
Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, TPL has also been shown to have strong anti-cancer ability both in vitro and in vivo. It could induce apoptosis in a variety of cancer cell lines. However, the clinical applications of TPL are limited by its narrow therapeutic window and severe toxicity. Thus, the focus of study about TPL has been switched to the ability of low dose of TPL in enhancing the drug-induced apoptosis of cancer cells. KG1a cell line is a kind of cell line which has been demonstrated to have a certain proportion of leukemia stem cells which are responsible for the relapse of leukemia. Usage of this kind of cell line could resemble the relapse situation of patients. MDR1 and NF-ΚB are two important factors closely related to drug-resistance. Dow-regulation of these two genes could lead to the sensitization of cells to drugs.

Aims
This study aims to explore whether low-dose TPL could enhance the Idarubicin-induced apoptosis in KG1a cells through down-regulation of MDR1 and NF-κB.

Methods
Hoechst 33342 staining and flow cytometry analysis were used to determine the ability of TPL in enhancing the Idarubicin-induced apoptosis in KG1a cells. RT-PCR as well as western blotting analysis were used to determine whether TPL combined with Idarubicin could down-regulate MDR1 and NF-κB.

Results
Hoechst 33342 staining and flow cytometry analysis showed that low-dose of TPL((IC20F5.0nM)) could significantly enhance the Idarubicin-induced apoptosis in KG1a cells(Idarubicin alone vs Idarubicn combined with TPL: 15.13%: 60.17%; P<0.05). TPL combined with Idarubicin could down-regulate expression of MDR1 both at protein and mRNA level with the down-regulation of NF-κB at protein level.

Conclusion
Low-dose Triptolide could enhance the Idarubicin-induced apoptosis IN KG1a cells through down-regulation of MDR1 and NF-κB

Disclosures:
No relevant conflicts of interest to declare.