bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting Bromodomains in Myeloma
Authors: Bradner James E. 
Published: October 2019

Abstract
Abstract SCI-6
In cancer, epigenetic proteins are among the most promising and intently pursued targets in drug discovery. This relates to the growing appreciation that cancer growth pathways are centrally influenced by proteins that modify chromatin. Already, inhibitors of DNA methyltransferases and histone deacetylases have demonstrated substantial clinical efficacy, leading to regulatory approval for use in hematologic malignancies. These events have triggered intense competition to develop inhibitors of the chromatin-modifying enzymes known as epigenetic “writers” and “erasers.” Perhaps owing to perceptions regarding the feasibility of targeting protein-protein interactions, small-molecule inhibitors of chromatin-binding modules or epigenetic “readers” have received considerably less attention. Recently, we reported the first potent small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional coactivators. Of broad relevance to cancer, we demonstrated that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate downregulation of c-Myc expression, leading to suppression of a transcriptional program associated with proliferation, survival, and metabolic adaptation. In translational models of MYC-dependent hematologic malignancies, in particular multiple myeloma, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable small molecules. In addition, we have developed a high-throughput platform for modeling mechanism-based, drug-drug combinations to achieve synergy in translational model systems of multiple myeloma. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 350 academic, governmental, and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer.

Disclosures:
Bradner: SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML
Authors: Rao Arati V. , Fedoriw Yuri D. , Richards Kristy L. , Sun Zhen , Love Cassandra L , Dave Sandeep 
Published: October 2019

Abstract

Background
Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML.

Methodology
We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform.

Results
We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs.

Conclusion
This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival.

Disclosures:
No relevant conflicts of interest to declare.
Peripheral Arterial Occlusive Disease (PAOD) In Chronic Phase Chronic Myeloid Leukemia Patients Treated With Nilotinib
Authors: Jeon Young-Woo , Lee Sung-Eun , Choi Soo-Young , Kim Soo-Hyun , Park Jin-Eok , Jeon Hye-Rim , Jang Eun-Jung , Kim Dong-Wook 
Published: October 2019

Abstract

Introduction
ENESTnd trial, nilotinib showed superior efficacy and good tolerability compared with standard dose of imatinib in chronic phase chronic myeloid leukemia (CP CML). However, serious vascular adverse events such as ischemic heart disease (IHD) and peripheral arterial occlusive disease (PAOD) have been reported. Therefore, we have prospectively evaluated for emergence of PAOD in all (CML patients treated with nilotinib.

Materials and methods
Between May 2005 and April 2013, a total of 98 CP CML patients on nilotinib treatment were tested with laboratory studies, electrocardiography and ankle brachial index (ABI) + pulse wave velocity (PWV). Abnormal ABI index was defined with below 1.0 and abnormal PWV as over 1200∼2000 cm/sec adjusted by age. and patients who had abnormal ABI or PWV were performed with computed tomography angiography.

Results
So far, 88 of total 98 patients (57 male) were evaluated with screening test. 17 patients of 88 showed abnormal ABI or PWV. Finally, 3 patients (3.4%) of 17 were diagnosed with overt PAOD by CT angiography (2 upper extremity, 1 lower extremity). The median age was 46 year old (range, 19∼72). The median duration of disease was 49 months (range,1.2 ∼ 65) and 51.3 months (range,36 ∼ 115) in the patients treated with first-line and second-line nilotinib, respectively. The median mean daily dosage of nilotinib was 710mg (range, 656∼800mg) and 737 mg (range, 500∼800mg) in each group. The duration of nilotinib treatment was 46.3 months (range, 0.7∼64.2) and 25.0 months (range, 2.8∼55.8) in each group. Comparing first and second-line nilotinib treated patients, an abnormal ABI or PWV (1 versus 16 patients) and overt PAOD (0 versus 3 patients) was more frequent in patients with second-line nilotinib (p=0.042, p< 0.001, p<0.001).

Conclusion
Our preliminary data suggest an importance of PAOD evaluation in patients with long-term nilotinib treatment (p=0.042) and a routine assessment of PAOD including ischemic heart disease (IHD) is strongly recommended on regular basis.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation Of hOCT1expression In Patients With Chronic Myeloid Leukemia (CML) Treated With Imatinib In First Line
Authors: Couto Mascarenhas Cintia Do , Almeida Maria Helena , Miranda Eliana C M , Virgilio Bruna , Delamain Marcia Torresan , Duarte Gislaine O. , Duarte Vagner O , De Souza Carmino A , Pagnano Katia B. 
Published: October 2019

Abstract

Introduction
The majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP), present satisfactory response to imatinib treatment. However, 25-30% of these pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The human organic cation transporter 1 (hOCT1, SLC22A1), an influx transporter, is responsible for the uptake of imatinib into chronic myeloid leukemia (CML) cells The aim of this study was to analyze hOCT-1 levels at diagnosis of CML patients and correlate with cytogenetics and molecular responses.

Methods
hOCT-1 expression was evaluated in 58 newly diagnosed CML pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis and RNA was obtained from total leucocytes. For cDNA synthesis, 3 ug of RNA was used. hOCT-1 expression was evaluated by real-time PCR with TaqMan probe SLC22A1 (Applied Biosystems) and endogenous GAPDH control. The results were analyzed using 2-ΔΔCT. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RT-PCR (RQ-PCR). Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1%.

Results
58 CML pts, 60% male, median age of 46 years (19-87) were evaluated, 71% in chronic phase (CP), 21% in accelerated phase (AP) and 5% in blast crisis (BC). The mean and median of hOCT-1 transcript levels in the total group was 2.03 and 0.961 respectively (0.008–19.039) and CP pts was 1.86 and 1.00 (0.008-10.34).The median duration of imatinib treatment was 27 months (1-109) and 96.6% achieved complete hematological response, 79.3% complete cytogenetic response and 69% major or complete molecular response. The regression analysis showed correlation between higher transcript levels of hOCT-1 and BCR-ABL transcripts<10%) at 3 months analysis (p<0.0001). Albeit, there was no influence of the hOCT-1 transcript levels at diagnosis in the achievement of cytogenetic and molecular response at 24 months of treatment.

Conclusions
In this report, we found that high hOCT-1 expression was predictive of BCR-ABL transcripts<10% at 3 months, although we did not find correlation between hOCT-1 levels at diagnosis and the achievement of molecular response at 24 months, studies show that there is correlation between BCR-ABL log reduction in the first months of treatment and the achievement of molecular response.

Disclosures:
No relevant conflicts of interest to declare.
Mutations In Transcription Factor NF-E2 Confer Resistance To Interferon Treatment In MPN Patients
Authors: Jutzi Jonas S. , Gründer Albert , Kaiser Sandra , Nikoloski Gorica , Jansen Joop H. , Pahl Heike L. 
Published: October 2019

Abstract
Recently, we described insertion and deletion mutations in transcription factor NF-E2 in a subset of patients with Myeloproliferative Neoplasms (MPN), most frequently patients with primary or secondary myelofibrosis (PMF or sMF, respectively) as well as patients with polycythemia vera (PV). These mutations lead to the formation of truncated NF-E2 proteins that lack DNA binding activity. Nonetheless, mutant NF-E2 proteins augment wild-type NF-E2 activity both in vitro and in vivo in a murine bone marrow transplant model. Mice expressing mutant NF-E2 develop erythrocytosis and thrombocytosis.
Here, we demonstrate that, in contrast to mice expressing wild-type NF-E2, treatment of mice expressing mutant NF-E2 with interferon-alpha does not lower red blood cell numbers (RBC), white blood cell counts (WBC) or platelet numbers. While these parameters are significantly decreased by interferon-alpha (IFN) in mice expressing wild-type NF-E2, mice expressing mutant NF-E2 showed no response to interferon treatment in these counts over a four month period.
Two PV patients carrying truncating mutations in NF-E2 received IFN treatment. Blood samples were available both from before treatment as well as from 3 and 4 years after the beginning of therapy, respectively. Purified granulocytes from these samples were analyzed for mutant NF-E2 allele burden by GeneScan analysis. In both patients, the mutant NF-E2 allele burden rose during IFN treatment, increasing from 1% to 30% in one patient and from 24% to 100% in the second.
We have demonstrated that acquisition of an NF-E2 mutation confers a proliferative advantage on cells already carrying the JAK2V617F mutation. Using colony assays from primary MPN cells, we showed that cells carrying both mutations outcompete cells carrying only the JAK2V617F mutation in vivo. Cells expressing both mutant NF-E2 and JAK2V617F are statistically significantly more frequently in the S-phase of the cell cycle and express significantly higher levels of several proteins that promote G1/S transition, cyclin D3, CDK4 and CDK6. The data presented here suggest that IFN treatment is insufficient to counteract the proliferative drive conferred by mutant NF-E2.

Disclosures:
No relevant conflicts of interest to declare.
Identification Of FOXO3A As a Key Downstream Target For The Oncogenic JAK2V617F
Authors: Kobayashi Masaki , Araki Marito , Hironaka Yumi , Ohsaka Akimichi , Komatsu Norio 
Published: October 2019

Abstract
A gain-of-function mutant JAK2 has been demonstrated to play a causal role in myeloproliferative neoplasms (MPN) development. The mutant JAK2, typically a substitution of position 617 valine (V) to phenylalanine (F) (hereinafter called JAK2V617F), activates STAT, Akt, and ERK pathways, which subsequently promotes cell proliferation. Although these pathways are well documented, their downstream effectors in MPN are still poorly understood.
FOXO3A, a subfamily of Forkhead transcription factors, paradoxically regulates cell death and survival in a variety of cells. We and others have previously shown that an oncogene product Bcr-Abl tyrosine kinase suppresses FOXO3A activity by phosphorylation and subsequently represses cell death in chronic myelogenous leukemia (CML), proposing a potential therapeutic strategy for CML treatment. Thus, defining FOXO3A function and uncovering its regulation in MPN are important for the understanding of this disease and the development of a novel therapeutic strategy.
To demonstrate this, we employed a model cell line Ba/F3/EpoR that requires IL-3 for survival but becomes independent of the cytokine when JAK2V617F is expressed. When JAK2V617F-expressing Ba/F3/EpoR cells were treated with a JAK2 inhibitor, FOXO3A phosphorylation diminished in a dose-dependent manner. The trend of diminution paralleled to the reduction of phospho-STAT5, a major JAK2V617F substrate, suggesting that FOXO3A phosphorylation is regulated by JAK2V617F activity. Removal of JAK2 inhibitor from the media induced a massive FOXO3A phosphorylation within 20 minutes in JAK2V617F-expressing but not in the wild-type JAK2-expressing Ba/F3/EpoR cells. This observation indicates that at least in this model system, JAK2V617F positively modulates the phosphorylation of FOXO3A.
To further investigate the function of FOXO3A in JAK2V617F-expressing Ba/F3/EpoR cells, we knocked down FOXO3A expression by shRNA. When treated with a JAK2 inhibitor to induce apoptotic cell death, FOXO3A-knocked down cells were more resistant to the inhibitor than the control cells. This strongly suggests that when JAK2V617F activity is blocked and subsequently FOXO3A is dephosphorylated, FOXO3A induces apoptosis in JAK2V617F-expressing cells.
Finally, we elucidated that the inhibition of JAK2V617F activity results in a reduction of FOXO3A phosphorylation and the induction of apoptosis in CD34-positive cells obtained from MPN patients. Taken together, we propose a model that shows JAK2V617F induces FOXO3A phosphorylation and subsequently blocks FOXO3A activity to repress apoptosis in JAK2V617F positive MPN cells.

Disclosures:
Kobayashi: Sysmex Corporation: Employment. Komatsu:Sysmex Corporation: Research Funding.
CD8+ /ICOS+ /CXCR5+ Tumor Infiltrating T-CELLS (CD8fc) Sharing Functional Similarities With TFH CELLS ARE Present In Classical Hodgkin’S Lymphoma Tissues Displaying A Specific “MIXED NODULARITY” Pattern
Authors: Le Thi Suong , Broussais Florence , Bouabdallah Reda , gondois-Rey Françoise , Xerri Luc , Olive Daniel 
Published: October 2019

Abstract
We have previously reported that some classical Hodgkin’s Lymphoma (cHL) tissues display a gene signature evocative of a Th1 immune reaction. In order to better characterize this process, immune cell subsets were isolated from cHL tissue samples (n=21) using a powerful multicolor flow cytometry method, in parallel with cell sorting. Fresh tissue samples from follicular B cell lymphoma (FL, n=8), diffuse large cell B cell lymphoma (n=8) and reactive lymphadenitis (n= 5) were used as controls.
In 4 cLH cases, we observed a significant proportion of activated CD8+ T-cells expressing ICOS and CXCR5 at high levels. The presence of either CD8+/ICOS+/CXCR5- T cells or CD8+/ICOS +/ CXCR5+ T-cells was a specific feature of HL tissues since it was absent from B-cell lymphomas, T-cell lymphomas and reactive tissues. In contrast, CD8+/CXCR5+ T-cells were found not only in cHL, but also in most other samples analyzed.
Further phenotypic characterization showed that the CD8+/ICOS +/ CXCR5+ T cells expressed markers associated with CD4 TFH cells, like PD1, BTLA, bcl-6 and IL-21. Under stimulation, they expressed only low levels of IFNG, granzyme B and perforin, and thus do not fulfill the criteria of activated cytotoxic effectors. Co-culture experiments showed a dramatic enhancement of CD86 expression on stimulated B-cells in contact with CD8+/ICOS +/ CXCR5+ T cells. This effect was similarly observed after co-culture with CD4+TFH cells. The 4 cHL cases associated with CD8+/ICOS +/ CXCR5+ T-cells contained CD30+ CD15+ EBV+ Reed Sternberg cells (RSC). They were characterized a nodular non-sclerotic pattern reminiscent of the nodular lymphocyte-rich classical HL (NLRCHL) subtype, but also displayed a specific “mixed nodularity” feature. Various nodules were indeed observed, including reactive germinal centers (GC) partly colonized by RSC co-localizing with CD8+/ICOS+ T-cells, suggesting an early GC invasion triggering an intra-follicular CD8 T-cell reaction. Other nodules were composed of a high number of RS cells admixed with numerous CD8+/ICOS+ T-cells. This “mixed nodularity” pattern was absent in the other HL cases.
Altogether, our results point out a previously unrecognized intra- follicular CD8 T-cell subset sharing phenotypic and functional features with CD4 TFH, that we have thus considered as putative “follicular cytotoxic” CD8 T-cells (TFC). This cell subset appears to be specifically associated with EBV+ cHL tissues with unusual histo-phenotypic features, which may probably reflect a strong CD8 activation process.

Disclosures:
No relevant conflicts of interest to declare.
MCL-1 Critically Regulates Survival Of T-Lymphocyte Non-Hodgkin Lymphoma
Authors: Spinner Sabine , Gemma Kelly , Herold Marco , Pechloff Konstanze , Crispatzu Giuliano , Ruland Jürgen , Herling Marco , Peschel Christian , Strasser Andreas , Jost Philipp J. 
Published: October 2019

Abstract
The heterogeneous subgroup of T-lymphocyte non-Hodgkin lymphoma (T-NHL) exhibits an aggressive growth pattern and is relatively insensitive to chemotherapy. The molecular mechanisms of survival and resistance to therapy remain largely undefined. We hypothesized that BCL-2 proteins with anti-apoptotic properties protect T-NHL cells from cell death, cause resistance to therapy and therefore represent an attractive target for therapy. MCL-1 potently protects physiologically healthy T-lymphocytes from cell death, but its function in transformed T-cells is incompletely understood. We therefore dissected the role of MCL-1 using in silico meta-analyses on available gene expression data on human primary T-NHL samples. We identified MCL-1 as the primary BCL-2 family protein to be highly expressed across most major T-NHL subsets. Expression of MCL-1 was restricted to its anti-apoptotic full-length splice variant as opposed to its pro-apoptotic short isoform. To functionally characterize the requirement for MCL-1, we utilized a T-NHL mouse model, which is based on four consecutive low-dose whole-body γ-irradiations in 4 week-old mice resulting in the development of T-NHL mimicking human peripheral T-cell lymphoma. We utilized mice harbouring loxP-flanked Mcl-1 or loxP-flanked Bcl-x(L) in combination with inducible Cre recombinase to conditionally delete the gene of interest after lymphoma induction. Interestingly, conditional deletion of only one allele of Mcl-1 in fully established primary T-NHL cells ex vivo led to a significant and specific loss of viability, whereas survival remained unaffected by full deletion of Bcl-x(L). Reduced MCL-1 levels resulted in substantially elevated sensitivity to standard chemotherapeutics such as anthracyclins, cyclophosphamide and etoposide. In addition, mono-allelic deletion of Mcl-1 in vivo prolonged survival of lymphoma-bearing mice. Together, these data argue that anti-apoptotic MCL-1 is the single most important BCL-2 family member involved in the sustained survival of T-NHL.

Disclosures:
Strasser: Genentech Inc: Consultancy.