bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lenalidomide Enhances Anti-Tumor Effect of γδ T Cells against Mantle Cell Lymphoma
Authors: Gaidarova Svetlana , Corral Laura G , Gleizer Emilia , Young David , Brady Helen , Bennett Brydon , Lopez-Girona Antonia 
Published: April 2020

Abstract
Introduction: Mantle cell lymphoma (MCL) has had one of the poorest prognoses of all NHL subtypes, and is characterized by a relatively short duration of response, progression-free survival, and time-to-progression, with most patients relapsing. The immunomodulatory drug lenalidomide has shown promising activity in MCL and currently is being investigated further in the clinic for treatment of this tumor. In previous experiments, lenalidomide stimulated proliferation as well as activation of γδ T cells in vitro. Potential mechanisms of lenalidomide’s anti-tumor activity in MCL were investigated here.
Methods: As γδ T lymphocytes mediate anti-tumor effects through antigen recognition and intracellular signaling, we assessed the effect of lenalidomide on tumor cell expression of co-stimulatory and antigen-presenting molecules, as well as other immune response molecules, using microarray analysis. We then assessed the impact of these tumor cell changes on tumor-cell recognition by γδ T lymphocytes. We treated peripheral blood mononuclear cells (PBMCs) from healthy donors with vehicle or lenalidomide. We next purified γδ T cells from control or lenalidomide treated PBMCs and co-cultured them with MCL cells.
Results: Lenalidomide upregulated the expression of several genes involved in the immune response, cell adhesion, cytokine-cytokine receptor interaction, cell cycle and apoptosis by at least 2-fold. In the immune response category, co-stimulatory and antigen-presenting molecules were strongly up regulated including CD86, CD40, CD58, and CD1c. The lenalidomide-induced up-regulation of CD1c was confirmed in MCL lines by quantitative RT-PCR as well as flow cytometry and was also observed in primary B-CLL cells and normal B cells treated with lenalidomide ex-vivo. CD1c, a member of the CD1 family of MHC-like molecules, is expressed mainly in professional antigen-presenting cells, such as B cells, and mediates the presentation of lipid antigens to γδ T cells. Lenalidomide strongly promoted expansion of the Vδ1 subpopulation of γδ T cells and enhanced production of interferon-g. Treatment of MCL cells and γδ T cells with lenalidomide induced changes in the organization of the actin cytoskeleton, re-localization of surface markers and enhanced the number of γδ T-MCL synapses. Furthermore, gd T cells, expanded in the presence of lenalidomide, had enhanced killing activity against MCL cells.
Conclusion: Our studies show that lenalidomide might provide dual activity against MCL cells by inducing expression of CD1c in the tumor cells and by enhancing γδ T cell-mediated cytotoxicity. The therapeutic implications, as well as the prospects for novel biomarker development will be discussed further.
Antileukemia Activity of JNJ-26481585, a Potent Second-Generation Histone Deacetylase Inhibitor
Authors: Tong Weigang , Wei Yue , Stevenson William , Kuang Shao-Qing , Arts Janine , Garcia-Manero Guillermo 
Published: April 2020

Abstract
Background: Histone Deacetylases (HDACs) play key roles in tumor cell proliferation, survival and angiogenesis. HDAC inhibitors have been shown to induce cell cycle arrest, terminal differentiation and/or apoptosis in broad spectrum of human tumors and xenograft animal models. JNJ-26481585 is a hydroxamic acid derivative second-generation pan-HDAC inhibitor with nM potency in pre-clinical studies. In animal models, no evidence of cardiac toxicity has been observed, which suggests a better therapeutic index than other HDAC inhibitors.
Aim: We evaluated the anti-leukemia and molecular activity of JNJ-26481585 in human leukemia cell lines (HL60, MOLT4, THP1 and U937) and primary leukemia cells.
Results: JNJ-26481585 potently inhibited proliferation of leukemia cells, with IC50 between 25 nM and 250 nM after 24-hour of treatment. In contrast, analysis of other HDAC inhibitors, including vorinostat and MGCD0103, had IC50 in the μM range. JNJ-26481585 also induced dose-dependent apoptosis (aprox. 25–35% at concentration of 25 nM) of leukemia cell lines by Annexin V staining after 24-hour treatment. A synergistic apoptotic effect was observed by co-treating with hypomethylating agent 5-aza-2′-deoxycytidine (25% with JNJ-26481585 25 nM, 5% with decitabine 1 μM, and 70% for the combination). Acetylation of histones H3, H4 and phosphorylation of H2AX was also observed after exposure to JNJ-26481585 at concentration of 12.5 nM or higher. We also studied fresh primary leukemia cells from 9 patients (ALL N=1, MDS N=1, AML N=7). Six of the 9 patients (67%) had relapsed/refractory disease. Their median white cell counts were 32×103/L (range 6–407×103/L), with 25% to 95% blasts in the peripheral blood. Treatment with JNJ-26481585 for 24-hours induced significant growth arrest and apoptosis (20–50% apoptosis at 25 nM) in a dose-dependent manner (between 12.5–500 nM), but not in mononuclear cells from healthy donors (N=2). It also induced acetylation of histones H3, H4, phosphorylation of H2AX, induction of p21 and HSP70, activation of caspase-3, cleavage of PARP in these primary leukemia cells after 24-hour treatment at concentrations of 12.5–500 nM.
Conclusion: JNJ-26481585 is a potent second generation pan-HDAC inhibitor with activity in human leukemias. It is a promising new agent for future clinical studies either as single agent or combination epigenetic therapy with DNA hypomethylating agents and should be tested in clinical trials.
Small Molecule Inhibition of Cdc7, a Key Cell Cycle Regulator and Novel Therapeutic Target, Successfully Inhibits Leukemia Cell Growth in Vitro and in Vivo
Authors: Frattini Mark G. , Shum David , O’Dwyer Kristen M , Brentjens Renier J. , Yeh Ray , Maslak Peter , Heaney Mark Lawrence , Jurcic Joseph , Djaballah Hakim , Kelly Thomas J 
Published: April 2020

Abstract
Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.
Diagnosis and Classification of Amyloidosis in Abdominal Subcutaneous Fat Aspiration Specimens Using Mass Spectrometry Based Proteomics
Authors: Vrana Julie A. , Zeldenrust Steven R. , Theis Jason D. , Gamez Jeffrey D. , Kurtin Paul J. , Dogan Ahmet 
Published: April 2020

Abstract
Abdominal subcutaneous fat aspiration is one of the most practical, sensitive and specific methods for the diagnosis of systemic amyloidosis. One limitation of this method, compared to more invasive tissue biopsy based approaches, remains the technical difficulties in further classification of the amyloidosis as commonly used methods, such as immunohistochemistry, are not readily applicable to fat aspiration specimens. To overcome these difficulties we developed a method using nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) that could identify amyloid subtypes in freshly obtained Congo Red positive fat aspirate specimens with great accuracy.
Abdominal subcutaneous fat aspirate specimens were obtained from 73 patients with clinical suspicion for systemic amyloidosis. One half of the specimen was stained with Congo red and used for diagnosis of amyloidosis and the other half was processed and enzyme digested for LC-MS/MS analysis. The resulting LC-MS/MS data was correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Of the 73 cases studied, 41 were positive for Congo red consistent with systemic amyloidosis. In Congo red positive cases, LC-MS/MS peptide profiles consistent with AL-lambda (28/31), AL-kappa (6/7), and ATTR (2/3) were observed. Only one case in the Congo red negative control group (31/32) gave a kappa light chain profile which was attributed to a high level of kappa in the serum (285 mg/dL). Of the 35 out of 41 cases of systemic amyloidosis successfully classified by LC-MS/MS, additional clinical and pathology data validating the amyloid type was available. In each of these cases the MS/MS results accurately predicted the amyloid type.
In conclusion, LC-MS/MS proteomic analysis of abdominal subcutaneous fat aspiration specimens involved by amyloidosis provides a highly specific (97% specificity) and sensitive (&gt;85% sensitivity) method for diagnosis and classification of amyloidosis. The method is rapid and readily applicable in a clinical setting and will greatly improve the clinical management of amyloidosis patients.
A Novel Targeted Therapeutic Approach to Multiple Myeloma Using Tumor Specific Anti-Sense RNA Sequences
Authors: Li Zhi-Wei , Shalitin Dror , Shen Jing , Share Michael , Li Mingjie , Wang Cathy S , Sanchez Eric , Steinberg Jeffrey A , Chen Haiming , Bonavida Benjamin , Berenson James R. 
Published: April 2020

Abstract
Despite recent advances in the therapy of multiple myeloma (MM), the disease remains incurable. Current drugs for MM target not only MM but also normal cells. This limits the efficacy of these agents and may lead to significant morbidities for patients. Therefore, novel strategies are needed that allow targeting of tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells of these patients. We have explored the possibility of using a unique sequence from this transcript, the complementarity determining region (CDR) gene sequence, to direct drug release into MM cells. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe, our results have demonstrated that quenched fluorescent signals of this RPMI8226-specific oligonucleotide were released upon binding to the CDR3 gene sequence of RPMI8226, but not to oligo(dT), random sequences or the CDR3 gene sequence from another MM cell line, U266. Similarly, using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from U266 as a probe, our results have also shown that quenched fluorescent signals of the U266-specific oligonucleotide were released upon binding to the CDR3 gene sequence of U266, but not to oligo(dT), random sequences or the CDR3 gene sequence from RPMI8226. In addition, transfection of 8226- or U266-specific oligonucleotides into 8226 and U266 cells indicated that the oligonucleotides specifically discriminated the two cell lines. Furthermore, these oligonucleotides are stable in PBS or in transfected cells for at least 48hrs at 37°C, long enough for killing of MM cells with most currently available anti-MM drugs. Currently, we are evaluating the efficacy of this targeted approach to specifically identify tumor cells, and developing therapeutic modification of the oligonucleotide to allow drug delivery specifically to the tumor cells. Our studies validate a new strategy for MM treatment that may introduce a new targeted therapeutic approach in MM as well as other B cell malignancy, without impacting nonmalignant cells, which should allow much higher concentrations of drugs to be delivered into the tumor cells. Updated results using this novel targeted treatment approach will be reported at the meeting.
Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SRC-Dependent Manner
Authors: Gorantla Sivahari P. , von Bubnoff Nikolas , Peschel Christian , Duyster Justus 
Published: April 2020

Abstract
FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Also, F604S and F604S+D842H showed strong activation of Stat5, ERK and Akt compared to wt and D842H. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Moreover, SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. We hypothesized that F604S might interfere with FIP1L1-PDGFRA protein stability, and that SRC might be involved in this process. GST pull down experiments using SRC-SH2 domain showed lesser binding of FIP1L1-PDGFRA/F604S compared to wt. Similarly, using a GST-PDGFRA fragment, more SRC was precipitated with wt compared to F604S. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Also, co-expression of SRC in 293T cells augmented degradation of wt, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Similar results were obtained with an exchange in near proximity to F604. Kinase-defective SRC had no effect on FIP1L1-PDGFRA stability, indicating that kinase activity of SRC is necessary for its effect on FIP1L1-PDGFRA stability. Moreover, kinase defective FIP1L1-PDGFRA (G610R) was not degraded indicating that kinase activity of FIP1l1-PDGFRA is necessary for its own degradation. Taken together, imatinib resistance screening in FIP1L1-PDGFRA identified a novel class of resistance mutations, that do not act by impeding drug binding to the target, but rather increase target protein levels by interfering with its SRC mediated degradation.
Lyl1 and Scl Dosage Is Critical for Adult Hematopoietic Stem Cell Function and Survival
Authors: Souroullas George , Salmon Jessica , Curtis David J. , Goodell Margaret 
Published: April 2020

Abstract
The Stem Cell Leukemia gene (Scl) and the Lymphoblastic Leukemia gene 1 (Lyl1) genes encode two related basic-helix-loop-helix transcription factors that are involved in T-cell acute lymphoblastic leukemia via chromosomal translocations or aberrant expression due to other genetic or epigenetic abnormalities. Previous studies have shown that Scl is necessary for embryonic hematopoiesis and erythropoiesis, but dispensable for adult hematopoietic stem cell (HSC) function. Lyl1 on the other hand is important for adult HSC function and B-cell development. In order to elucidate in more detail the functions of Lyl1 and Scl in adult HSCs, we generated double knock-out mice and studied the function of their HSCs. Double knock-out mice demonstrate a striking genetic interaction between the two genes, with a clear dose-dependence for the presence of Scl or Lyl1 alleles for HSC function.
Specifically, we carried out competitive fetal liver transplantation assays using mice with different combinations of Lyl1 and Scl alleles and analyzed their potential to repopulate the bone marrow of recipient mice. Peripheral blood chimerism of recipient mice shows that HSC function is lost with progressive loss of Lyl1 or Scl dosage, suggesting an overlap of function of the two genes in the HSCs (Figure)
We have further investigated the function of Lyl1 and Scl in adult hematopoiesis by generating Lyl1/Scl-conditional double knock-out mice and studied adult HSC function measured by bone marrow repopulation assays. Donor engraftment dramatically decreased after deletion of the floxed Scl allele, demonstrating rapid loss of adult HSC function. Furthermore, Scl deletion in a Lyl1−/− background causes hematopoietic progenitors to rapidly undergo apoptosis in a cell-intrinsic fashion. These results demonstrate that at least one allele of either Lyl1 or Scl must be present for adult HSC survival, and show that their functions partly overlap in HSCs, suggesting that Lyl1 and Scl are crucial regulators of adult, as well as embryonic, hematopoiesis.
Figure Figure
Human T-Cell Leukemia Virus Type 1 Tax Activates Caveolin-1 Gene Expression
Authors: Sawada Shigeki , Ishikawa Chie , Tanji Hiroe , Mori Naoki 
Published: April 2020

Abstract
Caveolae are plasma membrane invaginations that function as regulators of signal transduction. Caveolins are a class of oligomeric structural proteins that are both necessary and sufficient for caveolae formation. Caveolin-1 has been shown to regulate multiple cancer-associated processes including cellular transformation, tumor growth, cell migration and metastasis, cell death and survival, multidrug resistance, and angiogenesis. Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). We examined expression of caveolin-1 and caveolin-2 in HTLV-1-infected T-cell lines and primary ATL cells. These cells expressed high levels of caveolin-1 mRNA compared with uninfected T-cell lines and normal PBMCs. However, caveolin-2 mRNA expression was not restricted to HTLV-1-infected T cells. Immunohistochemical staining of caveolin-1 in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for caveolin-1. In vitro infection of a human T-cell line with HTLV-1 induced caveolin-1 mRNA expression. The viral protein Tax transcriptionally activated caveolin-1 gene. Using Tax mutants, we demonstrated that Tax-induced caveolin-1 expression required the NF-kappaB and CREB signaling pathways. TGF-beta is an inhibitor of T-cell proliferation and cytotoxicity. HTLV-1-infected T-cell lines and ATL cells are known to be resistant to TGF-beta-induced growth inhibition. We demonstrated that caveolin-1 is able to suppress TGF-beta signaling at the transcriptional level in T cells. Caveolin-1 colocalized with TGF-beta type I receptor in an HTLV-1-infected T-cell line. Thus, we describe a new function of Tax, as a repressor of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.