bluuebunny/crossref_metadata_2025_split huggingface dataset:
Treatment with Hsp90 Inhibitor, 17-AAG Overcomes Resistance to Small Molecule FLT3-Inhibitors in FLT3/ITD-Positive Leukemia Cells Harboring N676K-Mutation.
Authors: Minami Yosuke , Nomura Yuka , Abe Akihiro , Tanizaki Ryohei , Kuwatsuka Yachiyo , Kiyoi Hitoshi , Naoe Tomoki 
Published: April 2020

Abstract
The receptor tyrosine kinase FLT3 is mutated in ~30% of acute myeloid leukemia (AML) and small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3-inihibitors have shown measurable clinical responses. However, most of these responses were transient and in some cases an additive single amino acid substitution (such as N676K) within FLT3 kinase domain was reported to be a possible cause for the temporary response. We previously showed a novel FLT3 inhibitor, FI-700, selectivity suppresses the growth of leukemia cells with FLT3 mutations (Kiyoi, et al., Clin Cancer Res 07). In order to examine how we can overcome drug resistance due to additional kinase mutations, we cultured MOLM-13 (FLT3/ITD-positive leukemia cell line) cells with increasing doses of FI-700 to generate several FI-700-resistant sublines. In 6 of 6 clones with resistance to more than 1 μM FI-700, FLT3N676K mutation was detected in one allele, and these were also resistant to small molecule FLT3-inhibitors such as AG1295 and AG1296. In a drug-screening for this kind of subline (MOLM-13/FLT3N676K), sub-molar treatment with molecular chaperon Hsp90 inhibitor, 17-AAG, inhibited growth as revealed by MTT-assays, and a combination of 17-AAG and FI-700 synergistically induced cell death as indicated by DNA-histograms and PI/Annexin-V assays. Expression of FLT3 by FACS and phosphorylation of FLT3 and its substrate molecules (such as STAT5), were significantly inhibited by the combined treatment of FI-700 and 17-AAG. To reveal the detailed mechanisms underlying these synergistic effects, we are currently investigating the binding of FLT3 and Hsp90, the degradation and the localization of FLT3 in these cells. We are also investigating the in vivo-effects of combined treatment in NOD/SCID mice inoculated with MOLM-13/FLT3N676K. These results imply that treatment of FLT3/ITD-positive leukemia cells with 17-AAG is a promising strategy for overcoming the drug resistance of cells with mutations in the FLT3 kinase domain.
GF-15, a Novel Inhibitor of Centrosomal Clustering, Suppresses Tumor Growth in Vivo.
Authors: Raab Marc S , Breitkreutz Iris , Rebacz Blanca , Nguyen Thu , Hayden Patrick J. , Clausen Mads H , Larsen Thomas O. , Fruehauf Johannes , Anderson Kenneth C. , Kraemer Alwin 
Published: April 2020

Abstract
The lack of tumor-specific targets that allow for selective eradication of malignant cells without affecting healthy tissues is a major drawback of cancer chemotherapy. In contrast to normal cells, tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. In many tumor types, mitotic stability is regained after clonal selection by coalescence of multiple centrosomes into two functional spindle poles (centrosomal clustering). To identify potent and selective inhibitors of centrosomal clustering, we recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res2007; 67: 6342–6350). We here describe the novel small molecule GF-15, which is a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas and multiple myeloma) as well as solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1–5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized tumor cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, short term treatment with GF-15 was also associated with inhibition of cytokine-triggered cell migration. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors.
Familial Aggregation of Multiple Myeloma and Its Precursor Monoclonal Gammopathy of Undetermined Significance (MGUS): A Population-Based Study in Sweden.
Authors: Kristinsson Sigurdur Y , Bjorkholm Magnus , Goldin Lynn R. , Blimark Cecilie , Mellqvist Ulf-Henrik , Wahlin Anders , Turesson Ingemar , Landgren Ola 
Published: April 2020

Abstract
Background: Evidence from multiply affected families, case-control and population-based registry studies implicate a role for genetic factors in multiple myeloma (MM). The aim of this large population-based familial case-control study was to quantify risks of MM, monoclonal gammopathy of undetermined significance (MGUS), and other lymphoproliferative disorders among first-degree relatives of MM patients.
Methods: We identified 13,963 MM patients diagnosed in Swedish hospitals 1958–2005, with linkable relatives. Using the population-based central Population- and Multigenerational registries, we obtained 54,610 matched controls and first-degree relatives of MM patients (n=37,838) and controls (n=151,068). Relatives of MM patients and controls were linked with hospital-based outpatient registries and the central Swedish Cancer Registry to define occurrence of MGUS and lymphoproliferative malignancies. Measures of familial aggregation were calculated by a marginal survival model using relatives as the cohort.
Results: First-degree relatives of MM patients had a significantly increased risk of developing MM [relative risk (RR)=2.1; (95% confidence interval (CI), 1.6–2.9)] and MGUS [2.1 (1.5–3.1)]. The risk estimates were very similar when we conducted analyses by gender of proband, by type of first-degree relative (parent, sibling, offspring), and by age at MM diagnosis (below/above 65 yrs) for probands. Among relatives of MM patients, we found no excess risk of chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, or lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia.
Conclusions: In this large population-based study, we found relatives of MM patients to have a 2-fold excess risk of developing MM and MGUS compared with relatives of controls. Our findings support the theory that there are common, shared susceptibility genes that predispose to MM and MGUS. Better characterization of early genetic lesions mediating monoclonal plasma-cell proliferation, survival, and migration in the bone marrow microenvironment will potentially provide clues to pathogenesis and allow identification of novel molecular targets.
Regulatory T Cells (Treg) Are Depressed in Patients with Relapsed/Refractory Multiple Myeloma (MM) and Increases towards Normal Range in Responding Patients Treated with Lenalidomide (LEN).
Authors: Quach Hang , Ritchie David , Neeson Paul , Harrison Simon , Tai Tsin , Tainton Kellie , Lynch Kevin , Prince Miles 
Published: April 2020

Abstract
CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a group of immune-suppressive T cells responsible for the maintenance of self-tolerance and autoimmunity. Increased Tregs have been implicated in suppression of tumour-immunity, and are associated with poor prognosis in gastric, oesophageal and ovarian cancers. Conversely, reduced Tregs are associated with aggressive disease and poor prognosis in lymphoproliferative disorders such as follicular lymphoma and Hodgkin lymphoma. Meanwhile, conflicting literature exists with regards to the role of Tregs in the pathogenesis of multiple myeloma (MM). Lenalidomide (LEN) is an immunomodulatory drug (ImiD™) that increases T cell numbers and function in vitro but the impact on Tregs in vivo is unknown. As part of an ongoing clinical trial of low-dose lenalidomide, peripheral blood lymphocyte subsets (LSS) and Treg numbers were examined in patients [n=18, median age 67 years (49–77)] with relapsed/refractory MM at baseline and at various time points during treatment with combination LEN (15mg/o/d d1–21q28)-dexamethasone (dex) (20mg/o/d d1–4,9–12,17–20 q28), in order to measure changes induced by LEN-dex treatment. Results were compared to 10 age matched healthy donors. LSS and Tregs were assessed by flow cytometry, with Tregs strictly defined as CD4+CD25hiCD127dimFOXP3+. At baseline, MM patients had depressed B cells (median 0.76 vs. 3.2 ×105/ml; p=0.02) and CD3+ T cells (median 7.2 vs. 12.1 ×105/ml; p&lt;0.001) compared to healthy donors, with an inverse CD4:CD8 T cell ratio (0.7:1 vs. 2.9:1, p=0.008). Tregs in MM patients were markedly reduced at baseline compared to healthy donors [median 0.40 (range: &lt;0.01–1.9) vs. 4.8 (range 2.4–7.1) ×104/ml; p=0.0005]. In 11 patients completing at least 4 treatment cycles, no recovery of B cells (7.6 vs. 2.3 ×105/ml, p=0.053), NK cells (2.1 vs. 2.7×105/ml, p=0.1) or T cells (5.6 at baseline vs. 7.5 × 105/ml, p=0.45) were seen despite all achieving at least a PR (one with IF negative CR). In contrast, median Treg numbers progressively increased towards normal range, from a baseline of 0.3 to 1.3 × 104/ml (p=0.005) and 2.0 × 104/ml (p=0.009) after 4 (n=11) and 6 (n=7) cycles of LEN-dex respectively (figure 1). Patients with relapsed/refractory MM have profoundly disturbed LSS including marked decreased in Treg numbers. Normalisation of Treg numbers in responding patients treated with LENdex suggest that, as in other tumours, Tregs are actively involved in the pathophysiology of MM and their modulation may be an important component of the immunomodulatory mechanisms of LEN.
Figure 1: Change in regulatory T cell (Treg) numbers with each cycle of LEN-dex in MM patients Figure 1:. Change in regulatory T cell (Treg) numbers with each cycle of LEN-dex in MM patients
Loss of Chromosome 1p21 Band Is Associated with Disease Progression and Poor Survival in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation.
Authors: Chang Hong , Qi Connie , Jiang Allan , Xu Wei , Trieu Young , Reece Donna E. 
Published: April 2020

Abstract
Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies are largely unknown. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we investigated the prevalence and prognostic significance of del(1p21) in a cohort of 235 MM patients undergoing highdose therapy and autologous stem cell transplant at our institution. Fluorescence in situ hybridization combined with cytoplasmic light chain detection (cIg-FISH) was used to evaluate clonal plasma cells for 1p21 deletion as well as other myeloma-associated chromosomal abnormalities. In addition, 1p21 status was evaluated in 16 patients with of monoclonal gammopathy of undetermined significance (MGUS) and 41 patients with plasma cell leukemia (PCL). CIg-FISH detected hemizygous 1p21 deletions in 18% of the MM, 34% of PCL but none of the MGUS cases. In MM, the median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with 1q21amplification (p=0.01), and marginally correlated with del(p53) (p=0.05) or t(4;14) (p= 0.06), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. The median follow-up in this cohort was 36 months. Patients with 1p21 deletions had significantly shorter progression-free (median 14.2 vs. 25.4 months, p&lt;0.001) and overall survivals (median 39.4 vs. 82.3 months, p=0.001) than those without such deletions. In multivariate analysis, del(1p21) was an independent risk factor for progression free (p= 0.003) and overall survivals (p=0.013) after adjusting for del(13q), del(p53), t(4;14) and 1q21 amplification. 1q21 amplification was significant risk factor in univariant analysis for either progression-free or overall survivals, but did not remain as an independent risk factor in multivariant model adjusting for other genetic risk factors. Our results indicate that del(1p21) is an independent poor prognostic factor associated with disease progression in MM. We recommend include this genetic aberration as part of a routine work-up in the risk-stratification of MM patients receiving autologous stem cell transplantation.
siRNA, a Potential Therapy for AL Amyloidosis.
Authors: Hovey Beth M. , Ward Jennifer , Seldin David C. 
Published: April 2020

Abstract
AL amyloidosis is a rare, sporadic disorder characterized by the accumulation of misfolded monoclonal immunoglobulin light chains (LC) as systemic amyloid deposits. Current therapies primarily include chemotherapies targeted at killing the plasma cells, often having toxic side effects for the patients. We have designed siRNAs targeted at either the variable (VL) or the constant regions (CL) of the LC to reduce amyloidogenic precursor protein expression in the producing cells. The strategy of targeting the unique VL is to develop sequence-specific siRNAs; alternatively, by targeting shared sequences in CL, siRNAs may be effective for sets of patients sharing the same LC family. Using picomolar concentrations of siRNAs, we have reduced LC protein levels in transfected cells in vitro in a dose-dependent fashion. In an in vivo tumor transplant mouse model of AL amyloidosis we have shown we can decrease the local and circulating LC levels. In this model, SP2/0 plasmacytoma cells stably expressing human amyloidogenic LCs are injected subcutaneously, and allowed to form a tumor. Once the tumor is the appropriate size, In Vivo Electroporation (IVEP) is used to deliver the siRNA oligos into the cells. By examining tumor and serum samples 48 hours post treatment, we have shown the siRNAs can significantly decrease the amount of LC in a sequence-specific manner. This model system highlights the therapeutic potential of siRNA for this disease. In conclusion, siRNA appears to be a viable potential therapy for AL amyloidosis.
Identification of DJ-1/Park-7 as a Determinant of Tnfα-Induced and Stroma-Dependent Apoptosis in Leukemia Using Mass Spectometry and Phosphopetide Analysis.
Authors: Marcondes A. Mario Q. , Xiang Li , Milless Brian P , Deeg H. Joachim 
Published: April 2020

Abstract
The bone marrow microenvironment provides essential signals for the fate of normal hematopoietic and for leukemic cells. Contact with marrow stroma, which is part of the microenvironment, is generally thought to convey anti-apoptotic signals to (clonal) leukemia cells. Patients with low-grade myelodysplastic syndrome (MDS) early in the disease course show high rates of apoptosis in normal and clonal marrow cells, mediated by tumor necrosis factor alpha (TNFα) and other cytokines. As MDS advances and evolves to leukemia, clonal cells tend to become apoptosis resistant. We showed previously that the leukemia-derived cell line KG1a was resistant to TNFα-mediated apoptosis, but TNFα did induce caspase-3 activation and apoptosis in KG1a cells when co-cultured with the human marrow stroma cell line HS5 (derived from healthy marrow). Apoptosis was contact dependent and required expression of TNF receptor 1 on KG1a cells. Identical results were obtained in co-cultures with primary stroma cells. Gene expression profiling of KG1a cells showed that stroma contact resulted in significant upregulation of genes involved in apoptosis, including PYCARD and p53. To further dissect the relevant signaling pathways, we used a PhosphoScan proteomic LC-MS (Liquid chromatography-mass spectrometry) method to identify proteins that were phosphorylated in response to stroma contact. In parallel to KG1a we examined the parent cell line KG1, which is sensitive to TNFα mediated apoptosis. We determined the phosphorylation sites in proteins within the leukemic cell lines using MS2 and MS3 scans. Database searches were performed with X! Tandem and Mascot and results analyzed by PeptideProphet using data from a synthetic doubly-phosphorylated peptide as control. In KG1a cells cultured without stroma support, the peptide DJ-1/Park-7 was highly phosphorylated, and expression of p53 was inhibited as indicated by decreased levels of p53 mRNA and protein. In co-culture with stroma, KG1a cells expressed higher levels of p53 protein, and levels of phosphorylated DJ-1/ Park-7 were undetectable over a time course of 30 min to 24 hours. In apoptosis-sensitive KG1 cells constitutive DJ-1/Park-7 phosphorylation (in the absence of stroma contact) was undetectable, and p53 was expressed at higher levels than in KG1a cells, consistent with the observed activation of caspase-3 and induction of apoptosis in KG1 cells. Taken together, these data suggest that phosphorylation of DJ-1/Park-7, originally identified as an oncogene product involved in cellular transformation, oxidative stress responses, and transcriptional regulation, was associated with repression of p53 and resistance to TNFα-mediated apoptosis. The relevance of DJ-1/Park-7 (and other genes identified by the PhosphoScan proteomic method) in primary MDS cells is currently being investigated at the molecular and functional levels.
Smad4 as a Therapeutic Target in Nup98-HoxA9-Induced Leukemia.
Authors: Quere Ronan , Karlsson Goran , Andradottir Silja , Rissler Marianne , Olsson Karin , Hertwig Falk , Reckzeh Kristian , Cammenga Jorg , Karlsson Stefan 
Published: April 2020

Abstract
Hematopoietic stem cells (HSC) are subject to complex regulation by many signal transduction pathways and transcription factors. In leukemia, these regulatory circuits are abnormal and generate dysregulated growth of HSC and their progeny. Among the prominent transcription factors that are dysregulated in leukemia are the homeobox (Hox) transcription factors or fusion-proteins derived from Hox proteins. The Nup98- HoxA9 oncogenic fusion protein is found to enforce expression of HoxA9 target genes that immortalize and block the differentiation of HSC. TGFβ inhibits Nup98-HoxA9 transformation of bone marrow cells, through direct interaction between Smad4 and the fusion protein, in a fashion which is surprisingly independent of active Smad signaling (EMBO J, 2006). Our hypothesis is that an interaction between the TGFβ and the Hox pathways is involved in leukemogenesis. To directly address this issue, we used our knockout mouse model (J.Exp Med, 2007) to determine HSC behavior in Smad4-/- cells and analyze interconnection between the TGFβ signaling pathway and the HoxA9 oncogenic activity. Using retroviral vectors to generate enforced expression of Nup98- HoxA9 in Smad4 null HSC, the transforming function was found increased approximately 4-fold in vitro. Transplantation of these transduced cells into recipient mice confirms that a block of the TGFβ pathway leads to a fast development of leukemia. At the molecular level, a 5-fold accumulation of Smad4 protein is found in wt HSC transduced by the Nup98-HoxA9, suggesting that the oncogenic potential involves a sequestration of Smad4 and a block of the TGFβ pathway. To reactivate the TGFβ pathway, one therapeutic strategy would be to induce a re-allocation of the Smad4 protein accumulated in these cells. To directly address this issue, we generated retroviral constructs that enable expression of different portions of the MH1 part of the Smad4 protein, which has been described to interact physically with the Nup98-HoxA9 oncoprotein. The retroviral transduction of one of this portions encoding a 20aa-peptide, allows release of the endogenous sequestrated- Smad4 by competition, which leads to reactivation of the TGFβ pathway and the expansion of leukemic cells is prevented in secondary recipient mice. While, administration of TGFβ remains inconceivable, because of secondary effects, the findings from these studies might consequently be used to design novel therapies for leukemia induced by abnormal Hox proteins.
Transgenic Expression of Wild Type JAK2 Suppresses Myeloproliferative Disorder Phenotypes Induced by Mutant JAK2V617F in Mice
Authors: Xing Shu , Zhao Wanming , Ho Wanting Tina , Zhao Zhizhuang Joe 
Published: April 2020

Abstract
JAK2V617F, a mutant form of tyrosine kinase JAK2, is found in the majority of patients with myeloproliferative disorders (MPDs). It displays increased kinase activity and causes MPD phenotypes in transgenic mice in a transgence dosage-dependent manner. Following our initial generation and characterization of JAK2V617F transgenic mice, we further generated transgenic mice expressing wild type JAK2 by using the same vav promoter employed for JAK2V617F. Three lines of JAK2 transgenic mice were generated. Real time PCR analyses revealed transgene copy numbers of 38, 2, and 1. All these mice are viable and fertile, and they displayed normal blood cell counts. This proves that the V617F mutation but not gene overexpression per se caused MPD phenotypes in JAK2V617F transgenic mice. We then crossed the JAK2 and JAK2V617F transgenic mice to generate JAK2/JAK2V617F double transgenic hybrids. Interestingly, these hybrid mice developed no or mild MPD phenotypes with only a slight increase in blood counts in contrast to the striking elevation observed in JAK2V617F transgenic mice. Expression of wild type JAK2 also blocked the constitutive activation of signal transduction components caused by JAK2V617F. Our data indicates that over-expression of wild type JAK2 suppresses the pathogenic function of mutant JAK2V617F. Therefore, JAK2V617F is not a typical dominant oncogene. Homozygous mutation or in the case of heterozygous mutation, its amplification with concurrent deletion or suppression of wild type JAK2, is required to produce MPD phenotypes. Our transgenic mouse models will serve as an invaluable tool to study the interplay of JAK2 and JAK2V617F and the mechanism by which specific MPD phenotypes develop.