bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clofarabine Combinations in Acute Myeloid Leukemia (AML) Salvage: A Dose-Finding Phase I Study of Clofarabine Plus Idarubicin and Clofarabine/Idarubicin Plus Cytarabine (ara-C).
Authors: Faderl Stefan , Ferrajoli Alessandra , Wierda William , Ravandi Farhad , Estrov Zeev , Verstovsek Srdan , Thomas Deborah A. , Garcia-Manero Guillermo , Kwari Monica , Kantarjian Hagop M. 
Published: April 2020

Abstract
Clofarabine is a second-generation nucleoside analog with single agent activity in acute leukemias. To try and improve efficacy, various combination trials are being conducted. In studies of clofarabine plus ara-C we reported overall response rates of 41% (CR 24%) in AML salvage and 60% (CR 52%) in untreated elderly AML with acceptable toxicity profile. To explore additional clofarabine combinations in AML we conducted a phase I study of clofarabine (C) with idarubicin (I) [CI] alone and with ara-C (A) [CIA] in pts with relapsed AML and high-grade MDS. Dose-limiting toxicities (DLT) were defined as ≥ grade 3 drug-related toxicities. Maximum tolerated dose (MTD) was determined by “3+3” method. Thirty-three patients (18 on CI and 15 on CIA) have been treated and are evaluable. Of 18 pts on CI, 6 were primary refractory and 12 in first relapse (median first remission duration [CRD1] 2 mos. [range 0–9]. Eleven pts had abnormal cytogenetics. Fourteen pts received prior ara-C-based regimens, 2 relapsed from allogeneic transplant (SCT). Median age: 57 yrs (range 24–71). Four dose levels have been explored. When C was given at 22.5mg/m2 i.v. daily x 5d and I at 12mg/m2 i.v. daily x 3d, 2 ≥ gr. 3 toxicities (diarrhea, rash, ↑ bili) occurred necessitating dose de-escalation. Subsequent levels included C at 15mg/m2 x 5d/I at 8mg/m2 x 3d (6 pts, 1 ≥ gr.3 toxicity [↑ bili]), C at 18 mg/m2 x 5d, I at 10mg/m2 x 3 d (3 pts, no DLT), and C at 22.5mg/m2 x 5d, I at 10mg/m2 x 3d (3 pts, no DLT). Three (17%) responses (2 CRp, 1 CR) occurred. Of 15 pts on CIA, 4 were primary refractory and 11 in first relapse. Median CRD1 was 9 mos (0–24). Eight pts had an abnormal karyotype. Seven pts received prior ara-C-based regimens and 2 failed unrelated donor SCT. Median age: 58 yrs (23–78). Three dose levels were evaluated. At C 22.5mg/m2 i.v. daily x 5d, I 8mg/m2 i.v. daily x 3d, A 1g/m2 i.v. daily x 5d, 2 of 3 pts developed ≥ gr.3 toxicities (↑ bili, diarrhea) necessitating dose de-escalation. Subsequent levels included C at 15mg/m2 x 5d, I at 6mg/m2 x 3d, A at 0.75g/m2 x 5d (6 pts, 1 with ≥ gr. 3 rash, ↑ bili), and C at 22.5mg/m2 x 5d, I at 6mg/m2 x 3d, and A at 0.75g/m2 x 5d (6 pts, 1 with ≥ gr. 3 ↑ bili). Nine (60%) responses (8 CR, 1 CRp) occurred. Further dose escalation of clofarabine is planned in both trials. The preliminary results indicate feasibility of the combinations. The higher response rates with CIA need to be evaluated in view of different pt. characteristics between the two trials.
Valproic Acid Induces Cell Cycle Arrest and Differentiation of t(15;17) Positive Leukemic Cells Independent from ATRA Sensitivity and Has No Effect on CD34pos Cells.
Authors: Drescher Bettina , Goerlich Kerstin , Reile Dagmar , Doehring Axel , Ganser Arnold , Heil Gerhard , Wagner Katharina , Krauter Juergen 
Published: April 2020

Abstract
The translocation t(15;17) in acute promyelocytic leukemia results in the PML/RARα fusion protein. PML/RARα recruits histone deacetylases (HDAC) and represses target genes of wild-type RARα. Recently, the anticonvulsant drug valproic acid (VPA) has been described as an HDAC inhibitor. In order to evaluate the role of VPA in the treatment of acute promyelocytic leukemia, the effect of VPA on the t(15;17) positive cell line NB4 was examined. To exclude toxicity on non malignant stem cells, CD34pos cells were analyzed. In NB4 cells, VPA led to an increase of acetylated histone H4. Histone acetylation was associated with a dose dependent inhibition of proliferation. Furthermore, inhibition of NB4 clonogenic growth was observed. In contrast, clonogenic growth of CD34pos cells was not affected by the presence of VPA. The VPA induced inhibition of cell growth in malignant cells was not caused by altered apoptosis. Rather an arrest at the G1/G0 phase was observed in the presence of VPA. In agreement with p21 expression of leukemic cell lines not harboring the t(15;17) translocation, VPA induced p21 mRNA expression also in t(15;17) positive cells. However, no induction of p21 was observed in normal CD34pos cells. VPA triggered the differentiation of NB4 cells measured by NBT test and surface expression of CD11b and CD11c. Flow cytometry of colonies from CD34pos cells showed an increased fraction of CD34pos cells in the presence of VPA. Thus, VPA does not induce differentiation in non malignant hematopoiesis. To study whether VPA and ATRA induced differentiation are mediated via identical pathways, the process of differentiation was studied in NB4 cells and ATRA resistant NB4-R2 cells. While differentiation in the presence of ATRA led to induction of C/EBPβ and C/EBPε, no change in the expression of these transcription factors was observed after VPA treatment. Downregulation of c-myc was detected upon ATRA as well as VPA treatment, an additive effect was seen after the combination. Resistance to ATRA did not interfere with effects of VPA on cell differentiation. However, upon ATRA treatment no c-myc downregulation was observed in the NB4-R2 cell line. Taken together, VPA acts as an HDAC inhibitor in t(15;17) positive cells and induces myeloid differentiation by mechanisms distinct from ATRA and independent of ATRA-resistance. Moreover, VPA does not interfere with normal hematopoiesis. Therefore, this substance might be helpful in patients with APL - especially in ATRA-resistant disease.
Downregulation of Bub1 and a Defective Spindle Checkpoint in Acute Myeloid Leukemia.
Authors: Waesch Ralph , Schnerch Dominik 
Published: April 2020

Abstract
Chromosomal aberrations are common in leukemia. The spindle assembly checkpoint (SAC) is a surveillance mechanism responsible to ensure proper chromosome segregation. Unattached chromosomes to the mitotic spindle or lack of spindle tension are sensed by the SAC leading to metaphase arrest. The activated SAC inhibits the ordered proteolysis of regulatory proteins and thereby prevents mitotic progression providing time for the repair of spindle defects. Ubiquitin-dependent proteolysis of cell cycle regulators is a central mechanism of cell cycle control. The anaphase-promoting complex (APC) is an essential ubiquitin-ligase responsible for mitotic progression. The activated SAC inhibits the APC preventing degradation of cyclin B1 and the anaphase inhibitor securin and thereby delaying chromosome separation. We found this mitotic delay mechanism to be defective in acute myeloid leukemia (AML). Leukemia cells that were exposed to the microtubule disrupting agent nocodazole were not capable of preventing anaphase onset. This is indicated by a low mitotic index and degradation of the APC targets securin and cyclin B1 in nocodazole treated Kasumi-1 cells compared to the SAC competent burkitt lymphoma cell line DG-75. Thus, unattached chromosomes were unable to induce prolonged metaphase arrest in these cells. They undergo apoptosis instead due to the complete absence of the mitotic spindle. Similar results with defective mitotic arrest and enhanced apoptosis were obtained for several other leukemia cell lines. In vivo SAC malfunction in AML can favor the generation of aneuploidy, consistent with the numerical aberrations observed in these cells. In order to identify the underlying defect we screened for mutations or altered expression of checkpoint proteins and identified the SAC component Bub1 to be posttranscriptionally downregulated in all tested AML cell lines. Our studies suggest that impaired SAC function is an important mechanism to create genetic instability and thus may contribute to rise and progression of AML.
High Resolution Genome-Wide Array-Based Comparative Genome Hybridization Reveals Cryptic Chromosome Changes in AML and MDS Cases with Trisomy 8 as the Sole Cytogenetic Aberration.
Authors: Paulsson Kajsa , Heidenblad Markus , Strombeck Bodil , Borg Ake , Fioretos Thoas , Johansson Bertil 
Published: April 2020

Abstract
Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), next to nothing is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML and MDS, cryptic - and possibly primary - genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. To date, however, no such hidden anomalies have been reported. We performed a high resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of ten AML/MDS cases with isolated +8, using an array set containing &gt;30,000 BAC and PAC clones. Array CGH revealed intra-chromosomal imbalances, not corresponding to known genomic polymorphisms, in 5/10 cases, comprising ten duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. Most notably, a 1.8 Mb hemizygous deletion at 7p14.1, which had occurred prior to the +8, was identified in one MDS transforming to AML. Furthermore, a hemizygous deletion at 12p13.2, including ETV6, was present in one case. The remaining eight imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML and MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.
A LDI-PCR Based Method Allows the Identification of Any MLL Rearrangement.
Authors: Meyer Claus , Schneider Bjoern , Dingermann Theo , Klingebiel Thomas , Marschalek Rolf 
Published: April 2020

Abstract
Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (&gt;80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.
Activation of a Novel Proteasomal Independent Bcr/Abl Degradation Pathway by WP1130 Induces Apoptosis in CML Cells.
Authors: Bartholomeusz Geoffrey A. , Donato Nichalos , Estrov Zeev , Priebe Waldemar , Talpaz Moshe 
Published: April 2020

Abstract
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells caused by 9:22 reciprocal chromosomal translocation resulting in expression of a highly stable, constitutively active tyrosine kinase, Bcr/Abl. Inhibition of Bcr/Abl with imatinib mesylate, a potent Abl-specific tyrosine kinase inhibitor, is a highly effective therapy for this disease. However, clinical resistance occurs particularly in the later stages of the disease due mainly to the occurrence of point mutations in the Abl kinase domain and continual Bcr/Abl signaling. Alternative and supplemental therapies are still needed and of great clinical interest. Screening a library of small molecular weight compounds for their ability to induce the degradation of critical transcription factors and tyrosine kinases led to the design and synthesis of WP1130, a compound that reduces Jak2 and Bcr/Abl protein stability. Treatment with WP1130 caused the rapid destruction of Bcr/Abl in human CML cell lines (K562, BV173) as well as in clinical CML specimens obtained from patients. This treatment also resulted in dephosphorylation of pSTAT5, pHck and pCrkL, three known targets of Bcr/Abl. Loss of Bcr/Abl protein following WP1130 treatment was similar to that caused by Bcr/Abl silencing (siRNA), and both treatments lead to the induction of apoptosis. Treatment with WP1130 caused rapid degradation of both wild-type and mutant (T315I) Bcr/Abl protein in BaF3 cell expressing these proteins and in a CML cell line expressing wildtype (BV173) and T315I mutant BCR-ABL (BV-173R). Treatment with WP1130 also strongly inhibited colony formation in soft agar of human CML cells from imatinib resistant patients expressing the T315I mutation. The degradation of Bcr/Abl in cells treated with WP1130 was rapid, (1h) and independent of the proteasomal system and HSP90, a chaperone associated with Bcr/Abl stability. Interestingly, WP1130-induced Bcr/Abl degradation was blocked in the presence of vanadate, a tyrosine phosphatase inhibitor. Together, these observations suggest that WP1130-induced Bcr/Abl degradation is mediated by a unique, proteasomal independent pathway in CML cells. Further development of WP1130 is underway with the goal of developing an effective therapy for treating CML by targeting Bcr/Abl protein expressed in early CML progenitors or stem cell populations.
Interferon Regulatory Factor 4 Is Not Required for Induction of Chronic Myeloid Leukaemia-Like Myeloproliferative Disease by Bcr/Abl in Mice.
Authors: Illert Anna Lena , Miething Cornelius , Grundler Rebekka , Schmidt Manuel , Burchert Andreas , Neubauer Andreas , Mugler Claudia , Peschel Christian , Duyster Justus 
Published: April 2020

Abstract
Interferon regulatory factors (IRF) are activating and/or repressing transcription factors induced by treatment with type I and II Interferon (IFN), other cytokines, receptor cross-linking and viral infection. In contrast to IRF-1 and IRF-2, which are widely expressed, IRF-4 and IRF-8 are tissue-restricted factors. IRF-8 is expressed mainly in cells of haematopoietic origin and has recently been shown to inhibit mitogenic activity of p210 Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. IRF-4 is most homologous with IRF-8 (approximately 70% overall homology) and its expression is highly restricted to lymphocytes of the B-cell type (pre-B, B, and plasma cells), mature T-cells and macrophages. Furthermore IRF-4 expression is significantly impaired in CML and AML patient samples predominately in T-cells. To examine a potential role of IRF-4 in Bcr/Abl mediated transformation we used a bone marrow transplant model (BMT). We transduced IRF-4 knockout (KO) bone marrow with retrovirus expressing p210 Bcr/Abl and transplanted it into lethally irradiated recipient C57/bl6 mice. For proper control we transplanted also wildtype (WT) bone marrow transduced with Bcr/Abl and mock transfected IRF-4 KO bone marrow (BM). All recipients transplanted with Bcr/Abl transduced BM (regardless of which IRF-4 KO or WT) developed rapidly a myeloproliferative disorder characterized by leukocytosis and expression of the myeloid lineage markers CD11b and Gr1. Surprisingly, IRF-4 KO Bcr/Abl infected BM recipient mice survived slightly longer than the control group transplanted with WT p210 BM (12 vs. 19 days). Histopathologic studies of the affected organs (spleen/lung) revealed extramedullary haematopoiesis in the spleens of both groups and a distinct infiltration of the tumor cells in the lung of WT Bcr/Abl transduced BM recipient mice, resulting in massive punctuated bleedings. Interestingly, preliminary analysis suggest a significantly reduced lung infiltration with almost no pulmonary bleedings in IRF-4 KO Bcr/Abl infected BM recipient mice, which we assume to be the reason for the differences in the overall survival. Taken together our data demonstrate that IRF-4 is not required for the induction of a myeloproliferative disorder by Bcr/Abl in vivo and for its ability to transform BM cells in vitro, but IRF-4 deficiency seems to have an impact on the fulminant pulmonary haemorrhage occurring in the murine CML-like disease.
Enhanced Phosphorylation of Nbs1, a Member of the DNA Repair/Checkpoint Activation Complex Rad50/Mre11/Nbs1, Prolongs Cell Cycle S Phase and Contributes to Drug Resistance in BCR/ABL-Positive Leukemias.
Authors: Rink Lori , Stoklosa Tomasz , Skorski Tomasz 
Published: April 2020

Abstract
Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.
BCR/ABL Regulates the Expression and Interacts with Werner Syndrome Helicase/Exonuclease To Modulate Its Biochemical Properties.
Authors: Slupianek Artur , Jozwiakowski Stanislaw , Gurdek Ewa , Nowicki Michal O. , Skorski Tomasz 
Published: April 2020

Abstract
A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.