bluuebunny/crossref_metadata_2025 huggingface dataset:
A 3′ RACE-PCR Method of Identification of Unknown MLL Gene Partner and Break-Point at RNA Level.
Authors: Formisano-Tréziny Christine , Hermitte Fabienne , Quilichini Benoît , Chambost Hervé , Michel Gérard , Gabert Jean
Published: April 2020

Context: Chromosomal translocations involving the MLL gene at 11q23 are particularly common in infant acute leukaemia (AL) (80% of all cases) and are also associated with secondary leukemias. Monitoring minimal residual disease (MRD) is important in predicting efficacy of treatment and detecting early relapse. In case of MLL translocation, detection of MLL fusion transcript by real time PCR is a powerful technique to investigate MRD. Currently approximately 50 different fusion partners of the MLL gene have been characterized at the molecular level and at least 36 remain to be identified. In addition, MLL chromosomal translocations are routinely investigated by FISH. This cytogenetics method allows determination of the presence of a chromosomal translocation involving the MLL gene and the chromosomal location of the partner gene. In this context, only the most frequent and well characterised MLL partner genes are identified, then the corresponding fusion transcripts are analysed by real time PCR.

Rational: Here we describe a useful and simple method of identifying any unknown MLL gene partner and its specific break-point at RNA level.

Methods: This method is based on 3′ RACE-PCR follow-up by cloning and sequencing of resulting PCR products. Briefly, a reverse transcription is performed on total RNA with an anchor random primer. The resulting cDNA is subjected to a long range PCR (PCRI) allowing amplification of normal MLL transcripts and MLL fusion transcripts. Primers used are: a sense primer specific to an MLL region upstream of the break-point cluster region, and an antisense primer specific to the anchor of the random primer used at the reverse transcription step. PCRI products are amplified again with nested primers (PCRII), and PCRII products are cloned. Recombinant clones are screened by 3 different PCR in order to differentiate clones corresponding to the normal MLL transcript from those corresponding to the MLL fusion transcript. Finally clones of interest are sequenced.

Results:. A first case of an infant (AL) was investigated in our laboratory. We identified an infrequent t(11;19)(q23;p13) involving the MYO1F gene, associated with exon 10 deletion of the non translocated MLL transcript. This infant AL case is now routinely followed up with a specific real time PCR assay which we developed. Currently a study of a second case with an uncharacterised MLL translocation involving chr. 7 is in progress.

Conclusion: This method is especially convenient because of its simplicity, speed, and universality. Furthermore, this technique not only allows identification of the MLL fusion transcript sequence in order to choose a specific real time PCR assay, but it also permits immediate lay out of the plasmid which can be used as real time PCR calibrator. Thus, this method can be introduced in molecular therapy management of MLL rearranged patients with rare partner gene or molecular break-point.
Near-Tetraploidy Can Mask the Presence of Chromosome 13q Deletions in Plasma Cells of Patients with Multiple Myeloma.
Authors: Koren-Michowitz Maya , Hardan Izhar , Berghoff Janina , Yshoev Galina , Amariglio Ninette , Rechavi Gideon , Nagler Arnon , Trakhtenbrot Luba
Published: April 2020

Ploidy status and chromosomal aberrations are important prognostic features in multiple myeloma (MM). In particular, non-hyperdiploidy i.e. hypodiploid, pseudodiploid and near-tetraploid (NT), and chromosome 13q aberrations are associated with a worse outcome. We have used combined I-FISH and cell morphology analysis with the Duet™ system for the diagnosis of del 13q and IgH aberrations in bone marrow (BM) samples from MM patients (pts). Using this method we suspected that del 13q can be masked in plasma cell populations due to NT chromosome number.

We studied 125 consecutive BM samples from MM pts for the presence of NT. Mean pts’ age was 56±11 years, sixty percent were men, sixty-seven percent had stage III disease and forty-eight percent had IgG myeloma.

Two commercial probes were used in all FISH experiments: LSI 13 (RB1) and LSI IGH dual-color, break-apart rearrangement probe. For further clarification other probes of Vysis were used.

The measurement of plasma cells DNA content (DI) was done by determining the DAPI intensity using the Volocity Grid Confocal system controlled by the Volocity Acquisition software. To examine the accuracy of measurements DI of 2 MM cell lines: U266 and NCI-H929 were determined. DI in the two cell lines deviated only by 3.2% and 3.6%, respectively, therefore supporting accuracy of ploidy measurements. The criteria for the diagnosis of NT included:

Duplication of hybridization patterns in both 13q and IgH Normal hybridization pattern in 13q and duplication of hybridization patterns in IgH and another probe Normal hybridization pattern in IgH and duplication of hybridization patterns in 13q and another probe If normal hybridization signals were seen in both 13q and IgH probes but plasma cells had large nuclei, at least two other probes with duplication of hybridization patterns were required for NT determination.

NT was diagnosed in 45/125 BM samples (36%), median plasma cells with NT 12.5%, range 2–100% of the plasma cells population.

DI was determined in 10 BM samples suspected for NT. All the samples studied were verified as NT with a mean DI of 1.7 ± 0.14 (range 1.58–1.90).

Chromosome 13q status was normal in only 8 NT samples (18%), i.e. four hybridization signals were detected. In 32/45 (71%) NT samples there were two hybridization signals in a portion of plasma cells population, representing a duplication of del 13q in the presence of NT. Importantly, in 5 samples (11%) all plasma cells had two hybridization signals for del 13q. These cases could actually be overlooked unless NT was specifically searched for and ruled out, therefore potentially loosing important prognostic information.

In conclusion, we found a high prevalence of NT in MM pts using a sensitive novel assay. The determination of NT is important as del 13q can be masked in BM samples harboring NT DNA content.
Non-Coding RNA without ORF Is Overexpressed in Diffuse Large B-Cell Lymphoma with t(1;14)(p33;q32) Translocation.
Authors: Suzuki Ritsuro , Nakamura Shigeo , Seto Masao
Published: April 2020

Background: Several oncogenes, such as cyclin D1, BCL2, BCL6, and c-Myc have been identified to be translocated with immunoglobulin gene In B-cell lymphomas. These genes are overexpressed by the promoter/enhancer activity of immunoglobulin genes, and play important roles in lymphomagenesis. We investigated a translocation partner of immunoglobulin heavy chain gene in a case of diffuse large B-cell lymphoma (DLBCL) with t(1;14)(p33;q32) translocation.

Materials and methods: The patient is a 58-year-old female with stage 3AE gastric DLBCL with t(1;14)(p33;q32) translocation. High molecular weight DNA was extracted from resected specimens. Long distance inversed PCR was performed to identify the breakpoint of translocation partner. Cloned fragments were sequenced and the sequence was searched in the database. RT-PCR and 5′- and 3′-RACE (rapid amplification of cDNA end), and were performed to identify the full length of cDNA which is overexpressed as a result of translocation.

Results: The breakpoint was located in the 1p34 region. An EST located 20kb downstream from the breakpoint was overexpressed. This EST was not expressed in normal lymphocyte and most of the lymphoma samples and cell lines without t(1;14)(p33;q32). No other known genes including ESTs and hypothetical genes within 1Mb from the breakpoint was overexpressed. Therefore, we concluded that a gene including this EST is the target of t(1;14)(p33;q32) translocation. 5′- and 3′-RACE and RT-PCR revealed that a non-coding RNA without open reading frame of 20kb length was overexpressed. Several palindrome-like sequences existed in the non-coding RNA, but the expression of short length (17–25mer) RNA sequence was not detected.

Conclusion: We identified a previously unrecognized type of translocation partner activated by immunoglobulin heavy chain gene in t(1;14)(p33;q32) translocation. Although the intimate mechanism is not clarified, the cloned novel non-coding RNA plays a role in lymphomagenesis.
Donor-Cell Acute Leukemia after Sibling Marrow Grafting for Aplastic Anemia: Another Type of Familial Monosomy 7?.
Authors: Benattar Laurence K. , Bouffard Laurence , Coignet Lionel J.
Published: April 2020

We report here the case of a bone marrow transplant (BMT) patient who received a graft from her 1-year-old HLA-identical brother for severe aplastic anemia (AA) with severe thrombopenia at the age of 4, followed by myelodysplasia at the age of 6 that transformed to AREBt/AML type M6 at the age of 7. She died at the age of 8.

Two years after the BMT, the brother developed hypoplasia that rapidly transformed to AML-M2 and he died rapidly during chemotherapy induction.

Cytogenetic results showed that the brother initially developed an AML-M2 with a t(16;21) translocation and, 3 years later, the patient developed an AREBt/AML-M6 with a monosomy 7 and a donor profile (45,XY,−7). Retrospective examinations did not allow the detection of the t(16;21) in any samples of the patient nor monosomy 7 in any available sample from her brother.

This observation shows that the same cells (the donor cells) in another microenvironment, even HLA-identical, can transform to different acute leukemia types with different chromosomal abnormalities. In both, the chromosomal abnormalities seem to appear at a late stage of the disease and can be considered a secondary event. Familial MDS/AML with monosomy 7 are well described: heterozygous deletion and nonpreferential deletion of parental chromosome suggest that the inherited gene(s) might not be located on chromosome 7. Other familial hematological disorders such as familial platelet disorders present a AML1 translocation or mutation. In our case, on one hand, two hematological disorders in siblings (one AA and one AML at diagnosis) can be considered a familial case even if the past medical history of the other members of the family is unremarkable. On the other hand, the same cells (the brother’s cells) have provided a translocation involving AML1 in the donor and a monosomy 7 in the recipient, the two genetic abnormalities being involved in familial hematological disorders. We could hypothesize that those two events might have been induced by the same inherited gene(s) that could be responsible at the same time for familial monosomy 7 and for other familial hematological disorders.
Proteomics Analysis of Bone Marrow Cells of Acute Myeloid Leukemia (M2a) and Its Prognosis Significance.
Authors: Meng Fan Yi , Tian Shuai , Tang Jia-ming
Published: April 2020

Objective:The distinct proteins of leukemic cells were investigated by proteomics technology between AML-M2a patients before inductive treatments with evidently different duration of first continuous complete remission(CCR1) and AML-M2a patients at replase in order to find their relations with prognosis of AML-M2a.

Methods:The bone marrow mononuclear cells(BMMNCs) from 17 cases of AML-M2a patients before inductive treatment were grouped with different duration of CCR1: group A with CCR1 duration exceeded 12 months(11 cases), group B within 6 months(6 cases), and group C was composed of 3 patients at replase among group B. The proteins of BMMNCs from all the patients were separated by two-dimensional electrophoresis, and the part of differentially-expressed proteins were identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).

Results: 6 differentially-expressed proteins were identified between group A and B by MALDI-TOF-MS: tubulin-specific chaperone B, myeloperoxidase, Solution Structure Of The Ch Domain Of Human Transgelin-2, glutathione S-transferase, RING zinc finger protein, glyceraldehyde-3-phosphate dehydrogenase.3 differentially-expressed proteins were identified in group C: NAD(P)H dehydrogenase, hypothetical protein, HES1.

Conclusion: The distinct proteins of leukemic cells of AML-M2a patients before inductive treatments were involved in prognosis, and the proteins of BMMNCs from patients at replase have changed.
Livin Expression in Normal Hematopoietic Cells and in Hematologic Malignancies.
Authors: Abd-Elrahman Ihab , Bucholtz Vered , Hershko Klilah , Amir Gail , Perlman Riki , Ben-Yehuda Dina
Published: April 2020

Livin is a member of the Inhibitor of Apoptosis Proteins (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin is unique among the IAP members as upon strong apoptotic stimuli, it is specifically cleaved by caspases to produce a large C-terminal subunit. This subunit has a paradoxical pro-apoptotic activity. Thus, Livin is not merely an inhibitor of apoptosis. Rather, it is a regulator of apoptosis that can protect against apoptosis but upon continuous apoptotic signals it helps to assure cell death. We showed that Livin plays a major role in melanoma. The level of the Livin protein is directly correlated to the resistance of melanoma cells to chemotherapy and to the survival rate of melanoma patients. Livin was also shown to be over expressed in other solid tumors such as nasopharyngeal, neuroblastoma, colorectal and lung cancers. In this work we studied Livin expression in normal hematopoietic cells as well as hematologic malignancies. Using immunohistochemistry staining for Livin we evaluated its expression in reactive lymph nodes (LN) and showed that in contrast to Bcl2, Livin was detected in highly proliferating germinal centers. In normal bone marrow Livin was detected in Megakaryocytes and immature myeloid precursors. In peripheral blood mononuclear cells, using quantitative RT-PCR, we found that Livin expression was down regulated in activated monocytes and T cells while in B cells, Livin was upregulated upon activation. We studied bone marrow and LNs from 84 patients (pts) with hematologic malignancies. Positive immunohistochemistry staining was found in the malignant cells of all pts with DLBC NHL (31 pts), follicular lymphoma (12 pts) and multiple myeloma (15 pts). Peripheral blood samples from 28 B-CLL pts were compared with healthy controls’ B cells. High mRNA levels were detected in 43% of the pts, in correlation with older age (p<0.05). On the other hand, Livin was not expressed in Hodgkin’s disease (4 pts and 4 cell lines) and only 6/29 pts with AML had high levels of Livin in RT-PCR without any clinical correlation. Our data demonstrate that Livin is over expressed in activated normal B cells both in peripheral blood and LN as well as in most B cell lymphoproliferative diseases. Further investigation will establish the role of Livin over expression in hematologic malignancies.
Upregulation of SOCS-1 and Downregulation of miR-214 Correlate with Disease Course in Chronic Lymphocytic Leukemia.
Authors: Jones Kimberly A. , Phillips John D. , Mendell Joshua T. , Glenn Martha G. , Yu Margaret K.
Published: April 2020

SOCS-1, suppressor of cytokine signaling, is a negative regulator of the JAK2/STAT signal transduction pathway. This pathway, often deregulated in myeloproliferative disorders, has not yet been implicated in chronic lymphocytic leukemia (CLL). We demonstrated SOCS-1 upregulation and miR-214 downregulation with clinical response in a patient treated with cladribine. Similar changes were not seen in a treated patient with progressive disease. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate expression of other genes by complementary pairing at the seed sequence in the 3′-UTR of the target mRNA. Some patients with CLL reportedly regulate bcl-2 expression by downregulating miR-15a and miR-16-1. Although no experimentally verified targets have been demonstrated for miR-214, complete inhibition of miR-214 by anti-sense inhibitors prevents apoptosis in Hela cells. Of the 44 predicted human miRNAs that target SOCS-1 (http://microrna.sanger.ac.uk), we determined the expression of 24 of these by real-time PCR using the Early Access Human Panel from Applied Biosystems. MiR-214 was the only downregulated miRNA after treatment. The other 23 miRNAs were either upregulated or unchanged. We demonstrated SOCS-1 protein upregulation by two fold after cladribine in the “responsive” patient without a concomitant increase in the transcript level as measured by real time PCR. SOCS-1 expression is often silenced by DNA promoter methylation in hematologic and solid tumor malignancies. We report another mechanism of regulation through miRNA translational repression. This suggests involvement of the JAK2/STAT pathway in B-cell survival in CLL.
Aberrant Transcriptional Regulation of the MLL Fusion Partner EEN Gene by AML1-ETO and Its Implication in Leukemogenesis.
Authors: Ma Li-Heng , Liu Han , Xiong Hui , Chen Bing , Fang Yi , Zhang Xiao-Mei , Wang Yue-Ying , Huang Qiu-Hua , Zhang Qing-Hua , Li Bo-Liang , Chen Zhu , Chen Sai-Juan
Published: April 2020

EEN (extra eleven nineteen) gene located on chromosome 19p13 was cloned as a fusion with MLL from an acute myeloid leukemia (AML) patient with translocation t(11;19)(q23;p13). In this study, we characterized the genomic structure of EEN gene including its 5′ regulatory region and transcription start site (TSS). We found that Sp1 could bind to the GC-stretch of EEN promoter and was critical for the normal EEN expression, while the leukemia associated fusion protein AML1-ETO could aberrantly transactivate EEN gene through an AML1 binding site. Of note, overexpressed EEN showed oncogenic properties, such as transforming potential in NIH3T3 cells, stimulating cell proliferation and increasing the activity of transcriptional factor AP-1. Retroviral transduction of EEN increased self-renewal and proliferation of murine hematopoietic progenitor cells. Moreover, Kasumi-1 and HL60 cell growth was inhibited with down regulation of EEN by RNAi. These findings demonstrate that EEN might be a common target in two major types of AML associated with MLL or AML1 translocations, and over-expression of EEN may play an essential role in leukemogenesis.
TGFβ Activates p53 Via PP2A in Hematopoietic Cells.
Authors: Takimoto Rishu , Kato Junji , Takada Koichi , Takahari Daisuke , Niitsu Yoshiro
Published: April 2020

Dysfunction of TGFβ signal transduction has been shown to result in leukemogenesis. It has been generally accepted that Smads mediate the TGFβ signaling to inhibit cell proliferation, while several Smad4-impaired cell lines retain certain levels of responsiveness to TGFβ, suggesting an additional TGFβ signaling pathway for cell cycle arrest. Here we demonstrate evidence for a novel signal pathway for G1 arrest linking TGFβ and p53/Protein phosphatase 2A (PP2A) in CD4+ T lymphocyte and megakaryocytes. TGFβ induced growth suppression, associated with increment of PP2A activity was clearly observed in cells with impaired Smad signal. In these cells, upon TGFβ stimulation, PP2A was transiently recruited by heteromeric complex of TGFβ receptors (TβRs), released into cytosol, and activated. Subsequently, PP2A dephosphorylated p53 to import it into nucleus by interacting with importin-α/β followed by p21Waf1 induction. The recruitment of PP2A by TβRs and TGFβ-induced growth suppression as well as nuclear import of p53 were inhibited by introduction of siRNA for PP2A-Bα. Based on these preliminary observation, we next used human peripheral CD4+ T lymphocytes, which are reportedly impaired in Smad signaling, but sensitive to TGFβ when stimulated with PHA and IL-2. Growth suppression induced by TGFβ was almost completely abolished by siRNA for p53 (siRNA-p53), but not by siRNA for Smad4 (siRNA-Smad4). Further, nuclear translocation of p53 was inhibited by siRNA for PP2A-Bα(siRNA-Bα). These results suggest that growth suppressive activity of TGFβ in certain immunological responses could be ascribed to PP2A/p53 signal transduction. Thus these results define a novel pathway of TGFβ-induced growth suppression linking p53-PP2A, and suggest that loss of function of p53 which is frequently observed in hematological malignancies may result in the escape from growth inhibitory signal of TGFβ.
Significance of Ubiquitin Ligase Subunit SKP-2 Proto-Oncogene and Proliferative Marker Ki67 Expression in Diffuse Large B-Cell Lymphoma.
Authors: Uddin Shahab , Hussain Azhar R. , Bavi Prashant P. , Al-Kuraya Khawla S.
Published: April 2020

S-phase kinase-associated protein 2 (SKP-2) is a proto-oncogene that has been shown to be expressed in a number of tumors. A number of studies have shown that SKP-2 plays a role in the degradation of tumor suppressor genes by increased proteosome-dependent degradation. SKP-2 overexpression is highly representative of intrinsic biological aggressiveness of certain cancers including breast, non-small cell lung cancer and gastric carcinoma, how ever its role in hematological malignancies have not yet been explored. Therefore, in this study we examined 100 clinical samples of diffuse large B-cell lymphoma (DLBCL) to study the expression pattern of ubiquitin ligase subunit SKP-2 proto-oncogenes and its relation to proliferative index marker protein Ki67 to correlate tumor aggressiveness. The expression of SKP-2 and Ki67 were examined by immunohistochemistry using specific antibodies on formalin-embedded tissue sections of DLBCL patients. Our data showed that SKP-2 was over expressed in majority of DLBCL and was associated with expression pattern of the proliferating index marker Ki67 protein. Since increased proteosome-dependent degradation of tumor suppressor genes play a critical role in the etiology of various tumors and proteosome inhibition is a novel approach for treating malignancies and has been approved for clinical use, we sought to determine whether inhibition of proteosome by MG132, a specific proteosome inhibitor induces apoptosis in a panel of DLBCL cell lines. Our data showed that treatment of DLBCL cell lines by MG132 induced apoptosis in a dose dependent manner. Inhibition of proteosome also decreased the expression of SKP-2 leading to subsequent disruption of mitochondrial membrane potential causing release of cytochrome c into the cytosol. Release of cytochrome c resulted in activation of caspase-3 and cleavage of PARP ultimately leading to apoptosis. These data suggests that SKP-2 expression plays a major role in the oncogenesis of DLBCL and overexpression of SKP-2 can be used as useful prognostic marker. Furthermore, proteosome inhibitors can be used as future therapeutic modality in treating DLBCL.
A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway.
Authors: Stone Stacie , Sobeck Alexandra , Landais Igor , LaChapelle Alexis , Hoatlin Maureen E.
Published: April 2020

Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds.

We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.
Anaplastic Lymphoma Kinase Is Activated through the Pleiotrophin/Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ Signaling Pathway: A Unique Mechanism of Activation of Receptor Protein Tyrosine Kinases.
Authors: Perez-Pinera Pablo , Zhang Wei , Wang Zhaoyi , Berenson James R. , Deuel Thomas F.
Published: April 2020

Anaplastic Lymphoma Kinase (ALK) is a receptor-type transmembrane tyrosine kinase (RTK) of the insulin receptor superfamily that structurally is most closely related to leukocyte tyrosine kinase. It was first discovered as a chimeric protein (NPM-ALK) of nucleophosmin and the C-terminal (kinase) domain of ALK in anaplastic large cell lymphomas (ALCL). NPM-ALK is constitutively active and generates the oncogenic signals that are the pathogenic mechanisms of these highly malignant cancers. The full-length ALK also is believed to have an important role in the pathogenesis of other human malignancies, since its expression is found in rhabdomyosarcomas, neuroblastomas, neuroectodermal tumors, glioblastomas, breast carcinomas, and melanomas. Recently it was proposed that pleiotrophin (PTN the protein, Ptn the gene) is the ligand that stimulates ALK to transduce signals to activate downstream targets. However, this proposal contrasted with earlier studies that demonstrated Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ is the functional receptor for PTN. PTN was shown to inactivate RPTPβ/ζ and thereby permit the activity of different tyrosine kinases to increase tyrosine phosphorylation of the substrates of RPTPβ/ζ at the sites that are dephosphorylated by RPTPβ/ζ in cells not stimulated by PTN. Subsequent studies identified β-catenin, β-adducin, Fyn, GIT1/Cat-1, P190RhoGAP, and histone deacetylase 2 (HDAC-2) as downstream targets of the PTN/RPTPβ/ζ signaling pathway and demonstrated that their levels of tyrosine phosphorylation increase in PTN-stimulated cells. This diversity of PTN-regulated targets is one basis for the pleiotrophic activities of PTN. We now demonstrate that tyrosine phosphorylation of ALK is increased in PTN-stimulated cells through the PTN/RPTPβ/ζ signaling pathway. It is furthermore shown that ALK is activated in PTN-stimulated cells when it is expressed in cells without its extracellular domain, that β-catenin is a substrate of ALK, that the tyrosine phosphorylation site in β-catenin phosphorylated by ALK is the same site dephosphorylated by RPTPβ/ζ, and that PTN-stimulated tyrosine phosphorylation of β-catenin requires expression of ALK. The data suggest a unique mechanism to activate ALK; the data support a mechanism in which β-catenin is phosphorylated in tyrosine through the coordinated inactivation of RPTPβ/ζ, the activation of the tyrosine kinase activity of ALK, and the phosphorylation of β-catenin by ALK at the same site regulated by RPTPβ/ζ in PTN-stimulated cells. Since PTN often is inappropriately expressed in the same malignancies that express ALK, the data suggest a mechanism through which ALK signaling may contribute to those malignancies that express full length ALK through the activity of PTN to signal constitutively the same pathways as NPM-ALK in ALCL.
Identification of Chemotherapy-Resistant Mechanisms in Childhood T-ALL Cell Lines: Correlation with Genetic Profiles in Patients Having Early Treatment Failure.
Authors: Estes David A. , Lovato Debbie M. , Khawaja Hadya M. , Winter Stuart S. , Larson Richard S.
Published: April 2020

Acquisition of drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) is a major cause of treatment failure. Drug resistant cell lines are effective tools for elucidating mechanisms of acquired drug resistance. In this study, we developed novel drug resistant cell lines that could be employed to identify mechanisms of drug resistance. We established three new drug resistant T-ALL cell lines: 2 resistant to L-asparaginase (L-asp), and one resistant to daunorubicin (DNR). The DNR-resistant line (Jurkat) acquired a 117-fold increase in EC50 to DNR, from 20.6 nM to 2.4 μM, while the L-asp resistant cells (Jurkat and Sup T1) showed respective increases in resistance of 320-fold (0.003 IU/mL to 0.962 IU/mL) and 29-fold (0.042 IU/mL to 1.22 IU/mL). The DNR resistant cell line acquired a multidrug resistant phenotype, showing 310 and 120-fold increase in resistance to vincristine and prednisolone, respectively. Resistance to L-asp was unchanged. Microarray analysis showed that ABCB1 (MDR1, P-glycoprotein) was significantly upregulated (567-fold) in DNR resistant cells. siRNA experiments that reduced ABCB1 mRNA levels by 74% restored DNR sensitivity. In L-asp resistant T-ALL cells (Jurkat and Sup T1), two notable genes were upregulated, asparagine synthetase (ASNS) which catalyzes synthesis of asparagine (41-fold and 1.5-fold) and argininosuccinate synthase (ASS)(32-fold and 6.5-fold), respectively. Reduction of ASNS with siRNA restored drug sensitivity in both cell lines. Interestingly, siRNA suppression of ASS in conjunction with ASNS achieved an exaggerated restoration of drug sensitivity compared to ASNS alone. We next examined the microarray profiles of drug resistant cells with those of 86 T-ALL patients; of which 8 failed induction (IF). Interestingly, these 3 key genes are upregulated in 25–62% of IF cases. Although ABCB1 overexpression has been shown to be a mechanism of DNR resistance in many cancers, there is a paucity of resistant T-ALL cell lines to adequately model the effect of stage of differentiation and genetic heterogeneity underlying drug resistance in T-ALL. With the establishment of 3 new T-ALL cell lines in this report, there exist 5 T-ALL drug resistant cell lines, representing the spectrum of T-cell differentiation (pre-T, cortical T, and mature T cells). Finally, this is the first report of the potential contribution of ASS in addition to ASNS to L-asp resistance in leukemia cells.
Risk Factors for Outcome after Haploidentical Hematopoietic Stem Cell Transplant in Children with Very High Risk Acute Lymphoblastic Leukemia: Impact of Centre Experience. A Survey on Behalf of the ALWP and PDWP of the EBMT.
Authors: Klingebiel Thomas , Cornish Jacqueline , Labopin Myriam , Locatelli Franco , Balduzzi Adriana , Owoc Joanne , Fagioli Franca , Engelhard Dan , Peters Christina , Dini Giorgio , Rocha Vanderson
Published: April 2020

In the absence of an HLA identical donor, T-cell depleted haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) is an alternative option to treat children with very high risk acute lymphoblastic leukemia (ALL). However, little data is available in children. We have analyzed 118 children (≤16 years old) with ALL transplanted with a myeloablative Haplo-HSCT from 1995 to 2004 in Europe. Only transplants with 2 or more HLA disparities out of 6 (A, B and DRB1) were included. The median age was 8.5 years and median follow-up 56 months (8–116). At transplant, 21 (18%) were in CR1, 72 (61%) in CR2 or CR3 and 25 (21%) in more advanced phase. The 3-year leukemia free survival was 32±10%, 28±5% and 0%, respectively. Therefore we restricted the analysis to patients in remission (n=93) transplanted in 30 EBMT centers. Thirty-four (37%) patients were treated in centres performing more than 10 Haplo-HSCT in the study period (3 centres). The median age of recipient and donor was 8.7 and 37 years, respectively. Fifty four (65%) received a full Haplo-HSCT and 95% of the donors were parental. Twenty seven (29%) had t(9;21) or t(4;11). The majority of patients did not receive drugs for GVHD prophylaxis, ATG/ALG was used in conditioning in 74%, and 73% received TBI. The Clinimacs® device was used in 74% of selections. The median number of CD34+ cells infused was 12.8 106/Kg. Cumulative incidence with competing risk and KM estimates were used to calculate outcome probabilities. The median days of neutrophil recovery was 15 days (8–55) and 90% of patients had signs of engraftment. Acute GVHD II–IV was observed in 24% of the patients. In univariate analysis for LFS there was a trend towards better results for patients receiving higher CD34 cell dose (p=0.08) and a significant difference for patients transplanted in centres performing more Haplo-HSCT (49% versus 17%, p=0.002). Relapse incidence (RI) and non relapse mortality NRM) tended to be different between the experienced and less experienced centres. In less experienced centres NRM was 41% vs 27% (p=0.13) and RI 41% vs. 24% (p=0.10). There were patient, donor and transplant related differences between less and more experienced centres, specifically related to donor sex, Philadelphia positivity, year of transplantation, use of TBI, ATG and DLI and previous autograft. In conclusion, Haplo-HSCT is an alternative option to treat children with very high risk ALL in the absence of HLA identical donor. Centres with more experience have better LFS.
Ionizing Radiation Up-Regulates Telomerase Activity in Cancer Cell Lines; Mechanisms and Possible Clinical Relevance.
Authors: Lahav Meir , Ram Ron , Uziel Orit , Fenig Eyal , Beery Einat , Nordenberg Yardena
Published: April 2020

Background: Activation of telomerase has been recently recognized as one of the pivotal events in malignant transformation. Up-regulation of this enzyme is probably necessary for transformation and, more importantly, for perpetuation of the malignant clone. Down regulation of telomerase activity may compromise the viability of cancer cells. Therefore, telomerase is considered to be a central target for future anti cancer therapies. Indeed, others and we have shown that telomere shortening by inhibition of telomerase sensitizes malignant cells to several cytotoxic drugs. Very little is known about the effect of irradiation, an important anticancer modality, on telomerase activity. The available data is sketchy and controversial. In this study we characterize the effect of irradiation on telomerase activity in malignant cells and its molecular mechanism.

Methods: Several cancer cell lines were exposed to a range of radiation doses. Telomerase activity was assessed at various time points. All levels of telomerase regulation were evaluated: transcriptional, alternative splicing, posttranslational phosphorylation and dephosphorylation and intracellular compartmentalization of the enzyme. Subsequently, the involvement of several signal transduction pathways triggered by irradiation and relevant to telomerase regulation was evaluated by point inhibition of numerous signaling compounds.

Results: In all cell lines, irradiation caused a fourfold increase in telomerase activity with a maximal effect at 24 hours at a dose of 400 Rad. This elevation was mediated by posttranslational modification of the enzyme as phospho-AKT; the enzyme that phosphorylates telomerase was increased after irradiation. Transcriptional activity and intracellular compartmentalization of telomerase were not affected. PI3K/AKT pathway mediates the irradiation-induced upregulation of telomerase activity, as wortmanin decreased that effect. RAS oncogene was the upstream effector of PI3K, since its inhibition by FTS decreased irradiation dependent telomerase activity. PTEN was not involved in regulating that effect. In addition, several membrane receptors known to be activated by radiation including Erb1, Erb2 andIGF1R were not involved in the signaling cascade leading to the up regulation of telomerase.

Conclusions: Radiation causes posttranslational upregulation of telomerase in cancer cells lines. Telomerase activation probably enables non-specific “chromosomal healing” in certain low irradiation dosages. This activation was mediated via the PI3K/AKT signaling pathway activated by Ras. The membrane receptors classically activated by irradiation were not involved in this process. These finding have potential clinical implication, since the upregulation of telomerase by irradiation in cancer cells may interfere with future cytotoxic treatment and necessitate concomitant inhibition of the enzyme.
FoxO Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress.
Authors: Tothova Zuzana , Kollipara Ramya , Huntly Brian J. , Lee Benjamin H. , Castrillon Diego H. , Passegue Emmanuelle A. , Cullen Dana E. , McDowell Elizabeth P. , Lazo-Kallanian Suzan , Williams Ifor R. , DePinho Ronald A. , Gilliland D. Gary
Published: April 2020

Hematopoietic development and long-term homeostasis are tightly regulated by a dynamic balance of stem cell self-renewal and differentiation. To understand the role of the FoxO family of transcription factors in these processes, we studied the impact of somatic deletion of all FoxO genes in the adult hematopoietic system of mice engineered with the interferon-inducible Mx-Cre transgene and various conditional FoxO alleles: FoxO1L/L; FoxO3 L/L; and/or FoxO4 L/L. Cre-mediated excision of FoxO1, FoxO3 and FoxO4 (FoxO1/O3/O4L/L), resulted in myeloid lineage expansion, lymphoid developmental abnormalities and a marked decrease of the lineage-negative, Sca-1+, c-Kit+ (LSK) compartment, containing the hematopoietic stem cell (HSC) population, but normal numbers of myeloid progenitors. Furthermore, FoxO1/O3/O4L/L null bone marrow cells were defective in competitive and noncompetitive repopulation assays. This FoxO deficient defect correlated with enhanced cell cycling and increased apoptosis of HSC cells. Notably, there was an HSC-restricted elevation in the level of reactive oxygen species (ROS) that was not observed in committed progenitors, and was associated with decreased catalase and MnSOD expression. Furthermore, in vivo treatment with the anti-oxidative agent N-acetyl-cysteine (NAC) resulted in complete reversion of the FoxO deficient HSC phenotype. Taken together, these results demonstrate that, in the HSC compartment, FoxO proteins are mediators of quiescence and enhanced survival and play essential regulatory roles in the response to physiologic oxidative stress, a function that may contribute to the long-term regenerative potential of the hematopoietic stem cell compartment.
Histone Deacetylase Inhibitor, PCI-24781, Induces Growth Inhibition and Apoptosis in Hematopoietic Tumor-Derived Cell Lines and Primary Leukemia Cells.
Authors: Buggy Joseph , Balasubramanian Sriram , Ramos Jason , Sirisawad Mint , Naumovski Louie
Published: April 2020

Histone deacetylase (HDAC) inhibitors are a new class of anticancer drugs that have shown promising activity in clinical trials. PCI-24781 (formerly CRA-024781) is a novel, hydroxamic acid-based HDAC inhibitor that is currently under evaluation in phase I clinical trials in patients with refractory solid malignancies and lymphomas. In this study, we show that PCI-24781 inhibits growth and induces apoptosis in a variety of hematopoietic cell lines derived from B-, T- and myeloid malignancies. Growth inhibition and apoptosis were noted at drug concentrations < 0.125 μM and were accompanied by known biochemical markers of HDAC inhibition including histone and tubulin hyperacetylation. PCI-24781 was also found to be active in animal xenograft models of hematopoietic disease displaying favorable drug-like pharmacokinetic and pharmacodynamic profiles, both intravenously and orally. To further establish the potential clinical utility of PCI-24781 in hematologic malignancies, primary leukemia samples were isolated from patients and screened for resistance to PCI-24781 in vitro. Of these 19 leukemia samples (10 acute myelogenous leukemia (AML) and 9 acute lymphocytic leukemia (ALL) some of which were derived from patients who had failed standard therapy), none were resistant to PCI-24781 at 0.5 μM and only 2 (1 AML and 1 ALL) were considered resistant at 50 nM. Gene expression analysis of PCI-24781 activity in these primary samples was consistent with HDAC inhibition and suggested potential downstream mechanisms of action for this compound in leukemia. These results demonstrate that PCI-24781 is active in hematopoietic tumor-derived cell lines in vitro as well as in vivo in preclinical models. Furthermore, the sensitivity of primary leukemic tumors to treatment with PCI-24781 in vitro, coupled with the predicted pharmacokinetics in humans suggests that PCI-24781 could be a valuable agent for the treatment of hematopoietic tumors.
Bortezomib Combined with Harringtonine or Arsenious Acid Induced Apoptosis in HL-60 Cells and Its Mechanism.
Authors: Meng Fan Yi , Fu Yun-bi , Sun Qi-xin , Xie Jun , Zhou Guang-biao
Published: April 2020

Objective: To explore the apoptosis effect induced by bortezomib combined with harringtonine or arsenious acid in HL60 cell line and the mechanism.

Methods: Cell’s apoptosis was demonstrated by MTT assay and Hoechst33342 staining. Expression of Bcl-2, Caspase-9, Caspase-3 and PARP protein was detected by western blotting.

Results: HL60 cells’ apoptosis could be induced by bortezomib, harringtonine and arsenious acid respectively, and the apoptosis effect was inhanced significantly when bortezomib combined with harringtonine or arsenious acid. Western blotting showed Bcl-2 protein was down-regulated and Caspase-9, Caspase-3 and PARP proteins were all cleaved activation when cells were treated by 15uM arsenious acid alone, but only cleaved activation of PARP and down-regulation of Bcl-2 protein be detected when cells were treated with 30nM harringtonine alone, expression of Caspase-9 and Caspase-3 has no change compared with the control. The changes of associated proteins were paralleled with the cell’s apoptosis when treated with combined drugs.

Conclusion: HL60 cells’ apoptosis could be inhanced significantly when treated by bortezomib combined with harringtonine or arsenious acid compared with treated by bortezomib alone. Arsenious acid and bortezomib can inhibit caspase signaling pathway and down-regulate the expression of Bcl-2 protein together, but harringtonine and bortezomib can only down-regulate the expression of Bcl-2 protein and induce cleaved activation of PARP together.
Soluble CD30 Can Distinguish Disease Progression from Acute GVHD in Patients with Adult T Cell Leukemia/Lymphoma Who Received Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Yoshimitsu Makoto , Fujiwara Hiroshi , Aoki Noriko , Ozaki Atsuo , Hamada Heiichiro , Matsushita Kakushi , Arima Naomichi , Tei Chuwa
Published: April 2020

At present, allogeneic hematopoietic stem cell transplantation (HCST) has been an only curative modality for the treatment of adult T cell leukemia/lymphoma (ATLL). Disease relapse and GVHD are the major causes of mortality after allo-HSCT for ATLL. To achieve better outcome, early detection of disease relapse after allo-HSCT is absolutely crucial for decision-making of interventions such as donor lymphocyte infusion and/or adjuvant chemotherapy, while tumor burden still remains in the level of minimal residual disease (MRD). Serum soluble interleukin-2 receptor alpha (sIL-2R) has been known as a marker of ATLL tumor burden in affected patients, however it increases in patients with acute and chronic GVHD as well. Here we propose a new strategy to distinguish disease progression of ATLL and GVHD at an early stage by measuring serum levels of soluble CD30 antigen (sCD30) and sIL-2R. The surface CD30 is normally expressed by activated B or T cells. It has been known that Hodgkin cells, anaplastic large cell lymphoma and ATLL cells also express surface CD30. The soluble form of CD30 is produced by metalloprotease cleavage. Acute type ATLL patients whose tumor cells express surface CD30 (CD30+ATLL), who underwent allo-HSCT, were included in this study. As control, the serum levels of sCD30 were measured in a healthy donor, an asymptomatic HTLV-I carrier and acute type ATLL patients whose tumor cell were negative for surface CD30 (CD30−ATLL), all were below measurable range. In addition, the level of sCD30 was undetectable in a CD30− ATLL patient with grade II gut acute GVHD as well. Confirming these low backgrounds, both of sCD30 and sIL2R levels were sequentially measured from induction chemotherapy and throughout allo-HSCT. During chemotherapy, the levels of sCD30 and sIL2R are well-correlated along with tumor burden as previously reported, and both markers increase at disease relapse after allo-HSCT as well. In contrast, during GVHD, sIL-2R levels increase along with GVHD status, while sCD30 remained lower or undetectable. In conclusion, serum levels of sCD30 and sIL-2R may predict not only disease progression at an early stage after allo-HSCT, but also distinguish that from GVHD. These findings may help to introduce an early intervention for ATLL progression with DLI and/or adjuvant chemotherapy at the level of minimal residual disease.

Figure Figure