The Acute Promyelocytic Leukemia-Oncoprotein PML-Raralpha Blocks Senescence and Disrupts The Atrx/Daxx Chromatin Remodeling Complex To Promote Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1267-1267
Author(s):  
Katharina Korf ◽  
Harald Wodrich ◽  
Alexander Haschke ◽  
Ron M. Evans ◽  
Thomas M. Sternsdorf
Keyword(s):  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Cell Cycle Regulators ◽  
Murine Bone Marrow ◽  
Pml Nuclear Bodies

Abstract Although Acute Promyelocytic Leukemia (APL) has become a curable disease due to in-depth understanding of the underlying molecular processes, its investigation has provided unique and valuable insights into the processes involved in leukemogenesis. Therefore we use it as a model disease. 99% of APL-patients express a PML-RAR fusion protein. While involvement of RAR has proven indispensable for oncogenicity, the role of the PML domain is far less clear. In our previous study (Sternsdorf et al., Cancer Cell, 2006) we found that substitution of PML with heterologous self-interaction domains suffices to induce leukemias, but drastically decreases oncogenic potency of the resulting fusion proteins. In this study, we have chosen the inverse strategy: we have modified the PML domain to create a more active artificial model oncoprotein by adapting PR to its biological environment: As the typical model organism for APL studies is the mouse, we have replaced the human PML domain with the murine PML domain. This oncoprotein (mPR) creates APL-type leukemias in mice with higher penetrance and shorter latency than its human counterpart, hPR. We have used this system to study immediate early effects of expression of the model oncoprotein. While proliferating murine bone marrow cells go into senescence ex vivo, expression of mPR prevents this and robustly immortalizes murine bone marrow from every mouse strain tested so far. Senescence-associated upregulation of the cell-cycle regulators p21 and p19 was efficiently blocked by mPR expression. In mouse cells, mPR exhibits higher potency in disrupting the PML-associated Daxx/ATRX complex than hPR. Knockdown of ATRX, but not Daxx ameliorated ATRA-induced growth suppression and p21 upregulation in the human APL model cell line NB4. These data suggest, that PML-RAR promotes leukemogenesis by disrupting the Daxx/ATRX complex, which assembles at PML nuclear bodies during the onset of senescence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of residual leukemic cells in patients with acute promyelocytic leukemia by the fluorescence in situ hybridization method: potential for predicting relapse

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 495-499 ◽  
Author(s):  
L Zhao ◽  
KS Chang ◽  
EH Estey ◽  
K Hayes ◽  
AB Deisseroth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Low Frequencies

Abstract The translocation between chromosomes 15 and 17, t(15;17)(q22–24;q11– 21), is present in the bone marrow cells of most patients with acute promyelocytic leukemia (APL). Although conventional cytogenetic methods are useful for diagnosing this disease, difficulties are experienced in detecting residual disease among those patients who have achieved remission. In this study, we used the fluorescence in situ hybridization (FISH) method to attempt to detect residual leukemic cells in 10 APL patients in clinical remission. The duration of remission ranged from 2 to 93 months at the time of study. Multiple bone marrow samples were analyzed by FISH in most patients. In 6 patients, no cell with t(15;17) was found. These patients remain in complete remission at present (approximately 25 to 33 months since first studied by FISH). In 4 patients, low frequencies of cells with t(15;17) were observed in at least one bone marrow sample examined. All of these patients relapsed within 1 to 14 months. No cell with t(15;17) was identified by the conventional G-banding method in any sample. The FISH results correlated well with that of a two-round nested reverse transcription polymerase chain reaction assay that was performed on the same samples. Thus, our study suggests that FISH is potentially a useful tool for detecting residual APL cells and for identifying patients at high risk of relapse.

scholarly journals Immunocytochemical Diagnosis of Acute Promyelocytic Leukemia (M3) With the Monoclonal Antibody PG-M3 (Anti-PML)

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4046-4053 ◽  
Author(s):  
Brunangelo Falini ◽  
Leonardo Flenghi ◽  
Marta Fagioli ◽  
Francesco Lo Coco ◽  
Iole Cordone ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Detection System ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Nuclear Staining ◽  
Wild Type ◽  
Amino Terminal ◽  
Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes, leading to the expression of the PML/RARα fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARα fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARα fusion protein (usually ≥50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARα. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.

Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3973-3973
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloproliferative Disease ◽  
Murine Bone Marrow ◽  
Hematologic Disorder ◽  
Almost All ◽  
Marrow Cells

Abstract Abstract 3973 Poster Board III-909 Purpose Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q. Results We transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells. Conclusion Thus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of residual leukemic cells in patients with acute promyelocytic leukemia by the fluorescence in situ hybridization method: potential for predicting relapse

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 495-499
Author(s):  
L Zhao ◽  
KS Chang ◽  
EH Estey ◽  
K Hayes ◽  
AB Deisseroth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Low Frequencies

The translocation between chromosomes 15 and 17, t(15;17)(q22–24;q11– 21), is present in the bone marrow cells of most patients with acute promyelocytic leukemia (APL). Although conventional cytogenetic methods are useful for diagnosing this disease, difficulties are experienced in detecting residual disease among those patients who have achieved remission. In this study, we used the fluorescence in situ hybridization (FISH) method to attempt to detect residual leukemic cells in 10 APL patients in clinical remission. The duration of remission ranged from 2 to 93 months at the time of study. Multiple bone marrow samples were analyzed by FISH in most patients. In 6 patients, no cell with t(15;17) was found. These patients remain in complete remission at present (approximately 25 to 33 months since first studied by FISH). In 4 patients, low frequencies of cells with t(15;17) were observed in at least one bone marrow sample examined. All of these patients relapsed within 1 to 14 months. No cell with t(15;17) was identified by the conventional G-banding method in any sample. The FISH results correlated well with that of a two-round nested reverse transcription polymerase chain reaction assay that was performed on the same samples. Thus, our study suggests that FISH is potentially a useful tool for detecting residual APL cells and for identifying patients at high risk of relapse.

A Novel t(3;17) Variant of Acute Promyelocytic Leukemia with Rearrangement of the RARA Locus.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4428-4428
Author(s):  
Robert L. Redner ◽  
Lydia C. Contis ◽  
Carol Evans ◽  
Maureen E. Sherer ◽  
Sofia Shekhter-Levin
Keyword(s):  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Control Culture ◽  
Promyelocytic Leukemia ◽  
Dna Probe ◽  
Chromosome 17 ◽  
Fish Analysis ◽  
Dual Color

Abstract The vast majority of patients with Acute Promyelocytic Leukemia (APL, FAB M3) have the t(15;17)(q12;q21) chromosomal translocation. This introduces the gene for PML into the retinoic acid receptor alpha (RARA) locus, which leads to expression of a PML-RARA fusion. There is convincing evidence that expression of PML-RARA underlies the APL phenotype. Yet, there have been identified rare cases of APL that do not manifest t(15;17). Many of these cases exhibit cryptic rearrangements of PML and RARA. However, in a number of cases it has clearly been shown that a fusion protein different than PML-RARA is expressed. These include the t(11;17)(q23;q21) that expresses a PLZF-RARA fusion; t(5;17)(q35;q21) that encodes NPM-RARA; t(11;17)(q13;q21) that encodes NUMA-RARA; and der(17) with duplication of 17q21.3-q23 that fuses STAT5b to RARA. We report here a novel case of APL with t(3;17) with rearrangement of RARA, but not PML. A 72 year old man presented with leukocytosis, anemia, and thrombocytopenia: wbc 20.4 X10E+9/L; hgb 10.3 g/L; PLT 22 x10E+9/L. The wbc differential showed 20% polys, 4% bands, 15% lymphocytes, 19% monocytes, 34% blasts, 1% promyelocyte, 6% myelocyte, 1% metamyelocytes. Auer rods were seen. The bone marrow was hypercellular (approximately 80%), with 88% blasts, 1.7% promyelocytes, 0.3% myelocyte, 0.3% polys, 0.3% eosinophile, 3% monocytes, 0.3% pronormoblasts, 3.7% normoblasts, and 2.3% lymphocytes. The blasts demonstrated prominent cytoplasmic granulation, Flow cytometric analysis showed the blasts to be CD117 positive, myeloperoxidase positive, CD13/33 positive, but lacking CD34 or HLA-DR expression, consistent with a diagnosis of APL. Cytogenetic studies indicated a mosaic abnormal analysis with an apparent normal cell line and one that demonstrated a 47,XY,t(3;17)(p25;q12-21), +8 karyotype. Analysis for PML-RARA expression by RT-PCR was indeterminate, owing to poor quality of the extracted RNA. Fluorescence In Situ Hybridization (FISH) was therefore performed on two hundred unstimulated cells, primarily in interphase, using the Vysis t(15;17) dual color DNA probe. 98.5% of the cells were negative for PML-RARA rearrangement (the value of 1.5% positivity is within the laboratory’s control range for false positives). To confirm that the t(3;17) rearrangement involved the RARA locus, we scored 203 unstimulated cells using the LSI RARA dual color DNA probe. 100% were positive for the RARA gene rearrangement (split signal). Four metaphase cells each showed one fused red/green signal on the normal chromosome 17, one red signal on der (17), and one green signal on the distal arm of chromsome 3. The FISH analysis therefore indicated rearrangement of the RARA, but not the PML locus. The patient expired before treatment could begin. To determine whether the t(3;17) blasts could differentiate (a hallmark of t(15;17) APL), we cultured the bone marrow cells in RPMI 1640 with 10% FCS and 10E-6 M ATRA. At 10 days 58% of the cells resembled metamyelocytes, bands, or mature polys, compared with none in the control culture. This indicates that t(3;17) retains its ability to differentiate in the presence of ATRA, consistent with its classification as a novel variant of APL.

scholarly journals PML Mutation in PML-Rarα Alters PML Nuclear Body Organization and Induces ATRA Resistance in Acute Promyelocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3923-3923 ◽  
Author(s):  
Hong-Hu Zhu ◽  
Congying Wu ◽  
Yujie Sun ◽  
Jiong Hu
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Single Molecule ◽  
Structural Changes ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Response To Therapy ◽  
Nuclear Bodies ◽  
Cross Resistance ◽  
Drug Induced

Abstract Acute promyelocytic leukemia (APL) has become a highly curable disease using target drugs including all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) with or without chemotherapy. However, APL patients with acquiredPML mutationsin PML-RARα had dismal outcomes and showed resistance not only to ATO but also to ATRA.In this study, we confirmed in the clinic that APL patients with mutations in the PML moiety of PML-RARα showed different resistanceto ATRA (cross-resistance). We demonstrated that critical mutations are responsible for this "cross-resistance". Super-resolution microscopy and electron microcopy were employed to examine the fine structural changes of PML/PML-RARα nuclear bodies (NBs) in response to therapy. We observed drug-induced acceleration of NB movement, which was absent in the mutant cells. Furthermore, NB doublets were revealed, and fusion events were captured upon drug treatment. We proposed that SUMOylation contributed to increased NB dynamics, leading to more fusion incidents and thustaking functional NBs for oncoprotein degradation. Different protein density in the mutant NBswas identified by single molecule quantification, indicating reduced permeability and recruitment of clients. Taken together, our results revealed that critical sites in the PML moiety of the PML-RARα fusion gene disrupted NB scaffold organization and perturbed fusion protein degradation in response to ATRA.Our work will largely improve the mechanistic understandings of APL pathology and therapy and will shed new light on unveiling the structure and function of PML NBs. Disclosures No relevant conflicts of interest to declare.

scholarly journals The PML/RARα-Regulated MiR-181a/b-Cluster Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2195-2195
Author(s):  
Daniela Braeuer-Hartmann ◽  
Jens-Uwe Hartmann ◽  
Dennis Gerloff ◽  
Christiane Katzerke ◽  
Alexander Arthur Wurm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Acute Promyelocytic Leukemia ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Knock Down ◽  
Functional Studies

Abstract In acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify the genomically clustered miR-181a and miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. In addition, the expression of the miR-181a/b-cluster was strongly reduced in bone marrow samples of APL patient while ATRA-based therapy. Furthermore, we showed the transcriptional induction of miR-181a and miR-181b by the APL-associated PML-RARα oncogene in vitro and in vivo. In PR9 cells, carrying a zinc-driven PML/RARα construct, and in PML/RARα-knock in mice the expression of the fusion gene leads to upregulation of the microRNA-cluster expression. Analysis of bone marrow samples of APL patients showed an enhanced expression of miR-181a and miR-181b in comparison to AML patient samples with normal karyotype, whereas other AML subgroups show no significant regulation. Based on siRNA experiments we could propose AP-1 and GATA-2 as potential co-activators for the PML/RARα-dependent regulation of the miR-181a/b-cluster. In functional studies in NB4 cells we observed after lentiviral knock down of miR-181a and miR-181b a significant reduction of colony size and number as well as proliferation rate. In contrast, transient overexpression of miR-181a and miR-181b led to an inhibition of ATRA-induced expression of the differentiation marker CD11b. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181a and miR-181b. In functional studies we showed that enforced expression of miR-181a and miR-181b reduces the protein level of RASSF1A by binding to the 3´UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. Overexpression of RASSF1A in NB4 cells strongly induced apoptosis. Additional, we could show by western blot that the miR-181a/b-cluster and RASSF1A modulate cell cycle via regulation of cyclin D1. In conclusion, we identified the miR-181a/b-cluster as an important player in the PML/RARα associated APL. Moreover, we firstly described the miR-181a/b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated microRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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