ALBERT

Description: The t(10;11)(p12;q14) is a recurring chromosomal translocation that is found in acute myeloid and acute lymphoblastic leukemia as well as in malignant lymphoma. This translocation results in the fusion of AF10, a putative zinc finger transcription factor containing an N-terminal LAP/PHD zinc finger motif, a nuclear localization signal, an AT-hook domain, and a leucine zipper and with the CALM gene (Clathrin assembly protein lymphoid myeloid leukemia gene) that encodes a clathrin assembly protein. In the monocytic cell line U937 both the CALM/AF10 and the AF10/CALM fusion mRNAs can be identified. The CALM/AF10 fusion mRNA codes for the CALM/AF10 fusion protein which contains almost the complete CALM protein fused in frame to about 90% of the AF10 protein without the N terminal two PHD zinc fingers. CALM/AF10 is highly leukemogenic with its expression in primary bone marrow cells leading to the development of an aggressive acute leukemia with a short latency of 10 weeks in a murine bone marrow transplant model. We set out to identify immediate target genes of CALM/AF10. The fusion gene was cloned into pRTS-1, an episomally replicating tetracycline/doxycycline inducible (tet-on) expression vector. The construct was stably transfected into the Burkitt’s lymphoma B cell line DG75. The expression of CALM/AF10 after induction was confirmed by RT-PCR. Gene expression profiling experiments were conducted using the Affymetrix® Human Genome U133 Plus 2.0 Array analyzing non-induced and induced (24 and 72 hours after induction) samples. As controls, DG75 cells transfected with the pRTS-1 vector without the CALM/AF10 fusion gene were used. The results were analyzed using the R statistical package and dChip (DNA Chip Analyzer) software. The expression of 1237 genes was found to be changed at least 2 fold 24 hours after the induction of CALM/AF10. 594 (48%) genes were downregulated, while 643 (52%) were upregulated. Downregulated genes included genes involved in DNA repair (DDB2, TOP2A, BRCA1), cell cycle check point control (CCNE2, CHEK1, CDC2), and chromosome maintenance (MCM3, MCM7, MCM10). Upregulated genes included signal transduction molecules like RAB8B (a member of the RAS oncogene family) and STAT family members (STAT1 and STAT2), chromatin remodeling factors like BAZ2A (bromodomain adjacent to zinc finger domain, 2A), and MAML3 (master mind like 3), a positive regulator of Notch signaling. Pathway analysis using KegArray showed an enrichment of differentially regulated genes in processes like cell cycle regulation and DNA replication and repair. Interestingly, no significant upregulation of Hox genes was observed, as was previously reported in a study of CALM/AF10 positive patient samples (Dik et al., 2005). The changes in the expression levels of some selected genes were confirmed using a Taqman® Low Density Array (LDA). This analysis showed a statistically significant correlation (r = 0.78, p = 0.001) between the real time PCR results and the expression levels obtained from the Affymetrix arrays. Our results demonstrate that the expression of the leukemogenic CALM/AF10 fusion protein leads to a severe deregulation of critical cellular processes giving a first hint at the direct target genes of this fusion protein.

Description: The focus of our research group is the study of the t(10;11)(p13;q14) translocation that leads to the fusion of the proteins CALM and AF10. This translocation can be found in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL) and also in malignant lymphomas. In some patients the t(10;11) is the only cytogenetic abnormality which indicates that the CALM/AF10 fusion is a causal event during leukemogenesis. Previous studies of our group have shown that the expression of CALM/AF10 in hematopoietic stem cells triggers the development of an aggressive leukemia in a murine bone marrow transplantation model. CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) has a function in Clathrin mediated endocytosis. AF10, a putative transcription factor with a PHD-Motive (Plant Homeo Domain) and a Leucine Zipper domain, was initially identified as fusion partner of MLL. The underlying mechanism of CALM/AF10 dependent leukemogenesis, however, remains mostly unknown. Recently we could show that AF10 interacts with the transcription factor Ikaros (ZNFN1A1) in yeast-two-hybrid assays. Interestingly, Ikaros is a key regulator of hematopoesis, required for normal differentiation and proliferation of B- and T-lymphocytes. The structure of the protein is characterized by a DNA-binding and an oligomerisation domain. Through interaction with many factors in the cell nucleus, Ikaros can act both as activator and repressor of transcription. In various forms of ALL as well as chronic myeloid leukemia (CML) an aberrant expression pattern of Ikaros has been found. In a murine model the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. Using various AF10 deletion mutants in the yeast, the Ikaros interaction domain of AF10 was mapped to the Leucine Zipper domain of AF10 which has also been shown to be required for malignant transformation by the MLL/AF10 fusion protein. Overexpression of fluorescently labelled proteins reveals a similar distribution pattern of AF10 and Ikaros in the nucleus, whereas in the presence of CALM/AF10 Ikaros appears to be localized predominately in the cytoplasm. The interaction between AF10 and Ikaros has been confirmed by GST-pull-down assays. In order to further study this interaction and its role in leukemogenesis we have raised monoclonal antibodies against the C-terminus of AF10. These antibodies are currently established for Western Blot analysis and Immunoprecipitation experiments. Reporter gene assays are carried out to measure the impact of CALM/AF10 on Ikaros’ function as repressor or activator of transcription. These studies may provide new insights into the mechanism of CALM/AF10 induced leukemia and thereby facilitate the development of new therapies.

Description: The CALM/AF10 fusion, which is the result of the t(10;11)(p12;q14), is associated with various hematological malignancies including acute myeloid leukemia (AML), T cell acute lymphoblastic leukemia (ALL) and malignant lymphoma, and has usually a poor prognosis. We established a CALM/AF10 knock-in mouse model, which allows tissue-specific expression of the fusion gene. The CALM/AF10 fusion gene, preceded by a loxP site flanked transcriptional stop cassette, was knocked into the Rosa26 locus (R26LSLCA strain). Tissue-specific CALM/AF10 expression was achieved by crossing R26LSLCA mice with three Cre inducer lines expressing the Cre recombinase under the control of defined promoters (Vav-Cre, Mb1-Cre, CD19-Cre). Acute leukemia developed in all (n=23) Vav-Cre/R26LSLCA mice with a median latency of 12 months. In the Vav-Cre line, the Cre recombinase is expressed in all hematopoietic cells including stem cells. Leukemias were either myeloid or had a combination of myeloid and lymphoid features with the expression of the B cell marker B220. The leukemia in these mice was characterized by leukocytosis, splenomegaly and bone marrow as well as multi organ infiltration of myeloid blast like cells. In contrast, none of the mice with the Mb1-Cre/R26LSLCA (n=25) or the CD19-Cre/R26LSLCA (n=20) genotype, which expressed the CALM/AF10 from the early B cell progenitor stage, developed leukemia, even though the B cells of these mice expressed the CALM/AF10 transcript at comparable levels to the levels observed in the bone marrow and spleen cells of the leukemic mice. Affymetrix gene expression profiling (GEP) of leukemic and pre-leukemic bone marrow cells of Vav-Cre/R26LSLCA mice revealed that high expression of Hoxa cluster genes and the Hox co-factor Meis1 occurred before the onset of overt leukemia. The B cells from Mb1-Cre/R26LSLCA mice did not show higher expression of Hoxa cluster genes or of Meis1 compared to B cells from wild type mice. The long latency to leukemia development in the Vav-Cre/R26LSLCA mice suggested that additional genetic lesions were required to cooperate with the CALM/AF10 fusion to lead to malignant transformation. To identify these lesion, we performed whole exome sequencing (WES) on the DNA from the leukemic cells of 8 Vav-Cre/R26LSLCA mice and compared the sequence to the corresponding germ line DNA and a pool of 10 germline control WES datasets. We identified between 1 and 6 somatic point mutations and indels per sample in the 5 exomes with the highest product of percent exome coverage at more than 10x and blast percentage (10x coverage: range 22 to 91%, median 86%). There was a median of 4 somatic nonsense and missense mutations per exome in the 5 exomes, with a strong tendency for more mutations being identified in the exomes with higher coverage and higher blast percentages. Even though only a small number of leukemias was analyzed by WES, two leukemia exomes had recurring mutations in the same gene (4930595M18Rik), and two other leukemias had mutations in known leukemia drivers involved in cellular proliferation pathways. One leukemia carried an activating mutation in the tyrosine kinase domain of Flt3, and in another exome a mutation in the catalytic domain of the intracellular protein tyrosine phosphatase Ptpn11 was found. PTPN11 is a downstream effector of the Ras pathway and mutations in PTPN11 is repoted in juvenile myelomonocytic leukemia (JMML) and Noonan sydnrome. There was no obvious correlation between the mutations and the type of leukemia (myeloid or myeloid with B220 expression) observed in the mice. Our results strongly suggest that leukemia only develops if CALM/AF10 is expressed in hemaptoietic stem cells. Expression of CALM/AF10 in B cells is not sufficient for transformation. Presumably, the expression of CALM/AF10 in long-lived hematopoietic stem cells allows for the acquisition of additional, cooperating mutations, which are required for full leukemic transformation.The reproducibility and relatively long latency of leukemia development in the Vav-Cre/R26LSLCA mice should make them a good model for the study of clonal evolution and collaborating events in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3536 ΔNp73 is an alternative TP73 gene transcript lacking the transactivation (TA) domain that is generated via alternative splicing and/or P2 promoter. The encoded protein acts as a potent transdominant inhibitor of wild type TP53 and full-length TAp73. In several human malignancies the unbalanced expression of transcriptionally active (TAp73) and inactive (ΔNp73) variants correlates with treatment outcome. We have previously reported that higher ΔN/TA isoform expression ratio was associated with poorer prognosis and resistance to cytarabine induced apoptosis in patients with acute myeloid leukemia (AML) (Lucena-Araujo et al., 2008). In acute promyelocytic leukemia (APL), both isoforms are expressed, but the clinical significance remains unknown. The aim of this study was to determine whether the ΔN/TA expression ratio was associated with treatment outcome of APL patients and to investigate the mechanisms by which ΔNp73 may contribute to PML-RARa+ cell survival. Using isoform-specific probes for ΔNp73 and TAp73, their expression was analyzed in 166 APL patients by Real-time quantitative polymerase chain reaction (RQ-PCR). Patients were divided into tertiles for ΔN/TA expression ratio (median value=23.62; 33rd/66th percentiles=12.8/42.3) and their clinical and laboratory characteristics were compared. Patients in the highest tertile presented higher white blood cells (WBC) counts than those in intermediate/lower tertiles (p

Description: The t(10;11)(p13;q14) is a recurring translocation associated with the CALM/AF10 fusion gene which is found in undifferentiated leukemia, acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma with poor prognosis. The CALM/AF10 fusion protein was reported to be the most common fusion protein in T-ALL with TCR gamma delta rearrangement. We have analyzed samples from 9 patients with different types of leukemia: case 1 (AML M2), case 2 (AML M0), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), case 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), with a t(10;11) translocation suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM mRNA by RT-PCR and sequence analysis. All these patients were found positive for the CALM/AF10 fusion. In addition, we analyzed a series of twenty-nine patients with T-ALL with gamma delta rearrangement. Among these patients, four were positive for CALM/AF10 transcripts, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. We found three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 11). There was no correlation between disease phenotype and breakpoint location. The patients were 5 to 46 years old (median 25). Ten CALM/AF10 positive patients were further analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent of the pattern seen in leukemias with rearrangements of the MLL gene, and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. It is known that alhambra, the Drosophila homologue of AF10 can act on polycomb group responsive elements, which play a critical role in the regulation of the HOX gene clusters. It is thus conceivable that the CALM/AF10 fusion proteins acts in a dominant negative fashion on wild type AF10 function relieving the repression that is presumably normally exerted by AF10 on the expression of HOX genes.

Description: The CATS gene was first identified in a yeast two hybrid screen as CALM interacting protein expressed in thymus and spleen. The CATS interaction region of CALM is contained in the leukemogenic fusion protein CALM/AF10, which is found in acute myeloid leukemia, malignant lymphoma and acute lymphoblastic leukemia. We performed co-immunoprecipitation experiments to confirm CALM-CATS interaction found in the yeast system. RT PCR analysis showed CATS expression in patient cells and cell line carrying the CALM/AF10 rearrangement. Further expression analysis showed CATS expression in different cell subpopulation from the murine thymus (CD4+, CD8+ and CD4+/CD8+) and bone marrow (cKit+, Sca 1+/CKit+, Ter119, Gr1, CD8+, Mac1) but nearly absent in B220 positive cells. Interestingly, there was a high CATS expression in CALM/AF10 expressing B220+ leukemic cells in a CALM/AF10 murine bone marrow transplant model. Several monoclonal antibodies against the C-terminus of human CATS were generated. These antibodies recognize both the human and the murine CATS protein. Using these antibodies we could show a high expression of CATS in different human leukemia, lymphoma and solid tumor cell lines, as well as in normal proliferating cell lines (HEK293 and WI38), but not in T-cell lines (TYRF8 and JB4). Using protein lysates from cell cycle synchronized cells (Hela and U2OS) a clear cell cycle dependent regulation of CATS protein levels was demonstrated. Moreover, Western Blot analysis shows that serum stimulation after serum starvation leads to increased expression of CATS in the glioblastoma cell line T98G. Immunofluorescence shows that CATS is localized mainly to the nucleus. Coexpression of CFP-CATS with YFP tagged CALM or CALM/AF10 was able to markedly increase the nuclear localization of both CALM and the CALM/AF10 fusion protein. This effect of CATS is stronger on the YFP-CALM/AF10 fusion protein than on the CALM protein. When fused to a GAL4 DNA binding domain, CATS acts as a strong repressor of transcription in reporter gene assays. Our results indicate that the subcellular localization of CALM and CALM/AF10 could depend in part on the presence of CATS with a greater fraction of CALM or CALM/AF10 being present in the nucleus in cells with high CATS expression (e.g. lymphoid cells). The CALM-CATS interaction might thus play an important role in CALM/AF10 mediated leukemogenesis.

Description: The identification of cancer stem cells is a major step towards the understanding of the pathogenesis of solid and hematological neoplasias and might have direct implications for the development of innovative therapeutic strategies aiming at the eradication of the tumor propagating cell. Here we describe that acute myeloid leukemia (AML), induced by the CALM/AF10 fusion gene, is propagated by a transformed lymphoid progenitor in a murine bone marrow (BM) transplantation model of t(10;11)(p13;q14) positive AML. When mice were transplanted with BM cells retrovirally engineered to express the C/A fusion, all animals (n=13) died from AML showing DJ rearrangement of the heavy chain of the IgH locus after a median of 110 days post transplantation. Diseased mice showed an accumulation of myeloid Gr1+/Mac1+ cells in the peripheral blood and spleen and a multi-organ infiltration by myeloperoxidase and chloracetate esterase positive cells in immunohistochemical sections. In the leukemic mice only a minor population counting for 6.7 % (± 2.1) cells in the BM displayed the B220 lymphoid antigen and lacked myeloid markers (on average 9.4 % ± 3). The majority of cells expressed myeloid markers (on average 82.9 % (± 8.6) Mac1+ cells, 86.4 % (± 3.7) Gr-1+ cells). Additionally, in the leukemic mice an average of 26.0 % (± 8.6) and 32.5 % (± 13.2) of these cells co-expressed B220 and Mac1 or B220 and Gr1, respectively, compared to 2.1 % (± 0.7) and 1.3 % (± 0.3), respectively, in GFP controls. Importantly, in vitro only the B220+/Mac− cell population had growth potential at the single cell level (seeding efficiency 29 %) compared to the B220+/Mac+ (1%) and B220−/Mac+ cells (1%). When the frequency of leukemia propagating cells (LPC) of the three different populations isolated from primary leukemic mice was determined by limiting dilution transplantation and Poisson statistics the frequency of the LPC was more than 380 fold higher in the ‘B220+/Mac1−’ population (1 in 36 cells) than in the ‘Mac1+/B220−’ bulk population (1 in 13906 cells) and more than 12fold increased compared to the B220+/Mac+ cells (1 in 437 cells). In vitro a single B220+/Mac1− cell isolated from a leukemic mouse was able to give rise to the B220+/Mac1+ as well as the Mac1+/ B220− population, both populations showing the identical genomic DJ rearrangement at the IgH locus as the initial B220+/Mac1− cell, demonstrating its capacity to differentiate into the myeloid lineage at the single cell level. The B220+/Mac1− population displayed a CD43+/AA4.1+/HSA+/CD19−/IL-7R− phenotype, was promiscuous in its transcription profile with positivity for EBF, but also MPO and lacked Pax5. Taken together, this murine leukemia model indicates that AML can be propagated from an early transformed lymphoid progenitor cell. The transformation of an early lymphoid cell, which is re-directed into the myeloid lineage by appropriate oncogenes, could explain recurrent observations of immunoglobulin rearrangements in patients with AML and provide a rationale for therapies, aiming at the elimination of the leukemia propagating cell with lymphoid characteristics, but sparing normal HSCs.

Description: The balanced chromosomal translocation t(10;11)(p13;q14) results in the CALM/AF10 fusion gene. This translocation is found in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL) and malignant lymphoma. The CALM/AF10 fusion gene has recently been shown to cause an aggressive biphenotypic leukemia in a murine bone marrow transplant model. The CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) gene product is a clathrin assembly protein which plays a role in clathrin mediated endocytosis and trans Golgi network trafficking. AF10 is a putative transcription factor most likely involved in processes related to chromatin organization and has polycomb group gene like properties. To learn more about the function of the CALM/AF10 fusion protein, we searched for protein interaction partners of CALM. In a yeast two hybrid screen the four and a half LIM domain protein FHL2 was identified as putative CALM interacting partner. The CALM FHL2 interaction was confirmed by co-transformation assay in yeast and by GST-pulldown experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294 to 335 of CALM using the yeast two hybrid assay. In co-localization studies with transiently expressed fluorescent protein tagged CALM and FHL2, both proteins showed cytoplasmatic localization. Expression analysis (Affymetrix based) in different AML subtypes showed a significantly higher expression of FHL2 in AML with complex aberrant karyotypes compared to AML with normal karyotypes or balanced chromosomal translocations like the t(8;21), inv(16) or t(15;17). FHL2, which is also known as DRAL (downregulated in rhabdomyosarcoma LIM protein), is a TP53 responsive gene known to interact with numerous proteins in both the nucleus and the cytoplasm and can function as a transcriptional cofactor. Known FHL2 interactors include TP53, BRCA1, PLZF (promyelocytic leukemia zinc finger protein), the proto-oncogene SKI1 and beta-catenin. High expression of FHL2 in breast cancer has recently been shown to be associated with an adverse prognosis. CALM has been shown to shuttle between the nucleus and the cytoplasm because inhibition of CREM-mediated nuclear export by leptomycin B leads to the accumulation of CALM in the nucleus. Reporter gene assays using a GAL4-DNA binding domain CALM fusion protein and a GAL4 responsive luciferase reporter were able to demonstrate a transcriptional activation function of CALM. We are currently investigation the effect of FHL2 co-expression on this aspect of the CALM function. It is thus conceivable that FHL2 is playing an important role in CALM/AF10-mediated leukemogenesis by tethering the CALM/AF10 fusion protein to various nuclear transcription factor complexes.

Description: The objective of this study was to evaluate the effects of Bacillus subtillis PB6, chromium propionate or a combination of the two on the performance, egg and eggshell quality, nutrient metabolizability and serum biochemistry of layer breeders. White Plymouth Rock and Red Rhodes Island breeder hens at 55 weeks of age were allocated in individual cages using a completely randomized block design with 16 replicates. Hens were fed control, control + probiotic (500 g/ton of Bacillus subtilis PB6), control + CrProp (50 g/ton of chromium propionate) and control + probiotic + CrProp diets from 55 to 70 weeks of age. Productive parameters and eggshell quality as well as cortisol and blood biochemistry were grouped each 28 d as well as for the overall period. The metabolizability of nutrients and energy was determined at 70 weeks of age. In the overall period, hens fed the control + probiotic or control + probiotic + CrProp diets had significantly higher egg production, egg mass, shell percentage, thickness and shell strength. The metabolizability of dry matter, nitrogen and energy increased in hens that were fed the control + probiotic + CrProp diet. In conclusion, diets supplemented with Bacillus subtillis PB6 and chromium propionate resulted in improved productive performance, eggshell quality and nutrient metabolizability of layer breeders, without modifying serum cortisol, albumin and triglycerides.