Leukemic Tissues Research Articles

Role of ARHGEF3 as a GEF and mTORC2 Regulator.

Guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating the release of guanosine diphosphate to permit the binding of guanosine triphosphate. ARHGEF3 or XPLN (exchange factor found in platelets, leukemic, and neuronal tissues) is a selective guanine nucleotide exchange factor for Rho GTPases (RhoGEFs) that activates RhoA and RhoB but not RhoC, RhoG, Rac1, or Cdc42. ARHGEF3 contains the diffuse B-cell lymphoma homology and pleckstrin homology domains but lacks similarity with other known functional domains. ARHGEF3 also binds the mammalian target of rapamycin complex 2 (mTORC2) and subsequently inhibits mTORC2 and Akt. In vivo investigation has also indicated the communication between ARHGEF3 and autophagy-related muscle pathologies. Moreover, studies on genetic variation in ARHGEF3 and genome-wide association studies have predicted exciting novel roles of ARHGEF3 in controlling bone mineral density, platelet formation and differentiation, and Hirschsprung disease. In conclusion, we hypothesized that additional biochemical and functional studies are required to elucidate the detailed mechanism of ARHGEF3-related pathologies and therapeutics.

Leukemic Cytokines co-opt Regulatory T cell and Th17 immunity to promote chronic myeloid leukemia

Abstract Non-immune roles of Regulatory T cells (Tregs) have been described across various tissues. However, the mechanisms by which T cells regulate the bone marrow in myeloproliferative neoplasms lack resolution. A hallmark of leukemic progression is the increase in pro-inflammatory cytokines, cellular inflammation, and an expansion of CD4 T cells. In this study, we demonstrate that this inflammatory milieu promotes pathogenic skewing of both regulatory and effector T cells. We characterized the altered CD4+ T cell compartment in chronic myeloid leukemia (CML) and we found that Tregs are unstable and dysregulated across leukemic tissues. Specifically, the CML environment effectively co-opts Treg function and stability. This contributes to a phenotype of immune activation as well as an increase of pathogenic inflammatory T cells. Notably, our findings reveal that Treg/Th17 ratios are disrupted in leukemia and that loss of Tregs and activation of Th17 cells influence disease progression. Additionally, we found that Treg-secreted IL-10 directly influences myeloid and neutrophil differentiation of leukemic progenitors has beneficial anti-inflammatory effects. We demonstrate that restoring Treg function and expanding their IL-10 production can be used therapeutically to limit leukemic progression. Our results support the hypothesis that Tregs are compromised during leukemic progression as a consequence of pro-inflammatory cytokines from the myeloid associated disease. These findings highlight how the Treg/Th17 balance regulates disease progression. Furthermore, these studies provide an avenue by which Treg secreted IL-10 can be used for therapeutic intervention in the context of hematopoietic malignancies.

Energy-Transfer Phenomena in Thermoresponsive and pH- Switchable Fluorescent Diblock Copolymer Vesicles.

We describe the development of a polymeric vesicle that not only selectively fluoresces at low pH, a condition prevailing in cancer cells, but also can potentially monitor the thermoresponsive release of a drug even if the drug is nonfluorescent. The developed fluorescence resonance energy transfer (FRET)-based thermoresponsive vesicular nanocarriers are composed of a new poly(PEGMA)-b-poly(NIPA-r-R6GMED) block copolymer, which undergoes pH-switchable superior turn on-off fluorescence characteristics. The block copolymer was synthesized using the RAFT technique, and its solution properties and self-assembly behavior were investigated by turbidity measurements, fluorescence spectroscopy, 1H NMR, dynamic light scattering, and transmission electron microscopy. The block copolymer self-assembled to form nanostructured vesicles above the critical aggregation temperature under physiologically relevant conditions. Steady-state and time-resolved fluorescence spectroscopy were utilized to study the FRET process between encapsulated hydrophobic guest C-153 (donor) and polymer-bound R6GMED units (acceptor) in the thermoresponsive vesicles. The FRET rate and efficiency were found to vary as a result of the pH-dependent changes in the quantum yield of the acceptor molecules. The occurrence of a highly efficient FRET in this polymeric vesicular nanocarrier at acidic pH, a condition similar to the cytoplasm and cell nucleus in leukemic tissues, and the ability to encapsulate hydrophilic and hydrophobic molecules and their temperature-controlled release make it potentially useful in imaging guided real-time monitoring of drug-delivery vehicles.

Revisiting thymus function.

OPINION article Front. Immunol., 28 August 2014Sec. T Cell Biology Volume 5 - 2014 | https://doi.org/10.3389/fimmu.2014.00411

Loss Of p53 Induces Leukemic Transformation In a Murine Model Of JAK2V617F-Induced Polycythemia Vera

Myeloproliferative neoplasms (MPN) have an inherent tendency toward leukemic transformation, but its mechanisms remain largely unknown. Recently, TP53 mutation is reported to be frequently found in cases with post-MPN leukemia. Here, to address the contribution of p53 loss to leukemic transformation from MPN in vivo, we retrovirally transduced c-kit+ bone marrow (BM) cells from p53 knockout (p53-/-) and littermate mice (p53+/+) with either wild-type Jak2 (Jak2WT) or Jak2V617F respectively, and transplanted them into lethally irradiated mice. At 3 weeks after transplantation, both recipients of Jak2V617F/p53-/- and Jak2V617F/p53+/+ cells developed a polycythemia vera-like disease characterized by high WBC count and elevated hemoglobin (Hb) level. Jak2V617F/p53+/+ mice survived and continued to have elevated Hb level, whereas 5 weeks after transplantation, Jak2V617F/p53-/- recipients developed cachexia, and their Hb level declined. Eventually, these mice developed fatal leukemia with a median survival of 46.5 days after transplantation, suggesting loss of p53 cooperates with Jak2V617F mutation to promote leukemic transformation from MPN.To characterize these leukemias, we analyzed leukemic tissues from moribund Jak2V617F/p53-/- mice. Peripheral blood smears and BM specimen from Jak2V617F/p53-/- recipients showed a marked increase of erythroid precursors with dysplastic features, leading to suppression of normal hematopoiesis. Notably, Jak2V617F/p53-/- mice displayed marked hepatosplenomegaly and extensive pulmonary hemorrhage. Consistent with the histopathologic findings, Jak2V617F/p53-/- animals exhibited a remarkable accumulation of erythroid precursors (CD71+), and especially more immature progenitors (Ter119-/CD71+) in the BM and spleen, compared with Jak2V617F/p53+/+ animals. These data suggest Jak2V617F/p53-/- recipients developed infiltrative disease with accumulation of immature erythroid cells, fulfilling the Bethesda Criteria of erythroleukemia in mice.To assess the transplantability of Jak2V617F/p53-/- leukemia, we injected unfractionated BM cells from Jak2V617F/p53-/- mice into lethally irradiated mice. In all cases, lethal leukemia developed earlier than in primary recipients. Moreover, there was a significant increase in erythroid progenitors in secondary recipients, suggesting the erythroid component is the predominant lineage involved in this leukemia model. As Jak2V617F/p53-/- leukemic tissues contained three major populations: CD71+ erythroid progenitors, Mac1+ mature myeloid cells, and lineage-negative (CD71-/Mac1-) primitive leukemic cells, we purified and transplanted these subfractions into secondary recipients to evaluate their leukemia-initiating potential. As a result, both lineage-negative (CD71-/Mac1-) cells and CD71+ erythroid progenitors possessed leukemia- initiating capacity, but Mac1+ myeloid cells could not reconstitute the disease. In addition, these two fractions had different capacities to induce leukemias; recipients of CD71+ cells rapidly developed erythroleukemia, whereas lineage-negative cells caused lethal leukemia after the polycythemic state. Moreover, hematopoietic tissues in recipients transplanted with CD71+ cells mainly consisted of erythroid lineages, whereas lineage-negative cells produced both erythroid and myeloid lineages, suggesting lineage-negative cells are more immature than CD71+ erythroid precursors. Furthermore, subsequent fractionation of lineage-negative cells revealed leukemia-initiating cells were enriched in Lin-/Sca-1+/c-kit+ (LSK) cells. To further characterize two types of leukemia-initiating cells in Jak2V617F/p53-/- leukemia, we assessed their sensitivity to a JAK2 inhibitor, INCB18424, in vitro. Interestingly, INCB18424 treatment significantly reduced CD71+ cell proliferation, whereas LSK cells were able to expand in the presence of INCB18424, indicating different leukemia-initiating cells existing in post-MPN leukemia have different responsiveness to JAK2 inhibiton.In summary, these results demonstrate p53 loss is sufficient for inducing leukemic transformation in JAK2V617F-postive MPN and offers an in vivo model to assess novel therapeutic approaches for post-MPN leukemia. In addition, we revealed leukemia-initiating cells at different differentiation stages could exist in post-MPN leukemia. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Use of CALM-AF10 and NUP98-HOXD13 fusions to Identify Oncogenic Pathways That Collaborate with HOXA9 Overexpression

AbstractAbstract 2499Acute myeloid leukemias (AMLs) are thought to result from a series of complementary mutations that affect several vital pathways, including differentiation, self-renewal, apoptosis, and proliferation. Several mutations associated with AML (MLL fusions, NUP98 fusions, CALM-AF10 fusion, and NPM1 mutations) are thought to impair hematopoietic differentiation by dysregulation of target genes; one of the most consistently dysregulated target genes is HOXA9. Mice that express a CALM-AF10 (CA10) or NUP98-HOXD13 (NHD13) fusion gene overexpress HOXA9 and develop AML with a delayed onset and incomplete penetrance, suggesting the need for complementary mutations. We have used three techniques including candidate gene resequencing, retroviral insertional mutagenesis (RIM), and gene expression profiling to compare and contrast the complementary mutations that occur in these mice. The frequency of Ras pathway mutations is similar for both CA10 (20%) and NHD13 (32%) leukemic mice, and previously reported RIM studies identified common integration sites near Mn1 and Evi1 in both models, suggesting that dysregulation of similar collaborative pathways can lead to leukemia in both models. However, there were significant differences between these models. The aforementioned RIM studies identified Zeb2 as the most frequent insertion site for CA10 leukemias, suggesting that Zeb2 collaborated with CA10, whereas no Zeb2 insertions were found in the NHD13 RIM study. Another distinction between the NHD13 and CA10 leukemias is the expression of the HOXA9 co-regulatory factor Meis1. Meis1 is markedly upregulated in bone marrow from clinically healthy, pre-leukemic CA10 mice and CA10 leukemic tissues compared to wildtype bone marrow. Conversely, Meis1 expression was decreased in bone marrow from clinically healthy NHD13 mice compared to wildtype, and only occasionally overexpressed in NHD13 leukemias. Interestingly, RIM analysis of NHD13 mice identified Meis1 as the most frequent insertion site, suggesting that Meis1 overexpression can collaborate with NHD13 during leukemic transformation. Flt3, which is frequently mutated in patients with AML, has been shown to be transcriptionally activated by Meis1. Of note, Flt3 is consistently overexpressed in CA10 leukemias but is overexpressed only in those NHD13 leukemias that have upregulated Meis1. Intriguingly, the frequency of Flt3 activating mutations in CA10 leukemias (which overexpress Flt3) was 25%, but none were found in the NHD13 leukemias (which do not overexpress Flt3). Lastly, using gene expression arrays, N-myc was identified as one of the most differentially overexpressed mRNAs, suggesting that it may cooperate with CA10 during leukemic transformation. Furthermore, N-myc has recently been shown to promote AML in mice, and our preliminary experiments suggest that N-myc and CA10 collaborate and lead to increased cell number and replating potential in bone marrow colony formation assays. In conclusion, we have taken several genetic approaches to identify key similarities and differences in the biology of two leukemic models (CA10 and NHD13), both of which overexpress HOXA9. Disclosures:No relevant conflicts of interest to declare.

Abstract B38: Identification of metabolic markers of acute myeloid leukemia in MLL-AF9 transgenic mice

Abstract Manifestation of the malignant potential of the neoplastic cell requires metabolic transformation to support the bioenergetic, synthetic, and catabolic requirements of malignancy. Metabolic phenotypes have been associated with every type of cancer and have been used as indicators of disease status, prognosis, and response to therapy. However, few studies investigating changes in metabolism associated with acute leukemia have been reported. We used ex vivo magnetic resonance spectroscopy to investigate changes in metabolite levels in the bone marrow, spleen, and/or blood of leukemic MLL-AF9 transgenic (Tg) mice relative to wild-type littermates. These studies identified differences in more than 20 cellular metabolites and metabolic pathways in leukemic tissues. Significant differences in the levels of metabolites related to glucose metabolism and in flux through glucose metabolic pathways (theWarburg effect) were observed in all tissues examined. Decreased levels of glutamine and glutamate suggest increased utilization of glutaminolysis as an alternative carbon source for macromolecular synthesis. Significant changes in the levels of phospholipid precursors and increased levels of lipids and phospholipids were observed in the bone marrow, suggesting increased flux through lipid biosynthetic pathways and/or decreased lipid breakdown. These changes may allow for increased macromolecular and membrane synthesis to support the hyperproliferative phenotype of leukemic blasts. A switch from high levels of GPC and low levels of PCho to low levels of GPC and high levels of PCho, as we observed here, has also been observed in patients with breast (Aboagye and Bhujwalla, 1999, Cancer Res 59:80) or ovarian cancers (Iorio et al., 2005, Cancer Res 65:9369). Thus, changes in lipid metabolism in leukemic MLL-AF9 Tg mice reflect similar changes in patients with leukemias and other tumors and suggest mechanisms by which metabolic changes may contribute to leukemogenesis in a physiologically relevant context. In contrast to the wide-spread changes in lipid metabolism observed in blood and bone marrow, only a small subset of lipid metabolites were significantly different in the spleens of leukemic mice. The vast majority of metabolic differences in leukemic mice were tissue-specific, being restricted to either the bone marrow or spleen with the largest number and broadest spectrum of metabolic changes detected in the spleen. These data indicate that leukemia cell metabolism is critically dependent on cellular environment, underscoring the importance of development of robust animal models for metabolic studies, as we have described here. Many of the metabolic changes observed in tissues are also reflected in the blood, which may allow for ex vivo assessment of these markers using samples that can be routinely obtained with minimal discomfort to patients. Because this is the first comprehensive study of the “metabolomic” profile associated with leukemia, several markers that have not been previously associated with leukemogenesis and/or that have not been previously associated with any tumor type were identified in this study, revealing novel insights into leukemia cell biology and leukemogenesis. These studies also identify metabolic changes that may be clinically relevant for prognostication, as indicators of therapeutic efficacy, and/or as novel therapeutic targets. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B38.

An Insertional Mutagenesis Screen for Genes That Cooperate with Mll-AF9 in Leukemogenesis.

Abstract 3962Poster Board III-898By altering the activity of specific transcription complexes, the MLL-AF9 fusion oncogene can initiate the process of acute myeloid leukemia (AML) development. However, all the genetic pathways that can cooperate with MLL-AF9 expression to cause full-blown AML are unknown. These pathways will provide therapeutic targets for MLL-AF9-associated AML. Mice with constitutive expression of the Mll-AF9 fusion oncoprotein under the control of the endogenous promoter develop AML but only after a prolonged latency. This model thus provides a system for understanding the evolution of AML initiated by an MLL fusion oncoprotein. We hypothesized that infection with a recombinant Murine Leukemia Virus, abbreviated M4070, could cooperate with MLL-AF9 expression to accelerate the onset of leukemia by causing the secondary mutations required for cancer progression. We bred Mll-AF9 heterozygous males to wild type females, and the offspring were injected at three days of age with M4070 virus (n=211) or were mock infected (n=68). All mice were genotyped and observed for disease progression. Virally infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virally infected wild type (WT) mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. Histopathology, immunohistochemical staining, analysis of the surface immunophenotype by flow cytometry, and Southern blot analysis of T and B cell receptor rearrangement indicated that infected Mll-AF9/+ animals developed primarily myeloid leukemia (myeloperoxidase positive, Mac1 or Mac1/Gr1 positive, CD3 negative) while infected WT animals developed mostly lymphoid leukemia (CD3 positive, CD4 and/or CD8 positive, myeloperoxidase negative). Retroviral insertion sites were cloned from 167 leukemic tissues from the accelerated leukemia mice using two different restriction enzymes in a shotgun-based, linker-mediated, cloning protocol to identify the genes most frequently mutated in Mll-AF9 positive leukemia. More than 4,100 independent insertions were isolated and 101 common insertion sites (CIS), defined as genomic locations with several proviral insertions from at least 3 mice, were identified. The majority of the CIS harbored proviral insertions in both Mll-AF9/+ and wild type mice, but a subset of CIS were found in only one group or the other. Some of the genes closest to the CIS have been identified as target genes in other proviral screens and some are known cancer genes. We studied a subset of the CIS-associated genes for aberrant expression in leukemic tissues. There was elevated expression of Mn1, and a trend towards increased expression of Bcl11a and Fosb, in our Mll-AF9 murine leukemia samples with proviral insertions nearby these genes. Moreover, elevated expression of MN1, FOSB, and BCL11A has been observed in microarray studies of human patients with AML. We have completed a bone marrow transduction/transplantation experiment to seek functional evidence of cooperation with Mll-AF9. Mice transplanted with Mll-AF9/+ bone marrow that had been transduced with a retrovirus encoding the candidate gene MN1 succumb to myeloid malignancy faster than mice transplanted with wild type bone marrow transduced with MN1, or Mll-AF9/+ bone marrow transduced with a retrovirus encoding just the Green Fluorescent Protein gene. This data suggests that MN1 can cooperate with Mll-AF9 to accelerate myeloid leukemia in a mouse model. We are currently using shRNA knockdown strategies in human cell lines to confirm cooperation of more candidate genes with MLL-AF9 in AML development. Thus, CIS-associated genes from leukemias accelerated by M4070 in Mll-AF9/+ mice may help define important genetic pathways that are altered during progression of AML induced by MLL fusion oncogenes. Disclosures:No relevant conflicts of interest to declare.

Haploinsufficiency and acquired loss of Bcl11b and H2AX induces blast crisis of chronic myelogenous leukemia in a transgenic mouse model

Chronic myelogenous leukemia (CML) is a hematological malignancy that begins as indolent chronic phase (CP) but inevitably progresses to fatal blast crisis (BC). p210BCR/ABL, a chimeric protein with enhanced kinase activity, initiates CML CP, and additional genetic alterations account for progression to BC, but the precise mechanisms underlying disease evolution are not fully understood. In the present study, we investigated the possible contribution of dysfunction of Bcl11b, a zinc-finger protein required for thymocyte differentiation, and of H2AX, a histone protein involved in DNA repair, to the transition from CML CP to BC. For this purpose, we crossed CML CP-exhibiting p210BCR/ABL transgenic (BA(tg/-)) mice with Bcl11b heterozygous (Bcl11b(+/-)) mice and H2AX heterozygous (H2AX(+/-)) mice. Interestingly, p210BCR/ABL transgenic, Bcl11b heterozygous (BA(tg/-)Bcl11b(+/-)) mice and p210BCR/ABL transgenic, H2AX heterozygous (BA(tg/-)H2AX(+/-)) mice frequently developed CML BC with T-cell phenotype and died in a short period. In addition, whereas p210BCR/ABL was expressed in all of the leukemic tissues, the expression of Bcl11b and H2AX was undetectable in several tumors, which was attributed to the loss of the residual normal allele or the lack of mRNA expression. These results indicate that Bcl11b and H2AX function as tumor suppressor and that haploinsufficiency and acquired loss of these gene products cooperate with p210BCR/ABL to develop CML BC.

Direct transcriptional regulation of MDM2 by Fli-1

The Ets transcription factor, Fli-1, has been shown to play a pivotal role in the induction and progression of Friend Murine Leukemia Virus (F-MuLV)-induced erythroleukemia, with its overexpression leading to erythroblast survival, proliferation, and inhibition of terminal differentiation. P53 inactivation is an additional genetic alteration that occurs in late-stage leukemic progression associated with in vivo and in vitro immortalization. Since p53 protein expression levels are low, to undetectable, in primary erythroleukemic cells that express elevated levels of Fli-1, we investigated the potential regulation of p53 by Fli-1. We assessed whether the overexpression of Fli-1 could partially regulate p53 via modulation of its well-established regulator, MDM2. In this paper, we demonstrate that the promoter of MDM2 contains a consensus binding site for Fli-1 that is bound by this transcription factor in vitro and in vivo, resulting in MDM2 transcriptional regulation. We further substantiate these observations in vivo by demonstrating a positive correlation in the expression of Fli-1 and MDM2, and a negative correlation with p53 in leukemic tissues obtained from mice with Friend Disease. These observations depict a significant function of Fli-1 overexpression in the indirect control of p53, evidently capable of leading to an increasingly aggressive erythroleukemic clone in vivo.

TEL-JAK2 transgenic mice develop T-cell leukemia

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

TEL-JAK2 transgenic mice develop T-cell leukemia

AbstractWe previously reported a fusion between TEL and JAK2in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor–independent proliferation to the interleukin-3–dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2–induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EμSRα enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRβ locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells.

Association of v-Ha- ras Transgene Expression with Development of Erythroleukemia in Tg.AC Transgenic Mice

A transgenic mouse line (Tg.AC) carrying an activated v-Ha-ras oncogene fused to the embryonic zeta-globin promoter develops an array of spontaneous epithelial and mesenchymal neoplasms. In this report we describe the morphological, immunophenotypic, and molecular features of a unique hematopoietic neoplasm in these mice. The cardinal lesion of this disease is marked hepatomegaly due to leukemic proliferation and infiltration. In the peripheral blood, there is a marked increase in the number of metarubricytes and other less differentiated erythroid progenitor cells. Leukemic cells stain positively with an erythroid-associated nuclear transcription factor (GATA-1). Using a reverse transcription polymerase chain reaction assay, co-expression of GATA-1 and endogenous zeta-globin genes is detected in hematopoietic tissues of nonleukemic transgenic and nontransgenic mice. ras transgene expression is, however, detected only in normal bone marrow and leukemic tissues of transgenic mice, and 5' mapping experiments using S1 protection analysis of total RNA from leukemic tissue indicates that transcription of the transgene mRNA is initiated from the natural zeta-globin promoter start site, supporting the belief that the zeta-globin promoter directs v-Ha-ras expression in erythroid progenitor cells, ultimately leading to leukemic transformation.

Development of Acute Lymphoblastic Leukemia and Myeloproliferative Disorder in Transgenic Mice Expressing p210bcr/abl: A Novel Transgenic Model for Human Ph1-Positive Leukemias

AbstractThe Philadelphia (Ph1) chromosome can be detected in chronic myelogenous leukemia (CML) and a significant number of acute lymphoblastic leukemia (ALL) cases. Generation of p210bcr/abl, a chimeric protein with enhanced kinase activity, is thought to be involved in the pathogenesis of these diseases. To elucidate the biological properties of p210bcr/abl and to create an animal model for human Ph1-positive leukemias, we generated transgenic mice expressing p210bcr/abl driven by the promoter of the tec gene, a cytoplasmic tyrosine-kinase preferentially expressed in the hematopoietic lineage. The founder mice showed excessive proliferation of lymphoblasts shortly after birth and were diagnosed as suffering from ALL based on surface marker and Southern blot analyses. Expression and enhanced kinase activity of the p210bcr/abl transgene product were detected in the leukemic tissues. In contrast, transgenic progeny exhibited marked granulocyte hyperplasia with thrombocytosis after a long latent period and developed myeloproliferative disorders (MPDs) closely resembling human CML. Expression of p210bcr/abl mRNA in the proliferating granulocytes was detected by RT-PCR. In particular, one MPD mouse showed remarkable proliferation of blast cells in the lung, which might represent an extramedullar blast crisis. The results demonstrate that the expression of p210bcr/abl in hematopoietic progenitor cells in transgenic mice can contribute to two clinically distinct hematopoietic malignancies, CML and ALL, indicating that this transgenic system provides a novel transgenic model for human Ph1-positive leukemias.

Development of Acute Lymphoblastic Leukemia and Myeloproliferative Disorder in Transgenic Mice Expressing p210bcr/abl: A Novel Transgenic Model for Human Ph1-Positive Leukemias

The Philadelphia (Ph1) chromosome can be detected in chronic myelogenous leukemia (CML) and a significant number of acute lymphoblastic leukemia (ALL) cases. Generation of p210bcr/abl, a chimeric protein with enhanced kinase activity, is thought to be involved in the pathogenesis of these diseases. To elucidate the biological properties of p210bcr/abl and to create an animal model for human Ph1-positive leukemias, we generated transgenic mice expressing p210bcr/abl driven by the promoter of the tec gene, a cytoplasmic tyrosine-kinase preferentially expressed in the hematopoietic lineage. The founder mice showed excessive proliferation of lymphoblasts shortly after birth and were diagnosed as suffering from ALL based on surface marker and Southern blot analyses. Expression and enhanced kinase activity of the p210bcr/abl transgene product were detected in the leukemic tissues. In contrast, transgenic progeny exhibited marked granulocyte hyperplasia with thrombocytosis after a long latent period and developed myeloproliferative disorders (MPDs) closely resembling human CML. Expression of p210bcr/abl mRNA in the proliferating granulocytes was detected by RT-PCR. In particular, one MPD mouse showed remarkable proliferation of blast cells in the lung, which might represent an extramedullar blast crisis. The results demonstrate that the expression of p210bcr/abl in hematopoietic progenitor cells in transgenic mice can contribute to two clinically distinct hematopoietic malignancies, CML and ALL, indicating that this transgenic system provides a novel transgenic model for human Ph1-positive leukemias.

BCR/ABL-induced Leukemogenesis Causes Phosphorylation of Hef1 and Its Association with Crkl

BCR/ABL is considered responsible for the development of Philadelphia chromosome-positive leukemia. Experimental animal models, such as transgenic mice, have demonstrated unambiguously that Bcr/Abl is capable of inducing leukemogenesis. The adaptor molecule Crkl is a major in vivo substrate of the deregulated Bcr/Abl tyrosine kinase and functions as a molecular link with other signaling proteins. While associated in vivo with Bcr/Abl through its SH3 domain, Crkl can interact simultaneously via its SH2 domain with other tyrosine-phosphorylated proteins. Here we report the identification of prominently tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa, which bind specifically to the Crkl SH2 domain in leukemic tissues of P190BCR/ABL transgenic mice. We demonstrate that these proteins are identical to Hef1/Cas-L, which is related to p130(Cas). The proto-oncoprotein p120(Cbl) and Hef1, but not p130(Cas), were detectably phosphorylated on tyrosine in P190Bcr/Abl-expressing leukemic cells and were found in complex with Crkl, showing the existence of protein complexes in P190Bcr/Abl leukemic cells, consisting of P190Bcr/Abl, Crkl, and Hef1 or p120(Cbl). This supports a model in which Crkl acts as mediator between Bcr/Abl and downstream effectors. Since Hef1 is involved in the beta1-integrin signaling pathway, our study demonstrates that Bcr/Abl could specifically interfere with normal beta1-integrin signaling.

Dissemination and tissue invasiveness in murine acute leukemia associated with acquisition of p53 mutation and loss of wild-type p53.

Approximately 60% of mice treated with split-dose radiation develop leukemias that disseminate widely through the body, whereas 40% of the treated mice incur leukemias that are contained entirely within the thymus. We studied the status of p53 in non-cultured samples of thymic leukemias and in cell lines established from these leukemias. In those mice with disseminated disease, primary samples were also obtained from visceral leukemic organs, and cell lines were established from these leukemic organs for further study. Using single-strand conformation polymorphism (SSCP), nucleic acid sequencing, and immunochemical analysis, we found that mutation of both p53 alleles occurred in leukemic cell lines developed from nine of 10 disseminated leukemias; mutation of one p53 allele with the other remaining wild-type occurred in one disseminated leukemia. A p53 mutation unique for each mouse was found in all cell lines established from the different leukemic organs of each mouse. The same mutation was also found in the non-cultured leukemic tissues of each mouse, indicating that the mutations originated in vivo and were clonal. Seven of seven non-disseminating thymomas possessed wild-type p53 only. Hence, in vivo dissemination and tissue invasiveness were associated with the loss of wild-type p53 by mutation of both alleles or by mutation and loss of heterozygosity, as revealed by studies of cell lines established from them. The selective in vivo dissemination of leukemia cells possessing p53 mutations had a parallel in vitro. Leukemia cell lines from mice harboring disseminating leukemia were established more readily (success rate greater than 80%) than lines from mice harboring thymic nondisseminating leukemia (success rate less than 10%). Additionally, while mice with disseminating leukemia harbored a mixture of wild-type and mutant p53-encoding thymoma cells, only cell lines possessing mutant p53 became established in culture. Mutations found in thymoma cell lines were always detectable by SSCP and sequencing of DNA extracted from non-cultured thymoma tissue. However, in non-cultured leukemic tissue of visceral organs, the clonal p53 mutations found in cell lines established from them were often not detectable by SSCP or sequencing but were detectable by immunochemical analysis or polymerase chain reaction amplification. This indicates an unexpected degree of masking of mutant genes by wild-type genes present in the leukemic tissue. Masking was evident even in leukemic organs that were grossly larger than normal organs. Hence, routine screening of leukemic tissue by SSCP and sequencing may result in a highly significant underestimation of the incidence of p53 mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of p210bcr/abl by Metallothionein Promoter Induced T-Cell Leukemia in Transgenic Mice

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice.

Env-derived gp55 gene of Friend spleen focus-forming virus specifically induces neoplastic proliferation of erythroid progenitor cells.

A group of retroviruses carrying truncated viral genes has recently been suggested as the cause of new patterns of diseases. One such virus is the replication defective component of the Friend murine leukemia virus (F-MuLV) complex, called Friend spleen focus forming virus (F-SFFV). This virus induces erythroblastosis, and a virion envelope-related glycoprotein, gp55, encoded by F-SFFV has been suggested as the pathogenic gene. The role of the gp55 gene is, however, yet unclear in the apparently multistep erythroleukemogenesis. By separately producing transgenic mice harboring the whole F-SFFV DNA, the gp55 gene alone under the control of the retroviral long terminal repeat (LTR) and the gp55 gene under the control of cytoplasmic beta actin transcriptional regulatory unit, we show here that the gp55 gene is capable of inducing neoplastic proliferation of erythroid progenitor cells specifically in the absence of helper virus and other F-SFFV sequences. Under the control of the viral LTR the gp55 expression was detected only in leukemic tissues, but under the control of cytoplasmic beta-actin regulatory sequences, the gp55 was also expressed in a variety of normal tissues including preleukemic normal spleens. The development of erythroleukemia was suppressed under the genetic background of C57B1/6 mouse (resistant to F-MuLV; Fv-2rr), and required additional events even under the background of DDD mouse (susceptible to F-MuLV; Fv-2ss). The p53 and Spi-1 genes were frequently aberrant in transplanted tumors and cell lines derived from them, but were not in primary leukemic spleens.

Intervention in potential leukemic cell migration pathway affects leukemogenesis.

Several factors are involved in the high frequency of T-cell lymphomas of AKR mice, which appear mainly in the thymus at the age of 6–12 months [1]. The thymus is considered to play a major role in the disease since its removal prevents the development of T-cell lymphoma [2], while retransplantation of thymic epithelium to thymectomized AKR reconstitutes the high frequency of lymphoma [3], Although the AKR/J strain has the predisposition to develop the disease since birth [4], the mean latent period is delayed until the age of 8 months. The long latent period has been attributed to the delayed formation of the leukemogenic dual tropic virus (DTV) with the MCF characteristic [5, 6], formed in the thymus as a consequence of recombination within the envelope gene of ecotropic and xenotropic murine leukemia virus (MuLV). DTVs are detected only in preleukemic thymus and leukemic tissues of strains of mice prone to develop high incidence of leukemia [7]. DTVs enhance leukemia development whereas endogenous ecotropic or xenotropic viruses are usually nontumorigenic. Exceptional is the ecotropic virus isolate SL3 with the enhancing activity on T-cell lymphomagenesis [8]. These observations support the assumption that DTVs are proximal transforming agents of thymocytes and thereby responsible for high incidence of T lymphoma in AKR mice. Cloyd [9] proposed specific cellular tropism of two subclasses of MCF virus, and claimed that oncogenicity is closely linked to cellular differentiation. MCF isolated from lymphomatous thymus was replicating in the thymus and T peripheral cells, while nonlymphomagenic MCF isolated from leukemic spleen of NFS mice did not replicate in the thymus but rather in bone marrow cells, spleen, and lymph node B-lymphocytes.