ALBERT

Description: The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC. As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen. To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.

Description: While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in the chronic phase of chronic myeloid leukemia, these drugs are inefficient for BCR/ABL associated B-cell acute lymphocytic leukemia (B-ALL). Therefore, it is necessary to identify molecular targets downstream of BCR/ABL to develop additional therapeutic approaches. Cells transformed by BCR/ABL are resistant to a wide variety of apoptotic stimuli and therapeutic strategies aimed at reinstating the apoptotic pathway appear as an attractive concept. Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and studies employing cell lines, as well as primary cells have linked BCR/ABL expression with increased levels of Bcl-xL, resulting in resistance to chemotherapeutic agents. To define the role of Bcl-xL in BCR/ABL associated B-ALL, we generated two inducible transgenic mouse models. In the first model, BCR/ABL and loss of Bcl-x expression are co-induced, and in the second model, leukemia is induced with expression of Bcl-xL protein well above the levels found in wildtype lymphoblasts. Surprisingly, we found that deletion of Bcl-xL did not inhibit leukemogenesis or affect apoptosis. Bcl-x deficient B-ALL mice rapidly succumbed to a B-ALL like disease with Bcl-x deficient B-ALL animals being moribund as early as 17 days after induction. By day 28, all mice (n=10) had died or had to be euthanized. Necropsy of animals suffering from Bcl-x deficient leukemia revealed massive lymphadenopathy, pleural effusion, and splenomegaly. While loss of Bcl-x in our B-ALL model led to a more severe phenotype with considerable tumor burden, no statistically significant difference was found between the survival time in Bcl-x deficient and wild type B-ALL animals due to development of pleural effusion in both models. The most prominent difference was the presence of mitotic figures in the peripheral blood, lymph node, and spleen of Bcl-x deficient B-ALL animals, suggestive of increased proliferation of Bcl-x deficient lymphoblasts. Cell cycle analysis of leukemic cells isolated from pleural effusion and spleen of Bcl-x deficient B-ALL mice demonstrated a significant increase of cells in S/G2/M phase (p ≤ 0.05) compared to wildtype lymphoblasts. Thus, loss of Bcl-xL results in increased passage through the cell cycle, while expression of the protein limits the proliferation rate. To test this hypothesis, we generated a second model in which Bcl-xL is expressed at higher levels than in wild type lymphoblasts. Overexpression of Bcl-xL in BCR/ABL positive mice led to reduced proliferation as significantly fewer leukemic cells were present in the S phase than in controls substantiating a role for in Bcl-xL proliferation of lymphoblasts. Initial studies performed to determine the mechanisms by which loss of Bcl-x leads to increased proliferation suggest that the protein may indirectly regulate stability of p27Kip1. Our data show that cells from Bcl-x deficient B-ALL mice in G1 and S phase contain less p27, as a consequence of proteosomal degradation. Clearly, our model systems demonstrate an unexpected and novel role for Bcl-xL in the context of BCR/ABL associated B-ALL. Ongoing studies are aimed at the identification of the mechanism and molecules through which Bcl-xL is linked to cell cycle and proliferation of BCR/ABL transformed lymphoblasts.

Description: Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre–B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

Description: Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may reestablish functional hematopoiesis in these patients. We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on promonocytic U937 cells and Ficoll-enriched blast cells from patients with AML and analyzed their impact on cell growth and viability (assessed by trypan blue exclusion), surface marker expression (FACS analysis of CD11b, C14), and the expression and function of target molecules (Northern blot, Western blot, EMSA). Doses ranging from 0.1 to 1 uM of decitabine induced CD11b and CD14 surface expression 3- to 18-fold and 3- to 4-fold, respectively. 3 to 100nM of VD3 induced CD11b and CD14 expression 1.5- to 10-fold and CD14 1.5- to 8-fold, resp. Interestingly, decitabine synergized with VD3 to increase CD11b and CD14 surface marker expression up to 73-fold and 11-fold, resp., and to induce morphologic differentiation of U937 cells along the monocytic lineage. The mechanisms of decitabine- and VD3-induced monocytic differentiation are currently unclear. Therefore, we investigated the effects of the two drugs on several transcription factors that have been implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Monocytic target promoters such as the CD11b promoter have been shown to be activated by a complex containing PU.1 and c-jun. Using electromobility shift assays, we now demonstrate increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition to the effects seen with the CD11b promoter, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis. In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.

Description: The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3′ enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5′ and 17 kb of 3′ human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.

Description: Allogeneic stem cell transplantation is the most potent form of effective adoptive immunotherapy. The graft-versus-leukemia (GVL) effect mediated by the allogeneic graft, however, is typically coexpressed with graft-versus-host disease (GVHD), which is the major complication of allogeneic stem cell transplantation. In this study, we used genetic and antibody-based strategies to examine the effect that blockade of interleukin 23 (IL-23) signaling had on GVH and GVL reactivity in murine transplantation recipients. These studies demonstrate that the selective protection of the colon that occurs as a consequence of inhibition of IL-23 signaling reduces GVHD without loss of the GVL effect. The separation of GVH and GVL reactivity was noted in both acute and chronic hematologic malignancy models, indicating that this approach was not restricted by the kinetic profile of the underlying leukemia. Furthermore, a potent GVL response could be mounted in the colon under conditions where tumor cells migrated to this site, indicating that this organ did not serve as a sanctuary site for subsequent systemic relapse in GVHD-protected animals. These studies demonstrate that blockade of IL-23 signaling is an effective strategy for separating GVH and GVL responses and identify IL-23 as a therapeutic target for the regulation of alloresponses in humans.

Description: In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

Description: The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent “priming” of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.