Normal Myeloid Progenitors Research Articles

Myelopoiesis Requires a Noncatalytic, Ras-Independent Function of SHP-2.

Activating mutations in PTPN11, which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in juvenile myelomonocytic leukemia (JMML). Other etiologies of JMML include activating mutations in NRAS or KRAS2 and inactivation of the tumor suppressor NF1. These and other observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1. Ras proteins are signal switch molecules that respond to extracellular stimuli by cycling between inactive GDP-bound and active GTP-bound conformations. Oncogenic alleles encode proteins that preferentially accumulate in the GTP-bound form. While NF1 encodes a GTPase activating protein for Ras that directly modulates Ras-GTP levels, the biochemical relationship between SHP-2 phosphatase activity and Ras signaling remains unclear. Most mammalian systems place SHP-2 upstream of Ras activation, but the mechanism is not known. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. We tested the hypothesis that the essential function of SHP-2 in primary hematopoietic cells is to activate Ras. To do this, we determined if Ras activation by expression of an oncogenic Kras2 allele could eliminate the requirement for SHP-2. We used conditional alleles of Kras2 (LSL-KrasG12D) and Ptpn11 (Ptpn11flox/flox) coupled with the inducible Mx1-Cre transgene. Juvenile mice were injected with polyI:polyC, resulting in expression of K-RasG12D and inactivation of Ptpn11. Although these mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre, LSL-KrasG12D mice (Braun et al., PNAS 101(2):597–602), myeloid progenitors invariably retained an intact Ptpn11 allele despite uniform activation of the conditional KrasG12D allele. These data suggested that there was strong selective pressure to retain a functional Ptpn11 allele despite oncogenic K-Ras expression. To test this hypothesis directly, we enumerated myeloid progenitor colonies in methylcellulose medium immediately after inactivating Ptpn11 and activating KrasG12D via retroviral transduction. This confirmed a strong dependence on SHP-2 for formation of myeloid colonies either in the presence or absence of KrasG12D. Infecting Ptpn11flox/flox, LSL-KrasG12D cells with a Ptpn11-IRES-Cre virus fully restored the aberrant growth phenotype of KrasG12D mutant cells. Remarkably, alleles encoding phosphatase-deficient SHP-2 proteins also rescued CFU-GM growth. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro. Our data support a model in which SHP-2 has essential hematopoietic functions that are independent of Ras activation and do not require SHP-2 phosphatase activity. The presence of protein-protein interaction domains in SHP-2 suggests that it may have a noncatalytic adaptor function. Because transformation by leukemogenic Ptpn11 alleles requires catalytic activity, our data imply that inhibition of SHP-2 catalysis will selectively target neoplastic hematopoietic progenitors.

Identification of Genes Abnormally Expressed in Both Human and Murine MLL-ELL and/or MLL-ENL Leukemia.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. Their abnormally expressed genes identify specific markers for their clinical diagnosis. Important biological properties are often conserved across species. However, although genetically engineered mouse leukemia models are well-established, few systematic studies have validated the genes that exhibit similar abnormal expression patterns in both human and mouse leukemia models. MLL-ELL and MLL-ENL fusion genes resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3), respectively, are frequently involved in human acute leukemia, and in retrovirus-mediated mouse leukemia models. We used the SAGE technique to compare gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both human and mouse. We analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL or MLL-ENL fusions, and a leukemia cell line with an MLL-ELL fusion. 484,303 SAGE tags were identified from the nine samples, yielding 103,899 unique tags in human and 60,993 in mouse samples. We identified 40 candidate genes that appear to be abnormally expressed in both human and murine MLL-ELL leukemias (2 up- and 38 down-regulated), and 72 in both human and murine MLL-ENL leukemias (23 up and 49 down). 25 candidate genes are down-regulated in both types of leukemias, and many of them can bind with and/or regulate other candidate genes in the candidate list. For example, LCN2 can bind directly with and positively regulate MMP9; MMP9 and TMSB4X may positively regulate FOS; FOS and JUNB can bind directly and positively regulate each other. JUNB may inhibit proliferation and promote apoptosis, and it was reported that inactivation of JunB in LT-HSC leads to MPD while its inactivation in committed myeloid progenitors also predisposes to leukemia evolution. LCN2 may also positively regulate apoptosis. Meanwhile, some important candidate genes are observed only in one type of leukemia. For example, both PXN and ARHGEF1 are down-regulated only in MLL-ELL leukemias. PXN can bind directly with ARHGEF1, and the latter may inhibit proliferation. Similarly, MYB is significantly upregulated only in MLL-ENL leukemias, which was reported to play a role in MLL-ENL-mediated transformation. Taken together, some common pathways may exist in the development of both types of leukemias, whereas each may also have their own pathway. The deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes, and are studying the functions and pathways of the validated candidate genes. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute myeloid leukemia, which may lead to more effective therapy for these leukemias.

Analysis of the CD33-related siglec family reveals that Siglec-9 is an endocytic receptor expressed on subsets of acute myeloid leukemia cells and absent from normal hematopoietic progenitors

CD33 (Siglec-3) is expressed on most acute myeloid leukemia (AML) cells and is currently being exploited as a therapeutic target. The purpose of this study was to investigate the expression pattern and potential utility of the seven recently described CD33-related siglecs as markers in AML. Besides CD33, Siglec-9 was the most highly expressed, particularly on AML cells with features of monocytic differentiation that also expressed Siglecs-5 and -7. Siglec-9 was absent from normal bone marrow myeloid progenitors but present on monocytic precursors. Using primary AML cells or transfected rat basophilic leukemia cells, Siglec-9 mediated rapid endocytosis of anti-Siglec-9 mAb. In contrast to CD33 and Siglec-5, levels of soluble Siglec-9 were low or undetectable in bone marrow plasma from AML patients and serum from normal donors. These features suggest that Siglec-9 provides not only a useful marker for certain subsets of AML, but also a potential therapeutic target.

Murine Embryonic Stem Cells Secrete Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis and Stimulate Colony Formation of Murine Hematopoietic Progenitor Cells

Stromal cell-derived factor (SDF)-1/CXCL12, released by murine embryonic stem (ES) cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells. Conditioned medium (CM) from murine ES cells growing in the presence of leukemia inhibitory factor (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-1alpha, IL-10, IL-11, macrophage-colony stimulating factor (CSF), oncostatin M, stem cell factor, vascular endothelial growth factor, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins, results which may be of physiological and/or practical significance.

Gene expression profiles in acute myeloid leukemia with common translocations using SAGE

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.

Constitutively Active β-Catenin Confers Multilineage Differentiation Potential on Lymphoid and Myeloid Progenitors

Beta-catenin-mediated Wnt signaling may contribute to the self-renewal of hematopoietic stem cells and proliferation in some malignancies. We now show that expression of constitutively active beta-catenin in normal lymphoid or myeloid progenitors generated uncommitted cells with multilineage differentiation potential. Inappropriate gene expression occurred in cells destined to produce either cell type and caused corresponding changes in their characteristics. For example, forced activation of beta-catenin quickly increased C/EBPalpha while reducing EBF and Pax-5 in lymphoid progenitors that then generated myeloid cells. Inversely, EBF dramatically increased in transduced myeloid progenitors and lymphocytes were produced. The results indicate that ectopic activation of beta-catenin destabilizes lineage fate decisions and confers some, but not all, stem cell properties on committed progenitors.

Effects of 5-aza-2′-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells

The demethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) have been shown to induce differentiation and inhibit growth of leukemic myeloid cells at low concentrations. However, the effect of DAC in changing the differentiation and proliferation behavior of normal human myeloid progenitors has rarely been investigated. Therefore, we established an in vitro model of normal hematopoietic differentiation, using CD34+ cells from mobilized peripheral blood, to study proliferation and colony formation, expression of several myeloid maturation markers and of the inhibitor of cyclin-dependent kinases p15/INK4b. Upon DAC treatment, cell growth was significantly decreased in a dose-dependent manner, without an increase in cytotoxicity. DAC treatment also resulted in a substantial increase of lysozyme-positive cells, which could be enhanced by G-CSF, a modest increase of myeloperoxidase+ and CD15+ cells, as well as an increase of colony-forming cells (CFU-GM) compared to control cells. p15/INK4b protein expression was strongly upregulated upon myeloid maturation, and additional DAC treatment did not change p15 expression or the methylation status of the p15 promoter at the noncytotoxic concentrations used. Taken together, these data indicate a role of DAC in changing myeloid progenitor cell expansion and differentiation. This model appears suitable also for global analyses of multiple differentially methylated genes.

Mutational Analysis of the CBFβ-SMMHC Assembly Competence Domain Identifies a Surface Critical for Multimerization and Inhibition of RUNX1/AML1.

CBFβ complexes with RUNX1/AML1 to form Core Binding Factor. CBFβ-SMMHC is expressed from the inv(16) or t(16;16) chromosome in 8% of AML cases. This fusion protein contains the majority of CBFβ linked to the α-helical rod domain of smooth muscle myosin heavy chain. CBFβ-SMMHC is thought to contribute to leukemogensis by dominantly inhibiting RUNX1/AML1. Inhibition of AML1 depends upon the integrity of a 28 amino acid region near the C-terminus of the SMMHC segment termed the Assembly Competence Domain (ACD). A homologous region is present in multiple myosins and is required for optimal multimerization of their respective rod domains. The ACD is located within a 63 residue "extended" ACD, which includes 12 residues N-terminal and 23 residues C-terminal to the ACD. The extended ACD was noted to have a more neutral charge than other segments of myosin rods. We have now carried out a mutagenic analysis of individual α-helices within or near the extended ACD and have assessed the effect of these mutations on the ability of CBFβ-SMMHC to multimerize in vitro and to inhibit endogenous AML1 activities in the Ba/F3 cell line and in normal murine myeloid progenitors. The 7 amino acids constituting a single turn of the rod domain α-helix are designated abcdefg. The a and d residues form a hydrophobic surface that mediates coiled-coil dimerization, the e and g residues often form salt bridges that stabilize the dimer, and the b, c and f residues are on the outer surface of the helix and are the best candidates for mediating multimerization. We have therefore mutated the bcf residues as a group in ten helices, N3, N1, A, B, C, D, E, F, G, and H. A–D constitutes the core, 28 residue ACD. N3 and N1 are three or one helix N-terminal to helix A. Mutation of N3 or N1 did not affect multimerization in low ionic strength or the ability of CBFβ-SMMHC to inhibit AML1-mediated G1 to S cell cycle progression in Ba/F3 cells. In contrast, mutation of helices A, B, C, D, E, F, G, or H both impaired multimerization in vitro and prevented cell cycle slowing in Ba/F3 cells. Mutants A–E are each located predominantly in the cell nucleus. In transduced murine myeloid progenitors, mutant N3 again behaved similar to intact CBFβ-SMMHC, mutant A also markedly slowed proliferation, mutant B had an intermediate effect, and mutants C, D, or E did not slow proliferation, each in three independent experiments. The increased activities of mutants A or B in the latter setting may reflect the fact that Ba/F3 cells accumulate three times faster than myeloid progenitors and so perhaps are more sensitive to subtle effects. Sin3A, a co-repressor shown to interact with CBFβ-SMMHC, retained the ability to bind mutants A–E. Analysis of mutants N1 and F–H for mSin3A binding, nuclear localization, and their effects on normal progenitor proliferation is in progress. Together, these findings indicate that a surface near the C-terminus of the CBFβ-SMMHC rod domain, encompassing much of the "extended ACD", is required for multimerization and inhibition of AML1. Helices N1 and H demarcate the boundaries of this surface, with helix H been the very last helix of the rod domain. Further characterization of the molecular interactions which allow this surface to mediate SMMHC multimerization may enable the rationale design of drugs for the therapy of AML associated with inv(16).

IL-3 Inhibits Terminal Granulocytic Differentiation by Inducing the Binding of Stabilizing Proteins to a 300 Base Pair Region within p21cip1/waf1 Messenger RNA.

Elevated levels of the molecular adaptor protein p21cip1/waf1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demo−nstrated that p21 undergoes dynamic changes in expression levels and subcellular compartmentalization during cytokine-induced granulocytic differentiation, suggesting that p21 may play an important role in myeloid development. Based on our observation that p21 protein levels decrease during granulocytic differentiation of CD34+ human progenitor cells, we hypothesized that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 is required to maintain the undifferentiated state in murine 32Dcl3 cells and has been shown to slow the kinetics of differentiation of normal human myeloid progenitors (Hevehan DL, 2000). Given that 32Dcl3 myeloblasts express high basal levels of p21, we also hypothesized that IL-3 inhibition of 32Dcl3 differentiation is mediated in part by p21. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3 that are normally not abundant in cells maintained as myeloblasts by IL-3. Rescue of p21 knockdown myeloblasts with human p21 suppressed aberrant expression of granule proteins. The upregulation of myeloperoxidase and proteinase-3 occurred at a posttranscriptional level. These findings indicated that p21 prevented premature expression of primary granule proteins and may contribute to maintenance of the myeloblast phenotype. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF, indicating that p21 antagonized the entire differentiation process rather than only suppressing primary granule proteins. We then determined how IL-3 maintains p21 expression in myeloblast cells. We demonstrated that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3′ untranslated region (UTR) of the p21 transcript. IL-3 also rescued the decrease in p21 mRNA stability noted during G-CSF-induced differentiation. This has been shown to coincide with differentiation blockade. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3′ UTR and are regulated by IL-3 and G-CSF signaling. Association of a ~60–65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a ~40–42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.

C/EBPα and C/EBPα Myeloid Oncoproteins Induce Bcl-2 Via Interaction of Their Basic Regions with NF-κB p50.

The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases, and wild-type CEBPA levels are reduced in additional AMLs, e.g. as a result of AML1-ETO or flt3ITD mediated inhibition of transcription or bcr-abl mediated inhibition of translation. One consequence of diminished C/EBPα activity or expression is likely inhibition of myeloid differentiation. We now find, via analysis of data from two published microarray studies, that CEBPA and Bcl-2 RNA levels correlate highly in low risk human AMLs (P = 0.003 and 0.001), suggesting that inhibition of apoptosis via induction of bcl-2 by C/EBPα or its mutant variants also contributes to transformation. Lack of correlation in other risk groups may reflect the presence of additional mutations that inhibit apoptosis. Among the low-risk patients, those with AML1-ETO had the lowest, CBFβ-SMMHC intermediate, and PML-RARα the highest levels of CEBPA (P < 0.05). Patients with CEBPA mutations were mainly in the intermediate-risk, normal karyotype group, and within this group the average CEBPA level was higher than those having wild-type alleles (P = 0.0002), suggesting a selective advantage to enable dominant-inhibition of remaining wild-type C/EBPs and/or induction of bcl-2. The two categories of C/EBPα mutant alleles associated with AML are C/EBPα p30, lacking an N-terminal transactivation domain, and C/EBPα LZ, carrying in-frame mutations in the leucine zipper (LZ) that prevent DNA-binding. Each of this oncoproteins induced bcl-2 in hematopoietic cell lines, and wild-type C/EBPα also induced bcl-2 when transduced into normal murine myeloid progenitors or in the splenocytes of H2K-C/EBPalpha]-Eμ transgenic mice. Remarkably, C/EBPα LZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA-binding, and C/EBPα p30 also activated the promoter. C/EBPα and the C/EBPα oncoproteins cooperated with NF-κB p50, but not p65, to induce bcl-2 promoter transcription. Endogenous C/EBPα preferentially coimmunoprecipitated with p50, versus p65, in myeloid cell extracts. Mutation of residues 297–302 in the C/EBPα basic region (BR) prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65 when co-expressed in 293T cells, whereas C/EBPα p30 or a C/EBPα LZ oncoprotein retain the ability to co-ip with p50. These findings offer an explanation for preferential in-frame rather than out-of-frame mutation of the LZ with sparing of the BR in the C/EBPα LZ oncoproteins that constitute one-third of CEBPA mutations in AML. C/EBPα or its mutant variants may tether to a subset of NF-κB targets genes, including Bcl-2, via p50 to facilitate gene activation. Targeting interaction between the C/EBPα basic region and NF-κB p50 may contribute to the therapy of AMLs expressing either mutant or residual wild-type C/EBPα , by increasing susceptibility to apoptosis. Targetting this interaction may also be relevant to the therapy of other malignancies expressing C/EBPs, not only via down-regulation of bcl-2 but also by preventing their cooperative activation of genes mediating inflammation and angiogenesis, which have recently been shown to contribute to the pathogenesis of several solid tumors in animal models.

Oct-4 Is Crucial for Murine Embryonic Stem Cell Secretion of Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis of Murine ES Cells and Bone Marrow Hematopoietic Progenitor Cells.

Murine embryonic stem (ES) cells may be of potential use for cell replacement and gene therapy. Maintenance of ES cells in an undifferentiated and proliferative state depends on cytokines either secreted by ES cells and/or added to the medium. By understanding the production and release of cytokines in ES cell culture, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that SDF-1/CXCL12, secreted by ES cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells (Guo et al, Stem cells, in press, 2005). To evaluate whether other cytokines were produced by murine ES cells, we generated conditioned medium (CM) from these cells in the presence of LIF, while the ES cells were in an undifferentiated Oct-4 expressing state, and assayed the CM for cytokines, chemokines, and other growth modulatory factors. ES cell CM enhanced survival in vitro of ES cells subjected to delayed addition of serum to ES cell cultures. Without serum, ES cells didn't grow in low cell density. However, with CM, ES cells formed colonies at about 63% of the growth of the ES cells in the presence of serum. ES cell CM also enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased the rate of apoptosis in murine bone marrow c-kit+lin− cells as assessed by Annexin V assay. Our data showed ES cell CM contained IL-1α, IL-10, IL-11, M-CSF, OSM, SCF, VEGF, as well as a number of chemokines and other proteins. For a number of these proteins, we have already verified that the mRNA for them is expressed in the ES cells. This indicates that ES cells produce and secrete these cytokines. Some of these cytokines are known to have an enhanced survival/antiapoptosis effect on progenitors. IL-6, FGF-9, and TNF-a, which were not detected prior to irradiation of the ES cells, were seen after ES cells were irradiated. Irradiation of the ES cells enhanced release of some proteins and decreased release of others. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for cytokines released from ES cells which supported CFU-GM survival and antiapoptosis. Oct-4 conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. CM from the wild type ES cell line enhanced survival of CFU-GM similar to that of other ES cell lines, while the Oct-4 knockout ES cell line didn't. These results indicate that release of proteins involved in survival enhancement may be related to Oct-4 expression. We also found that the wild type cell line which expressed Oct-4 didn't initiate caspase 3 dependent apoptosis after mitotic stress. However ZHBTc4, the Oct-4 deleted cell line demonstrated caspase 3 dependent apoptosis. These results may be of physiological significance, although this has not yet been proven, and suggest the possibility of potential future applicability for use of irradiated ES cells as accessory cells for growth modulation in vitro and in vivo.

Oncogenic Ras Requires SHP-2 To Induce Myeloproliferative Disease (MPD).

Oncogenic mutations in NRAS or KRAS2 are found in 20–40% of myeloid malignancies including acute myeloid leukemia, high risk subtypes of myelodysplastic syndrome, and in juvenile myelomonocytic leukemia (JMML) and other types of MPD. Mutant Ras proteins accumulate in the active, GTP-bound state due to impaired intrinsic GTPase activity and resistance to GTPase activating proteins (GAPs). Alternative genetic mechanisms, such as BCR-ABL fusion and inactivation of the NF1 tumor suppressor, also deregulate Ras signaling in myeloid malignancies. Moreover, the association of germline mutations in NF1 with a markedly increased risk of JMML and studies of KrasG12D and Nf1 mutant mice argue strongly that hyperactive Ras can initiate MPD in vivo. Activating mutations in PTPN11, which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in JMML. As is the case for NF1, germline PTPN11 mutations impart an increased risk of developing JMML. These observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1. While NF1 encodes a GAP for Ras that directly reduces Ras-GTP levels, data from various systems have shown that SHP-2 is a positive effector of Ras activation. However, exactly how depohsophorylation of SHP-2 substrates regulates Ras output and how other SHP-2 target proteins modulate its effects are incompletely understood. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. To test the hypothesis that Ras is strictly downstream of SHP-2 in primary hematopoietic cells, we used the conditional alleles LSL-KrasG12D and Ptpn11flox/flox coupled with the interferon-inducible Mx1-Cre transgene. Juvenile mice were injected with pI:pC, resulting in expression of K-RasG12D and inactivation of Ptpn11. These mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre, LSL-KrasG12D mice (Braun et al., PNAS 101(2):597–602), although with slightly increased latency. Importantly, however, myeloid progenitors invariably retained at least one functional Ptpn11 allele despite uniform activation of the conditional KrasG12D allele. These data suggested that there was strong selective pressure to retain at least one functional Ptpn11 allele, even in the presence of oncogenic K-Ras expression. To address this hypothesis directly, we infected LSL-KrasG12D, Ptpn11flox/flox, and compound mutant fetal liver cells with a Cre-expressing retrovirus and enumerated myeloid progenitor colonies in methylcellulose medium. Remarkably, no granulocyte-macrophage colonies developed from Cre-expressing Ptpn11flox/flox fetal liver cells either in the presence or absence of KrasG12D. Infecting compound mutant cells with a Ptpn11-IRES-Cre virus fully restored the aberrant growth phenotype of KrasG12D mutant cells. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro. Our data support a model in which SHP-2 has essential Ras-independent functions in hematopoiesis (i.e. that Ras is not strictly downstream of SHP-2). SHP-2 may be required either for efficient activation of canonical Ras effector pathways and/or to regulate signaling parallel to Ras. Our data imply that SHP-2 inhibition will ablate both normal and neoplastic hematopoietic progenitors. This may have a therapeutic role in preparing patients with JMML and other high risk MPDs for hematopoietic stem cell transplantation.

Exogenous C/EBPα Favors the Monocytic Commitment and Maturation of Normal Murine Myeloid Progenitors.

C/EBPα (−/−) cells do not effectively generate the granulocyte-monocyte progenitor (GMP) from the common myeloid progenitor (CMP). However, the role of C/EBPα in the further commitment of the GMP remains uncertain. Transduction of murine marrow or fetal liver cells with KRAB-C/EBPα-ER, an estradiol-regulated, dominant inhibitor of C/EBPs, suppresses formation of CFU-M, CFU-G, and CFU-GM without affecting formation of BFU-E, suggesting a role for C/EBPα in both myeloid lineages. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells and several other cell lines, but induces monocytic development from transduced B cell progenitors. We have now assessed the effect of C/EBPα on the myeloid commitment and maturation of murine myeloid progenitors. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPα-ER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient and only then subjected to lineage-depletion, just prior to culture +/− estradiol. This protocol, combined with rapid assessment of lineage maturation aims to avoid biases due to effects on proliferation or differentiation during transduction. C/EBPα-ER was expressed approximately 4-fold above endogeous levels. By 48 hrs, 80% of the lin- cells had acquired myeloid surface markers. Mac1+/Gr1+ or Mac1+/MPO+ cells, assessed by FACS, were granulocytes and Mac1+/Gr1− or Mac1+/MPO− cells were monocytes, as confirmed by staining cytospins. Activation of C/EBPα-ER with estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold at 48 hrs in five assessments (P < 0.002). A similar effect was seen when the estradiol dose was reduced from 1.0 to 0.3, 0.1, 0.03, or 0.01 μM, suggesting that observed effects on myeloid maturation occur near a physiologic activity level. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68, a 2-fold increase in M-CSFR and PU.1, and a 4-fold decrease in NE by 48 hrs. Estradiol had little effect on the myeloid development of vector-transduced cells or those expressing a leucine zipper mutant. Interestingly, transduction with the BR3 basic region mutant favored granulopoiesis in 5 independent experiments (P < 0.005), potentially reflecting dominant inhibition of endogenous C/EBPα or the the inability of this variant to interact with NF-κB p50. To assess effects on more immature progenitors, transduced cells were plated in methylcellulose. If estradiol was included, a 3- to 5-fold reduction in colony number precluded assessment of lineage commitment. However, if transduced progenitors were cultured with reduced concentrations of estradiol for 24 hrs and then plated without estradiol, C/EBPα-ER only reduced CFU numbers 1.2 to 2-fold. In two independent experiments, culture in IL3/IL6/SCF or in GM-CSF after transient activation of C/EBPα-ER resulted in a 2.6- to 5-fold increase in the ratio of CFU-M to CFU-G, indicating an effect on monocyte commitment at an early progenitor stage. For example, in one experiment no estradiol gave an average of 111 CFU per dish in IL3/IL6/SCF, culture for 24 hrs with 0.1 μM estradiol prior to plating gave 93 CFU, and pretreatment with estradiol increased the ratio of CFU-M to CFU-G from 1.1 to 3.9. We suggest a model of myelopoiesis in which C/EBPα first directs commitment of stem cells to the GMP by inducing PU.1 transcription. C/EBPα then has the capacity to direct monocytic commitment and maturation, likely dependent on cytokine conditions and availability of cooperating factors such as AP-1 or NF-κB.

Genes Similarly Abnormally Expressed in Both Human and Murine MLL-Associated Leukemia.

Although more than 50 different loci translocate to the MLL gene at chromosome band 11q23, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), no unifying property is shared by all partner genes. The translocations result in a functional fusion of the N-terminal part of MLL gene and the C-terminal part of each partner gene, presumably leading to changes in the expression of the normal target genes, most of which have not been identified. Although genetically engineered mouse leukemia models have been widely used, few systematic studies have evaluated whether such models are valid equivalents of human leukemia. We used serial analysis of gene expression (SAGE) to obtain genome-wide gene expression profiles in normal myeloid progenitor cells from human CD15+ and mouse Gr-1+ cells. We also analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL [t(11;19)(q23;p13.1)] or MLL-ENL [t(11;19)(q23;p13.3)] fusions, and a cell line from a leukemia mouse transduced with an MLL-ELL fusion. MLL-ELL and MLL-ENL fusions are frequently involved in human AML, while MLL-ENL is also seen in human ALL. 484,303 SAGE tags were identified from the nine samples (40,000 to 100,000 tags per sample), yielding 103,899 unique SAGE tags in the human and 60,993 in the mouse samples. Analysis of the SAGE data identified 43 candidate genes that appear to be abnormally expressed in both human and mouse myeloid leukemia progenitor cells with either MLL-ELL or MLL-ENL fusions (9 up-regulated and 34 down-regulated; Table 1). Increasing evidence suggests that endogenous antisense RNAs may play critical roles in gene regulation and cancer. Natural antisense RNAs include cis-encoded antisense RNAs transcribed from the opposite strand of the same genomic locus as the sense target genes, and trans-encoded antisense RNAs such as microRNAs (miRNAs) transcribed from a genomic locus different from the sense target genes. 26 of the 43 candidate genes have antisense partners (with a total of 7 cis-encoded antisense RNAs and 36 trans-encoded miRNAs) and thereby might be regulated by endogenous antisense RNAs. We are currently validating the expression pattern of the 43 candidate genes in at least 30 different human and mouse leukemia and normal control samples with quantitative RT-PCR, and measuring the level of expression of all known miRNAs via microarray in these samples. Our studies on the abnormally expressed genes and their potential antisense partners will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute leukemia, which may lead to more effective therapy for these leukemias.Table 1Genes deregulated in both human and mouse leukemiasaTotal numberUp-regulated genesDown-regulated genesGenes with antisense partner(s)In MLL-ELL fusions2112012In MLL-ENL fusions3382521In both types of fusions110117Total unique genes4393426aThe genes have at least 3 fold difference in expression with a significance P < 0.05 between each leukemia sample and the normal control sample.

On the TRAIL of a new therapy for leukemia

The cytokine TRAIL (tumor necrosis factor alpha-related apoptosis-inducing ligand) as well as agonistic antibodies that bind to the TRAIL receptors, death receptor 4 (DR4) and DR5, are undergoing preclinical and early clinical evaluation as potential therapeutic agents for a variety of hematological and nonhematological malignancies. Here, we briefly review the normal biological function of TRAIL, the mechanism of cytotoxicity of TRAIL receptor ligands, and their effects on normal myeloid progenitors, myelodysplastic marrow and leukemic cells, including acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL), in vitro. Recent observations suggesting that DR4 is the predominant receptor for the cytotoxic effects of TRAIL in CLL and that histone deacetylase inhibitors synergize with TRAIL in CLL in vitro are described and discussed. Collectively, the reviewed studies not only illustrate the potential therapeutic usefulness of TRAIL and the agonistic antibodies, but also highlight the need for additional preclinical evaluation of these agents.

CCAAT/Enhancer Binding Protein α (C/EBPα) and C/EBPα Myeloid Oncoproteins Induce Bcl-2 via Interaction of Their Basic Regions with Nuclear Factor-κB p50

The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein alpha (C/EBPalpha) or its mutant variants contributes to transformation. C/EBPalphap30, lacking a NH2-terminal transactivation domain, or C/EBPalphaLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPalpha induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPalpha-Emu transgenic mice. C/EBPalpha protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPalphaLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPalphap30 also activated the promoter. C/EBPalpha and the C/EBPalpha oncoproteins cooperated with nuclear factor-kappaB (NF-kappaB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPalpha preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPalpha basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPalpha or its mutant variants tether to a subset of NF-kappaB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPalphaLZ oncoproteins. Targeting interaction between C/EBPalpha basic region and NF-kappaB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.

CBFβ-SMMHC slows proliferation of primary murine and human myeloid progenitors

CBFbeta-SMMHC is expressed in 8% of acute myeloid leukemias and inhibits AML1/RUNX1. In this study, murine marrow or human CD34(+) cells were transduced with retroviral or lentiviral vectors expressing CBFbeta-SMMHC or two mutant variants. CBFbeta-SMMHC reduced murine or human myeloid cell proliferation three- to four-fold in liquid culture relative to empty vector-transduced cells, during a period when vector-transduced cells accumulated five-fold and human cells 20-fold. CBFbeta-SMMHC decreased the formation of myeloid, but not erythroid, colonies two- to four-fold, and myeloid colonies expressing CBFbeta-SMMHC were markedly reduced in size. However, CBFbeta-SMMHC did not slow differentiation to granulocytes or monocytes. Neither CBFbeta-SMMHC(Delta2-11), which does not bind AML1, nor CBFbeta-SMMHC(DeltaACD), which does not multimerize or efficiently bind corepressors, slowed proliferation or reduced myeloid colonies. CBFbeta-SMMHC increased the G1/S ratio 1.4-fold. AML1 had an effect opposite to CBFbeta-SMMHC, stimulating proliferation of murine myeloid progenitors 2.0-fold in liquid culture. Thus, CBFbeta-SMMHC directly inhibits the proliferation of normal myeloid progenitors via inhibition of AML1 and dependent upon the integrity of its assembly competence domain. These findings support the development of therapeutics that target the ability of CBFbeta-SMMHC to interact with AML1 or to multimerize via its assembly competence domain.

Prognostic progenitor profiling in chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a unique myeloproliferative disease characterized by marrow dysplasia and an increase in monocytes. The median survival of patients with CMML is short, in part, because CMML is frequently resistant to therapy. A better understanding of the basic biology of CMML progenitors is needed in order to reduce regimen related toxicity and provide more effective CMML targeted therapies. To this end, a mouse transgenic model of CMML was created in which human bcl-2 overexpression was targeted to the myeloid lineage by the myeloid promoter hMRP8. Transgenic hMRP8-bcl2 mice developed a myeloproliferative disorder characterized by a monocytosis in all hematopoietic tissues and splenomegaly. Myeloid progenitor analysis of hMRP8-bcl-2 bone marrow revealed a marked expansion of human bcl-2 expressing granulocyte-macrophageprogenitors compared with control mice. In order to see if thiscorrelated with human CMML, we used FACS analysisand recently identified phenotypic markers to identify phenotypicand functional differences between normal and CMML bonemarrow hematopoietic stem cells and myeloid progenitors. CMML marrow was typified by a reduction in CD34+CD38−CD90+Lin− hematopoietic stem cells and an expansion of CD34+CD38−CD90−Flk2+Lin− cells relative to normal bone marrow. In addition, there was a two-fold expansion in common myeloid progenitors (CMPs) and a corresponding decrease in megakaryocyte-erythroid progenitors (MEPs) suggesting that there was a skew in differentiation toward the myeloid lineage. In contrast to normal bone marrow derived CMPs, CMML CMPs gave rise to myeloid but not erythroid colonies. Moreover, real time quantitative RT-PCR analysis of highly purified FACS-sorted CMML CMPs demonstrated increased expression of two key regulators of myelomonocytic differentiation, PU.1 and c-jun, compared with normal bone marrow. A more detailed understanding of the basic biology of CMML progenitors and the genes that work in concert to expand them may aid in identifying novel molecular targets for CMML.

Exogenous C/EBPα Increases the Monocytic Maturation of Normal Murine Myeloid Progenitors.

Within hematopoiesis C/EBPα is expressed specifically in both the granulocytic and monocytic myeloid lineages. Mice lacking C/EBPα have reduced neutrophil and monocyte production, due in part to diminished expression of the G-CSF, M-CSF, and GM-CSF receptor mRNAs. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells, and induction of late markers of granulopoiesis by C/EBPα-ER in 32Dcl3 cells was dependent upon its ability to slow G1 to S progression. We have now assessed the effect of C/EBPα-ER on the myeloid maturation of murine marrow progenitors. Regulation of the transgene using estradiol avoids biasing cell populations during transduction. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPαER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient, subjected to lineage-depletion, which removed 98% of Mac1+ and Gr1+ cells, and placed in liquid or methylcellulose culture with IL3/IL6/SCF +/− estradiol. By 48 hrs, 80% of the cells had acquired myeloid surface markers. Mac1+/Gr1+ cells were granulocytes and Mac1+/Gr1− cells were monocytes, as confirmed by Wright’s-Giemsa staining of cytospins. Estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold in 5 independent assessments. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68/macrosialin RNA, a 2-fold increase in M-CSF receptor and PU.1 RNA, and a 4-fold decrease in NE RNA by 48 hrs. In contrast, estradiol had little effect on the development of monocytes or granulocytes from vector, pBabePuro-transduced cells. Consistent with these findings, in an initial experiment we observed increased granulocytes when progenitors from floxxed C/EBPα mice (kindly provided by P. Johnson) were transduced with pBabeNeo-CRE, compare with pBabeNeo, subjected to lineage-depletion, and cultured in IL3/IL6/SCF. Loss of C/EBPα was demonstrated by PCR and Western blotting. During the first 48 hrs after transfer of transduced wild-type cells to estradiol, C/EBPα-ER slowed the proliferation of myeloid progenitors 2.6-fold, increased their G1/S ratio 2.8-fold, and reduced myeloid CFUs 5.1-fold. CFU-M, CFU-G, and CFU-GM were affected equally. Transduction with C/EBPαL12V-ER, harboring a defective leucine zipper, or with C/EBPαBR3-ER, mutant in 4 basic region residues, slowed proliferation in liquid culture 2-fold, reduced myeloid CFUs 1.5-fold, and had little effect on the G1/S ratio, perhaps indicating a role for DNA-binding in the regulation of the cell cycle by C/EBPα in normal myeloid progenitors (CFUs) and precursors. The L12V variant did not affect lineage output, whereas C/EBPαBR3 reduced monocytic maturation 1.4-fold and increased granulocyte formation 1.4-fold, in 4 experiments. C/EBPαp30, expressed in a subset of AML cases and lacking an N-terminal transactivation domain, increased the G1/S ratio and reduced myeloid CFUs 1.6-fold and had no effect on myeloid maturation. In summary, exogenous C/EBPα increases the monocytic and reduces the granulocytic maturation of transduced myeloid progenitors cultured in cytokines whose receptors are not regulated by C/EBPα. Cell cycle inhibition may play a role in this lineage bias, as the the L12V and p30 variants had both a reduced ability to slow G1 progression and to induce monocytes. Induction of PU.1 may also favor monocytic maturation. Finally, results with BR3 compared with L12V, neither of which bind DNA, suggest that alteration of a protein interaction with the non-DNA contact surface of the C/EBPα basic region favors granulocyte formation.

C/EBPα and C/EBPα Myeloid Oncoproteins Inhibit Apoptosis and Induce Bcl-2 Via DNA-Binding Dependent and Independent Mechanisms.

C/EBPα dimerizes via its leucine zipper domain and then binds DNA via the adjacent basic region. One or both copies of the C/EBPα gene harbors point mutations in 5–8% of human AMLs. One common group of mutations leads to expression of C/EBPαp30, lacking an N-terminal transactivation domain. In addition, insertions and deletions commonly occur in the first α-helix of the leucine zipper, preventing DNA-binding - these alterations are typically in-frame, suggesting that the C/EBPαLZ mutants retain a role in transformation. We have now found that C/EBPα, C/EBPαp30, and four C/EBPαLZ mutants inhibit the apoptosis which occurs when the 32Dcl3 or HF1 myeloid or Ba/F3 or FL5.12 lymphoid cell lines are withdrawn from IL-3; for example, 30% of Ba/F3-αER cells survived at 24 hrs and 0–2% at 48 hrs in the absence of estradiol, and 180%, 80%, and 40% survived at 1, 2, or 3 days in its presence. However, TNF death receptor-mediated apoptosis was not affected. C/EBPα-ER did not alter expression of STAT3, P-STAT3, STAT5, P-STAT5, MAPK, P-MAPK, PI3K, AKT, c-Jun, JunB, bcl-xL, or bim in Ba/F3 cells. However, bcl-2 protein levels, which diminish several-fold after IL-3 withdrawal, were elevated 5-fold at 16 hours by C/EBPα-ER, but not in vector-transfected cells exposed to estradiol. Northern blotting demonstrated induction of transcription from the bcl-2 P2 promoter. Induction of bcl-2 by C/EBPα was previously seen in t(14;18) lymphoma lines, in which the bcl-2 gene is fused to the immunoglobulin heavy chain promoter, and C/EBPα was shown to transactivate P2-luciferase, in part via interaction with a Cdx:A-Myb complex. We have now also identified a consensus C/EBP-binding site, using EMSA, in the P2 promoter. C/EBPα-ER also induced endogenous bcl-2 in NIH 3T3 cells, in transduced normal murine myeloid progenitors, and in the splenocytes of H2K-Eμ-C/EBPα transgenic mice. C/EBPα enhanced survival of splenocytes exposed to low-dose ionizing radiation. The ability of additional C/EBPα mutants to induce bcl-2 and inhibit apoptosis was assessed. The BRM2 mutant, which does not bind E2F, Δ3–8, lacking transactivation domains, GZ, having the GCN4 leucine zipper, and L12V, which cannot dimerize, each retained these activities. In contrast, BR3, with 4 mutant residues in the basic domain, did not induce bcl-2 and only weakly prevented cell death. The BR3 mutations were then studied individually. Only R300G prevented DNA-binding; this mutant did not induce bcl-2 and only weakly inhibited cell death. K298T and K302N each potently induced bcl-2 and prevented apoptosis. Finally, R297G only weakly induced bcl-2 and had an intermediate effect on survival. Perhaps R300G alters the conformation of R297, explaining R300G's inability to induce bcl-2. In summary, C/EBPα stimulates cell survival, in part via direct activation of the bcl-2 P2 promoter. This induction is optimal when C/EBPα retains its ability to bind DNA, but can also occur via protein:protein interaction, perhaps mediated in part by R297 with the Cdx:A-Myb complex or with other proteins which regulate the bcl-2 gene, such as NF-κB. Targeting this interaction with the outer surface of the C/EBPα basic region may be an effective means to identify novel therapeutics for AMLs expressing C/EBPα oncoproteins.