Factor-independent Colony Research Articles

Myelomonocytic skewing in chronic myelomonocytic leukemia: phenotypic, molecular and biologic features and impact on survival.

BackgroundMyelomonocytic skewing is considered as a key pathophysiologic phenomenon in chronic myelomonocytic leukemia (CMML), but its prevalence and potential correlation with phenotypic, genotypic, and clinical features are poorly defined.MethodsSkewed differentiation toward the myelomonocytic over erythroid commitment as indicated by an inverse ratio of myelomonocytic/erythroid colonies was investigated in 146 patients with CMML by semisolid in vitro cultures.ResultsThere was a high prevalence of myelomonocytic skewing in patients with CMML (120/146, 82%); whereas, this phenomenon was rare in normal individuals (1/98, 1%). Patients with CMML with myelomonocytic skewing had higher white blood cell and peripheral blast cell counts, and lower platelet values. The number of mutations in genes of the epigenetic and/or splicing category was higher in CMML patients with as compared with patients without skewing. Patients with myelomonocytic skewing had more frequently mutations in RASopathy genes and higher growth factor independent myeloid colony formation. Interestingly, the lack of myelomonocytic skewing discriminated patients with CMML with a particularly favorable prognosis (60 vs 19 months, P = .003) and a minimal risk of transformation.ConclusionMyelomonocytic skewing as determined by semisolid cultures can discriminate subgroups of patients with CMML with a different phenotype, a different genotype, and a different prognosis.

Genetic Depletion of Fc Gamma Receptor 2b Affects CML Stem Cell Biology

Chronic myeloid leukemia (CML) is caused by the acquisition of the Philadelphia chromosome t(9;22) at the hematopoietic stem cell (HSC) level. The resulting Bcr-Abl protein loses the Abl autoinhibitory function and gains the Bcr oligomerization domain resulting in the constitutive activation of the Abl tyrosine kinase. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, various mechanisms such as the acquisition of tyrosine kinase domain mutations or kinase-independent alterations have been described to result in therapy resistance. Using an inducible transgenic SCLtTAxBcr-Abl CML mouse model, we previously demonstrated that Bcr-Abl inhibition leads to eradication of mature leukemic cells but spares disease-driving leukemic stem cells (LSC) due to a lack of oncogene-addiction (Schemionek et al., BLOOD 2010; Hamilton, Helgason, Schemionek et al., BLOOD 2012). Aiming to identify new Bcr-Abl targets that could overcome therapy resistance of stem cells, we previously performed a microarray analysis using murine LSC. Expression of FcγRIIb (Fc gamma receptor 2b; CD32) was significantly increased 2.79-fold in Bcr-Abl positive vs. control Lin-Sca1+c-kit+(LSK) cells. The FcγRIIb contains an immuno receptor tyrosine-based inhibitory motif (ITIM) and inhibits intracellular signaling upon activation. Phosphorylation of the receptor is mainly mediated via Src kinase Lyn and enables the receptor to recruit Src homology 2 domain-containing phosphatases.Using qRT-PCR, we confirmed 2.83-fold upregulation of FcγRIIb within the LSK population of CML mice. Retroviral infection of 32D cells and primary BM cells with Bcr-Abl significantly increased FcγRIIb expression level (90.3-fold and 13.2-fold, resp., p<0.005 each vs. emtpy vector). TKI treatment of Bcr-Abl positive 32D cells did not affect FcγRIIb mRNA levels or surface expression. Bcr-Abl infection of lineage-depleted BM cells decreased growth factor independent colony forming capability (5.5-fold, p<0.05) and the proliferation potential of FcγRIIb-/- lineage negative bone marrow (cells) compared to wild type (wt) in the absence of cytokines. Transplantation of Bcr-Abl infected FcγRIIb-/- BM cells significantly reduced white blood cell count (WBC) of the recipients as compared to wt Bcr-Abl positive cells by 2.4-fold (p<0.05). Interestingly, WBC of Bcr-Abl positive FcγRIIb-/- mice was similar to FcγRIIb-/- empty vector control animals (p=0.875) as well as wt control mice (p=0.241). CML development was analyzed upon autopsy by FACS of BM and spleen cells and showed a 2.0-fold reduction (p<0.05) of leukemic c-kit positive cells as well as a reduction in spleen weight in recipients of Bcr-Abl transduced FcγRIIb-/- cells (293mg ± 30) vs. Bcr-Abl transduced wt controls (381mg ± 13; p<0.05). Moreover, total numbers of leukemic LSK cells were significantly decreased by 3.5-fold in BM and 3-fold in spleen of Bcr-Abl transduced FcγRIIb-/- BM recipients compared to recipients of Bcr-Abl transduced wt cells. Transplantation of empty vector transduced FcγRIIb-/- vs. empty vector transduced wt control cells did not result in a decrease of knock-out cells showing that the observed effects were specific for impairment of Bcr-Abl transforming activity by loss of FcγRIIb but not due to FcγRIIb depletion alone.In conclusion, we show that FcγRIIb is upregulated in the stem cell compartment of CML mice and that this is due to Bcr-Abl expression. The absence of FcγRIIb impairs leukemic cell growth in vitro and in vivo. Moreover, genetic depletion of the receptor decreases LSC numbers and affects CML development in vivo suggesting that FcγRIIb plays an important role in Bcr-Abl leukemogenesis, making it an attractive novel stem cell specific target for CML therapy. (*Authors contributed equally) DisclosuresBrümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Koschmieder*:Novartis: Consultancy, Honoraria, Other, Research Funding.

Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis

AbstractPTPN11, which encodes the tyrosine phosphatase SHP2, is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis, we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11D61Y. Expression of Ptpn11D61Y in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin−Sca1+cKit+ (LSK) compartment is expanded and “right-shifted,” accompanied by increased stem cell factor (SCF)–evoked colony formation and Erk and Akt activation. However, repopulating activity is decreased in diseased mice, and mice that do engraft with Ptpn11D61Y stem cells fail to develop MPD. Ptpn11D61Y common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) produce cytokine-independent colonies in a cell-autonomous manner and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Ptpn11D61Y megakaryocyte-erythrocyte progenitors (MEPs) yield increased numbers of erythrocyte burst-forming units (BFU-Es), but MEPs and erythrocyte-committed progenitors (EPs) produce fewer erythrocyte colony-forming units (CFU-Es), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cell-autonomous and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.

Expression of Leukemogenic Ptpn11 Causes a Fatal Myeloproliferative Disorder by Affecting Multiple Stages of Hematopoiesis

The Src homology-2 (SH2) domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is an ubiquitously expressed non-receptor protein tyrosine phosphatase (PTP) that positively regulates Ras/Erk activation in many receptor protein tyrosine kinases (RTK) and cytokine receptors. Mutations in PTPN11 are found in ~35% of patients with juvenile myelomonocytic leukemia (JMML) and at lower incidence in other neoplasms. To model JMML pathogenesis, we generated knock-in mice that conditionally express the leukemia-associated mutant Ptpn11D61Y. Ptpn11D61Y expression in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly with extramedullary hematopoiesis, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin−Sca1+cKit+ (LSK) compartment is expanded and “right-shifted”, accompanied by increased stem cell factor (SCF)-evoked colony formation and Erk and Akt activation. However, stem cell activity is decreased in diseased mice, and mice engrafted with Ptpn11D16Y stem cells fail to develop MPD. Ptpn11D16Y common myeloid (CMP) and granulocyte-monocyte (GMP) progenitors produce cytokine-independent colonies in a cell-autonomous manner, and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-SCF) stimulation. Ptpn11D61Y megakaryocyte-erythrocyte progenitors (MEP) yield increased numbers of erythrocyte burst-forming units (BFU-E), but MEP and erythrocyte-committed progenitors (EP) produce fewer erythrocyte colonyforming units (CFU-E), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cellautonomous, and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.

Oncogenic mutations of the PIK3CA gene in head and neck squamous cell carcinomas

Phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases that regulate cellular activities such as proliferation, survival, motility and morphology. Recent studies reported that the p110alpha (PIK3CA), catalytic subunit of PI3-kinase is somatically mutated in human cancers. Hot- spot mutations (E542K, E545K and H1047R) are reported to have higher oncogenic potential. Although PIK3CA mutations were reported in head and neck squamous cell carcinomas (HNSCC) of limited ethnicity, the functional consequences of HNSCC-associated PIK3CA mutations have not been examined. Status of PI3K signaling related genes (PTEN-RAS-EGFR) in the presence of PIK3CA mutation have not been reported. In this study, we analyzed exons 9 and 20 of PIK3CA in 54 samples, including 17 HNSCC cell lines, 19 Indian and 18 Vietnamese primary tumors. We found mutations in 29.4% (5/17) of HNSCC cell lines, 10.5% (2/19) of Indian tumors and no mutation (0/18) in Vietnamese tumors. Two homozygous PIK3CA mutations were found in cell lines and a novel insertion mutation with oncogenicity in Indian tumor. Analysis of PI3K signaling related genes showed that PIK3CA and PTEN mutations were mutually exclusive, though PTEN mutation is uncommon in HNSCC. However, PIK3CA mutation coexisted with H-RAS mutation. Furthermore, PIK3CA mutations were mutually exclusive to EGFR amplification. All the 5 mutants that we found in HNSCC, showed increased PI3 kinase activities, followed by growth factor independent higher colony forming efficiency, changes in morphology, higher rates of migration and invasion compared with PIK3CA wild-type. Our study is the first to examine the oncogenic potential of PIK3CA mutants associated with HNSCC and report on PIK3CA mutations in Indian and Vietnamese ethnicity. These results suggest that PIK3CA mutations in HNSCC are likely to be oncogenic and may significantly contribute to HNSCC carcinogenesis and pave attractive target for therapeutic prevention.

Molecular Mimicry in the Chronic Myeloproliferative Disorders: Reciprocity between JAK2 V617F Genotype and Mpl Expression.

Polycythemia vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocytosis (ET) are clonal myeloproliferative disorders (MPD) that share in common increased blood production, megakaryocyte dysplasia, growth factor-independent colony formation and similar cytogenetic defects. Until recently, a dearth of molecular markers hindered analysis of the interrelationship of PV, IMF and ET. Mpl protein expression is decreased in most PV patients and the degree of impairment correlated with disease duration and extent. Impaired Mpl expression was also present in many IMF and ET patients. The recent discovery of the JAK2 V6217F mutation in PV recapitulated this behavior since JAK2 V617F was present in many classical IMF and ET patients. Importantly, JAK2 is an obligate Mpl chaperone required for Mpl surface expression (JBC 280: 27251, 2005). Since JAK2 V617F is constitutively active, we hypothesized that both its presence and gene dosage could influence Mpl expression. Therefore, we examined the concordance of the JAK2 V617F genotype and platelet Mpl protein expression in 78 PV, 13 IMF and 82 ET patients, using allele-specific, real-time PCR screening of granulocyte DNA for JAK2 V617F and quantitative densitometry for platelet Mpl with a cutoff of ≤ 60% for reduced and ≥ 120% for overexpression. JAK2 V617F prevalence was 91% in PV (40% homozygous), 46% in IMF (66% homozygous) and 41% in ET (none homozygous). JAK2 V617F-negative patients (7 PV, 7 IMF and 58 ET) were indistinguishable clinically from JAK2 V617F-positive patients. Mean disease duration was similar for homozygous JAK2 V617F (11 years, range 1–25) and heterozygous JAK2 V617F (8 years, range 1–22) PV patients. Mean disease duration was also similar for JAK2 V617F-positive (7.5 years, range 1–18) and JAK2 V617F-negative (6 years, range 1–22) ET patients. Mean Mpl expression was 31% in PV patients (range 1–227, reduced in 89%), 7% in IMF (range 1–22, reduced in 100%) and 72% in ET (range 1–246, reduced in 52%). Of the JAK2 V617F-positive patients, mean platelet Mpl was 27% in PV (range 1–86%, reduced in 92%), 5% in IMF (range 1–11%, reduced in 100%) and 38% in ET (range 1–127%, reduced in 81%). JAK2 V617F genotype significantly inversely correlated (p<0.004) with platelet Mpl expression: in JAK2 V617F homozygotes, mean platelet Mpl expression was 12% (range 1–44, 100% reduced); in JAK2 V617F heterozygotes, mean Mpl expression was 35% (range 1–127, 86% reduced), while in JAK2 V617F-negative patients, mean Mpl was 82% (range 1–246%, 44% reduced). JAK2 V617F-negative PV and IMF patients had either markedly reduced Mpl (8 patients, mean 6%) or markedly elevated Mpl (2 patients, mean 200%). In ET, JAK2 V617F-positive patients had reduced platelet Mpl (38% of normal, range 1–127%) compared to JAK2 V617F-negative patients (92% of normal, range 1–246%) (p=0.004). However, JAK2 V617F-negative ET patients were heterogeneous; 42% had reduced, 24% normal and 33% elevated platelet Mpl expression. These data indicate that JAK2 V617F expression and gene dosage directly influence platelet Mpl expression in PV, IMF and ET. Less commonly however, impaired Mpl expression, often marked, also occurred in the JAK2 V617F-negative setting, suggesting that other genetic or epigenetic lesions can create the same molecular abnormality. We conclude that Mpl protein expression abnormalities, whether associated with JAK2 V617F or not, provide a common denominator for the MPD phenotype. JAK2 V617F genotype does not necessarily predict MPD phenotype, but the degree of Mpl reduction does.

Mutations Required for Lymphoid Leukemogenesis Are Not Required for c-Myc to Rapidly Induce Acute Myeloid Leukemia.

Dysregulation of c-Myc is one of the critical oncogenic events required for tumorigenesis in many tissue types. Although c-Myc has been shown to block differentiation of leukemic and normal myeloid cells and is activated by oncogenes commonly found in leukemia, its exact role in myeloid leukmogenesis remains elusive. When c-Myc is over-expressed in the lymphoid compartment, Eμ-Myc transgenic mice develop B-cell lymphomas only after a prolonged latency and require additional mutations to inhibit apoptosis, most commonly by disrupting the Arf-Mdm2-p53 pathway. Although this model has been well used to dissect pathways of Myc-induced lymphomagenesis, there is currently no efficient model with which to study the role of c-Myc in AML. In this study, we utilized a retroviral gene transfer system (MSCV-Myc) to introduce c-Myc into unfractionated murine bone marrow that was subsequently transplanted into syngeneic mice. Surprisingly, 100% of transplanted mice (N=110) from three different strains, developed acute myeloblastic leukemia (AML) characterized by rapid onset, hemiparesis, splenomegaly and infiltration of the marrow and spleen with myeloblasts. c-Myc protein level was comparable to the Eμ-Myc tumors. Disease latency was short (median survival=38 days). MSCV-Myc-induced tumors were oligoclonal, did not display significant cytogenetic abnormalities by SKY analysis, and were exclusively of myeloid origin. These tumors contained germline configuration of the Ink4A locus and wild type coding region of the p53, Arf and Ink4a. Also, the p53 target gene p21 in Myc expressing cells responded normally to gamma irradiation. These data demonstrate that c-Myc induced AML development without disrupting the Arf-p53 pathway and suggest that cooperating mutations were not required for the development of AML. In contrast, using the same retroviral system, lymphoid leukemia developed only if c-Myc and Bcl-2 were co-expressed or if Ink4a was mutated. Tumors from Bcl-2/c-Myc transplanted mice were comprised of a mixture of myeloid and lymphoid leukemic cells. Similarly, mice transplanted with c-Myc transduced Ink4a−/− bone marrow developed leukemia that displayed a mixed phenotype of myeloid and lympoid lineages. Lastly, factor independent methylcellulose colonies induced by Myc were exclusively of myeloid lineage suggesting as intrinsic difference between myeloid and lymphoid cells in response to Myc. Our data imply that dysregulation of c-Myc is sufficient to induce AML and provide direct evidence that Myc is an important downstream target in myeloid leukemogenesis.

Mechanism of Oncogenesis by Leukemia-Associated Mutants of Shp2 (PTPN11).

Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder of childhood that is principally characterized by proliferation of cells of the granulocytic and monocytic lineages. Previous studies showed that Ras or Nf1 mutations contribute to ~65% cases of JMML. More recently, mutations in the protein tyrosine phosphatase Shp2 (PTPN11) have been found in ~35% of sporadic JMML, and at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and childhood B-cell acute lymphoblastic leukemia (B-ALL). Interestingly, germline mutations of Shp2 cause the autosomal dominant genetic disorder Noonan Syndrome (NS) and NS patients may have an increased incidence of JMML. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and JMML mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activated. We have compared the biochemical and biological effects of NS and JMML mutations. When produced as recombinant proteins in bacteria, nearly all NS mutations studied showed increased PTP activity. However, the JMML mutations E76K and D61Y have the highest activity, resembling the fully active mutant E76A that we defined earlier. Retroviral-mediated expression of E76K or D61Y, but not wild type (WT) Shp2 in murine bone marrow (BM) cells results in cytokine-independent myeloid colony outgrowth, as well as hypersensitivity to both IL-3 and GM-CSF. Notably, NS- associated mutants (e.g., D61G or N308D) also could transform BM cells to factor-independence, but yielded far fewer colonies. Transformation by the leukemia-associated E76K mutant required the phosphatase activity and intact FLVRE motifs in the Shp2 SH2 domains. Gab2, a major Shp2 SH2 domain binding protein, also was required for transformation. When retrovirally transduced BM cells were transplanted into lethally irradiated recipients, E76K and D61Y but not WT Shp2 evoked fatal myeloproliferative disease (MPD) in 30–40% of recipients characterized by splenomegaly, leukocytosis, neutrophilia and monocytosis with occasional anemia and thrombocytopenia. Another ~20% showed less severe MPD at early stage and ultimately succumbed to T-ALL at later times. Histological analysis of mice dying MPD revealed BM and spleen packed with myeloid cells and livers with perivascular myeloid infiltrates. Flow cytometry on BM and spleen confirmed the presence of MPD. Moreover, BM from E76K and D61Y transplanted recipients exhibited enhanced factor-independent colony formation. Furthermore, mast cells derived from E76K and D61Y transplanted mice exhibit increased proliferation and enhanced activation of Erk, Akt and Stat5 in response to IL-3. In contrast, mice with a knock-in mutation of NS (D61G) displayed only a mild MPD. These data support a model in which leukemia-associated mutants of Shp2 are strong hypermorphs that enhance signaling via βc cytokines, whereas NS mutants are less severe gain of function alleles. Our results provide the first evidence that Shp2 mutants have a causal role in leukemogenesis and therefore, Shp2 is the first bona fide PTP proto-oncogene.

Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease.

Oncogenic ras alleles are among the most common mutations found in patients with acute myeloid leukemia (AML). Previously, the role of oncogenic ras in cancer was assessed in model systems overexpressing oncogenic ras from heterologous promoters. However, there is increasing evidence that subtle differences in gene dosage and regulation of gene expression from endogenous promoters play critical roles in cancer pathogenesis. We characterized the role of oncogenic K-ras expressed from its endogenous promoter in the hematopoietic system using a conditional allele and IFN-inducible, Cre-mediated recombination. Mice developed a completely penetrant myeloproliferative syndrome characterized by leukocytosis with normal maturation of myeloid lineage cells; myeloid hyperplasia in bone marrow; and extramedullary hematopoiesis in the spleen and liver. Flow cytometry confirmed the myeloproliferative phenotype. Genotypic and Western blot analysis demonstrated Cre-mediated excision and expression, respectively, of the oncogenic K-ras allele. Bone marrow cells formed growth factor-independent colonies in methylcellulose cultures, but the myeloproliferative disease was not transplantable into secondary recipients. Thus, oncogenic K-ras induces a myeloproliferative disorder but not AML, indicating that additional mutations are required for AML development. This model system will be useful for assessing the contribution of cooperating mutations in AML and testing ras inhibitors in vivo.

Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis.

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor-dependent myeloid precursor cells, STAT5 activation-deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor-independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor-independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation-defective BCR/ABL SH3+SH2 mutants to induce growth factor-independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor-independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras.

A hammerhead ribozyme directed against oncogenic N-ras (N13-ras) was introduced into a retroviral vector and its activity evaluated in vitro and in cell lines. The catalytic efficiency of the ribozyme embedded within a 2618 nucleotides in vitro-generated transcript was not significantly affected by the length of non-base pairing flanking sequences. A sensitive assay based on N-ras/luciferase fusion transcripts as a reporter system was used to assess ribozyme activity in mammalian cells. More than 95% reduction in luciferase activity was observed in cells transduced with a retrovirus containing the active form of the ribozyme, whereas no significant reduction was observed with the inactive form of the same ribozyme. In order to assay the activity of the retrovirally encoded ribozyme in a biological setting, the IL-3-dependent cell line TF-1 was transformed with N13-ras. Expression of N13-ras in these cells induced factor-independent colony growth and a dose-dependent proliferative response to erythropoietin (Epo). Retrovirus-mediated expression of the active form of the ribozyme in these cells restored factor-dependent colony growth and abolished the proliferative response to Epo. The reversion of the transformed phenotype correlated with a reduction in the amount of N13-ras mRNA.

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway.

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3-kinase (PI-3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI-3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI-3k activation failed to stimulate Akt kinase, a recently identified PI-3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI-3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI-3k specific inhibitor at low concentrations, suppressed BCR/ABL-dependent colony formation of murine marrow cells, and that a kinase-deficient Akt mutant with dominant-negative activity inhibited BCR/ABL-dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation-defective SH2 domain BCR/ABL mutants to induce growth factor-independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c-Myc and Bcl-2; in contrast, expression of a constitutively active Akt mutant induced Bcl-2 and c-Myc expression, and stimulated the transcription activation function of c-Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI-3k activation and document the essential role of the PI-3k/Akt pathway in BCR/ABL leukemogenesis.

Possible Mechanisms Accounting for the Growth Factor Independence of Hematopoietic Progenitors From Umbilical Cord Blood

AbstractHematopoietic progenitors obtained from the bone marrow of healthy adults fail to undergo clonogenic maturation in vitro if a source of hematopoietic growth factors is not included in the culture dishes. In contrast, a fraction of similarly purified progenitors obtained from umbilical cord blood undergo clonogenic maturation even in the absence of added growth factors. We postulated that production of hematopoietic growth factors within the culture dishes containing the progenitors of umbilical cord blood origin might be responsible. We postulated further, that this production might be by non-progenitor cells co-plated along with the progenitors, or alternatively by CD34+ cells themselves, or by cells clonally derived from CD34+ cells. To test these possibilities we first assessed the effect of including in the cultures neutralizing antibody directed against various growth factors. Inclusion of anti-granulocyte macrophage colony-stimulating factor (GM-CSF) and anti-interleukin-3 (IL-3) (but not anti-IL-2) significantly reduced the growth factor independence of cord blood progenitors (P < .005 and P < .01). Inclusion of both anti-GM-CSF and anti-IL-3 almost completely ablated the spontaneous colony growth (P < .001). Inclusion of IL-10 also reduced, in a concentration-dependent fashion, the spontaneous generation of umbilical cord blood-derived colonies. Transcripts for GM-CSF and IL-3 were detected, by reverse transcriptase-polymerase chain reaction (RT-PCR), in the CD34+ cells from cord blood and from adult marrow. When plated without added growth factors, however, the CD34+ cells of adult marrow origin failed to produce colonies, whereas 6% of cord blood CD34+ cells similarly cultured did so. When these growth factor independent colonies were plucked from culture, transcripts for GM-CSF and IL-3 were identified in all. We conclude that production of GM-CSF and IL-3 occurs within culture dishes containing hematopoietic progenitors of umbilical cord origin, and that this explains some of their apparently unique features of in vitro growth.

High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library

Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirus-producing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral beta-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-IL3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized high-efficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.

MURINE BONE MARROW LONG-TERM CULTURE AFTER INFECTION WITH V-ABL-CONTAINING RECOMBINED RETROVIRUS

Murine long-term bone marrow cultures that support myelocyte cell development were infected at initiation with a recombinant retrovirus containing v-abl and neo genes. Other cultures were uninfected controls. Harvested nonadherent cells were tested weekly for granulocyte macrophage colony formation (CFU-GM) , factor independent colony formation and anti-G418 CFU-GM. Cultures infected by virus with v-abl demonstrated a significant increase in cumulative nonadherent cell and CFU-GM in first 1 ~2 week cultures. There was anti-G418 CFU-GM, and factor independent colony formation in the supernatant nonadherent cells in the infected cultures. Cells from control cultures did not form anti-G418 CFU-GM and factor independent colonies. The data supported the conclusion that v-abl expression in long-term marrow cultures stimulated hematopoietic stem cell proliferation and differentiation. Key words: oncogene; recombined retrovirus; cell culture

Formation of Factor-Independent Hematopoietic Multilineage Colonies After Abelson Virus Infection

Yolk-sac-derived hematopoietic cells were infected with a helper-free stock of Abelson virus (A-MuLV). After infection, cells were plated in a clonogenic methylcellulose culture in the absence of exogenous growth factors such as interleukin 3 (IL 3) and erythropoietin (Epo). No colonies were observed in cultures without viral infection, whereas factor-independent colonies were consistently observed with virus-infected cultures. The number of colonies was linearly correlated with the number of cells plated. Erythroid-mix colonies consisting mostly of erythroblasts, macrophages, and mast cells could be observed. Tumorigenic, continuously growing mast cell lines could be generated at high frequency from these erythroid-mix colonies after they were initially passaged in the presence of an irradiated feeder layer for 4 to 8 weeks. Southern blot analysis of the DNA from five of these lines examined were all shown to contain integrated A-MuLV proviral DNA. These data are evidence that A-MuLV can directly infect embryonic multipotent hematopoietic progenitor cells and drive them to differentiate to various progeny cells without exogenous growth factors.

Formation of factor-independent hematopoietic multilineage colonies after Abelson virus infection

Yolk-sac-derived hematopoietic cells were infected with a helper-free stock of Abelson virus (A-MuLV). After infection, cells were plated in a clonogenic methylcellulose culture in the absence of exogenous growth factors such as interleukin 3 (IL 3) and erythropoietin (Epo). No colonies were observed in cultures without viral infection, whereas factor-independent colonies were consistently observed with virus- infected cultures. The number of colonies was linearly correlated with the number of cells plated. Erythroid-mix colonies consisting mostly of erythroblasts, macrophages, and mast cells could be observed. Tumorigenic, continuously growing mast cell lines could be generated at high frequency from these erythroid-mix colonies after they were initially passaged in the presence of an irradiated feeder layer for 4 to 8 weeks. Southern blot analysis of the DNA from five of these lines examined were all shown to contain integrated A-MuLV proviral DNA. These data are evidence that A-MuLV can directly infect embryonic multipotent hematopoietic progenitor cells and drive them to differentiate to various progeny cells without exogenous growth factors.

Transformation of single myeloid precursor cells by the malignant histiocytosis sarcoma virus (MHSV): generation of growth-factor-independent myeloid colonies and permanent cell lines.

Direct single-cell assays for oncogenic transformation are available for fibroblasts but not for other cell types. Using malignant histiocytosis sarcoma virus (MHSV), a member of the ras family of retroviruses, in vivo-infected granulocyte/macrophage and macrophage precursor cells lost the requirement for externally added hematopoietic growth factors. Factor-independent growth was demonstrated by colony-transfer experiments. More than 25% of the independent colonies were established as permanent macrophage cell lines following a phase of adaptation to tissue culture conditions. Factor-independent colony growth was also obtained by in vitro infection of single cells. As many as 50% of all myeloid precursor cells were target cells for MHSV as measured by this assay. About 2 x 10(-3) of these colony-forming cells acquired growth factor independence and immortality after in vitro infection. Cell lines derived from these colonies did not require adaptation to tissue culture conditions.