Scientific Resources

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia,bone marrow (BM) erythroblastopenia,and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however,the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis,we compared highly purified multipotential,erythroid,and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes,such as TNFRSF10B and FAS,transcriptional control genes,including the erythropoietic transcription factor MYB (encoding c-myb),and translational genes were greatly dysregulated,mostly in diseased erythroid cells. Cancer-related genes,including RAS family oncogenes and tumor suppressor genes,were significantly dysregulated in all diseased progenitors. In addition,our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes,as well as downregulation of genes involved in translation in DBA cells. In conclusion,the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression,which causes complete failure of fetal liver erythropoiesis in knockout mice,suggests a link between RPS19 mutations and reduced erythropoiesis in DBA. View Publication

Somatic activation of a conditional targeted Kras(G12D) allele induces a fatal myeloproliferative disease in mice that closely models juvenile and chronic myelomonocytic leukemia. These mice consistently develop severe and progressive anemia despite adequate numbers of clonogenic erythroid progenitors in the bone marrow and expanded splenic hematopoiesis. Ineffective erythropoiesis is characterized by impaired differentiation. These results demonstrate that endogenous levels of oncogenic Ras have cell lineage-specific effects and support efforts to modulate Ras signaling for therapy of anemia in patients with myelodysplastic syndromes and myeloproliferative disorders. View Publication

Most patients with acute promyelocytic leukemia (APL) express PML-RAR alpha,the fusion product of t(15;17)(q22;q11.2). Transgenic mice expressing PML-RAR alpha develop APL with long latency,low penetrance,and acquired cytogenetic abnormalities. Based on observations that 4% to 10% of APL patients harbor oncogenic ras mutations,we coexpressed oncogenic K-ras from its endogenous promoter with PML-RAR alpha to generate a short-latency,highly penetrant mouse model of APL. The APL disease was characterized by splenomegaly,leukocytosis,extramedullary hematopoiesis (EMH) in spleen and liver with an increased proportion of immature myeloperoxidase-expressing myeloid forms; transplantability to secondary recipients; and lack of cytogenetic abnormalities. Bone marrow cells showed enhanced self-renewal in vitro. This model establishes a role for oncogenic ras in leukemia pathogenesis and thus validates the oncogenic RAS signaling pathway as a potential target for therapeutic inhibition in leukemia patients. This mouse model should be useful for investigating signaling pathways that promote self-renewal in APL and for testing the in vivo efficacy of RAS signaling pathway inhibitors in conjunction with other targeted therapies such as ATRA (all trans retinoic acid) and arsenic trioxide. View Publication

Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations textgreater or =5 microM rapidly (i.e.,within 4 h) induced cytochrome c release,Bax mitochondrial translocation,and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction analysis revealed pronounced down-regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore,cotreatment with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked SU9516-mediated Mcl-1 down-regulation,implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome c release,Bax translocation,and apoptosis,whereas knockdown of Mcl-1 by small interfering RNA potentiated SU9516 lethality,confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production,which was diminished,along with cell death,by the free radical scavenger N-acetylcysteine (NAC). We were surprised to find that NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2,reductions in Mcl-1 mRNA levels,and Mcl-1 down-regulation. Together,these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level,culminating in mitochondrial injury and cell death. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders,we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months,mice developed polycythemia,macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months,polycythemia regressed,abnormally shaped red blood cells and platelets were seen in circulation,and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia,thrombocytopenia,high neutrophilia,and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. View Publication

Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha,a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation,inhibited proliferation in vitro and in mice,and suppressed leukemogenesis. Moreover,activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells,respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis,as indicated by the effects of p42 C/EBPalpha,which were more potent than those of K298E C/EBPalpha,a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC,including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation,also induced granulocyte differentiation. Thus,these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors,and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. View Publication

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers,although antitumor responses are often heterogeneous,and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug,we studied the action of imatinib (STI-571,Gleevec) against murine cells and leukemias expressing BCR-ABL,an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53,which can accompany disease progression in human CML,impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly,p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution. View Publication