Mutations in the catalytic subunit of class IA PI3K confer leukemogenic potential to hematopoietic cells.

Constitutive activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway is observed in up to 70% of acute myelogenous leukemia. To investigate the relevance of an intrinsic PI3K-AKT pathway activation in hematopoietic malignancies, we analysed the effect of point mutations in the catalytic (p110alpha) and regulatory (p85alpha) subunit of class IA PI3K. We demonstrated that mutations in the helical (E542K, E545A) and kinase domain (H1047R) of p110alpha constitutively activate the PI3K-AKT pathway and lead to factor-independent growth of early hematopoietic cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using either PI3K or AKT inhibitors. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not antiapoptotic signaling of mutant p110alpha. In a syngenic mouse model, hematopoietic cells expressing mutated p110alpha induced a leukemia-like disease characterized by anemia, neoplastic infiltration of hematopoietic organs and 90% mortality within 5 weeks, whereas activated mutants of the receptor tyrosine kinase c-KIT led to 100% mortality within 10 days. Our data show that point mutations in the p110alpha subunit of class IA PI3K confer factor independence to hematopoietic cells in vitro and leukemogenic potential in vivo, but have lower transforming activity than a deregulated class III receptor tyrosine kinase.

Long-term follow-up of chronic hepatitis C patients with sustained virological response to various forms of interferon-based anti-viral therapy.

BACKGROUND: Combination anti-viral therapy achieves a sustained virological response (defined as HCV-RNA negativity 6 months after the end of therapy) of 56% of patients with chronic hepatitis C. Little is known about long-term durability of HCV-RNA negativity in patient treated with pegylated interferon. AIM: To evaluate the durability of virologic response in patients with sustained virological response to anti-viral therapy treated at our centre. METHODS: A total of 187 sustained virological responses (50% genotype 1, 42% genotype 2 or 3 and 8% genotype 4; 20% with cirrhosis) with a follow-up of >12 months post-therapy were studied. Twelve patients received monotherapy with interferon-alpha2a or -2b. One hundred and seventy-five received combination therapy with ribavirin and standard interferon-alpha (n = 73) or pegylated interferon-alpha2a or 2b (n = 102). Qualitative serum HCV-RNA was tested by COBAS AMPLICOR HCV test, v2.0. RESULTS: Median follow-up time was 29 months (range 12-172). Recurrence of HCV infection was not observed in any of the 187 sustained virological responders. Alanine aminotransferase values were normal in 90% and two patients showed minimal elevation of alpha-fetoprotein levels. CONCLUSIONS: No recurrence of HCV infection was seen in any patient. Thus, long-term prognosis in chronic hepatitis C patients with a sustained virological response to therapy with pegylated interferon +/- ribavirin is promising, but long-term studies need to continue.

Downregulation of c-kit (stem cell factor receptor) in transformed hematopoietic precursor cells by stroma cells.

We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.

Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line.

Retroviral insertion mutagenesis has been used extensively in vivo but not in vitro to induce and identify critical mutations during oncogenic progression and differentiation. We have developed a tissue culture system using the human, growth factor-dependent, hematopoietic precursor cell line TF-1 that permits the use of retroviral vectors to induce a large (up to 28-fold) increase in the mutation frequency to growth factor independence and thus the isolation of many mutants. The mutation frequency, as expected, is directly proportional to the number of retroviral insertions (2.2 x 10(-7) mutants per insertion). The mutant phenotypes can be subdivided into mutants that release growth factors and those that do not ("autonomous" mutants). The majority of growth factor-producing mutants release an unidentified ligand. A subset of the autonomous mutants shows alterations in expression of the alpha subunit of either the GM-CSF or the IL-3 receptor. One mutant expresses neither GM-CSF nor IL-3 alpha receptor chains, thus showing coordinate regulation of the alpha receptor subunits.

Autocrine stimulation after transfer of the granulocyte/macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway.

Autocrine stimulation of cells by aberrant synthesis of growth factor may lead to malignant transformation, either as a direct consequence of endogenous factor production or as a first step of a series of successive events. Introduction of the granulocyte/macrophage colony-stimulating factor (GM-CSF) cDNA clone into a vector based on the myeloproliferative sarcoma virus allowed efficient transfer and expression of GM-CSF in factor-dependent myeloid cell lines (FDC-P1 and FDC-P2). Factor-independent growth was acquired when the vector was introduced into the GM-CSF-responsive FDC-P1 cell line but not the multi-CSF-dependent FDC-P2 line. Nonlinear clonability in the absence of exogenous growth factor and growth inhibition by GM-CSF antiserum support a model of autocrine stimulation that requires interaction of factor and receptor at the outer membrane. However, many, but not all, infected FDC-P1 cells acquired subsequently a second mutation that abrogated the requirement of GM-CSF secretion and external interaction. The nature of the second step, which presumably leads to tumorigenicity of these cells, is not well understood, but its frequency could be correlated with the level of GM-CSF released by an individual cell clone.

Point mutations in the U3 region of the long terminal repeat of Moloney murine leukemia virus determine disease specificity of the myeloproliferative sarcoma virus.

The myeloproliferative sarcoma virus (MPSV) is made up entirely of sequences derived from the Moloney murine leukemia virus (Mo-MuLV) and the cellular mos oncogene. As other members of the Moloney murine sarcoma virus (Mo-MuSV) family, MPSV transforms fibroblasts in vitro and causes sarcomas in vivo. In addition, however, MPSV also causes an acute myeloproliferative disease in adult mice. The mos oncogene is essential for its transforming capacity, but sequences specific to the long terminal repeat (LTR) U3 region of MPSV account for its expanded target specificity as compared to Mo-MuSV (C. Stocking, R. Kollek, U. Bergholz, and W. Ostertag, Proc. Natl. Acad. Sci. USA 82, 5746-5750 (1985)). The U3 region of the LTR of MPSV is, however, closely related to that of the Mo-MuLV, and it appeared likely that the difference between MPSV and Mo-MuSV was caused by a divergent evolution of Mo-MuSV LTRs. In this paper, we show that this is not the case. The few nucleotide differences in the LTR between Mo-MuLV and MPSV are crucial for the expanded host range of MPSV. Moreover, Mo-MuLV-related gag sequences retained in MPSV are not essential for the distinctive biological properties of MPSV.

The myeloproliferative sarcoma virus retains transforming functions after introduction of a dominant selectable marker gene.

The dominant neomycin resistance gene (neoR) was introduced into the genome of the myeloproliferative sarcoma virus (MPSV), a replication-defective retrovirus carrying the mos oncogene. The resulting selectable neoR-MPSV virus did not lose its acute transforming property, unlike the results of attempts by other groups to insert marker genes into oncogenic viruses. NeoR-MPSV DNA was used to generate infectious virus by transfection followed by rescue with Friend or Moloney murine leukaemia virus. Infection of fibroblasts with this virus resulted in morphologically transformed cells which were resistant to the neomycin analogue G418. Segregation of the two functions (transformation and G418 resistance) was not observed in more than 500 independent viral transfers to fibroblasts. Furthermore, neoR-MPSV retained the leukaemogenesis-inducing properties of the wild-type virus. Myeloproliferation and G418-resistance transfer did not segregate after passage in mice.

Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.

The myeloproliferative sarcoma virus not only transforms fibroblasts but also causes extensive expansion of the hematopoietic stem cell compartment on infection of adult mice. Similar to the Moloney sarcoma virus, it carries the mos oncogene. Moloney sarcoma virus, however, does not induce myeloproliferation and leukemia in adult mice. The difference between the two viruses was explored by using their molecularly cloned genomes and the cellular mos oncogene to construct recombinant genomes. It was shown that the U3 region of the viral long terminal repeat (LTR) has a decisive function in determining the target cell specificity of the myeloproliferative sarcoma virus. Any mos gene, whether of cellular or viral origin, is sufficient in conjunction with the proper LTR to induce myeloproliferation. Our results indicate that the pathogenicity of acutely transforming viruses is determined not only by the oncogene but also by sequences in the viral LTR.