Human FANCC is hypomorphic in murine Fancc-deficient cells.

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

TAT-mediated intracellular delivery of NPM-derived peptide induces apoptosis in leukemic cells and suppresses leukemogenesis in mice.

Nucleophosmin (NPM) is frequently overexpressed in leukemias and other tumors. NPM has been reported to suppress oncogene-induced senescence and apoptosis and may represent a therapeutic target for cancer. We fused a NPM-derived peptide to the HIV-TAT (TAT-NPMDeltaC) and found that the fusion peptide inhibited proliferation and induced apoptotic death of primary fibroblasts and preleukemic stem cells. TAT-NPMDeltaC down-regulated several NF-kappaB-controlled survival and inflammatory proteins and suppressed NF-kappaB-driven reporter gene activities. Using an inflammation-associated leukemia model, we demonstrate that TAT-NPMDeltaC induced proliferative suppression and apoptosis of preleukemic stem cells and significantly delayed leukemic development in mice. Mechanistically, TAT-NPMDeltaC associated with wild-type NPM proteins and formed complexes with endogenous NPM and p65 at promoters of several antiapoptotic and inflammatory genes and abrogated their transactivation by NF-kappaB in leu-kemic cells. Thus, TAT-delivered NPM peptide may provide a novel therapy for inflammation-associated tumors that require NF-kappaB signaling for survival.

Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells.

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G(1)-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.

Impaired type I IFN-induced Jak/STAT signaling in FA-C cells and abnormal CD4+ Th cell subsets in Fancc-/- mice.

The Fanconi anemia (FA) group C protein, FANCC, interacts with STAT1 following stimulation with IFN-gamma and is required for proper docking of STAT1 at the IFN-gamma receptor alpha-chain (IFN-gammaRalpha, IFN-gammaR1). Consequently, loss of a functional FANCC results in decreased activation of STAT1 following IFN-gamma stimulation. Because type I IFN receptors influence the function of type II receptors, and vice versa, we conducted experiments designed to determine whether type I IFN-induced activation of other STAT proteins is compromised in FA-C cells and found that activation of STAT 1, 3, and 5 is diminished in type I IFN-stimulated cells bearing Fancc-inactivating mutations. We also determined that the reduced activation of STATs was accompanied by significant reduction of type I IFN-induced tyrosine kinase 2 and Jak1 phosphorylation. Because tyrosine kinase 2 plays a role in differentiation of Th cells, we quantified cytokine secretion from CD4+ cells and in vitro generated CD4+ Th cell subsets from splenocytes of Fancc null mice to that of heterozygous mice and discovered reduced CD4+ IFN-gamma secretion in the Fancc-/- mouse, indicating impaired Th1 differentiation. We suggest that Fancc mutations result in a subtle immunological defect owing to the failure of FANCC to normally support Jak/STAT signaling.

The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded RNA-dependent protein kinase-mediated death signaling pathway requires the Fanconi anemia protein, FANCC.

Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents. A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR). Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex. By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR both in vivo and in vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins. Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR. We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function.

Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR.

In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.