Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

Mouse models of non-Hodgkin lymphoma reveal Syk as an important therapeutic target.

We have generated mouse models of non-Hodgkin lymphoma (NHL) that rely on the cooperation between MYC overexpression and B-cell antigen receptor (BCR) signaling for the initiation and maintenance of B-cell lymphomas. Using these mouse models of NHL, we have focused on the identification of BCR-derived signal effectors that are important for the maintenance of NHL tumors. In the present study, we concentrate on Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase required to transduce BCR-dependent signals. Using a genetic approach, we showed that Syk expression is required for the survival of murine NHL-like tumors in vitro and that tumor cells deficient in Syk fail to expand in vivo. In addition, a pharmacologic inhibitor of Syk was able to induce apoptosis of transformed B cells in vitro and led to tumor regression in vivo. Finally, we show that genetic or pharmacologic inhibition of Syk activity in human NHL cell lines are generally consistent with results found in the mouse models, suggesting that targeting Syk may be a viable therapeutic strategy.

The B cell antigen receptor and overexpression of MYC can cooperate in the genesis of B cell lymphomas.

A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR) in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL), whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL). We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.

Small-molecule inhibitors of Bcl-2 family proteins are able to induce tumor regression in a mouse model of pre-B-cell acute lymphocytic lymphoma.

The overexpression of prosurvival members of the Bcl-2 family is commonly associated with the enhanced malignancy of hematological tumors. There has been great interest in a novel set of agents that are able to mimic the function of the BH3 domain by binding to the groove of Bcl-2-like proteins and initiating the cell death sequence. We sought to examine the efficacy of BH3 mimetics in a spontaneous mouse model of B-cell neoplasia. We evaluated the ability of the BH3 mimetics to preferentially target tumor cells while sparing normal cells. In addition, we examined the contributions of Bim and Puma to the sensitivity of tumor cells to the BH3 mimetics. We report here that two BH3 mimetics (HA-14-1 and BH3-I-2') were able to induce apoptosis of murine B-cell lymphoma cells in vitro and in vivo. Tumors that arose from transplantation of primary lymphoma cells regressed following 7 days of treatment with BH3-mimetic drugs. The long-term benefits of the transient treatment of tumor-bearing mice with the BH3 mimetics, however, could not be properly evaluated, due to the high levels of toxicity we observed in vivo with these drugs. Decreased expression of either Bim or Puma from B-cell tumor cells was able to protect these cells from the apoptosis induced by these BH3 mimetics, suggesting that they function through other means. We conclude that while the BH3-mimetic drugs are effective at inducing cell death of lymphoma cells in vitro and in vivo, their unclear molecular specificity and their ability to kill normal cells may limit their therapeutic uses in humans.

The protooncogene MYC can break B cell tolerance.

The protooncogene MYC has been implicated in both the proliferation and programmed cell death of lymphoid cells, and in the genesis of lymphoid tumors. Here, we report that overexpression of MYC, as found in many lymphomas, can break immune tolerance. Mice that would otherwise be tolerant to a transgenic autoantigen mounted an immune response to the antigen if MYC was vigorously expressed in the B cell lineage. The responsive B cells converted to an activated phenotype and produced copious amounts of autoantibody that engendered immune complex disease of the kidney. MYC was required to both establish and maintain the breach of tolerance. These effects may be due to the ability of MYC to serve as a surrogate for cytokines. We found that the gene could mimic the effects of cytokines on both B cell proliferation and survival and, indeed, was required for those effects. These findings demonstrate a critical role for MYC in the response of B cells to antigen and expand the potential contributions of MYC to the genesis of lymphomas.

Substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine CEACAM1a by the murine coronavirus MHV-A59.

The host range of the murine coronavirus (MHV) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (S) with its receptor, mCEACAM1a. We identified five residues in S (S33, L79, T82, Y162 and K183) that are conserved in the receptor-binding domain of MHV strains, but not in related coronaviruses. We used targeted RNA recombination to generate isogenic viruses that differ from MHV-A59 by amino acid substitutions in S. Viruses with S33R and K183R substitutions had wild type growth, while L79A/T82A viruses formed small plaques. Viruses with S33G, L79M/T82M or K183G substitutions could only be recovered from cells that over-expressed a mutant mCEACAM1a. Viruses with Y162H or Y162Q substitutions were never recovered, while Y162A viruses formed minute plaques. However, viruses with Y162F substitutions had wild type growth, suggesting that Y162 may comprise part of a hydrophobic domain that contacts the MHV-binding site of mCEACAM1a.

Receptor-dependent coronavirus infection of dendritic cells.

In several mammalian species, including humans, coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in murine coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the murine coronavirus receptor, murine CEACAM1a, are susceptible to murine coronavirus infection by a receptor-dependent pathway.