Brian Curtis Turner

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.

B-cell receptor signaling in the genesis and maintenance of B-cell lymphoma

Lymphomas constitute a number of different diseases that have been subdivided into two broad categories, Hodgkin’s and non-Hodgkin’s lymphoma (NHL). Hodgkin’s disease comprises a uniform set of malignancies primarily defined by the presence of Reed–Sternberg giant cells, whereas NHL is a heterogeneous set of clinical entities. The number of cases of NHL is almost tenfold that of Hodgkin’s disease [1]. In addition, the number of newly diagnosed cases of NHL has increased by almost 80% in the last 25 years. This dramatic increase does not correlate with age, gender or infectious agents, and cannot be accounted for by the onset of HIV-associated B-cell lymphomas [2]. As a result, NHL are currently the fifth most common form of cancer in the USA, after breast, prostate, lung and colon cancer [101]. NHL is one of the few cancers whose incidence and mortality rates have risen in the past 35 years. Despite the increase in the incidence of NHL, the etiology of these lymphomas remains elusive, and current therapeutic approaches rely on traditional, nonspecific chemotherapeutic approaches. The diagnosis of NHL encompasses different clinical entities. Approximately 85–90% of all NHL in the USA consist of B-cell lymphomas [3]. Among the B-cell NHLs, aggressive lymphomas account for 45–50% of new diagnoses. The two most common forms of aggressive NHL are diffuse large B-cell lymphoma and Burkitt’s lymphoma. These two types of malignancies involve neoplastic B cells that have a surface phenotype that is consistent with that of mature, activated B cells. Specifically, they express B-cell antigen receptors (BCR) on their surface, which contain mutations consistent with the process of affinity maturation during a germinal center reaction [4]. These cells also express other molecules that are normally expressed by postgerminal B cells [5]. There are other B-cell NHLs that have a similar cellular composition. These include follicular lymphomas, mucosal-associated lymphoid tissue lymphomas and mantle-cell lymphomas [3]. All of these B-cell NHL share a B-cell surface phenotype, including BCR expression. The nature of additional mutations, possibly involving oncogenes and tumor suppressor genes, has been postulated to explain their biological differences [6]. The one characteristic they have in common, namely BCR expression and evidence of antigen-dependent activation, may suggest that the BCR and an antigen may have an important role in the genesis of these tumors. Dameshek and Schwartz originally proposed that antigenic stimulus could contribute to the development of lymphomas nearly 50 years ago [7]. Throughout the years, a large amount of circumstantial evidence has implicated chronic inflammation in lymphomagenesis. There are prior hints that antigenic stimulus can play a role in lymphomagenesis. First, retroviral infection of mice elicits T-cell lymphomas only in those strains of mice that can mount an immune response to the virus [7,8]. Second, infection with Helicobacter pylori is an apparent cause of human lymphomas in mucosal-associated lymphoid tissue and gut-associated lymphoid tissue [9]. Treatment with antibiotics to eradicate infection elicits remission of these tumors, as if they might have been sustained by antigenic stimulus from the microbe [10,11]. Third, mice with graft versus host disease consequent to bone marrow transplantation frequently develop T-cell lymphomas; immunosuppression of the mice prevents the tumors [12]. Fourth, chronic antigenic stimulation by infection may contribute to the genesis of Burkitt’s lymphoma [13,14]. Fifth, the gene-expression profiles of diffuse large B-cell lymphomas resemble those of B cells that have mounted a response to antigen [15], and the tumor cells display high-affinity antigen receptors on their surface, as if they had been subjected to the selective pressure of an antigen [4,16–18]. Sixth, our own studies demonstrate a causal relationship between BCR-derived signals and elevated levels of MYC in the genesis of B-cell lymphomas in mice [19]. These findings prompt the hypothesis that an antigenic stimulus may cooperate with other tumorigenic influences in the genesis of lymphoma.

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.