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Question: What is the molecular basis of the interaction between antigen receptor signaling and dysregulated oncogenes?
Answer: Understanding the mechanistic details that underlie the interaction between antigen receptor signals and dysregulated oncogenes may allow us to identify novel targets for the development of therapeutics for Non-Hodgkin's lymphomas. We will address this issue by answering five interrelated questions.
Question: Can other oncogenes also cooperate with chronic BCR signals?
Answer: The experiments that the overexpression of MYC in the context of chronic BCR signals yielded a Burkitt's-like (BL) tumor. Those neoplasms usually carry a translocation that juxtaposes the MYC locus to the Ig heavy or light chain genes (t8:14, or t8:22). We will test whether other oncogenes that have been identified in chromosomal translocations involving the Ig loci can also cooperate with continuous BCR signaling. These genes will be selected based on their ability to affect either cell proliferation (e.g., Bcl-1/CyclinD1), cell survival (e.g. Bcl-3/IkB-like), or both (e.g. Rel-NRG, or abl). The hope here is to broaden the conclusion and establish chronic BCR signaling as a common cooperating event in lymphoid neoplasia.
Question: What is the nature of the signals derived from the BCR that cooperate with elevated levels of MYC?
Answer: Our Burkitt's-like mouse model also showed that commonly used immunosuppressive drugs were able to inhibit tumor formation, and lead to tumor regression. We will first confirm that the known protein targets of these drugs (e.g., Calcineurin for CsA and FK506, and mTOR for Rapamycin) in T cells are being affected in the transformed B cells in the BL-like tumors. Once these known targets are confirmed, we plan to construct retroviral vectors that encode dominant negative forms of these proteins, in bicistronic vectors that also express GFP. The idea is to retrovirally transduce tumor cells in vitro and transplant these into syngeneic mice. The ability of these cells to compete with their non-transduced counterparts will be used as a measure of the effect of these molecules to block the BCR signals that cooperate with MYC. In addition, we plan to analyze the effect of altering the activities of molecules that are acting upstream, or downstream of these known targets. This initial set of experiments will indicate whether the molecules in question are necessary for the BCR dependent signal to cooperate with MYC. One additional set of experiments we plan to pursue is to generate retroviruses that express constitutively active forms of the signaling molecules that appear to be necessary for the BCR dependent signal that cooperates with MYC. These viruses will be used to transduce B cells from Eµ—MYC mice that express a transgenic BCR on their surface, and transplant these into syngeneic mice. These experiments aim to define whether the molecules in question are sufficient to mediate the BCR signal that cooperates with MYC in lymphomagenesis. Along these lines, and in collaboration with Dr. Kenneth Field, we are currently testing the ability of dominant negative and constitutive active forms of small GTP-binding proteins (Ras, Rho, Rap) in BCR dependent oncogenesis. We also plan to examine whether these molecules can affect the proliferation or survival of cell lines derived from human Burkitt's lymphoma tumors. One additional aspect of this set of experiments is the role of costimulatory and negative regulators of antigen receptor signaling. We plan to initially use inhibitory antibodies to cell surface molecules that participate in antigen receptor signaling (e.g. CD28 for T cells, and CD23 for B cells), or to negative regulators of the antigen receptor signals (e.g. CTLA4 for T cells, and FC?RII for B cells). The initial screen with antibodies will be followed up by genetic experiments involving the breeding of knockout alleles to our triple transgenic combination. The hope here is to identify specific signaling mediators that mediate the functional cooperation of BCR dependent signals with MYC. There is a chance that many molecules may qualify for this category, which broadens the number of potential targets we can further pursue for the development of small molecule inhibitors.
Question: Studies on the biochemical nature of tonic BCR signals that cooperate with elevated levels of MYC.
Answer: The nature of the signals that are involved in tonic signaling by the BCR is a largely understudied area in immunology, but one which may prove to be of great potential for the identification of drug targets for lymphoid tumors. We plan to use some of the same approaches described earlier for the analysis of the cognate antigen triggered signals involved in the tumorigenic cooperation with MYC, with two important differences. We plan to develop lentiviral constructs to transduce naive, resting B cells. This method of genetic manipulation will allow us to study the signaling requirements for naive B cell survival and maintenance of quiescence in vivo. These studies may also unveil potential molecules that can prevent the onset of cognate antigen triggered B cell activation.
Question: Does the interaction between a dysregulated oncogene and chronic antigen receptor signaling also occur in T cells?
Answer: One prediction from our results in B cells is that a similar interaction may be important for the development of T cell tumors. We are currently introducing T cell receptor (TCR) transgenes into a tetracycline-regulated MYC background. In analogy to the experiments we have pursued in the B cell compartment, the TCR transgene will fix the antigenic specificity of the majority of the T cells in these mice, and render them susceptible to stimulation by a known antigen. The antigen can be introduced by immunization, or transgenesis. The biological differences between T and B cells may turn out to be important for determining the parameters for an interaction with dysregulated oncogenes. These differences can potentially unveil some important differences in the nature of the molecules needed to target in the two compartments.
Question: Does the timing of oncogene dysregulation alter the nature of the tumor?
Answer: There are many B cell malignancies that appear to be antigen dependent (e.g. DLBCL, follicular B cell lymphoma, mantle cell lymphoma, etc). In order to test whether the key difference among these tumors is the time at which the oncogenes become dysregulated, We plan to breed the BCR-HEL and s-HEL transgenes onto a tetracytline-regulated background, that over expresses MYC in a B cell specific manner. These mice will be useful for determining whether the timing at which MYC is overexpressed alters the nature of the tumors obtained. They are also useful for determining whether the overexpression of MYC is required for maintaining these tumors, or alternatively, study the details underlying tumor regression.
Question: What is the nature of the signals that mediate the proliferation of lymphocytes by MYC, downstream of cytokine receptors?
Answer: Understanding the biochemical pathway by which cytokine receptors result in the regulation of MYC function may allow us to establish functional associations between MYC and other cellular protoconcogenes. Such functional interactions may provide some insights into treatment modalities for tumors with associated mutations.
Question: What is the nature of the genes that are transcriptionally regulated by MYC in lymphocytes?
Answer: We will compare the transcriptional profiles of primary lymphocytes that will be obtained from either mice that overexpress MYC in a tetracytcline-regulated fashion, or can delete their MYC loci by Cre/LoxP directed methods. These two sets of data may allow us to gain some perspective of the nature of the genes involved in MYC induced lymphocyte proliferation and survival. Candidate genes will be tested for their ability to replace MYC, in their constitutively active form, by transducing these genes into T or B cells obtained from mice in which the MYC loci is flanked by loxP sites, and can inducibly express Cre (Mx-Cre transgenic). If the genes are able to replace the proliferative or survival function of MYC, the transduced lymphocytes should behave different than their non-transduced counterparts. In addition, the transduction of T or B cells from tetracycline-regulated MYC overexpressing mice with dominant negative forms of these candidate genes may allow us to confirm their involvement in the MYC dependent proliferative and survival pathways. This system may also allow us to test whether these proto-oncogenes can lead to the development of tumors in a MYC independent fashion in vivo.
Question: Does MYC regulate proliferation and survival in IL-2 family cytokines, or in a wide range on growth factors?
Answer: We are interested in determining whether MYC is a key determinant of proliferation and survival in different cytokines. This is important to determine in the context of rendering transformed cells cytokine independent in the process of tumorigenesis. In collaboration with Dr. Warren Pear, Dr. Refaeli has shown that MYC is a critical regulator of Notch-1 dependent proliferation and survival in T cells, and is a negative regulator of Notch-1 dependent apoptosis in B cells. Further work in this area will attempt to determine if the molecular signals linking MYC to various cytokine receptors is common across different receptor families.
Question: What is the role of host-inflammatory pathways in the establishment and maintenance of lymphoid malignancies?
Answer: Dr. Refaeli's work has shown that IFN-g is important for the development of tumor associated clinical signs in our BL-like tumor model. In addition, Dr. Refaeli has shown that IRF-1, a transcription factor that regulates IFN-type I (a/b) expression, is critical for the engraftment of BL-like tumors in vivo. We plan to further explore the requirements of inflammation for the initiation, establishment and maintenance of lymphoid malignancies in vivo. We have obtained mice in which the genes encoding the type I IFN receptor (INF a/b Receptor b), or type II IFN receptor (IFN g Receptor b) have been disrupted. We plan to breed these mutations onto our triple transgenic combination to further dissect the role or inflammatory mediators in lymphomagenesis. In addition, we plan to compare these experiments to tumor transplantation experiments. Along these lines, we have also used Cox-2 inhibitory drugs to explore the role of the downstream effectors of inflammation in tumor engraftment. The preliminary experiments Dr. Refaeli has conducted show an important role for Cox-2 in the engraftment of BL like tumors in vivo. We plan to further pursue this line of experiments with Cox-1 and Cox-2 deficient mice. In addition, the downstream targets of Cox-2 include VEGF. A potential relationship between inflammation and angiogenic events may turn out to be an important source of new targets for the prophylactic treatment of lymphoid tumors.
Future Directions
Question: Studies on the biochemical nature of tonic BCR signals that cooperate with elevated levels of MYC.
Answer: The nature of the signals that are involved in tonic signaling by the BCR is a largely understudied area in immunology, but one which may prove to be of great potential for the identification of drug targets for lymphoid tumors. We plan to use some of the same approaches described earlier for the analysis of the cognate antigen triggered signals involved in the tumorigenic cooperation with MYC, with two important differences. We plan to develop lentiviral constructs to transduce naive, resting B cells. This method of genetic manipulation will allow us to study the signaling requirements for naive B cell survival and maintenance of quiescence in vivo. These studies may also unveil potential molecules that can prevent the onset of cognate antigen triggered B cell activation.
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