• Identifying Myc target genes in cell types corresponding to naturally occurring tumors whose pathogeneses include Myc overexpression, and then
• Establishing the functional significance of these target genes for tumorigenesis in vivo.

Our effort began with the discovery that Myc overexpression leads to down-regulation of thrombospondin-1, a secreted glycoprotein ( Journal of Biological Chemistry, 1996). Surprisingly, this down-regulation is based on the shortening of the thrombospondin-1 mRNA half-life, not promoter inactivation ( Nucleic Acids Research, 2000). Since thrombospondin-1 was known to inhibit angiogenesis, we hypothesized that by reducing its levels, Myc can promote the ingrowth of blood vessels. At that point, the role of Myc in tumor angiogenesis was not recognized. To put our hypothesis to a test, we demonstrated that transient overexpression of Myc indeed confers upon rodent fibroblasts the angiogenic phenotype ( Cell Growth & Differentiation, 2000). The effects of Myc on neovascularization were independently observed by several Myc laboratories, and recently it has been shown that down-regulation of thrombospondin-1 by Myc underlies the pro-angiogenic activity of Ras, another potent oncoprotein. The question still remained whether Myc can trigger the angiogenic switch in genetically complex tumors.

Myc promotes angiogenesis in murine colon carcinomas. To assess the angiogenic effects of Myc in a bona fide tumor context, we established a new mouse model of colon cancer. In this model, primary colonocytes deficient in the p53 tumor suppressor are sequentially transformed by Ras and Myc oncoproteins. We discovered that Ras/p53-null cells are very weakly angiogenic on their own. This was surprising, since mutations in both Ras and p53 had been reported to promote vascularization. However, in our system microvascular densities and overall growth rates became robust only after overexpression of Myc ( Dews et al, manuscript submitted). Interestingly, the pro-angiogenic effects of Myc correlated with down-regulation of several proteins with thrombospondin-1 type repeats (TSR), including thrombospondin-1 itself and clusterin. The latter was only recently assigned to the TSR superfamily and its function is still not firmly established. We discovered that down-regulation of clusterin by Myc boosts both proliferation and neovascularization of primary colonocytes. In addition, clusterin attenuates neoplastic transformation of epithelial cells during skin carcinogenesis ( Cancer Research, 2004). These findings demonstrate how Myc shapes the angiogenic phenotype of solid tumors. However, leukemias and lymphomas, whose reliance on blood vasculature is less obvious, are also known to overexpress Myc. This poses a question: what are crucial Myc targets in hematopoietic cells?

Myc regulates B-cell differentiation markers of therapeutic importance. To determine the contribution of Myc to hematological malignancies, we developed another non-transgenic mouse model based on transduction of p53-null bone marrow cells with Myc-encoding retroviruses. We demonstrated that overexpression of Myc combined with inactivation of p53 suffices for B-lymphomagenesis ( Oncogene, 2002). We also determined that some Myc- transformed cells possess dual B-myeloid potential and differentiate into macrophage-like cells following spontaneous down-regulation of the Pax5 transcription factor ( Blood, 2003). Interestingly, Pax5 appears to be an oncogene in its own right and cooperates with Myc in many non-Hodgkin lymphomas. Moreover, the propensity of some of our cell lines to silence Pax5 afforded a unique opportunity to study, in collaboration with other laboratories, the role of Pax5 in B-cell differentiation. One such study co-authored by Kathryn Calame’s ( Columbia), David Schatz’s (HHMI-Yale) and our laboratories, was published last year ( Nature Immunology, 2004). In that paper, we established that Pax5 controls commitment to the B cell lineage via the loss of histone 3 methylation in the V(H) immunoglobulin locus.

To gain a better understanding of the role of Myc in lymphomagenesis, we used a conditional mutant of Myc (MycER), which requires the presence of a synthetic estrogen for its activity. MycER/p53-null lymphomas were generated, and the role of Myc in tumor sustenance was studied using estrogen-deprivation. We discovered that inactivation of Myc does not cause overt tumor regression, as observed in one-hit transgenic systems. Instead, it merely suppresses cell cycle progression, leading to stasis and eventual relapse. However, the hallmark of surviving Myc OFF cells is overexpression of the interleukin-10 receptor and CD20, two well-known therapeutic targets. Thus, targeting Myc, while moderately effective on its own, shapes the phenotype of quiescent neoplastic cells and sensitizes them to other molecular therapies ( Cancer Research, 2005).

This experimental system proved to be very useful in the analysis of other Myc targets first identified in cultured cells. Already it has spawned numerous collaborations and joint publications with investigators interested in gene regulation by Myc, for example Steven McMahon (Wistar Institute) (revised manuscript in preparation) and Wafik el-Deiry (PENN) ( Molecular & Cellular Biology, 2004). In these two studies, the MycER system allowed us to establish the role of Myc as a major regulator of the metastasis-associated protein 1 and of resistance to TNF-related apoptosis-inducing ligand (TRAIL), respectively.

Myc targets and anti-tumor surveillance. Our studies on Myc-induced lymphomas led to the conclusion that Myc profoundly down-regulates responses to several tumor-suppressive cytokines, including interferon gamma (IFN g). This prompted us to investigate the mechanisms underlying anti-neoplastic effects of IFN g and to re-assess its therapeutic potential. We discovered that production of retrovirally encoded ( Cancer Letters, 2001) or infection-induced interferon gamma strongly inhibits tumor angiogenesis and/or neoplastic growth ( Cancer Biology & Therapy, 2003). Importantly, it is this inhibition of angiogenesis, not bystander immunity, that underlies well-documented resistance to tumors during infection. This became apparent when we observed tumor resistance in acutely infected mice lacking major cytotoxic responses, for instance T- and tumoricidal NK-cells ( Journal of Immunology, 2001). Our discovery represented a major development in the field of tumor surveillance and was featured in numerous commentaries (Lancet, Drug Discovery Today, ScientificAmerican.com, etc). One provocative corollary of our work is that anti-tumor vaccines should be evaluated not only for the ability to elicit cytotoxic immunity but also for their anti-angiogenic effects.

In the future, we will continue to develop and refine in vivo mouse models that recapitulate genetic defects in naturally occurring human neoplasms. Our overall hypothesis is that in order to be clinically beneficial, inactivation of Myc would have to be combined with adjuvant molecular therapies. With this in mind, we hope to exploit biologically significant targets of Myc (such as thrombospondin-1 and interleukin-10 receptor) for the development of new cancer therapeutics.

2.   C.Ngo, M.Gee, N.Akhtar, D.Yu, O.Volpert, R.Auerbach and A.Thomas-Tikhonenko (2000) "An in vivo function for the transforming myc protein: elicitation of the angiogenic phenotype", Cell Growth & Diff, 11:201-210

3.   A.Janz, C. Sevignani,, K.Kenyon, C.Ngo and A.Thomas-Tikhonenko (2000) "Activation of the Myc oncoprotein leads to increased turnover of the thrombospondin-1 mRNA", Nucl Acids Res, 28:2268-2275

4.   C.Hunter, D.Yu, M.Gee, C.Ngo, C.Sevignani, M.Goldschmidt, T.Golovkina, S.Evans, W.M.Lee, and A.Thomas-Tikhonenko (2001) "Cutting edge: systemic inhibition of angiogenesis underlies resistance to tumors during acute toxoplasmosis", J Immunol., 166: 5878-5881

5.   D.Yu and A.Thomas-Tikhonenko (2001) "Intratumoral delivery of an interferon gamma retrovirus-producing cells inhibits growth of a murine melanoma by a non-immune mechanism", Cancer Lett, 173:145-154

6.   D.Yu and A.Thomas-Tikhonenko (2002) "A non-transgenic mouse model for B-lymphoma: in vivo infection of p53-null bone marrow progenitors by a Myc-encoding retrovirus", Oncogene, 21:1922-1927

7.   A.Thomas-Tikhonenko (2002) “Poisoning the messengers: could tumor endothelial cells acquire drug resistance?” Cancer Biol & Therapy, 1(3):266-267

8.   D.Yu, D.Allman, J.G.Monroe, M.H.Goldschmidt, M.L. Atchison, and A.Thomas-Tikhonenko (2003) "Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/reactivation of the EBF/Pax5 pathway”, Blood, 101:1950-1955

9.   A.Thomas-Tikhonenko and C.Hunter (2003) “Infection and cancer: the common vein”, Cytokines & Growth Factor Rev, 14(1):67-77      

10.   E.B.Rankin, D.Yu, J.Jiang, H.Shen, E.Pearce, M.Goldschmidt, D.Levy, T.Golovkina, C.Hunter, and A.Thomas-Tikhonenko (2003) "An important role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth", Cancer Biol & Ther, 2:687-693

11.   A.Thomas-Tikhonenko, I.Viard-Leveugle, M.Dews, P.Wehrli, C.Sevignani, D.Yu, S.Ricci, W.El-Deiry, B.Aronow, G.Kaya, J.-H.Saurat, and L.E. French (2004) "Myc-transformed epithelial cells down-regulate clusterin which inhibits their growth in vitro and carcinogenesis in vivo", Cancer Res, 64:3126–3136

12.   A.Thomas-Tikhonenko and M.L.Iruela-Arispe (2004) “Whence thrombospondin?”, Cancer Biol & Therapy, 3(4):406-407

13.   K.Johnson, D.L.Pflugh, D.Yu, D.G.T.Hesslein, K.-I.Lin, A.L.M.Bothwell, A.Thomas- Tikhonenko, D.G.Schatz, and K.Calame (2004) “B-cell specific loss of histone 3 lysine 9 in the VH locus depends on Pax5", Nature Immunology, 5(8):853-861

14.   M.S. Ricci, Z.Jin, M.Dews, D.Yu, A.Thomas-Tikhonenko, D.T.Dicker and W.S.El-Deiry  (2004) “Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity”, Mol Cell Biol, 24(19):8541-55

15.   D.Yu, M.Dews, A.Park, J.W.Tobias, and A.Thomas-Tikhonenko (2005) “Inactivation of Myc in murine two-hit B-lymphomas causes dormancy with elevated levels of interleukin-10 receptor and CD20: implications for adjuvant therapies”, Cancer Res, 65(12):5454-5461

16. D.Yu, D.Cozma, A.Park, and A.Thomas-Tikhonenko (2005) "Functional validation of genes implicated in lymphomagenesis: an in vivo selection assay using a Myc-induced B-cell tumor", Ann N Y Acad Sci, 1059

17. X.Zhang, L.M.DeSalle, J.H.Patel, A.J.Capobianco, D.Yu, A.Thomas-Tikhonenko and S.B.McMahon (2005) "Metastasis-associated protein 1 (MTA1) is an essential downstream effector of the c-MYC oncoprotein", Proc Natl Acad Sci USA, 102(39):13968-13973