Scientific Foundation

The mission of CBLI is to build upon discoveries of the molecular mechanisms controlling apoptotic cell death to manipulate this process for therapeutic gain. The key concept underlying CBLI’s technology is that normal and tumor cells die by different mechanisms when exposed to stresses such as radiation (reviewed in [1]) ( See Figure 1). Death of normal cells occurs through a specific, highly regulated mechanism called apoptosis or “programmed cell death.” Apoptosis is critical for both normal development (triggered by cellular developmental cues) and for elimination of defective and potentially tumorigenic cells (triggered by cues resulting from cell stress, such as DNA damage). Complex intracellular pathways regulate apoptosis which ultimately involves cleavage of critical cellular proteins and fragmentation of chromosomal DNA. In contrast, apoptosis is rarely involved as a primary event in tumor cell killing since tumor cells frequently acquire genetic and epigenetic lesions that block programmed cell death as part of their progression. The sensitivity of tumor cells to radiation is usually realized in the form of mitotic catastrophe, necrosis or senescence indicative of impaired mechanisms of checkpoint control and DNA repair in tumor cells. Two general strategies are currently being pursued to exploit these differences between normal and tumor cells: 1) restoration of apoptosis in tumor cells as a cancer treatment, and 2) inhibition of apoptosis in normal cells to prevent pathologies resulting from their loss in the face of stress.

Figure 1

Restoration of apoptosis: selective killing of cancer cells. Cancer cells frequently acquire defects in their apoptotic machinery, which contributes to their continued growth despite attempted intervention by the immune system and clinical treatments (Figure 2). In many tumors, this involves deregulation of two major cellular signaling pathways that control apoptosis: function of the pro-apoptotic molecule p53 is usually lost, while the anti-apoptotic molecule NF-kappaB becomes constitutively active. Moreover, CBLI researchers have discovered a novel mechanism of tumor resistance to apoptosis that involves interplay between these two pathways: functional repression of p53 by constitutively active NF-kappaB [2]. As a result of these defects, therapies that induce cell death in normal tissues may not be effective in cancer cells [1]. CBLI is using this knowledge to rationally design new therapeutic strategies aimed at restoring apoptotic mechanisms to allow enhanced killing of cancer cells. Through this approach, CBLI has identified a number of small molecules, named Curaxins, that selectively kill tumor cells through the unique mechanism of concerted p53 activation and NF-kappaB inhibition.

Figure 2

Suppression of apoptosis: protection of normal cells in the face of stress. CBLI is also taking the converse approach of protecting healthy cells from undergoing stress-induced apoptosis through temporary and reversible pharmacological imitation of the naturally occurring apoptotic-resistance of cancer cells.

Genotoxic stresses such as irradiation and most chemotherapeutics produce DNA damage, which triggers apoptosis in sensitive cells. Massive apoptotic cell loss, particularly in the hematopoietic (HP) and gastrointestinal (GI) systems, produces the pathologies of acute radiation syndrome and side effects of anti-cancer radiation and chemotherapies. Radiation-induced apoptosis of normal cells is dependent upon the p53 tumor suppressor protein, the major cellular sensor of genotoxic stress. Accordingly, p53-deficient mice can tolerate total body irradiation with up to 10 Gy, a dose that is lethal in 100% of wild type mice. Thus, while the conventional view of p53 as a drug target aims to restore its activity in order to induce tumor cell death, CBLI has forwarded the novel concept of inhibiting p53 in order to protect normal cells. The concept of therapeutic p53 inhibition was validated by isolation of a small molecule inhibitor of p53, pifithrin-alpha, that was found to be a powerful radio- and chemo-protectant [3]. Temporary suppression of p53 by pifithrin-alpha rescued wild type mice from radiation doses that normally cause death via HP syndrome [3]. These observations led us to consider suppression of p53 as a pharmacological approach to reducing the side effects of anti-cancer radiotherapy [4]. Loss or inactivation of p53 in the majority of human cancers restricts the radioprotective effect of p53 inhibition to normal tissues, suggesting that the side effects of cancer treatment might be eliminated while the anti-tumor effects are maintained. Unfortunately, while this approach demonstrated effective radioprotection of the HP system, it proved unsuitable for protection of the GI tract, where wild-type p53 was found to play the role of a survival factor [5]. We therefore switched focus to pharmacological targeting of another mechanism of apoptosis suppression frequently acquired by tumors – constitutive activation of NF-kappaB [6].

The NF-kappaB signaling pathway responds to a variety of extrinsic stresses to regulate critical cellular and organismal reactions including innate and adaptive immune responses (PMID: 17072327). Activation of the major branch of the NF-kappaB pathway leads to transcriptional induction of numerous responsive genes, many of which encode proteins with known roles in reactive oxygen species (ROS)-scavenging, apoptosis suppression or cytokine and chemokine production. Each of these responses could contribute to radioprotection by either reducing the amount of damage (ROS scavengers), preventing cell loss through apoptosis or promoting tissue regeneration (cytokines). Indeed, the importance of NF-kappaB signaling for radioprotection was demonstrated by the increased radiosensitivity of the GI tract in mice with a genetic defect in the NF-kappaB signaling pathway [7].

Pharmacological activation of NF-kappaB can be achieved by a variety of natural factors mediating immune responses. Some well-known and effective activators of NF-kappaB such as interleukins, interferons and tumor necrosis factor alpha (TNFa) were shown to possess radioprotective properties [8, 9]. However, radioprotective doses of these cytokines induced acute inflammatory responses that prevented their development into useful radioprotectants. CBLI, therefore, is taking the unique approach of exploring naturally occurring inducers of NF-kappaB produced by benign microorganisms of the human microflora that rely upon inhibition of apoptosis in their host cells as part of their survival strategy [10]. The natural activity of these factors which have undergone long-term evolutionary adaptation, suggests that they are likely to be both potent and safe inhibitors of apoptosis.

Activation of NF-kappaB in response to specific microbial molecular patterns (e.g., bacterial cell wall components, bacterial DNA or viral RNA) occurs in large part via a family of Toll-like receptors (TLRs) expressed on the host cell surface. TLRs function as the key sensor elements of the innate immune system and all induce NF-kappaB signaling following binding of their specific ligands [11-13]. A genetic defect in Myd88, a common mediator of NF-kappaB induction downstream of TLRs, was shown to increase the sensitivity of the small intestine of mice to external stresses including radiation [10]. Moreover, there are indications that bacterial endotoxin (lipopolysacharide or LPS), the ligand of TLR4, is radioprotective in mice; however, this has not been explored further due to its high toxicity [14, 15]. These findings provide support for the principle of using TLR agonists as radioprotectants, although it is clear that the targeted receptor and agonist need to be carefully chosen and/or engineered in order to develop a strategy that is not only effective, but also non-toxic and non-immunogenic. CBLI is generating optimized derivatives of TLR agonists that fulfill these requirements and therefore have a broad spectrum of potential clinical applications.

References

1. Gudkov, A.V. and E.A. Komarova, The role of p53 in determining sensitivity to radiotherapy. Nat Rev Cancer, 2003. 3(2): p. 117-29.
2. Gurova, K.V., et al., Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-kappaB-dependent mechanism of p53 suppression in tumors. Proc Natl Acad Sci U S A, 2005. 102(48): p. 17448-53.
3. Komarov, P.G., et al., A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science, 1999. 285(5434): p. 1733-7.
4. Gudkov, A.V. and E.A. Komarova, Prospective therapeutic applications of p53 inhibitors. Biochem Biophys Res Commun, 2005. 331(3): p. 726-36.
5. Komarova, E.A., et al., Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice. Oncogene, 2004. 23(19): p. 3265-71.
6. Karin, M., Nuclear factor-kappaB in cancer development and progression. Nature, 2006. 441(7092): p. 431-6.
7. Wang, Y., et al., Activation of nuclear factor kappaB In vivo selectively protects the murine small intestine against ionizing radiation-induced damage. Cancer Res, 2004. 64(17): p. 6240-6.
8. Neta, R., et al., Role of cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor beta) in natural and lipopolysaccharide-enhanced radioresistance. J Exp Med, 1991. 173(5): p. 1177-82.
9. Riehl, T.E., et al., TNFR1 mediates the radioprotective effects of lipopolysaccharide in the mouse intestine. Am J Physiol Gastrointest Liver Physiol, 2004. 286(1): p. G166-73.
10. Rakoff-Nahoum, S., et al., Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell, 2004. 118(2): p. 229-41.
11. Akira, S. and K. Takeda, Toll-like receptor signalling. Nat Rev Immunol, 2004. 4(7): p. 499-511.
12. Iwasaki, A. and R. Medzhitov, Toll-like receptor control of the adaptive immune responses. Nat Immunol, 2004. 5(10): p. 987-95.
13. Kaisho, T. and S. Akira, Toll-like receptors as adjuvant receptors. Biochim Biophys Acta, 2002. 1589(1): p. 1-13.
14. Riehl, T., et al., Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism. Gastroenterology, 2000. 118(6): p. 1106-16.
15. Hofer, M., et al., Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis. Physiol Res, 1992. 41(2): p. 157-61.

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