FUNDED PROJECTS
PROJECT ONE
Cancer therapy-induced long-term bone marrow toxicity (Supported by R01CA122023 from NCI/NIH, PI: Zhou):
Many patients receiving chemotherapy and/or ionizing radiation (IR) develop acute and residual (or long-term) bone marrow (BM) injury that limits the success of cancer treatment and adversely affects their quality of life. Acute myelosuppression is the result of the induction of apoptosis in the rapidly proliferating hematopoietic progenitor cells (HPCs) and to a lesser degree in the relatively quiescent hematopoietic stem cells (HSCs). Its clinical manifestations have been successfully managed by the use of various hematopoietic growth factors. In contrast, residual BM injury has been largely attributed to the induction of HSC senescence. However, neither the molecular mechanisms by which chemotherapy and/or IR induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate residual BM injury. Recent studies from our laboratory and others provide new insights into HSC damage. First, we have found that exposure of mice to a sublethal dose of total body irradiation (TBI) perturbs the balance of reduction/oxidation (redox) reactions ONLY in HSCs, leading to a persistent and prolonged increase in reactive oxygen species (ROS) production. Second, HSCs are more sensitive to ROS-induced oxidative damage than HPCs and other hematopoietic cells. Moreover, it appears that ROS injure HSCs not by a nonspecific cytotoxic effect as previously hypothesized. Instead, the damage is at least partially mediated by induction of cellular senescence through redox-dependent activation of the p38 mitogen-activated protein kinase (p38)-p16Ink4a (p16) pathway. Based on these novel findings, we hypothesize that chemotherapy and IR cause residual BM injury by SELECTIVELY inducing HSC senescence through ROS-mediated activation of the p38-p16 pathway. Thus, we predict that antioxidants can be used to effectively mitigate residual BM injury. Moreover, antioxidant therapy provides additional benefits to cancer patients by suppressing chemotherapy- and IR-induced genetic instability, a primary cause of secondary tumors and a contributing factor to the development of tumor resistance. Therefore, this strategy offers the promise of significantly improving the quality of life and increasing the efficacy of chemotherapy and radiotherapy for cancer patients.
PROJECT TWO
Radiation-induced lymphoid and hematopoietic toxicity (Supported by R01CA86860 from NCI/NIH, PI: Zhou):
Bone marrow (BM) suppression is the primary cause of death after accidental or intentional exposure to a moderate or high dose of radiation and a common side effect of cancer therapy. The mechanisms whereby IR induces BM suppression have been largely attributed to the induction of apoptosis in hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We have provided the foremost direct evidence showing that HSCs and HPCs die by apoptosis, not necrosis, after exposure to a moderate dose of IR. Treatment of HSCs and HPCs in vitro with a broad-spectrum caspase inhibitor, such as z-VAD, inhibited IR-induced apoptosis (without conversion to necrotic cell death) and preserved their function. Furthermore, mice treated with z-VAD exhibited a significant reduction in IR-induced mortality, demonstrating that the preserved HSCs had the ability to reconstitute damaged BM in vivo. In contrast, various tumor cells die not by apoptosis but by reproductive cell death in response to IR and inhibition of caspase activity with a caspase inhibitor fails to protect these cells from IR-induced clonogenic cell death. Based on these novel findings, we hypothesize that activation of caspases mediates IR-induced BM suppression by the induction of HSC and HPC apoptosis. Furthermore, caspase inhibitors are novel radioprotectants that have the potential to be developed as new mechanism-based therapeutic agents to selectively protect BM but not tumor cells from IR-induced damage. To test our hypothesis, we will determine: (1) the sequence of IR-induced caspase activation and the role of individual caspases in IR-induced apoptosis in HSCs and HPCs in vitro; (2) the radioprotective effects of a pan caspase inhibitor and/or G-CSF on total body irradiation (TBI)-induced BM suppression; and (3) the effects of the caspase inhibitor on IR-induced tumor cell killing and mutagenesis and genetic instability in normal cells in a mouse model. We expect that the proposed experiments will provide new insights into the role of caspases in IR-induced myelosuppression. This will allow us to develop novel and mechanism-based interventions to circumvent IR-induced BM toxicity, which are urgently needed as new medical countermeasures against nuclear terrorism. In addition, these new interventions could markedly improve the therapeutic efficacy of conventional cancer therapy by reducing normal tissue injury induced by radiation and chemotherapy.
PROJECT THREE
Role of p38 MAPK in HSC self-renewal and radiation-induced bone marrow injury (Supported by R01AI080421 from NIAID/NIH, PI: Zhou):
Nuclear terrorism is an increasing threat in the United States. Many people may receive doses of radiation that can cause significant casualties due to radiation-induced normal tissue damage. Among various tissues, the bone marrow (BM) is the most radiosensitive tissue in the body. Severe damage to BM occurs when hematopoietic stem cell (HSC) self-renewal is impaired by radiation, which can lead to BM failure, the primary cause of death after exposure to a substantial dose of radiation.
To achieve this goal, we have extensively investigated the mechanisms by which IR causes HSC damage. Our studies demonstrate that transplantation of a single HSC into a lethally irradiated mouse is sufficient to reconstitute its entire hematopoietic system and maintain the steady-state hematopoiesis of the recipient for the lifetime. In contrast, HSCs from irradiated donors were less capable of engraftment after transplantation than those from normal controls. These findings indicate that an intrinsic damage to HSCs by IR is likely the primary cause for the loss of HSC self-renewal. In addition, recent studies from us and others provide new insights into IR-induced intrinsic damage to HSCs. Results from these studies show that IR induces not only HSC/HPC apoptosis but also HSC senescence. HSC senescence represents an irreversible loss of proliferation capacity of HSCs, which is primarily responsible for the loss of HSC self-renewal and HSC premature exhaustion under various pathological conditions. Also important, the induction of HSC senescence by IR was associated with a sustained activation of the p38 mitogen-activated protein kinase (p38) pathway. Inhibition of p38: 1) attenuated IR-induced suppression of HSCs in vitro by selectively protecting HSCs from IR-induced senescence but not apoptosis; and 2) reduced IR-induced BM suppression in vivo by promoting hematopoietic regeneration after TBI. Based on these novel findings, we hypothesize that p38 is a negative regulator of HSC self-renewal and its activation can mediate IR-induced HSC injury and BM suppression. This will allow us to develop novel and mechanism-based therapies to reduce radiation-induced BM damage to save lives in a nuclear event and to increase long-term survival of the nuclear victims, which are urgently needed as new medical countermeasures against nuclear terrorism.
PROJECT FOUR
D609 – A novel cytoprotectant and selective antitumor agent (Supported by R01 CA10255 from NCI/NIH, PI: Zhou):
Tricyclodecan-9-yl-xanthogenate (D609) was originally developed as an antitumor and antiviral agent. Recently, we have discovered that D609 is a novel and potent antioxidant and cytoprotectant that has the ability to protect mice from ionizing radiation (IR)-induced lethality. Similar to the other known nucleophilic sulfur antioxidants, D609 has a –SH group presented as a xanthate (-C[=S]S-/-C[=S]SH) moiety that scavenges free radicals in a manner similar to that of the sulfhydryl (-SH) moiety of glutathione (GSH) and WR1065 (the active compound of amifostine). However, unlike all other known nucleophilic sulfur antioxidants, D609 is unique because it is also a selective tumor cytotoxic agent, possibly by inhibition of sphingomyelin synthase (SMS). Inhibition of SMS by D609 increases the intracellular level of ceramide and decreases that of diacylglycerol (DAG) thus favoring the induction of cell cycle arrest, senescence, and apoptosis in tumor cells in a cell type dependent manner. The xanthate group of D609 appears to be part of the SMS inhibitory pharmacophore, since blocking this group by S-alkylation abolishes that activity. However, this moiety is oxidatively unstable and is thought to undergo facile oxidation in biological systems, contributing to the poor antitumor activity of D609 observed in vivo. We hypothesize that S-modification of D609 through a metabolically labile linkage (alkoxyphosphoryl or alkoxylacyl) will protect the pharmacophore (-C[=S]SH) and lead to prodrugs with increased oxidative stability, improved pharmacokinetics and enhanced therapeutic efficacy. To test this hypothesis, we will pursue the following specific aims: 1). To rationally design, synthesize and select lead D609 prodrugs with optimal oxidative stability, appropriate hydrolytic properties and superior pharmacokinetics for in vivo therapeutic evaluation; 2). To assess the therapeutic efficacy of select D609 prodrugs for radiation protection and for tumor therapy with or without IR in a mouse model. We believe that D609 prodrugs have the potential to be developed as a unique double agent for cancer therapy. When used as an adjuvant with IR and/or chemotherapy, D609 prodrugs may provide dual therapeutic benefits against cancer, e.g. protection of normal tissues and enhanced tumor cell killing.