1. ZAK: A novel therapeutic target for treating Shiga toxin and ricin mediated disease? 1 D.M. Jandhyala, 2 J. Wong, 1 S.M. Stone, 3 N.J. Mantis, 2 B.E. Magun, and 1 C.M. Thorpe. 1 Division of Geographic Medicine and Infectious Diseases, Department of Medicine, Tufts Medical Center, 2 Department of Cell and Developmental Biology, Oregon Health and Science University, 3 Division of Infectious Diseases at the Wadsworth Center, New York State Department of Health. Introduction Shiga toxins are thought to promote disease following infection by Shiga toxin producing Escherichia coli (STEC) by promoting intestinal inflammation followed by subsequent endothelial damage and induction of a thrombotic microangiopathy termed the hemolytic uremic syndrome (HUS). While the mechanism(s) by which Shiga toxins cause disease are not fully understood, activation of PMNs, and changes in the levels of cytokines such as IL-8, MCP-1, CCL3, and fractaline are associated with disease in humans. Similarly, exposure to ricin can result in multi-organ failure in humans, and animal models have demonstrated that ricin can cause intestinal damage, acute respiratory distress syndrome, and HUS-like pathologies. These models have also demonstrated that ricin-induced pathology involves PMN infiltration, increases in pro-inflammatory cytokine levels, and involves macrophage-mediated IL-1  signaling. Shiga toxins and ricin are two deadly toxins sharing a common mode of action on cellular targets, specifically the N-glycosidase mediated depurination of a single adenine from the  -sarcin loop of the 28S ribosomal RNA. In addition to the halt in global protein synthesis, damage to the 28S rRNA also results in a MAPKinase dependent up-regulation of pro-inflammatory and pro-apoptotic signaling cascades termed the ribotoxic stress response (RSR). Although the RSR is activated by certain inhibitors of protein synthesis, protein synthesis inhibition itself does not activate the RSR. Instead it appears that the RSR occurs through sensing of damage to actively translating ribosomes. This RSR signal is transduced through the MAP3Kinase ZAK. While siRNA knockdown and chemical inhibition of ZAK have been shown to block Shiga toxin- and ricin-mediated activation of the RSR, little is known concerning the role of ZAK in the promotion of Shiga toxin and ricin- mediated disease in vivo. Based on in vitro data, we hypothesize that inhibition of ZAK signaling in vivo may prevent or lessen Shiga toxin- and ricin-mediated pathologies via blocking toxin-induced inflammation and apoptosis. We are currently testing this hypothesis using two different approaches. The first approach is to use chemical inhibitors of ZAK to prevent Shiga toxin- and ricin-mediated pathology in rabbit and murine models of disease. Our second approach involves evaluating toxin-mediated damage in a zak-/- mouse vs a zak+/+ mouse. Herein we present some of our preliminary findings concerning the role of ZAK in Shiga toxin- and ricin-mediated disease. Figure 2. To demonstrate N-glycosidase activity is required for intestinal damage in vivo, infant New Zealand white rabbits 1-2 days old were given oral 100  g/kg Shiga toxin 2, Shigat toxin 2 A-subunit mutant toxin with decreased N-glycosidase activity, or heat-inactivated Shiga toxin 2 daily, for two consecutive days, followed by euthanization and necropsy. H&E stained tissue sections were blindly scored on a scale of 0-4 for heterophil (rabbit PMNs) infiltration, epithelial defects, edema, and congestion and hemorrhage. The data shows that A-subunit N-glycosidase activity is indeed necessary for detectable intestinal damage. Shiga toxin mediated intestinal damage in the infant rabbit is A-subunit dependent ZAK MAP2Kinases ERKs, p38, and JNKs Stx or ricin mediated 28S ribosomal damage InflammationApoptosis The ribotoxic stress response Figure 1. Following damage to the 28S rRNA by Shiga toxin or ricin, the ribotoxic stress response is activated. The RSR is characterized by the activation of one or more MAPKinases following treatment by certain protein synthesis inhibitors. It should be noted that the RSR is not activated by protein synthesis inhibition, and that the RSR requires inactivation of an actively translating ribosome. ZAK is the MAP3Kinase that transduces the Shiga toxin- and ricin-mediated RSR in vitro. Shiga toxin activates the RSR in the colon of the infant rabbit Stx2 Mutant ( Y77S, E167Q) Stx2 wt Heat inactivated Stx2 Stx2 Mutant ( Y77S, E167Q) Stx2 wt A. B. Figure 3. Infant rabbits were orally treated with Stx2. Immunohistochemical staining for phospho-p38 (A.) or phospho-ERKs demonstrate that Stx2-treatments results in actvation of these two MAKinases in the colon of infant rabbits. Imatinib inhibits Stx2 induced p38 and JNKs phosphorylation in vitro Figure 4. Imatinib and dasatinib are known BCR-Abl inhibitors that also have affinity for ZAK (imatinib K d = 2.0  M). Confluent HCT-8 cells were pretreated with 200  M imatinib or dasatinib followed by Stx2 treatment. Imatinib pretreatment results in a decrease in Stx2-induced phospho-JNKs and phospho-p38. Pre-treatment with imatinib reduces Stx2-induced heterophil infiltration of the colon Figure 5. Infant rabbits were treated with 125 mg/kg 2 times daily beginning 12 hours prior to treatment with Stx2 or heat-inactivated Stx2. Imatinib-treated animals had a significant decrease in colonic heterophil (rabbit PMNs) infiltration P = 0.0068. Bone marrow derived macrophages from zak-/- do not initiate the RSR Figure 6. Bone marrow derived macrophages (BMDMs) were isolated from wild type or zak-/- mice and treated with 10 ng/ml ricin. The western blot in A. shows that unlike wild type mice, zak-/- mice do not activate JNKs or p38 MAPKinases in response to ricin treatment. zak-/- mice are non-lethal and have no other apparent phenotypes. B. zak-/- mice still respond normally to LPS suggesting that ZAK signaling is specific to the RSR. zak-/- mice and mice pretreated with certain BCR-Abl inhibitors have decreased ricin-induced pro-inflammatory gene expression Figure 8. BMDMs from wild type mice untreated, pretreated with the BCR-Abl inhibitors nilotinib, sorafinib or ponatinib (K d for ZAK not available), or zak-/- mice were treated with ricin (10 ng/ml) for 4 hours. Real-time PCR was used to access gene expression for IL-1 , IL-6, and CXCL1. zak-/- mice as well as mice treated with the BCR-Abl inhibitors had highly reduced expression of said cytokines in response to ricin treatment. Nilotinib and sorafinib block the ricin-induced RSR in murine BMDMs. Figure 7. Murine BMDMs were pretreated with nilotinib or sorafiniby (K d for ZAK = 8-10 nM, 6.3 nM respectively) followed by treatment with 10 ng/ml ricin for 3 hours. Western blots show that nilotinib and sorafinib blocked ricin-induced activation of JNKs and p38. Based on their affinity for ZAK, these data suggest that niltonib and sorafinib are indeed inhibitors of ZAK signaling. Figure 9. zak-/- or zak +/+ littermates (murine) were orally dosed with 10 mg/kg ricin. After 24 hours duodenal tissue was harvested andfixed and H&E sections were blindly scored for degree of tissue damage on a scale from 0-13 with 13 signifying the most severely damaged tissue. P=0.017 zak-/- mice have decreased intestinal damage following oral ricin treatment comparedwith wild type littermates Summary Oral administration of Shiga toxin to infant rabbits results in damage to the distal colon. This damage is dependent on Stx2A- subunit activity, and these damaged tissues also show activation of p38 and ERKs MAPKinases suggesting that ribotoxic stress is being initiated in vivo. That the RSR is being initiated by Stx2 in the infant rabbit colon is further supported by the observation that imatinib, a compound with affinity for ZAK and which inhibits the RSR in vitro, also reduces Stx2 associated infiltration of PMNs in the colon of infant rabbits. Ricin which has a similar N-glycosidase activity as Shiga toxin is unable to intitiate the RSR in BMDMs from zak-/- mice. The inability of zak-/- BMDMs to activate MAPKinase activation is specific to the RSR as treatment with LPS or treatment with double- stranded RNA (data not shown) do not induce MAPKinase activation in these cells. Also, zak-/- BMDMs have lower levels of pro- inflammatory cytokine message following ricin treatment as compared with wild type BMDMs. Similarly, treatment of wild type BMDMs with compounds known to inhibit ZAK block ricin-mediated MAPKinase activation and subsequent pro-inflammatory gene expression. Finally, following oral administration of ricin, zak-/- mice develop less intestinal damage compared with their zak+/+ littermates. Together these data suggest that Stx2 and ricin induce the RSR in vivo, and that the RSR contributes to toxin-induced pathology. As nilotinib, sorafinib, and imatinib are clinically available compounds approved for use in humans, targeting ZAK with these compounds may be a reasonable strategy for treating Shiga toxin and ricin mediated illnesses for which there are currently no specific therapies outside supportive care. Acknowledgments: We would like to thank the National Institutes of Health, Bethesda, MD, USA for its support of our work through the following grants: AI-59509 (C.M.T), AI0883360O1A1 (C.M.T and D.M.J), and AI1059335 (B.E.M).