Summary Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here, we identify SMO mutations in 50% (22/44) of resistant BCCs and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket mutations defining sites of inhibitor binding and four variants confering constitutive activity and inhibitor resistance, illuminating pivotal residues that ensure receptor autoinhibition. In the presence of a SMO inhibitor, tumor cells containing either class of SMO mutants effectively outcompete cells containing the wild type SMO. Finally, we show that both classes of SMO variants respond to aPKC-ι/λ or GLI2 inhibitors that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists.

SUMMARY Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMOD477G, confer resistance to these inhibitors. We report here that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMOD477G medulloblastoma.

Background-Current treatment for acute myocardial infarction (AMI) focuses on reestablishing blood flow (reperfusion).Paradoxically, reperfusion itself may cause additional injury to the heart. We previously found that ␦-protein kinase C (␦PKC) inhibition during simulated ischemia/reperfusion in isolated rat hearts is cardioprotective. We focus here on the role for ␦PKC during reperfusion only, using an in vivo porcine model of AMI. Methods and Results-An intracoronary application of a selective ␦PKC inhibitor to the heart at the time of reperfusion reduced infarct size, improved cardiac function, inhibited troponin T release, and reduced apoptosis. Using 31 P NMR in isolated perfused mouse hearts, we found a faster recovery of ATP levels in hearts treated with the ␦PKC inhibitor during reperfusion only. Conclusions-Reperfusion injury after cardiac ischemia is mediated, at least in part, by ␦PKC activation. This study suggests that including a ␦PKC inhibitor at reperfusion may improve the outcome for patients with AMI. (Circulation. 2003;108:2304-2307.)Key Words: reperfusion Ⅲ cardioprotection Ⅲ kinases C urrent treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of the ischemic period by disrupting the occlusion in the coronary artery. However, no therapeutic treatment is available to prevent reperfusion injury, which occurs after these interventions. 1,2 We previously developed several isozyme-selective inhibitor and activator peptides of protein kinase C (PKC). 3 Recently, we found that treatment with a ␦PKC-selective inhibitor during ischemia/reperfusion reduced cardiac damage in isolated perfused rat hearts. 3,4 Here, we show that the ␦PKC inhibitor prevented reperfusion injury in an in vivo porcine model of AMI. Methods Peptide SynthesisThe ␦PKC inhibitor peptide ␦V1-1 was synthesized and conjugated to Tat-derived peptide 5 via a cysteine S-S bond as described. 3 In Vivo Local Occlusion, Peptide Delivery, and Pathological AssessmentWe applied a balloon catheter into the mid left anterior descending coronary artery of female juvenile Yorkshire pigs (35 to 40 kg) under anesthesia (1% isoflurane) and inflated the balloon to produce a total occlusion for 30 minutes. The guide wire was removed, and Tat alone (Tat) or Tat-␦V1-1 conjugate (␦V1-1) was infused via the lumen of the balloon catheter only for the last 1 minute of ischemia (250 ng/kg, 1 mL/min). Left ventriculograms were performed to determine cardiac function. Hearts were harvested 4 hours or 5 days after ischemia. Double staining with Evans blue dye and TTC marked areas at risk for ischemia and infarcted areas, respectively, as described previously. 6 Troponin T levels in blood, as an indicator of cardiac cytolysis, were also determined after 24 hours of reperfusion.Wedge biopsies of liver, spleen, lung and kidney were fixed in 10% buffered neutral formalin and embedded in paraffin, and 8-m-thick sections were stained with hematoxylin and eosin for pathological examination. Biochemical Analysis of Porcine Cardiac TissueH...