Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors. Cancer Res; 71(2); 435-44. Ó2010 AACR.

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-β1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to β1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5β1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

IntroductionThe receptor tyrosine kinase (RTK) c-kit is essential for the development of normal hematopoietic cells and has been proposed to play a functional role in acute myeloid leukemia (AML). 1,2 c-Kit is a member of the class III family of RTKs, characterized by an extracellular ligand binding region containing 5 immunoglobulin repeats, a hydrophobic transmembrane domain, and an intracellular kinase domain split by an insert. 3 Binding of the c-kit ligand, stem cell factor (SCF), initiates a signal transduction cascade that includes receptor autophosphorylation and subsequent phosphorylation on numerous intracellular substrates. c-Kit and its ligand play a pivotal role in normal hematopoiesis, as evidenced by naturally occurring murine mutations at the Sl locus, which encodes SCF, as well as in the c-kit receptor itself. These mutations result in varying degrees of macrocytic anemia and a loss of mast cells in addition to deficiencies in gametogenesis and melanogenesis. [4][5][6] In addition to its role in normal hematopoiesis, c-kit is expressed in leukemic blasts in approximately 60% to 80% of AML patients, as assessed by surface immunostaining using antibodies specific to c-kit or by expression of c-kit messenger RNA. 1,7,8 Supporting a functional role for c-kit in AML, increased tyrosine phosphorylation of the receptor, as well as a proliferative response upon SCF stimulation, has been demonstrated in leukemic blasts in most AML cases that were c-kit ϩ . 1,2 The proliferative response to SCF has been shown to be synergistic with granulocytemacrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-3, both of which are known to promote the growth of leukemic cells in vitro.SU5416 and SU6668 are small-molecule inhibitors of RTKs such as Flk-1/KDR that have structural and sequence similarity to c-kit. SU5416 is a more selective and potent inhibitor of the Flk-1/KDR receptor; in contrast, SU6668 exhibits a broader inhibitory target profile, with effects on platelet-derived growth factor (PDGF) receptor and fibroblast growth factor (FGF) receptor in addition to Flk-1/KDR. 9,10 Both compounds have been shown to exhibit selectivity with respect to other tyrosine kinases, for example, with inhibitory concentration of 50% (IC 50 ) above 10 M for epidermal growth factor (EGF) receptor, Src, Met,10 In cell-based and preclinical animal models, both compounds have also been shown to exhibit antiangiogenic properties. SU5416 inhibits vascular endothelial growth factor (VEGF)-induced and SU6668 VEGF-and FGF-induced proliferation of human umbilical vein endothelial cells in culture; however, neither compound potently inhibits the growth of tumor cells grown in culture. 9,10 In addition, both compounds inhibit the growth of a variety of tumor cells grown as subcutaneous xenografts in athymic mice; furthermore, SU6668 has been shown to regress established xenograft tumors in mice. 9,10 Intravital fluoresence videomicroscopy in mouse tumor xenograft models has demonstrated that SU5416 and SU6668 also inhibit tumor...

Purpose: Vismodegib (GDC-0449) is a potent and selective inhibitor of the Hedgehog (Hh) pathway that shows antitumor activity in preclinical models driven by mutational or ligand-dependent activation of the Hh pathway. We wished to characterize the pharmacokinetic-pharmacodynamic (PK/PD) relationship of vismodegib in both model systems to guide optimal dose and schedule for vismodegib in the clinic.Experimental Design: Preclinical efficacy and PK/PD studies were carried out with vismodegib in a Ptch þ/À allograft model of medulloblastoma exhibiting mutational activation of the Hh pathway and patient-derived colorectal cancer (CRC) xenograft models exhibiting ligand-dependent pathway activation. Inhibition of the hedgehog pathway was related to vismodegib levels in plasma and to antitumor efficacy using an integrated population-based PK/PD model. Results: Oral dosing of vismodegib caused tumor regressions in the Ptch þ/À allograft model of medulloblastoma at doses !25 mg/kg and tumor growth inhibition at doses up to 92 mg/kg dosed twice daily in two ligand-dependent CRC models, D5123, and 1040830. Analysis of Hh pathway activity and PK/PD modeling reveals that vismodegib inhibits Gli1 with a similar IC 50 in both the medulloblastoma and D5123 models (0.165 mmol/L AE11.5% and 0.267 mmol/L AE4.83%, respectively). Pathway modulation was linked to efficacy using an integrated PK/PD model revealing a steep relationship where > 50% of the activity of vismodegib is associated with >80% repression of the Hh pathway. Conclusions: These results suggest that even small reductions in vismodegib exposure can lead to large changes in antitumor activity and will help guide proper dose selection for vismodegib in the clinic.

Apoptosis, or programmed cell death, is a genetically regulated process with critical roles in development and homeostasis in metazoans (1). Deficient apoptosis leads to the absence of normal cell death and contributes to the development and progression of human cancers (2). Apoptotic cell death can be initiated through the engagement of cell surface proapoptotic receptors by their specific ligands or by changes in internal cellular integrity (3, 4). Both of these pathways converge at the activation of caspases, cysteine-dependent aspartyl-specific proteases that comprise the effector arm of apoptotic cell death (5, 6). The intrinsic or mitochondrial pathway is initiated by developmental cues or cellular stress signals. These signals activate Bcl-2 homology 3 (BH3) 3 proteins, leading to neutralization of the antiapoptotic proteins, such as Bcl-2, Bcl-x L , or Mcl-1, activation of proapoptotic proteins Bax and Bak, and subsequent disruption of mitochondrial membrane potential (7). The resulting release of cytochrome c from the mitochondria into the cytoplasm leads to Apaf-1-mediated caspase-9 activation and consequent activation of effector caspases-3 and -7 and culminates in cell death.The extrinsic apoptotic pathway is triggered when proapoptotic receptors such as Fas or death receptor 5 (DR5) are engaged by their respective ligands, resulting in recruitment of the adaptor protein FADD and the apical caspases 8-or -10 (3). Incorporation of these caspases into the receptorassociated death-inducing signaling complex causes their autoactivation and leads to ensuing activation of effector caspases-3 and -7. In most cell types (type II cells), amplification of extrinsic pathway signaling through caspase-8-mediated activation of the BH3-only protein Bid is critical for efficient execution of apoptosis (8, 9); in type I cells direct activation of effector caspases by caspase-8 is sufficient. Bid plays an important role in a number of cellular pathways including regulation of Fas-and TNFR1-mediated hepatocellular injury (9 -13). In addition to stimulation by their respective ligands, proapoptotic receptors can be engaged by agonistic antibodies (14). DR5 agonist antibody (PRO95780) binds DR5 tightly and selectively, triggering apoptosis in various types of cancer cells and inhibiting tumor xenograft growth in vivo (15,16).IAP proteins represent the ultimate line of defense against cellular suicide by regulating caspase activity and preventing caspase activation (17). c-IAP1 and c-IAP2 are components of TNF receptor (TNFR) complexes where they modulate apoptotic signaling and caspase-8 activation (18 -20). X chromosome-linked IAP (XIAP) is the only true endogenous inhibitor of caspases because other IAP proteins exhibit weak binding to and inhibition of caspases (21). XIAP inhibits caspases-3 and -7 using the linker region between its baculoviral IAP-repeat (BIR) domain 1 (BIR1) and BIR2 as well as the BIR2 domain, whereas inhibition of caspase-9 relies on the binding of the BIR3 domain to an N-terminal