LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor, AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions where vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 out of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R, SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced and growth in 3D-culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combatting resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma.

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho-signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho-signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer-acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues. (Cancer Sci 2012; 103: 522-527) T he super-chaperone system is involved in the folding and maturation of newly synthesized proteins.(1,2) HSP90 in particular aids in the folding and maturation of a distinct subset of proteins, which includes kinases, cell surface receptors and transcription factors. (3,4) The N-terminal domain ATPase activity of HSP90 is essential for this function.(5) Inhibition of this domain induces remodeling of the HSP90 chaperone complex, resulting in the recruitment of ubiquitin ligases, polyubiquitination and subsequent proteasomal degradation of HSP90 client proteins.(6,7) Through this mechanism, the inhibition of a single target enzyme can have a wide effect on the stability and, hence, the function of a large set of client proteins. As many oncogenic proteins are HSP90 clients, HSP90 inhibition has been found to have broad antitumor effects.(8-10) In contrast to more recent targeted therapies, where the appearance of new driver mutations or resistance mutations result in a loss of efficacy, client protein mutation increases dependence on HSP90 chaperoning activity as these mutations tend to render the proteins less stable. (11)(12)(13) Previous studies have also demonstrated that the constitutively activated mutant forms of EGFR are particularly dependent on HSP90 both in vitro and in vivo, (14)(15)(16) indicating an HSP90 inhibitor may be particularly efficacious in mutant EGFR tumors.After HSP90 inhibiti...

Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR), and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear if HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels, but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I-II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. .