Emergence of resistance to tyrosine kinase inhibitors in non-small-cell lung cancer can be delayed by an upfront combination with the HSP90 inhibitor onalespib.

BACKGROUND: Tyrosine kinase inhibitors, such as crizotinib and erlotinib, are widely used to treat non-small-cell lung cancer, but after initial response, relapse is common because of the emergence of resistance through multiple mechanisms. Here, we investigated whether a frontline combination with an HSP90 inhibitor could delay the emergence of resistance to these inhibitors in preclinical lung cancer models. METHODS: The HSP90 inhibitor, onalespib, was combined with either crizotinib or erlotinib in ALK- or EGFR-activated xenograft models respectively (H2228, HCC827). RESULTS: In both models, after initial response to the monotherapy kinase inhibitors, tumour relapse was observed. In contrast, tumour growth remained inhibited when treated with an onalespib/kinase inhibitor combination. Analysis of H2228 tumours, which had relapsed on crizotinib monotherapy, identified a number of clinically relevant crizotinib resistance mechanisms, suggesting that HSP90 inhibitor treatment was capable of suppressing multiple mechanisms of resistance. Resistant cell lines, derived from these tumours, retained sensitivity to onalespib (proliferation and signalling pathways were inhibited), indicating that, despite their resistance to crizotinib, they were still sensitive to HSP90 inhibition. CONCLUSIONS: Together, these preclinical data suggest that frontline combination with an HSP90 inhibitor may be a method for delaying the emergence of resistance to targeted therapies.

Epigenetic Priming by Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-cell Lymphoma.

Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from patients with peripheral TCL treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in IFN signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation. SIGNIFICANCE: The IAP antagonist tolinapant can induce necroptosis, a key immune-activating event, in TCL. Combination with DNA hypomethylation enhances tolinapant sensitivity and primes resistant cells by re-expressing necrosome proteins. In addition, this combination leads to increases in genes involved in IFN signaling and neoantigen expression, providing further molecular rationale for this novel therapeutic option.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1. RESULTS: Our results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts. CONCLUSION: AT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC.

The heat shock protein 90 inhibitor, AT13387, displays a long duration of action in vitro and in vivo in non-small cell lung cancer.

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.

Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells.

Despite promising clinical results from imatinib mesylate and second-generation ABL tyrosine kinase inhibitors (TKIs) for most BCR-ABL(+) leukemia, BCR-ABL harboring the mutation of threonine 315 to isoleucine (BCR-ABL/T315I) is not targeted by any of these agents. We describe the in vitro and in vivo effects of AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea), a potent inhibitor of several protein kinases, including Aurora A, Aurora B, Janus kinase 2 (JAK2), JAK3, and ABL on diverse imatinib-resistant BCR-ABL(+) cells. AT9283 showed potent antiproliferative activity on cells transformed by wild-type BCR-ABL and BCR-ABL/T315I. AT9283 inhibited proliferation in a panel of BaF3 and human BCR-ABL(+) cell lines both sensitive and resistant to imatinib because of a variety of mechanisms. In BCR-ABL(+) cells, we confirmed inhibition of substrates of both BCR-ABL (signal transducer and activator of transcription-5) and Aurora B (histone H3) at physiologically achievable concentrations. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3/BCR-ABL, human BCR-ABL(+) cell lines, or primary patient samples expressing BCR-ABL/T315I or glutamic acid 255 to lysine, another imatinib-resistant mutation. These data together support further clinical investigation of AT9283 in patients with imatinib- and second-generation ABL TKI-resistant BCR-ABL(+) cells, including T315I.

Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma.

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders.

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi-targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2-dependent systems. AT9283 potently inhibited proliferation and Jak2-related signalling in Jak2-dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6-JAK2 (TEL-JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.

Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa.

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 (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.

Phase 1 study of the HSP90 inhibitor onalespib in combination with AT7519, a pan-CDK inhibitor, in patients with advanced solid tumors.

PURPOSE: We conducted a phase 1 trial of the HSP90 inhibitor onalespib in combination with the CDK inhibitor AT7519, in patients with advanced solid tumors to determine the safety profile and maximally tolerated dose, pharmacokinetics, preliminary antitumor activity, and to assess the pharmacodynamic (PD) effects on HSP70 expression in patient-derived PBMCs and plasma. METHODS: This study followed a 3 + 3 trial design with 1 week of intravenous (IV) onalespib alone, followed by onalespib/AT7519 (IV) on days 1, 4, 8, and 11 of a 21-days cycle. PK and PD samples were collected at baseline, after onalespib alone, and following combination therapy. RESULTS: Twenty-eight patients were treated with the demonstration of downstream target engagement of HSP70 expression in plasma and PBMCs. The maximally tolerated dose was onalespib 80 mg/m2 IV + AT7519 21 mg/m2 IV. Most common drug-related adverse events included Grade 1/2 diarrhea (79%), fatigue (54%), mucositis (57%), nausea (46%), and vomiting (50%). Partial responses were seen in a palate adenocarcinoma and Sertoli-Leydig tumor; a colorectal and an endometrial cancer patient both remained on study for ten cycles with stable disease as the best response. There were no clinically relevant PK interactions for either drug. CONCLUSIONS: Combined onalespib and AT7519 is tolerable, though below monotherapy RP2D. Promising preliminary clinical activity was seen. Further benefit may be seen with the incorporation of molecular signature pre-selection. Further biomarker development will require the assessment of the on-target impact on relevant client proteins in tumor tissue.

Dexamethasone modulates Salmonella enterica serovar Typhimurium infection in vivo independently of the glucocorticoid-inducible protein annexin-A1.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice.

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth.

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 vivo 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 in vitro, 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. Mol Cancer Ther; 17(7); 1381-91. ©2018 AACR.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

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 whether 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. Cancer Res; 76(9); 2731-42. ©2016 AACR.

Inhibition of HSP90 by AT13387 delays the emergence of resistance to BRAF inhibitors and overcomes resistance to dual BRAF and MEK inhibition in melanoma models.

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 in which 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 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 and 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, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating 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.

The HSP90 inhibitor, AT13387, is effective against imatinib-sensitive and -resistant gastrointestinal stromal tumor models.

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib.

Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages.

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.