N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.

A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.

The NFIB-ERO1A axis promotes breast cancer metastatic colonization of disseminated tumour cells.

Metastasis is the main cause of deaths related to solid cancers. Active transcriptional programmes are known to regulate the metastatic cascade but the molecular determinants of metastatic colonization remain elusive. Using an inducible piggyBac (PB) transposon mutagenesis screen, we have shown that overexpression of the transcription factor nuclear factor IB (NFIB) alone is sufficient to enhance primary mammary tumour growth and lung metastatic colonization. Mechanistically and functionally, NFIB directly increases expression of the oxidoreductase ERO1A, which enhances HIF1α-VEGFA-mediated angiogenesis and colonization, the last and fatal step of the metastatic cascade. NFIB is thus clinically relevant: it is preferentially expressed in the poor-prognostic group of basal-like breast cancers, and high expression of the NFIB/ERO1A/VEGFA pathway correlates with reduced breast cancer patient survival.

Deciphering mechanisms of response and resistance in large-scale mouse cancer screens.

Acquired resistance is a major limitation for the successful treatment of cancer patients. Although numerous efficacious cancer therapeutics have been developed in the past decades, resistance arises due to a variety of reasons including tumoral genetic alterations, or modulation of factors in the tumor environment. Understanding the mechanistic reasons for tumor relapse supports the identification of novel combination therapies that could lead to more durable responses. Here, we will review large-scale in vivo screens in pre-clinical cancer models that employed genetic and pharmacological agents toward elucidating acquired drug resistance and informing on beneficial combinations to be tested in clinical trials.

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf-/- mouse model.

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

The HSP90 inhibitor NVP-AUY922 potently inhibits non-small cell lung cancer growth.

Heat shock protein 90 (HSP90) is involved in protein folding and functions as a chaperone for numerous client proteins, many of which are important in non-small cell lung cancer (NSCLC) pathogenesis. We sought to define preclinical effects of the HSP90 inhibitor NVP-AUY922 and identify predictors of response. We assessed in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines. We evaluated gene expression changes induced by NVP-AUY922 exposure. Xenograft models were evaluated for tumor control and biological effects. NVP-AUY922 potently inhibited in vitro growth in all 41 NSCLC cell lines evaluated with IC50 < 100 nmol/L. IC100 (complete inhibition of proliferation) < 40 nmol/L was seen in 36 of 41 lines. Consistent gene expression changes after NVP-AUY922 exposure involved a wide range of cellular functions, including consistently decreased dihydrofolate reductase after exposure. NVP-AUY922 slowed growth of A549 (KRAS-mutant) xenografts and achieved tumor stability and decreased EGF receptor (EGFR) protein expression in H1975 xenografts, a model harboring a sensitizing and a resistance mutation for EGFR-tyrosine kinase inhibitors in the EGFR gene. These data will help inform the evaluation of correlative data from a recently completed phase II NSCLC trial and a planned phase IB trial of NVP-AUY922 in combination with pemetrexed in NSCLCs.

Targeting conserved water molecules: design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization.

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.

Preclinical antitumor activity of the orally available heat shock protein 90 inhibitor NVP-BEP800.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed molecular chaperone with ATPase activity involved in the conformational maturation and stability of key signaling molecules involved in cell proliferation, survival, and transformation. Through its ability to modulate multiple pathways involved in oncogenesis, Hsp90 has generated considerable interest as a therapeutic target. NVP-BEP800 is a novel, fully synthetic, orally bioavailable inhibitor that binds to the NH(2)-terminal ATP-binding pocket of Hsp90. NVP-BEP800 showed activity against a panel of human tumor cell lines and primary human xenografts in vitro at nanomolar concentrations. In A375 melanoma and BT-474 breast cancer cell lines, NVP-BEP800 induced client protein degradation (including ErbB2, B-Raf(V600E), Raf-1, and Akt) and Hsp70 induction. Oral administration of NVP-BEP800 was well tolerated and induced robust antitumor responses in tumor xenograft models, including regression in the BT-474 breast cancer model. In these tumor models, NVP-BEP800 modulated Hsp90 client proteins and downstream signaling pathways at doses causing antitumor activity. NVP-BEP800 showed in vivo activity in a variety of dosing regimens covering daily to weekly schedules, potentially providing a high degree of flexibility in dose and schedule within the clinical setting. Overall, given the mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, NVP-BEP800 is an exciting new oral Hsp90 inhibitor warranting further development. Mol Cancer Ther; 9(4); 906-19. (c)2010 AACR.

Toward the optimization of stent-based treatment for coronary artery disease.

Coronary artery disease consists of obstruction (stenosis) of the coronary arteries by the deposition of atherosclerotic plaques, resulting in an insufficient supply of oxygen to the heart muscle. Treatment options include the insertion of a stent - a metal mesh tube - into the obstructed vessel to keep the artery open, thus preventing acute occlusion and restenosis. The occlusion of vessels resulting from subacute stent thrombosis and late in-stent restenosis are potential complications after successful revascularization. However, the rate of stent thrombosis has been reduced dramatically by means of adequate antiplatelet therapy, and in-stent restenosis has been addressed successfully with drug-eluting stents. These drug-eluting stents are engineered to release bioactive agents into the affected blood vessels, plaques or tissues adjacent to the stent. Various antimitotic, anti-inflammatory, and anticoagulant immunosuppressive agents have been attached to stents, including sirolimus and tacrolimus. Future opportunities include the use of gene therapies released from gene-eluting stents. These advances highlight some of the opportunities for optimizing stent-based treatment for coronary artery disease.

Hsp90 inhibitors: clinical development and future opportunities in oncology therapy.

The development of small molecular mass Hsp90 inhibitors has become an increasingly competitive field of research in recent years. Progress in preclinical and clinical research has provided increasing evidence that Hsp90 represents a promising molecular target for the treatment of cancer and other diseases. Although many challenges remain, recent clinical trial results for the most clinically advanced inhibitors indicate that clinical proof of concept in oncology therapy is achievable, and that Hsp90 inhibitors have the potential to contribute to the collection of anticancer treatments available to clinicians and patients in the near future.

(89)Zr-trastuzumab PET visualises HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a human tumour xenograft.

NVP-AUY922, a potent heat shock protein (HSP) 90 inhibitor, downregulates the expression of many oncogenic proteins, including the human epidermal growth factor receptor-2 (HER2). Because HER2 downregulation is a potential biomarker for early response to HSP90-targeted therapies, we used the (89)Zr-labelled HER2 antibody trastuzumab to quantify the alterations in HER2 expression after NVP-AUY922 treatment with HER2 positron emission tomography (PET) imaging. The HER2 overexpressing human SKOV-3 ovarian tumour cell line was used for in vitro experiments and as xenograft model in nude athymic mice. In vitro HER2 membrane expression was assessed by flow cytometry and a radio-immuno assay with (89)Zr-trastuzumab. For in vivo evaluation, mice received 50mg/kg NVP-AUY922 intraperitoneally every other day. (89)Zr-trastuzumab was injected intravenously 6d before NVP-AUY922 treatment and after 3 NVP-AUY922 doses. MicroPET imaging was performed at 24, 72 and 144h post tracer injection followed by ex-vivo biodistribution and immunohistochemical staining. After 24h NVP-AUY922 treatment HER2 membrane expression showed profound reduction with flow cytometry (80%) and radio-immuno assay (75%). PET tumour quantification, showed a mean reduction of 41% (p=0.0001) in (89)Zr-trastuzumab uptake at 144h post tracer injection after NVP-AUY922 treatment. PET results were confirmed by ex-vivo (89)Zr-trastuzumab biodistribution and HER2 immunohistochemical staining. NVP-AUY922 effectively downregulates HER2, which can be monitored and quantified in vivo non-invasively with (89)Zr-trastuzumab PET. This technique is currently under clinical evaluation and might serve as an early biomarker for HSP90 inhibition in HER2 positive metastatic breast cancer patients.

Combining hit identification strategies: fragment-based and in silico approaches to orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models.

INTRODUCTION: Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the post-translational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies. METHODS: The concentrations of the HSP90 inhibitor NVP-AUY922 required to reduce cell numbers by 50% (GI50 values) were established in a panel of breast cancer cell lines and patient-derived human breast tumors. To investigate the properties of the compound in vivo, the pharmacokinetic profile, antitumor effect, and dose regimen were established in a BT-474 breast cancer xenograft model. The effect on HSP90-p23 complexes, client protein degradation, and heat shock response was investigated in cell culture and breast cancer xenografts by immunohistochemistry, Western blot analysis, and immunoprecipitation. RESULTS: We show that the novel small molecule HSP90 inhibitor NVP-AUY922 potently inhibits the proliferation of human breast cancer cell lines with GI50 values in the range of 3 to 126 nM. NVP-AUY922 induced proliferative inhibition concurrent with HSP70 upregulation and client protein depletion--hallmarks of HSP90 inhibition. Intravenous acute administration of NVP-AUY922 to athymic mice (30 mg/kg) bearing subcutaneous BT-474 breast tumors resulted in drug levels in excess of 1,000 times the cellular GI50 value for about 2 days. Significant growth inhibition and good tolerability were observed when the compound was administered once per week. Therapeutic effects were concordant with changes in pharmacodynamic markers, including HSP90-p23 dissociation, decreases in ERBB2 and P-AKT, and increased HSP70 protein levels. CONCLUSION: NVP-AUY922 is a potent small molecule HSP90 inhibitor showing significant activity against breast cancer cells in cellular and in vivo settings. On the basis of its mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, the compound recently has entered clinical phase I breast cancer trials.

Targeting Hsp90 for the treatment of cancer.

Heat shock protein (Hsp)90 is a molecular chaperone that is responsible for the correct folding of a large number of proteins, which allows these proteins to achieve their functional conformation. Client proteins of Hsp90 include many overexpressed or mutated oncogenes that are known to be critical for the transformed phenotype observed in tumors. The compounds 17-AAG (Kosan Biosciences Inc/National Cancer Institute) and 17-DMAG (Kosan Biosciences Inc/National Cancer Institute) are Hsp90 inhibitors that are derived from the prototypical ansamycin natural product Hsp90 inhibitor geldanamycin. These compounds have demonstrated preclinical efficacy in mouse xenograft models, and are now undergoing phase II and I clinical trials, respectively. Preclinical efficacy studies of these compounds are collated and discussed in this review. More recent disclosures of small-molecule Hsp90 inhibitors include purine and resorcinol analogs, and the first small-molecule Hsp90 compounds showing oral efficacy have been described. Inhibition of Hsp90 not only results in the degradation of client proteins, but also results in the induction of another chaperone, Hsp70. Hsp70 is known to be anti-apoptotic, and therefore the induction of Hsp70 may ultimately limit the efficacy of Hsp90 inhibitors under certain circumstances. Histone deacetylase inhibitors have recently been demonstrated to exert some of their effect through modulation of Hsp90 chaperoning activity, and some mechanistic aspects of this control are also discussed herein.

E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes.

The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7 results in suppression of E2F target genes and accumulation of cells in G1. Furthermore, E2F7 associates with E2F-regulated promoters in vivo, and this association increases in S phase. Interestingly, however, E2F7 binds only a subset of E2F-dependent promoters in vivo, and in agreement with this, inhibition of E2F7 expression results in specific derepression of these promoters. Taken together, these data demonstrate that E2F7 is a unique repressor of a subset of E2F target genes whose products are required for cell cycle progression.

Reduced hepatic tumor incidence in cyclin G1-deficient mice.

Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53.