Loss of function mutations in Kelch Like ECH Associated Protein 1 (KEAP1) or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) are common in non-small cell lung cancer (NSCLC) and is associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ~400,000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to the Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs such as doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells compared to single agents alone. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant anti-tumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c؉ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c؉ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML IntroductionNucleophosmin (NPM1 or B23.1) is a ubiquitously expressed, nucleolar phosphoprotein that functions as a molecular chaperone, shuttling between the nucleolus and the cytoplasm. 1-3 NPM1 plays multiple roles in cell growth and proliferation by participating in diverse biologic processes, including ribosome biogenesis and transport, centrosome duplication, DNA repair, transcriptional regulation and histone chaperoning. 4-7 Intracellular NPM1 is predominantly oligomeric and binds to other proteins, including the tumor suppressor proteins p14ARF and p53. 1,[8][9][10] Multifunctional characteristic of NPM1 appears to be dictated not only by its sub-cellular localization and its binding partners, but is also influenced by the various post translational modifications in NPM1, including acetylation, phosphorylation, poly-ubiquitination and sumoylation. [11][12][13][14] Wild-type (WT) NPM1 contains distinct structural domains that account for its ability to act as a multifunctional protein. 1,15 NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain (residues, 1-110), which is common to all isoforms of NPM1 and critical for its chaperone activity. [1][2][3] Recently, NSC348884 was identified as a small molecule inhibitor that disrupts NPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. 16 Oncogenic fusion proteins created by chromosomal translocation involving NPM1 gene, or mutations in NPM1 are observed in leukemia and lymphoma. 17 Notably, NPM-ALK fusion protein is found in CD30ϩ anaplastic large-cell lymphoma, 18 while leukemia related NPM1 fusion proteins include NPM-MLF1 and NPM-RAR␣. 17,19,20 These chimeric fusion proteins contain the N-terminal NPM1 oligomerization domain and a C-terminal fragment of the other protein. 17 NPM1 gene is also mutated in one third of adult acute myeloid leukemia (AML), especially those with the normal karyotype. 21 NPM1 mutations are heterozygous and, in the majority, localized to exon 12 of the gene. 21,22 Approximately 50 different types of mutations have been found, all creating the cytoplasm-dislocated mutant (Mt) NPM1 (NPM1cϩ) protein. 21,22 The most common is the type-A mutation, accounting for 75% of cases, which consists of TCTG tetranucleotide tandem duplication at position 956-959 of the NPM1 coding sequence. [22][23][24] This mutation causes the loss of tryptophans 288 and 290 (or 290 alone) from the carboxy-terminus and the creation of an additional leucine-rich nuclear export motif in the NPM1 protein, which causes the aberrant cytoplasmic dislocation of NPM1cϩ. [22][23][24] Knock...

Purpose We determined the activity of heat shock protein (hsp) 90 inhibitor (HI), and/or JAK2 tyrosine kinase inhibitor (TKI) against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE1) or primary human CD34+ myeloproliferative neoplasm (MPN) cells. Experimental Design Following exposure to the HI AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined. Results Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, i.e., phospho (p)-STAT5, p-AKT and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1 and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone, and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Co-treatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34+ MPN versus normal hematopoietic progenitor cells. As compared to the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE1/TGR cells exhibited significantly higher IC50 values for JAK2-TKI (p <0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the HIs AUY922 and 17-AAG; accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT and Bcl-xL, with concomitant induction of BIM. Conclusions Findings presented here demonstrate that co-treatment with HI and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. Additionally, treatment with HI may overcome resistance to JAK2-TKI in human MPN cells.

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis, while promoting autophagy, which promotes cancer cell survival when apoptosis is compromised. Here, we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62, caused synergistic cell death of MB-231 and SUM159PT cells, and inhibited mammosphere formation in MB-231 cells, compared with treatment with each agent alone. Finally, in mouse mammary fat pad xenografts of MB-231 cells, a tumor size-dependent induction of heat shock response, ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively, our findings show that cotreatment with an autophagy inhibitor and pan-HDI, for example, chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins, exerts superior inhibitory effects on TNBC cell growth, and increases the survival of TNBC xenografts. Mol Cancer Ther; 11(4); 973-83. Ó2012 AACR.

Following DNA damage that results in stalled replication fork, activation of ATR-CHK1 signaling induces the DNA damage response (DDR) in transformed cells. In the present studies on human cervical and breast cancer cells, we determined the effects of hsp90 inhibition on the levels and accumulation of DNA damage/ repair-associated proteins following exposure to g-ionizing radiation (IR; 4 Gy). We show that hsp90 inhibition with 17-allylamino-demehoxygeldanamycin or the novel, nongeldanamycin analogue AUY922 (resorcinylic isoxazole amide; Novartis Pharma) dose-dependently reduced the levels of ATR and CHK1 without affecting ATM levels. AUY922-mediated depletion of ATR and CHK1 was associated with an increase in their polyubiquitylation and decreased binding to hsp90. Cotreatment with bortezomib partially restored AUY922-mediated depletion of ATR and CHK1 levels. Additionally, treatment with AUY922 reduced the accumulation of ATR, p53BP1, and CHK1 but not g-H2AX to the sites of DNA damage. Following exposure to IR, AUY922 treatment abrogated IR-induced phospho (p)-ATR and p-CHK1 levels, but significantly enhanced g-H2AX levels. AUY922 treatment also increased IR-induced accumulation of the cells in G 2 -M phase of the cell cycle, inhibited the repair of IR-induced DNA damage, and augmented IR-mediated loss of clonogenic survival. Short hairpin RNA-mediated depletion of ATR also inhibited IR-induced p-ATR and p-CHK1, but increased g-H2AX levels, sensitizing cancer cells to IR-induced apoptosis and loss of clonogenic survival. These findings indicate that ATR is a bona fide hsp90 client protein and post-IR administration of AUY922, by inhibiting ATR-CHK1-mediated DDR, sensitizes cancer cells to IR. Mol Cancer Ther; 10(7); 1194-206. Ó2011 AACR.

Genetic alterations activating K-RAS and PI3K/AKT signaling are also known to induce the activity of mTOR kinase through TORC1 and TORC2 complexes in human pancreatic ductal adenocarcinoma (PDAC). Here, we determined the effects of the dual PI3K and mTOR inhibitor, NVP-BEZ235 (BEZ235), and the pan-histone deacetylase inhibitor panobinostat (PS) against human PDAC cells. Treatment with BEZ235 or PS inhibited cell cycle progression with induction of the cell cycle inhibitory proteins, p21 waf1 and p27 kip1. BEZ235 and PS also dose dependently induced loss of cell viability of the cultured PDAC cells, associated with depletion of phosphorylated (p) AKT, as well as of the TORC1 substrates 4EBP1 and p70S6 kinase. While inhibiting p-AKT, treatment with PS induced the levels of the pro-apoptotic proteins BIM and BAK. Co-treatment with BEZ235 and PS synergistically induced apoptosis of the cultured PDAC cells. This was accompanied by marked attenuation of the levels of p-AKT and Bcl-xL but induction of BIM. Although in vivo treatment with BEZ235 or PS reduced tumor growth, co-treatment with BEZ235 and PS was significantly more effective in controlling the xenograft growth of Panc1 PDAC cells in the nude mice. Furthermore, co-treatment with BEZ235 and PS more effectively blocked tumor growth of primary PDAC heterotransplants (possessing K-RAS mutation and AKT2 amplification) subcutaneously implanted in the nude mice than each agent alone. These findings demonstrate superior activity and support further in vivo evaluation of combined treatment with BEZ235 and PS against PDAC that possess heightened activity of RAS-RAF-ERK1/2 and PI3K-AKT-mTOR pathways.

Aurora kinases (AKs) regulate multiple components of mitotic cell division in eukaryotic cells. Aurora A is frequently amplified or overexpressed in breast cancer cells leading to aberrant chromosome segregation, genomic instability, and activation of oncogenic pathways. In the present studies, we determined the effects of treatment with the pan-AK inhibitor MK-0457 and/or the pan-histone deacetylase inhibitor vorinostat against human breast cancer cells that were either ER-, PR-, and HER2- (MDA-MB-468 and MDA-MB-231) or exhibited Aurora A amplification (BT-474 and MDA-MB-231 cells). Treatment with MK-0457 depleted p-AKs levels and their activity, as well as induced G2/M accumulation, DNA endoreduplication, multipolar mitotic spindles, and apoptosis of the breast cancer cells. Similar apoptotic effects were observed with treatment with the Aurora A-specific inhibitor, MLN8237. Treatment with vorinostat induced hsp90 acetylation and inhibited its chaperone association with AKs, leading to depletion of AKs and Survivin. Exposure of the siRNA to AK A also induced apoptosis, which was augmented by co-treatment with MK-0457 and vorinostat. Co-treatment with vorinostat enhanced MK-0457-mediated inhibition of the activities of Aurora A and Aurora B, leading to synergistic in vitro activity against human breast cancer cells. Co-treatment with MK-0457 and vorinostat also caused greater tumor growth inhibition and superior survival of mice bearing MDA-MB-231 xenografts. These pre-clinical findings indicate that combined treatment with a pan-AK inhibitor or an Aurora A-specific inhibitor and vorinostat represents a novel therapeutic strategy for the treatment of Aurora A-amplified and/or triple negative breast cancers.

Supplementary Figures Legends 1-4 from Hsp90 Inhibitor–Mediated Disruption of Chaperone Association of ATR with Hsp90 Sensitizes Cancer Cells to DNA Damage