Restored Drug Sensitivity Research Articles

Glutamine addiction activates polyglutamine-based nanocarriers delivering therapeutic siRNAs to orthotopic lung tumor mediated by glutamine transporter SLC1A5

Many human cancer cells exhibit an oncogenetic-driven addiction to glutamine (Gln) as rapidly proliferating cancer cells consume Gln at a dramatically increased rate compared to normal cells. Tumor cells, therefore, compete with host cells for Gln, which causes Gln to flux from normal tissues to the tumor. We have developed and characterized a Gln macromolecular analog polyglutamine (PGS) for the delivery of gene regulators, such as siRNAs, in our previous works. Here, we hypothesize that PGS can utilize the Gln transporter SLC1A5 to specifically deliver therapeutic compounds to Gln-addicted cancer cells. Compared to human lung fibroblast HLF cells, cisplatin-resistant human lung adenocarcinoma A549/DDP cells significantly overexpress SLC1A5, which has a high binding affinity to PGS, as confirmed through molecular docking analysis. Due to the differences in Gln metabolism between malignant and normal cells, PGS/siRNA complexes were remarkably increased in cancer cells, especially when cells were deprived of Gln, which mirrors the conditions that are commonly found in a tumor microenvironment. Furthermore, we identified that chemical and genetic inhibition of Gln transporter SLC1A5 reduced the cellular internalization of PGS/siRNA complexes, suggesting a critical role for SLC1A5 in PGS uptake in cells. In turn, PGS upregulated SLC1A5 expression. Increased uptake of PGS complexes profoundly decreased intracellular Gln levels. Decreased Gln caused a moderate reduction in cell growth. To restore drug sensitivity and further enhance anti-tumor effects, the hybrid siRNAs anti-Survivin and anti-MDR1 (siSM), as model therapeutics, were administered through the PGS delivery system, which resulted in knockdown of Survivin and MDR1 and further sensitized cancer cells to the drug cisplatin (DDP). Since PGS complexes administered i.v. mostly accumulated in the lung parenchyma, a lung orthotopic tumor model was established to evaluate their inhibitory effects on tumors in the lungs. PGS/siSM comparably decreased the rate of tumor growth, while concurrent administration of PGS/siSM and DDP enhanced this effect and insignificantly improved life span. Consistent with our hypothesis, this study demonstrated that PGS mimicked Gln in the SLC1A5 pathway and selectively ferried therapeutics to Gln-addicted cancer cells. Our findings identified a new lung cancer targeting strategy based on Gln metabolism and can be used as a drug/gene delivery system.

Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation.

To cope with intrinsic and environmental stress, cancer cells rely on adaptive pathways more than non-transformed counterparts. Such non-oncogene addiction offers new therapeutic targets and strategies to overcome chemoresistance. In an attempt to study the role of adaptive pathways in acquired drug resistance in carcinoma cells, we devised a model of in vitro conditioning to three standard chemotherapeutic agents, cisplatin, 5-fluorouracil, and docetaxel, from the epithelial cancer cell line, HEp-2, and investigated the mechanisms underlying reduced drug sensitivity. We found that triple-resistant cells suffered from higher levels of oxidative stress, and showed heightened anti-stress responses, including the antioxidant Nrf2 pathway and autophagy, a conserved pleiotropic homeostatic strategy, mediating the clearance of aggregates marked by the adapter p62/SQSTM1. As a result, re-administration of chemotherapeutic agents failed to induce further accumulation of reactive oxygen species and p62. Moreover, autophagy proved responsible for chemoresistance through the avoidance of p62 accumulation into toxic protein aggregates. Indeed, p62 ablation was sufficient to confer resistance in parental cells, and genetic and pharmacological autophagic inhibition restored drug sensitivity in resistant cells in a p62-dependent manner. Finally, exogenous expression of mutant p62 lacking the ubiquitin- and LC3-binding domains, required for autophagic engulfment, increased chemosensitivity in TDR HEp-2 cells. Altogether, these findings offer a cellular system to investigate the bases of acquired chemoresistance of epithelial cancers and encourage challenging the prognostic and antineoplastic therapeutic potential of p62 toxicity.

Reversal of epigenetic aberrations associated with the acquisition of doxorubicin resistance restores drug sensitivity in breast cancer cells

Acquired resistance against doxorubicin is a major limitation in clinical treatment of breast cancer. The molecular mechanism behind the aberrant expression of genes leading to doxorubicin resistance is not clear. Epigenetic changes play an important role in the regulation of gene expression. Therefore, the objective of this study was to identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin-resistant cells were selected by repeated exposure of MCF-7 and MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin for 18 months. MTT assay, cell cycle analysis, colony formation, qRT-PCR, and Western blot analyses were used to characterize the epigenetic and molecular mechanism. Pyrosequencing was used to detect MSH2 promoter hypermethylation. Aberrant expression of epigenetic regulatory genes, a significant increase in H3 acetylation and methylation, as well as promoter hypermethylation-mediated inactivation of MSH2 gene were associated with the acquired resistant phenotype. Demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin. Findings of this study revealed that epigenetic aberrations including promoter hypermethylation-mediated inactivation MSH2 contribute to the acquisition of doxorubicin resistance in breast cancer cells. Additionally, our data suggest that some of these epigenetic aberrations are progressive during resistance development and therefore can potentially be used as biomarkers for early detection of resistance. These epigenetic aberrations, being reversible, can also serve as targets for epigenetic therapy to re-sensitize doxorubicin-resistant breast cancer cells. Epigenetic inactivation of mismatch repair gene MSH2 further suggests that loss of MMR-dependent apoptotic potential could be a novel mechanistic basis for the acquisition of doxorubicin resistance in breast cancer cells.

Abstract SY19-02: Novel mechanisms of acquired resistance to selective CDK4/6 inhibition

Abstract CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptor (ER)-positive breast cancer, so that understanding mechanisms of resistance is a pressing issue. We have identified identified increased CDK6 expression as a key determinant of acquired resistance after exposure to palbociclib in ER-positive breast cancer cells. Increased CDK6 in resistant cells is dependent on TGF-β pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediated transfer of the resistance phenotype between neighboring cell populations, causing previously sensitive cells to acquire CDK4/6 inhibitor resistance. We confirmed these data in pretreatment and postprogression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating that this resistance mechanism is clinically relevant. Additionally, this CDK4/6 inhibitor resistance phenotype can be reversed in vitro and in vivo by a prolonged drug holiday, which restored drug sensitivity. CDK4 is also emerging as a target in KRAS-mutant non-small cell lung cancer (NSCLC). We have demonstrated that KRAS-mutant NSCLC cell lines are initially sensitive to palbociclib, but readily acquire resistance associated with increased expression of CDK6, D-cyclins, and cyclin E. Resistant cells also demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating palbociclib resistance. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A genetically engineered mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2 and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 that confers palbociclib resistance, and indicate that CDK4/6 inhibition acts to promote MAP kinase dependence. Citation Format: Geoffrey I. Shapiro. Novel mechanisms of acquired resistance to selective CDK4/6 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY19-02.

Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer.

Trastuzumab-refractory, HER2 (human epidermal growth factor receptor 2)-positive breast cancer is commonly treated with trastuzumab emtansine (T-DM1), an antibody-drug conjugate of trastuzumab and the microtubule-targeting agent, DM1. However, drug response reduces greatly over time due to acquisition of resistance whose molecular mechanisms are mostly unknown. Here, we uncovered a novel mechanism of resistance against T-DM1 by combining whole transcriptome sequencing (RNA-Seq), proteomics and a targeted small interfering RNA (siRNA) sensitization screen for molecular level analysis of acquired and de novo T-DM1-resistant models of HER2-overexpressing breast cancer. We identified Polo-like kinase 1 (PLK1), a mitotic kinase, as a resistance mediator whose genomic as well as pharmacological inhibition restored drug sensitivity. Both acquired and de novo resistant models exhibited synergistic growth inhibition upon combination of T-DM1 with a selective PLK1 inhibitor, volasertib, at a wide concentration range of the two drugs. Mechanistically, T-DM1 sensitization upon PLK1 inhibition with volasertib was initiated by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, leading to caspase activation, followed by DNA damage through CDK1-dependent phosphorylation and inactivation of Bcl-2/xL. Furthermore, we showed that Ser70 phosphorylation of Bcl-2 directly regulates apoptosis by disrupting the binding to and sequestration of the pro-apoptotic protein Bim. Importantly, T-DM1 resistance signature or PLK1 expression correlated with cell cycle progression and DNA repair, and predicted a lower sensitivity to taxane/trastuzumab combination in HER2-positive breast cancer patients. Finally, volasertib in combination with T-DM1 greatly synergized in models of T-DM1 resistance in terms of growth inhibition both in three dimensional (3D) cell culture and in vivo. Altogether, our results provide promising pre-clinical evidence for potential testing of T-DM1/volasertib combination in T-DM1 refractory HER2-positive breast cancer patients for whom there is currently no treatment available.

Abstract A147: Synthetic lethality screening reveals ATR as responsible for oxaliplatin resistance in colorectal cancer cells

Abstract Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. The aim of this project is thus to identify new targets involved in resistance to oxaliplatin using a phenotypic high-throughput screening. We established oxaliplatin-resistant cellular clones from the colon carcinoma cell line HCT-116. Using a clone displaying mild resistance, HCT116-R1 (i.e., with an IC50 10-fold higher than the parental line), we performed a genetic screening based on short hairpin RNA (ShRNA) targeting genes of human kinome. The final aim of this procedure was to reveal genes whose silencing restores drug sensitivity, so displaying a synthetic lethal interaction with the drug. We identified ATR (ataxia-telangiectasia mutated and rad3 related), a protein playing a key role in DNA repair that is activated in response to persistent single-stranded DNA (ssDNA) indirectly induced by various DNA-damaging agents, as a determinant of oxaliplatin resistance. We showed that ShRNA-mediated repression of ATR in HCT 116-R1 sensitize them to the drug. Then, we evaluated the effect of combining oxalipaltin with VE-822, an ATR inhibitor. The analysis of interaction between oxaliplatin and VE-822 was addressed by mean of concentration matrix cytotoxic test and the Bliss equation for additivity. We demonstrated that coincubation of oxaliplatin and VE-822 led to a dramatic synergistic effect in oxaliplatin-sensitive CRC cell lines (HCT116 and SW48) and in oxaliplatin-resistant correlative clones (HCT116-R1, HCT116-R2, SW48-R1, and SW48-R2) both in 2D and 3D. We also showed that the synergistic effect of oxaliplatin and VE-822 was accompanied by an increase of ssDNA followed by DNA double-strand breaks, growth arrest, and apoptosis induction. The appearance of these DNA damages is correlated with activation of ATM pathway, p53, and inhibition of CDK2 activity. Finally, Ve-822 + oxaliplatin association is also efficient in vivo, on immunodepressed mice xenografted with oxaliplatin-resistant cells as well as on immunocompetent mice, with a higher synergistic effect indicating that immune cell death (ICD) is part of the mechanism of this drug combination. In conclusion, all these data confirm the results of our screen by demonstrating for the first time the functional role of ATR in sensitivity to oxaliplatin. Our results highlight the strong potential of ATR inhibition combined to oxaliplatin in vitro but also in vivo to sensitize cells to chemotherapy, creating dramatic DNA damages and stimulating ICD. Citation Format: Eve Combes, Augusto Faria Andrade, Diego Tosi, Flavie Coquel, Delphine Désigaud, Veronique Garambois, Arnaud Coquelle, Pierre Martineau, Maguy Del Rio, Philippe Pasero, Jerome Moreaux, Roderick Beijersbergen, Nadia Vie, Céline Gongora. Synthetic lethality screening reveals ATR as responsible for oxaliplatin resistance in colorectal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A147.

PP2A as the Main Node of Therapeutic Strategies and Resistance Reversal in Triple-Negative Breast Cancer.

Triple negative breast cancer (TNBC), is defined as a type of tumor lacking the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The ER, PR and HER2 are usually the molecular therapeutic targets for breast cancers, but they are ineffective for TNBC because of their negative expressions, so chemotherapy is currently the main treatment strategy in TNBC. However, drug resistance remains a major impediment to TNBC chemotherapeutic treatment. Recently, the protein phosphatase 2A (PP2A) has been found to regulate the phosphorylation of some substrates involved in the relevant target of TNBC, such as cell cycle control, DNA damage responses, epidermal growth factor receptor, immune modulation and cell death resistance, which may be the effective therapeutic strategies or influence drug sensitivity to TNBCs. Furthermore, PP2A has also been found that could induce ER re-expression in ER-negative breast cancer cells, and which suggests PP2A could promote the sensitivity of tamoxifen to TNBCs as a resistance reversal agent. In this review, we will summarize the potential therapeutic value of PP2A as the main node in developing targeting agents, disrupting resistance or restoring drug sensitivity in TNBC.

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation.

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus – Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT;ApcMin/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT;ApcMin/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;ApcMin/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

Abstract 1081: APC regulation of breast cancer therapeutic resistance

Abstract Resistance to chemotherapy is one of the leading causes of death from breast cancer. Our lab discovered that Adenomatous Polyposis Coli (APC) loss in breast cancer cells results in an elevation of tumor-initiating cells (TICs) and resistance to chemotherapy-induced apoptosis. Given that TICs are often most resistant to standard chemotherapeutic compounds, we sought to understand the mechanism responsible for APC-mediated TIC enhancement. Our hypothesis is that the molecular mechanism involved in chemotherapeutic resistance parallels that promoting the TICs. APC-mutant cells have amplified activation of signal transducer and activator of transcription 3 (STAT3). Interestingly, inhibition of STAT3 with a small molecule inhibitor A69 decreases the TIC population and restores drug sensitivity. Studies are ongoing in the laboratory to investigate other molecular signaling pathways involved in APC-mediated enhanced TIC population and therapy resistance. These data have begun to reveal the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance. Note: This abstract was not presented at the meeting. Citation Format: Anne Arnason, Monica VanKlompenberg, Jenifer R. Prosperi. APC regulation of breast cancer therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1081. doi:10.1158/1538-7445.AM2017-1081

LncRNA UCA1 in anti-cancer drug resistance.

The pivotal role of the long non-coding RNA (lncRNA) urothelial carcinoma associated 1 (UCA1) in anti-cancer drug resistance has been confirmed in many cancers. Overexpression of lncRNA UCA1 correlates with resistance to chemotherapeutics such as cisplatin, gemcitabine, 5-FU, tamoxifen, imatinib and EGFR-TKIs, whereas lncRNA UCA1 knockdown restores drug sensitivity. These studies highlight the potential of lncRNA UCA1 as a diagnostic and prognostic biomarker, and a therapeutic target in malignant tumors. In this review, we address the role of lncRNA UCA1 in anti-cancer drug resistance and discuss its potential in future clinical applications.

About P‐glycoprotein: a new drugable domain is emerging from structural data

P‐glycoprotein (P‐gp) has been considered an important molecular target in the reversal of multidrug resistance (MDR). As such, the development of P‐gp modulators able to restore drug sensitivity in resistant cells is still considered one of the most promising strategies for overcoming MDR. Since the identification of the P‐gp's role in MDR, several studies have been performed in order to develop effective P‐gp modulators and understand the efflux mechanism. However, no efflux modulator is still clinically available for treating multidrug‐resistant cancers. Nevertheless, recent experimental studies suggest that MDR can be surpassed by targeting a specific region within the ABC transporter structure rather than the polyspecific drug‐binding pocket. This article will focus on the information available about this new target region and on a brief overview of which scaffolds would be suitable for modulating P‐gp at this new location. WIREs Comput Mol Sci 2017, 7:e1316. doi: 10.1002/wcms.1316This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics

G protein-coupled KISS1 receptor is overexpressed in triple negative breast cancer and promotes drug resistance

Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor α, progesterone receptor and human epidermal growth factor receptor 2 (HER2). TNBC patients lack targeted therapies, as they fail to respond to endocrine and anti-HER2 therapy. Prognosis for this aggressive cancer subtype is poor and survival is limited due to the development of resistance to available chemotherapies and resultant metastases. The mechanisms regulating tumor resistance are poorly understood. Here we demonstrate that the G protein-coupled kisspeptin receptor (KISS1R) promotes drug resistance in TNBC cells. KISS1R binds kisspeptins, peptide products of the KISS1 gene and in numerous cancers, this signaling pathway plays anti-metastatic roles. However, in TNBC, KISS1R promotes tumor invasion. We show that KISS1 and KISS1R mRNA and KISS1R protein are upregulated in TNBC tumors, compared to normal breast tissue. KISS1R signaling promotes drug resistance by increasing the expression of efflux drug transporter, breast cancer resistance protein (BCRP) and by inducing the activity and transcription of the receptor tyrosine kinase, AXL. BCRP and AXL transcripts are elevated in TNBC tumors, compared to normal breast, and TNBC tumors expressing KISS1R also express AXL and BCRP. Thus, KISS1R represents a potentially novel therapeutic target to restore drug sensitivity in TNBC patients.

Hypomethylation agent decitabine restores drug sensitivity by depressing P-glycoprotein activity through MAPK signaling pathway.

The multidrug resistance (MDR) continues to be an obstacle in the treatment of both hematological and solid tumors. Hypomethylation agent, decitabine (5-Aza-dC), is an experimental agent in MDR therapy, while the mechanism is not very clear. In the present study, we demonstrated 5-Aza-dC may reverse MDR induced by P-glycoprotein (P-gp) coded by mdr1 gene in both hematologic K562/ADR cells and solid tumor MCF-7/ADR cells with time and dose-dependent manner. 5-Aza-dC significantly increased drug sensitivity in patients' leukemic cells which had higher expression of mdr1 gene. Both total protein and membrane P-gp expression were up-regulated with 5-Aza-dC treatment in K562/ADR and MCF-7/ADR cells. However, accumulation of adriamycin and rhodamine 123 were increased which suggested the depression of P-gp activity. Gene expression profiling was performed and activation of MAPK signaling pathway was identified as the most significant change affected by 5-Aza-dC. Inhibition of MAPK pathway could increase P-gp activity. Our data suggested that hypomethylation agent decitabine restores drug sensitivity in the P-gp-induced MDR phenotype by depressing of P-gp activity as drug pump partly through MAPK signaling pathway.

Polyphyllin I Overcomes EMT-Associated Resistance to Erlotinib in Lung Cancer Cells via IL-6/STAT3 Pathway Inhibition.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is the most important limiting factor for treatment efficiency in EGFR-mutant non-small cell lung cancer (NSCLC). Much work has linked the epithelial-mesenchymal transition (EMT) to the emergence of drug resistance, consequently, ongoing research has been focused on exploring the therapeutic options to reverse EMT for delaying or preventing drug resistance. Polyphyllin I (PPI) is a natural compound isolated from Paris polyphylla rhizomes and displayed anti-cancer properties. In the current work, we aimed to testify whether PPI could reverse EMT and overcome acquired EGFR-TKI resistance. We exposed HCC827 lung adenocarcinoma cells to erlotinib which resulted in acquired resistance with strong features of EMT. PPI effectively restored drug sensitivity of cells that obtained acquired resistance. PPI reversed EMT and decreased interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway activation in erlotinib-resistant cells. Moreover, addition of IL-6 partially abolished the sensitization response of PPI. Furthermore, co-treatment of erlotinib and PPI completed abrogation of tumor growth in xenografts, which was associated with EMT reversal. In conclusion, PPI serves as a novel solution to conquer the EGFR-TKI resistance of NSCLC via reversing EMT by modulating IL-6/STAT3 signaling pathway. Combined PPI and erlotinib treatment provides a promising future for lung cancer patients to strengthen drug response and prolong survival.

Acriflavine Inhibits Acquired Drug Resistance by Blocking the Epithelial-to-Mesenchymal Transition and the Unfolded Protein Response

Epithelial-to-mesenchymal transition (EMT) is linked to tumor invasion, drug resistance and aggressive disease and this is largely dependent on the cell's microenvironment. Acriflavine (ACF) is an old antibacterial drug recently also suggested as anticancer agent and HIF inhibitor. We wanted to study the effect of acriflavine on EMT in different human cancer models. Pancreatic cancer cells (Panc-1) were exposed to TGF-β1 or cobalt chloride (to mimick severe hypoxia) to induce EMT. For our third model we exposed HepG2 liver cancer cells to sorafenib which resulted in development of acquired drug resistance with strong features of EMT and aggressive behavior. These models were morphologically and functionally (invasion assay) characterized. Markers of EMT were determined using qRT-PCR and Western blotting. Transcriptome analysis was performed following gene expression determination and combining the iRegulon tool and Gene Set Enrichment Analysis (GSEA). We made the following observations: (1) acriflavine inhibited EMT based on changes in cell morphology, invasive capacities and markers of EMT (at protein and gene expression level). (2) Transcriptome analysis revealed potent inhibition of ATF4 target genes and of the unfolded protein response. We showed that acriflavine blocked eIF2a phosphorylation and reduced ATF4 translation thereby inhibiting the PERK/eIF2a/ATF4 UPR pathway. (3) ACF restored drug sensitivity of cells that obtained acquired resistance. Conclusions: We identified acriflavine as a potent inhibitor of EMT and the UPR, thereby re-sensitizing the cancer cells to antineoplastic drugs.

Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia.

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence

Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.

Machine learning-, rule- and pharmacophore-based classification on the inhibition of P-glycoprotein and NorA

The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acctrain-diverse = 84%; accinternal-test = 92% and accexternal-test = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.

Minireview: The Link Between ERα Corepressors and Histone Deacetylases in Tamoxifen Resistance in Breast Cancer.

Approximately 70% of breast cancers express the estrogen receptor (ER)α and are treated with the ERα antagonist, tamoxifen. However, resistance to tamoxifen frequently develops in advanced breast cancer, in part due to a down-regulation of ERα corepressors. Nuclear receptor corepressors function by attenuating hormone responses and have been shown to potentiate tamoxifen action in various biological systems. Recent genomic data on breast cancers has revealed that genetic and/or genomic events target ERα corepressors in the majority of breast tumors, suggesting that the loss of nuclear receptor corepressor activity may represent an important mechanism that contributes to intrinsic and acquired tamoxifen resistance. Here, the biological functions of ERα corepressors are critically reviewed to elucidate their role in modifying endocrine sensitivity in breast cancer. We highlight a mechanism of gene repression common to corepressors previously shown to enhance the antitumorigenic effects of tamoxifen, which involves the recruitment of histone deacetylases (HDACs) to DNA. As an indicator of epigenetic disequilibrium, the loss of ERα corepressors may predispose cancer cells to the cytotoxic effects of HDAC inhibitors, a class of drug that has been shown to effectively reverse tamoxifen resistance in numerous studies. HDAC inhibition thus appears as a promising therapeutic approach that deserves to be further explored as an avenue to restore drug sensitivity in corepressor-deficient and tamoxifen-resistant breast cancers.

Abstract 5045: Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC

Abstract Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents. Citation Format: Yoshitaka Yagi, Hiroaki Ozasa, Takahiro Tsuji, Yuichi Sakamori, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5045.