Critical Role In Drug Resistance Research Articles

The RNA helicase Ski2 in the fungal pathogen Cryptococcus neoformans highlights key roles in azoles resistance and stress tolerance.

The yeast SKI (superkiller) complex was originally identified from cells that were infected by the M 'killer' virus. Ski2, as the core of the SKI complex, is a cytoplasmic cofactor and regulator of RNA-degrading exosome. The putative RNA helicase Ski2 was highly conserved from yeast to animals and has been demonstrated to play a key role in the regulation of RNA surveillance, temperature sensitivity, and growth in several yeasts but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Ski2 protein, named SKI2, in the fungal pathogen C. neoformans. To obtain insights into the function of Ski2, we created a mutant strain, ski2Δ, with the CRISPR-Cas9 editing tool. Disruption of SKI2 impaired cell wall integrity. Further investigations revealed the defects of the ski2Δ mutant in resistance to osmotic stresses and extreme growth temperatures. However, significantly, the ability to undergo invasive growth under nutrient-depleted conditions was increased in the ski2Δ mutant. More importantly, our results showed that the ski2Δ mutant exhibited slightly lower virulence and severe susceptibility to anti-ribosomal drugs by comparison to the wild type, but it developed multidrug resistance to azoles and flucytosine. By constructing the double deletion strain ski2Δafr1Δ, we verified that increased Afr1 in ski2Δ contributed to the azole resistance, which might be influenced by nonclassical small interfering RNA. Our work suggests that Ski2 plays critical roles in drug resistance and regulation of gene transcription in the yeast pathogen C. neoformans.

Autophagy activation mediates resistance to FLT3 inhibitors in acute myeloid leukemia with FLT3-ITD mutation

BackgroundAutophagy plays a critical role in drug resistance in acute myeloid leukemia (AML), including the subtype with FLT3-ITD mutation. Yet how autophagy is activated and mediates resistance to FLT3 inhibitors in FLT3-ITD-positive AML remains unsure.MethodsWe detected the expression of autophagy markers in FLT3-ITD-positive leukemic cells after vs. before acquired resistance to FLT3 inhibitors; tested the stimulative effect of acquired D835Y mutation and bone marrow micro-environment (BME) on autophagy; explored the mechanism of autophagy mediating FLT3 inhibitor resistance.ResultsSorafenib-resistant cells markedly overpresented autophagy markers in comparison with sorafenib-sensitive cells or the cells before sorafenib treatment. Both acquired D835Y mutation and BME activated cytoprotective autophagy to mediate FLT3 inhibitor resistance. Autophagy activation decreased the suppression efficacy of FLT3 inhibitors on FLT3 downstream signaling and then weakened their anti-leukemia effect. Inhibition of autophagy with CQ significantly enhanced the suppressive effect of FLT3 inhibitor on FLT3 downstream signaling, in the end overcame resistance to FLT3 inhibitors.ConclusionsAutophagy might be stimulated by acquired mutation or BME, and bypass activate FLT3 downstream signaling to mediate FLT3 inhibitor resistance in FLT3-ITD-positive AML. Targeting autophagy could be a promising strategy to overcome resistance.

Roles of RNA alternative splicing during the occurrence and development of lung cancer

<p indent="0mm">In the human genome, more than 90% of the human genes undergo alternative splicing, which participates in the entire life activity. Abnormal RNA splicing is closely associated with human diseases, including cancer. Cancer is still a serious public health concern as the second leading cause of death. Every year, lung cancer ranks first in the number of new cancer cases and deaths. Abnormal alternative splicing occurs extensively in lung cancer, regulating tumorigenesis and lung cancer progression. Splicing factor mutations and aberrant expression are well-known drivers of lung cancer progression. Furthermore, alternative splicing plays a critical role in drug resistance, radiotherapy resistance, angiogenesis, and tumor metabolism.

High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. DisclosuresBlachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

MicroRNA-29b-3p promotes 5-fluorouracil resistance via suppressing TRAF5-mediated necroptosis in human colorectal cancer.

Drug resistance in colorectal cancer is a great challenge in clinic. Elucidating the deep mechanism underlying drug resistance will bring much benefit to diagnosis, therapy and prognosis in patients with colorectal cancer. In this study, miR-29b-3p was shown to be involved in resistance to 5-fluorouracil (5-FU)-induced necroptosis of colorectal cancer. Further, miR-29b-3p was shown to target a regulatory subunit of necroptosis TRAF5. Rescue of TRAF5 could reverse the effect of miR-29b-3p on 5-FU-induced necroptosis, which was consistent with the role of necrostatin-1 (a specific necroptosis inhibitor). Then it was demonstrated that miR-29b-3p was positively correlated with chemoresistance in colorectal cancer while TRAF5 negatively. In conclusion, it is deduced that miR-29b-3p/TRAF5 signaling axis plays critical role in drug resistance in chemotherapy for colorectal cancer patients by regulating necroptosis. The findings in this study provide us a new target for interfere therapy in colorectal cancer.

Effect of glutamine metabolism on chemoresistance and its mechanism in tumors.

The metabolic reprogramming of tumor cells is characterized by increased uptake of various nutrients including glutamine. Glutamine metabolism provides the required substances for glycolysis and oxidative phosphorylation and affects the homeostasis of carbohydrate，fat and protein metabolism to induce the chemoresistance of tumor cells. Combination of chemotherapeutic agents with inhibitors specific to different components of glutamine metabolic pathway has obtained favorable clinical results on various tumors. Glutamine metabolic pathway plays a role in drug resistance of tumor cells in various ways. Firstly，the dynamic change of glutamine transporters can directly affect intracellular glutamine content thereby causing drug resistance; secondly，tumor stromal cells including adipocyte，fibroblast and metabolite from tumor microenvironment would give rise to immune-mediated drug resistance; thirdly，the expression and activity of key enzymes in glutamine metabolism also has a critical role in drug resistance of tumors. This article reviews the effects of glutamine metabolic pathway in the development of tumor chemoresistance，in terms of transporters，tumor microenvironment and metabolic enzymes，to provide insight for improving the therapeutic efficacy for drug-resistant tumors.

Targeted Brain Tumor Therapy by Inhibiting the MDM2 Oncogene: In Vitro and In Vivo Antitumor Activity and Mechanism of Action.

There is a desperate need for novel and efficacious chemotherapeutic strategies for human brain cancers. There are abundant molecular alterations along the p53 and MDM2 pathways in human glioma, which play critical roles in drug resistance. The present study was designed to evaluate the in vitro and in vivo antitumor activity of a novel brain-penetrating small molecule MDM2 degrader, termed SP-141. In a panel of nine human glioblastoma and medulloblastoma cell lines, SP-141, as a single agent, potently killed the brain tumor-derived cell lines with IC50 values ranging from 35.8 to 688.8 nM. Treatment with SP-141 resulted in diminished MDM2 and increased p53 and p21cip1 levels, G2/M cell cycle arrest, and marked apoptosis. In intracranial xenograft models of U87MG glioblastoma (wt p53) and DAOY medulloblastoma (mutant p53) expressing luciferase, treatment with SP-141 caused a significant 4- to 9-fold decrease in tumor growth in the absence of discernible toxicity. Further, combination treatment with a low dose of SP-141 (IC20) and temozolomide, a standard anti-glioma drug, led to synergistic cell killing (1.3- to 31-fold) in glioma cell lines, suggesting a novel means for overcoming temozolomide resistance. Considering that SP-141 can be taken up by the brain without the need for any special delivery, our results suggest that SP-141 should be further explored for the treatment of tumors of the central nervous system, regardless of the p53 status of the tumor.

Enhancement of cisplatin-induced cytotoxicity against cervical cancer spheroid cells by targeting long non-coding RNAs

Cervical cancer (CVC) is one of the most common types of gynecologic malignant tumor in the world. Unfortunately, current treatments including chemo-/radiotherapy still show the limitation on CVC progress. It is well known that cancer stem cells (CSCs) plays a critical role in drug resistance and metastasis. Furthermore, dysregulation of long non-coding RNA (LncRNA) shows close association to tumorigenesis and development of multiple cancers. In this study, we investigated the cytotoxic effect of cisplatin, a clinical chemotherapeutic for cervical cancer treatment, on parental and spheroid CVC cells and surveyed the effect of LncRNA on drug-resistance. We found that spheroid CVC cells showed much more resistant to cisplatin-induced cytotoxicity compared with parental CVC cells. Furthermore, cisplatin significantly induced apoptotic cell death, while it induced cell cycle arrest in G0/G1 phase at the same dose (10 μg/ml). We also found the significant expression of EGFR in spheroid instead of parental CVC cells. Interestingly, we revealed that protruding target lncRNAs were up-regulated in cisplatin-treated spheroid CVC cells, and inhibition of these lncRNAs enhanced the cytotoxicity of cisplatin against spheroid CVC cells. These data suggest that LncRNA might act as a critical modulator on drug-resistant capability of cervical CSCs and would be a novel target for cervical cancer treatment.

Over-expression of miR-206 decreases the Euthyrox-resistance by targeting MAP4K3 in papillary thyroid carcinoma

PurposemicroRNAs (miRNAs) play a critical role in drug resistance of multiple cancers including papillary thyroid carcinoma (PTC), indicating the potential of miRNAs as chemoresistance regulators in cancer treatment. The aim of this paper is to explore the relationship between miR-206 and chemoresistance of PTC. MethodsqRT-PCR was conducted to examine the expression of miR-206 in PTC tissues, parental and TPC-1/euthyrox. The CCK-8 assay, EdU assay and flow cytometry were performed to test cells viability, proliferation and apoptosis, respectively. Luciferase reporter assay was used to confirm the potential target of miR-206. Western blotting analysis was performed to evaluate the expressions of related-proteins. ResultsmiR-206 was significantly down-regulated in PTC tissues, parental and TPC-1/euthyrox. Moreover, the expression of miR-206 was exceptionally lower in TPC-1/euthyrox cells than that in TPC-1 cells. Furthermore, we found that over-expression of miR-206 could notably decrease the IC50 values both in TPC-1 and TPC-1/euthyrox cells, which indicated that miR-206 played an essential role in the euthyrox resistance in PTC. In addition, up-regulation of miR-206 inhibited the proliferation, induced apoptosis, suppressed the expressions of multidrug resistance-related proteins, including p-gp, MRP, BCRP and LRP, in euthyrox-resistant PTC cells. Besides, over-expression of miR-206 could notably promoted the expression of NIS, an intrinsic membrane protein that mediates the active transport of iodide into the thyroid and other tissues, playing a critical role in the progress. Further, miR-206 was demonstrated to be able to bind to MAP4K3 and negatively regulated the expression of MAP4K3. Besides, MAP4K3 was clearly up-regulated in PTC tissues, parental and TPC-1/euthyrox cells, and down-regulation of miR-206 attenuated the effect of si-MAP4K3 on the euthyrox sensitivity in euthyrox-resistant PTC cells. Moreover, TPC-1/euthyrox cells transfected with miR-206 mimics could significantly inhibit the expressions of p-p38, p-JNK and p-Erk, which indicated that miR-206 might play an essential role in the euthyrox resistance in PTC by negatively regulating the p38 and JNK signaling pathway. ConclusionmiR-206 contributed to euthyrox resistance in PTC cells through blockage p38 and JNK signaling pathway by targeting MAP4K3, providing a potential therapeutic application for the treatment of patients with euthyrox-resistant PTC in the further.

Protein Arginine Methyltransferase 3 Enhances Chemoresistance in Pancreatic Cancer by Methylating hnRNPA1 to Increase ABCG2 Expression.

Pancreatic cancer is poorly responsive to chemotherapy due to intrinsic or acquired resistance. Our previous study showed that epigenetic modifying enzymes including protein arginine methyltransferase 3 (PRMT3) are dysregulated in gemcitabine (GEM)-resistant pancreatic cancer cells. Here, we attempt to elucidate the role of PRMT3 in chemoresistance. Overexpression of PRMT3 led to increased resistance to GEM in pancreatic cancer cells, whereas reduction of PRMT3 restored GEM sensitivity in resistant cells. We identified a novel PRMT3 target, ATP-binding cassette subfamily G member 2 (ABCG2), which is known to play a critical role in drug resistance. PRMT3 overexpression upregulated ABCG2 expression by increasing its mRNA stability. Mass spectrometric analysis identified hnRNPA1 as a PRMT3 interacting protein, and methylation of hnRNPA1 at R31 by PRMT3 in vivo and in vitro. The expression of methylation-deficient hnRNPA1-R31K mutant reduced the RNA binding activity of hnRNPA1 and the expression of ABCG2 mRNA. Taken together, this provides the first evidence that PRMT3 methylates the RNA recognition motif (RRM) of hnRNPA1 and promotes the binding between hnRNPA1 and ABCG2 to enhance drug resistance. Inhibition of PRMT3 could be a novel strategy for the treatment of GEM-resistant pancreatic cancer.

Down-regulation of miR-377 contributes to cisplatin resistance by targeting XIAP in osteosarcoma.

Chemoresistance is one of the obstacles for effective treatment of cancers, and recent evidence has shown that microRNAs play critical roles in drug resistance. In this study, we investigated the effect of miR-377 on cisplatin resistance in osteosarcoma (OS). Tumor tissues from chemoresistant and control OS patients were subjected to Real-time polymerase chain reaction (PCR) to assess miR-377 expression. The effect and mechanism of miR-377 on cisplatin resistance were assessed using Cell Counting Kit (CCK) 8, flow cytometry, Western blot, and luciferase assays in cisplatin-resistant OS cells lines. Down-regulation of miR-377 was found in chemoresistant OS tissues and cisplatin-resistant OS cell lines. Overexpression of miR-377 re-sensitizes cisplatin resistant OS cells to cisplatin-induced caspase-3 dependent apoptosis. MiR-377 directly represses X-linked inhibitor of apoptosis protein (XIAP) expression through binding to its 3' untranslated region (UTR) of mRNA. Overexpression of XIAP partially cancelled the cisplatin-sensitizing effect of miR-377. These data uncovered an essential function of miR-377/XIAP signaling axis in regulating cisplatin resistance of OS. MiR-377 may have potential therapeutic values in tackling OS chemoresistance.

Gemcitabine treatment causes resistance and malignancy of pancreatic cancer stem-like cells via induction of lncRNA HOTAIR.

Gemcitabine is the first-line chemotherapeutic agent for advanced adenocarcinoma of the pancreas, despite the high risk of chemoresistance as a major disadvantage. In the past few years, significant advances have been made in the field of pancreatic cancer stem-like cells (CSCs) and their critical roles in drug resistance, invasion and metastasis, which are tightly regulated by long non-coding RNAs (lncRNAs). The present study demonstrated that HOX antisense intergenic RNA (HOTAIR) is not different between the pancreatic cancer cell line PANC-1 and its enriched CSC sub-population. However, after gemcitabine treatment, the expression levels of HOTAIR in CSCs were induced, but not in PANC-1 cells. HOTAIR induced by gemcitabine failed to cause chemoresistance, but promoted the clonogenicity, proliferation and migration of the cells. By introducing HOTAIR using lentivirus, chemoresistance was induced and the self-renewal capacity, proliferation and migration were significantly promoted. By contrast, HOTAIR knockdown in PANC-1 CSCs treated with or without gemcitabine decreased the cell proliferation, altered the cell cycle progression and induced apoptosis, demonstrating its critical roles in regulating the malignant character of PANC-1 CSCs. In conclusion, the present study demonstrated that HOTAIR may be induced by gemcitabine and acts as a tumor promoter by inhibiting the chemosensitivity, and promoting the self-renewal capacity, proliferation and migration of PANC-1 CSCs, which supports its potential application as a novel therapeutic approach for pancreatic cancer.

Exosomes play an important role in the process of psoralen reverse multidrug resistance of breast cancer.

BackgroundRelease of exosomes have been shown to play critical roles in drug resistance by delivering cargo. Targeting the transfer of exosomes from resistant cells to sensitive cells may be an approach to overcome some cases of drug resistance.MethodIn this study, we investigated the potential role of exosomes in the process of psoralen reverse multidrug resistance of MCF-7/ADR cells. Exosomes were isolated by differential centrifugation of culture media from MCF-7/ADR cells (ADR/exo) and MCF-7 parental cells (S/exo). Exosomes were characterized by morphology, exosomal markers and size distribution. The ability of ADR/exo to transfer multidrug resistance was assessed by MTT and real-time quantitative PCR. The different formation and secretion of exosomes were detected by immunofluorescence and transmission electron microscopy. Then we performed comparative transcriptomic analysis using RNA-Seq technology and real-time quantitative PCR to better understand the gene expression regulation in exosmes formation and release after psoralen treatment.ResultsOur data showed that exosomes derived from MCF-7/ADR cells were able to promote active sequestration of drugs and could induce a drug resistance phenotype by transferring drug-resistance-related gene MDR-1 and P-glycoprotein protein. Psoralen could reduce the formation and secretion of exosomes to overcome drug resistance. There were 21 differentially expressed genes. Gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most significantly expressed genes were linked to PPAR and P53 signaling pathways which were related to exosomes formation, secretion and cargo sorting.ConclusionsPsoralen can affect the exosomes and induce the reduction of resistance transmission via exosomes might through PPAR and P53 signaling pathways, which might provide a novel strategy for breast cancer resistance to chemotherapy in the future.

Abstract LB-007: Interaction between integrin α4β1 and IGF1 and its role in IGF signaling in hematologic malignancies

Abstract Integrins are transmembrane αβ heterodimers that function as cell adhesion receptors. Integrins are aberrantly overexpressed in cancer cells and also in tumor-associated cells. They are involved in cancer cell proliferation, invasion and survival. It has been proposed that the interaction between integrin α4β1, a major integrin in hematologic cancer, and VCAM-1 and fibronectin is responsible for drug resistance in these malignances, since antagonists to this integrin reduce drug resistance. However these results are based on the assumption that VCAM-1 and fibronectin are the only ligands for α4β1. Insulin-like growth factor 1 (IGF1) signaling plays a critical role in drug resistance and radio resistance hematologic cancer. IGF1 is a polypeptide hormone that binds to tyrosine kinase receptor IGF1R and activates the PI3K/Akt signaling pathway. This pathway is involved in cell proliferation, survival, and inhibition of apoptosis. In various types of cancer IGF1 is over expressed and the PI3K/Akt pathway is activated at a much higher rate than in normal cells. This results in the growth of cancer cells and resistance to chemotherapy. IGF1 signaling is implicated in drug resistance, and is a good therapeutic target in hematologic cancers. It is unclear if α4β1 and IGF signaling work independently in drug resistance. Recently we established that IGF1 binds to integrins αvβ3 and α6β4, and subsequent integrin-IGF1-IGF1R ternary complex formation is a critical step in IGF signaling in non-hematologic cancer. We developed the R36E/R37E IGF1 mutant that is defective in integrin binding and a dominant-negative antagonist of IGF signaling. Here we show that IGF1 is a ligand for integrin α4β1 and induces a ternary complex with IGF1R and α4β1. Antagonists to integrin α4β1 and the R36E/R37E IGF1 mutant reduced intracellular signaling in Chinese hamster ovary cells expressing human integrin α4 (α4-CHO cells) and also in different hematologic cancer cells such as Reh, Molt-4, U937 and JY cells. These results suggest that integrin α4β1 is involved in IGF signaling through direct binding to IGF1 in hematologic cancer. The α4β1-IGF1 interaction is a novel therapeutic target for drug resistance in hematologic cancer. Citation Format: Dora M. Cedano Prieto, Jessica Yu, Yoko Takada, Yoshikazu Takada. Interaction between integrin α4β1 and IGF1 and its role in IGF signaling in hematologic malignancies. [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 LB-007.

MiR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET.

MicroRNAs (miRNAs) play a critical role in drug resistance and epithelial-mesenchymal transition (EMT). The aims of this study were to explore the potential role of miR-206 in governing cisplatin resistance and EMT in lung cancer cells. We found that both lung adenocarcinoma A549 cisplatin-resistant cells (A549/DDP) and H1299 cisplatin-resistant cells (H1299/DDP) acquired mesenchymal features and were along with low expression of miR-206 and high migration and invasion abilities. Ectopic expression of miR-206 mimics inhibited cisplatin resistance, reversed the EMT phenotype, decreased the migration and invasion in these DDP-resistant cells. In contrast, miR-206 inhibitors increased cisplatin resistance, EMT, cell migration and invasion in non-DDP-resistant cells. Furthermore, we found that MET is the direct target of miR-206 in lung cancer cells. Knockdown of MET exhibited an EMT and DDP resistant inhibitory effect on DDP-resistant cells. Conversely, overexpression of MET in non-DDP- resistant cells produced a promoting effect on cell EMT and DDP resistance. In lung adenocarcinoma tissues, we demonstrated that low expression of miR-206 were also correlated with increased cisplatin resistance and MET expression. In addition, we revealed that miR-206 overexpression reduced cisplatin resistance and EMT in DDP-resistant cells, partly due to inactivation of MET/PI3K/AKT/mTOR signaling pathway, and subsequent downregulation of MDR1, ZEB1 and Snail expression. Finally, we found that miR-206 could also sensitize A549/DDP cells to cisplatin in mice model. Taken together, our study implied that activation of miR-206 or inactivation of its target gene pathway could serve as a novel approach to reverse cisplatin resistance in lung adenocarcinomas cells.

Selinexor Is an Effective Cancer Treatment in Hypoxic Conditions and Synergizes with Proteasome Inhibitors to Treat Drug Resistant Multiple Myeloma

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) associated with lytic bone disease. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance. Low oxygenation (hypoxia) in the BM microenvironment, which was shown to decrease beyond physiologic BM conditions during the progression of MM, together with cellular and acellular components, plays a critical role in drug resistance. Increased levels of the nuclear exporter of tumor suppressors, exportin 1 (XPO1), was demonstrated in MM, and XPO1 inhibition showed a broad anti-tumor activity. In this study, we tested inhibition of XPO1 using the novel oral, covalent, slowly reversible inhibitor of nuclear export selinexor currently in Phase I/II clinical trials on sensitizing the hypoxia-inducible drug resistant MM cells both in vitro and in vivo.Methods: MM cell lines (MM1s, H929, OPM2 and U266) were treated with increasing concentrations of selinexor in normoxic (21% O2) and hypoxic (1% O2) conditions with or w/o bortezomib or carfilzomib. Assays included MTT proliferation Annexin V/PI staining for apoptosis, PI staining and immuniblots for cell cycle analysis.We tested the effects of selinexor as a single agent on tumor initiation in vivo, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and mice were treated instantaneously with selinexor (15mg/kg) or vehicle (PVP + F-68) three times a week. We also tested the effect of selinexor (15mg/kg) as a single agent on tumor progression and survival in mice with established MM tumor. Furthermore, we studied the effects of selinexor on sensitization to bortezomib-resistant MM cells in vivo. MM1s-Luc-GFP cells were injected IV into SCID mice and tumors were allowed to grow for three weeks. Animals were then treated with a high-dose of bortezomib (1.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were then randomly divided into 3 groups treated with (1) bortezomib (0.5mg/kg), (2) KPT-330 (10mg/kg), and (3) a combination of selinexor and bortezomib. Selinexor was administered by oral gavage three times a week (days 1, 3 and 5) and bortezomib was injected intraperitoneally twice a week (days 1 and 4). Tumor progression was imaged using bioluminescence imaging (BLI).Results: Selinexor, as a single agent, decreased proliferation, induced G1 cell cycle arrest and increased apoptosis of MM cells in both hypoxia and normoxia in a dose-dependent manner. Moreover, we found that the combination of selinexor with bortezomib or carfilzomib reversed the hypoxia-induced resistance in MM cells to proteasome inhibition and induced a synergistic effect on the inhibition of cell proliferation, G1 arrest and apoptosis, which was confirmed by response of respective cell signaling pathways as PI3K, MAPK and JNK.In vivo studies revealed that selinexor alone delayed tumor initiation and decreased by half existing tumor size as a single agent (p=0.0397), and significantly extended mice survival (53 days on vehicle versus 62 days on selinexor, p=000375). In addition, MM-bearing mice which developed resistance to bortezomib were resensitized by treatment of selinexor combined with bortezomib, decreasing tumor burden and showed considerably extended survival (half of the mice were alive at day 52) compared to mice treated with either selinexor (half of the mice were alive at day 49) or bortezomib alone (half of the mice were alive at day 40) (bortezomib versus selinexor p=0.0014, bortezomib versus combination treatment p=0.0004 as analyzed by Student t-test).Conclusions: The orally available inhibitor of nuclear export, selinexor, decreased survival of MM cells in vitro and delayed tumor initiation and tumor progression in vivo as a single agent. In addition, selinexor overcame hypoxia-induced resistance to proteasome inhibitors and resensitized MM cells to therapy both in vitro and in vivo, extending mice survival. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by inhibition of XPO1 with selinexor as well as combination studies of selinexor and proteasome inhibitors. A study of selinexor and carfilzomib is currently ongoing and early results that were published last year showed marked activity for this combination treatment in heavily pretreated MM patients. DisclosuresLandesman:Karyopharm: Employment. Azab:Karyopharm: Research Funding; Selexys: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Cell Works: Research Funding.

Epithelial-mesenchymal transition plays a critical role in drug resistance of hepatocellular carcinoma cells to oxaliplatin.

Drug resistance is one characteristic of hepatocellular carcinoma (HCC) and can affect the prognosis of patients directly. To explore drug resistance well, we established an oxaliplatin (OXA)-resistant cell line Bel-7402/OXA by exposure to gradually increased concentration of OXA. Some biological characters, such as proliferation, migration, and invasion, were studied. Drug sensitivity and the mechanisms of drug resistance were also investigated. We found that the resistant index of Bel-7402/OXA was 8.3. In comparison with Bel-7402, the percentages of cells in S and G2/M phase were increased. The nature apoptosis rate and drug-after apoptosis rate were all decreased in Bel-7402/OXA, as compared to Bel-7402. Bel-7402/OXA acquired increased migration and invasion ability with epithelial-mesenchymal transition (EMT) phenotype. Knockdown of EMT transcription factor Snail could reverse EMT and sensitized Bel-7402/OXA cells to OXA. EMT was one mechanism of drug resistance and may be a novel target of treatment for drug resistance.

Silencing of TGM2 reverses epithelial to mesenchymal transition and modulates the chemosensitivity of breast cancer to docetaxel

Epithelial to mesenchymal transition (EMT) plays a critical role in drug resistance. The aim of the present study was to further elucidate its role by examining the effect of tissue transglutaminase (TG2) on EMT and drug resistance in breast cancer. An antisense lentiviral (LV) short hairpin (sh)RNA construct specific to the TG2 gene (TGM2) was designed, synthesized and stably transfected into MDA-MB-231 cells to silence TGM2 by RNA interference (RNAi). The transfected cells expressed low levels of TG2 and constituted the RNAi (TGM2-shRNA) group. A control (NC) group was also established by transfecting MDA-MB-231 cells with scrambled shRNA. The expression levels of TG2, E-cadherin, vimentin and B-cell lymphoma (Bcl)-2 in the cells were examined via western blotting. The transfected MDA-MB-231 cells were divided into four groups, two of which were treated with doxetaxel (TXT): NC, RNAi, TXT and RNAi + TXT groups,. Cell proliferation was analyzed by MTT assay and cell apoptosis was detected by flow cytometry. An in vivo assay was also conducted, in which MDA-MB-231 cells transfected with scrambled shRNA or TGM2-shRNA were subcutaneously implanted into nude mice. After 2 weeks, TXT or vehicle was intraperitoneally administered at a dose of 10 mg/kg on day 1 of every week and tumor growth was monitored. Following the silencing of TGM2 in the MDA-MB-231 cells, the cells showed changes in morphology, indicating that an increased expression of TG2 was associated with a mesenchymal morphology. Transfection of the cells with TGM2-shRNA affected the expression of TG2, E-cadherin, vimentin and Bcl-2. In the MTT assay, the proliferation of MDA-MB-231 cells was significantly inhibited in the RNAi group compared with the control group (P<0.05), and the inhibitory effect increased in a time-dependent manner. Following treatment with TXT for 48 h, apoptosis was significantly promoted in the RNAi + TXT group compared with that in the other groups (P<0.05). Measurement of the tumors in the nude mice indicated that the combination of RNAi and TXT brought about a stronger antitumor effect than either treatment alone. These results suggest that the downregulation of TG2 reversed EMT and modulated the chemosensitivity of breast cancer to TXT. TG2 may be an important predictive and prognostic factor for the treatment efficacy of chemotherapy in patients with breast cancer.

Abstract 4842: The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes

Abstract The bone marrow (BM) microenvironment plays a critical role in drug resistance of malignant cells. Although both, direct contact as well as soluble factors have been proposed, the mechanisms responsible for this microenvironment-mediated protection remain unclear. We have recently shown that BM stroma protects human hematopoietic stem cells from the pro-differentiating effects of retinoid via cytochrome P450 (CYP) 26 mediated degradation (Ghiaur G et al., PNAS 2013). Here, we hypothesized that drug metabolism by BM stroma via CYP enzymes is a general cell-extrinsic mechanism of protection of malignant cells from chemotherapy. First, we have designed a qPCR expression array platform and found that human BM stroma expresses a variety of drug metabolizing enzymes to levels comparable to human hepatocytes. Next, using qRT-PCR, we confirmed that CYP3A4, a major drug metabolizing enzyme, is expressed in primary BM stroma but not in multiple myeloma (MM) or acute myeloid leukemia (AML) cells. We have found that BM stroma protects against Bortezomib mediated kill of MM cells [H929 (p&amp;lt;0.01)] and VP16 mediated kill of AML cells [KG1 (p0.01)] (Table). Since CYP3A4 plays a major role in the metabolism of aforementioned drugs, we studied if stromal CYP3A4 contributes to this protection. We found that ketoconazole, a potent CYP3A4 antagonist, overcomes bone marrow stroma-mediated resistance to Bortezomib (p=0.01), and VP16 (p=0.03). Ketoconazole had no effects in stroma free cultures. In addition, shRNA-mediated knockdown of stromal CYP3A4 renders these cells unable to protect malignant cells from chemotherapy (p=0.01). These studies open the possibilities that the stem cell niche serves as a drug-free sanctuary, where malignant stem cells can survive conventional chemotherapy. Targeting stromal P450 enzymes hold promise of improved efficiency of stem cell directed therapies and decrease rate of relapse in hematological malignancies. Table.Effects of VP16 and Bortezomib on cellular and clonogenic recovery of KG1 and H929 cells, respectively. “Stroma-CYP3A4” represents experiments performed on CYP3A4 knockdown stroma. Control shRNA infected stroma showed no differences compared to untransduced cells. Data are presented as mean±SEM from three independent experiments.VP16: KG1 cellular recoveryBortezomib: H929 clonogenic (CFU-C) recoveryLiquidStromaStroma+ketoconazoleStroma- CYP3A4LiquidStromaStroma +ketoconazoleStroma-CYP3A48.10 ± 1.5529.38 ± 0.515.7 ± 2.719.35 ± 0.8315.77 ± 3.289.5 ± 2.549.33 ± 860.35 ± 4.2 Citation Format: Salvador Alonso, Meng Su, Richard Jones, Gabriel Ghiaur. The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4842. doi:10.1158/1538-7445.AM2014-4842

IFI16 mis-localization can be a contributing factor to hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is a highly deadly cancer, with usually drug resistance. However the mechanisms responsible for this phenomenon are poorly understood. Interferon-γ inducible protein 16 (IFI16), a multifunctional protein, has roles in anti-proliferation, autophagy, cell senescence, anti-inflammation, and DNA sensor to trigger innate immunity. IFI16 physiologically absents in adult healthy hepatocyte, but exists in liver cancer cells. Interestingly, increasing evidences suggest that dysregulation or/and loss of IFI16 function have a critical role in drug resistance and tumor progression. Furthermore, interaction with DNA or other protein depends on IFI16 localization. In our study, to our knowledge, we first showed that IFI16 is a chromatin-binding protein in four HCC cell lines with different TP53 genotype, but not in fetal liver cell line, L02 cells. However, the function of IFI16 subcellular localization has not been determined in HCC. Therefore, we present our study and theoretical basis and presume that chromatin-bounding localization of IFI16 is associated with HCC progression. If we are able to acetylate or/and delete NLS of IFI16 with activated-p53 restoration, we may offer an alternative for HCC therapy.