Overcoming Resistance to 5-Azacytidine in Acute Myelogenous Leukemia

Abstract Abstract 1370 Background: The DNA methylation inhibitor 5-azacytidine (AZA), which is approved for treatment of myelodysplastic syndrome, is also a potential agent for treatment of leukemia; however, drug resistance is an ongoing problem, and mechanisms underlying developing resistance to AZA are poorly understood. Therefore, clarifying the resistance mechanisms is central to establish effective countermeasures. Methods: To probe the mechanisms of resistance to AZA and to develop an effective method for overcoming them, we first generated two AZA-resistant cell lines, THP-1/AR and HL60/AR, from the human acute myelogenous leukemia cell lines THP-1 and HL60. We then studied variations between the parental and resistant lines. Results: AZA increased the percentages of sub-G1 and G2/M-phase cells in the AZA-sensitive parental cell lines; whereas, it had no similar effect in the resistant lines. Consistent with these results, the AZA-induced increases in the levels of cleaved forms of caspase 3, caspase 7, caspase 9, and PARP seen in sensitive cells were diminished in resistant cells. Furthermore, AZA markedly elevated the level of phospho JNK/SAPK in sensitive cells, but not in resistant cells. These results suggest that AZA induced apoptosis as well as G2/M arrest due to activation of JNK/SAPK signaling, and that induction of these changes was prevented in resistant cells. We also found that the activity as well as protein levels of DNA methyltransferases (DNMTs), which are the main target molecules of AZA, were suppressed by AZA in sensitive cells. However, in resistant cells, this effect was abrogated; and accordingly, AZA-induced up-regulation of p16 gene expression was also negated. These findings thus suggest that resistance was acquired by a DNMT-dependent mechanism. There was no remarkable difference between resistant cells and sensitive cells in the levels of uridine-cytidine kinase 2 (UCK2), which is a key enzyme for conversion of AZA to active form. However, several point mutations were found restrictedly in exon 4 of the UCK2 gene in both resistant cells. These results raised the possibility that the AZA activation process was perturbed due to reduction of UCK activity; and consequently, AZA failed to suppress DNMT in resistant cells. In addition, by microarray analysis, we identified eleven genes that were expressed at significantly different levels in resistant cells versus sensitive cells. Finally, we showed that the histone deacetylase inhibitor romidepsin induced p16 gene expression and increased the levels of apoptosis-related molecules, while suppressing growth in both sensitive and resistant cell lines. An isobologram analysis demonstrated that simultaneous administration of AZA and romidepsin resulted in an additive inhibitory effect on both AZA-sensitive and AZA-resistant cell growth. These results suggest that romidepsin can overcome AZA resistance; therefore, the combination of AZA and romidepsin not only augments the anti-leukemia effect but also prevents acquisition of resistance to AZA. Conclusions: Newly established 5-azacytidine-resistant cell lines THP-1/AR and HL60/AR are good models to analyze the mechanisms of drug resistance to 5-azacytidine. Using these cell lines, we revealed that acquisition of resistance is primarily caused by a DNMT-dependent mechanism, which can be surmounted with addition of romidepsin. It is likely that the combination of AZA and romidepsin can prevent patients from acquiring resistance to AZA while augmenting its anti-leukemia therapeutic effect. Disclosures: No relevant conflicts of interest to declare.

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Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.113.113 ◽

2009 ◽

Vol 114 (22) ◽

pp. 113-113 ◽

Cited By ~ 1

Author(s):

Chad C. Bjorklund ◽

Deborah J. Kuhn ◽

Jairo A. Matthews ◽

Michael Wang ◽

Veerabhadran Baladandayuthapani ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Lines ◽

Expression Profiling ◽

Fold Increase ◽

Resistant Cell ◽

Refractory Disease ◽

Wild Type ◽

Myeloma Cells ◽

Resistant Cells

Abstract Abstract 113 Background: Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods: To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results: Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions: These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures: No relevant conflicts of interest to declare.

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Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404.

Blood ◽

10.1182/blood.v108.11.1826.1826 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1826-1826

Author(s):

Stuart S. Winter ◽

Hadya Khawaja ◽

Zeyu Jiang ◽

Timothy Griffin ◽

Barbara Asselin ◽

...

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Resistant Cell ◽

Acquired Drug Resistance ◽

Induction Failure

Abstract The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children’s Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.

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A New Form Of Therapeutic Resistance: Drug Glucuronidation Regulated By The Sonic Hedgehog Factor Gli1

Blood ◽

10.1182/blood.v122.21.821.821 ◽

2013 ◽

Vol 122 (21) ◽

pp. 821-821

Author(s):

Hiba A Zahreddine ◽

Biljana Culjkovic-Kraljacic ◽

Sarit Assouline ◽

Abdellatif Amri ◽

Patrick Gendron ◽

...

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Phase Ii ◽

Resistant Cell ◽

Type I ◽

Type Ii ◽

Protein Levels ◽

Gli 1 ◽

Type Ii Resistance ◽

Resistant Cells

Abstract Despite many recent successes in the treatment of cancer, the development of chemoresistance in many of the initially responding patients, and primary resistance in others, remains a major impediment in therapy development. Our studies provide evidence for a novel mechanism underlying drug resistance: Gli1 dependent drug glucuronidation. While carrying out a Phase II clinical trial of targeting the eukaryotic translation initiation factor eIF4E with ribavirin in M4/M5 subtypes of AML, we observed that all responding patients eventually became clinically and molecularly resistant. To understand the cause of this resistance, we generated ribavirin resistant cell lines. In these models, ribavirin no longer targeted eIF4E activity or impaired growth, and importantly, the ability of ribavirin to bind eIF4E was severely impaired. However, the eIF4E gene was not mutated and its protein levels were not altered. The cell lines could be divided into two groups: type I with a defect in drug uptake and type II with a normal uptake. In type I resistant cells, we observed a substantial reduction in levels of Adenosine Kinase (ADK) an enzyme that catalyzes the rate limiting step in the metabolic activation of ribavirin allowing its retention in the cells. We used RNA Sequencing to examine the molecular underpinnings of type II resistance. Our data revealed a drastic increase in the levels of Gli1. In stably overexpressing cells, Gli1 was sufficient to produce the same resistance phenotype that we observed for type II cell models, both molecularly and at the level of cell growth. In addition, Gli1 overexpression correlated with the loss of drug-to-target interaction, as observed by our eIF4E immunoprecipitation studies using 3H-Ribavirin, similarly to the resistant cell lines. Conversely, Gli1 knockdown in type II cells or its pharmacological inhibition with the FDA approved Gli1 inhibitor GDC0449/Vismodegib, restored the eIF4E-ribavirin interaction and re-sensitized these cells to ribavirin. Our subsequent studies revealed a close correlation between Gli1 expression and the protein levels of the UGT1A glucuronosyl transferase enzymes involved in phase II drug metabolism whereby xenobiotics or metabolites are modified by the addition of a sugar, glucuronic acid. Given these findings, we examined whether the loss of the eIF4E interaction in resistant cells was due to the glucuronidation of ribavirin. Using 13C/12C ribavirin and mass spectrometry, we observed glucuronidated forms of ribavirin in resistant cells and cells overexpressing Gli1 but not in parental cells and that ribavirin is glucuronidated on its triazole ring which binds eIF4E. Treatment of cells with the Gli1 inhibitor GDC0449 reduces UGT1A levels, and correlates with reduced levels of ribavirin-glucuronides and the re-emergence of ribavirin-eIF4E complexes. We further hypothesized that the type II resistant cells could be resistant to other drugs. We observe that our ribavirin resistant cell lines are also resistant to the cornerstone of AML therapy, cytarabine. GDC0449/Vismodegib treatment reverts resistance to cytarabine in these cells. Preliminary studies indicate that these cells are also resistant to azacytidine and cisplatin. This is particularly striking as these cells were never exposed to these compounds. Thus, this could represent a novel form of multi-drug resistance. To establish the clinical relevance of our findings to patients in our AML ribavirin trial, we examined features of type I and type II resistance. Out of 10 patient samples available for evaluation, all six responding patient specimens showed elevated Gli-1 mRNA levels, up to 26 fold, upon relapse relative to levels during response. For most, the ratio of Gli1 during response relative to at relapse was about 2-4 fold with some patients up to 10 fold. For the two patients examined that did not respond, both had highly elevated Gli-1 levels prior to treatment relative to healthy individuals, and this was not lowered after 28 days of ribavirin treatment. We also noted elevated UGT1A protein levels upon relapse in our patient population. Type I resistance was observed in only two patients whereas Gli1 and UGT1A were dysregulated at relapse in all patients examined. In summary, we identified a novel form of drug resistance: Gli1 dependent drug glucuronidation. Treatment with Gli1 inhibitors appears to be a promising avenue for overcoming this form of drug resistance. Disclosures: No relevant conflicts of interest to declare.

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A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

Blood ◽

10.1182/blood.v126.23.4266.4266 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4266-4266 ◽

Cited By ~ 1

Author(s):

Tzu-Chieh Ho ◽

Craig T Jordan ◽

Mark W. LaMere ◽

John M. Ashton ◽

Kristen O'Dwyer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cell Lines ◽

Small Molecule ◽

Acute Myelogenous Leukemia ◽

Research Funding ◽

Myelogenous Leukemia ◽

Molecule Inhibitor ◽

Acute Myelogenous ◽

Pre Treatment

Abstract Background Acute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach. Results While the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect. Conclusions We identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. Disclosures Wang: Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

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ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942

Oncogene ◽

10.1038/s41388-019-1008-y ◽

2019 ◽

Vol 39 (3) ◽

pp. 651-663 ◽

Cited By ~ 3

Author(s):

Georgina P. Sava ◽

Hailing Fan ◽

Rosemary A. Fisher ◽

Sabrina Lusvarghi ◽

Sunil Pancholi ◽

...

Keyword(s):

Cell Lines ◽

Abc Transporter ◽

Abc Transporters ◽

Cancer Therapeutics ◽

Resistant Cell ◽

Cross Resistance ◽

Common Mechanism ◽

Mechanism Of Resistance ◽

Resistant Lines ◽

Resistant Cells

Abstract The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.

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Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BMC Cancer ◽

10.1186/s12885-021-08607-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Kana Oiwa ◽

Naoko Hosono ◽

Rie Nishi ◽

Luigi Scotto ◽

Owen A. O’Connor ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Acquired Resistance ◽

3D Culture ◽

Resistance Mechanisms ◽

Resistant Cell ◽

Gene Set Enrichment Analysis ◽

Synergistic Activity ◽

Mechanisms Of Resistance ◽

Resistant Cells

Abstract Background Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. Methods To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. Results Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. Conclusions The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

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Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

Cancers ◽

10.3390/cancers13236058 ◽

2021 ◽

Vol 13 (23) ◽

pp. 6058

Author(s):

Vikas Patel ◽

István Szász ◽

Viktória Koroknai ◽

Tímea Kiss ◽

Margit Balázs

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Melanoma Cell ◽

Acquired Resistance ◽

Resistance Mechanisms ◽

Resistant Cell ◽

Differentially Expressed ◽

Molecular Alterations ◽

Melanoma Cell Lines ◽

Resistant Cells

Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.

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Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma

Frontiers in Pharmacology ◽

10.3389/fphar.2021.718675 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuzo Sato ◽

Masaru Tomita ◽

Tomoyoshi Soga ◽

Atsushi Ochiai ◽

Hideki Makinoshima

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Malignant Pleural Mesothelioma ◽

Quantitative Polymerase Chain Reaction ◽

Resistance Mechanism ◽

Parental Cell ◽

Resistant Cell ◽

Pleural Mesothelioma ◽

Pyrimidine Synthesis ◽

Resistant Cells

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5′-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

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Targeting Folate Metabolism In Acute Myelogenous Leukemia

Blood ◽

10.1182/blood.v122.21.3798.3798 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3798-3798

Author(s):

Yana Pikman ◽

Alexandre Puissant ◽

Gabriela Alexe ◽

Stacey M. Frumm ◽

Linda Ross ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Critical Role ◽

Gene Expression Signature ◽

Myelogenous Leukemia ◽

Data Sets ◽

Expression Signature ◽

Metabolic Gene Expression ◽

Acute Myelogenous ◽

The One

Abstract There is increasing evidence that deranged metabolism is an important mechanism of cancer pathogenesis. We conducted multiple genomic analyses of publicly available acute myelogenous leukemia (AML) data sets that revealed a critical role for one carbon and nucleotide metabolism, particularly mitochondrial, in a subset of AML samples. One carbon metabolism is a complex series of pathways involving several amino acids, the synthesis of purines, thymidylate, S-adenosylmethionine, and the support of cellular methylation reactions. SHMT2, MTHFD2, and MTHFD1L are the major enzymes functional in the one carbon folate pathway in the mitochondria. MTHFD2 is a NAD-dependent, mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase, derived from a similar trifunctional cytoplasmic protein. In the mitochondria, the formyltetrahydrofolate synthetase activity is performed by MTHFD1L. We noted that these enzymes are downregulated with suppression of MYC. Gene set enrichment analysis (GSEA) of cell lines treated with JQ1, a small molecule BET bromodomain inhibitor which suppresses MYC, showed a significant enrichment in genes of the one carbon pool by folate KEGG pathway. We show that treatment of AML cells with JQ1 causes a decrease in MTHFD2 and MTHFD1L levels. This is recapitulated with knockdown of MYC with four shRNAs in multiple AML cell lines. Analysis of ENCODE ChIP-Seq data revealed MYC binding at SHMT2, MTHFD2 and MTHFD1L promoters, which we confirmed with ChIP-qPCR in human AML cell lines. Moreover, Independent component analysis (ICA) of primary AML samples in The Cancer Genome Atlas (TCGA) showed a significant correlation between high MTHFD2 and high MYC expression and a metabolic gene expression signature. MTHFD2 is differentially expressed in transformed and non-differentiated cells, and is thus an attractive drug target given its limited expression in normal tissues. Knockdown of MTHFD2 with four shRNAs in five AML cell lines caused a decrease in cell proliferation as measured by BrdU incorporation and a decrease in colony formation in methylcellulose. MTHFD2 knockdown also induced myeloid differentiation, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. AML cells transduced with MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML at day 15 post-injection. We next deployed a doxycycline inducible shRNA system to demonstrate that shRNAs directed against MTHFD2 cause a decrease in AML burden in mice with established disease as measured by bioluminescence with an increase in survival. Metabolite profiling is currently underway to further elucidate the metabolic consequences of MTHFD2 loss in AML. In summary, in silico analyses of primary patient AML data sets revealed a subset of AML samples enriched for a metabolic gene expression signature. We demonstrate that MYC is a regulator of the one carbon folate pathway, modulating expression of SHMT2, MTHFD2 and MTHFD1L. In vitro and in vivo data strongly supports a critical role for MTHFD2 in AML pathogenesis and its potential as a new target for AML therapy. Disclosures: No relevant conflicts of interest to declare.

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Correlation of Non-Coding RNA Expression with Response to Proteasome Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3415.3415 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3415-3415

Author(s):

Ehsan Malek ◽

Tahir Latif ◽

Anil Goud Jegga ◽

Sajjeev Jagannathan ◽

Nikhil Vad ◽

...

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Plasma Cells ◽

Proteasome Inhibitors ◽

Resistant Cell ◽

Drug Resistant ◽

Patient Response ◽

Drug Naïve ◽

Rpmi 8226 ◽

Resistant Cells

Abstract Background: Multiple myeloma (MM) is a heterogeneous diseaseand there is an increased need for more accurate risk classification methods to improve treatment decision-making because of its high impact on clinical outcomes. Here, we demonstrate evidence to support the prognostic value of non-coding RNAs (ncRNAs) as newly discovered genetic biomarkers of drug-resistant and/or high-risk forms of MM. NcRNAs, e.g., long ncRNAs (lncRNAs) and microRNAs (miRNAs), act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. NcRNAs have been shown to play a role in both solid and hematological tumors. Stratification of MM based upon cytogenetic abnormalities and protein-coding gene signatures does not adequately correlate with the depth and durability of response to novel agents such as bortezomib. Therefore, ncRNAs as new class of molecular effectors may enhance the basic understanding of myelomagenesis and provide better stratification of myeloma subtypes. To investigate the role of ncRNAs in resistance to proteasome inhibitors (PIs), we compared global ncRNA profiling in drug-naïve cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. We hypothesized that ncRNAs commonly deregulated in the 3 resistant cell lines would yield a ncRNA signature and novel therapeutic targets. Experimental Procedures: RPMI 8226 cells resistant to PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib over a period of 6 months. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix3.0 microarray chips that contained nearly 40,000 miRNA and 13,300 lncRNA probes. NcRNA expression profiles from drug-resistant cells were compared to that of drug- naïve parental cells treated with vehicle alone using the same treatment algorithm. Housekeeping genes were used for log expression normalization. MM patients' bone marrow aspirates were obtained from patients after University of Cincinnati Institutional Review Board approval. Results: Bioinformatic analysis of the ncRNA profiles identified a panel of 87 lncRNAs and ~40 miRNAs that were significantly (>100-fold) deregulated in all three drug-resistant cell lines relative to drug- naïve parental cells. Strikingly, ~90% of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines. Twenty lncRNAs were deregulated > 1000-fold in all 3 resistant cell lines (Figure 1). RPMI 8226 cells carry a chromosomal (14,16) translocation. Interestingly, none of the deregulated lncRNAs detected here localized to chromosome 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. The lncRNA COL4A-2A was upregulated >5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Also, our microarray-based studies have identified ncRNAs deregulated in MM patient tumor samples relative to normal plasma cells from healthy age-matched individuals. A significant number of the deregulated ncRNAs between drug- naïve and drug resistant cells were also deregulated in normal plasma cells relative to myeloma cells. Studies are correlating the ncRNA patterns seen in drug-sensitive and drug-resistant cell lines with ncRNA patterns obtained from malignant plasma cells of patients currently receiving bortezomib-based therapy. Updated results to correlate ncRNA expression with myeloma patient response to bortezomib will be presented.Conclusions: Taken together, we have identified a curated panel of ncRNAs deregulated in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors. Ongoing studies will correlate ncRNA expression patterns from resistant cells with patterns generated from patients with monoclonal gammopathy of unknown significance (MGUS), Smoldering MM, newly diagnosed MM, refractory disease and plasma cell leukemia. In addition, ncRNA patterns will be generated based upon MM patient response to bortezomib. Further investigation is warranted to shed light on the role of these ncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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