TOP2A Knockdown Resensitizes Carfilzomib-Resistant Hmcls to Carfilzomib

Abstract Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified. Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays. Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the 'proliferation cluster' associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib. Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM. Disclosures No relevant conflicts of interest to declare.

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TOP2A a New Predictive Marker of Response to Carfilzomib in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4461.4461 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4461-4461

Author(s):

Antonia Reale ◽

Tiffany Khong ◽

Sridurga Mithraprabhu ◽

John Reynolds ◽

Malarmathy Ramachandran ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Anticancer Agents ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Predictive Marker ◽

Myeloma Cell ◽

Patient Response ◽

Escherichia Coli Infection

Abstract Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib (CZ), a second-generation proteasome inhibitor (PI) currently used in clinical settings, could be identified. Materials and Methods: Nine human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive or resistant to carfilzomib. Subsequently, the gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs were compared. GEP-identified differences were validated on a panel of 17 HMCLs with q-RT-PCR and siRNA knockdown assays. Topoisomerase 2-alpha (TOP2A) immunohistochemistry was performed on a panel of fully annotated trephine biopsy specimens acquired prior to treatment with PIs. Results: 206 genes were differentially expressed between the sensitive and resistant HMCLs. Gene ontology analysis identified two pathways that were significantly different: pathogenic Escherichia Coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A has also been noted to be overexpressed in the 'proliferation cluster' of published MM GEP profiles and associated with poor outcome. Following suppression of TOP2A by siRNA, carfilzomib-resistant HMCLs were re-sensitized to carfilzomib, moreover, the combination of carfilzomib with topoisomerase inhibitors known to target directly TOP2A, demonstrated synergistic cytotoxic effects against HMCLs. Trephine-derived TOP2A protein expression levels were shown to be higher in patients treated with PIs including bortezomib, carfilzomib, ixazomib, marizomib, failing to achieve a response when compared to responding patients. Finally, logistic regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.764, p=0.048). Conclusion: TOP2A status may be used as a predictive factor for patient response to PIs including carfilzomib. Disclosures No relevant conflicts of interest to declare.

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NCOR2 Mutations Are Associated with IMiD Resistance in Human Myeloma Cells

Blood ◽

10.1182/blood-2020-141968 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-4

Author(s):

Tomoaki Mori ◽

Cristina Panaroni ◽

Chukwuamaka Onyewadume ◽

Noopur S. Raje

Keyword(s):

Cell Lines ◽

Protein Interaction ◽

Enrichment Analysis ◽

Myeloma Cell ◽

Resistant Cell ◽

Ppi Network ◽

Myeloma Cells ◽

Knock Out ◽

Protein Protein Interaction ◽

Bet Inhibitor

The immunomodulatory drug thalidomide, and its analogs, lenalidomide, and pomalidomide (IMiDs) have significantly changed the treatment paradigm of multiple myeloma (MM). Despite this progress, IMiD resistance develops in the majority of patients resulting in the development of refractory disease. Cereblon (CRBN), a direct target, has been implicated in IMiD resistance. However, alternate mechanisms of IMiD resistance independent of CRBN remain largely unknown. To understand and study the mechanisms responsible for the development of IMiD resistance, we created lenalidomide-resistant (Len-R) and pomalidomide-resistant (Pom-R) human myeloma MM.1s cell lines, by continuous culture in the presence of lenalidomide or pomalidomide for 3 months. Whole genome sequencing of these 2 resistant cell lines compared with parental MM.1s revealed 172 genes with exonic mutations in both Len-R and Pom-R myeloma cells. Furthermore, a protein-protein interaction (PPI) network was constructed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PPI network demonstrated 8 genes that scored a high degree of protein-protein interaction. Among these genes, we identified NCOR2, a corepressor that negatively regulates gene expression, as a downregulated gene in resistant cell lines. To study this further, we created NCOR2 knock out MM.1s cell lines using CRISPR/cas9 gene modification. Our data demonstrates that depletion of NCOR2 confers IMiD resistance independent to CRBN. Interestingly, Len-R, Pom-R and NCOR2 knock out MM.1s showed increased MYC protein expression, which is essential for myeloma cell survival and proliferation. A BET inhibitor, known to disrupt the binding of BRD4 to chromatin, inhibited the proliferation of Len-R and Pom-R and NCOR2 knock out MM.1s by completely suppressing MYC expression. These results indicate that NCOR2 down regulation in IMiD resistant cells induces MYC upregulation which may in part result in IMiD resistance. Our findings reveal a novel molecular mechanism associated with IMiD resistance, independent of CRBN and suggest that NCOR2-MYC pathway may be a new target for IMiD refractory patients. Disclosures Raje: Celgene: Consultancy.

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Synthesis and anticancer activity of carbosilane metallodendrimers based on arene ruthenium(ii) complexes

Dalton Transactions ◽

10.1039/c6dt00465b ◽

2016 ◽

Vol 45 (16) ◽

pp. 7049-7066 ◽

Cited By ~ 38

Author(s):

Marta Maroto-Díaz ◽

Benelita T. Elie ◽

Pilar Gómez-Sal ◽

Jorge Pérez-Serrano ◽

Rafael Gómez ◽

...

Keyword(s):

Anticancer Activity ◽

Cell Lines ◽

Anticancer Agents ◽

Resistant Cell ◽

Micromolar Range

Carbosilane-based ruthenium dendrimers have been tested as anticancer agents. These metallodendrimers are active against a number of cisplatin resistant cell lines in the low micromolar range while showing a dendritic effect.

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Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽

10.1182/blood.v116.21.1915.1915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1915-1915

Author(s):

Unn-Merete Fagerli ◽

Thorsten Stühmer ◽

Toril Holien ◽

Randi Utne Holt ◽

Ove Bruland ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Factors ◽

Cell Lines ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Transcriptional Response ◽

Myeloma Cell ◽

Myeloma Cells ◽

Growth And Survival ◽

Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽

10.1182/blood.v116.21.4469.4469 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4469-4469

Author(s):

Hilmar Quentmeier ◽

Sonja Eberth ◽

Julia Romani ◽

Margarete Zaborski ◽

Hans G. Drexler

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemia Cell ◽

Resistant Cell ◽

Imatinib Resistance ◽

Imatinib Resistant ◽

Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

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PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Blood ◽

10.1182/blood.v118.21.2628.2628 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2628-2628

Author(s):

Hiromichi Yuki ◽

Shikiko Ueno ◽

Hiroaki Niiro ◽

Hiro Tatetsu ◽

Hiroyuki Hata ◽

...

Keyword(s):

Cell Growth ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Growth Arrest ◽

Myeloma Cell ◽

G1 Arrest ◽

Classical Hodgkin Lymphoma ◽

Lymphoma Cells ◽

Down Regulation

Abstract Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.3962.3962 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3962-3962

Author(s):

Xing-Ding Zhang ◽

Robert Z. Orlowski ◽

Lin Yang

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Hela Cells ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Cancer Center ◽

In Vivo Models ◽

Junction Protein ◽

Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

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Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.939.939 ◽

2012 ◽

Vol 120 (21) ◽

pp. 939-939

Author(s):

Esther Yoo ◽

Alex Vasuthasawat ◽

Danh Tran ◽

Alan Lichtenstein ◽

Sherie Morrison

Keyword(s):

Multiple Myeloma ◽

Fusion Protein ◽

Cell Lines ◽

Dna Content ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Inhibition Of Proliferation ◽

Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

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Metabolic Rewiring Drives Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v128.22.3956.3956 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3956-3956 ◽

Cited By ~ 2

Author(s):

Nithya Balasundaram ◽

Saravanan Ganesan ◽

Hamenth Kumar Palani ◽

Ansu Abu Alex ◽

Sachin David ◽

...

Keyword(s):

Glucose Uptake ◽

Acute Promyelocytic Leukemia ◽

Cell Lines ◽

Conflicts Of Interest ◽

Resistant Cell ◽

Promyelocytic Leukemia ◽

Leukemic Cells ◽

Synergistic Activity ◽

Normal Peripheral Blood ◽

Nb4 Cells

Abstract The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells. Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b). Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. Disclosures No relevant conflicts of interest to declare.

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Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2047.2047 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2047-2047

Author(s):

Nao Nishimura ◽

Shinya Endo ◽

Niina Ueno ◽

Shikiko Ueno ◽

Hiromichi Yuki ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Tumor Suppressor ◽

Cell Lines ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Suppressor Activity ◽

Enhancer Region ◽

Tumor Suppressor Activity

Abstract PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.

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