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Role of RECQ1 helicase in multiple myeloma drug resistance
Abstracts are ongoing to inform our efforts of clinically translating CAR T-cell therapy in this entity.
BP-037 Role of RECQ1 helicase in multiple myeloma drug resistance E. Viziteu,1 B. Klein,1,2,3 J. Basbous,1 Y.L. Lin,1 A. Seckinger,4 C. Gourzones,1 C. Grandmougin,1 A. Constantinou,1 H. Goldschmidt,4 P. Pasero,1 D. Hose,4 J. Moreaux1,2,3 1
Institute of Human Genetics, CNRS UPR 1142, Montpellier, France;
2
Laboratory for Monitoring Innovative Therapies, Department of Bio-
logical Hematology, CHRU Montpellier, France; 3Montpellier University, UFR Medecine, Montpellier, France; 4Medizinische Klinik V, Universitätsklinikum Heidelberg and Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany
Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.
In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1-high patients associated with a poor prognosis.
BP-038 A Plastic SQSTM1/p62-Dependent Autophagic Reserve Maintains Protein Homeostasis and Determines Proteasome Inhibitor Susceptibility in Multiple Myeloma Cells T. Perini,1,2,3 E. Milan,1,2 M. Resnati,1 U. Orfanelli,1 L. Oliva,1 A. Raimondi,4 P. Cascio,5 A. Bachi,6 M. Marcatti,3 F. Ciceri,3 S. Cenci1,2 1
San Raffaele Scientific Institute, Division of Genetics and Cell Biology,
Milano, Italy; 2Università Vita-Salute San Raffaele, Milano, Italy; 3San Raffaele Scientific Institute, Department of Oncohematology, Hematology and Bone Marrow Transplantation Unit, Milano, Italy; 4San Raffaele Scientific Institute, Imaging Research Center, Milano, Italy; 5
Department of Veterinary Sciences, University of Torino, Italy; 6IFOM,
FIRC Institute of Molecular Oncology, Milano, Italy
Background: Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. A possible mechanism of primary resistance and an attractive target to enhance sensitivity is (macro)autophagy, the main alternative catabolic pathway, which we recently found essential to bone marrow plasma cells, the normal counterpart of MM. Methods: Here, using MM cell lines and primary cells, we integrated comparative proteomics with electron microscopy, fluorescent imaging, lentiviral engineering, and molecular biology techniques to address the role of autophagy in the maintenance of protein and organelle homeostasis in MM cells, and the underlying mechanisms. Results: We found that in MM cells abundant autophagy alleviates proteasomal load by constitutively disposing of substantial amounts of ubiquitinated proteins and by selectively targeting the endoplasmic reticulum (ER), a chief source of proteasome substrates, disclosing a co-operation between the ubiquitinproteasome system (UPS) and autophagy closer than previously thought. Indeed, autophagy inhibition or stimulation greatly sensitizes to, or protects from, PI-induced protein aggregation and cell death. Moreover, we found such co-operation to depend on the autophagic cargo receptor/adapter protein SQSTM1/p62. Lentiviral engineering revealed that p62 is essential for MM cell survival and affords specific PI resistance. Under PIs, myeloma cells selectively enhance p62 de novo expression and reset its vast endogenous interactome, diverting p62 from signaling partners to maximize its association with ubiquitinated proteins for autophagic degradation. Of clinical relevance, saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested p62+ aggregates, neatly discriminates patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. Conclusions: We disclosed an essential role for autophagy in protein and organelle
15th International Myeloma Workshop, September 23-26, 2015
- e67
BP-037 Role of RECQ1 helicase in multiple myeloma drug resistance E. Viziteu,1 B. Klein,1,2,3 J. Basbous,1 Y.L. Lin,1 A. Seckinger,4 C. Gourzones,1 C. Grandmougin,1 A. Constantinou,1 H. Goldschmidt,4 P. Pasero,1 D. Hose,4 J. Moreaux1,2,3 1
Institute of Human Genetics, CNRS UPR 1142, Montpellier, France;
2
Laboratory for Monitoring Innovative Therapies, Department of Bio-
logical Hematology, CHRU Montpellier, France; 3Montpellier University, UFR Medecine, Montpellier, France; 4Medizinische Klinik V, Universitätsklinikum Heidelberg and Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany
Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.
In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1-high patients associated with a poor prognosis.
BP-038 A Plastic SQSTM1/p62-Dependent Autophagic Reserve Maintains Protein Homeostasis and Determines Proteasome Inhibitor Susceptibility in Multiple Myeloma Cells T. Perini,1,2,3 E. Milan,1,2 M. Resnati,1 U. Orfanelli,1 L. Oliva,1 A. Raimondi,4 P. Cascio,5 A. Bachi,6 M. Marcatti,3 F. Ciceri,3 S. Cenci1,2 1
San Raffaele Scientific Institute, Division of Genetics and Cell Biology,
Milano, Italy; 2Università Vita-Salute San Raffaele, Milano, Italy; 3San Raffaele Scientific Institute, Department of Oncohematology, Hematology and Bone Marrow Transplantation Unit, Milano, Italy; 4San Raffaele Scientific Institute, Imaging Research Center, Milano, Italy; 5
Department of Veterinary Sciences, University of Torino, Italy; 6IFOM,
FIRC Institute of Molecular Oncology, Milano, Italy
Background: Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. A possible mechanism of primary resistance and an attractive target to enhance sensitivity is (macro)autophagy, the main alternative catabolic pathway, which we recently found essential to bone marrow plasma cells, the normal counterpart of MM. Methods: Here, using MM cell lines and primary cells, we integrated comparative proteomics with electron microscopy, fluorescent imaging, lentiviral engineering, and molecular biology techniques to address the role of autophagy in the maintenance of protein and organelle homeostasis in MM cells, and the underlying mechanisms. Results: We found that in MM cells abundant autophagy alleviates proteasomal load by constitutively disposing of substantial amounts of ubiquitinated proteins and by selectively targeting the endoplasmic reticulum (ER), a chief source of proteasome substrates, disclosing a co-operation between the ubiquitinproteasome system (UPS) and autophagy closer than previously thought. Indeed, autophagy inhibition or stimulation greatly sensitizes to, or protects from, PI-induced protein aggregation and cell death. Moreover, we found such co-operation to depend on the autophagic cargo receptor/adapter protein SQSTM1/p62. Lentiviral engineering revealed that p62 is essential for MM cell survival and affords specific PI resistance. Under PIs, myeloma cells selectively enhance p62 de novo expression and reset its vast endogenous interactome, diverting p62 from signaling partners to maximize its association with ubiquitinated proteins for autophagic degradation. Of clinical relevance, saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested p62+ aggregates, neatly discriminates patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. Conclusions: We disclosed an essential role for autophagy in protein and organelle
15th International Myeloma Workshop, September 23-26, 2015
- e67