RECQ1 helicase is involved in replication stress survival and drug resistance in multiple myeloma

Abstract

Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA unwinding enzymes involved in the maintenance of chromosome stability. Here we show that RECQ1 is significantly overexpressed in MMCs compared to normal plasma cells and that increased RECQ1 expression is associated with poor prognosis in three independent cohorts of patients. Interestingly, RECQ1 knockdown inhibits cells growth and induces apoptosis in MMCs. Moreover, RECQ1 depletion promotes the development of DNA double-strand breaks, as evidenced by the formation of 53BP1 foci and the phosphorylation of ataxia-telangiectasia mutated (ATM) and histone variant H2A.X (H2AX). In contrast, RECQ1 overexpression protects MMCs from melphalan and bortezomib cytotoxicity. RECQ1 interacts with PARP1 in MMCs exposed to treatment and RECQ1 depletion sensitizes MMCs to poly(ADP-ribose) polymerase (PARP) inhibitor. DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. Altogether, these data suggest that association of DNA damaging agents and/or PARP inhibitors with DNMT inhibitors may represent a therapeutic approach in patients with high RECQ1 expression associated with a poor prognosis.

Figure 1

RECQ1 expression in MM. (a) RECQ1 gene expression in normal BMPCs, patients’ MMCs and HMCLs. Data are MAS5-normalized Affymetrix signals (U133 plus 2.0 microarrays). Statistical difference was tested using a Student t-test. (b) Western blot showing RECQ1 expression in seven different HMCLs, normal in vitro-generated plasma cells and purified primary MMCs of four patients. (c) High RECQ1 expression in MMCs could predict for shorter overall and event-free survival. Patients of the Heidelberg–Montpellier cohort (N=206) were ranked according to increasing RECQ1 expression and a maximum difference in OS and EFS was obtained using the Maxstat R function.

Figure 2

RECQ1 depletion in MMC results in cell growth inhibition and apoptosis induction. (a) Doxycycline-mediated inhibition of RECQ1 protein expression in three different HMCLs transduced with a RECQ1 shRNA inducible vector. Data are those of one experiment out of three. Protein load was assayed using β-actin quantification. (b) The expression of RECQ1 protein was quantified with ImageJ software, normalized to β-actin level, and data are the mean normalized values of RECQ1 expression. * indicates a significant decrease compared to doxycycline (dox)-untreated cells using a Wilcoxon test for pairs (P⩽0.05) (three independent experiments). (c) Inducible depletion of RECQ1 delays myeloma cell growth. The HMCLs transduced with the TR lentivirus or with both TR and RECQ1 shRNA (shRECQ1) lentiviruses were exposed to doxycycline and cell viability analyzed by trypan blue assay. Results are those of one experiment representative of five. Statistical significance was tested using a Wilcoxon test for pairs. (d) Caspase activity was evaluated by luminescence assay in XG19-TR-shRECQ1 5 days after doxycycline treatment. The activity of caspase-9 and caspase-3/7 was significantly elevated in RECQ1-depleted cells (* indicates a P-value <0.01). Data are those of one experiment representative of three. Statistical significance was tested using a Wilcoxon test for pairs.

Figure 3

RECQ1 depletion induces defects in cell cycle progression of HMCL and mortality of primary MM cells from patients. (a) Cell cycle of RECQ1-depleted MM cell lines was analyzed by flow cytometry using BrdU incorporation and labeling with an anti-BrdU antibody and DAPI. Results are representative of five independent experiments. * indicates a significant decrease compared to doxycycline (dox)-untreated cells (P<0.05; Wilcoxon test for pairs). ** indicates a significant increase compared to dox-untreated cells (P<0.05, Wilcoxon test for pairs). (b) Mononuclear cells from seven patients with MM were transduced with shRECQ1 or shRNA scramble lentiviruses. At day 4 of culture, the viability and total cell counts were assessed and the percentage (i) CD138⁺ viable plasma cells and (ii) bone marrow non-myeloma cells were determined by flow cytometry. Results are median values of the numbers of myeloma cells in the culture wells. Results were compared with a Wilcoxon test for pairs. * indicates a significant decrease compared to scramble shRNA (P<0.01).

Figure 4

RECQ1 depletion induces DDR in MM cells. (a) Alkaline comet assay was used to analyze DNA breaks formation after RECQ1 depletion in XG19 cell line. Tail length and tail moment (fraction of total DNA in the tail × tail length) were analyzed with ImageJ software. Data are calculated from measurement of 50 comets for each sample. Statistical difference was tested using a Student t-test. (b) 53BP1 staining was used as marker for DNA damage. The number of 53BP1 foci found in each cell was enumerated 3 days after doxycycline treatment. At least 300 cells were counted for each group. The percentage of cells with more than 10 53BP1 foci per cell is displayed in the histograms. Statistical difference was tested using a Student t-test. (c) HMCL-TR-shRECQ1 cells were cultured for 7 days with or without doxycycline (dox) and protein detection was assayed using western blot analysis. Membranes were stained with anti-phospho-ATM, anti-ATM, anti-phospho-p53 (Ser15 or Ser20) and anti-p53, anti-Phospho-Chk2, anti-Chk2 and anti-γH2AX. A mouse monoclonal anti-β-actin antibody was used as control. (d) XG19-TR-shRECQ1 cell line was treated or not with doxycycline for 5 days and replication fork progression was determined by DNA fiber spreading after double labeling with IdU and CldU. Statistical difference was tested using a Student t-test.

Figure 5

Inducible overexpression of RECQ1 protects MMC from melphalan and bortezomib-induced toxicity. (a) HMCL apoptosis induction was assessed with Annexin V, by flow cytometry after treatment or not with 4 μM of melphalan for 4 days. * indicates a significant decrease compared to doxycycline-untreated cells using a Wilcoxon test for pairs (P⩽0.05). Results are those of one experiment representative of five. (b) HMCL-TR-RECQ1 cells were treated or not with doxycycline. Cell cycle of non-apoptotic cells was analyzed by flow cytometry using BrdU incorporation and labeling with an anti-BrdU antibody and DAPI after 3 days of treatment with 4 μM of melphalan. Results are representative of five independent experiments. * indicates a significant difference using a Wilcoxon test for pairs (P<0.05). (c) MMC overexpressing or not RECQ1 were treated with 4 μM of melphalan (Mel) for 72 h and analyzed using alkaline comet assay. Tail length and tail moment (fraction of total DNA in the tail × tail length) were analyzed with ImageJ software. Data are calculated from measurement of 50 comets for each sample. Results are representative of three independent experiments. Statistical difference was tested using a Student t-test. (d) XG19 MM cell line overexpressing or not RECQ1 were treated with 10 nM of bortezomib for 4 days. Cell cycle was analyzed by flow cytometry using BrdU incorporation and labeling with an anti-BrdU antibody and DAPI. Results are those of one experiment representative of three. Statistical difference was tested using a Wilcoxon test for pairs (i) and apoptosis induction was analyzed with Annexin V APC staining by flow cytometry. Results are those of one experiment representative of three. Statistical difference was tested using a Wilcoxon test for pairs (ii). High RECQ1 expression is associated with a shorter OS in a cohort of 188 patients at relapse treated with bortezomib monotherapy (Mulligan cohort) (iii).

Figure 6

RECQ1 depletion enhances the sensibility of HMCL to PARP inhibitor. XG19 TR and XG19-TR-shRECQ1 (a) or XG7 TR and XG7 TR shRECQ1 (b) HMCLs were cultured for 4 days in 96-well flat-bottom microtiter plates in RPMI 1640 medium, 10% fetal calf serum, 2 ng/ml IL-6 culture medium (control) and graded PJ34 hydrochloride concentrations. Data are mean values±s.d. of five experiments determined on sextuplet culture wells. * indicates a significant decrease compared to doxycycline (dox)-untreated cells using a Wilcoxon test for pairs (P⩽0.05).

Figure 7

DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. (a) 5-Azacitidine treatment leads to increased miR-203 expression in HMCL. XG7 and LP1 were treated for 7 days with 1 μM 5-azacitidine. miR-203 expression levels were determent by RT-qPCR analysis and normalized to miR-16 expression. Data are mean values±s.d. of three experiments. (b) miR-203 inhibition results in RECQ1 overexpression in HMCL. XG7 and LP1 cell lines were transfected with anti-miR-203 miRNA inhibitor; RECQ1 expression was assayed by RT-qPCR and normalized by GAPDH expression. Data are mean values±s.d. of three experiments.

Figure 8

Model of RECQ1 expression regulation and functions in MM cells. (a) Aberrant methylation of miR-203 is associated with abnormal RECQ1 overexpression in MM cells conferring resistance to replicative stress and treatment. (b) DNMTi treatment induces a significant upregulation of miR-203 expression downregulating RECQ1 expression in MM cells in association with replicative stress, cell death and hypersensitivity to treatments.