The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance

Abstract

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom's syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan.

Keywords: BLM; DNA damage; drug resistance; multiple myeloma; replication stress.

Figure 1

BLM expression in MM. (A) BLM expression analysis by Affimetrix microarrays from normal BMPCs from 5 healthy donors (median: 1133; range: 863-1328), MMCs from 206 MM patients from the HM cohort (median: 935; range: 113-5206), and 42 HMCLs (median: 1848; range: 246-10901). * marks outliers. Statistical analysis used a Student’s t-test of paired samples. (B) Gene expression profiling of MMCs of the patients of UAMS-TT2 cohort were used. Patients (n = 250) were classified in the 7 molecular groups of MM. PR: cell cycle and proliferation, LB: low bone disease, MSET: MMSET overexpression, HY: hyperdiploid signature, CDNN1: Cyclin D1 overexpression, CDNN2: Cyclin D2 overexpression, MAF: overexpression of MAF and MAFB genes. Small asterisks mark outliers in each group. Big red asterisks indicate that BLM expression is significantly higher in the group compared to all the patients of the cohort (P < 0.05) (Student’s t-test). (C) BLM expression is significantly higher at relapse compared to diagnosis in a longitudinal cohort of 18 paired patient’s samples (paired Student’s t-test). * p-value < 0.05.

Figure 2

High BLM expression is associated with a poor outcome in MM. Correlation between BLM expression and overall survival was analyzed using Maxstat R package in independent cohorts of MM patients: including patients at diagnosis treated by HDT and ASCT (A, left panel) HM cohort; (B, left panel) UAMS-TT2 cohort; (C) TT3 cohort; a cohort of patients at relapse treated by Anti-CD38 antibody (Daratumumab) (D) Mtp cohort anti-CD38 and a cohort of patients at diagnosis non eligible to HDT and ASCT (E) Mtp cohort non eligible HDT. (A, B, right panels) High expression of BLM is associated with shorter event free survival in the HM, TT2 and Mtp cohort non eligible HDT cohorts.

Figure 3

Effect of ML216 in HMCLs. (A) 10 HMCLs were treated with increasing doses of ML216 (0.78 – 100 μM). At day 4, cell viability was assessed using CellTiter-Glo Luminiscent Cell Viability Assay. IC50 for each cell line was calculated using GraphPrism software. Upper panel shows the non-linear regression curve of growth inhibition for all HMCLs. Low panel shows the IC50 value for each HMCL. Data are based on at least 3 independent experiments. (B) BLM expression and sensitivity to ML216 (IC50) do not correlate after applying a Spearman’s test. (C) Western blot analysis of the protein level of the RECQ helicases and basal DDR markers in 10 HMCLs. Cell lines are ranked from left to right in order of increasing IC50 for ML216. Note that in most cell lines protein levels do not correlate with BLM expression levels determined by RNAseq in Figure 3B. (D) Mutational status of MM frequently mutated genes in the 10 HMCLs used for the other experiments in this figure. “HMCL classification” refers to the MM molecular group classification. PR (cell cycle and proliferation), LB (low bone disease), MSET (aberrant expression of FGFR3 and MMSET genes), HY (hyperdiploid signature), CDNN1 (overexpression of Cyclin D1), CDNN2 (overexpression of Cyclin D2), and MAF (overexpression of MAF and MAFB genes) (72). Data extracted from (66).

Figure 4

ML216 selectively induces apoptosis in MMCs. (A) XG19, XG2 and XG1 cell lines were treated for 48 and 96 hours with the indicated concentrations of ML216. Apoptotic cells were detected as Annexin V+ cells by flow cytometry. Results are the average of 3 independent experiments. * indicates a significant increase in apoptosis compared to DMSO controls using a Student’s t-test. (B, C) BM cells extracted from 7 MM patients were cultured with recombinant IL-6 and 3, 6 or 10 μM ML216 for 96 hours. Cytotoxicity was assessed by flow cytometry. (B) MM plasma cells (CD138+) were detected using an anti-CD138 antibody and (C) all CD138- cells were analyzed as non-myeloma cells. Number of cells in each condition was normalized with respect to the control. P-values indicate the significance of the observed differences after applying a Wilcoxon test for pairs. NS, not significant. * p-value < 0.05, ** p-value < 0.01.

Figure 5

ML216 effect on cell cycle distribution in HMCLs. XG19, XG2 and XG1 cell lines were treated for 48 and 96 hours with the indicated concentrations of ML216. BrdU (10 μg/ml) was added during the last 1.5 hours of treatment. Cells were fixed and processed to detect BrdU incorporation and total DNA (see Materials and Methods for more details). BrdU+ cells were assigned to S-phase. BrdU- cells were assigned to G0/G1 or G2/M phases based on their DNA content. * indicates a significant difference compared to DMSO treated (control) cells after applying a Student’s t-test for pairs. Results are the mean of 3 independent experiments. ** p-value < 0.01.

Figure 6

ML216 treatment synergizes with melphalan to inhibit HMCLs proliferation. Dose-response matrixes to measure synergy of ML216 and Melphalan co-treatment. Synergy scores are shown using a continuous pseudo-color scale ranging from bright-green (=antagonism) to bright-red (=synergism). XG19, XG2 and XG1 were treated with increasing concentrations of ML216 (0.78125 – 25 μM), and of the alkylating agent melphalan (0.78125 – 50 μM), for 4 days. Cell viability was assessed using the CellTiter-Glo Luminiscent Cell Viability Assay and was normalized to untreated conditions. Matrixes show the average of 3-4 independent experiments.

Figure 7

Effect of combination of ML216 and melphalan. (A) XG19 and XG2 cells were treated with ML216 and melphalan as indicated for 96 hours. Apoptotic cells were detected as Annexin V+ cells by flow cytometry. Results are the average of 3 independent experiments. * indicates a significant increase in apoptosis compared to DMSO controls using a Student’s t-test. (B) XG19 and XG2 cells were treated for 96 hours with the indicated concentrations of ML216 and melphalan. BrdU (10 μg/ml) was added during the last 1.5 hours of treatment. Cells were fixed and processed to detect BrdU incorporation and total DNA (see Materials and Methods for more details). BrdU+ cells were assigned to S-phase. BrdU- cells were assigned to G0/G1 or G2/M phases based on their DNA content. Asterisks indicate a significant difference compared to DMSO treated (control) cells after applying a Student’s t-test: * p-value < 0.05, ** p-value < 0.01. Results are the mean of 3 independent experiments. (C) XG19 cells treated as in (B) were processed to quantify γH2AX intensity by flow cytometry (see Materials and Methods for more details). * indicate a significant difference compared to DMSO treated (control) cells after applying a Student’s t-test for pairs: p-value < 0.05. (D) XG2 cells were treated with the indicated doses of ML216 and 1 μM melphalan for 48 hours. Cells were harvested and γH2AX levels were analyzed by western blot. One representative experiment out of three is shown. The graph shows the quantification of the γH2AX signal (a.u.: arbitrary units) with respect to tubulin, normalized to the control condition (DMSO) in 3 independent experiments. *** p-value < 0.001.

Figure 8

BLM depletion sensitizes MM cells to melphalan. XG2 cells were transduced with control lentiviruses (Ctrl) or miRNA against BLM to stably knock-down its expression (KD BLM). Both cell lines were treated with 1 μM melphalan for 96 hours and samples were collected to analyze (A) BLM depletion and protein levels of the other RECQ helicases; (B) cell cycle and DDR markers by western blot; (C) PARP and caspases cleavage as apoptosis markers by western blot; (D) apoptotic cells (Annexin V+) by flow cytometry; (E) cell cycle distribution using BrdU incorporation and DAPI staining as in Figures 5 and 7 . Asterisks indicate a significant difference after applying a Student’s t-test for pairs. * p-value < 0.05, ** p-value < 0.01. All experiments in this figure were repeated 3 times independently.

Figure 9

ML216 re-sensitizes Melphalan-resistant MM cells to Melphalan. Dose-response matrixes to measure synergy of ML216 and Melphalan co-treatment. Synergy scores are shown using a continuous pseudo-color scale ranging from dark-green (=antagonism) to dark-red (=synergism). (A) XG7 parental cells and XG7 MR (Melphalan Resistant) (Melphalan IC50: 0.625 μM and 7.5 μM respectively) were treated for 4 days with the indicated doses of ML216 and melphalan. Cell viability was assessed using CellTiter-Glo Luminiscent Cell Viability Assay and was normalized with respect to untreated conditions. Matrixes show the mean of 3-4 independent experiments. (B) Same as in (A) but comparing XG2 parental and XG2 MR (Melphalan Resistant) (Melphalan IC50: 0.625 μM and 2 μM respectively). Matrixes show the mean of 3 independent experiments. (C) Treatment of MM cells with melphalan produces DNA damage. Cells that overexpress BLM can cope better with the drug-induced DNA damage and therefore survive to the treatment, showing a resistant phenotype. On the contrary, BLM inhibition in combination with melphalan increases DNA damage to levels that tumoral plasma cells cannot efficiently repair, leading to cell cycle arrest and eventually to cell death, overcoming melphalan-resistance. Figure was created with BioRender.com.