Whole-exon sequencing of human myeloma cell lines shows mutations related to myeloma patients at relapse with major hits in the DNA regulation and repair pathways

Background Human myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully. Their genetic background is mostly undiscovered, and no comprehensive study has ever been conducted in order to reveal those details. Methods We performed whole-exon sequencing of 33 HMCLs, which were established over the last 50 years in 12 laboratories. Gene expression profiling and drug testing for the 33 HMCLs are also provided and correlated to exon-sequencing findings. Results Missense mutations were the most frequent hits in genes (92%). HMCLs harbored between 307 and 916 mutations per sample, with TP53 being the most mutated gene (67%). Recurrent bi-allelic losses were found in genes involved in cell cycle regulation (RB1, CDKN2C), the NFκB pathway (TRAF3, BIRC2), and the p53 pathway (TP53, CDKN2A). Frequency of mutations/deletions in HMCLs were either similar to that of patients (e.g., DIS3, PRDM1, KRAS) or highly increased (e.g., TP53, CDKN2C, NRAS, PRKD2). MAPK was the most altered pathway (82% of HMCLs), mainly by RAS mutants. Surprisingly, HMCLs displayed alterations in epigenetic (73%) and Fanconi anemia (54%) and few alterations in apoptotic machinery. We further identified mutually exclusive and associated mutations/deletions in genes involved in the MAPK and p53 pathways as well as in chromatin regulator/modifier genes. Finally, by combining the gene expression profile, gene mutation, gene deletion, and drug response, we demonstrated that several targeted drugs overcome or bypass some mutations. Conclusions With this work, we retrieved genomic alterations of HMCLs, highlighting that they display numerous and unprecedented abnormalities, especially in DNA regulation and repair pathways. Furthermore, we demonstrate that HMCLs are a reliable model for drug screening for refractory patients at diagnosis or at relapse. Electronic supplementary material The online version of this article (10.1186/s13045-018-0679-0) contains supplementary material, which is available to authorized users.


Background
Human myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully [1]. HMCLs are mainly derived from refractory patients, mostly presenting with extramedullary disease and having thus received numerous classes of drugs inducing DNA damage, proteasome inhibition, immunomodulation, and anti-inflammation (e.g., melphalan, bendamustine, Velcade, Revlimid, and dexamethasone). However, HMCLs harbor the 14q32 abnormality, which occurs early at the MGUS stage, and display frequent mutations in NRAS and KRAS, as observed in patients at diagnosis (approximately 50% of patients) [2,3]. By contrast, HMCLs display very frequent deletion and mutation in the TP53 gene that are associated with resistance to treatments [4]. Indeed, it is well known that hits in the TP53 gene (deletion and/or mutation) at diagnosis are associated with resistance and shortened survival and that their frequency increases with relapse [4,5]. Thus, HMCLs are a mixture of abnormalities occurring both early and late in the time course of disease. Besides hits in the TP53 and RAS genes, HMCLs have not been widely characterized for their global mutation profile and gene deletion. In the present work, using wholeexon sequencing (WES) in 33 HMCLs, we report common gene mutations and deletions. We analyzed the frequency of mutations/deletions in comparison with patients at diagnosis and relapse. We further identified hits preferentially associated with 14q32 translocations and analyzed responses to conventional and nonconventional drugs in relation to a mutation and/or deletion profile.
Whole-exon sequencing DNA sample processing was performed according to Agilent Technologies (Santa Clara, CA, USA) using the sureselect target enrichment system kit (Human all exon v6, library version 1.6). Sequencing was performed on HiSeq 2500, High Output in paired-end 2 × 100 bp. The reads were aligned (BWA-v0.7.10-r789) to the GRCh37 human reference genome. Duplicated reads were marked by the Picard tool (v1.119), indels were realigned around capture (± 500 bp), and base quality recalibration was finally performed (Genome Analysis Toolkit [GATK-v3. 2.2]). In the absence of germline DNA, variants were called by the GATK unified genotyper. Variants were processed through vcf2maf-1. 6 Variants that were present more than three times were removed, as well as variants with Global Allele Frequency in ExAC databases over 1% (with respect to ethnicity frequencies when known). Finally, clinically benign mutants, as annotated by ClinVar, were removed ("benign" or "likely benign"). Only protein-coding variants were used for subsequent analyses, and structural protein coding genes (actin, myosin, collagen, fibronectin, vitronectin, tenascin, laminin, titin, obscurin, plectin, aggrecan, and mucins) were removed.
Exon loss was estimated from the read depth using ExomeCOPY and CANOES. The results were validated by visual inspection of the BAM read depth in Integrative Genomics Viewer (IGV; Broad Institute). Genes with frequent variants were selected and were assessed by direct Sanger sequencing on cDNA.

Statistical analyses
Analyses were performed under R 3.4.4. Fisher's test was carried out with the resampling of parameters for robustness. The somatic interaction plot code was adapted from Gerstung et al. [15]. Enrichment analyses were carried out by ReactomePA and clusterProfiler [16,17], p values were adjusted for multiple testing by the false discovery rate (q = 0.05). For the Reactome determination, KEGG and GO annotations were used. MAF manipulation was performed using the maftools packages [18]. Oncoprints, heatmaps, and Chord-Diagrams were performed with ComplexHeatmap R-package. Considering the number of samples, the linear regressions between scores and drug responses were calculated by robust a linear regression using a M-estimator (rlm, MASS package) in order to discard outliers. Coefficients were further bootstrapped by Boot function (car package), with 5000 replicates (seed = 22,062,016) and considered significant if the 95% confidence interval (95% CI) did not overlap with zero, only β1 coefficients are presented in the text.

Metrics and variant filtering
WES was performed in 33 HMCLs of European, American or Asian origin, 19 having been derived in the presence of exogenous IL6 (Additional file 1: Table S1). After global SNP enrichment analysis on 609,585 bi-allelic SNPs (SNPRelate package [19]), three groups of HMCLs were identified: a group gathering HMCLs of Pacific/Japanese origin (AMO-1, KMM1, KMS12PE, KMS11, NAN8, OPM2) and a cluster encompassing all other HMCLs except MM1S, which was individualized as African ethnicity (Additional file 1: Figure S1). To remove ethnic-related SNPs, HMCLs were filtered with Global Allele Frequencies, plus East Asian frequencies for the Pacific/Japanese cluster and African frequencies for MM1S. Because of the lack of normal DNA from patients from whom the HMCLs were derived, we could not easily discriminate the constitutive SNPs from the tumor-associated mutations. Thus, we excluded variants shared by more than 3 HMCLs of the 33: indeed, the most mutated genes in HMCLs and myeloma patients [20], i.e., RAS and TP53, never displayed more than three identical variants across the HMCL collection. For NRAS, the most frequent variant was c.38G-A (Gly12Asp) in JJN-3, Karpas620, and Nan7, while the only TP53 shared variant was 406G-A (Karpas620, XG11). Variant effect predictions were carried out as described in the "Methods" section. We further removed variants of genes uniformly low expressed across the collection (maximum of the considered gene inferior to the first quartile mean expression of the microarray). After filtering, we retained 15,602 variants, spanning over 7641 genes (Maf file, Additional file 2). Most mutated samples were KMM1 and KMS12PE with 916 and 755 variants, respectively. The most frequent variant was missense (n = 14,309; 92%), while frameshifts occurred in 273 variants (1.7%), insertions or deletions without frameshifts occurred in 226 cases (1.4%), and 482 variants (3.1%) were nonsense mutations (Additional file 1: Figure S2). Single mutations were mainly C > T transitions (63%, Additional file 1: Figure S3), corresponding to spontaneous deamination of 5-methyl cytosine. HMCLs age was not associated with a particular mutation (Fisher test, FDR > 0.05), but younger cell lines displayed a lower mutation load (β = 4.29, 95% CI = [1.07; 9.47]). Mutations were confirmed in 18 genes by direct sequencing of RT-PCR products as previously reported for RAS and TP53 [1,9] (Additional file 1: Table S1). Although amplification of genes was not assessed because of the high number of chromosome abnormalities across the HMCL collection, exon losses were reported as described in the "Methods" section. Main variants are presented in Lollipop Plots (Additional file 3).
HMCLs display alterations similar to those in MM cells Figure 1 shows the most frequently altered genes across the collection and recurrent in MM [20][21][22]. Residues modified by mutants are provided in Additional file 1: Figure S4. Five genes, i.e., TP53, KRAS, NRAS, CDKN2C and PRKD2 were altered in at least 21% and up to 67% of HMCLs.
WES revealed that HMCLs displayed frequent muta- ) as well as in helicases (such as RECQL4, 15%, and BLM, 15%) and epigenetic modifiers (e.g., TET2, 15% and SETD2, 6%). FANC family genes were recently reported to be mutated mostly in patients at relapse [24,30,31], suggesting that these mutations did not occur in vitro in continuously replicating cells but in vivo. Mutations in epigenetic modifiers were recently described as being more frequent at relapse [20,22,24,32], such as histone methyl-transferases (6.9% vs 17%, in DMM and RMM, respectively) and DNA methylation modifiers (1.9% vs 8.3%, respectively). On the other hand, genes involved in apoptotic pathways (extrinsic, intrinsic, execution) displayed few mutations or deletions, showing that cell death resistance was not associated with major defects in the apoptotic machinery.
To provide a comprehensive landscape of mutations/ deletions, we next performed global analysis of altered pathways based on the whole data of the mutated genes.
HMCLs harbor the signatures of dysregulation in Rho GTPase, the cell cycle, and DNA replication Gene Ontology (GO) enrichment analysis showed that most of the dysregulated biological processes were related to Rho GTPase signal transduction, Cell cycle/DNA replication and DNA damage (check-points before replication, DNA repair, DNA unwinding) (Additional file 1: Figure  S6A). GO molecular functions such as helicase activity, nuclease activity, and Rho GTPase activity were also highly enriched (Additional file 1: Figure S6B-C).
Reactome Pathway Enrichment analysis revealed oncogenic MAPK signaling. After relaxing the q value at 0.1, pathways involved in DNA repair, p53 regulation of Fig. 1 Oncoprint of the most frequently mutated and/or deleted genes in human myeloma cell lines. HMCLs were ranked according to the most frequent abnormalities. Several events affecting the same cell line (mutations and deletion) were represented in the same slot. The number of cumulative events per HMCL is indicated on the top of the graph activity, and DNA double helix-modifying pathways were highlighted, as well as defects in the SUMOylation of DNA replication proteins, DNA damage response and repair proteins, cell cycle regulation by p53, resolution of D-loop structures through Holliday junction intermediates, and DNA repair (Additional file 1: Figure S6D).
Additional file 1: Figure S7 summarizes hits in the most dysregulated pathways. While pathway dysregulations were globally well balanced among the recurrent translocation subgroups, genes encoding helicases were more frequently encountered in t(11;14) cell lines (p = 0.006). On the other hand, intrinsic apoptosis mutants were more frequent in t(4;14), 66% vs 12% in non t(4;14) cell lines (p = 0.004).
The extrinsic, intrinsic, and executive pathways of apoptosis are mostly unaltered in HMCLs Thirteen HMCLs displayed one or several mutations in the apoptotic pathway (extrinsic, intrinsic, and executive), which were heterozygous (except in LP1 that displayed a bi-allelic BCL2L11/BIM deletion) (Fig. 3). To assess the impact of mutations, we analyzed the cell death response through either the extrinsic, i.e., response to Fas/Trail-R agonist receptors (CH11, mapatumumab, or lexatumumab [9]) or the intrinsic pathway of Fig. 2 Comparison of the gene mutation/deletion frequency in human myeloma cell lines with multiple myeloma patients at diagnosis and relapse. The frequency of mutation/deletion at diagnosis (x-axis) was plotted against that at relapse (y-axis, blue). HMCL hit frequencies are represented in red dots. The dashed line represents the theoretical identical ratio between diagnosis and relapse apoptosis, i.e., response to BH3-mimetics (ABT-199, A-1210477, or A-1155463) [10,11,34,35] (Additional file 1: Table S2). Pathway hit scores were calculated according to the number of hits in each pathway. No correlation could be drawn between the sensitivity to Trail-R agonists or Fas ligands and extrinsic apoptosis hits. Similarly, ABT-199/-737 responses did not correlate with intrinsic apoptosis hits. Moreover, intrinsic pathway hits were not associated with BH3-profiling, i.e., cytochrome C release in response to BIM peptide (Additional file 1: Table S2), confirming that the heterozygous mutations did not affect the upstream apoptotic responses (Additional file 1: Figure S8).
HMCLs with RAS mutation are highly sensitive to trametinib Eighty-two percent of HMCLs displayed at least one variant of the MAPK pathway, with 60% of HMCLs bearing a K-/H-/N-RAS variant (Fig. 3 and Additional file 1: Figure  S9). FGFR3 mutations were present in 12% of HMCLs but in 50% of t(4;14) HMCLs with FGFR3 overexpression (KMS11, LP1, OMP2). Five HMCLs expressed a BRAF mutation with BCN displaying six non-silent mutations in the PKc-like domain (without evidence of a frameshift). BRAF mutations mostly occurred in the PKc-like domain (four of five mutants), outside of the V600 codon. Two samples displayed both NRAS and BRAF mutations (NAN10 and NAN3). As shown in Fig. 4a, RAS-mutated HMCLs displayed hypersensitivity to the MEK-1/2 inhibitor trametinib (Mann-Whitney test, p = 0.002), while the three FGFR3-mutated HMCLs did not display significant sensitivity. Two HMCLs with BRAF mutations (without concomitant RAS mutation) out of three displayed hypersensitivity to trametinib. Of note, the two BRAF mut HMCLs displaying high sensitivity to trametinib had variants around V600. Finally, the MAPK pathway hit score was associated with an increased sensitivity to trametinib (β 1 = − 1.17, 95% CI = [− 1.41; − 0.61]), which was mainly related to RAS mutations (Fig. 4b).

Overactivation of NFκB by genomic alterations does not confer oversensitivity to proteasome inhibitors
The NFκB pathway was altered in 45% of HMCLs, mostly by inactivating TRAF3 (frame-shift, non-sense mutation, insertion or deletion) or BIRC2/BIRC3 (homozygous deletion) as previously reported in primary myeloma cells [20,23]. Alterations in the NFκB pathway were associated with the overexpression of NFκB signature genes [37] (i.e., CD74, TNFAIP3, IL2RG, BIRC3, and PLEK), and we further identified that NFE2L3 (a downstream target of TNF-α signaling displaying a NFκB site in the promoter) was highly expressed in samples harboring NFκB pathway hits (Fig. 6). Furthermore, we found no correlations between NFκB pathway dysregulation and sensitivity to proteasome inhibitors (β = 0.2, 95% CI = [− 0.25; 0.65] for bortezomib and β = − 0.09, 95% CI = [− 0.5; 0.16] for carfilzomib, Additional file 1: Figure S8). p53 and DNA damage pathways are associated with shifts in response to myeloma alkylating drugs We previously reported that the sensitivity to alkylating drugs was impaired by p53 deficiency [12]. We further assessed whether hits in pathway(s) were associated with the response to drugs reported to be related to p53 deficiency. As shown in Fig. 7 On the other hand, hits in the p53 pathway were also associated with reduced sensitivity to lexatumumab (β = 0.71, 95% CI = [0.01; 1.95]) and with a trend for increased sensitivity to mapatumumab (β = − 0.79, 95% CI = [− 1.24; − 0.08]). These correlations were related to the direct and indirect p53-mediated regulation of TNFRSF10B and TNFRSF10A expression, as previously reported [9]. The sensitivity to trametinib is associated with RAS mutation. a Cells were cultured for 4 days with increasing concentrations of trametinib, and the sensitivity was determined by the area under the curve using the MTT assay and expressed as z-score. Analysis was performed as a function of mutations in the MAPK pathway (Mann-Whitney test). b Trametinib response associated with dysregulation in the MAPK pathway. Robust linear regression is displayed; regression line was drawn according to coefficients obtained after 5000 bootstrapped replicates. Points were jittered for clarity Correlations between other clinically commonly used drugs (Imids, dexamethasone, and proteasome inhibitors) and pathways were not significant (Additional file 1: Figure S8).

Deficiency in the DNA repair pathway does not predict the responses to RITA or CX-5461
We assessed whether efficacy of DNA targeting drugs could be related to specific alterations. We analyzed sensitivity to RITA and CX-5461, which induce DNA crosslinking and stabilize DNA G-quadruplex, respectively, and are known to involve DNA repair during DNA replication [13,38,39]. Since p53 is involved in DNA repair, we analyzed drug responses according to TP53 status of HMCLs. Sensitivity to RITA in TP53 wt HMCLs was enhanced by mutations in helicases (β = − 0.59, 95% CI = [− 1.04; − 0.01]) (Fig. 7). HMCL sensitivity to CX-5461 was not associated with any genes or pathway alterations (Additional file 1: Figure S8) although XG11, which displayed a homologous BRCA2 mutation, showed the highest sensitivity to CX-5461.

Discussion
WES was performed in 33 HMCLs, including 19 that had not been reported yet. HMCLs selected in this study were established between 1965 and 2015 in Europe, USA, or Japan and in the presence or absence of added recombinant IL6. While HMCLs' ages spanned from5 5 to 3 years old, mutation load was similar among those, even if more recent HMCLs display lower Fig. 5 The sensitivity to palbociclib is associated with the lack of RB1 deletion/mutation in CCND1+ human myeloma cell lines. a Cells were cultured for 24 h with palbociclib (500 nM); cell cycle modifications (a) and Rb phosphorylation (b) were assessed by propidium iodide staining and Western blotting, respectively. For HMCLs harboring a functional RB1, mean phase S/G2 reduction was 26% (95% CI = 16-34%) (paired t test, p = 0.03) and mean (pRb/Rb reduction was 63% (95% CI = 42-91%) (paired t test, p = .03). c, d, e Cells were cultured for 4 days with increasing concentrations of palbociclib, and sensitivity was determined by the area under the curve (AUC) using the MTT assay. c Correlation between S/G2 phase inhibition and palbociclib sensitivity (AUC z-score), bootstrapped robust linear regression. d, e Palbociclib sensitivity according to either the CDKN2A/CDKNC status or MM molecular classification. RB1 abnormal HMCLs are indicated (Mann-Whitney test) mutation load. Our analysis showed that mutated genes were shared between HMCLs and primary myeloma cells, whatever the organ origin of samples that gave rise to the cell lines. Although HMCLs always emerged from patients with extra-medullary disease, no strong comparison could be made with primary or secondary PCL because of the very low number of sequenced PCLs yet, except for del17p (46% in sPCL) [29]. We thus compared mutation frequency with primary cells at diagnosis and at relapse (without any indication of medullary or extramedullary disease). Although the number of HMCLs was low as compared with patients, we nevertheless identified genes with a very high tumor load suggesting that some of them are drivers. The HMCLs mutational landscape may thus provide a panorama of mutations in refractory patients. The frequency of mutated "myeloma" genes in HMCLs was identical, lower, or higher when compared to primary cells at diagnosis or relapse [20][21][22]. While mutation rate in KRAS was similar between HMCLs and primary myeloma cells, the frequencies of TP53 (67%), CDKN2C (33%), PRKD2 (21%), FAM46C (15%), and BRAF (15%) dramatically increased compared to primary myeloma cells, either in DMM or RMM. The high TP53 abnormality frequency (67%) in HMCLs identified by WES in our study (and confirmed by direct RT-PCR sequencing [1]) was not in good agreement with a previous WES study reporting a rate of 21% in HMCLs [40], which was highly underestimated: indeed, well-known TP53 mutations in L-363, LP-1, and SKMM-2 (COSMIC database and p53.iarc.fr, Release = 18) were not reported in this study and at least three "HMCLs" were not of myeloma origin (ARH77, MC-CAR, CTV-1) [41,42].
Our results clearly confirmed a major alteration in both proliferation control, with either loss of suppressor (TP53, CDKN2C, RB1) or acquisition of activator (BRAF, RAS) and in tumor suppression/drug response (TP53, FAM46C), as in most if not all cancers [43]. Because the loss of function of TP53, FAM46C, or CDKN2C are not directly targetable, drugs bypassing these proteins or exploiting their loss consequences are required. Indeed, as shown in Fig. 5, cells lacking CDKN2C expression were sensitive to the CDK4/6 inhibitor palbociclib, especially in the CCND1 group. This CCND1 impact was surprising because palbociclib is efficient against all CDK-CCND complexes, i.e., CDK4/CCND1, CDK4/ CCND3, and CDK6/CCND2 [44]. Of note, CCND2 myeloma cells overexpress CDK6 and CDK4 while CCND1 myeloma cells overexpress CDK4 but not CDK6, suggesting that CDK4 is "empty" of cyclin D in CCND2 myeloma cells (Additional file 1: Figure S10). This free CDK4 pool might explain the low efficiency of palbociclib in CCND2 HMCLs. Palbociclib has shown no global efficiency in MM patients without indication of their subgroup origin and their CCND1 expression [45]. However, since it is efficient (in combination) in patients with tumors overexpressing CCND1 such as mantle cell lymphoma or HR + breast cancer, it might be of interest for patients with t (11;14) without Rb deficiency [46,47]. Concerning TP53, we previously described p53 independent drugs, which were efficient whatever TP53 status, such as PRIMA-1 Met that targets glutathione or BH3 mimetics that target anti-apoptotic proteins [6,11]. We also reported that loss of p53 function favors measles virus replication and cell death in myeloma cells Fig. 6 Mutations/deletions in the NFκB pathway genes are correlated with the overexpression of NFκB target genes. a The expression of genes significantly associated with mutation/deletion in NFκB pathway genes was identified using the limma algorithm. Clustering was performed with the most significant genes. b Representation of the NFκB pathway hits according to HMCL classification in a [48]. FAM46C was recently been shown to encode for a non-canonical poly(A) polymerase and its over expression in MM cells induced cell death [49]. FAM46C is a type I IFN-stimulated gene, and it might modulate virus replication such as the yellow fever virus (YFV) and the Venezuelan equine encephalitis virus (VEEV) [50]. Of note, anti-viral type I IFN pathway appeared highly impaired, suggesting defects in infection defense that might be exploited using oncolytic viruses such as measles virus [48,51].
Concerning mutations with gain of function such as RAS mutations, we showed that sensitivity of 27 HMCLs to MEK1/2 inhibitor trametinib was associated to RAS mutations (70% of "RAS only" mutated HMCLs were sensitive), but not to FGFR3 (none sensitive HMCL out of three with "FGFR3 only" mutation). Concerning BRAF, four HMCLs out of five with BRAF mutation (and with NRAS mutations for two of them) were sensitive but the low number of "BRAF only" mutated HMCLs prevented definitive conclusions. Although all HMCLs without hit in RAS/BRAF/FGFR3 genes were resistant to trametinib, all HMCLs with NRAS mutation were not sensitive since four NRAS mutated HMCLs were resistant. These data collectively suggest that mutation in RAS/BRAF genes is required but not sufficient for eliciting response to trametinib. The BRAF/RAS impact will be assessed in an ongoing clinical trial (NCT03091257) evaluating dabrafenib and/or trametinib in patients with relapsed and/or refractory multiple myeloma patients according to their BRAF/RAS mutation.
The high percentage of altered genes in DNA/chromatin repair/regulation, Fanconi pathway, and chromatin/ DNA modification might be related to the frequency in relapsing patients [32]. Because of the lack of specific drugs, we could not directly assess the functionality/vulnerability of these pathways, which require a deep investigation. Of note, mutations in Fanconi genes were recently reported in patients at relapse, suggesting that drug escape might involve this pathway. HCLMs exhibiting such "BRCAness" will be a good model for assessing Fig. 7 Significant associations between dysregulated pathways and drug responses. The number of hits in each pathway was plotted against the drug z-score. Robust linear regression is displayed; regression line was drawn according to coefficients obtained after 5000 bootstrapped replicates. Only significant associations of tested drugs with the pathways of interest are displayed. Drugs responses were detailed in Methods, as durations and assessment of response differ according to drugs pharmacodynamical specificities. a Drug responses associated with dysregulation in the p53 pathway. b Drug responses associated with dysregulation in the DNA damage pathway. c Drug responses associated with dysregulation in the MAPK pathway. d RITA sensitivity correlated with the helicase hit scores in TP53 wt HMCLs. TP53 wt and TP53 Abn HMCLs are represented by black and red dots, respectively. The lethal-dose-50 of RITA was determined as previously described [13]. Asterisk indicates not statistically significant efficiency of drugs like USP1 and/or PARP inhibitors [23,31,52].
On the other hand, no major alteration was found in apoptosis pathway, either extrinsic/intrinsic or executive, showing that resistance to cell death was rather upstream of the mitochondria. In good agreement with the low number of alterations in apoptosis pathway, HMCLs were highly primed for death as shown by their BH3 profiling and their high response rate to BH3 mimetics [11,53] (Additional file 1: Table S2). Considering the huge difference between cell responses to DNA damaging drugs and BH3 mimetics, loss of response was not on the mitochondrial side, and BH3 mimetics appear thus of major interest to target MM cells whatever their genomic alterations or responses to classical myeloma drugs.

Conclusions
In summary, WES suggests that HMCLs harbor enriched mutations and defects in cell cycle, p53, recombination/ DNA repair, NFκB, and epigenetic genes. Importantly, some very early pathogenic events such as IgH translocations and MAPK pathway mutants are stable over time and are not enriched by in vitro long-term culture, thus making HMCLs a reliable drug screening model for refractory patients at diagnosis or relapse. What is more, detection at diagnosis of mutations/deletions in genes associated with progression and HMCLs (i.e., CDKN2C, FAM46C, TRAF3, PRKD2) might identify particularly aggressive sub-clones warranting adapted treatment strategies and surveillance. WES results suggest that in addition to target apoptosis using BH3 mimetics and the antiviral deficiency using oncolytic viruses, targeting DNA damage, recombination/DNA repair, and epigenetic modifiers should be further investigated and might offer significant options for high-risk and refractory patients, including extramedullary diseases.