# VDAC2 primes myeloma cells for BAK-dependent apoptosis and represents a novel therapeutic target

Ophélie Champion, Sophie Maïga, Chloé Antier, Christelle Dousset, Agnès Moreau-Aubry, Céline Bellanger, Géraldine Descamps, Jose Antonio Moreno, Ohyun Kwon, Nicoletta Libera Lilli, et al.

► To cite this version:

Ophélie Champion, Sophie Maïga, Chloé Antier, Christelle Dousset, Agnès Moreau-Aubry, et al. VDAC2 primes myeloma cells for BAK-dependent apoptosis and represents a novel therapeutic target. Leukemia, 2025, 10.1038/s41375-025-02523-8. hal-04965584

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1 VDAC2 primes Myeloma cells for BAK-dependent apoptosis and represents a novel

2 therapeutic target

3

4 Ophélie Champion¹,², Sophie Maïga¹,², Chloé Antier¹, Christelle Dousset¹,², Agnès Moreau-Aubry¹,²,

5 Céline Bellanger¹,², François Guillonneau¹,²,³, Géraldine Descamps¹,², Jose Antonio Moreno⁴, Ohyun

6 Kwon⁴, Nicoletta Libera Lilli⁵, Philippe Moreau ¹,², David Chiron¹,², Catherine Pellat-Deceunynck¹,²,

7 Cyrille Touzeau¹,², Patricia Gomez-Bougie¹,²

8

9 1.Nantes Université, CHU Nantes, CNRS, INSERM, CRCI2NA, Université d'Angers, F-44000 Nantes, France

10 2.SIRIC-ILIAD (Imaging and Longitudinal Investigations to Ameliorate Decision-making)

11 Nantes-France.

12 3. Prot'ICO proteomics facility, Institut de Cancérologie de l'Ouest (ICO), F-49055 Angers, France

13 4.Department of Chemistry and Biochemistry, University of California—Los Angeles, Los Angeles, CA, USA.

14 5. Hematology Department, CHU Nantes, Nantes, France

15

16 19 Corresponding authors: Patricia Gomez-Bougie, Centre de Recherche en Cancérologie et

20 Immunologie Intégrée Nantes Angers, INSERM, CNRS, Institut de Recherche en Santé Nantes

21 Université (IRS-UN), 8 quai Moncousu, BP70721, F-44007. 33+22808318 Nantes-France. E-mail

22 patricia.gomez@inserm.fr ; and Catherine Pellat-Deceunynck, Centre de Recherche en Cancérologie et

23 Immunologie intégrée Nantes Angers, INSERM, CNRS, IRS-UN, 8 quai Moncousu, BP70721, F-44007

24 Nantes-France. 33+22808298 E-mail catherine.pellat-deceunynck@univ-nantes.fr

25

26

27 All authors declare no conflict of interest

28

29

30

31 Running title: VDAC2 controls mitochondrial priming in myeloma

32
33 **TO THE EDITOR**

34 BAX and BAK, key executors of apoptosis from the BCL2 family (1), are also regulated by
35 non-BCL2 family proteins, such as the mitochondrial channel VDAC2 (2). Although well-
36 recognized for its role in metabolite and ion flux (3), VDAC2 has drawn particular attention for
37 its involvement in the regulation of apoptosis. VDAC2 was reported as being necessary for
38 efficient BAX-dependent apoptosis (4), while inhibiting BAK apoptotic function (5). BAX, but
39 not BAK, is a well-known p53 target gene, and p53-mediated regulation of BAX is required
40 for an optimal response to BH3-mimetics targeting BCL2 or MCL1 (6,7). In p53-deficient
41 myeloma cells with low BAX expression, apoptosis is expected to rely on BAK expression and
42 regulation. These data prompted us to investigate whether targeting VDAC2 would overcome
43 the mitochondrial resistance of p53-deficient cells.

44 To investigate how VDAC2 influences mitochondrial apoptosis in multiple myeloma (MM),
45 we performed transient silencing of VDAC2 in TP53-deficient human myeloma cell lines
46 (HMCLs). Additionally, we evaluated the pharmacological targeting of VDAC2 in both MM
47 cell lines and primary MM patient cells.

48 We first analyzed RNA-seq data to evaluate the expression of *VDAC2* in MM cells from 32
49 patients at diagnosis and plasma cells from 9 healthy donors (HD) (8), as well as in 17 HMCLs.
50 *VDAC2* was ubiquitously expressed and significantly higher in myeloma cells compared to non-
51 malignant plasma cells (Figure 1A).

52 We next silenced VDAC2 in two p53-deficient, BAK-dependent HMCLs, KMS12PE and
53 KMM1, which rely on BAK for their response to BH3-mimetics (Figure 1B). Importantly,
54 VDAC2 silencing, which resulted in a decrease in VDAC2 expression by 41% in KMS12PE
55 and 65%, in KMM1 cells (Figure 1C), significantly increased global mitochondrial priming, as
56 evidenced by a 35% (p<0.0001) and 19% (p=0.0026) enhancement in cytochrome c release
57 triggered by BIM-derived peptide in KMS12PE and KMM1, respectively (Figure 1D).
Furthermore, VDAC2 silencing heightened the dependence on MCL1 and BCL2, as evidenced by the enhanced cytochrome c release triggered by MS1 peptide, with a 53% increase in KMS12PE (p<0.0001) and 29% in KMM1 (p=0.0002). Additionally, a 26% increase in cytochrome c release was observed upon Venetoclax in KMS12PE (p=0.0003; Figure 1D).

Accordingly, cell death induced by the MCL1 mimetic S63845 or Venetoclax was significantly increased upon VDAC2 knock-down. The S63845 LD₅₀ was dramatically decreased from 92 nM to 21 nM (4.4-fold decrease) in KMS12PE and from 85 nM to 28 nM (3.0-fold decrease) in KMM1. Similarly, Venetoclax LD₅₀ dramatically decreased from 35.25 nM to 2.6 nM in KMS12PE (13.6-fold decrease, Figure 1E). Notably, cell death induced by recombinant TRAIL was not modified upon VDAC2 silencing (Figure 1E), indicating that VDAC2 silencing specifically impacts the intrinsic apoptotic pathway. Furthermore, the efficient reduction of VDAC2 expression did not modify the protein levels of the effectors BAK or BAX (Figure 1F).

Taken altogether, these data indicate that VDAC2 silencing enhances mitochondrial priming, rendering cells more sensitive to apoptosis induced by BH3-mimetics.

These findings led us to pharmacologically target VDAC2, aiming to achieve a similar apoptotic response to that induced by VDAC2 transient silencing. Efsevin was identified through library screening for chemicals that are capable of reversing the arrhythmia phenotype in the tremblor zebrafish model (9). Recently, this activity was shown to be dependent on VDAC2, and Efsevin was found bound to VDAC2 (10). Nevertheless, the role of Efsevin in apoptosis has never been reported.

Efsevin itself did not induce apoptosis in HMCLs (Figure 2A, Supplementary Figure 1A) but significantly enhanced mitochondrial priming, as evidenced by the increased cytochrome c release induced by BIM, with a 38% increase in KMS12PE (p=0.0029) and 24% increase in KMM1 (p=0.0003). Efsevin also heightened dependence on MCL1 and BCL2, as evidenced by the enhanced cytochrome c release triggered by the MS1 peptide, with a 25% increase in
83 KMS12PE (p= 0.047) and a 13% increase in KMM1 (p=0.0187), as well as by Venetoclax,
84 with 52% increase in KMS12PE (p<0.0001; Figure 2B).

85 To confirm the impact of Efsevin on mitochondrial priming, we combined Efsevin with S63845
86 or Venetoclax in 9 HMCLs. Although the efficacy of this combination was observed as early as
87 24 hours of treatment (Supplementary Figure 1B), we opted to determine LD₅₀ values after 96
88 hours, with S63845 or Venetoclax added for the last 24 hours. Figure 2C shows that Efsevin
89 significantly sensitized 4 out of 6 HMCLs (BCN, KMS12PE, MM1S and KMM1) to S63845,
90 leading to a 1.7- to 4.6-fold reduction in S63845 LD₅₀ (Supplementary Table 1A). Bliss synergy
91 scores in KMM1 (39.7 ± 2.9) and KMS12PE (34.3 ± 2.3) confirmed a strong synergy between
92 Efsevin and S63845 (Supplementary Figure 1C).

93 Similarly, we also found a significant synergy between Efsevin and Venetoclax in 4 t(11;14)
94 HMCLs (KARPAS620, MDN, XG5 and KMS12PE), resulting in a 1.8- to 6.8-fold decrease in
95 Venetoclax LD₅₀ (Figure 2D, Supplementary Table 1B), with a Bliss score of 39.7 ± 2.9 in
96 KMS12PE (Supplementary Figure 1C). Notably, Efsevin enabled the achievement of
97 Venetoclax LD₅₀ values well below 100 nM, which is the highest ex vivo concentration
98 associated with a favorable in vivo clinical response to Venetoclax (11).

99 Interestingly, the combination of Efsevin and BH3-mimetics was effective irrespective of TP53
100 status. Sensitization to MCL1 or BCL2 BH3-mimetics was observed in both TP53⁺⁺ and TP53⁻
101 /mut HMCLs (Supplementary Table 1A and 1B). Consistently, Efsevin reduced S63845 LD50
102 values from 80nM to 40nM in the isogenic NCI-H929 TP53⁻/⁻, although this remained higher
103 than the S63845 LD50 observed in the NCI-H929 TP53⁺⁺ cells (Supplementary Figure 1D). The
104 decrease in LD50 values in p53-deficient cells, along with the observed BAK dependence for
105 the BH3-mimetic response in the p53-deficient KMS12PE and KMM1 cells, strongly suggests
106 that mitochondrial depolarization relies on BAK in this context. Indeed, in KMM1 cells, while
107 Efsevin similarly decreased S63845 LD50 in control cells (from 84 to 33 nM, p= 0.0025) and
108 *BAX-KO* cells (from 167 to 65 nM, p= 0.0023), it failed to overcome the S63845 resistance in
109 *BAK-KO* cells (from 400 to 334 nM, p=0.0746; Figure 2E, Supplementary Figure 1E),
110 underscoring the essential role of BAK in mediating the Efsevin response in this setting.

111 Since the Efsevin-induced increase in mitochondrial priming and sensitivity to BH3-mimetics
112 was not associated with any changes in the expression levels of VDAC2 or BCL2 family
113 members (Supplementary Figure 1F), we hypothesized that the enhanced sensitivity to S63845
114 might depend on the levels of MCL1-BAK complexes. Indeed, co-immunoprecipitation assays
115 of MCL1 in KMM1 cells pre-treated with Efsevin revealed a significant ~2-fold increase
116 (p=0.0121) in the amount of BAK bound to MCL1 compared to untreated cells (Figure 2F).

117 Since MM is mainly dependent on MCL1 for survival (12,13), we assessed the effect of Efsevin
118 (15 µM) in combination with S63845 (25 nM) in myeloma cells from 27 consecutive patients,
119 14 at diagnosis and 13 at relapse from the MYRACLE cohort (14) (Supplementary Table 2).

120 Median cell death induced by Efsevin, S63845 and their combination was 6.9%, 31.7%, and
121 54.7%, respectively (Figure 2G, left panel). Because it was not possible to perform synergy
122 assays in patient samples, we analyzed the differences between the expected and the observed
123 values (Figure 2G, right panel). The combination of S63845 and Efsevin was indeed effective
124 in 96% of the samples, being additive (-10%< observed minus expected death <10%) in 16
125 samples (59%) and synergistic (observed minus expected death >10%) in 10 samples (37%).

126 The combination efficacy was independent of 1q21 gain (median cell death 84% in 1q21 gain
127 vs. 58% in no 1q21, respectively, p= 0.4603; Supplementary Figure 1G).

128 In summary, we demonstrated that VDAC2 plays a critical role in the apoptotic response to
129 BH3-mimetics by regulating BAK function in *TP53*-deficient MM cells. Targeting VDAC2
130 thus offers a potential strategy to overcome apoptosis resistance associated with p53/BAX
deficiency. Specifically, VDAC2 knock-down or its pharmacological targeting, achieved for the
131 first time with Efsevin, led to increased mitochondrial priming and enhanced sensitivity to both
132
133 S63845 and Venetoclax. The increase in the amount of BAK bound to MCL1 upon Efsevin treatment, suggests a potential strategy to use Efsevin in combination with agents that directly or indirectly target MCL1. Moreover, the reported *in vivo* tolerability of Efsevin in a murine model (15), suggests that targeting VDAC2 with this modulator could represent a promising strategy with potential clinical applications to enhance the apoptotic response to BH3-mimetics such as Venetoclax. Further investigation is warranted to determine whether Efsevin could also enhance the response to other current therapeutic agents.

140 REFERENCES

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144 2. Moldoveanu T, Czabotar PE. BAX, BAK, and BOK: A Coming of Age for the BCL-2 Family Effector Proteins. Cold Spring Harb Perspect Biol. 2020 Apr 1;12(4):a036319.

146 3. Törnroth-Horsefield S, Neutze R. Opening and closing the metabolite gate. Proc Natl Acad Sci U S A. 2008 Dec 16;105(50):19565-6.

148 4. Chin HS, Li MX, Tan IKL, Ninnis RL, Reljic B, Scicluna K, et al. VDAC2 enables BAX to mediate apoptosis and limit tumor development. Nat Commun. 2018 Nov 26;9(1):4976.

150 5. Cheng EHY, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science. 2003 Jul 25;301(5632):513-7.

152 6. Thijssen R, Diepstraten ST, Moujalled D, Chew E, Flensburg C, Shi MX, et al. Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias. Blood. 2021 May 20;137(20):2721-35.

155 7. Durand R, Descamps G, Bellanger C, Dousset C, Maïga S, Alberge JB, et al. A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics. Blood. 2024 Mar 28;143(13):1242-58.

158 8. Alameda D, Goicoechea I, Vicari M, Arriazu E, Nevone A, Rodriguez S, et al. Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development. Blood. 2021 Oct 28;138(17):1583-9.

161 9. Shimizu H, Schredelseker J, Huang J, Lu K, Naghdi S, Lu F, et al. Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity. Elife. 2015 Jan 15;4:e04801.

164 10. Wilting F, Kopp R, Gurnev PA, Schedel A, Dupper NJ, Kwon O, et al. The antiarrhythmic compound efsevin directly modulates voltage-dependent anion channel 2 by binding to its
166 inner wall and enhancing mitochondrial Ca2+ uptake. Br J Pharmacol. 2020
167 Jul;177(13):2947-58.

168 11. Matulis SM, Gupta VA, Neri P, Bahlis NJ, Maciag P, Leverson JD, et al. Functional profiling
169 of venetoclax sensitivity can predict clinical response in multiple myeloma. Leukemia.
170 2019 May;33(5):1291-6.

171 12. Gong JN, Khong T, Segal D, Yao Y, Riffkin CD, Garnier JM, et al. Hierarchy for targeting
172 prosurvival BCL2 family proteins in multiple myeloma: pivotal role of MCL1. Blood. 2016
173 Oct 6;128(14):1834-44.

174 13. Gomez-Bougie P, Maiga S, Tessoulin B, Bourcier J, Bonnet A, Rodriguez MS, et al. BH3-
175 mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma
176 treatment. Blood. 2018 Dec 20;132(25):2656-69.

177 14. Benaniba L, Tessoulin B, Trudel S, Pellat-Deceuninck C, Amiot M, Minvielle S, et al. The
178 MYRACLE protocol study: a multicentric observational prospective cohort study of
179 patients with multiple myeloma. BMC Cancer. 2019 Aug 29;19(1):855.

180 15. Schweitzer MK, Wilting F, Sedej S, Dreizehnter L, Dupper NJ, Tian Q, et al. Suppression
181 of Arrhythmia by Enhancing Mitochondrial Ca2+ Uptake in Catecholaminergic Ventricular
182 Tachycardia Models. JACC Basic Transl Sci. 2017 Dec;2(6):737-47.

183
184
185 FIGURE LEGENDS

186 **Figure 1.** Transient inhibition of VDAC2 enhances mitochondrial priming and sensitivity
187 **to BH3-mimetics.**

188 A) VDAC2 expression in MM cells. VDAC2 expression was assessed by 3'seq-RNA (3'SRP)
189 in 32 MM patients at diagnosis and plasma cells from 9 healthy donors (HD) obtained from a
190 publicly available dataset (7) and in 17 human myeloma cell lines (HMCLs). B) KM12PE and
191 KMM1 HMCLs were transfected with siRNA targeting BAK or BAX for 24 and 48 hours,
192 respectively and then treated 24 hours with BH3-mimetics targeting MCL1 (S63845, 125nM
193 for KMS12PE and 50nM for KMM1) or BCL2 (Venetoclax, 25nM). Cell death was assessed
194 by Annexin V staining and flow cytometry. Results are representative of 3 independent
195 experiments. Statistical analysis was performed using paired t test. C) VDAC2 was efficiently
196 knocked down. KMS12PE and KMM1 cell lines were transfected with siRNA targeting
197 VDAC2 as in (B). siRNA transfection efficacy was determined by western blotting, a
198 representative western blot from 3 independent experiments is shown. VDAC2 relative protein
199 levels were quantified and normalized to Tubulin or Actin. Statistical analysis was performed
200 using paired t test. D) Mitochondrial priming was increased under transient VDAC2 silencing.
201 BH3 profiling was performed on KMS12PE and KMM1 cell lines that were transfected with
202 siRNA CT or targeting VDAC2 as in (B). Cytochrome c release was measured by flow
203 cytometry after challenging cells with BIM (0.05 µM), MS1 peptides (10 µM for KMS12PE
204 and 5 µM for KMM1) and Venetoclax (0.5 µM). PUMA 2A (10 µM) was an inert peptide.
205 Values corresponded to the percentage of cytochrome c loss (mean ± SD) of 3 independent
206 experiments. Statistical analysis was performed using the 2-way ANOVA test. E) VDAC2
207 knock-down increased apoptosis induced by BH3-mimetics targeting MCL1 or BCL2. Cells
208 were transfected as in (B) and treated 24 hours with S63845, Venetoclax or TRAIL (tumor-
209 necrosis-factor related apoptosis inducing ligand). Cell death was determined by Annexin V
staining using flow cytometry. Dose response and the respective LD₅₀ values in KMS12PE (left) and KMM1 cells (right). Results represent the mean ± SD of 3 independent experiments.

Statistical analysis was performed using paired t test. F) BAK and BAX protein levels were not modified upon VDAC2 silencing. Cells were transfected as in (B). BAK and BAX relative protein levels were quantified and normalized to Tubulin or Actin, results corresponded to the mean ± SD and are representative of 3 independent experiments. Statistical analysis was performed using paired t test.

**Figure 2. The VDAC2 modulator Efsevin increases mitochondrial priming and sensitivity to BH3 mimetics in MM cell lines and primary cells**

A) Efsevin did not induce cell death. HMCLs (n=9) were treated with EF as indicated for 24 hours. Cell death was assessed by Annexin V staining and flow cytometry. Values corresponded to the mean ± SD of 3 independent experiments. B) Mitochondrial priming was increased under EF treatment. BH3 profiling was performed on KMS12PE and KMM1 cells pre-treated with EF (10µM) during 72h. Cytochrome c release was measured by flow cytometry after challenging cells with BH3-derived peptides and Venetoclax. BIM peptide (0.05 µM), MS1 peptide (10 µM for KMS12PE, 5 µM for KMM1) and Venetoclax (0.5 µM). PUMA 2A (10 µM) was an inert peptide. Values corresponded to the percentage of cytochrome c loss (mean ± SD) of 3 independent experiments. Statistical analysis was performed using the 2-way ANOVA test. C) and D) Efsevin increased sensibility to BH3-mimetics targeting MCL1 or BCL2. HMCLs were treated with EF (10 µM) for 72h and then treated with the combination of C) EF and S63845 or D) EF and Venetoclax for the next 24 hours. Cell death was assessed by Annexin V staining using flow cytometry. LD₅₀ values corresponded to the mean ± SD of 3 independent experiments. Statistical analysis was performed using paired t test. E) Efsevin-sensitizing effect was dependent on BAK. KMM1 CTR4, *BAX*-KO1 and *BAK*-KO11 cells were treated as in (C). Cell death was assessed by Annexin V staining. LD₅₀ values corresponded to the mean ± SD of
235 4 independent experiments. Statistical analysis was performed using paired t test. F)

236 BAK/MCL1 complexes were increased following Efsevin treatment. KMM1 cells were treated

237 or not with EF (10 µM) for 72 hours, cell pellets were lysed and MCL1 was

238 immunoprecipitated. Immunoprecipitation eluates were analyzed by western blotting to
239 determine MCL1 and BAK levels. Western blot results are representative from 3 independent

240 experiments (left). The graph represents the relative amount of BAK bound to MCL1 from 3
241 independent experiments. Statistical analysis was performed using unpaired t test. G)

242 sensitized primary MM cells to MCL1 BH3-mimetic. The left graph represents cell death
243 response of myeloma cells from patient samples (n=27) to EF (15 µM), S63845 (25 nM), or

244 their combination after 24 hours of treatment. The right graph represents the difference between
245 observed cell death (25 nM S63845 combined with 15 µM EF) minus expected cell death (sum

246 of cell death induced by 25 nM S63845 and by 15 µM EF) (right). Cell death was assessed after
247 24 hours by flow cytometry. Statistical analysis was performed using the Wilcoxon rank sum

248 test.

249

250
251 ACKNOWLEDGEMENTS

252 The authors acknowledge the Cytocell-Flow Cytometry and FACS core facility (Structure
253 Fédérative de Recherche (SFR) Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556,
254 Nantes, France), member of the Scientific Interest Group Biogenouest and the labex IGO-
255 program supported by the French National Research Agency (n°ANR-11-LABX-0016-01) for
256 its technical support.

257 The authors thank the Genomics Core Facility GenoA, member of Biogenouest and France
258 Genomique and the Bioinformatics Core Facility BiRD, member of Biogenouest and Institut
259 Français de Bioinformatique (ANR-11-INBS-0013) for the use of their resources and their
260 technical support.

261 AUTHOR CONTRIBUTION

262 OC participated in the design of the study, performed experiments, derived CRISPR/Cas9
263 clones, analyzed the data and participated in the writing of the manuscript; AMA and CD
264 designed CRISPR/Cas9 strategy and profiled CRISPR/Cas9 BAK1 and BAX cells; SM managed
265 the patient cohort and performed experiments; CB performed bioinformatic analysis and
266 analyzed 3'SRP sequencing; FG participated in the proofreading of the manuscript; CA and GD
267 performed experiments. JAM and OK synthetized Efsevin; NLL managed the patient cohort;
268 PM managed the patient cohort, CT participated in the design of the study and managed the
269 patient cohort; DC and CPD participated in the design of the study, in the writing and revision
270 of the manuscript; PGB designed the study, performed experiments, analyzed the results and
271 wrote the manuscript.

272 ETHICS STATEMENT

273 The studies involving human participants were reviewed and approved by the University
274 Hospital of Nantes (MYRACLE study; NTC03807128) (14). The patients provided their
275 written informed consent to participate in this study. All methods used in this study were
276 performed in accordance with the relevant guidelines and regulations.

277 **FUNDING**

278 This work was supported by grants from Ligue Contre le Cancer, Comité de Loire-Atlantique
279 (CD44) and Vendée (CD85), Action Cancer 44 and INCA (INCa-DGOS-INSERM_12558,
280 INCa-DGOS-INSERM-ITMO Cancer_18011). OC was supported by grants from HEMA-
281 NexT (Chaire NexT to CT; ANR-16-IDEX-0007) and Fondation Française pour la Recherche
282 contre le Myélome et les Gammapathies (FFRMG).

283 **DATA AVAILABILITY STATEMENT**

284 RNA-sequencing datasets are publicly available (8). Data that support the findings of this study
285 are included in this published article and in its supplementary information files, and are
286 available from the corresponding author on reasonable request

287

288
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SUPPLEMENTARY INFORMATION

This document contains Supplementary Material and Methods, Supplementary References, 2 Supplementary Tables and 1 Supplementary Figure.
# Supplementary Material and Methods

## Human myeloma cell lines and primary myeloma cells

Human Myeloma Cell Lines, as well as the generated *BAK-KO* and *BAX-KO* KMM1 clones were characterized and routinely identified, as previously described (1) and tested for mycoplasma contamination by PCR. BCN, MDN, XG7, XG11, NAN6, NAN3, NAN8 and NAN11 were derived in our laboratory and are cultured with RPMI1640 with 5% FCS and 3ng/ml recombinant IL-6. The KMM1 and KMS12PE cell lines were kindly provided by Dr. Otsuki (Kawasaki Medical School, Kurashiki, Japan); MM1S by Dr. S. Rosen (Northwestern University, Chicago, USA); JJN3, LP1, L363, NCI-H929 (NCI), U266, OPM2 HMCLs were from DSMZ (Braunsweig, Germany).

Bone marrow/blood samples from MM patients were obtained after informed consent at University Hospital of Nantes, Department of Hematology (MYRACLE study; NTC03807128) (2).

## Reagents and antibodies

Venetoclax (ABT-199) was purchased from Selleck Chemicals (Houston, TX, USA); S63845 from Chemietek (Indianapolis, USA). Efsevin was synthesized by Dr. Ohyun Kwon and Jose Antonio Moreno, (Department of Chemistry and Biochemistry, University of California, Los Angeles) and previously characterized (3). Digitonin was from Sigma-Aldrich (D-141). Anti-VDAC2 (PA5-28106) was purchased from Thermo Fisher (Waltham, Massachusetts, USA). Anti-BAK (12105S), anti-BAX (2772S), anti- BCLXL (2764S), anti-PUMA (12450S), anti-NOXA (14766) and anti-TUBULIN (3873S) from Cell Signaling (Ozyme, Saint-Cyr l'Ecole, France). BCLXL (sc-271121), MCL1 (sc-12756) from Santa Cruz Biotechnology (Heidelberg, Germany). ACTIN (MAB1501) and BIM (Ab17003) from Millipore (Molsheim, France). Anti-BCL2 from BD Transduction Laboratories (Le Pont de Claix, France).

## Cell death assays

Cell death was assessed by AnnexinV-FITC staining (Beckman Coulter, Villepinte, France) in HMCLs or loss of CD138 staining in patient samples using flow cytometry as previously described (4,5).

## CRISPR-CAS9 genome editing

BAK1 or BAX knockout (KO) myeloma cells were generated by mutating a codon into a stop codon using KI CRISPR-CAS9. This approach is based on the specific cut followed by homologous recombination with the DNA matrix bearing the stop codon. It allows the
generation of identical clones, as the stop codon will always be at the same position and it ensures
the deletion of potential existing isoforms. 500,000 cells were transiently electroporated using
the Amaxa Transfection System (Lonza) in the presence of 44 pmol Cas9 electroporation
enhancer (IDT), with a RNP complex i.e., 36 pmol SpCas9 Nuclease pre-incubated with 44
pmol AATO550-conjugated tracerRNA and 44 pmol of the specific methylated crRNA, and a
DNA donor containing the stop codon (TAG). crRNA sequences were:

*BAK1* (5'-mAmGmACCTGAAAAATGGCTTCG-3') and *BAX* (5'-mAmGmTAGAAAAGGGCGACAACC-3'). The following codons were mutated to a stop codon (TAG), *BAK1* TCG (S3) and *BAX* CGG (R109).

Cells were then cultured in 96-well plates and cloned with a limiting dilution of 0.3 cells per well. Clones were characterized for BAK and BAX sequences by Sanger Sequencing (Supplementary Figure 3). Three clones *BAK-KO*, 3 clones *BAX-KO* and 3 CTR clones were selected for KMM1 cell line. Control clones were obtained using the same electroporation procedure, but without the addition of a target crRNA.

RNA-seq analysis

Genomic profiling of HMCLs was assessed by 3'-digital gene expression sequencing DGE-seq (3'seq-RNA), as previously described (6). For the analysis of publicly available data set and HMCLs, DESeq2 package was used to normalize expression. Normalized counts were transformed with vst (variance stabilized transformation) function from DESeq2.

siRNA transfection

KMM1 cell line was transfected with 100 pmol siRNA using Lipofectamine RNAimaxTM
Reagent (Life Technologies, St Aubin, France) according to the manufacturer's instructions.
KMS12PE cell line was transfected by nucleofection using the Amaxa Transfection System
(Lonza), the SF Solution and the CA 137 program. siRNAs ON-TARGETplus Control (D-
001810-10-20), BAX (L-003308-01-0005), BAK1 (L-003305-00-0005) and VDAC2 (L-
019766-00-0005) were from Dharmacon (Ilkirch, France).

Intracellular BH3 profiling

BH3 profiling assay was performed as previously described, using BH3 derived peptides and
Venetoclax (7). BIM peptide measured global priming, MS1 peptide measured MCL1
dependence, the inert peptide PUMA2A was included as a negative peptide control, Venetoclax
measured BCL2 dependence and DMSO was a control condition. Briefly, cells were permeabilized in DTEB buffer containing 0.002% of digitonin and exposed to peptides and Venetoclax for 1 hour at 27°C, followed by fixation with 8% formaldehyde at room temperature for 15 minutes. After addition of neutralizing buffer (Tris/Glycine buffer) for 5 minutes, cells were stained with anti-cytochrome c antibody Alexa 647 (BD Biosciences, Le Pont de Claix, France) in 0.1%Saponin/1%BSA/PBS overnight at 4°C. Cytochrome c release was analyzed by flow cytometry using a FACs CantOII (Becton Dickinson, Le Pont de Claix, France) and the quantification of cytochrome c loss induced by each peptide was calculated by gating the cytochrome c positive population.

## Immunoblotting

Cells pellets were lysed 40 minutes on ice in 0.5% NP40 containing lysis buffer. Lysates were then centrifugated 30 minutes at 10 000 rpm at 4°C and supernatants were collected. Total protein lysates (50µg per lane) were separated by SDS-PAGE using precast gels (Bio-Rad, California, USA or Thermo Fisher Scientific, Waltham, Massachusetts, USA) and then electrotransferred into a PVDF membrane using the Trans-blot Turbo transfer system at 2.5 A for 7 min (Bio-Rad, California, USA). Membranes were then blocked in 5% skim milk-TBS-0.01% Tween solution and further incubated overnight with the respective primary antibody Chemiluminescence was detected using Clarity-ECL detection reagent (Bio-Rad, California, USA) in a ChemiDoc Imaging System (Bio-Rad, California, USA). Protein expression levels were quantified using ImageLab and Image J software.

## Immunoprecipitation (IP)

Cell pellets were lysed in 1% digitonin-containing buffer and lysates were pre-cleared with protein A-sepharose beads. For MCL1 IP, 700 µg of cell lysate was incubated overnight with an agarose-conjugated monoclonal antibody anti-MCL1 from Santa Cruz Biotechnology (sc-12756AC). The immunoprecipitation eluates were analyzed by western blot to assess MCL1 and BAK levels and the BAK/MCL1 ratio was calculated to evaluate the relative abundance of the complex under each condition.

## Supplementary references

1. Maïga S, Brosseau C, Descamps G, Dousset C, Gomez-Bougie P, Chiron D, et al. A simple flow cytometry-based barcode for routine authentication of multiple myeloma and mantle cell lymphoma cell lines. Cytometry A. 2015 Apr;87(4):285-8.
2. Benaniba L, Tessoulin B, Trudel S, Pellat-Deceuninck C, Amiot M, Minvielle S, et al. The MYRACLE protocol study: a multicentric observational prospective cohort study of patients with multiple myeloma. BMC Cancer. 2019 Aug 29;19:855.

3. Shimizu H, Schredelseker J, Huang J, Lu K, Naghdi S, Lu F, et al. Mitochondrial $Ca^{2+}$ uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity. eLife. 2015 Jan 15;4:e04801.

4. Gomez-Bougie P, Maiga S, Tessoulin B, Bourcier J, Bonnet A, Rodriguez MS, et al. BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment. Blood. 2018 Dec 20;132(25):2656-69.

5. Durand R, Descamps G, Dousset C, Bellanger C, Maïga S, Alberge JB, et al. A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals major role of BAX in BH3 mimetics response. Blood. 2023 Dec 14;blood.2023021581.

6. Seiller C, Maiga S, Touzeau C, Bellanger C, Kervoëlen C, Descamps G, et al. Dual targeting of BCL2 and MCL1 rescues myeloma cells resistant to BCL2 and MCL1 inhibitors associated with the formation of BAX/BAK hetero-complexes. Cell Death Dis. 2020 May 5;11(5):316.

7. Dousset C, Maïga S, Gomez-Bougie P, Le Coq J, Touzeau C, Moreau P, et al. BH3 profiling as a tool to identify acquired resistance to venetoclax in multiple myeloma. Br J Haematol. 2017 Nov;179(4):684-8.
<table><caption>Supplementary Table 1A. HMCLs' characteristics and LD₅₀ values of S63845 +/- Efsevin</caption><thead><tr><th rowspan="2">HMCL</th><th rowspan="2">Translocation</th><th rowspan="2">Target Genes</th><th rowspan="2">TP53</th><th colspan="2">LD₅₀ (nM)</th></tr><tr><th>S63845</th><th>S63845 + EF</th></tr></thead><tbody><tr><td>BCN</td><td>t(14;16)</td><td>C-MAF</td><td>wt</td><td>370</td><td>79.7</td></tr><tr><td>KMS12PE</td><td>t(11;14)</td><td>CCND1</td><td>R337L/-</td><td>200</td><td>63</td></tr><tr><td>KMM1</td><td>t(6;14)</td><td>CCND3</td><td>C135F/S241F</td><td>83</td><td>32</td></tr><tr><td>MM1S</td><td>t(14;16)</td><td>C-MAF</td><td>wt</td><td>156.2</td><td>93.3</td></tr><tr><td>XG7</td><td>t(4;14)</td><td>MMSET</td><td>wt</td><td>51.5</td><td>60.3</td></tr><tr><td>L363</td><td>t(20;22)</td><td>MAFB</td><td>S261T/-</td><td>27.5</td><td>18.7</td></tr></tbody></table>

<table><caption>Supplementary Table 1B. HMCLs' characteristics and LD₅₀ values of Venetoclax +/- Efsevin</caption><thead><tr><th rowspan="2">HMCL</th><th rowspan="2">Translocation</th><th rowspan="2">Target Genes</th><th rowspan="2">TP53</th><th colspan="2">LD₅₀ (nM)</th></tr><tr><th>Ven</th><th>Ven + EF</th></tr></thead><tbody><tr><td>KMS12PE</td><td>t(11;14)</td><td>CCND1</td><td>R337L/-</td><td>6.8</td><td>1</td></tr><tr><td>MDN</td><td>t(11;14)</td><td>CCND1</td><td>wt</td><td>32.3</td><td>6.1</td></tr><tr><td>XG5</td><td>t(11;14)</td><td>CCND1</td><td>R282W/-</td><td>18.4</td><td>11</td></tr><tr><td>KARPAS620</td><td>t(11;14)</td><td>CCND1</td><td>C135Y/-</td><td>69.7</td><td>19</td></tr></tbody></table>

Cells were pre-treated or not during 72 hours with Efsevin (10µM), and then treated 24 hours with A) S63845 or B) Venetoclax (Ven). Cell death was assessed by Annexin V staining. LD₅₀ values corresponded to the mean of at least 4 independent experiments.
<table><caption>**Supplementary Table 2.** Cell death induced by S63845, Efsevin and their combination in MM primary cells</caption><thead><tr><th rowspan="2">Patient N°</th><th rowspan="2">Stage</th><th rowspan="2">Sample</th><th rowspan="2">Plasma cell %</th><th colspan="4">Characteristics of patients' samples</th><th colspan="3">Myeloma cell death (%)</th></tr><tr><th>1q21</th><th>del17p</th><th>t(4;14)</th><th>t(11;14)</th><th>Efsevin</th><th>S63845</th><th>Efsevin + S63845</th></tr></thead><tbody><tr><td>MM-01-0305</td><td>D</td><td>BM</td><td>25</td><td>1q21</td><td></td><td></td><td>t(11;14)</td><td>0</td><td>77</td><td>84</td></tr><tr><td>MM-01-0331</td><td>D</td><td>BM</td><td>25</td><td>1q21</td><td></td><td></td><td></td><td>6</td><td>37</td><td>67</td></tr><tr><td>MM-01-0348</td><td>D</td><td>PB</td><td>22</td><td></td><td></td><td></td><td>t(11;14)</td><td>0</td><td>46</td><td>55</td></tr><tr><td>MM-01-0370</td><td>D</td><td>BM</td><td>6</td><td>1q21</td><td></td><td>x</td><td></td><td>0</td><td>0</td><td>9</td></tr><tr><td>MM-01-0374</td><td>D</td><td>BM</td><td>16</td><td>1q21</td><td></td><td></td><td></td><td>2</td><td>8</td><td>29</td></tr><tr><td>MM-01-0377</td><td>D</td><td>BM</td><td>11</td><td>1q21</td><td></td><td>x</td><td></td><td>7</td><td>42</td><td>47</td></tr><tr><td>MM-01-0384</td><td>D</td><td>BM</td><td>26</td><td></td><td></td><td>x</td><td>t(11;14)</td><td>13</td><td>6</td><td>47</td></tr><tr><td>MM-01-0388</td><td>D</td><td>PB</td><td>26</td><td>1q21</td><td></td><td></td><td>t(11;14)</td><td>30</td><td>93</td><td>98</td></tr><tr><td>MM-01-0394</td><td>D</td><td>BM</td><td>26</td><td>1q21</td><td></td><td></td><td></td><td>7</td><td>77</td><td>95</td></tr><tr><td>MM-01-0421</td><td>D</td><td>BM</td><td>25</td><td></td><td></td><td></td><td>t(11;14)</td><td>8</td><td>54</td><td>70</td></tr><tr><td>MM-01-0431</td><td>D</td><td>BM</td><td>9</td><td>x</td><td>x</td><td>x</td><td>x</td><td>0</td><td>26</td><td>25</td></tr><tr><td>MM-01-0437</td><td>D</td><td>BM</td><td>25</td><td></td><td>x</td><td>x</td><td>x</td><td>24</td><td>33</td><td>73</td></tr><tr><td>MM-01-0434</td><td>D</td><td>BM</td><td>32</td><td>x</td><td>x</td><td>x</td><td>x</td><td>0</td><td>32</td><td>70</td></tr><tr><td>MM-01-0435</td><td>D</td><td>BM</td><td>6</td><td>x</td><td>x</td><td>x</td><td>x</td><td>14</td><td>24</td><td>51</td></tr><tr><td>MM-01-0264(3)</td><td>P</td><td>BM</td><td>18</td><td></td><td></td><td>x</td><td>t(11;14)</td><td>15</td><td>0</td><td>9</td></tr><tr><td>MM-01-0274(2)</td><td>P</td><td>BM</td><td>23</td><td>1q21</td><td>del17</td><td>x</td><td></td><td>3</td><td>78</td><td>86</td></tr><tr><td>MM-01-0143(2)</td><td>P</td><td>PB</td><td>32</td><td>1q21</td><td></td><td></td><td></td><td>0</td><td>83</td><td>88</td></tr><tr><td>MM-01-0059(2)</td><td>P</td><td>BM</td><td>9</td><td>1q21</td><td></td><td>x</td><td>x</td><td>0</td><td>81</td><td>100</td></tr><tr><td>MM-01-0167(4)</td><td>P</td><td>PB</td><td>41</td><td>1q21</td><td></td><td>x</td><td></td><td>15</td><td>4</td><td>35</td></tr><tr><td>MM-01-0319</td><td>R</td><td>PB</td><td>13</td><td>x</td><td>del17</td><td></td><td>x</td><td>0</td><td>11</td><td>43</td></tr><tr><td>MM-01-0361</td><td>R</td><td>BM</td><td>39</td><td></td><td></td><td></td><td>x</td><td>17</td><td>17</td><td>42</td></tr><tr><td>MM-02-0003(2)</td><td>R</td><td>BM</td><td>14</td><td></td><td></td><td></td><td></td><td>29</td><td>56</td><td>87</td></tr><tr><td>MM-01-0366</td><td>R</td><td>BM</td><td>16</td><td>1q21</td><td></td><td>x</td><td>x</td><td>2</td><td>16</td><td>14</td></tr><tr><td>MM-01-0391</td><td>R</td><td>PB</td><td>11</td><td>1q21</td><td></td><td>t(4;14)</td><td></td><td>11</td><td>86</td><td>95</td></tr><tr><td>MM-01-0110(2)</td><td>R</td><td>BM</td><td>7</td><td>x</td><td>del17</td><td></td><td></td><td>10</td><td>12</td><td>0</td></tr><tr><td>MM-01-0426</td><td>R</td><td>BM</td><td>6</td><td></td><td></td><td></td><td></td><td>0</td><td>56</td><td>47</td></tr><tr><td>MM-01-0170(2)</td><td>R</td><td>BM</td><td>6</td><td></td><td>del17</td><td></td><td>t(11;14)</td><td>27</td><td>91</td><td>93</td></tr></tbody></table>

Mononuclear cells isolated from bone marrow (BM) or peripheral blood (PB) from myeloma patients (MYRACLE cohort, NTC03807128) (2) were cultured 24 hours with S63845 (25nM), Efsevin (15µM) or their combination, and myeloma cell death was assessed by flow cytometry. Del17p, 1q21 gain, t(4,14) and t(11;14) were determined by FISH (threshold >50% of cells) D: Diagnosis. P: Progression; R: Relapse; x: not performed.
**Supplementary Figure 1 Legend**

A) HMCLs were treated with EF (10µM) for the indicated times. Cell death was assessed by Annexin V staining and flow cytometry. Values corresponded to the mean ± SD of 3 independent experiments. B) The combination of S63845 with EF (10 µM) was assessed in HMCLs after 24 hours of treatment. Cell death was measured as in (A). LD50 values corresponded to the mean±SD of ≥ 3 Independent experiments. Statistical analysis was performed using paired t test. C) Cells were pre-treated 72 hours with EF and then with the combination of EF and S63845 or Venetoclax for the next 24 hours, as indicated. BLISS scores were calculated using [https://synergyfinder.fimm.fi/](https://synergyfinder.fimm.fi/). Results represent the mean ± SD of 3 independent experiments. D) NCI-H929 NI#1 (TP53⁺/⁺) and NCI- H929 TP53KO#1 (TP53⁻/⁻) clones, previously generated in our laboratory (5) were pre-treated with EF (10µM) for 96 hours and then with the combination of EF with S63845 for the next 24h. Cell death was assessed by Annexin V staining. LD50 values corresponded to the mean ± SD of 3 independent experiments. Statistical analysis was performed using paired t test. E) BAK and BAX deletion in KMM1 *BAK-KO* and *BAX-KO* cells was evaluated by western blotting (upper panel). DNA sequences of clones *BAK1-KO* and *BAX-KO*. Sequences corresponded to the donor DNA strand (+). The generated stop codon is indicated in red (lower panel). Examples of chromatograms from one of the respective KO-generated clones are presented, with the stop codon identified in each case (lower panel). F) VDAC2 and BCL2 family protein expression in KMM1 and KMS12PE cell lines treated with 10µM of Efsevin (EF) for 72 hours was evaluated by western blotting. Results are representative of 2 independent experiments. G) Cell death response of myeloma cells from patient samples (n=22) to the combination of EF (15 µM) and S63845 (25 nM) after 24 hours of treatment according to 1q21 status. Statistical analysis was performed using Mann Whitney test.
Supplementary Figure 1

<figure><img src="image_33.png" /><figcaption>B)</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>C)</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>KMS12PE</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>D)</figcaption></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /><figcaption>E)</figcaption></figure>

<figure><img src="image_39.png" /></figure>

<table><thead><tr><th>Gene targeted</th><th>dsDNA donor sequence</th></tr></thead><tbody><tr><td>BAK1</td><td>5' CAGGCTGATCCCGTCCTCCACT<br>GAGACCTGAAAAATGGCTTAGGGC<br>CAAGGCCCAGGTCCCTCCAGGCAG<br>GAGTGCGGAGAG-3'</td></tr></tbody></table>

<table><thead><tr><th>Gene targeted</th><th>dsDNA donor sequence</th></tr></thead><tbody><tr><td>BAX</td><td>5' -GGCAGCTGACATGTTTTCTGA<br>CGGCAACTTCAACTGGGGCTAGGT<br>TGTCGCCCTTTTCTACTTTGCCAG<br>CAAACTGGTGCTCAAG-3'</td></tr></tbody></table>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>F)</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>G)</figcaption></figure>

<figure><img src="image_44.png" /></figure>