Multiple myeloma (MM) is a complex cancer of plasma cells, and despite advancements in treatment, drug resistance remains a significant hurdle. Venetoclax, a BCL-2 inhibitor, has emerged as a promising therapy, particularly for MM patients with the t(11;14) translocation. However, not all MM patients respond to venetoclax, highlighting the need to understand the mechanisms underlying venetoclax sensitivity and resistance. This study delves into the intricate relationship between cellular energetics, specifically the electron transport chain (ETC), and venetoclax response in multiple myeloma. Our findings reveal that venetoclax-sensitive MM cells exhibit a distinct metabolic profile characterized by reduced cellular respiration and diminished activity within the electron transport chain, in stark contrast to venetoclax-resistant counterparts. This metabolic vulnerability opens new avenues for therapeutic strategies aimed at overcoming venetoclax resistance and enhancing treatment efficacy in a broader MM patient population.
Figure 1: Venetoclax Sensitivity in Multiple Myeloma Correlates with Reduced Cellular Energetics and Electron Transport Chain Activity.
(a) Assessment of cell death via Annexin V/DAPI staining in a panel of multiple myeloma cell lines treated with or without 0.5 μM venetoclax for 24 hours. Cell lines are categorized by their sensitivity to venetoclax, demonstrating that t(11;14) translocation lines are significantly more sensitive. Data are presented as percentage of live cells normalized to vehicle control from three independent experiments (n=3), shown as mean ± SEM. Statistical significance was determined using a two-tailed Mann-Whitney test. (b-d) Mitochondrial stress tests using a Seahorse XFe96 analyzer evaluated basal, coupled, and maximal respiration in multiple myeloma cell lines. Oxygen consumption rates (OCR) are normalized to live cell number and presented as mean ± SEM from six replicate wells per cell line (except JJN3, n=11; U266, n=8; and KMS21BM, n=7). Venetoclax-sensitive MM cells exhibit significantly lower basal, maximal, and coupled OCR (p=0.0022), determined after sequential addition of oligomycin, FCCP, and antimycin/rotenone. Statistical significance was determined using a two-tailed Mann-Whitney test. (e) Spare respiratory capacity (SRC) was calculated by subtracting basal OCR from maximal OCR in venetoclax-sensitive and -resistant cells. Data are presented as mean ± SEM. Venetoclax-resistant lines are depicted in green, and venetoclax-sensitive lines in purple in panels (a-e). (f) Heat map illustrating gene expression of electron transport chain components in t(11;14) versus non-t(11;14) multiple myeloma patients from the CoMMpass trial RNAseq dataset. Statistical significance is indicated by adjusted p-values. Source data are available as a source data file.
To begin, we validated the differential sensitivity of multiple myeloma cell lines to venetoclax. Consistent with prior research, our analysis confirmed that venetoclax effectively induced cell death primarily in MM cell lines harboring the t(11;14) translocation (Fig. 1a). This heightened sensitivity was specific to venetoclax and was not observed with standard myeloma treatments like bortezomib and melphalan, or the MCL-1 inhibitor S63845 (Supplementary Fig. 1a, b).
To explore the metabolic underpinnings of these varying responses, we investigated energy metabolism in venetoclax-sensitive and -resistant cells using carbon isotope tracing with labeled U13C-glucose or U13C-glutamine. We selected resistant non-t(11;14) KMS11 and t(11;14) U266 cell lines, alongside sensitive t(11;14) KMS12PE and non-t(11;14) OCI-MY5 cell lines (Supplementary Fig. 2). Intriguingly, we detected lower levels of tricarboxylic acid (TCA) cycle metabolites in venetoclax-sensitive lines compared to resistant lines (Supplementary Fig. 2c, f). Specifically, the contribution of glucose-derived carbon to TCA cycle intermediates was diminished in sensitive cells. This was evident in reduced pools of citrate, succinate, fumarate, and malate, along with decreased 13C enrichment of these metabolites (citrate, α-ketoglutarate, succinate, fumarate, and malate) when cells were supplemented with U-13C-glucose (Supplementary Fig. 2a, d). In contrast, glutamine-derived carbon utilization remained comparable between sensitive and resistant lines (Supplementary Fig. 2b, e). These metabolic differences were not attributable to reduced nutrient uptake, mitochondrial content, or proliferation rates in venetoclax-sensitive lines, as these cells did not exhibit uniformly reduced glucose or glutamine consumption, proliferation (Supplementary Fig. 3a-c), or mitochondrial mass (Supplementary Fig. 4).
Next, we directly assessed mitochondrial function by measuring oxygen consumption rates (OCR) in the MM cell lines. Basal, maximal, and coupled OCR, as well as spare respiratory capacity (SRC), were significantly lower in all venetoclax-sensitive MM lines (Fig. 1b-e). Notably, while insensitive t(11;14) lines like U266 and KMS21BM displayed high respiration rates, the sensitive non-t(11;14) line OCI-MY5 exhibited lower respiratory parameters. This suggested that oxygen consumption and, by extension, electron transport chain (ETC) activity, could be a key differentiator between venetoclax-sensitive and -resistant MM cells. Crucially, SRC, which reflects the cell’s capacity to augment bioenergetics and ATP synthesis under stress, was also reduced in sensitive cells. These collective findings strongly indicated that electron transport chain (ETC) activity might be suppressed in venetoclax-sensitive multiple myeloma cells.
To further elucidate the basis for these OCR differences, we analyzed RNA sequencing data from the CoMMpass MM trial, using t(11;14) translocation as a proxy for venetoclax sensitivity, as patient venetoclax response data was not available in this dataset. Remarkably, this analysis revealed a significant downregulation of electron transport chain (ETC)-related genes in patients with t(11;14) MM (Fig. 1f). This genetic evidence corroborated our OCR findings in cell lines, reinforcing the link between reduced electron transport chain expression and venetoclax sensitivity.
Diminished Complex I and Complex II Activities within the Electron Transport Chain of Venetoclax-Sensitive Multiple Myeloma
Having established a correlation between venetoclax sensitivity and reduced overall electron transport chain function, we next focused on individual ETC complexes, specifically Complex I and Complex II. These complexes are crucial entry points for electrons into the electron transport chain. Complex I (NADH:Coenzyme Q oxidoreductase) receives electrons from NADH, while Complex II (Succinate dehydrogenase, SDH) receives electrons from FADH2. These electrons are then passed down the electron transport chain through a series of redox reactions, ultimately reducing oxygen to water and generating ATP.
To dissect the contribution of Complex I and Complex II, we directly measured their enzymatic activities. Complex I activity was assessed using an immunocapture-based assay, quantifying NADH oxidation and dye reduction. Complex II activities, specifically succinate dehydrogenase (SDH) and succinate ubiquinone reductase (SQR) activities, were measured in live, permeabilized cells with intact mitochondria. This approach allowed for selective measurement of Complex II activities by pretreating cells with inhibitors of Complex I, Complex III, and Complex IV. SDH activity was measured by electron transfer from succinate to a dye (MTT), while SQR activity was measured by electron transfer from succinate to decylubiquinone and then to another dye (DCPIP). These assays were validated using specific inhibitors and substrates (Supplementary Fig. 5).
Our results demonstrated that venetoclax-sensitive cells exhibited significantly lower Complex I activity (p=0.008), as well as reduced SQR and SDH activities (p=0.0007), compared to resistant lines, including t(11;14) U266 and KMS21BM (Fig. 2a-c). This reduction in Complex I and Complex II activities strongly correlated with venetoclax sensitivity. In contrast, the activity of citrate synthase, a TCA cycle enzyme, remained similar across sensitive and resistant lines (p=0.2824) (Fig. 2d), indicating that the TCA cycle itself was functionally intact, while specific electron transport chain complex activities were impaired. Interestingly, protein expression levels of Complex I subunit (NDUFS2) and SDH subunit (Supplementary Fig. 6a, b) did not directly correlate with the observed enzymatic activities (Fig. 2a-c). This highlights the importance of assessing enzymatic activity directly, rather than relying solely on subunit gene or protein expression levels, to understand functional contributions to the electron transport chain.
Figure 2: Venetoclax-Sensitive Multiple Myeloma Exhibits Reduced Complex I and Complex II Activity within the Electron Transport Chain.
(a) Complex I activity was measured in indicated multiple myeloma cell lines as described in the Materials and Methods section of the original publication. (b) SDH activity and (c) SQR activity were assessed in gently permeabilized whole cells supplemented with succinate and inhibitors of Complex I, III, and IV. (d) Citrate Synthase (CS) activity was measured as described in Materials and Methods. Complex I, SQR, and SDH activities were significantly lower in venetoclax-sensitive lines compared to resistant lines (p=0.008, 0.0007, and 0.0007, respectively), while CS activity did not show significant difference (p=0.2824). Data are presented as mean ± SEM from three independent experiments (n=3). Statistical significance was determined using a two-tailed Mann-Whitney test. Venetoclax-resistant lines are shown in green, and venetoclax-sensitive lines in purple in panels (a-d). Source data are available as a source data file.
To determine if this phenomenon extended beyond multiple myeloma, we examined diffuse large B-cell lymphoma (DLBCL) lines for both SQR activity and venetoclax sensitivity. However, in contrast to our MM findings, we did not observe a correlation between low SQR activity and venetoclax sensitivity in DLBCL (Supplementary Fig. 7). This suggests that the link between low SQR activity and BCL-2 dependence may be context-specific to plasma cell malignancies, a point further explored in the Discussion section of the original paper.
Sensitizing Resistant Multiple Myeloma to Venetoclax Through Selective SQR Inhibition within the Electron Transport Chain
Given the unique role of Complex II in linking the TCA cycle and the electron transport chain, we investigated the therapeutic potential of selectively inhibiting either SDH or SQR activities of Complex II to modulate venetoclax sensitivity. We tested the venetoclax-sensitizing effects of TTFA (SQR inhibitor) and 3NPA (SDH inhibitor) in eight venetoclax-resistant MM cell lines (Fig. 3a-h). 3NPA, a succinate analog, inhibits SDH activity by binding irreversibly to the active site, while TTFA specifically targets the Qp site of Complex II, inhibiting SQR activity.
Strikingly, inhibiting SQR activity with TTFA significantly increased venetoclax sensitivity in six out of the eight resistant MM lines tested (Fig. 3a-h). In contrast, 3NPA was less effective at sensitizing MM cells to venetoclax. We further validated the TTFA-venetoclax combination strategy using a colony-forming unit assay. In agreement with suspension culture results, TTFA treatment sensitized MM lines (L363 and KMS11) to a low dose of venetoclax (0.1 μM), significantly reducing cellular viability (Supplementary Fig. 8). These findings underscore the critical role of targeting the Qp site/quinone reductase activity of Complex II within the electron transport chain to induce BCL-2 dependence and sensitize resistant MM cells to venetoclax. To confirm the selective action of TTFA, we measured SQR, SDH, and citrate synthase (CS) activities in TTFA-treated KMS11 cells. TTFA treatment significantly reduced SQR activity (Fig. 3i) while leaving SDH (Fig. 3j) and CS (Fig. 3k) activities unaffected. Consistent with SQR inhibition, SRC was also reduced in TTFA-treated KMS11 cells but not in unsensitized U266 and KMS21BM cells (Supplementary Fig. 9).
Figure 3: Selective Inhibition of SQR within the Electron Transport Chain with TTFA Effectively Sensitizes Resistant Multiple Myeloma to Venetoclax.
(a-h) SQR inhibition using TTFA (100 μM) more effectively sensitizes indicated myeloma cell lines to venetoclax compared to SDHA inhibition with 3-NPA (1000 μM, except for RPMI-8226 treated with 250 μM) upon co-treatment with indicated doses of venetoclax for 24 hours. Cell viability was assessed by Annexin V/DAPI staining. Data are presented as mean ± SEM from three independent experiments (n=3). (i) SQR, (j) SDH, and (k) CS activities were evaluated, demonstrating selective inhibition of SQR activity upon 24-hour TTFA treatment. Data are presented as mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t-test. Data are from n=3 ± SEM. Source data are available as a source data file.
Genetic Disruption of SQR Function within the Electron Transport Chain Mimics Pharmacological Inhibition and Sensitizes Multiple Myeloma to Venetoclax
To further validate the direct role of SQR inhibition in inducing venetoclax sensitivity, we employed a genetic approach. We generated a Qp site mutant of SDHC (SDHC-R72C) that exhibits reduced ubiquinone binding affinity. This mutant was introduced into SDHC knockout (KO) L363 and KMS11 cell lines, as SDHC KO cells alone were not viable. We used a guide RNA targeting an intronic region of SDHC to avoid targeting the exogenously introduced SDHC-R72C mutant.
As expected, cells expressing the SDHC-R72C mutant exhibited selectively reduced SQR activity (Fig. 4a) while maintaining SDH (Fig. 4b) and CS (Fig. 4c) activities, compared to cells expressing wild-type SDHC (SDHC-WT). Complex I activity was also confirmed to be maintained in SDHC-R72C mutant-expressing KMS11 cells (Supplementary Fig. 10). Importantly, the expression of the SDHC-R72C mutant was sufficient to sensitize KMS11 and L363 resistant MM cells to venetoclax (Fig. 4d). This genetic evidence definitively demonstrates that Qp site/SQR inhibition within the electron transport chain is sufficient to induce venetoclax sensitivity, independent of SDH, CS, or Complex I activities. Consistent with reduced SQR activity, both KMS11 and L363 SDHC-R72C mutant cells exhibited a reduction in SRC (Fig. 4e), mirroring the metabolic phenotype of venetoclax-sensitive cell lines. In summary, these results solidify the conclusion that selective Complex II SQR inhibition within the electron transport chain increases BCL-2 dependence and venetoclax sensitivity in multiple myeloma.
Figure 4: Genetic SQR Inhibition via Qp Site SDHC-Mutant Introduction Sensitizes Multiple Myeloma to Venetoclax.
(a) SQR, (b) SDH, and (c) CS activities were measured in KMS11 and L363 SDHCKO cells expressing SDHC-WT or SDHC-R72C mutant constructs. Data are presented as mean ± SEM from four independent experiments (n=4), except for L363 SQR and SDH activity (n=3). Adjusted p-values were calculated using a two-way ANOVA with post-hoc Sidak’s multiple comparisons test. (d) SDHC-WT and SDHC-R72C mutant-expressing cells treated with or without 0.5 μM venetoclax for 24 hours were evaluated for viability using Annexin V/DAPI flow cytometric staining, demonstrating increased venetoclax sensitivity in SDHC-R72C cells. Adjusted p-values were calculated using a two-way ANOVA with post-hoc Tukey’s multiple comparisons test. (e) Spare respiratory capacity (SRC) was determined in indicated SDHC-WT and SDHC-R72C mutant-expressing cells, showing a reduction in SRC upon mutant introduction. Data are presented as mean ± SEM from six replicate Seahorse wells, except for KMS11 SDHC-R72C (n=4). Adjusted p-values were calculated using a two-way ANOVA with post-hoc Sidak’s multiple comparisons test. **** denotes p-value < 0.0001.
Unraveling the Downstream Mechanisms: ATF4, BIM, and NOXA Mediate TTFA-Induced Venetoclax Sensitivity
To dissect the molecular mechanisms underlying SQR inhibition-mediated venetoclax sensitization, we investigated the expression of ATF4, a transcription factor activated by cellular stress. We also examined the expression and interactions of BCL-2 family proteins. We observed increased expression of ATF4, NOXA, and variable induction of BIM and/or BCL-2 protein levels in MM cells treated with TTFA (Fig. 5a) and in SDHC-R72C expressing mutants (Fig. 5b).
To assess the functional role of ATF4, we knocked down ATF4 expression using siRNA in KMS11 and JJN3 cells (KD efficiency confirmed in Fig. 5e and Supplementary Fig. 11). ATF4 knockdown significantly reversed venetoclax sensitization induced by TTFA, rescuing cell viability in venetoclax and TTFA co-treated cells (Fig. 5c, d and Supplementary Fig. 11). This indicates a crucial role for ATF4 in mediating BCL-2 dependence upon SQR inhibition. Importantly, TTFA-induced NOXA expression was reversed by ATF4 knockdown (Fig. 5e), while BIM and BCL-2 induction showed variability. BIM induction was reduced in TTFA-treated KMS11 cells with ATF4 knockdown, but maintained in JJN3 cells (Fig. 5e). ATF4 knockdown also reduced BCL-2 and BAK induction in TTFA-treated JJN3 cells, suggesting context-specific roles for other BCL-2 family proteins in this sensitization process. The reduction in NOXA induction upon ATF4 knockdown (in both KMS11 and JJN3 cells treated with TTFA) points to a key role for NOXA in TTFA-induced venetoclax sensitivity. NOXA can facilitate BIM binding to BCL-2 by displacing BIM from MCL-1, thereby increasing BCL-2 dependence in MM.
Figure 5: ATF4, BIM, and NOXA Regulate TTFA-Induced Venetoclax Sensitivity in Multiple Myeloma.
(a) Expression levels of ATF4 and indicated pro- and anti-apoptotic proteins were assessed by immunoblotting in whole cell lysates of indicated cell lines treated with or without 100 μM TTFA for 24 hours. Actin was used as a loading control. Representative blots from one of three independent experiments are shown. (b) Protein expression levels were evaluated in SDHC-WT or SDHC-R72C mutant-expressing cells. (c, d) Control siRNA or ATF4 siRNA-transfected KMS11 or JJN3 cells were treated with venetoclax (0.5 μM), TTFA (100 μM), or the combination for 24 hours, and cell viability was assessed by Annexin V/DAPI flow cytometric staining. Data are presented as mean ± SEM from three independent experiments (n=3). (e) Cells from (c, d) were used to prepare lysates for immunoblot evaluation of indicated proteins. (f, g) CRISPR Cas9-generated L363 and KMS11 BIMKO (KO efficiency shown in (h) and (i)) cells were treated with or without TTFA (100 μM) and venetoclax (0.5 μM) for 24 hours, and cell death was evaluated by Annexin V/DAPI flow cytometric staining. Data are presented as mean ± SEM from three independent experiments (n=3). (j) Whole-cell lysates from KMS11 and KMS21BM cells treated or untreated with TTFA were evaluated for expression of indicated proteins by immunoblot analysis. (k) Cellular lysates from panel (j) were used for immunoprecipitation of BCL-2, and bound BIM was detected by immunoblotting. Representative blots from one of three independent experiments are shown. (l, n) Whole-cell lysates from RPMI-8226 and KMS18 NOXA KO cells treated or untreated with TTFA were evaluated for expression of indicated proteins by immunoblot analysis. Representative blots from one of three independent experiments are shown. (m, o) RPMI-8226 and KMS18 NOXA KO cells were treated with or without TTFA (100 μM) and venetoclax (0.5 μM) for 24 hours, and cell death was evaluated by Annexin V/DAPI flow cytometric staining. Data are presented as mean ± SEM from three independent experiments (n=3). Adjusted p-values were calculated using a two-way ANOVA with post-hoc Tukey’s multiple comparisons test. **** denotes p-value < 0.0001.
To further define the role of BIM, we generated BIM knockout (KO) KMS11 and L363 cell lines using CRISPR/Cas9. BIM KO reversed the synergistic cytotoxic effect of TTFA and venetoclax in both cell lines (Fig. 5f, g). This rescue of cell viability occurred despite maintained ATF4 expression (Fig. 5h, i), positioning BIM as a downstream effector of ATF4 in this context. These findings confirm BIM as a critical pro-apoptotic protein mediating TTFA/SQR inhibition-induced venetoclax sensitivity. Similarly, using NOXA KO lines (RPMI-8226 and KMS18), we demonstrated that NOXA induction is essential for TTFA-induced venetoclax sensitization. NOXA KO cells lacked TTFA-induced NOXA expression (Fig. 5l, n) and, importantly, lost TTFA-induced sensitivity to venetoclax (Fig. 5m, o). NOXA KO efficiency and expression of ATF4, BIM, and BCL-2 were confirmed (Fig. 5l, n).
We then investigated the regulation of ATF4, BIM, and NOXA in MM cells that were not sensitized by TTFA. We compared protein expression and BIM-BCL-2 binding in KMS11 (TTFA-sensitive) and KMS21BM (TTFA-insensitive t(11;14)) cells. U266 was excluded due to its lack of NOXA expression. We observed induction of BIM and NOXA upon TTFA treatment only in KMS11 cells, not in the resistant KMS21BM cells (Fig. 5j). Co-immunoprecipitation of BCL-2 revealed increased BIM binding to BCL-2 in TTFA-treated KMS11 cells, but not in resistant KMS21BM cells (Fig. 5k). Collectively, these results identify NOXA and BIM as key downstream effectors of ATF4, facilitating TTFA-induced venetoclax sensitization in multiple myeloma.
Clinical Relevance: SQR Activity in Patient Samples Inversely Correlates with Venetoclax Sensitivity and Predicts Response
To assess the clinical relevance of our findings, we examined multiple myeloma patient samples. We aimed to evaluate (1) the ability of TTFA to sensitize primary MM cells to venetoclax and (2) the correlation between SQR activity in purified MM cells and their ex vivo venetoclax sensitivity, as well as patient response to venetoclax therapy (using data from Trial NCT01794520). While Complex I activity is challenging to measure directly in patient samples, SQR activity can be readily assessed using a rapid, direct assay in permeabilized cells. Therefore, we focused on SQR activity in purified CD138+ MM cells from patient samples.
Bone marrow aspirates from 50 MM patients were treated with venetoclax, TTFA, or the combination. The combination of TTFA and venetoclax significantly increased cell death in primary myeloma cells compared to venetoclax alone (Fig. 6a). TTFA treatment sensitized 15 of 50 samples based on a venetoclax IC50 reduction criterion. However, when comparing IC50 values for venetoclax plus TTFA versus venetoclax alone, 46 of 50 samples (92%) showed increased venetoclax sensitivity, with 31 samples (62%) exhibiting more than a 50% reduction in IC50 (Fig. 6b). Of these 31 samples, 11 harbored the t(11;14) translocation. TTFA selectively sensitized CD38+ CD45− gated MM cells to venetoclax (Fig. 6c, d) with minimal impact on non-MM cells (Supplementary Fig. 12), suggesting selective synthetic lethality in MM cells.
Figure 6: SQR Activity Inversely Correlates with Venetoclax Sensitivity in Multiple Myeloma Patient Samples.
(a) Box plot representing cell death (Annexin V staining relative to vehicle control) in samples from 50 myeloma patient bone marrow aspirates treated with 0.1 μM venetoclax, 100 μM TTFA alone, or the combination for 24 hours. CD38-PE and CD45-APC-Cy7 were used to gate myeloma cells. Boxplots show median and quartiles with whiskers extending to the most extreme data point within 1.5 times the interquartile range. IC50 ± TTFA was calculated as indicated in Supplementary Table 1 of the original publication. (b) Scatter plot of IC50 values for patient samples treated with venetoclax alone (x-axis) versus venetoclax and 100 µM TTFA (y-axis). The diagonal line represents a one-to-one correspondence of IC50. Samples are colored by the change in IC50 relative to the venetoclax group (green: ≤50%; yellow: 50-100%; red >100%). The dashed box highlights patient samples with venetoclax IC50s > 100 nM and venetoclax + TTFA IC50s ≤ 100 nM. Statistical significance for IC50 p-values was determined using a paired Student’s t-test. (c, d) Flow cytometry plots of representative patient samples (exhibiting high and low SQR activity) and corresponding sensitivity of MM-gated cells to venetoclax and/or TTFA co-treatment are shown. (e) Venetoclax ± TTFA IC50, SQR activity, and FISH characteristics of purified CD138+ myeloma cells from 14 patient samples. Samples are further segregated based on >50% reduction in IC50. (f) Scatter plot showing a positive correlation between SQR activity and venetoclax IC50 (Spearman’s rank correlation (ρ) = 0.824, n=14, p=0.000466). Samples are colored by venetoclax IC50 (blue: ≤0.1 µM; red: >0.1 µM). Triangles represent t(11;14) samples, and circles represent non-t(11;14) samples. The dashed box indicates patient samples with venetoclax IC50 ≤ 0.1 µM and SQR activity ≤ 0.25 nmol min−1 mL−1. Source data are available as a source data file.
We further assessed SQR activity in purified CD138+ MM cells from 14 patient samples. We observed a significant positive correlation between SQR activity and ex vivo venetoclax resistance (Spearman’s rank correlation, ρ = 0.824, p < 0.0005) (Fig. 6f). Low SQR activity corresponded to ex vivo venetoclax sensitivity, while high SQR activity correlated with resistance. Interestingly, two non-t(11;14) patient samples with low SQR activity also exhibited ex vivo venetoclax sensitivity. Furthermore, patients from clinical trials (NCT02899052 and off-trial) with low SQR activity showed clinical responses to venetoclax.
Conversely, all ten resistant samples (based on ex vivo sensitivity) exhibited high SQR activity, and five of these harbored the t(11;14) translocation. This highlights the potential of SQR activity to identify venetoclax-resistant MM even within the t(11;14) subgroup. One patient sample (PS10001551-2) was from a post-venetoclax refractory patient, and their MM cells exhibited high SQR activity. Importantly, TTFA treatment reduced the venetoclax IC50 in all ten resistant samples, with seven showing a greater than 50% reduction. Notably, the relapsed, venetoclax-refractory t(11;14) patient (PS10001551-2) showed a greater than tenfold reduction in IC50 upon TTFA treatment. This suggests that SQR inhibition can overcome venetoclax resistance even in refractory cases. While the patient sample size is limited, the correlation between SQR activity and patient response warrants further investigation as a potential predictive biomarker.
Extending the Strategy: Complex I and Distal Electron Transport Chain Inhibition Also Sensitize Multiple Myeloma to Venetoclax
To investigate whether targeting other components of the electron transport chain could also sensitize MM cells to venetoclax, we tested the Complex I inhibitor IACS-010759, currently in clinical trials for AML and other cancers. IACS-010759 effectively sensitized resistant MM cell lines (KMS11, L363, and KMS21BM) to venetoclax (Fig. 7a). Similarly, another Complex I inhibitor, piericidin, also sensitized resistant MM cells to venetoclax (Supplementary Fig. 13a, b). Western blot analysis revealed that IACS-010759 treatment induced ATF4 and NOXA expression (Fig. 7b), similar to TTFA, suggesting a shared mechanism of sensitization through ATF4 and NOXA upregulation. Co-treatment with IACS-010759 significantly reduced venetoclax IC50 values (Fig. 7c).
We further explored whether inhibiting downstream complexes (Complex III, IV, and V) within the electron transport chain could also induce venetoclax sensitization. Inhibitors of Complex III (antimycin), Complex IV (sodium azide), and Complex V (oligomycin), at doses that reduced ATP levels by approximately 50%, also sensitized resistant MM cell lines to venetoclax (Supplementary Fig. 13e-g).
Figure 7: Inhibition of Complex I with IACS-010759 Sensitizes Resistant Multiple Myeloma to Venetoclax.
(a) Dose-response curves for co-treatment of indicated cell lines with 0.5 µM venetoclax and increasing doses of IACS-010759 for 24 hours. Cell viability was assessed by Annexin V/DAPI flow cytometric staining. (b) Expression levels of ATF4 and indicated pro- and anti-apoptotic proteins were evaluated by immunoblotting in whole-cell lysates of indicated cell lines treated with or without 25 nM IACS-010759 for 24 hours. Actin was used as a loading control. Representative blots from one of two independent experiments are shown. (c) Dose-response curves for co-treatment of 25 nM IACS-010759 with increasing doses of venetoclax. Cell viability was assessed by Annexin V/DAPI staining. Data are presented as mean ± SEM from three independent experiments (n=3). (d) Box plot of IC50 values for venetoclax alone and venetoclax + IACS, and table with FISH characteristics of nine myeloma patient samples. Boxplots show median and quartiles with whiskers extending to the most extreme data point within 1.5 times the interquartile range. PS10001243, resistant to venetoclax ± IACS, is represented with an artificial IC50 of 100 µM. (e) Mechanistic model illustrating how Complex I and Complex II within the electron transport chain regulate BCL-2 dependence in multiple myeloma cells. IACS-010759 and TTFA inhibit Complex I and Complex II, respectively, leading to electron transport chain inhibition. This inhibition induces ATF4, which in turn upregulates NOXA. NOXA displaces BIM from MCL-1, promoting BIM binding to BCL-2 and elevating BCL-2 dependence, thereby sensitizing cells to venetoclax. Source data are available as a source data file.
In patient samples, IACS-010759 also enhanced venetoclax sensitivity in eight out of nine samples, with a greater than 50% reduction in venetoclax IC50 in five samples. Notably, IACS-010759 significantly sensitized a venetoclax-resistant t(11;14) patient sample. Similar to TTFA, the combination of IACS-010759 and venetoclax had minimal impact on non-MM cells (Supplementary Fig. 14). These results suggest that IACS-010759, and potentially other electron transport chain inhibitors, can overcome venetoclax resistance in both non-t(11;14) MM and venetoclax-resistant t(11;14) patients.
In conclusion, our findings reveal a critical link between the electron transport chain and venetoclax sensitivity in multiple myeloma. Reduced electron transport chain activity, particularly in Complex I and Complex II, characterizes venetoclax-sensitive MM cells. Pharmacological or genetic inhibition of SQR within Complex II, as well as inhibition of Complex I and distal ETC complexes, can induce venetoclax sensitivity in resistant MM. This sensitization is mediated by ATF4-dependent upregulation of NOXA and BIM, leading to increased BCL-2 dependence (Fig. 7e). Furthermore, SQR activity in patient samples shows promise as a predictive biomarker for venetoclax response. The synergistic combination of electron transport chain inhibitors, such as IACS-010759 or TTFA, with venetoclax holds significant translational potential for overcoming venetoclax resistance and improving outcomes for a broader spectrum of multiple myeloma patients.
