Despite a significant improvement in the survival of patients with multiple myeloma (MM), the disease remains incurable and innovative strategies are needed. Interactions among BCL-2 family proteins mainly determine cellular fate decision in response to drug therapy. Thus, anti-apoptotic members such as BCL-2, BCL-XL or MCL-1 represent an attractive target for therapy.1 BH3 profiling is a functional assay that identifies the tumor cell’s addiction to these anti-apoptotic members.2 The oral BCL-2-specific BH3 mimetic ABT-199 demonstrated very promising results in BCL-2-dependent malignancies such as chronic lymphoid leukemia and mantle cell lymphoma.3–5 To date, there has been no determination of what proportion of MM cases are likely to be BCL-2 dependent. The present study demonstrated that MM is a heterogeneous disease with respect to BCL-2, BCL-XL or MCL-1 dependence. Moreover, identification by BH3 profiling of BCL-2 dependence predicted in vitro sensitivity to BH3 mimetics.
Dependence on BCL-2, BCL-XL or MCL-1 was measured in eight human myeloma cell lines. After cell permeabilization, mitochondria were exposed to standardized amounts of BAD, HRK or MS-1 peptides. The affinity of these peptides for the anti-apoptotic proteins are summarized in Figure 1a.2, 5, 6 The release of cytochrome c induced by each peptide was quantified by fluorescence-activated cell sorting analysis. Dependence on individual anti-apoptotic proteins was found to be very heterogeneous from one cell line to another (Figure 1b). One cell line (LP-1) was found to be relatively purely MCL-1 dependent. XG-5 and SKMM2 were found to be relatively BCL-2 dependent and MM1-S was found to be relatively purely BCL-XL dependent. The remaining cell lines were characterized by co-dependency on anti-apoptotic proteins: BCL-2 and BCL-XL (KMS12-PE) or BCL-XL and MCL-1 (NCI-H929 and RPMI-8226) and BCL-2 and MCL-1 (L-363). We then determined the in vitro sensitivity of MM cell lines to the BH3 mimetics ABT-199 and ABT-263 (Figure 1c). The mitochondrial response to BAD peptide predicted the in vitro sensitivity to ABT-263 (Figure 1d). The BAD minus HRK mitochondrial response was used to reflect the specific BCL-2 dependency and significantly predicted sensitivity to ABT-199. (Figure 1d) Finally, the cytochrome c release in response to ABT-199 and ABT-263 strongly correlated with the in vitro sensitivity to the drug.
We then determined the mitochondrial priming of primary plasma cells obtained from 14 different patients with a diagnosis of de novo or relapsed MM. Patient characteristics are summarized in Figure 2a. As observed with cell lines, patient samples showed a heterogeneous dependence on anti-apoptotic proteins (Figure 2a). Of note, mitochondria of five patient samples were found to be relatively independent of BCL-2, BCL-XL or MCL-1. We also determined the in vitro sensitivity to ABT-199 and ABT-263. Three and five samples were found sensitive (LD50⩽ 100 nM) to ABT-199 and ABT-263, respectively. As shown in Figure 2b, BH3 profiling using the BAD peptide correlated with in vitro sensitivity to ABT-199 and 263.
BH3 profiling is a unique, functional method to measure the dependence to the anti-apoptotic proteins in live cancer cells. The present study demonstrates that MM is a heterogeneous disease regarding its dependence on anti-apoptotic proteins and cannot be considered as monolithically BCL-2, BCL-XL or MCL-1 dependent. MCL-1 is expressed in MM cells at levels higher than normal plasma cells and its expression level has been shown to affect clinical outcome.7, 8 Here, mitochondria from half of MM cell lines (4/8) and from almost one-third of primary samples (4/14) were found to be MCL-1 dependent. These results are consistent with the requirement for MCL-1 for survival of many myeloma cells. BH3 profiling also identified a subset of BCL-2 and/or BCL-XL-dependent MM cells with relatively less dependence on MCL-1 and correctly predicted sensitivity to the BH3 mimetics ABT-199 and 263. In our series, one MM cell line (MM1-S) and two primary samples (#7 and #11) were found to be sensitive to ABT-263 but insensitive to ABT-199, that linked to their BCL-XL dependence determined using BH3 profiling. Even if these findings indicate that BCL-XL could be an attractive target for MM, the BCL-XL-related platelet toxicity has impaired the clinical development of ABT-263.9 Previous studies identified MM cells sensitive to BH3 mimetics based on their BCL-2/MCL-1 mRNA ratio10, 11 or interactions of BCL-XL and BCL-2 with BIM.12 From a practical point of view, BH3 profiling can be performed in just 3 h with fewer cells (5 × 104 plasma cells to determine response to the BAD BH3 peptide). This consideration is of importance because bone marrow samples from MM patients usually contain a low percentage of plasma cells (the median percentage of plasma cell in BM aspirate was 9% in our series). It has been previously reported that sensitivity to ABT-199 was restricted in MM patients with t(11;14) translocation.11 Here, among the three ABT-199 sensitive samples, two were found to be t(11;14). By including previous data from Touzeau et al.,11 sensitivity to ABT-199 from 29 different primary MM samples was analyzed. Overall, seven samples (24%) were found to be sensitive to the drug (LD50<100 nM). Interestingly, six of these ABT-199 sensitive samples carried the t(11;14) translocation. Of note, the remaining sensitive sample was found to be BCL-2 dependent according to BH3 profiling. Moreover, one sensitive patient sample was found to be negative for the t(11;14) translocation, suggesting that BCL-2 dependence may exist beyond this cytogenetic subgroup. The positive and negative predictive values of BH3 profiling to predict ABT-199 sensitivity were 75% and 100%, respectively (Supplementary Table).
Overall, and as previously demonstrated in other hematologic malignancies,2, 13, 14 BH3 profiling identifies BCL-2 dependence in myeloma cells and predicts sensitivity to BH3 mimetics. Phase 1 clinical trials evaluating ABT-199 as a single agent (NCT01794520) or in combination with bortezomib plus dexamethasone (NCT01794507) in relapsed MM patients are ongoing.15, 16 Preliminary results of these trials demonstrated significant anti myeloma response with a favorable safety profile.15, 16 The promising predictive value of BH3 profiling needs to be confirmed in the context of prospective clinical studies. Because BCL-2 dependency was found only in a subset of myeloma patients, future clinical trials with an efficacy objective should integrate biomarkers such BH3 profiling to stratify potential candidates for ABT-199 therapy.
Letai AG . Diagnosing and exploiting cancer’s addiction to blocks in apoptosis. Nat Rev Cancer 2008; 8: 121–132.
Certo M, Del Gaizo Moore V, Nishino M, Wei G, Korsmeyer S, Armstrong SA et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 2006; 9: 351–365.
BCL-2 inhibitor yields high response in CLL and SLL. Cancer Discov 2014; 4: OF5.
Davids MS, Roberts AW, Anderson MA, Pagel JM, Kahl BS, Gerecitano JF et al. The BCL-2-specific BH3-mimetic ABT-199 (GDC-0199) Is active and well-tolerated in patients with relapsed Non-Hodgkin lymphoma: interim results of a Phase I study. ASH Annu Meet Abstr 2012; 120: 304.
Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013; 19: 202–208.
Foight GW, Ryan JA, Gullá SV, Letai A, Keating AE . Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells. ACS Chem Biol 2014; 9: 1962–1968.
Munshi NC, Hideshima T, Carrasco D, Shammas M, Auclair D, Davies F et al. Identification of genes modulated in multiple myeloma using genetically identical twin samples. Blood 2004; 103: 1799–1806.
Wuillème-Toumi S, Robillard N, Gomez P, Moreau P, Le Gouill S, Avet-Loiseau H et al. Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival. Leukemia 2005; 19: 1248–1252.
Roberts AW, Seymour JF, Brown JR, Wierda WG, Kipps TJ, Khaw SL et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol Off J Am Soc Clin Oncol 2012; 30: 488–496.
Bodet L, Gomez-Bougie P, Touzeau C, Dousset C, Descamps G, Maïga S et al. ABT-737 is highly effective against molecular subgroups of multiple myeloma. Blood 2011; 118: 3901–3910.
Touzeau C, Dousset C, Le Gouill S, Sampath D, Leverson JD, Souers AJ et al. The Bcl-2 specific BH3 mimetic ABT-199: a promising targeted therapy for t(11;14) multiple myeloma. Leukemia 2013; 28: 210–212.
Morales AA, Kurtoglu M, Matulis SM, Liu J, Siefker D, Gutman DM et al. Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1-expressing myeloma cells. Blood 2011; 118: 1329–1339.
Pan R, Hogdal LJ, Benito JM, Bucci D, Han L, Borthakur G et al. Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. Cancer Discov 2014; 4: 362–375.
Chonghaile TN, Roderick JE, Glenfield C, Ryan J, Sallan SE, Silverman LB et al. Maturation stage of T-cell acute lymphoblastic leukemia determines BCL-2 versus BCL-XL dependence and sensitivity to ABT-199. Cancer Discov 2014; 4: 1074–1087.
Kumar S, Vij R, Kaufman J, Mikhael J, Facon T, Moreau P et alPhase I Interim Safety and Efficacy of Venetoclax (ABT-199/GDC-0199) Monotherapy for Relapsed/refractory Multiple Myeloma. J Clin Oncol 2015; 33 (suppl: abstract 8576)..
Touzeau C, Chanan-Khan A, Roberts AW, Agarwal A, Facon T, Lebovic D et alPhase 1b Interim Results: Venetoclax (ABT-199/GDC-0199) in Combination with Bortezomib and Dexamethasone in Relapsed/Refractory Multiple Myeloma. J Clin Oncol 2015; 33 (suppl): abstract 8580..
Ryan J, Letai A . BH3 profiling in whole cells by fluorimeter or FACS. Methods 2013; 61: 156–164.
We gratefully acknowledge support from NIH grants R01CA129974. CT was supported by the Fondation Française pour la Recherche contre le Myélome et les Gammapathies monoclonales (F.F.R.M.G.).
AL and CT designed the research and wrote the manuscript. CT performed the experiments. CT, AL, JR, TNC analyzed the data. PR, KA, MA, SLG and PM provided myeloma cells. All the authors critically reviewed the manuscript.
AL has been a paid advisor to AbbVie. AL’s laboratory has received sponsorship for research with AbbVie.
Supplementary Information accompanies this paper on the Leukemia website
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Touzeau, C., Ryan, J., Guerriero, J. et al. BH3 profiling identifies heterogeneous dependency on Bcl-2 family members in multiple myeloma and predicts sensitivity to BH3 mimetics. Leukemia 30, 761–764 (2016). https://doi.org/10.1038/leu.2015.184
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