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Editorials
inhibitors, and this was found to be true in DLBCL as well.12 Using co-immunoprecipitation, the authors showed BIM and BAX bound to BCL2 in BCL2-depen- dent/venetoclax-sensitive cell lines and BAX release upon drug treatment. In BCLXL-dependent/A-1331852-sensi- tive DLBCL lines, BIM, BAX, and BAK were bound to BCLXL and all three were released following treatment. The pro-apoptotic proteins BIM and BAK were bound to MCL1 and both displaced upon treatment with S63845 in MCL1-dependent cell lines.
BCL2 family expression data in the six resistant lines showed that five expressed some degree of all three anti- apoptotic proteins and one line expressed two. Furthermore, all six lines expressed BAK and/or BAX, with some variability in BIM expression. Co-immunopre- cipitation assays performed in two of the resistant lines showed BIM binding to at least one anti-apoptotic pro- tein but minimal binding of BAX or BAK. Displacement data following drug treatment in all six lines are necessary to gain a better understanding of the protein interactions driving this resistance. However, it remains unclear how a cell could be resistant to all three inhibitors. One possi- bility is that resistant cells utilize more than one BCL2 family member, requiring multiple inhibitors to be used in combination. Alternatively, the cells could be depend- ent on an anti-apoptotic BCL2 protein that was not test- ed. For example, BCL-w was recently reported to be over- expressed in DLBCL and investigating the role of this anti-apoptotic protein or that of BCL2A1 (A1/Bfl1) may reveal that it is implicated in cell survival in DLBCL.13 While there is not currently a selective inhibitor for BCL- w, testing cell lines that are resistant to venetoclax and A- 1331852 with ABT-737, which was reported to inhibit BCL-w, may provide indirect evidence of dependency.14
The authors used both siRNA and CRISPR to discern the contribution of the displaced proteins to the initiation of apoptosis. BIM is necessary for S63845-induced apop- tosis in the two cell lines tested, whereas its role in vene- toclax- and A-1331852-induced apoptosis is cell-line dependent. BAK and BAX are involved to some degree in the apoptotic response to all three inhibitors, however the contribution of each in initiation versus amplification of the apoptotic signal is not entirely clear.
The authors suggest that activated BAX released from BCL2 directly activates BAK; however, previous studies indicate that BAX is a poor activator of BAK.15 An alterna- tive explanation would be that displaced BIM is responsi- ble for activating BAK. In the two BCLXL-dependent cell lines examined only one was protected from A-1331852- induced apoptosis when BAX was silenced and the apop- totic response was not altered in either in response to the silencing of BAK. Furthermore, CRISPR knockout of BAK appeared to have a minor, A-1331852 dose-independent, effect on apoptosis in the one cell line shown. Repeating these experiments on a larger panel of cell lines is neces- sary to understand the relative importance of these pro- teins in A-1331852-induced apoptosis. The BAX siRNA data support the authors’ assertion that BAX is required for S63845-induced apoptosis; however, it remains unclear what protein(s) released from MCL1 are required to activate BAX. It should be noted that incomplete silencing of genes occurred in some experiments, which
could influence the interpretation of the results. Performing the mechanistic experiments with CRISPR knockouts of BIM and BAX, along with BAK, would pro- vide further insight into the role of each of these proteins in BH3-mimetic-induced apoptosis in DLBCL. Regardless, the studies clearly point to the importance of several BCL2 family members in the survival of DLBCL cells and provide insights into a potential means of target- ing these vulnerabilities.
The potential of ex vivo testing as a means to deliver pre- cision medicine based on functional testing instead of genotype has been reported in multiple myeloma with venetoclax and also in acute myeloid leukemia.16,17 Given the data presented here, one could envisage a way this type of assay could be used in DLBCL. While venetoclax was not effective as a sole agent in DLBCL, it is being test- ed in combination with current therapeutics. Recently pub- lished data from the CAVALLI phase Ib trial demonstrate the benefit of combining venetoclax with R-CHOP or G- CHOP (obinutuzumab plus cyclophosphamide, doxoru- bicin, vincristine, and prednisone) in BCL2-positive, MYC- positive DLBCL, with seven of eight patients (87.5%) reaching complete remission.18 Additionally, a clinical trial evaluating the MCL1 inhibitor S64315 (MIK665) in MYC- positive DLBCL is currently recruiting (NCT02992483).19 Ex vivo testing of patients prior to therapy initiation or enrollment on a clinical trial could provide guidance on treatment and spare the patient from ineffective therapy.
References
1. Freedman AS, Jacobson CA, Ng A, Aster JC. Non-Hodgkin lym- phoma. In: DeVita, Hellman, and Rosenberg’s Cancer Principles & Practice of Oncology. 11th Edition, 2019. pp 1671-1707. Editors: DeRita VT Jr, Lawrence TS, Rosenberg SA. Published by Wolters Kluwer.
2. LiuY,BartaSK.DiffuselargeB-celllymphoma:2019updateondiag- nosis, risk stratification, and treatment. Am J Hematol. 2019;94(5):604-616.
3. Ni Chonghaile T, Sarosiek KA, Vo TT, et al. Pretreatment mitochon- drial priming correlates with clinical response to cytotoxic therapy. Science. 2011;334(6059):1129-1133.
4. Smith VM, Dietz A, Henz K, et al. Specific interactions of BCL-2 family proteins mediate sensitivity to BH3-mimetics in diffuse large B-cell lymphoma. Haematologica. 2020;105(8):2150-2163.
5. Certo M, Del Gaizo Moore V, Nishino M, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL2 family members. Cancer Cell. 2006;9(5):351-365.
6. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL2 family reunion. Mol Cell. 2010;37(3):299-310.
7. DavidsMS,LetaiA.TargetingtheB-celllymphoma/leukemia2fam- ily in cancer. J Clin Oncol. 2012;30(25):3127-3135.
8. Souers AJ, Leverson JD, Boghaert ER, et al. ABT-199, a potent and selective BCL2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013;19(2):202-208.
9. Willis TG, Dyer MJ. The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood. 2000;96(3):808-822.
10. IqbalJ,NeppalliVT,WrightG,etal.BCL2expressionisaprognostic marker for the activated B-cell-like type of diffuse large B-cell lym- phoma. J Clin Oncol. 2006;24(6):961-968.
11. Davids MS, Roberts AW, Seymour JF, et al. Phase I first-in-human study of venetoclax in patients with relapsed or refractory non- Hodgkin lymphoma. J Clin Oncol. 2017;35(8):826-833.
12. Morales AA, Kurtoglu M, Matulis SM, et al. Distribution of Bim determines MCL1 dependence or codependence with BCLXL/BCL2 in MCL1-expressing myeloma cells. Blood. 2011;118(5):1329-1339.
13. Adams CM, Mitra R, Gong JZ, Eischen CM. Non-Hodgkin and Hodgkin lymphomas select for overexpression of BCLW. Clin Cancer Res. 2017;23(22):7119-7129.
14. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor of BCL2
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