BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Abstract

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Figure 1

Single-agent BCL-2 family inhibitor activity in AML cell lines. Seven AML-derived cells lines (TF-1, HEL, THP-1, U937, ML-2, HL-60 and MDS-L) are plotted for ABT-737, whereas 11 are plotted for ABT-199 activity (UKE-1, SET-2, M07e, TF-1, HEL, THP-1, ML-2, OCI-AML3, OCI-AML2, HL-60 and MDS-L). Six cell lines were tested with both compounds (TF-1, HEL, THP-1, ML-2, HL-60 and MDS-L).

Figure 2

ABT-737 compared with ABT-199 in vitro synergy with 5-Azacytidine. (a) Maximal 5-Aza EC₅₀ fold-shifts for ABT-737 and ABT-199 are shown side-by-side for each cell line. These EC₅₀ fold-shifts are a ‘one-sided' measurement of 5-Aza enhancement. (b) Corresponding ABT-737 or ABT-199 doses at which maximal 5-Aza EC₅₀ fold-shifts occurred are shown side-by-side. (c) *CalcuSyn Combination Index (CI) values corresponding to the greatest synergy (thus doses shown in b) nearest to the 5-Aza EC₅₀ dose are listed. It is important to note that CI values are a ‘two-sided' measurement of drug synergy for two specific doses, thus a single CI value is not a universal characteristic of the interaction between two drugs because interactions can be dose dependent. See Supplementary Figure 2B for an extensive data set of CI values across multiple dose combinations plotted against the corresponding Fractional Effect, where 0=no effect and 1.0=maximal effect. CI values <0.8 indicate synergy, whereas CI values >1.1 indicate antagonism.

Figure 3

BCL-XL, MCL-1 and BCL-2 protein expression in primary specimens determined by Reverse Phase Protein Array. 577 primary AML patient samples are shown grouped by AML FAB classification.

Figure 4

5-Azacytidine and ABT-737 synergistically combine in primary myeloid malignancy specimens (N=11). Combination Index (CI) versus Fractional Effect (FE) plots were calculated using CalcuSyn Software Version 2.1. The numbers in the tables (Figure 4 key) correspond to the dose combinations shown on the CI versus FE plots for the indicated primary specimens. CI values <0.8 indicate synergy, whereas CI values >1.1 indicate antagonism.

Figure 5

BH3-profiling metrics correlate with clinical 5-Azacytidine-based response. Clinical response to 5-Aza-based therapy (N=22) is plotted against % priming by BH3 profiling for the indicated BH3 peptide(s). Clinicopathologic variables are shown in Supplementary Table 1.