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.

Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients

“…Owing to the high level of heterogeneity of human MDS and in accordance with previous publications on myeloid malignancies, 40 we found inter-individual differences in drug sensitivity. 27 Sample two and three proved to be highly sensitive to ABT-737, with~50% of cells undergoing cell death at doses as low as 10 nM. Sample one was more resistant showing a drop in viability at higher doses (Supplementary Figure 2A).…”

“…These inter-individual differences in BCL-2 protein expression were also found in AML, where BCL-2 expression varied substantially between samples from a given French-AmericanBritish subgroup. 27 Given the critical role of MCL-1 as a resistance biomarker, the detection of protein expression by intracellular flow cytometry might serve as a reasonable way of screening patients before BH3-mimetic treatment.…”

“…23 currently holds great promise for the treatment of B-cell neoplasias 24,25 and initial data also suggest efficacy in de novo AML. 26,27 Here, we studied the effect of ABT-737 and ABT-199 on BM cells from a large cohort of primary human MDS patients and compared it with a cohort of healthy age-matched controls. We found that ABT-737 and ABT-199 efficiently killed primary stem/ progenitor cells as well as more differentiated BM cells from highrisk MDS/sAML patients, whereas low-risk MDS or healthy controls remained unaffected.…”

Inhibition of Mcl-1 with the pan–Bcl-2 family inhibitor (–)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia

“…43 Because the functional redundancy of Bcl-2 antiapoptotic proteins is the major cause of resistance to selective Bcl-2 antagonists (eg, ABT-199), concomitant inhibition of multiple Bcl-2 proteins can presumably overcome this resistance. This notion is supported by our finding that AML cells with high Mcl-1 expression were highly resistant to Bcl-2/Bcl-xL inhibitor ABT-737, whereas all AML cell lines tested showed similar sensitivity to the pan-Bcl-2 inhibitor (-)BI97D6.…”

Therapeutically targeting SELF‐reinforcing leukemic niches in acute myeloid leukemia: A worthy endeavor?

“…However, the observation that these LSCs, especially those harboring a IDH1/2 mutation [111], are characterized by an aberrant overexpression of BCL-2 which mediates mitochondrial respiration (the only source of their low energy requirement) makes them suitable targets for BCL-2 inhibitors [83]. Various studies have demonstrated that these inhibitors have modest activity when employed as single agents, but are much more effective when combined with daunorubicin, PI3K inhibitors [112], 5-Azacytidine [113] and mTOR inhibitors [114]. In addition, BCL-2 proteins might be more effectively inhibited by pan-Bcl-2 inhibitors that in contrast to ABT compounds block the expression of Mcl-1, an anti-apoptotic protein with a critical role in leukemic cell survival and drug resistance [115,116].…”