Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells

Abstract

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLX L •BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

Interrogating the relevance of mitochondrial apoptosis for vertebrate development and postnatal tissue homeostasis

“…Overall, this suggests that sequestration of BAK by BCLXL is much more critical for keeping cells alive than preventing BAK from becoming directly activated by BH3-only proteins. These findings are consistent with other studies proposing a unifying or hierarchical model of BCL2 protein function (Llambi et al 2011;Chen et al 2015) and also a recent report by Dai et al (2015), who observed that drug sensitivity of hematopoietic cancers correlates well with the oligomeric state and the amount of BAK present in a cell rather than with the ability of BAK to engage direct activators, such as BIM or PUMA. Perhaps all of these interactions between BH3-only proteins and prosurvival BCL2 proteins serve only one higher purpose: facilitating direct interactions of BAX with BAK.…”

Measurement of BH3-only protein tolerance

“…Plasmid encoding BakΔTM (GenBank BC004431, residues 1-186) in pET29b(+) 57 was a kind gift from Qian Liu and Kalle Gehring (McGill University, Montreal, Canada). Procedures to purify His 6 -tagged PUMA, BAD, tBID, NOXA and BAKΔTM 20,21,37 as well as BCL2ΔTM, BCLX L ΔTM and MCL1ΔTM 24,49 have been previously described. The EGFP open reading frame was cloned into pGEX-4 T-1 to yield a construct with EGFP fused at its N-terminus to GST.…”

“…Contrary to the recent suggestion that all antiapoptotic BCL2 family members might have similar effects, 46 we observed that wild-type BCL2 and MCL1 increased the tolerance for BIM EL about equally (Figures 4b and e), whereas BCL2 preferentially increased PUMA tolerance ( Figure 4c) and MCL1, which Figure 4a) although BCL2 has a stronger affinity for BAD. 29 This paradoxical observation might reflect the finding that partially activated BAK is extensively bound to BCLX L in Jurkat cells 24 and, upon displacement by BAD, would be buffered better by MCL1. 49 When the impact of BIM siRNA was assessed, BIM knockdown by 90% (Figure 4f To further assess the impact of antiapoptotic BCL2 family members, we examined K562 and HCT116 cells, two lines with higher endogenous BCLX L and MCL1 levels ( Figure 5a).…”

“…Genetic 2,17 as well as biochemical experiments [18][19][20][21][22][23] have indicated that the BH3-only proteins BIM, PUMA, a protease generated BID fragment (tBID), and, in some studies, NOXA can directly bind and activate BAK and/or BAX. To counteract these effects, antiapoptotic BCL2 family members such as BCL2, BCLX L and MCL1 bind and neutralize activated BAX and/or BAK 2,6,16,24 as well as activated or overexpressed BH3-only family members, [25][26][27][28][29][30] thereby preserving MOM integrity.…”

Effect of acadesine on breast cancer cells under hypoxia

“…AMPK фос-форилирует p53 по серину 15, что, в свою очередь, приводит к стабилизации белка и его накоплению в митохондриях [16][17][18]. Фосфорилированный p53 индуцирует высвобождение проапоптотического белка BAK из комплекса с Bcl-xL с последующим усилением апоптоза и, соответственно, чувствительности клетки к химиотерапевтическим агентам [19,20].…”