Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells

Abstract

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K D ) of 3.4 nM for Mcl-1, 70 nM for Bcl-x L , and 250 nM for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x L antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.Regression of sensitive tumors such as lymphomas after effective chemotherapy is thought to reflect the induction of apoptosis (1). Chemotherapy-induced apoptosis results largely from activation of the mitochondrial or intrinsic apoptotic pathway (2, 3), which is regulated by Bcl-2 family members (4 -8). This group of proteins consists of three functionally distinct subfamilies. The multidomain proapoptotic proteins Bax and Bak oligomerize upon death stimulation to induce mitochondrial outer membrane permeabilization, thereby allowing release of cytochrome c and subsequent caspase activation. Anti-apoptotic family members, including Bcl-2, Bcl-x L , Bcl-w, Mcl-1, and A1, prevent mitochondrial outer membrane permeabilization. Conversely, BH3-only proteins 3 such as Bim, Puma, and Noxa, which share only limited sequence homology with other Bcl-2 family members in a single 15-amino acid region known as the BH3 domain (9), serve as sensors of various cellular stresses and facilitate apoptosis when activated (6, 9 -14).Although it is clear that BH3-only proteins are activated through transcriptional up-regulation or post-translational modification (7, 10), the manner in which these proteins subsequently initiate apoptosis has been controversial. Two models have emerged to explain this process (15,16). The direct a...

Measurement of BH3-only protein tolerance

“…4,6 Second, the observation that antiapoptotic BCL2 family members preferentially impact BH3-only tolerance provides an explanation for the ability of a short-lived BCL2 family member like MCL1 to modulate apoptosis so effectively. 4,55 Third, the observation that individual antiapoptotic BCL2 paralogs affect tolerances of various BH3-only proteins somewhat differently (Figure 4e) provides additional support for the notion that antiapoptotic BCL2 family members have somewhat specialized functions (e.g., Noxa binding preferentially to MCL1 over BCL2) 29,37 rather than being equivalent. Finally, our observation that apoptotic tolerances are affected more by overexpression of antiapoptotic BCL2 family members than by downregulation of BH3-only family members also provides a potential biochemical explanation for the observation that amplification of the BCLX and MCL1 genes is much more common than mutation or deletion of genes encoding BH3-only proteins during cancer development.…”

“…These independent determinations of BH3-only protein sensitivity ( Figure 3) and expression (Figures 1 and 2) are consistent with our observation that 66 ± 7% of Jurkat cells are apoptotic 24 h after addition of 15 nM bortezomib (Supplementary Figure S1 and ref. 37 ). Moreover, the fact that analyses involving endogenous Noxa (Figures 1 and 2) and EGFP-Noxa (Figure 3) yield comparable quantative results suggests that the actions of EGFP-Noxa and native endogenous Noxa are similar.…”

“…After identifying drug concentrations that induce apoptosis in the sensitive Jurkat cell line (Supplementary Figure S1), we examined changes in BH3-only proteins previously shown to be critical for killing by these agents. 34,35,37,38 The proteasome inhibitor bortezomib induced 3-fold increases in BIM and 8-to 16-fold increases in NOXA ( Figure 1a); the Nedd8-activating enzyme inhibitor pevonedistat-induced negligible to 3-fold increases in BIM and 4-fold increases in NOXA ( Figure 1b); the mTOR dual inhibitor TAK-228 induced 2-to 3-fold increases in BIM and 2-to 10-fold increases in PUMA ( Figure 1c); and the farnesyltransferase inhibitor tipifarnib induced 2-to 3-fold increases in BIM in the sensitive Jurkat line but no discernible increase in BIM or NOXA in the resistant SKW6.4 line (Figure 1d). Given the critical role of these BH3-only protein increases in drug-induced killing, 34,35,37,38 these results suggested that cells can only tolerate a certain degree of BH3-only protein upregulation before dying.…”

“…For example, p53-dependent and -independent transcriptional processes upregulate NOXA and PUMA, 31,32 inhibition of mitogen activated protein kinase signaling enhances BIM stability, 33,34 inhibitors of the mechanistic target of rapamycin (mTOR) induce both BIM and PUMA, 35 and inhibition of the proteasome pathway upregulates NOXA through multiple mechanisms. [36][37][38] Importantly, downregulation of the indicated BH3-only proteins impairs killing by each of the indicated treatments, [32][33][34][35]37,38 indicating the critical role of BH3-only protein upregulation in the cytotoxicity of these stresses. Despite extensive qualitative observations such as these, however, quantitative information regarding the extent of BH3-only protein upregulation required to induce apoptosis and the ability of different cells to survive this upregulation is limited.…”

An update on molecular biology and drug resistance mechanisms of multiple myeloma

“…Moreover, different types of effector caspases (caspase 3, -6, and -7) were downregulated in corticosteroid, vincristine and melphalan resistant U-266 MM cells indicating their role in drug resistance (Mutlu et al, 2014). On the other hand, Bcl-2 overexpression and Noxa/Bcl-2 protein interactions conribute to bortezomib resistance in human lymphoid cells (Smith et al, 2011).…”