Death by a thousand knives: Multiple BH3-only proteins are required for maximal apoptosis triggered through the BCR

ABSTRACT

The B-cell receptor (BCR) represents a key driver of B-cell development. Consequently, multiple mechanisms link inappropriate BCR signaling to apoptosis. Recently, we characterized the molecular regulators involved in lymphoma cells, confirming a major role for Bcl-2 interacting mediator of cell death (Bim) and supplementary roles for Bcl-2 interacting killer (Bik) and Noxa, and showing that all 3 proteins are required for maximal apoptosis.

KEYWORDS:

• Apoptosis
• B-cell receptor (BCR)
• BH3-only
• BCL2
• Bim
• Bik
• mitochondrial outer membrane permeabilization (MOMP)
• Noxa

Abbreviations

BLNK	=	B-cell linker protein
Btk	=	Bruton’s tyrosine kinase
DAG	=	diacylglycerol
GRB2	=	growth factor receptor bound-2
IP3	=	inositoltrisphosphate
MEK	=	dual specificity mitogen-activated protein kinase kinase
MEKK	=	mitogen-activated protein kinase/ERK kinase kinase
MKK	=	mitogen-activated kinase kinase
MOMP	=	mitochondrial outer membrane permeabilization
mTORC2	=	mammalian target of rapamycin complex 2
NFAT	=	nuclear factor of activated T cells
NF-κB	=	nuclear-factor kappa B
PDK1	=	phosphoinositide-dependent kinase-1
PI3-K	=	phosphatidylinositol-3 kinase
PIP2	=	phosphatidylinositol-4,5-bisphosphate
PIP3	=	phosphatidylinositol-3,4,5-trisphosphate
PLC γ2	=	phospholipase-Cγ2
PKC	=	protein kinase-C
Ras	=	rat sarcoma
SoS	=	son of sevenless

Currently, targeted inhibitors of B-cell receptor (BCR) signaling are generating some exceptional clinical responses in hematologic malignancies.¹ However, the precise molecular mechanisms linking BCR inhibition to therapeutic response remain poorly defined. Therefore, a clearer understanding of BCR signaling networks and how they influence cellular responses is required.

Imprecise recombination of BCR-encoding genes during B-lymphopoiesis generates a hugely diverse pool of unique BCRs with the potential to recognize the disparate antigens vital for ensuring effective humoral immunity.² Furthermore, expression of a signal-competent BCR is essential for B-cell survival, since inducible BCR ablation leads to the collapse of B-cell populations.³ This prosurvival function of the BCR is also implicated in the pathogenesis and maintenance of malignant B cells, most notably in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL).⁴

However, in addition to driving proliferation and survival, BCR signaling can also trigger the negative responses of anergy or apoptosis. For example, the random recombination of BCR-encoding genes often culminates in the formation of potentially pathogenic autoreactive BCRs. Consequently, multiple mechanisms delete or silence B-cell clones with inappropriate BCR specificity, including cell death via apoptosis.

In mammalian cells, BCR-induced apoptosis occurs predominantly via the intrinsic apoptotic pathway.⁵ This pathway activates cysteine-aspartyl proteases (caspases), which degrade key cellular components via release of apoptogenic factors from the mitochondria during a process known as mitochondrial outer membrane permeabilization (MOMP).

MOMP is subject to tight regulation by the B-cell lymphoma 2 (Bcl-2) protein family⁶ and appears to be driven by homo-oligomerization of proapoptotic Bcl-2 family members (Bcl-2 associated X protein [Bax], Bcl-2 antagonist/killer [Bak], and possibly Bcl-2-related ovarian killer [Bok]) at the outer mitochondrial membrane. This process requires a subset of Bcl-2 homology-3 (BH3)-only Bcl-2 family members, of which 8 exist in mammalian systems: Bcl-2 associated death protein (Bad), BH3 interacting death domain agonist (Bid), Bcl-2 interacting killer (Bik), Bcl-2 interacting mediator of cell death (Bim), Bcl-2 modifying factor (Bmf), Harakiri (Hrk), Noxa (Latin for damage), and p53-upregulated mediator of apoptosis (Puma). These proteins trigger the oligomerization of Bax-like proteins. Under normal circumstances, the activities of both BH3-only proteins and Bax/Bak are repressed by the prosurvival Bcl-2 family proteins (Bcl-2, B-cell lymphoma extra-large Bcl-XL, apoptotic regulator Bcl-w (Bcl-w), myeloid cell leukemia-1 [Mcl-1], and Bcl-2 related protein expressed in fetal liver-1 [Bfl-1]).

It has long been appreciated that BCR-induced apoptosis is dependent on the BH3-only protein Bim.⁷ However, since genetic loss of Bim fails to impart complete resistance toward BCR-induced apoptosis, roles for additional BH3-only proteins are implied. Over the years many BH3-only proteins have been implicated in this processes including Bad, Bik, Bmf, and Puma; however, conclusive proof has not been forthcoming.

Recently, we comprehensively characterized the molecular mechanisms linking BCR signaling to apoptosis in a murine model of B-cell lymphoma, the Eμ-Myeloctytomatosis (Myc) model.⁸ Our studies revealed that apoptosis represents the primary in vivo mechanism of clonal deletion in this Myc-driven lymphoma model and that 3 BH3-only proteins were transcriptionally upregulated following BCR activation—Bim, Bik, and Noxa. This pattern was also conserved in human lymphoma cell lines and normal murine lymphocytes. We subsequently determined that transactivation of Bim was dependent on signaling through spleen tyrosine kinase (SYK) and extracellular signal regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK) or phosphatidylinositol-3 kinase (PI3-K), whereas upregulation of both Bik and Noxa occurred by alternative means. This upregulation of multiple BH3-only transcripts resulted in increased expression at the protein level and correlated with caspase-independent degradation of Bcl-2 and Mcl-1. This reduction in Mcl-1 is most likely attributable to the ability of Noxa to target Mcl-1 for polyubiquitination and proteasomal degradation.⁹ These effects culminated in enhanced neutralization of prosurvival Bcl-2 family proteins, MOMP, and caspase-dependent cellular execution.

To assess the roles of individual BH3-only proteins downstream of the BCR, panels of Eμ-Myc lymphoma cell lines deficient in each BH3-only protein were screened for sensitivity to BCR-induced cell death. In agreement with previous studies, genetic loss of Bim imparted the greatest level of resistance toward BCR-induced cell death. However, reductions in BCR-induced cell death were also evident in Bik^-/- and Noxa^-/- lymphomas, whereas Bad^-/-, Bid^-/-, Bmf^-/-, and Puma^-/- lymphoma cells exhibited comparable levels of death to wild-type cells.

To further dissect the roles of Bik and Noxa downstream of the BCR we generated panels of Eμ-Myc lymphoma cell lines deficient in both Bim and Bik, or Bim and Noxa. In this setting, we observed that a deficiency in both Bim and Noxa generated greater resistance to BCR-induced cell death than loss of Bim alone. Furthermore, this resistance was statistically indistinguishable from that produced upon Bcl-2 overexpression (blocking all intrinsic apoptotic cell death). However, deficiencies in both Bim and Bik expression had no additional effect over single loss of Bim.

These studies demonstrate that Bim, and to a lesser extent Noxa, represents a key driver of BCR-induced cell death in the Eμ-Myc lymphoma model, whereas Bik plays a key sensitization role that in isolation is unable to drive cellular death responses. Interestingly, transcriptional upregulation of Bik appeared kinetically later in normal lymphocytes, explaining the normal levels of sensitivity toward BCR stimulation exhibited by non-malignant Bik^-/- lymphocytes.¹⁰ These observations suggest that malignant and non-neoplastic B-cells utilize different cellular execution responses to BCR stimulation. Therefore, we propose a model whereby transcriptional upregulation of Bim, Bik, and Noxa initially sensitizes malignant B cells toward BCR-induced killing by neutralizing prosurvival Bcl-2 family members via both direct interaction and targeting for degradation. Following these effects, a Bim- and Noxa-dependent MOMP drives caspase activation culminating in apoptotic cell death (Fig. 1).

Figure 1. B-cell receptor (BCR)-signaling-induced cell death upregulates multiple Bcl-2 homology-3 (BH3)-only proteins. Antigen engagement of the B-cell receptor (BCR) and the associated cluster of differentiation (CD)19/CD21 complex culminates in the generation of numerous second messengers and activation of a wide range of kinases including mitogen-activated protein kinases (MAPKs: extracellular-signal regulated kinase [ERK], 38 kDa MAPK [p38], and c-Jun N-terminal kinase [JNK]), Akt, and protein kinase-C (PKC), which together coordinate downstream cellular responses. Activation of spleen tyrosine kinase (SYK) and ERK promotes transcription of the BH3-only protein Bcl-2 interacting mediator of cell death (Bim); other, currently unclear, signals increase the transcription of Bcl-2 interacting killer (Bik) and Noxa. Together, these proteins serve to bind and inhibit the prosurvival members of the BCL-2 family, releasing Bcl-2 associated X protein (Bax) and Bcl-2 associated killer (Bak) from repression. In addition, Bim (and potentially Noxa) directly activates Bax/Bak, driving their homo-oligomerization and promoting the formation of pores in the outer mitochondrial membrane. Formation of these pores precipitates the release of cytochrome c from the mitochondria, eliciting caspase activation and cellular destruction.

These findings suggest that agonists as well as inhibitors of BCR-inducible kinases implicated in the apoptotic response to stimulation, such as ERK, may prove therapeutically efficacious in the treatment of B-cell malignancies. Furthermore, it is hypothesized that rationally selected combinations of these agonists and inhibitors could selectively reprogram malignant BCR signaling toward proapoptotic responses and break the protective effect of BCR signaling that is critical for malignant cell survival.

Disclosure of potential conflict of interest

No potential conflicts of interest were disclosed.

Financial disclosure statements

No financial disclosures are made.

Funding

This work was supported by an MRC studentship to M.J.C. as well as Leukemia & Lymphoma Research grants to M.S.C. (04006 and 07048)