Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

No abstract

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Members of the B cell lymphoma 2 (BCL-2) gene family have a central role in regulating programmed cell death by controlling pro-apoptotic and anti-apoptotic intracellular signals. In cancer, apoptosis evasion through dysregulation of specific BCL-2 family genes is a recurring event; accordingly, selective inhibition of specific anti-apoptotic BCL-2 family proteins represents an exciting therapeutic opportunity. A combination of nuclear magnetic resonance (NMR)-based screening and structure-based drug design has yielded the first bona fide BCL-2 homology 3 (BH3) mimetics, including the BCL-2 and BCL-X dual antagonist navitoclax, which is the first BCL-2 family inhibitor to show efficacy in patients with cancer. Clinical experience with navitoclax prompted the generation of the highly selective BCL-2 inhibitor venetoclax, which is now approved in the United States for the treatment of patients with chronic lymphocytic leukaemia with 17p deletion who have received at least one prior therapy. Recent advances have also been made in the development of potent and selective inhibitors of BCL-X and myeloid cell leukaemia 1 (MCL1), which are additional BCL-2 family members with established anti-apoptotic roles in cancer. Here we review the latest progress in direct and selective targeting of BCL-2 family proteins for cancer therapy.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

Cellular IAP12 and IAP2 (c-IAP1 and c-IAP2) were identified in a search for proteins associated with TNF receptors (TNFRs) (1). Through binding to TNFR-associated factor 2 (TRAF2), c-IAP1 and c-IAP2 are recruited to TNFR signaling complexes, where they regulate the activation of caspase-8 (1, 2). c-IAP1 and c-IAP2 were also proposed to modulate activation of the canonical NF-B pathway, although most of these studies relied on overexpression (3, 4). In contrast, however, targeted deletion of c-IAP1 or c-IAP2 genes in mice did not reveal any abnormalities in TNF␣-induced NF-B (nuclear factor B) activation (5, 6). The absence of any appreciable phenotype in single c-IAP knock-out mice has been attributed to the putative redundancy of c-IAP1 and c-IAP2 due to their high level of sequence and functional similarities (7). Thus, combined deficiency of cellular IAPs might expose their role in this signaling pathway. In support of this possibility, a null mutation in the sole cellular IAP in zebrafish results in severe defects in NF-B activation (8). c-IAP1 and c-IAP2 are also RING domain-containing ubiquitin ligases capable of promoting ubiquitination of several of their binding partners, including TRAF2 and SMAC (second mitochondrial activator of caspases) (4, 5, 9 -12).TNFR1 mediates activation of several signaling pathways, among them the canonical NF-B pathway (13). Binding of TNF␣ to TNFR1 induces recruitment of the adaptor protein TNFR-associated death domain (TRADD) to the death domain of the receptor (14). Through its death domain and amino-terminal region, TRADD recruits RIP1 (receptor-interacting protein), TRAF2, and through its interaction with TRAF2, c-IAP1 and c-IAP2 (13). Following binding to TRADD, TRAF2 was thought to mediate non-degradative Lys-63-linked polyubiquitination of RIP1 via its RING E3 ligase domain (15,16). This RIP1 modification induces assembly of two RIP1-associated kinase complexes, TAK1-TABs (transforming growth factor ␤-activated kinase 1-TAK1-binding proteins) and IB kinase (IKK) (17)(18)(19). Binding of these two complexes to Lys-63-linked polyubiquitin chains on RIP1 leads to phosphorylation of IKK␤ and subsequent phosphorylation and proteasomal degradation of IB (20). Loss of IB allows translocation of p50/RelA dimer to the nucleus and induction of gene expression (20).In the present study, we investigate the role of c-IAP1 and c-IAP2 in TNF␣-induced NF-B activation. We discover that c-IAP proteins are important mediators of canonical NF-B signaling and demonstrate that the absence of c-IAPs severely attenuates TNF␣-induced NF-B activation. Finally, we show that c-IAPs are ubiquitin ligases capable of promoting polymerization of Lys-63-linked polyubiquitin chains on the critical adapter in the canonical NF-B signaling pathway, RIP1. was from Genentech, Inc. The primary antibodies against mouse c-IAP1 were kindly provided by Drs. John Silke and David Vaux; anti-human c-IAP1 antibodies were purchased from R&D (affinity-purified goat antibody) or Protein Tech Group Inc.; pan c-IAP1/2 ...

The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein–protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1–BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.