Sarah G. Hymowitz

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets

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-XL), which has shown clinical efficacy in some BCL-2–dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-XL 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.

Therapeutic antibody targeting of individual Notch receptors.

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the γ-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although γ-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

IL‐17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL‐17F, and implications for receptor binding

The proinflammatory cytokine interleukin 17 (IL‐17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL‐17 homolog, IL‐17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL‐6, IL‐8 and granulocyte colony‐stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL‐17F reveals that IL‐17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL‐17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL‐17s and neurotrophins with respect to receptor recognition.

Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non–small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine–galactose–sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.

Triggering Cell Death: The Crystal Structure of Apo2L/TRAIL in a Complex with Death Receptor 5

Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains. We report the crystal structure of the complex between Apo2L and the ectodomain of DR5. The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand. The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top. Both patches contain residues that are critical for high-affinity binding. A comparison to the structure of the lymphotoxin-receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily.

K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Loss of the Tumor Suppressor BAP1 Causes Myeloid Transformation

Identifying BAP1 Targets Inactivating mutations in the deubiquitinating enzyme BAP1 have been associated with cancer. Dey et al. (p. 1541, published online 9 August; see the Perspective by White and Harper) reveal molecular targets of the enzyme and show evidence for a role in leukemia. Mice specifically lacking the target of BAP1, HCF-1, in the bone marrow developed myeloid leukemia. BAP1 appears to be part of a complex that regulates modification of histones and gene expression important for normal hematopoiesis and tumor suppression. The deubiquitinating enzyme BAP1 is implicated in myelodysplastic syndrome. De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor–1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.

A20: from ubiquitin editing to tumour suppression

Clinicians have suspected for hundreds of years that chronic activation of the immune system contributes to the development of cancer. However, the molecular mechanisms that mediate this precarious interplay are only now being elucidated. Recent reports have identified A20 as a crucial tumour suppressor in various lymphomas. A20 is a ubiquitin-editing enzyme that attenuates the activity of proximal signalling complexes at pro-inflammatory receptors. In this Review we summarize the evidence linking chronic inflammation with tumorigenesis and consider how A20 modulates inflammatory signalling cascades, thereby providing a mechanism to explain how deregulation of ubiquitylation can promote tumorigenesis.

Specific Btk inhibition suppresses B cell– and myeloid cell–mediated arthritis

Brutons tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.