Craig J. Thomas

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of ‘chronic active’ BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-κB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-κB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton’s tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-κ, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt’s lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses

The glycolytic metabolism of cancers differs from normal tissues, allowing tumor cells to survive under oxidative stress. Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4+ T cells. CP-690,550 inhibited IL-4–dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.

Noncanonical TGFβ signaling contributes to aortic aneurysm progression in Marfan syndrome mice.

Transforming growth factor–β promotes aortic aneurysm formation through activation of its “noncanonical” signaling pathway. Transforming growth factor–β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal–regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase–1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Exploiting Synthetic Lethality for the Therapy of ABC Diffuse Large B Cell Lymphoma

Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and amplify prosurvival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.

Cooperative Epigenetic Modulation by Cancer Amplicon Genes

Chromosome band 9p24 is frequently amplified in primary mediastinal B cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced after JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases.

Identification of oxadiazoles as new drug leads for the control of schistosomiasis

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends almost exclusively on praziquantel. Millions of people are treated annually with praziquantel, and drug-resistant parasites thus are likely to evolve. Phosphinic amides and oxadiazole 2-oxides, identified from a quantitative high-throughput screen, were shown to inhibit a parasite enzyme, thioredoxin glutathione reductase (TGR), with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of schistosome-infected mice with 4-phenyl-1,2,5-oxadiazole-3-carbonitrile-2-oxide led to marked reductions in worm burdens from treatments against multiple parasite stages and egg-associated pathologies. The compound was active against the three major schistosome species infecting humans. These protective effects exceed benchmark activity criteria set by the World Health Organization for lead compound development for schistosomiasis.

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.

Significance The treatment of cancer is highly reliant on drug combinations. Next-generation, targeted therapeutics are demonstrating interesting single-agent activities in clinical trials; however, the discovery of companion drugs through iterative clinical trial-and-error is not a tenable mechanism to prioritize clinically important combinations for these agents. Herein we describe the results of a large, high-throughput combination screen of the Bruton’s tyrosine kinase inhibitor ibrutinib versus a library of nearly 500 approved and investigational drugs. Multiple ibrutinib combinations were discovered through this study that can be prioritized for clinical examination. The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug–drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell–like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton’s tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.