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Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45β, in multiple myeloma (MM). We showed that the GADD45β/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45β/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Immunohistochemistry (IHC) is a technique aimed at detecting specific antigens on tissue sections by the use of targeting reagents labeled with reporter molecules. This technique allows a snapshot of the structure of tissue and determines the cellular and subcellular localization of a target antigen. This chapter describes how to identify and localize NF-κB proteins in human tissue using immunohistochemical staining on formalin-fixed paraffin-embedded and frozen tissue.

Graphical abstract, Highlights • DTP3 eliminates any viable MM cells in mice upon i.v. bolus administration. • DTP3 exhibits highly favourable PK and ADME profiles, with long plasma half life. • DTP3 had no adverse effect on vital organ systems in GLP safety pharmacology studies. • DTP3 was tolerated in repeat-dose 28-day toxicity studies with wide exposure margins., Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)β/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)β/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers.

NF-κB and MYC are found co-deregulated in human B and plasma-cell cancers. In physiology, NF-κB is necessary for terminal B-to-plasma cell differentiation, whereas MYC repression is required. It is thus unclear if NF-κB/MYC co-deregulation is developmentally compatible in carcinogenesis and/or impacts cancer cell differentiation state, possibly uncovering unique sensitivities. Using a mouse system to trace cell lineage and oncogene activation we found that NF-κB/MYC co-deregulation originated cancers with a plasmablast-like phenotype, alike human plasmablastic-lymphoma and was linked to t(8;14)[MYC-IGH] multiple myeloma. Notably, in contrast to NF-κB or MYC activation alone, co-deregulation rendered cells addicted to IL6 for survival and phenotypic stability. We propose that conflicting oncogene-driven differentiation pressures can be accommodated at a cost in poorly-differentiated cancers.

Objective Growth arrest and DNA damage–inducible protein 45β (GADD45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45β deficiency exacerbates K/BxN serum–induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45β plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45β regulates autoimmunity, we evaluated collagen-induced arthritis in GADD45β–/– mice. Methods Wild-type (WT) and GADD45β–/– DBA/1 mice were immunized with bovine type II collagen (CII). Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. Expression of cytokines and matrix metalloproteinases in the joint and spleen was determined by quantitative polymerase chain reaction. The in vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+ Treg cells and Th17 cells were quantified using flow cytometry. Results GADD45β–/– mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP-3 and MMP-13 expression was also markedly reduced in GADD45β–/– mice. However, serum anti-CII antibody levels were similar in both groups. FoxP3 and interleukin-10 (IL-10) expression was increased 2–3-fold in splenocytes from arthritic GADD45β–/– mice compared with those from WT mice. Flow cytometric analysis showed greater numbers of CD4+CD25+ Treg cells in the spleen of GADD45β–/– mice than in the spleen of WT mice. In vitro studies showed that interferon-γ and IL-17 production by T cells was significantly decreased in GADD45β–/– mice. Conclusion Unlike passive K/BxN arthritis and EAE, GADD45β deficiency in CIA was associated with lower arthritis severity, elevated IL-10 expression, decreased IL-17 production, and increased numbers of Treg cells. The data suggest that GADD45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.

The liver plays a central role in the transformation and degradation of endogenous and exogenous chemicals, and in the removal of unwanted cells such as damaged, genetically mutated or virus-infected cells. Because of this function, the liver is susceptible to toxicity caused by the products generated during these natural occurrences. Hepatocyte death is the major feature of liver injury. In response to liver injury specific intracellular processes are initiated to maintain the liver integrity. Inflammatory cytokines including tumor necrosis factor (TNF)alpha and interleukin-6 (IL-6) are key mediators of these processes as they can activate different cellular response like proliferation, survival and death. TNFalpha induces specific signaling pathways in hepatocytes leading to the activation of either pro-survival mediators or effectors of cell death. While the activation of transcription factor NF-kappaB promotes survival, the induction of c-Jun N-terminal kinases (JNKs) and caspases represent the strategic effectors of cell death in the TNFalpha-mediated signaling pathway. This review summarizes recent advances in the mechanisms of TNFalpha-induced hepatotoxicity, suggesting that NF-kappaB plays a protective role against JNK-induced hepatocyte death. The identification of mechanisms regulating the interplay between the NF-kappaB and JNK pathways is required to identify novel targets for the treatment of liver disease, including hepatitis and hepatocellular carcinoma.

NF-kappa B is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-kappa B. We have generated mice deficient in both the p50 and p52 subunits of NF-kappa B to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings no iv establish critical roles for NF-kappa B in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.

p52 is a subunit of nuclear factor (NF)-κB transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the IκB family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

NF-κ B is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-κ B. We have generated mice deficient in both the p50 and p52 subunits of NF-κ B to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-κ B in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.