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Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development., (© 2023. The Author(s).)

The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has been suggested to inhibit as well as to promote substrate degradation. Contrasting these findings, proteome-wide stability measurements failed to detect any correlation between Nt-acetylation status and protein stability. Accordingly, by analysis of protein stability datasets, we found that predicted Nt-acetylation positively correlates with protein stability in case of GFP, but this correlation does not hold for the entire proteome. To further resolve this conundrum, we systematically changed the Nt-acetylation and ubiquitination status of model substrates and assessed their stability. For wild-type Bcl-B, which is heavily modified by proteasome-targeting lysine ubiquitination, Nt-acetylation did not correlate with protein stability. For a lysine-less Bcl-B mutant, however, Nt-acetylation correlated with increased protein stability, likely due to prohibition of ubiquitin conjugation to the acetylated N-terminus. In case of GFP, Nt-acetylation correlated with increased protein stability, as predicted, but our data suggest that Nt-acetylation does not affect GFP ubiquitination. Similarly, in case of the naturally lysine-less protein p16, Nt-acetylation correlated with protein stability, regardless of ubiquitination on its N-terminus or on an introduced lysine residue. A direct effect of Nt-acetylation on p16 stability was supported by studies in NatB-deficient cells. Together, our studies argue that Nt-acetylation can stabilize proteins in human cells in a substrate-specific manner, by competition with N-terminal ubiquitination, but also by other mechanisms that are independent of protein ubiquitination status., (© 2023. The Author(s).)

During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

All 6 human prosurvival Bcl-2 proteins can drive cancer development and contribute to therapy resistance. However, their relative abilities to protect cells against cancer therapy were not examined previously. We report that Bcl-2, Bcl-xL, or Bcl-w consistently protected leukemic cells better than Bcl-B, Bfl-1, or Mcl-1 against a wide variety of anticancer regimens. Current thinking would attribute this to differences in their ability to bind to BH3-only proteins, Bax, and Bak. To address this, we established the first complete, quantitative cellular interaction profile of all human prosurvival Bcl-2 proteins with all their proapoptotic relatives. Binding was unexpectedly promiscuous, except for Bad and Noxa, and did not explain the differential antiapoptotic capacity of the Bcl-2 proteins. Rather, Bcl-B, Bfl-1, or Mcl-1 proved less potent due to steady-state or drug-induced proteasomal degradation. All 6 Bcl-2 proteins similarly protected against the diverse anticancer regimens when expressed at equal protein levels, in agreement with their broad interaction profile. Therefore, clinical diagnostics should include all family members and should be performed at the protein rather than at the messenger RNA level. In drug development, targeting the ubiquitination machinery of prosurvival Bcl-2 proteins will complement and potentially improve on targeting Bcl-2 protein interactions with BH3 mimetics.

Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and may convey resistance to anti-cancer regimens. Therefore, they are important targets for novel therapeutics, particularly Bcl-2 homology (BH)3 mimetics. Bcl-B (BCL-2-like protein-10) is a relatively understudied member of the Bcl-2 protein family. Its physiological function is unknown, but it has been proven to have an anti-apoptotic activity and to act as a tumor promoter in mice. In human, high Bcl-B protein expression levels correlate with poor prognosis in various carcinomas and predict treatment resistance in acute myeloid leukemia. We here report that protein expression level and anti-apoptotic activity of Bcl-B are dictated by its ubiquitination. We demonstrate that Bcl-B is polyubiquitinated at steady state, in a unique loop between the BH1 and BH2 domains. Mutagenesis identified lysine (K)128 as an acceptor site for polyubiquitin chains, and K119 and K120, but not K181, as potential ubiquitination sites. Mass spectrometry confirmed K128 as a ubiquitination site and defined the polyubiquitin chains as K48-linked, which was confirmed by linkage-specific antibodies. Accordingly, Bcl-B proved to be an instable protein that is subject to ubiquitin-dependent proteasomal degradation at steady state. At equal mRNA expression, protein expression of a lysineless, nonubiquitinated Bcl-B mutant was fivefold higher than that of wild-type Bcl-B, demonstrating that ubiquitination is a key determinant for Bcl-B protein expression levels. Ubiquitination controlled the anti-apoptotic capacity of Bcl-B, in response to a variety of conventional and novel anti-cancer drugs. Certain anti-cancer drugs, known to reduce Mcl-1 protein levels, likewise downregulated Bcl-B. Together, these data demonstrate that polyubiquitination and proteasomal turnover dictate the expression level and anti-apoptotic capacity of Bcl-B.

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can therefore only be administered i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b(-/-) mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a(-/-) (12-fold) or Mdr1a/1b(-/-) (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b(-/-) mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows.

CYP3A4 is an important determinant of drug-drug interactions. In this study, we evaluated whether cytochrome P450 3A knockout mice [Cyp3a(-/-)] and CYP3A4 transgenic (CYP3A4-Tg) mice can be used to study drug-drug interactions in the liver and intestine. Triazolam was used as a probe drug because it is a highly specific CYP3A substrate and not a P-glycoprotein substrate. Triazolam metabolism was profoundly reduced in Cyp3a(-/-) mice both in vitro and in vivo. In vitro studies revealed clear species differences in humans and mice, but triazolam metabolism in microsomes derived from CYP3A4-Tg "humanized" mice closely resembled that in human microsomes. It is interesting to note that studies with tissue-specific CYP3A4-Tg mice revealed that intestinal CYP3A4 has a major impact on oral triazolam exposure, whereas the effect of hepatic CYP3A4 was limited. To mimic a drug-drug interaction, we coadministered triazolam with the prototypical CYP3A inhibitor ketoconazole, which increased triazolam exposure in all the CYP3A-proficient mouse strains but not in Cyp3a(-/-) mice. We further found that the anticancer drug gefitinib is a potent stimulator of 1'-OH triazolam formation in vitro. It is noteworthy that we could also show in vivo stimulation of triazolam metabolism by gefitinib, resulting in a lower oral triazolam exposure. To our knowledge, this is the first in vivo example of direct stimulation of CYP3A4 activity after oral drug administration. Overall, this study illustrates how Cyp3a(-/-) and CYP3A4-Tg mice can be used to study drug-drug interactions. The data clarify that for drugs that are not P-glycoprotein substrates, intestinal metabolism also can be more important than hepatic metabolism after oral administration.

Purpose: Various proapoptotic agents are currently being explored to improve the outcome of radiotherapy. We have evaluated whether APO010-a novel recombinant ligand of the Fas/CD95 death receptor-enhanced the cytotoxic effect of radiation on lymphoid and solid tumor cell types., Experimental Design: A Bcl-2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line (HCT116), and a mesothelioma cell line were used as model systems in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by tumor growth delay using bioluminescence and palpation in vivo., Results: Whereas the three cell lines resisted apoptosis induction by irradiation and APO010 alone, combined treatment greatly enhanced their apoptotic response. In clonogenic survival assays, APO010 reduced the outgrowth of Jurkat-Bcl-2 and HCT116 cells and sensitized the mesothelioma cell line to radiation. In vivo, systemic treatment with APO010 alone caused tumor growth delay in Jurkat-Bcl-2 and HCT116 cells. However, APO010 did not improve the efficacy of radiotherapy in any of the model systems at the selected single dose, which had moderate and reversible systemic toxicity., Conclusions: Although APO010 and radiation had a clear combined cytotoxic effect on tumor cells in vitro, a combined therapeutic effect was not achieved on the same cells subcutaneously grafted in mice, at APO010 doses approximating the maximally tolerable level. These findings suggest that it will be difficult to identify a therapeutic window for this combined modality approach in a clinical setting.

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up-regulated in Cyp3a(-/-) mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a(-/-) mice. Induction studies with prototypical inducers demonstrated an important role for the nuclear receptors PXR and CAR in the up-regulation of CYP2C55. Subsequent diet-switch experiments revealed that food-derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a(-/-) mice. Our data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a(-/-) mice and subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies with tissue-specific CYP3A4 transgenic Cyp3a(-/-) mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food-derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3A activity and could have profound implications for the prediction of drug exposure.