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Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.Key words irciniastatin A; extracellular signal-regulated kinase; translation inhibitor; tumor necrosis factor receptor 1; ribotoxic stress response; p38 mitogen-activated protein kinase Irciniastatin A, also known as psymberin (Fig. 1A), is a marine natural product originally isolated from the marine sponges Ircinia ramose and Psammocinia.

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Allantopyrone A is a fungal metabolite that uniquely possesses two α,β-unsaturated carbonyl moieties. We recently reported that allantopyrone A inhibited the nuclear factor-κB (NF-κB) signaling pathway induced by tumor necrosis factor (TNF)-α in human lung carcinoma A549 cells. In the present study, the mechanism by which allantopyrone A inhibits the TNF-α-induced signaling pathway was investigated in more detail. Allantopyrone A blocked extensive modifications to receptor-interacting protein 1 (RIP1) in the TNF receptor 1 (TNF-R1) complex. Allantopyrone A augmented the high-MW bands of TNF-R1, TNF receptor-associated factor 2, RIP1, the NF-κB subunit RelA and inhibitor of NF-κB kinase β in A549 cells, suggesting that it binds to and promotes the crosslinking of these proteins. The extracellular cysteine-rich domains of TNF-R1 were crosslinked by allantopyrone A more preferentially than its intracellular portion. The present results demonstrate that allantopyrone A interferes with multiple components of the TNF-R1 complex and blocks RIP1 modifications in the TNF-α-induced NF-κB signaling pathway.

Background: We previously established the safety and antitumor efficacy of regionally delivered mesothelin-targeted M28z chimeric antigen receptor (CAR) T cells combined with programmed death-1 (PD-1) antibody (NCT02414269). As a next step, we developed next-generation CAR T cells equipped with a modified CD3z signaling domain with loss-of-function mutations within 2 of 3 ITAM motifs (1XX), and a PD-1 dominant negative receptor (PD1DNR) that provides T-cell intrinsic checkpoint blockade (M28z1XXPD1DNR CAR T cells). Herein, we provide evidence of the preclinical safety and enhanced antitumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells. Methods: Comparative cytotoxicity, proliferation, and cytokine secretion of human T cells engineered to express M28z or M28z1XXPD1DNR CAR were assessed by chromium-release, accumulation, and Luminex assays, respectively. The antitumor efficacy of a single dose (1x105 CAR T cells; E:T 1:1000) of intrapleurally administered M28z or M28z1XXPD1DNR CAR T cells was investigated in NSG mice with orthotopic pleural mesothelioma by serial bioluminescence imaging and by comparing survival. Following tumor eradication, functional persistence of CAR T cells was tested by repeated tumor challenge (increasing doses of 2x106 to 10x106 tumor cells). Results: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and effector cytokine secretion (table). In vivo, a single dose of M28z1XXPD1DNR CAR T cells led to tumor eradication, mice exhibited enhanced survival with weight gain, and resistance to tumor reestablishment upon 10 tumor rechallenges (table) versus a single dose of M28z CAR T cells. Table.In vitro and in vivo characteristics of M28z and M28z1XXPD1DNR CAR T-cell constructsM28zM28z1XXPD1DNRTargetMesothelinMesothelinCostimulatory domainCD28CD28CD3zNo mutations2 ITAM mutations (1XX)T-cell intrinsic checkpoint blockade (PD1DNR)NoYesIn vitro resultsHuman T-cell transduction, range25%-82%30%-89%PD-1 extracellular domain mRNA expression compared to untransduced, fold4158Cytotoxicity, rangeE:T 10:135%-45%25%-51%E:T 5:128%-44%20%-38%E:T 2:117%-32%14%-24%Accumulation, range, fold110-39053-622Effector cytokines (E:T 1:1, 24 h), rangeIL-214-23 ng/mL9-19 ng/mLTNF-α545-977 pg/mL380-852 pg/mLIFN-γ8-11 ng/mL6-15 ng/mLIn vivo resultsTumor eradication26 days19 daysMedian survival56 daysNot reachedTumor progression as measured by bioluminescence imaging following rechallengeRechallenged 3 times over 15 days+1 log+0.2 logsRechallenged 10 times over 52 days+3-4 logs+0.5 logsCurrent statusIn clinical trialIND submission pending Conclusion: Supported by the safety, tumor eradication, and functional persistence, M28z1XXPD1DNR CAR T cells will advance to IND submission and initiation of a phase I clinical trial in patients with pleural mesothelioma, and further extend our investigation to other mesothelin-expressing solid tumors. Citation Format: Stefan Kiesgen, Camille Linot, Hue T. Quach, Jasmeen Saini, Rebecca Bellis, Srijita Banerjee, Zhaohua Hou, Navin K. Chintala, Michel Sadelain, Prasad S. Adusumilli. Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-378.