Ihms, E. A. | Search bioRxiv

bioRxiv2018

The integrated stress response mediates type I interferon driven necrosis in Mycobacterium tuberculosis granulomas

Necrosis in the tuberculous granuloma is a hallmark of tuberculosis that enables pathogen survival and transmission. Susceptibility to tuberculosis and several other intracellular bacteria is controlled by a mouse genetic locus, sst1, and mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human TB granulomas. Our previous work established that increased disease severity in sst1S mice reflects dysfunctional macrophage effector or tolerance mechanisms, but the molecular mechanisms have remained unclear. Here we demonstrate that sst1S macrophages develop aberrant, biphasic responses to TNF characterized by super-induction of stress and type I interferon pathways after prolonged TNF stimulation with this late-stage response being initiated by oxidative stress and Myc activation and driven via a JNK - IFN{beta} - PKR circuit. This circuit leads to induction of the integrated stress response (ISR) mediated by eIF2 phosphorylation and the subsequent hyper-induction of ATF3 and ISR-target genes Chac1, Trib3, Ddit4. The administration of ISRIB, a small molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden revealing that induction of the ISR and the locked-in state of escalating stress driven by type I IFN pathway in sst1S macrophages plays a causal role in the development of necrosis. Our data support a generalizable paradigm in intracellular pathogen-host interactions wherein host susceptibility emerges within inflammatory tissue due to imbalanced macrophage responses to growth, differentiation, activation and stress stimuli. Successful pathogens such as M. tuberculosis may exploit this aberrant response in susceptible hosts to induce necrotic lesions that favor long-term pathogen survival and transmission. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel therapeutic strategies.

immunology↗

bioRxiv2018

A second generation IL-2 receptor-targeted diphtheria fusion toxin exhibits anti-tumor activity and synergy with anti-PD-1 in melanoma

Denileukin diftitox (DAB1-389-IL-2, Ontak(R)) is a diphtheria toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells (Tregs) and was approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB1-386-IL-2(V6A), which elicits 50-fold less HUVEC monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB1-386-IL-2 Additionally, to overcome aggregation problems, we developed a novel production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully-folded, biologically active, monomeric s-DAB1-386-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB1-386-IL-2(V6A) could provide a superior activity window. In a sequential dual therapy study in tumors that have progressed for 10 days both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 given prior to checkpoint inhibition with anti-PD-1 antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB1-386-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.\n\nSignificance StatementRegulatory T cells (Tregs) infiltrate tumors in various cancers and promote an immunosuppressive microenvironment that hinders anti-tumor immunity. Denileukin diftitox, a diphtheria toxin-based fusion protein that depletes Tregs, was approved for the treatment of T cell malignancies, but its clinical use was limited due to the presence of protein aggregates and toxicity associated with vascular leakage. Here we report the production of a second generation IL-2 receptor-targeted, fully-folded, monomeric diphtheria fusion toxin, and a V6A mutant variant which showed reduced vascular leak in vitro and reduced lethality in mice. In a mouse model of melanoma, we found significant decrease in tumor growth associated with reduction in Tregs when the protein was tested as monotherapy or in combination with checkpoint blockade.

cancer biology↗