Allosteric Inhibition of MALT1 Is Efficacious in a Model of Chronic Graft-Versus-Host Disease and Modulates Immunometabolic Activation

Introduction

Graft-versus-host disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation. In its chronic form, GVHD causes prolonged post-transplant morbidity and significant mortality. There is a paucity of approved therapies and the current standard of care results in incomplete responses that leave significant unmet need for novel agents. Mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a bi-functional protein that acts as a signal-propagation scaffold and protease for selective innate and adaptive immune cell receptor signaling. The established link between MALT1 and receptor-mediated activation of multiple immune cell types involved in cGVHD argues for it as a therapeutic target for cGVHD. Furthermore, we have identified MALT1 signaling to be important in linking receptor-initiated signaling to changes in cellular metabolism necessary for immune cell effector function and propose that immunometabolic profiling of immune cells from cGVHD patients may provide novel biomarkers of both disease pathophysiology and associated MALT1 activity.

Methods, Results, Conclusion

To assess MALT1 as a novel drug target in cGVHD, we first characterized the effectiveness of allosteric inhibition on protease, scaffolding, and metabolic activity in primary human T cells. MALT1 inhibition of T-cell receptor (TCR) activated CD4 ⁺ T cells reduced protease activity by >90% and scaffolding activity by ∼50%, downstream of TCR activation. Consistent with disruption of the TCR signaling cascade, MALT1 inhibition reduced T _H1 and T _H17 cytokines, and the T _FH cytokine IL-21 in a concentration dependent manner. B-cell receptor (BCR) driven proliferation of human B cells and C-type-lectin receptor and immune complex driven cytokine production from human macrophages were also inhibited.

We next tested MALT1 inhibition in a sclerodermatous rodent model of cGVHD (scGVHD). Allogeneic bone marrow and splenocytes from LP/J donor mice were transferred to irradiated C57BL/6 recipient mice. Starting on day 21 after transfer, animals were dosed orally once daily with a MALT1 inhibitor, or twice daily with the JAK1/2 inhibitor ruxolitinib as a comparator. All animals were monitored daily for weight change, survival and GVHD score. On a modified scGVHD scoring scale, animals treated with a MALT1 inhibitor had significantly more progression-free survival throughout the study than vehicle or ruxolitinib-treated animals. No differential effect was observed on body weight and overall survival. MALT1 inhibitor treatment, but not ruxolitinib, reduced frequencies of splenic T _FH cells and germinal center B cells compared with vehicle-treated mice. Importantly, MALT1 inhibition had no effect on the frequency or numbers of splenic regulatory T cells. Additionally, a delayed-type hypersensitivity (DTH) response was abrogated with MALT1 inhibition, showing that T-cell-driven responses in the skin, an important target tissue in cGVHD, can be abrogated with MALT1 blockade.

Given recent studies implicating MALT1 activity as a link between immunoreceptor signaling and activation-dependent metabolic activity in T cells, we performed non-targeted metabolomics on plasma and spleens from scGVHD animals. Clustering of splenic metabolite intensities across treatment groups revealed a subset of metabolites responsive to MALT1 inhibition. This motivated us to explore the metabolic activity of cells from GVHD patients. By developing a method to infer metabolic pathway activity from transcriptional profiles of whole blood, we were able to utilize publicly available RNA-seq data. Using a data set containing 8 healthy controls and longitudinal samples from 25 GVHD patients, this approach segregated GVHD patients into four subsets, with the two largest subsets showing oppositional activity in most major metabolic pathways. Collectively, these data reveal a breadth of MALT1 activity across diverse cell types implicated in the initiation and progression of cGVHD and support development of small-molecule inhibitors of MALT1. These data also define novel heterogeneity among GHVD patients based on immunometabolic activity and motivate development of metabolic biomarkers of MALT1 activity for clinical use.

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