Role of Antiapoptotic Protein Mcl1 in Graft-Versus-Host Disease

Introduction

Graft-versus-host disease (GvHD) is the leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Induced myeloid leukemia cell differentiation protein (Mcl1) is an antiapoptotic protein and a member of the Bcl2 family protein, which regulates the intrinsic mitochondrial pathway of apoptosis. Additionally, Mcl1 is the only antiapoptotic protein that plays a significant role in the cell cycle. The expression and interaction of Bcl2 family proteins in T lymphocytes varies depending on their differentiation and activation status. Mcl1 drastically increases its expression following activation and is the essential antiapoptotic protein of lymphocytes following activation. Furthermore, Mcl1 is described as a mechanism of resistance to glucocorticoids, which are the gold standard in the treatment of GvHD. Despite this, the role of Mcl1 in the pathophysiology of GvHD remains completely unexplored.

Methods

PBMCs form healthy donors were cultured with anti CD3/CD28 for 72h to activate human T cells. First, we measured Mcl1 expression in activated and non-activated MACS-isolated human T cells by western blot and confirmed it by flow cytometry. Next, we explored the effect of the Mcl1 inhibitor UMI-77 in vitro on activated human T lymphocytes, and assessed cell viability and proliferation using flow cytometry. For in vivo models, we employed the Mcl1-Flox-CD4-UBC-CRE-ERT2 genetic model to achieve Mcl1 deletion following Tamoxifen treatment, using this model as donor for both acute GvHD and graft-versus-leukemia effect (GvL) assays. This approach included administering Tamoxifen in vivo to recipient animals. In addition, to avoid any potential adverse effect of Tamoxifen, a second model was generated in which donor cells were pre-treated with 4-hydroxy-Tamoxifen in vitro for 1 hour prior to trasplantation. Acute GvHD models were established by intravenously-tail injecting 5x10^6 total bone marrow cells and 5x10^6 splenocytes or 1x10^6 AutoMACS-purified T cells (from C57BL/6 to BALB/c). GvL models were established by injecting 5x10^5 A20-Luciferase cells into immunocompromised NSG mice, followed by the injection of 5x10^6 splenocytes pre-treated in vitro with 4-hydroxy-Tamoxifen for 1 hour after 5 days. Bioluminiscence-based tumor growth was monitored by IVIS-Lumina system.

Results

We confirmed the increased expression of Mcl1 upon T cell activation (p value 0.0286, Relative Mcl1 expression Non activated 1 ; activated 7.11; n=4). In vitro treatment with UMI-77 significantly reduced the viability (Control: 100%, UMI-77 34%; p-value 0.0001, n=3) and proliferation index (C: 1, UMI-77: 0.87; p-value 0.0115; n=6) of activated human T cells. In vivo, Mcl1 deletion improved the survival of recipient animals that received CRE+ cells compared to those that received CRE- cells following in vivo Tamoxifen treatment (median survival CRE-: 7 days, CRE+: 10 days; p-value 0.0241; n=24/group). Additionally, in vitro treatment of T-lymphocytes with Tamoxifen prior to transplantation significantly decreased GvHD and improved survival in recipient animals (median survival of control group: 8.5 days, Tmx: 100% survival at day +45 postrasplantation; p-value 0.0005; n>6/group). Despite this, the GVL model demonstrated that T-cells pre-treated with Tamoxifen retained their anti-leukemic effect, albeit at a slower rate than the control treatment. Finally, we confirmed that the combined treatment with UMI-77 and glucocorticoids exhibited a synergistic effect compared to individual treatments in terms of activated human T cells viability (CI 0.220, Strong Synergism).

Conslusions

Our findings from Mcl1 gene deletion in preclinical models demonstrate that Mcl1 expression in donor lymphocytes is crucial for the development of acute GvHD. This study identifies for the first time the anti-apoptotic protein Mcl1 as a novel therapeutic target for GvHD. These results suggest that Mcl1 inhibitors, currently under clinical investigation for various hematologic malignancies, could be used as prophylaxis or in combination with corticosteroids for the treatment of GvHD.