424 The battle within: AML´s p53 strategies to evade T-cell attack

<h3>Background</h3> Despite the success of bispecific T-cell recruiting antibody constructs (BsAbs) in B-cell malignancies, the translation to a myeloid setting has been less successful so far, independently of the target antigen. Several reports demonstrate that the diverse genetic makeup of AML confers resistance to T-cell based immunotherapy. Mutations of <i>TP53</i> occur in 5–10% of patients with <i>de novo</i> AML and up to 50% in older patients with tAML, highlighting its role as a potential resistance mechanism. We hypothesize, that genetic aberrations of <i>TP53</i> in AML contribute to cell intrinsic and extrinsic resistance against T-cell based immunotherapy. <h3>Methods</h3> To study the impact of <i>TP53</i> aberrations on T-cell based immunotherapy, MV4-11 p53 WT or p53 KD were cocultured with healthy donor T cells at an effector to target ratio of 1:6 with or without BsAbs. <h3>Results</h3> As expected, BsAb-mediated cytotoxicity was reduced against p53 KD vs p53 WT MV4-11. However, BsAb-mediated T-cell proliferation was also significantly decreased in cocultures with p53 KD. In line with this, we observed a significant decrease in secretion of the proinflammatory cytokines IFN-γ, TNF and IL-2. RNA-Seq analysis of T cells after coculture with MV4-11 p53 KD vs WT revealed a decrease in the expression of genes associated with mitosis, providing a potential explanation for the reduction in T-cell proliferation after coculture with p53 KD. To dissect if the observed effects were due to the AML surfacome or secretome, transwell assays were performed. Indeed, the negative impact of p53 KD AML cells on T-cell function was also observed without direct cell-cell contact. To gain further insights into the secretome, coculture supernatant was analyzed using the Olink^® proteomics platform. The analysis revealed significantly higher secretion of IL-18 and LAP TGF-β1 (TGF-β1 latency associated protein, cleaved from TGF-β before its activation) in the coculture with p53 KD vs p53 WT. The relevance of TGF-β on T-cell fitness was confirmed, as addition of TGF-β to the coculture with p53 WT decreased the specific lysis significantly and was now comparable to the lysis of p53 KD. Confirmatory studies in primary AML samples with or without p53 aberrations further validated our findings with a reduction of BsAb-mediated cytotoxicity, T-cell proliferation and secretion of proinflammatory cytokines. <h3>Conclusions</h3> We demonstrated that AML cells with p53 aberrations are less susceptible to BsAb-mediated cytotoxicity. Surprisingly, immune evasion was mediated by the AML secretome, which reduced T-cell proliferation and cytokine secretion. Secretome analysis identified TGF-β as a cytokine impairing T-cell function. <h3>Ethics Approval</h3> Samples from healthy donors and samples from AML patients were collected with written consent in accordance with the Declaration of Helsinki and with approval from the Institutional Review Board of LMU Munich.