Allison, Kristen;
Verneris, Michael R;
Cruz Cruz, Joselyn;
Lee-Sherick, Alisa
Inhibiting Efferocytosis in Acute Myeloid Leukemia Decreases Checkpoint Blockade through Decreased CCL5/STAT6 Signaling and Increases Activation through NF-Kb
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Medientyp:
E-Artikel
Titel:
Inhibiting Efferocytosis in Acute Myeloid Leukemia Decreases Checkpoint Blockade through Decreased CCL5/STAT6 Signaling and Increases Activation through NF-Kb
Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Efferocytosis is a tolerogenic wound-healing process carried out by macrophages including phagocytosis of cellular debris and production of T cell suppressive cytokines. MerTK, the prototypic efferocytic receptor, is expressed on macrophages in tissues that harbor high leukemic burden, or "leukemia-associated macrophages" (LAMs), and therefore efferocytosis is co-opted by leukemia for immune evasion. We previously reported that in acute myeloid leukemia (AML) models blocking efferocytosis through inhibition of MerTK led to increased serum [IL-18] and downregulation of checkpoint ligands PD-L1 and PD-L2 on LAMs, which was not attributable to decreased [INF-γ] given that INF-γ was actually increased with MerTK inhibition.</jats:p>
<jats:p>To evaluate a potential mechanism of PD-L1/PD-L2 downregulation when efferocytosis is inhibited, we evaluated for IL-4, IL-10 and IL-6 - all previously associated with checkpoint ligand expression - in serum samples of C57Bl/6 mice inoculated with a syngeneic MLL-ENL AML, and treated with a MerTK efferocytosis inhibitor (MRX-2843, n=15) or vehicle (PBS, n=14). There was no appreciable [IL-4] or [IL-10] detected in either treatment group. There was decreased serum [IL-6] in some MRX-2843-treated samples (mean: 45.4pg/ml) compared to controls (mean: 379.1pg/ml, p=0.3), however nearly all treated and untreated samples were &lt;40pg/ml. We then evaluated several additional serum cytokines from these mice. CCL5/RANTES, known to promote M2 macrophage polarization and modulate PD-L1/PD-L2, was significantly decreased in mice treated with MRX-2843 (12.92pg/ml) compared to vehicle (28.63pg/ml, p&lt;0.001). To validate this as relevant signaling pathway in LAMs, we evaluated STAT6 phosphorylation by western blot analysis in bone marrow derived macrophages (BMDM) from C57Bl/6 mice co-cultured with AML cells and treated with MRX-2843 or vehicle. When MerTK was inhibited, STAT6 phosphorylation was consistently decreased compared to vehicle-treated BMDM. This CCL5/STAT6 axis alteration may be a potential mechanism of PD-L1/PD-L2 downregulation when efferocytosis is inhibited.</jats:p>
<jats:p>We next sought to evaluate whether there was LAM autocrine cytokine signaling within tissues of high leukemic burden, which were not detected by serum cytokine analysis. BMDM co-cultured with AML cells, and treated with MRX-2843 or vehicle were harvested after 12 hours and RT-qPCR was performed. mRNA levels of IFN-β and IL-1β in BMDM treated with MRX-2843 were twice that of controls, suggesting classic macrophage activation. These BMDM did not amplify appreciable levels of IL-4, IL-10, or TGF-β mRNA.</jats:p>
<jats:p>Given the increased IL-1β mRNA and previously reported increased IL-18 associated with MerTK inhibition in LAMs, we evaluated NF-κB activation and subsequent inflammasome assembly. By RT-qPCR we did not detect any consistent alteration in p105 (precursor of p50) or RelA (p65), however when BMDM co-cultured with AML cells and treated with MRX-2843 or vehicle, western blot analysis consistently demonstrated increased phosphorylation of p65 as well as increased NLRP3 when MerTK was inhibited.</jats:p>
<jats:p>In conclusion, blocking efferocytosis through MerTK in LAMs decreased CCL5/STAT6 signaling, which likely contributes to decreased LAM PD-L1 and PD-L2 expression. Furthermore, MerTK inhibition led to activation of NF-κB p65, inflammasome assembly, and production of IL-1β and IL-18. Taken together, these data demonstrate a mechanism by which MerTK inhibition alters antigen presentation through decreased co-inhibition and augmented activating cytokine production. Given that MerTK inhibitors are currently in clinical trials for relapsed/refractory malignancies, these data are relevant, timely, and could provide additional justification for their use in acute leukemias.</jats:p>
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<jats:title>Disclosures</jats:title>
<jats:p>Verneris: Fate Therapeutics: Consultancy; Novartis: Other: advisory board; jazz: Other: advisory board.</jats:p>
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