Lc3-Associated Phagocytosis In Bone Marrow Macrophages Suppresses Aml Progression Through Tim-4 Mediated Sting Activation.

Abstract Acute myeloid leukemia (AML) is a tumor dependent on its interactions within the bone marrow (BM) microenvironment. LC3-associated phagocytosis (LAP) maintains tissue homeostasis by regulating immune responses, including in tumor immunity. Here we investigate the function of LAP in the AML BM microenvironment. We used two syngeneic leukemia models (HOXA9/Meis1 and MN1)to investigate the role of LAP on AML proliferation. AML cells were injected into LAP deficient (LAP-/-; ATG16E230-/-) and wild-type (WT) mice. LAP-/- mice had increased AML engraftment in the BM compared to WT mice, as well as reduced animal survival. Flow cytometry (Annexin+) of the BM microenvironment showed an increase in apoptosis in the BM compartment of mice engrafted with AML. This was further increased in LAP-/- mice with AML. The number of BM macrophages (MØ) (CD45+, GR1-, F4/80+ CD115INT) did not differ between the WT and LAP-/- mice. Next, we quantified MØ numbers in the BM of WT and LAP-/- mice with AML. We found increased numbers of tumor associated CD206+ BM MØ in LAP-/- mice with AML compared to WT animals with AML. Gene expression analysis showed up-regulation of type I interferons (IFNs) relating to the STING pathway in the WT engrafted mice. Inhibition of the STING pathway reversed the LAP dependent AML suppression of inflammatory cytokines, suggesting LAP processing of apoptotic cells is important for STING activation. AML has high mtDNA content compared to non-malignant cells, and as mtDNA can activate the STING pathway via cGAS we studied the effects of AML cells without mtDNA (AML ρ0) on STING pathway activation. Induction of apoptosis in AML ρ0 cells followed by co-culture with BM derived MØ (BMDM) for 24 hours did not activate the STING pathway in the MØ. In contrast, MØ co-culture with mitochondria containing apoptotic AML cells did activate the MØ STING pathway. STING pathway activation via type I IFNs induces recruitment of cytotoxic T cells, but no increase in CD8+ T cell numbers or activation (Granzyme-B and IFN-γ) was observed in the BM between LAP-/- and WT animals engrafted with AML. Type I IFNs produced by MØ have been shown to act in an autocrine manner, we therefore investigated MØ phagocytic capacity in AML. Ex-vivo analysis showed enhanced phagocytosis and LAP processing of Zymosan fluorescent beads and LC3 association in MØ from AML engrafted mice compared to controls. Engulfment of apoptotic cells by MØ requires recognition of phosphatidylserine by surface TIM-4 which mediates LAP. We found TIM-4 inhibition increased AML proliferation in vivo. Finally, ex vivo analysis of TIM-4 inhibited MØ confirmed reduced LAP. We report that BM MØ process apoptotic AML cells via LAP through TIM-4. Furthermore, AML apoptotic bodies containing mtDNA initiate MØ STING activation and inhibit AML proliferation. Citation Format: Jamie Aaron Moore, Jayna J. Mistry, Charlotte Hellmich, Aisha Jibril, Tom Wileman, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. LC3-associated phagocytosis in bone marrow macrophages suppresses AML progression through TIM-4 mediated STING activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2752.