Macrophage Activation By Dual PI3K-δ/γ Inhibition Enhances Anti-CD47-Mediated Phagocytosis and Prolongs Survival in DLBCL

Intro: Relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) is associated with poor outcomes, and remains an area of unmet clinical need. Emerging data indicate that a key macrophage immune checkpoint termed CD47 can be effectively targeted in r/r DLBCL. CD47 is a potent "don't eat me" signal and impairs tumor cell phagocytosis by engaging the inhibitory macrophage receptor, SIRPα. Importantly, early clinical studies demonstrate that blocking CD47-SIRPα interactions with an anti-CD47 antibody (magrolimab) yields promising activity in r/r DLBCL patients, particularly when combined with rituximab, which further potentiates lymphoma cell phagocytosis via its F_c domain. Moving forward, it will be crucial to identify combinatorial strategies that improve the efficacy of anti-CD47 therapy in r/r DLBCL.

Tumor-associated macrophages (TAMs) contribute to an immunosuppressive environment in cancer through secretion of cytokines, metabolites, and expression of immune checkpoints. Sustained PI3K-γ signaling is critical to maintain immune suppressive TAM functions, and PI3K-γ inhibition polarizes TAMs into a pro-inflammatory state that culminates in potent anti-tumor T cell responses, sensitizing cancers to PD-1 blockade therapy. We hypothesized that activating TAMs through PI3K-δ/γ inhibition would also augment their inherent phagocytic capacity and synergize with phagocytosis-targeting treatments such as CD47 blockade. Thus, we investigated the impact of the dual PI3K-δ/γ inhibitor, duvelisib, on expression of key phagocytic receptors and pathways in human macrophages, as well as its effect on enhancing lymphoma cell phagocytosis with the anti-CD47 antibody, magrolimab.

Results: RNA sequencing of duvelisib-treated human monocyte-derived macrophages revealed that PI3K-δ/γ inhibition induced broad transcriptional changes suggestive of an activated macrophage state (Fig 1A), including upregulation of interferon response genes (IRF9) and costimulatory molecules (CD86), while decreasing expression of immune suppressive cytokines (IL10) and inhibitory ligands (PDL1). Moreover, duvelisib upregulated transcription of canonical pro-phagocytic receptors (LRP1, FCGR2A,CD44), lysosomal degradation components (LYZ, CTSS) and phosphatidylserine-bridging genes (MFG-E8, GAS6), while inhibiting metabolic pathways, most notably mTOR, each of which has been associated with increased and more efficient phagocytosis.

Given these findings, we evaluated the effect of dual PI3K-δ/γ inhibition on macrophage phagocytosis of DLBCL cells in the context of CD47 blockade in vitro. Across a broad panel of human DLBCL cell lines and multiple monocyte donors, pre-treatment of macrophages with duvelisib significantly increased lymphoma cell phagocytosis by magrolimab (Fig 1B-C). This effect reflected a direct increase in the phagocytic capacity of treated macrophages, as lymphoma cells were not exposed to duvelisib. In separate experiments, we found that duvelisib rarely induced full apoptosis of DLBCL cells, but did promote the expression of "eat me" signals, such as calreticulin and phosphatidylserine on many of the cell lines examined (Fig 1D). Collectively, these results suggest that PI3K-δ/γ inhibition enhances the inherent phagocytic capacity of macrophages, while also sensitizing lymphoma cells for engulfment. Similarly, potent effects were not observed with PI3K-δ- or PI3K-γ-specific inhibitors, suggesting that combined PI3K-δ/γ inhibition is critical to mediating these beneficial effects. Finally, to determine whether duvelisib impacted the efficacy of magrolimab in vivo, we treated human DLBCL xenografts and observed that while duvelisib had minimal impact on lymphoma growth, the combination of duvelisib and magrolimab induced complete tumor rejection in a high proportion of mice, leading to increased survival compared to mice treated with magrolimab alone (Fig 1E).

Conclusion: Collectively, our findings suggest that targeting the PI3K-δ/γ axis favorably induces key macrophage phagocytic pathways, while simultaneously upregulating important "eat me" signals on lymphoma cells. Together, these alterations lower the phagocytic threshold of TAMs and dramatically increase the efficacy of anti-CD47 therapy in DLBCL xenograft models. This work paves the way for early-phase studies of duvelisib and magrolimab in r/r DLBCL and other lymphomas.

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