BACKGROUND: Immune checkpoint blockade of CD47 has shown promising results in lymphoid malignancies, with its effects attributed to enabling tumor-cell phagocytosis. However, alternate cytotoxic cell death mechanisms have been reported, potentially contributing to the overall anti-tumor activity. Although previous studies have highlighted a mechanism of caspase-independent cell death, this mechanism has yet to be well-characterized, thereby warranting further investigation to comprehensively understand the anti-tumor mechanism of CD47 blockade to facilitate optimal drug partner selection for combination therapy. METHODS: The fully humanized anti-CD47 monoclonal antibodies, SRF231, magrolimab, as well as a mouse monoclonal anti-CD47 antibody, B6H12, were used. Multiple cell death mechanisms were evaluated including apoptosis, autophagy and necroptosis by using customized Hoechst/Annexin V, the precision medicine technique BH3 profiling, as well as standard experimental techniques – flow cytometry, siRNA and CRISPR Cas9 genetic manipulation, Western blotting, and immunohistochemistry. These techniques were used on a comprehensive range of lymphoid malignant models including diffuse large B-cell lymphoma, Burkitt lymphoma, and T-acute lymphoblastic leukemia cell lines, patient primary chronic lymphocytic leukemia cells, as well as lymphoid cell-line derived and patient-derived xenograft mice, to elucidate the mechanism of cell death by CD47 blockade and to identify the optimal drug partners for treatment combination. RESULTS: We demonstrate that the anti-CD47 antibodies SRF231, magrolimab, and B6H12 eliminated tumor cells from various in vitro and in vivo lymphoid malignant models via the activation of the RIPK1/MLKL/necroptotic pathway. Moreover, the BH3 profiling technique distinguished two different lymphoid malignant models that respond differently to the BCL-2 inhibitor venetoclax when combined with SRF231; one highlighting the effective yet distinct mechanisms of SRF231-induced necroptosis and venetoclax-induced apoptosis in models that were specifically and/or highly dependent on BCL-2 for survival, while the other implicating venetoclax as a counterproductive partner with SRF231 in models that were not dependent on BCL-2 for survival or were not responsive to venetoclax treatment. CONCLUSION: Collectively, this study unravels a novel, non-canonical cell death mechanism of targeting CD47 by activating necroptosis, and provides evidence and rationale for further evaluation of a therapeutic strategy of combining CD47 blockade with and without apoptotic inducers for suitable patients with lymphoid malignancies. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13045-025-01774-3.
