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Background <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusion <p>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.</p> Graphical Abstract <p></p>

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CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月3日

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  • Stephen J. F. Chong,
  • Rebecca Valentin,
  • Jing Wang,
  • Fen Zhu,
  • Prafulla C. Gokhale,
  • Benjamin K. Eschle,
  • Filip Garbicz,
  • Kartini Iskandar,
  • Tomasz Sewastianik,
  • Brienne C. Y. Toh,
  • Johany Penailillo,
  • Marisa O. Peluso,
  • Jeremy Zhang,
  • Liam Hackett,
  • Mary C. Collins,
  • Timothy Z. Lehmberg,
  • Ammar Adam,
  • Li Zhang,
  • Caroline M. Armet,
  • Matthew Rausch,
  • Benjamin H. Lee,
  • Pamela M. Holland,
  • Vito J. Palombella,
  • Alison M. Paterson,
  • Li Ren Kong,
  • Elisa ten Hacken,
  • Jennifer L. Guerriero,
  • Charles Herbaux,
  • Catherine J. Wu,
  • Wee Joo Chng,
  • Shazib Pervaiz,
  • Carsten U. Niemann,
  • Ruben D. Carrasco,
  • Matthew S. Davids

摘要

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