CD47 blockade induces necroptosis and complements the effects of BCL-2 inhibition in hematologic malignancies

Introduction: CD47 is a macrophage checkpoint protein that acts as a “ don't-eat-me ” signal to prevent cell phagocytosis. Its blockade has shown promising results in clinical trials of lymphomas. Interestingly, CD47 blockade-induced cell death beyond phagocytosis, has also been reported, potentially contributing to the overall anti-tumor activity. This cell death mechanism has yet been well-characterized, thus warranting investigation to comprehensively unravel the mechanism of CD47 blockade and to facilitate the identification of optimal drug partners for combination therapy. Method: Anti-CD47 monoclonal antibodies (mAb), SRF231, magrolimab, B6H12, were evaluated for cell death mechanisms such as apoptosis, autophagy or necroptosis. Techniques used include BH3 profiling, Annexin V, siRNA/CrisprCas9, Western blot and immunohistochemistry. Diffused large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML) cell lines were used. In vitro results were used to select for appropriate drug to combine with CD47 blockade. Results were validated ex vivo in leukemia patient samples and in vivo in cell line and patient-derived mouse models. Result: Anti-CD47 mAbs consistently killed tumor cells from 10 cell lines, 24 patients, 3 mouse models by activating necroptosis, while sparing healthy immune cells. Necroptosis was confirmed via increased phospho(p)-RIPK and p-MLKL, which were rescued by necroptosis inhibitors or CD47/MLKL silencing. We further ascertained that PLCgamma activation is upstream of necroptosis, as inhibiting PLCgamma prevented p-MLKL and necroptosis. Moreover, apoptosis or autophagy was not involved, as inhibiting these pathways did not rescue SRF231-induced cell death. Given that necroptosis is the primary mechanism, we proceeded to leverage on apoptosis as an additional pathway to enhance cell death. Using BH3 profiling, a technique that informs cellular sensitivity to apoptotic inducers - BH3 mimetics, we identified the BCL-2 inhibitor venetoclax as an effective partner for SRF231 against hematologic malignant cells that depend highly on BCL-2 for survival ( i.e. Cell survival: DMSO - 100%, (50nM) VEN - 71.39%, SRF231 - 57.01%, Combo - 19.53%, P < 0.0001). SRF231 and venetoclax combination completely eliminated tumor burden and prolonged progression free survival in BCL-2 dependent DLBCL and AML mouse models ( i.e. Mice survival at day 70: Control - 0%, VEN - 0%, SRF231 - 58.3%, Combo - 100%; P < 0.0001). Importantly, SRF231 was equally effective against non-BCL-2 dependent, venetoclax-resistant DLBCL mouse models ( i.e. Mice survival at day 200: Control - 0%, SRF231 - 62.5%, P < 0.0001). Conclusion: Our study unravels a novel cell death mechanism of CD47 blockade through necroptosis, thereby permitting the inclusion of venetoclax-induced apoptosis, a complementary combination worthy of further study against BCL-2 dependent hematologic malignancies in the clinic.