CD47 Agonist Peptides Induce Programmed Cell Death in Refractory Chronic Lymphocytic Leukemia B Cells via PLCγ1 Activation: Evidence from Mice and Humans

Abstract

BackgroundChronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides.Methods and FindingsIn peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease.ConclusionsOur work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.

Chronic lymphocytic leukemia: Time to go past genomics?

“…Together with the novel anti-CD20 obinutuzumab [152] and other promising agents (the BCL2 inhibitor ABT-199 [153], the XPO1 inhibitor selinexor [154], CD47 agonist [155]), these treatments may exert different pressure on CLL clonal evolution and may modify prognostic stratification and evolutionary trajectories, as evaluated until now.…”

The CD47‐SIRPα signaling axis as an innate immune checkpoint in cancer

“…Finally, when considering targeting of CD47 or SIRPα it should be mentioned that at least some agents have been shown to exert direct effects on cancer cell growth. For instance, triggering CD47 on the cell membrane of several hematological cancers resulted in a direct inhibition of cancer cell growth or lead to the induction of caspase‐dependent or ‐independent apoptotic cell death in vitro(see Table ). Finally, we showed that an antibody directed against SIRPα triggered caspase‐independent apoptosis in SIRPα‐expressing acute myeloid leukemic cells, and this antibody also synergized with various chemotherapeutics for triggering cell death …”

CD47 agonist peptide PKHB1 induces immunogenic cell death in T‐cell acute lymphoblastic leukemia cells

“…More recently, CD47 activation by peptides derived from the C‐terminal domain of TSP‐1 induces a caspase‐independent and calcium‐dependent cell death in different cancer cell lines . Indeed, CD47 engagement to PKHB1, the first‐described serum‐stable soluble CD47‐agonist peptide, induced changes in ER morphology, CRT exposure, reactive oxygen species (ROS) production and dissipation of the mitochondrial membrane potential in cells from patients with CLL …”