Chronic 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 Findings
In 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.
Our 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.
In this study, Santos Susin and colleagues demonstrate that a serum-stable CD47 agonist peptide is highly effective at inducing apoptosis in chronic lymphocytic leukemia B-cells and mice.
Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia (cancer of the white blood cells). It accounts for about a third of all adult leukemias, and about one in 200 people will develop CLL during their lifetime, usually in old age. White blood cells (including T and B lymphocytes) are made by the bone marrow (the soft center of certain bones in the body) and help the body fight infections. Leukemia begins when a bone marrow cell acquires genetic changes (mutations and chromosomal abnormalities) that make it grow and develop abnormally. Over time, the abnormal cells accumulate in the bone marrow, blood, and lymphoid organs (lymph nodes and spleen) (malignant B lymphocytes accumulate in the case of CLL) and stop the marrow producing healthy blood cells. CLL does not usually cause any symptoms during its early stages and is often diagnosed as a result of a routine blood test. Some patients, however, develop painless swellings in the lymph nodes in the neck, armpit, or groin. Other symptoms of CLL include tiredness, frequent infections, and weight loss. Treatments for CLL, which are not given unless symptoms develop, include chemotherapy, immunotherapy, and targeted therapies, which attack cancer cells without harming normal cells.
Why Was This Study Done?
CLL develops very slowly, and people with CLL usually live for many years after their diagnosis. However, there is no cure for CLL, and treatment fails in up to a quarter of patients because resistance to chemotherapy develops. The outlook (prognosis) is particularly bad for patients whose cancer cells contain a dysfunctional TP53 gene (TP53 encodes a protein that normally regulates cell division and cell death). Although some new therapies have recently been approved in the US and Europe, additional approaches to the treatment of CLL are still needed. One potential target for new treatments is the cell surface receptor CD47. In CLL cells, activation of CD47 by a small fragment (peptide) of thrombospondin-1 (a protein that binds to CD47) called 4N1K rapidly induces caspase-independent programmed cell death (PCD), a type of cell death in which the cell uses specialized cellular machinery to kill itself. (PCD normally controls cell numbers and eliminates cells that threaten an animal’s survival, but cancer cells often resist PCD.) Unfortunately, peptides are usually degraded quickly in the human body, which limits their usefulness as therapeutic agents. Here, therefore, the researchers investigate whether PKHB1, a variant of 4N1K designed to be stable in the body (a “serum-stable” agonist peptide), might provide a new approach to the treatment of CLL.
What Did the Researchers Do and Find?
The researchers tested the ability of PKHB1 to kill malignant CLL B lymphocytes grown from blood samples collected from 80 patients with CLL. PKHB1 efficiently killed these cells, including those from individuals with a dysfunctional TP53 gene but, importantly, did not kill either normal T and B lymphocytes collected from healthy donors or the residual normal T and B lymphocytes present in the patient samples. Genetic and molecular analyses undertaken by the researchers indicated that the differential response of normal and leukemic B lymphocytes to PKHB1 was the result of sustained activation of a protein called phospholipase C gamma-1 (PLCγ1) in the malignant CLL B lymphocytes. Other experiments showed that PLCγ1 activation drove the induction of a new PCD pathway (a calcium-mediated, caspase-independent pathway) that was not down-regulated by survival stimuli provided by the lymphocyte microenvironment. Finally, the researchers report that injection of PKHB1 reduced the tumor burden in a mouse model of CLL.
What Do These Findings Mean?
Taken together, these findings suggest that serum-stable CD47 agonist peptides represent a potential new treatment for CLL that could circumvent the problem of chemotherapy resistance and eliminate malignant CLL B lymphocytes while sparing residual normal B and T lymphocytes. These findings also demonstrate that PKHB1, the serum-stable CD47 agonist peptide tested in this study, directly induces a novel PCD pathway, thereby overcoming the innate avoidance of apoptosis by CLL cells. Before peptide-based strategies for the treatment of CLL can enter clinical trials, however, further studies are needed to develop peptides with increased stability and increased ability to bind to and activate CD47. Moreover, because the mouse CLL model used here incompletely recreates the development of CLL in people, the effectiveness and safety of this pioneer approach needs to be tested much more rigorously in animals before any clinical trials are initiated.
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001796.
The US National Cancer Institute provides information about cancer and how it develops, about targeted cancer therapies, and about CLL and its treatment (in English and Spanish)
Cancer Research UK, a not-for-profit organization, provides general information about cancer and how it develops and detailed information about CLL
The UK National Health Service website also provides information about CLL
The American Society for Hematology provides general information about blood cancer and detailed information about leukemia, including CLL
The French Society for Hematology provides general information about CLL (in French)
The not-for-profit Force Hemato organization provides general information about lymphoproliferative disorders and resources for patients and families (in French)
The not-for-profit organization HealthTalkOnline provides personal stories about living with CLL
Wikipedia provides information about programmed cell death and about CD47 (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)