Cell death induction by CD47 peptide-triggering: a new therapeutic strategy in the management of chronic lymphocytic leukemia – PeptiDeath

Starting from 4N1K, a well-known TSP-1-derived peptide, we have designed novel serum stable peptides. The affinity of these new peptides to CD47 has been assessed.
The pro-apoptotic efficacy of these new peptides has been verified in purified B-lymphocytes from a large panel of CLL patients, including CLL cells resistant to the current chemotherapeutics: “Proof of concept” in vitro.
The best responder peptide, named PKHB1, has been selected and the “proof of concept” in vivo has been performed in a CLL-xenograft mice model developed in the NOD/scid gamma strain.

Supported by a significant cohort of CLL patients, which incorporated individuals with positive and adverse prognostic features, our project demonstrated that the targeting of CD47 by serum-stable TSP1-derived peptides as a novel and efficient therapeutic approach against CLL. This approach is based on the induction of a caspase-independent programmed cell death (PCD) pathway that, sparing the normal T-lymphocytes and the residual B-cells from the patient, efficiently kills the tumor CLL cells, including those from drug-refractory patients (e.g., with TP53 dysfunction). Our recent work also identified the molecular mechanisms regulating this PCD, which involves a sequence of events initiated by the activation of the signal transduction protein PLC[gamma]1, a protein over-expressed in CLL. PLC[gamma]1 over-activation provokes endoplasmic reticulum stress, Ca2+ overload, mitochondrial transmembrane potential loss, calreticulin exposure, and tumor cell death. Finally, our work demonstrated that the intraperitoneal injection of serum-stable CD47 agonist peptides significantly reduces the in vivo tumor burden in a CLL-xenograft mice model, highlighting the potential of this peptide-based therapeutic approach.

Overall, our research program provides substantial progress in four major areas: (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL; (ii) understanding of the molecular events regulating a novel PCD pathway that overcomes the apoptotic avoidance that is characteristic of CLL; (iii) identification of PLC[gamma]1 as a marker of CLL gravity; and (iv) a description of a new anti-CLL strategy with a potential medical application.

Articles
PLoS Medicine, vol 12, pp. e1001796. (Impact Factor : 14,4)

Brevets
1.- New prognostic method for patients suffering of a cancer. PCT/EP2014/077335.
2.- Method and pharmaceutical composition for the use in the treatment of cancer. PCT/EP2013/061727 (WO2013182650 A1).

Programmed cell death (PCD) is essential for the development and maintenance of immune cell homeostasis. Aberrations in PCD signaling pathways result in a variety of pathological conditions and are common in cancer cells. In fact, cellular changes leading to inhibition of PCD play an essential role in tumor development. The activation of the PCD pathways is thus an important area that may provide insight into the causes of drug resistance and facilitate the development of novel anticancer therapies.

The core of the programmed death program (PCD) in mammals consists of a proteolytic cascade involving a family of Cys proteases called caspases. Since the induction of PCD through the use of caspase activators may theoretically constitute a treatment for defective death, initial pro-apoptotic trials have focused on caspase activity. Unfortunately, most of these studies are still in preclinical development because of their low efficacy. This may be partly due to the fact that PCD can proceed even when the caspase cascade is blocked. Alternative caspase-independent models of PCD have therefore been proposed. The activation of caspase-independent pathways opens therefore a new possibility for the development of new PCD approaches to both the research community and the pharmaceutical industry.

Chronic lymphocytic leukemia (CLL), a perfect example of a human malignancy caused by an imbalance between proliferation and programmed cell death (PCD), is the most common form of leukemia in Western countries. Drug resistance remains a major cause of treatment failure in CLL and its inevitable fate due to the prolonged natural course of disease and the repeated treatments. Theses treatments induce cytotoxicity in CLL cells via a caspase-dependent mechanism with a quite variable outcome. Indeed, as leukemic B cells present molecular defects that make them particularly resistant to the caspase-dependent PCD pathway, a significant group of CLL patients are refractory to these chemotherapeutics. For that reason, the introduction of new drugs that induce PCD via caspase-independent PCD pathways could open the way to novel therapeutic strategies in CLL treatment.

In this way, our data demonstrate that CD47 ligation by a soluble peptide named 4N1K induces caspase-independent PCD rapidly (1 h post-treatment) and with a higher efficacy in CLL cells than in normal B cells. And that, even in CLL cells from drug refractory patients. Moreover, our new results indicate that it is possible to enhance the biological PCD activity of 4N1K by generating a more important metabolic stability.

The present research program is designed to generate a series of soluble peptides that specifically eliminate CLL cells, including B-cells with a dysfunction in the TP53 gene (drug refractory patients). A proof of concept of these peptides will be performed both in vitro (in a large panel of purified B-lymphocytes) and in vivo in a mouse xenograft model. To do that, we have gathered a group with the required knowledge and skills, ranging from specialists on caspase-independent programmed cell death (SA Susin, Centre de Recherche des Cordeliers) and CLL (H Merle-Béral, Service d'hématologie hôpital Pitié-Salpêtrière) to specialists on peptide synthesis (P Karoyan, ENS-Paris) and mouse xenograft models (P Launay, UMRS699). This partnership contains biologists, chemists, and clinicians, allowing for a productive cross talk from “bench to bedside”. The scientific project is thus developed in close interaction with chemists/biologists/clinicians, each one taking advantage of the other’s expertise.