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Cell Death and Drug Resistance in Lymphoproliferative Disorders

 

Created in 2009 at the Cordeliers Center the team focused on the study of the physiopathology of B-cell malignancies, with a special interest in the regulation of programmed cell death (PCD) in chronic lymphocytic leukemia (CLL). Santos A. Susin and Florence Nguyen-Khac lead actually the team

-Scientific production. The team is a reference in the field of hematological malignancies, with 170 peer-reviewed publications in the last 5 years, receiving 2435 citations (~400 citations/year). Of the 46 reviews, a number of opinion articles and exhaustive reviews have become references in the field; 4 Editorial comments, 12 technical papers, 1 book, 7 book chapters, and 4 patents with international extension complete our scientific production.

-Academic reputation. Members of the team got relevant recognition on an international level with 65 abstracts/oral communications, 75 communications in international and national conferences published in prestigious hematological journals, and 106 seminars/presentations invites.

- Since 2012, the PIs of the team participated in 29 grants from governmental and private foundations.

- The members of the team attracted during the last 5 years 14 PhD students, presenting at least 1 first authorship paper, and trained over 12 master and 3 Erasmus students. Moreover, our team manages medical students in the lab (4 external/10 residents/year) and in the clinical department (15 external/8 residents/year). As to teaching, the team contributes substantially to medical studies (2d, 3d, 5th year), Master 1, Master 2, Diplômes universitaires, and Diplôme d’études spécialisées en Hématologie: 204 h/year.

- Impact on the public health. The research activity of our group has important impact on the management of patients suffering from hematological diseases. On the one hand, the discoveries of the team, based on studies performed in large cohorts of patients, identify new biomarkers that are important for the diagnosis and set-up of therapeutic strategies. On the other hand, our basic findings propose the introduction of new drugs to specifically eliminate the malignant cells. Our team is also involved in translating new technological developments into the field of innovative diagnostics in hematology, by ensuring assay standardization as well as organizing workshops and quality certification plans for many laboratories worldwide.

Our research projects:

1. Genomic alteration characterizing resistant CLL. The example of 2p

Among the highly heterogeneous landscape of genomic abnormalities characterizing CLL, our team has focused in 2p , a recurrent chromosomal abnormality associated with progressive disease. We have initially demonstrated that 2p was associated with XPO1, TTC27, BCL11A, REL, AHSA2, and USP34 overexpression, and that XPO1 plays a pivotal role in drug resistance (Cosson et al., Leukemia 2017). Our future work will unravel the specific role of these genes in CLL drug resistance in order to determine whether they cooperate with XPO1 in the apoptotic avoidance characterizing 2p . Using a CRISPR-cas9 method, we will deactivate the different genes in a 2p drug resistant B-cell line (JVM-3). We also plan to overexpress the 2p genes in two 2p- CLL cell lines, OSU-CLL and HG3, using a classical dead-Cas9-VP64 transcriptional activation strategy. The consequences on drug resistance and cell proliferation will be assessed by classical flow cytometry approaches. Overall, by expanding the understanding of the key genes associated to a recurrent chromosomal abnormality in CLL, our work will have an important impact, resulting in gene biomarkers and strategies for precision medicine. This work will be carried out with several collaborations, in particular Dr. O Bernard’s team at IGR (Villejuif).


 


 

 

 

2. Impact of the BCR structure on CLL drug resistance

The B-cell receptor (BCR) is a vital structure for the development and homeostasis of normal B-lymphocytes, playing an important role in the life of neoplastic B cells. About a third of CLL patients carry BCR with quasi-identical IG sequences, also referred to as stereotyped BCR (Agathangelidis A et al., Blood 2012). Patients expressing the same stereotyped BCR often share similar genomic aberrations and clinical course of the disease. Here, we propose to better understand the impact of the BCR structure on CLL drug resistance through 2 main approaches: (a) characterization of CLL IG repertoire using next generation sequencing (NGS). Within a European collaborative project (www.euroclonality.org), we will use a molecular barcoding strategy; improve our bioinformatics pipeline by introducing Big data-derived algorithmic approaches (collaboration with Dr Bernardes, Lab of Computational and Quantitative Biology, UMR 7238, UPMC); and develop graphic vizualization tools (collaboration with M Giraud, INRIA & CNRS, Lille). With this, we will be able to fully appreciate the clonal architecture of the CLL cells at the IG loci level and we will analyze its evolution during disease progression; (b) Impact of BCR antigen-binding specificity on leukemic cells behavior. We postulate that editing the IG variable region should alter the cellular response following BCR engagement. We will therefore use the CRISPR/Cas9 technology to edit the native IG variable region of CLL cell lines with those derived from stereotypic BCR CLL cases having opposite clinical behavior (indolent vs aggressive). These isogenic variants will be evaluated for their response to BCR stimulation.



3. Assessment of the key role of TP53 dysfunction in CLL drug-resistant patients

The TP53 abnormalities are, today, the only genetic alteration requiring a specific therapy. Given this clinical relevance, our team has recently developed an efficient and reliable p53 functional assay fully validated in a clinical prospective. Thus, each CLL patient from GHPS is now screened by three “TP53 approaches”: del17p by FISH, TP53 mutations by NGS, and p53 protein function by our test. This broad exploration has uncovered a representative population of discordant CLLs who presents dysfunctionality in the p53 PCD pathway without disruption in the TP53 gene. Thus, we plan to analyze in detail whether this discrepancy is predictive of drug-resistance (flow cytometry in vitro assessments). A complementary NGS approach will allow us to search for genetic alterations in the members of the p53-pathway, including p21. A key alternative application of our p53 functional assay will be to test its efficiency in the assessment of lesser-known TP53 mutations associated to drug resistance (in collaboration with Dr. Soussi, CR Cordeliers, the FILO group, E. Verhoeyen and F.L. Cosset, ENS Lyon). We plan to apply our test in a cohort of 340 CLLs containing 450 different TP53 mutations. A comparison of functional data may help to determine the prognostic and drug resistance impact of each mutation.

 

 

4. CD47-mediated PCD: a promising approach against refractory CLL

We have recently demonstrated that the targeting of CD47 with thrombospondin-1 (TSP-1) agonist peptides (e.g., PKHB1) induces PCD in CLL B-cells, including those from high-risk individuals with a dysfunctional TP53. We have also demonstrated that PKHB1 induces PCD specifically in the B malignant cells and that the treatment with the peptide eliminates the CLL cells in vivo in a xenografted NSG mice model (PLoS Medicine, 2015, 12: e1001796). In the forthcoming period, we will validate the potential use of CD47 agonists in CLL treatment by focusing on PKHB1-derivatives. Note that peptides are usually easier to produce and their effective cost is much lower when compared to therapeutic antibodies. Moreover, peptides have several advantages over small molecule drugs, e.g. they have higher affinity and specificity to interact with their target, while their toxicity remains low. To reach our objective, we will generate new CD47 peptide agonists with improved affinity to CD47 (collaboration with Pr. Karoyan UPMC). We plan to introduce non-natural amino-acids in the PKHB1 sequence and to generate a derivative homotrimer, which will mimic the natural interaction between TSP-1 and CD47. Then, we will validate the efficacy of the peptides on purified B-lymphocytes from a large panel of CLL cells, including those resistant to the current chemotherapeutics. We will monitor the induction of PCD in CLL cells and we will verifiy that the peptides are unable to induce cytotoxicity in other immune subsets. Finally, we test the hit peptides in the TCL-1 CLL mice model (Johnson et al. Blood 2006, 108:1334).

 

 

 

Team Leader:  Santos A. SUSIN (Dr)

Team Members: Brigitte BAUVOIS (Dr), Elise CHAPIRO (Dr), Myrto COSTOPOULOS (Dr), Frédéric DAVI (Pr), Magali LE GARFF-TAVERNIER (Dr), Hélène MERLE-BERAL (Pr), Florence NGUYEN-KHAC (Pr), Damien ROOS-WEIL (Dr).
Sandrine BOUCHET (Eng), Marie-Noëlle BRUNELLE-NAVAS (Eng), Clémentine GABILLAUD (Tech, GH Pitié-Salpêtrière), Claire QUINEY (Eng, GH Pitié-Salpêtrière), Christine MUAMBA (Tech).
Nabila BELHOUACHI (PhD), Audrey BERTAUX (PhD), Nadia BOUGACHA (PhD), Elodie PRAMIL (PhD).

Administration:
  Véronique BARRAUD  

Contact details : tel: 33 1 44 27 81 93 / 33 1 44 27 90 38  
Fax: 33 1 44 27 90 36
Email: veronique.barraud@crc.jussieu.fr

Web site: http://susinlab.com

 

Selected Publications

  • Cabon L, Bertaux A, Brunelle-Navas, M-N, Namazanyy I, Scourzic L, Delavallee L, Vela L, Baritaud M, Bouchet S, Lopez C, Quang Van V, Garbin K, Château D, Gilard F, Sarfati M, Mercher T, Bernard OA, and Susin SA. AIF loss deregulates hematopoiesis and reveals a different metabolic reprogramming in bone marrow cells and thymocytes. Cell Death Differ. 2018, in press.
  • Cosson A, Chapiro E, Bougacha N, Lambert J, Herbi L, Cung HA, Algrin C, Keren B, Damm F, Gabillaud C, Brunelle-Navas MN, Davi F, Merle-Béral H, Le Garff-Tavernier M, Roos-Weil D, Choquet S, Uzunov M, Morel V, Leblond V, Maloum K, Lepretre S, Feugier P, Lesty C, Lejeune J, Sutton L, Landesman Y, Susin SA*, Nguyen-Khac F*. Gain in the short arm of chromosome 2 (2p ) induces gene overexpression and drug resistance in chronic lymphocytic leukemia: analysis of the central role of XPO1. Leukemia. 2017 Jul;31(7):1625-1629.
  • Martinez-Torres AC, Quiney C, Attout T, Boullet H, Herbi L, Vela L, Barbier S, Chateau D, Chapiro E, Nguyen-Khac F, Davi F, Le Garff-Tavernier M, Moumné R, Sarfati M, Karoyan P, Merle-Béral H, Launay P, Susin SA. 2015. CD47 agonist peptides induce programmed cell death in refractory chronic lymphocytic leukemia B cells via PLCγ1 activation: evidence from mice and humans. PLoS Medicine, 12(3):e1001796
  • Damm F, Mylonas E, Cosson A, Yoshida K, Della Valle V, Mouly E, Diop M, Scourzic L, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Kikushige Y, Davi F, Lambert J, Gautheret D, Merle-Beral H, Sutton L, Dessen P, Solary E, Akashi K, Vainchenker W, Mercher T, Droin N, Ogawa S, Nguyen-Khac F*, Bernard OA*. 2014. Acquired initiating mutations in early hematopoietic cells of CLL patients. Cancer Discovery, 4(9):1088-101. * Senior co-authorship.
  • Le Garff-Tavernier M, Herbi L, de Romeuf C, Nguyen-Khac F, Davi F, Grelier A, Boudjoghra M, Maloum K, Choquet S, Urbain R, Vieillard V, Merle-Béral H. Antibody-dependent cellular cytotoxicity of the optimized anti-CD20 monoclonal antibody ublituximab on chronic lymphocytic leukemia cells with the 17p deletion. Leukemia. 2014 Jan;28(1):230-3.
  • Nguyen-Khac F, Lambert J, Chapiro E, Grelier A, Mould S, Barin C, Daudignon A, Gachard N, Struski S, Henry C, Penther D, Mossafa H, Andrieux J, Eclache V, Bilhou-Nabera C, Luquet I, Terre C, Baranger L, Mugneret F, Chiesa J, Mozziconacci MJ, Callet-Bauchu E, Veronese L, Blons H, Owen R, Lejeune J, Chevret S, Merle-Beral H, Leblond V. Chromosomal aberrations and their prognostic value in a series of 174 untreated patients with Waldenstrom's macroglobulinemia. Haematologica 2013 Apr;98:649-654.
  • Cabon L, Galan-Malo P, Bouharrour A, Delavallée L, Brunelle-Navas MN, Lorenzo HK, Gross A, and Susin SA. 2012. BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation. Cell Death and Differentiation, vol 19, pp. 245-256.
  • Artus C, Boujrad H, Bouharrour A, Brunelle MN, Hoos S, Yuste VJ, Lenormand P, Rousselle JC, Namane A, England P, Lorenzo HK, and Susin SA. 2010. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO Journal, vol 29, pp. 1585-1599.
  • Chapiro E, Russell LJ, Struski S, Cave H, Radford-Weiss I, Valle VD, Lachenaud J, Brousset P, Bernard OA, Harrison CJ, Nguyen-Khac F. 2010. A new recurrent translocation t(11;14)(q24;q32) involving IGH@ and miR-125b-1 in B-cell progenitor acute lymphoblastic leukemia. Leukemia, vol. 24, pp. 1362-1364.
  • Merle-Béral H, Barbier S, Roué G, Bras M, Sarfati M, and Susin SA. 2009. Caspase-independent type III PCD: a new means to modulate cell death in chronic lymphocytic leukemia cells. Leukemia, vol. 23, pp. 974-977.

 

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