AccueilAccueil>Santos A. SUSIN

Cell death and Drug Resistance in
Hematological Disorders

The main goal of our team is to analyze in detail the molecular mechanisms associated to drug resistance in different hematological malignancies (see our research projects below) and to propose original approaches to overcome this therapeutic blockade. To develop this program, we have gathered a group with the required knowledge and skills, ranging from researchers specialized on programmed cell death to clinicians specialized on the physiopathology of lymphoproliferative and mast cell malignancies. Thus, the main strength of our project is that it involves close interaction between scientists and clinicians, each one taking advantage of the other’s expertise. This cultural and technological exchange entails a multidisciplinary approach that should enable us to meet a two-fold objective. Fundamental: increase the existing knowledge of biochemical and molecular mechanisms associated to drug resistance. Applied/Translational: develop new therapeutic approaches to treat haematological malignancies.
     We have international expertise on the biological characterization of samples from patients suffering from hematological disorders. These analyses, performed through classical and high-throughput approaches, include morphological, immunophenotypical, cytogenetical, and molecular approaches. Moreover, other than leveraging the patients with lymphoproliferative and mast disorders from the Groupe Hospitalier Pitié-Salpétrière (GHPS), our team is involved in various national and international clinical trials that make it possible to access large cohorts.

-Scientific production. The team is a reference in the field of hematological malignancies, with 180 peer-reviewed publications in the last 5 years, receiving more than 2500 citations. 4 patents with international extension complete our scientific production.

-Academic reputation. Members of the team got relevant recognition on an international level with 75 abstracts/oral communications, 90 communications in international and national conferences published in prestigious hematological journals, and 106 seminars/presentations invites.

- Since 2013, the PIs of the team participated in 32 grants from governmental and private foundations.

- The members of the team attracted during the last 5 years 16 PhD students, presenting at least 1 first authorship paper, and trained over 15 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.

Our research projects:

1. Genomic alteration characterizing resistant CLL.
(i) The example of 2p (Pr. Nguyen-Khac, Dr. Chapiro)
. 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.

 

(ii) Genomic characterization of B-cell prolymphocytic leukemia (B-PLL) and analysis of the drug response (Pr. Nguyen-Khac, Dr. Chapiro, Dr. Roos-Weil, Dr. Susin). B-PLL is a very rare mature lymphoid neoplasm that occurs in elderly people. Molecular pathogenesis of B-PLL is poorly understood and the patients have generally a dismal prognosis with high levels of chemotherapy refractoriness. An understanding of the mechanisms of drug resistance and the development of new therapeutic approaches appears crucial. Our project proposes a comprehensive analysis of the oncogenetic events underlying B-PLL. We have collected a national cohort of 36 B-PLL cases. Cytogenetic analyses show that abnormalities leading to up-regulation of the MYC gene are the most frequent (76%). We are currently identifying the somatic mutations associated to B-PLL by whole exome and RNA sequencing. We will develop a methylome analysis to investigate the epigenetic alterations and to precisely define the maturation stage of the leukemic cells by comparison to normal B-cell sub-populations. Additionally, we will test, on primary B-PLL cells, the efficacy of the drugs used in the treatment of the B-cell malignancies combined with inhibitors of the bromodomain extra-terminal proteins that modulate MYC signalization pathways. The correlations between drug sensibility and genomic and epigenetic abnormalities would unravel the mechanisms of B-PLL drug resistance.

2. Impact of the BCR structure on CLL drug resistance (Pr. Davi).
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 (Dr. Le Garff-Tavernier, Dr. Costopoulos)
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 programmed cell death (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. 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 (Dr. S A Susin, Dr. B Bauvois).
We have recently demonstrated that the targeting of CD47 with thrombospondin-1 (TSP-1) C-terminal derived agonist peptides (e.g., PKHB1) induced PCD in CLL B-cells, including those from high-risk individuals with a dysfunctional TP53. We have also demonstrated that PKHB1 induced PCD specifically in the B malignant cells and that the treatment with the peptide eliminates the CLL cells in vivo in a xenografted CLL mice model (Martinez-Torres et al., PLoS Medicine 2015). Now, we plan to validate the potential use of CD47 agonists in CLL treatment. To reach our objective, we will generate new CD47 peptide agonists with improved affinity to CD47 (collaboration with Pr. Karoyan, Sorbonne Université). 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 verify that the peptides are unable to induce cytotoxicity in other immune subsets. Finally, we test the hit peptides in our CLL mice model.

 

5. Intraocular and cerebral lymphomas (Dr. Le Garff-Tavernier, Dr. Costopoulos, Pr. Davi).
As active members of the LOC network, our fields of expertise also include the diagnostic work-up of central nervous system lymphomas, which are very rare diseases with a pejorative prognosis. Our previous publications have set-up the diagnosis of these diseases by testing the IL-10 levels in ocular (Costopoulos et al., Ophthalmology 2016) and cerebrospinal fluids (Nguyen-Them L et al., Eur J Cancer 2016). These soluble biomarker quantifications have constituted a breakthrough in the identification of these disorders. We will pursue this work by evaluating other soluble biomarkers, like the recently identified prognostic indicators sCD19, CXCL13, and sCD25. We will also search for clone-specific IG gene rearrangements as well as cancer-associated gene mutations in circulating tumor DNA from cerebrospinal and ocular fluids.

 

6.- Mast cell disorders (Pr. Arock).
Our major scientific objective here is to find new treatments for patients presenting an aggressive systemic mastocytosis (ASM), a rare and incurable disease, characterized by the presence of a KIT D816V activating mutation in the neoplastic mast cells (NMCs). To find drugs active on the mutant receptor, and taking advantage of the availability of our proprietary model of human KIT D816V neoplastic MC line, ROSAKIT D816V, we have screened a panel of tyrosine kinase inhibitors (TKIs) and identified three new inhibitors which selectively prevent the constitutive phosphorylation of the mutant receptor. These drugs selectively inhibited proliferation and induced PCD. Our future projects are: (a) to validate the effect of the 3 compounds identified on NMCs obtained from bone marrow (BM) samples of KIT D816V ASM patients; (b) to corroborate the effect of the 3 compounds identified in vivo  (injection of ROSA cells in NSG mice gives rise to an ASM-like disease in 10 weeks); (iii) to investigate the in vitro effects of combinations of the TKIs with inhibitors of other targets activated in ASM patients (e.g., STAT5 or AKT): we will search for a potent combination able to eradicate NMCs.

 

Team Leaders:  Santos A. SUSIN (DR; CNRS) and Florence NGUYEN-KHAC (PU-PH; Sorbonne Univ)


 

Team Members:

Researchers: Michel AROCK (PU; Univ Paris Sud), Brigitte BAUVOIS (DR; Inserm), Elise CHAPIRO (MCU-PH; Sorbonne Université), Myrto COSTOPOULOS (PH; AP-HP), Frédéric DAVI (PU-PH; Sorbonne Univ), Magali LE GARFF-TAVERNIER (DPH; AP-HP, Hélène MERLE-BERAL (PU-PH em; Sorbonne Univ), Damien ROOS-WEIL (MCU-PH; Sorbonne Univ).

Technical Staff: Sandrine BOUCHET (IE; AP-HP), Marie-Noëlle BRUNELLE-NAVAS (IE; Inserm), Clémentine GABILLAUD (Tech, AP-HP), Claire QUINEY (IR; AP-HP), Christine MUAMBA (Adj Tec; Inserm)

Young Researchers: Nadia BOUGACHA (PhD Student), Justine CHIVOT (PhD Student), Ludovic JONDREVILLE (PhD Student), Elodie PRAMIL (PhD Student).

Students: Anaïs ROUSSEAUX (Master Student), Floriane THEVES (Master Student).

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

Publications

  • Peter B, Bibi S, Eisenwort G, Wingelhofer B, Berger D, Stefanzl G, Blatt K, Herrmann H, Hadzijusufovic E, Hoermann G, Hoffmann T, Schwaab J, Jawhar M, Willmann M, Sperr WR, Zuber J, Sotlar K, Horny HP, Moriggl R, Reiter A, Arock M, Valent P. Drug-induced inhibition of phosphorylation of STAT5 overrides drug resistance in neoplastic mast cells. Leukemia. 2018, 32(4):1016-1022.
  • 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, 25(5):983-1001.
  • 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.

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