Single-Cell Profiling of Signal Transduction Pathways in Pediatric T-Cell Acute Lymphoblastic Leukemia By Mass Cytometry:Dissecting JAK/STAT and PI3K/Akt/mTOR Active Signalling

Background Acute leukemia is the most common malignancy of childhood. Most of pediatric acute leukemias are derived from B-cell precursor, and about 15% are from T-cell origin and lead to T-cell acute lymphoblastic leukemia (T-ALL). Despite recent progress in treatment, the prognosis of T-ALL patients is worse, and ~30% of cases relapse (Pui CH, Seminars in hematology 2009). Despite the need for targeting molecules to treat resistant and high risk pediatric T-ALLs, in vitro drug testing may not be predictive of efficacy in vivo due to the complexity of the signalling network and to the cell heterogeneity within the individual's sample. High-resolution analysis of signalling profile at single-cell level may represent an innovative tool in the comprehension of signalling network and its role in the response to drugs (Bodenmiller B et al, Science signaling 2010). Currently mass cytometry (CyTOF) technique allows the measurement of more than 30 parameter per single cell (Bendall SC et al, Science 2011), rendering this approach an ideal method to investigate the complex biology of T-ALL (Girardi T et al, Blood. 2017) and to assay drug candidates for cellular targeting.

Methods Using single-cell mass cytometry we developed panel of 39 metal-labeled monoclonal antibodies (moAbs) identifying T-ALL blasts and non-malignant T-cells. Using phospho-specific moAbs and moAbs targeting proliferation and apoptosis we detected signal transduction upon in vitro treatment of 17 diagnostic and 5 relapse T-ALL samples with IL-7 (Jak/STAT5 pathway activator) , BEZ-235 (inhibitor of PI3K and mTOR), and Pervanadate (inhibitor of tyrosine phosphatases). We have used sample barcoding by anti-CD45 antibodies to unify sample preparation and acquisition for all treatment conditions and we resolved the cells of interest by manual gating. We evaluated both, the individual change sin p-kinases and the overall changes using dimensionality reduction approach.

Results T-ALL cells showed constitutive activation of various signaling pathways as well as proliferation markers compared to residual bone marrow T-cells and T-cells isolated from healthy donors. Up-regulated activity of PI3K-mTOR pathway (p4E-BP1, pAkt, pS6), proliferation rate (pRb, Ki-67), MAPK pathway (p-p38, pErk1/2), translation (pCREB) in T-ALL cells was detected, whereas lower levels of anti-apoptotic protein Bcl2 in T-ALL cells were detected. Interleukin 7 (IL-7) activates three main signalling pathways such as STAT5, PI3K/Akt/mTOR and MEK/Erk, leading to the promotion of leukemia cell viability, cell cycle progression and growth. Thus we interrogated T-ALLs in their ability to respond to IL -7 in vitro . We used an hierarchical clustering analysis (with Euclidean distance metrics and an average linkage) and we were able to divide T-ALL samples in IL-7 responder (6 out of 17) and IL-7 non responder (11 out of 17). Of note, no significant differences in IL7Ra (CD127) expression was observed between the two groups. Interestingly IL-7 responders had higher levels of pRb and Ki-67 proliferation markers as compared to both IL-7 non-responders and non-malignant T-cells. Moreover IL-7 non-response correlate with poor response in vivo to prednisone and higher level of minimal residual disease (MRD) at day15 of remission induction treatment. Finally T-ALL cells were treated ex vivo with PI3K/AkT/mTOR dual inhibitor BEZ-235. Of the 17 T-ALL patients tested, 9 responded to BEZ-235 but not to IL-7 , by contrast 4 did not respond to BEZ-235 being IL-7 responders, two patients responded both to IL-7 activation and BEZ-235 inhibition. One single patient did not respond neither to IL-7 nor to BEZ-235

Conclusions In summary we characterized pediatric T-ALL samples demonstrating the feasibility of CyTOF-based single-cell profiling of signal transduction pathways in this setting. We detected constitutively active pathways in T-ALL blasts as compared to residual non-malignant T-cells. Importantly we identified by functional read outs distinct clusters of IL-7 and BEZ-235 T-ALL responders patients, supporting the notion of a mutual exclusivity between JAK-STAT (or Ras) pathway genomic alterations and PI3K-AKT pathway alterations (Liu et al. Nat Genet. 2017). Our observation can contribute to the better understanding of the complex signalling network governing T-ALL behaviour and response to therapy.