Session: 618. Acute Lymphoblastic Leukemia: Biology, Cytogenetics, and Molecular Markers in Diagnosis and Prognosis II
Hematology Disease Topics & Pathways:
cell regulation, Biological Processes, pathways, signal transduction
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.
Disclosures: No relevant conflicts of interest to declare.
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