Figures and data
Summary of the experimental strategy.
A) Manual curation of FLT3-ITD-specific Prior Knowledge Network (PKN). B) Multiparametric analysis of signaling perturbations in TKI sensitive and TKI resistant cells. C) Model generation through the CellNOptR tool. D) Prediction of combinatorial treatments restoring TKI sensitivity. E) In vitro validation of novel combinatorial treatments. F) In silico prediction of co-treatment outcome in AML patients.
Generation of the training dataset.
A) Schematic representation of the FLT3-ITD PKN manual curation.
B) Schematic representation of the experimental design: FLT3 ITD-JMD and FLT3 ITD-JMD cells were cultured in starvation medium (w/o FBS) overnight and treated with selected kinase inhibitors for 90 minutes and IGF1 and TNFa for 10 minutes. Control cells are starved and treated with PKC412 for 90 minutes, while “untreated” cells are treated with IGF1 100ng/ml and TNFa 10ng/ml with PKC412 for 90 minutes. As an activity readout, we measured the phosphorylation levels of activatory (red) or inhibitory (blue) residues of the listed sentinel proteins.
C) Principal Component Analysis (PCA) of FLT3 ITD-JMD and FLT3 ITD-JMD cells treated upon different perturbations.
D) Unsupervised, hierarchical clustering of the intensity of the measured sentinel proteins discriminates samples according to the treatment.
E) Unsupervised, hierarchical clustering allows the classification of the sentinel proteins according to their role (red) and pathway (gray, purple and blue).
Optimized Boolean models recapitulate the different sensitivity of FLT3 ITD-JMD and FLT3 ITD-JMD cells to TKI.
A) Color-coded representations of the experimental activity modulation (T90 – T0) of sentinel proteins used to train the two Boolean models (upper panel) and the average prediction of protein activities in the family of 100 best models (central panel). The protein activity modulation ranges from −1 to 1 and is represented with a gradient from blue (inhibited) to red (activated). The absolute value of the difference between experimental and simulated protein activity modulation (lower panel) is reported as a gradient from light yellow (error < 0.5) to red (1.85).
B-C) FLT3 ITD-JMD (B) and FLT3-ITD TKD (C) high-confidence Boolean models. Perturbed proteins in the experimental setup are marked in red or green if inhibited or stimulated, respectively. Sentinel proteins are reported in blue. The edges’ weight represents their frequency in the family of 100 models and only the high-confidence ones (frequency > 0.4) are reported. Orange edges are cell-specific links.
D) Cartoon of the in silico conditions simulated to analyze the different TKI sensitivity of the FLT3 ITD-JMD and FLT3 ITD-TKD Boolean models. Untreated condition: TNFa+IGF1; FLT3i: TNFa+IGF1+FLT3 inhibition.
E) Heatmaps (left) report the activation level of positive and negative phenotype regulators present in the two Boolean models. Bar plots (right) showing the proliferation activation and apoptosis inhibition levels in untreated and FLT3i conditions in the steady states of FLT3 ITD-JMD (yellow) and FLT3 ITD-TKD (blue) Boolean models.
In silico simulation of the FLT3 ITD-TKD logic model allows the prediction of novel combinatorial treatment reverting TKI resistance.
A) Cartoon of the in silico simulation conditions.
B-C) Bar plot showing the in silico simulation of proliferation activation (B) and apoptosis inhibition (C) levels in untreated and FLT3i conditions in combination with knock-out of each of 10 crucial kinases in FLT3 ITD-JMD (blue) and -TKD (yellow) cells.
D-E) In FLT3 ITD-JMD (yellow) and -TKD (blue) cells treated with 100nM midostaurin and/or 10uM SP600125 (JNK inhibitor) for 24h, the percentage of Annexin V positive cells (D) and the absorbance values at 595nm (E), normalized on control condition, are reported in bar plots.
F) Cytofluorimetric cell cycle analysis of DAPI stained FLT3 ITD-JMD (yellow) and -TKD (blue) cells treated with 100nM midostaurin and/or 10uM SP600125 (JNK inhibitor) for 24h.
G) Cytofluorimetric analysis of phospho-H3 (S10) levels FLT3 ITD-JMD (yellow) and -TKD (blue) cells treated with 100nM midostaurin and/or 10uM SP600125 (JNK inhibitor) for 24h. Each bar represents the mean ± SE of the data obtained from three independent experiments. **p < 0.01; ANOVA test
H) In FLT3 ITD-JMD (yellow) and -TKD (blue) cells treated with 100nM midostaurin and/or 10uM SP600125, followed by 10’ of TNFa 10ng/ml, the protein levels of phospho-JNK (T183/Y185), JNK, phospho-CDK1 (Y15), phospho-CDK1 (T161), CDK1, phospho-CDK2 (T160), CDK2, CyclinB1, CycinE2, and Vinculin were evaluated by western blot analysis.
FLT3-ITD patient-specific Boolean models
A) Schematic representation of our computational approach to obtain personalized logic models.
B) Hierarchical clustering of patients according to their clinical characteristics (response to chemotherapy, vital status, and AML recurrence). Resistant, alive or deceased responders, and deceased with general or FLT3-ITD AML recurrence patients are reported in purple, light or dark green, and light or dark brown, respectively.
C-D) Hierarchical clustering of patients according to their mutational profile (B) and their expression profile of 262 genes (C).
E) Heatmap representing patient-specific in silico apoptosis inhibition (left panel) and proliferation levels (right panel) upon each simulation condition.
F) Patient-specific (JMD1 and TKD2) Boolean models. In the JMD1 model (left panel), nodes’ activity has been simulated in control (bottom-left part) and FLT3 inhibition conditions (upper-right part). In the TKD2 model (right panel), nodes’ activity has been simulated in FLT3 inhibition (bottom-left part) and FLT3 and JNK co-inhibition conditions (upper-right part).
