EHA Library - The official digital education library of European Hematology Association (EHA)

 

Pediatric T-cell acute lymphoblastic leukemia patients harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways.

 

We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of T-ALL patients. Additionally, we were interested to know whether these mutations occur in a mutually exclusive manner. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors—individually and in combinations—on downstream signaling and cytotoxicity for each of the mutations using our expression system in Ba/F3 cells.

 

We screened 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1-3, TYK2) and hotspot regions of N-RAS and K-RAS. The three-dimensional JAK1 model was superimposed on the TYK2 pseudokinase-kinase crystallographic structure. We developed a doxycycline-inducible system by adapting the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software.

 

IL7Ra, JAK, RAS, AKT and PTEN mutations are present in 44% of T-ALL patients and occur in a predominantly mutually exclusive fashion, suggesting they represent a single mutant signal transduction route. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. Our novel three-dimensional model of JAK1 provides insight on how these JAK mutations result in constitutive kinase activity. Amino acid residues that regulate or facilitate direct interactions between the pseudokinase and the kinase domains are frequent targets of JAK1 and JAK3 mutations in T-ALL. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes—in contrast to the wild type genes—transforms Ba/F3 cells by supporting IL3-independent growth, and by activating the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as Ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. However, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibition of either of these downstream pathways alone is insufficient. Inhibitor combinations of both pathways completely block IL7R signaling irrespective of the level of activation by specific mutations. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are insensitive to JAK inhibition.

 

We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. Since leukemia often depends on one or both pathways, the cytotoxic efficacy of synergistic MEK and PI3K inhibition should be further explored in clinical trials for IL7Ra mutant leukemia.