Interleukin-7 receptor α mutational activation can initiate precursor B-cell acute lymphoblastic leukemia

Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.


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Animal experiments: 1-In order to evaluate the impact of aberrant IL7R (overexpression or mutant) expression in hematopoietic development, offspring littermates from different genotypes were analyzed at given ages in order to compare the relevant hematopoietic compartments (these are dependent on the exact Cre line). For these experiments, due to their fundamental nature and to the vast possibilities of relevant results, a power analysis is not always possible and thus we used the alternative "Resource equation" method, leading us to consider groups of 10 animals/ genotype/age in the calculations. Dividing query into specific questions (two-sided t-tests, assuming effect size 1.4-5, power 0,8 and 5% significance) calculations led to numbers/ group in the order of 9-10. 2-Transfer of leukemic cells into immune-deficient hosts to study leukemia expansion. In order to confirm leukemic nature of primary tumors. Leukemic cells recovered from disease carrying animals are recovered, sorted and transferred into secondary hosts, in order to confirm the capacity to transfer disease and phenotype stability and evaluate disease aggressiveness. For these experiments a minimal number of 3-4 animals was considered. Pilot experiments showed that all original leukemias (2x10e5 cells) were able to transfer disease into each recipient mice. Thus, we used the minimum number of animals that allowed to perform standard deviation analysis when required. Our subsequent experiments confirmed pilot data. 3-Transfer of leukemic cells into immune deficient hosts in order to study response to therapeutic agents. In these experiments, we try to test compounds in parallel whenever possible, in order to use same control groups and limit numbers of animals. We also performed power analysis for detecting effects, to detect a difference between means of 0.2 and, st deviation 0.15 and alpha 0.05 and power 0.8 we are calculating groups of 10 animals per condition. Our experience (Lonetti et al, Leukemia 2014) indicates we can achieve statistical significance with slightly lower numbers (>=6).
In order to answer questions related to leukemogenesis and associated molecular mechanisms, cohorts of offspring animals were followed in time, up to a limit of 104 weeks, evaluating penetrance and incidence in pediatric, adult and old age corresponding groups. Leukemias arising in these animals were a fundamental resource as source of leukemias for further dissection of molecular cooperating axis with the mutation/ aberration we introduced. In the leukemia incidence cohorts some animals become sick (mostly when >80 weeks of age) without evidence of leukemia in blood and were not considered in the analysis. In some of them, we could perform necropsy and confirm lack of signs for leukemia. Also, a very low number of animals was discarded from the analysis as result of handling accidents. Animals used in further breeding (to originate Homozygous mutant animals) were also not considered for the cohort. Results clearly state leukemia-free survival to account for spurious death not related to the experiment. In drug treatment, one animal died when administering anaesthesia, which was withdrawn from the data.
The experimental findings were reliably reproduced as validated by at least two independent experiments. In vitro experiments were performed in triplicate whenever possible.
Randomization was performed in drug treatment experiments. At time of transfer, hosts were numbered, distributed into groups with webbased randomization tool (Graphpad Quickcalcs). In all other experiments, animals were compared with co-housed litter-mate controls.