Abstract
Background
In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines.
Aim
Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib.
Methods
This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels.
Results
JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS).
Conclusion
JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL.
Disclosures
No relevant conflicts of interest to declare.
Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods
