Novel Drugs to Target JAK2 Rearrangements in Pediatric Acute Lymphoblastic Leukemia

Background: Risk-based treatment is curative for 85% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), however relapse remains a leading cause of mortality, urging the need of novel molecular targets. JAK/STAT alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non- receptor tyrosine kinase fundamental for hematopoiesis and its mutations have been widely studied, whereas JAK2 fusion genes are still poorly characterized.

Aim: This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients, developing a target strategy in preclinical models.

Methods: RNA Next Generation Sequencing was applied to find JAK2 fusions in a cohort of high risk BCP-ALL pediatric patients. In vivo expansion of patients' cells has been carried out in NSG mice. A high throughput ex-vivo screening of about 174 FDA-approved drugs, through a luminescence assay (CellTiter glo) was performed. After ex-vivo and in-vivo treatments with single selected drugs, phosphoflow and apoptosis assays were done.

Results: We identified 11 pediatric cases carrying a JAK2 fusion with different partners, where PAX5 gene was the only recurrent. We expanded cells from 3 cases in mice, carrying PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2. We demonstrated the basal activation of pY1007-1008-JAK2 compared both to wild type JAK2 (+70%, two-tailed P value 0.03) and to P2RY8::CRLF2 rearrangements as well as to JAK2 mutation (+40%, two-tailed P value 0.16). JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated.

CHZ868, a new class-II tyrosine kinase inhibitor (TKI), was used to target JAK2, appreciating a mean inhibition of -62% of pY1007-1008 JAK2 in PAX5::JAK2, -22% in ATF7IP::JAK2 and -35% in ZEB2::JAK2 after 30 minutes till 48h. Moreover, we observed a decrease of pS727-STAT3(-35-50%) and pY694-STAT5 (-15-50%) and reduction of phosphorylation on PI3K pathway, downregulating PDPK1, AKT, 4pEBP1 and pS6.

After 48h monotherapy treatment by CHZ868, we detected decreased cell viability (20-75% at IC50), which increased in the combination with dexamethasone.

In PAX5::JAK2, we also performed treatments with BIBF1120/Nintedanib, LCK inhibitor (activated downstream to PAX5 fusions) evaluating a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, ruxolitinib caused autophagy with higher levels of LC3- II compared to untreated cells (+45%, p<0.01) and reduction of apoptosis. Indeed, active caspase 3 increased after ruxolitinib and chloroquine (autophagy inhibitor) combination (+20% vs ruxolitinib alone, p<0.01). Instead, CHZ868 alone or with chloroquine did not induce autophagy, with no effect on LC3-II and active caspase 3 levels.

Finally, we demonstrated the in vivo efficacy of CHZ868 in PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2 patient-derived- xenografts. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in BM (-43-85%), spleen (-72-89%), CNS (-13-62%) and PB (-46-80%). Moreover, CHZ868 significantly reduced the phosphorylation of pJAK2 (-18-46%), pSTAT5 (-23-71%) and pAKT(Ser473) (-18- 34%).

As CHZ868 in-vivo targeting showed not complete eradication of leukemia, with low effect on CNS, we decided to apply a high-throughput ex-vivo screening of a library of 174 FDA-approved drugs in early to late clinical trials, on 4 PDXs samples carrying JAK2 fusions (PAX5::JAK2, ATF7IP::JAK2, ZEB2::JAK2 and the recently expanded novel fusion GIT2::JAK2). We confirmed that Ruxolitinib was not effective on JAK2 cohort at the 6 different dosages that were tested (5nM-25uM), neither on MUTZ5, and five Lymphoblastoid cell lines (LCLs) used as healthy controls. Instead, AT9283 (p<0.001 vs LCLs), Fedratinib (p<0.01 vs LCLs) and Gandotinib (p<0.05 vs LCLs) were found to be potent, specific, and non-toxic JAK2 inhibitors. Additionally, this extended screening led us to identify drugs, not belonging to JAK inhibitors, specific and non-toxic for rearranged JAK2 cohort, such as Birinapant (Smac mimetic), JQ1 (BRD4 inhibitor), Fludarabine (Chemotherapy) among the others.

Summary/Conclusion: CHZ868 is a promising drug for the treatment of JAK2 fusions. Further studies will focus on effective and specific novel drugs found to be highly effective and specific on JAK2 rearrangements in BCP-ALL.

Biondi:Amgen: Honoraria; Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board.