Pre-Clinical Efficacy of the Novel Kinase Inhibitor Nintedanib on PAX5 Fusion Genes in Pediatric Ph-like B-Cell Precursor Acute Lymphoblastic Leukemia

Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120.

This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120.

We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement.

Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells.

In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent.