Targeting Hyperactivated Lck in PAX5 Rearranged Pediatric B-Cell Precursors Acute Lymphoblastic Leukemia

Despite intensive risk-based treatment protocols, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups.

We considered 135 B-other cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols without recurrent genetic rearrangements and excluding Down syndrome and High Hyper Diploid cases. By integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS, we identified 59 patients (43·7%) having a Ph-like signature; in addition to ERG-related (26%), High Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%).

CRLF2 rearrangements were almost exclusively found in the Ph-like group, and also the IKZF1-plus profile was prevalent in Ph-like cases (8/9) and associated with a poor outcome (5-year EFS (SE) 37·5% (17·1) vs. 65·5% (7·0) for non-IKZF1-plus cases). Interestingly, PAX5 gene lesions were mostly associated with the Ph-like signature, with CNAs and translocations in 43/131 B-other patients (32·8%), with 31/57 (54·3%) in Ph-like and 12/74 (16%) in non-Ph-like cases. Notably, all the 7 PAX5t were found in the Ph-like group. EFS (p<0·001) and OS (p=0·009) analyses according to the fusion classes reveal statistical significance in Ph-like cases, classified accordingly to different class fusions, such as ABL/JAK-class, PAX5-class, other fusions and negative cases after NGS analysis. In fact, the PAX5t cases have a poor EFS, similar to ABL/JAK-class patients (53% and 50%, respectively) and lower than Ph-like cases negative for any fusion gene (78%). Moreover, PAX5t OS was inferior compared to ABL/JAK-class and negative cases (about 53% vs. 83·3% and 88·9%).

These features of PAX5t cases prompted us to explore the feasibility of targeting this aberration. By applying the NetBID2 data-driven network interference algorithms, we constructed the BCP-ALL interactome and identified the hub drivers' activity (DA) based on network gene interaction on either PAX5 translocated patients versus all BCP-ALL excluding Ph-like or versus Ph-like. We also assessed a Differential Expression (DE) of genes belonging to these pathways. Interestingly, in the top NetBID2 drivers we identified the LCK signaling factor showing a significantly higher activity in PAX5t compared to other BCP-ALL subgroups. Furthermore, analysis of LCK activity in BCP-ALL patients with known PAX5 status revealed that it is an exclusive signature of PAX5t cases. Moreover, 22 genes resulted with a positive driver activity in PAX5t vs. Ph-like (p<0.05) and bioinformatic analysis to identify drug approved interactions found 7 targetable genes, including LCK.

As previously showed by us, we demonstrated the efficacy of the LCK-inhibitor Nintedanib, as single agent or in combination with conventional chemotherapy, both ex-vivo and in a patient-derived xenograft model, showing a synergistic effect with dexamethasone. We herewith applied a wider ex-vivo drug screening with 174 FDA-approved drugs, by comparative cellular viability of different sub-groups of leukemia PAX5t samples and control-B-lymphoblastoid cell lines (CTR-B-LCSs) in addition to NALL-1 cell lines (carrying PAX5/ETV6 fusion), measured by ATP-Glo based luminescent based assay after exposure of the depicted drugs, whereas Staurosporin was taken as a positive control. Drug sensitivity scores (DSS) are plotted as a clustered heat map, followed by unsupervised hierarchical clustering. From the drug screening, we selected Dasatinib, Bosutinib and Foretinib, known to be among the top 10 most potent LCK ligands. Here we demonstrated their efficacy in PAX5t cases by evaluating the differential DSS, i.e., by avoiding toxicity in CTR-B-LCSs.

This study provides new insights in the pathogenic mechanisms of poor risk Ph-like leukemia and identifies a potential novel therapy for targeting the PAX5t group. Moreover, in general this study opens the scenario to target LCK kinase activity in further BCP-ALL subgroups.

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