Towards Personalized Therapy in Pediatric Acute Lymphoblastic Leukemia; Ras Mutations and Prednisolone Resistance
Authors: Ariës Ingrid M. , van den Dungen Rosanna , Koudijs Marco , Cuppen Edwin , Voest Emile E. , Molenaar Jan J , Caron Huib N. , Pieters Rob , Den Boer Monique L. 
Published: October 2019

Abstract
Background:
The 5-year event-free survival of pediatric precursor-B acute lymphoblastic leukemia (BCP-ALL) has currently reached 80-90%. Targeted drugs are warranted to cure the remaining 10-20%. Prednisolone has been shown a pivotal drug and resistance remains an adverse prognostic factor in BCP-ALL. This study aimed to find new druggable targets that may modulate prednisolone resistance.
Methods:
Leukemic cells from children with newly diagnosed ALL were isolated from bone marrow aspirates, only samples with ≥ 90% leukemic blasts were used. Protein expression of patient leukemic cells before and after exposure to prednisolone was analyzed by reverse phase protein arrays. Deep sequencing of 26 BCP-ALL patients was performed on the Ion Torrent PGM using the Ion AmpliSeq Cancer Panel. Cytotoxicity of primary patients’ cells to Trametinib, Sorafenib, Crizotinib and AS1517499 in combination with or without prednisolone was determined by the MTT assay.
Results:
We compared phosphorylation levels of 18 key-signaling proteins in leukemic cells between in vitroprednisolone sensitive and resistant ALL patients. Basal phosphorylation levels of these proteins did not differ between prednisolone resistant and sensitive patients’ cells. Exposure to prednisolone for 48h induced discrepant changes in 7 out of 18 proteins between prednisolone resistant and sensitive cases. Exposure to prednisolone decreased phosphorylation of the RAS-RAF-MEK pathway, STAT6 and c-Met in prednisolone sensitive patients cells but not in resistant patients. These findings prompted us to investigate the presence of RAS-pathway and c-Met activating mutations. We investigated hotspot regions of mutations in the protein coding domains of BRAF, NRAS, HRAS, KRAS, PTPN11, FLT3 and c-Met with Ion Torrent deep sequencing (1000x read depth) in pediatric BCP-ALL patients. We observed that 42% of the BCP-ALL patients tested carried activating NRAS and/or KRAS, FLT3 or PTPN11 mutations, of which 6 cases harbored ≥2 RAS-mutations predominantly associated with presence of multiple minor clones (<30%). Interestingly, we detected more often RAS activating mutations in prednisolone resistant compared to sensitive patients (62% vs. 23%). We next evaluated the prednisolone sensitizing properties of inhibitors directed against MEK1/2 (Trametinib), BRAF (Sorafenib), STAT6 (AS1517499) and cMET (Crizotinib) in primary BCP-ALL patients samples. Remarkably, Trametinib was impressively potent in reversing prednisolone resistance eradicating all leukemic cells of the NRAS/KRAS-mutated BCP-ALL patients. In one NRAS/KRAS-mutated case Sorafenib but not Trametinib sensitized to prednisolone significantly.
Conclusion:
We observed a relative high frequency of RAS-pathway mutations in children with newly diagnosed BCP-ALL (42%). These mutations were more frequently found in prednisolone resistant cases compared to sensitive cases. Highly resistant leukemic patients’ cells harbouring K/N-RAS mutations were sensitized to prednisolone in the presence of the MEK1/2 inhibitor Trametinib or the BRAF inhibitor Sorafenib. Our data suggest that BCP-ALL patients with RAS-activating mutations may benefit from treatment with Trametinib/Sorafenib and strongly argue to implement these inhibitors in combination with prednisone.

Disclosures
No relevant conflicts of interest to declare.