Abstract PR005: Investigating mechanisms of resistance in Philadelphia chromosome-like acute lymphoblastic leukemia
Marieke Rozendaal, Louise Laramée, Aurelien Choblet, Maria Lucia. Marquez Chopite, Thai Hoa TranAbstract
Background:
Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is characterized by targetable kinase fusions with different tyrosine kinase inhibitors (TKIs) currently being investigated in clinical trials. While kinase domain (KD) mutations represent the primary mechanism of acquired resistance in Philadelphia chromosome-positive ALL, the mechanisms of resistance in Ph-like ALL remain largely unknown and warrant investigation.
Methods:
We conducted in vitro saturation mutagenesis screens to identify clinically-relevant TKI-resistant KD mutations in RCSD1::ABL2, SSBP2::CSF1R, PAX5::JAK2 and ETV6::NTRK3 Ph-like ALL. In brief, each plasmid containing the kinase fusion was propagated into DNA-repair-deficient E. Coli strain XL-1 Red to generate a library of random mutants. Mutagenized plasmid was transfected into 293T cells and viral supernatants were used to infect Ba/F3 cells. Transduced Ba/F3 cells were plated in 1.2% Bacto-agar and exposed to different TKIs. Genomic DNA from IL-3 independent and TKI-resistant colonies was isolated for KD mutational analysis. The relative sensitivity/resistance of identified KD mutations to different TKIs were assessed via CellTiter-Glo cell viability assays and PhosphoFlow assays.
Results:
The majority of TKI-resistant colonies identified in our in vitro screens using engineered Ba/F3 cells expressing RCSD1::ABL2, SSBP2::CSF1R and ETV6::NTRK3 harbored KD mutations. The most frequent KD mutations in RCSD1::ABL2 to 5μM imatinib were Y299H (64/85 colonies) or T361I (13/85 colonies); and the most common KD mutations to 25 nM dasatinib were ABL2 T361I (12/15 colonies) and Y363C (3/15 colonies). For SSBP2::CSF1R, the most frequent KD mutations to 5μM imatinib were CSF1R D802G/N (34/47 colonies) or T663M (3/47 colonies); and Y571C (13/22 colonies) or T663M (1/22 colonies) to 25 nM dasatinib. The most frequent resistant mutations in ETV6::NTRK3 to 500 nM larotrectinib were NTRK3 G623R/E (37/98 colonies) or F617L/S (42/98 colonies) and G623R/E (66/123 colonies) or F617L/S (21/123 colonies) to 25 nM selitrectinib. In contrast, only 2 JAK2 KD mutations (L884P and K999I) were detected among 41 PAX5::JAK2 resistant colonies to 250 nM ruxolitinib. In order to assess the relative sensitivity of KD mutations to different clinically-relevant TKIs, we then engineered Ba/F3 cells expressing the most frequently identified RCSD1::ABL2, SSBP2::CSF1R and ETV6::NTRK3 KD mutations from our in vitro screens. Using cell viability assays, we found significantly higher IC50 values for specific TKIs in mutant expressing cells vs. their wild-type counterparts. Furthermore, we showed decreased inhibition of downstream phosphoproteins in Ba/F3 cells harboring KD-mutated RCSD1::ABL2 or SSBP2::CSF1R to specific TKIs compared to their wild-type counterparts.
Conclusion:
Acquisition of KD mutations represents the predominant mechanism of acquired resistance in RCSD1::ABL2, SSBP2::CSF1R and ETV6::NTRK3 kinase fusions, whereas kinase-independent mechanisms are likely driving resistance in PAX5::JAK2.
Citation Format:
Marieke Rozendaal, Louise Laramée, Aurelien Choblet, Maria Lucia. Marquez Chopite, Thai Hoa Tran. Investigating mechanisms of resistance in Philadelphia chromosome-like acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Bridging Discovery and Clinical Impact in Pediatric Cancer; 2026 Sep 22-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(18_Suppl_1):Abstract nr PR005.