Abstract PR02: Investigating the use of tyrosine kinase inhibitors in Ph-like ALL.

Ph-like or BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (B-ALL) is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of the lymphoid transcription factor IKZF1, and poor outcome. The prevalence of Ph-like ALL rises with age from 10% in standard risk childhood ALL to over 25% in young adults with ALL. We recently performed genomic profiling of over 1700 B-ALL cases and next-generation sequencing of over 150 Ph-like ALL cases and identified alterations targeting 18 kinase or cytokine receptor genes, including rearrangement, sequence mutation and copy number alterations. The aim of this study was to determine the role of these kinase alterations in the development of Ph-like B-ALL, to define the signaling pathways that are activated, and to assess sensitivity to tyrosine kinase inhibitors (TKIs). To investigate the functional role of kinase fusions in Ph-like ALL, we introduced RCSD1-ABL2, SSBP2-CSF1R or PAX5-JAK2 into interleukin-7 dependent Arf-/- mouse pre-B cells expressing the dominant negative isoform of IKAROS, IK6. ABL2 (Abelson-related gene, ARG), a homolog of ABL1, has been implicated in the development of solid tumors, but rarely identified as a rearrangement partner in ALL. CSF1R (encoding the macrophage colony stimulating receptor) regulates the survival and differentiation of macrophages, and is not normally expressed in lymphoid cells. Each fusion tested conferred cytokine independent growth in vitro. Furthermore, recipient mice transplanted with pre-B cells expressing RCSD1-ABL2 or SSBP2-CSF1R developed ALL with a pre-B immunophenotype (CD43+, B220+, CD19+, BP-1+ and IgM-) and a median latency of 36 and 40 days respectively, implicating these kinases for the first time in the development of B-ALL. To assess activation of kinase signaling pathways, we performed phosphoflow cytometry analysis of Arf-/- pre-B cells or human leukemic cells harvested from xenografted mice expressing ETV6-ABL1, RANBP2-ABL1, PAG1-ABL2, RCSD1-ABL2, SSBP2-CSF1R, IGH-EPOR, ATF7IP-JAK2 and PAX5-JAK2. In both models we observed distinct patterns of signaling pathway activation and TKI sensitivity between the different fusions. Cells expressing ABL1-class kinase fusions (ABL1, ABL2, CSF1R and PDGFRB) showed activation of pSTAT5 that was inhibited with imatinib or dasatinib. Phosphorylation of CRKL, a known target of ABL1 and ABL2, was only observed in cells expressing these fusions. In contrast, leukemic cells harboring ATF7IP-JAK2, PAX5-JAK2 or IGH-EPOR showed activation of pSTAT5 that was only attenuated with the JAK2 inhibitor, ruxolitinib. This TKI response profile was confirmed by cytotoxicity assays, with ABL-class fusions being sensitive to dasatinib (IC50 range 1-2nM), whilst cases with ATF7IP-JAK2 and EPOR rearrangement responded to ruxolitinib, with IC50 values of 500nM and 850nM respectively. To further examine the utility of TKI therapy in Ph-like B-ALL, we investigated the in vivo efficacy of dasatinib in four xenograft models of ETV6-ABL1, RCSD1-ABL2, PAG1-ABL2 or SSBP2-CSF1R ALL. Dasatinib (20mg/kg/day p.o) or vehicle were commenced at measurable engraftment in peripheral blood (human CD45+/CD19+ cells >5%) and treatment ceased when leukemic burden exceeded 70% in vehicle treated mice. In all cases, dasatinib significantly reduced the leukemic burden and splenic weight compared to vehicle treated mice. Furthermore, phosphorylation of STAT5 was attenuated in cells harvested from dasatinib-treated mice compared to vehicle, indicating on target effect of the drug. Together, these data provide the first mouse models of Ph-like B-ALL, and show that despite the diversity of alterations identified, they activate distinct signaling pathways that can be targeted with a limited panel of FDA-approved agents. Prospective clinical trials that identify and direct Ph-like ALL patients to the appropriate TKI therapy may improve the treatment outcome for this high-risk subtype. Citation Format: Kathryn G. Roberts, Yung-Li Yang, Debbie Payne-Turner, Richard C. Harvey, I-Ming Chen, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl L. Willman, Stephen P. Hunger, Charles G. Mullighan. Investigating the use of tyrosine kinase inhibitors in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR02.