GNB1 K89M Drives TKI Resistance in ETV6-ABL1-Positive Leukemia

Leukemias harboring ETV6-ABL1 fusion represent a rare prognostically unfavorable subgroup of hematological malignancies. To a great extent they share biological and clinical characteristics with more common BCR-ABL1+ leukemias. Both fusion genes encode constitutively activated aberrant tyrosine kinases which can be targeted by specific tyrosine kinase inhibitors (TKI). The efficiency of TKI to inhibit ETV6-ABL1 kinase activity was proven in vitro and substantiated their use in clinical practice. However, there is still a lack of knowledge on potential induction and mechanisms of TKI resistance in ETV6-ABL1+ malignancies.

To address this issue we established a multi-TKI resistant ETV6-ABL1+ lymphoblastic cell line via a long term cultivation with TKI imatinib mesylate (IM) and investigated the mechanisms of the acquired TKI resistance. We excluded the most common mechanisms known from BCR-ABL1+ leukemias (kinase domain mutations, genomic amplification and enhanced kinase expression/activation) as causes of resistance in our model. Importantly, using shRNA mediated silencing we showed that unlike their sensitive counterparts, the resistant cells did not respond to the ETV6-ABL1 knockdown by growth suppression or cell death and, therefore, became independent on pro-survival signaling from the chimeric kinase.

To further elucidate molecular mechanisms underlying the TKI resistance, we compared biological profiles of resistant and parental sensitive cells. Molecular changes potentially associated with the TKI resistance were analyzed via genomic (single nucleotide polymorphism array, whole exome sequencing), gene expression and proteomic profiling. We integrated data from all platforms and identified K89M mutation in GNB1 gene as the top candidate causative lesion. A mutation affecting the same GNB1 codon, K89E, was recently identified in a blastic plasmacytoid dendritic cell neoplasm by Yoda et al. (Nature Medicine, 2015) and was shown to result in the upregulation of PI3K/Akt/mTOR and MAPK pathways signaling. Interestingly, Yoda et al. also identified functionally similar mutations of GNB1 and GNB2 in several TKI-resistant primary malignancies including a single case with BCR-ABL1+ leukemia.

In accordance with these findings we showed that unlike the parental sensitive cells, the resistant ETV6/ABL1+ cells were able to restore PI3K/Akt/mTOR and p44/MAPK signaling after IM treatment.

To further confirm the key role of GNB1 K89M mutation in our TKI resistance model we transduced the parental sensitive ETV6/ABL1+ cell line with wild type (WT) GNB1, GNB1-K89M, GNB1-K89E or with empty vector, and studied the impact on TKI sensitivity and kinase signaling. While the cells transduced with WT-GNB1 or with empty vector remained sensitive to TKI, the cells transduced with mutated GNB1 (both K89E and K89M)became resistant to IM, nilotinib, dasatinib and ponatinib. Similarly, while the repeated application of IM resulted in cell growth arrest and cell death in the cells with WT-GNB1 and empty vector, the cells transduced with mutated GNB1 showed only subtle changes in proliferation and viability. Administration of IM resulted in the inhibition of ETV6-ABL1-mediated signaling in all cell lines as demonstrated by diminished phosphorylation of the ETV6-ABL1 direct substrate - CRKL adaptor protein. However, while the IM induced a prolonged inhibition of p44/MAPK and PI3K/Akt/mTOR pathway signaling in the cells with WT-GNB1 and with empty vector, the cells transduced by mutated GNB1 (both K89E and K89M) were able to restore activation of both pathways within 24 hours after IM treatment (similarly to the original multi-TKI resistant cell line).

In summary, we showed that the long-term IM treatment of ETV6-ABL1+ leukemia resulted in a gain of TKI-resistance which was driven by the acquired GNB1 mutation activating pro-proliferative and pro-survival signaling, independent on the ETV6-ABL1 kinase activity. Importantly, leukemic cells with mutated GNB1 were resistant not only to IM, but also to the next generation TKI.

The frequency of GNB1/GNB2 mutations in primary ETV6-ABL1+ and BCR-ABL1+ leukemias and its clinical relevance is to be determined in the study currently initiated within an international collaboration.

Supported by grants GACR P302/12/G10 and GAUK 554214.