The Dark Side of NRF2: Upregulation of NRF2 as a Mechanism for Resistance to Imatinib In CML.

Abstract 3401

Chronic myeloid leukemia (CML) is a myeloproliferative disorder that results from a balanced translocation between chromosomes 9 and 22, creating the Philadelphia chromosome, and giving rise to a 210 kd (p210) fusion protein between Bcr and Abl. In addition to conferring anti-apoptotic properties, proliferative advantage and growth factor independence, BCR-ABL is associated with increased reactive oxygen species (ROS), DNA damage and altered repair that are proposed to play a role in genomic instability and disease progression. Imatinib mesylate, and other tyrosine kinase inhibitors (TKI)s, induce a substantial clinical response in patients with BCR-ABL-positive CML. However, resistance to TKIs through acquisition of mutations in BCR-ABL or BCR-ABL independent mechanisms is an increasing clinical problem. Thus, delineating mechanisms for disease resistance may allow the development of novel therapies for TKI-refractory disease.

Cells deal with high levels of intracellular ROS by transcriptionally activating a number of anti-oxidant pathways. In particular, acute increases in ROS levels activate the Nrf2 pathway. Since NRF2 is known to transcriptionally activate drug resistance genes, and some anti-oxidant genes are transcriptionally increased in imatinib-resistant cells, we characterized the role of NRF2 in imatinib resistance in CML. We studied two imatinib-resistant (IR) BCR-ABL-positive cell lines, P210Mo7eIR with no detectable mutations in BCR-ABL and Baf3P210IR with BCR-ABL mutation T315I, derived from isogenic imatinib-sensitive (IS; P210Mo7e and Baf3P210) cells and parental controls (Mo7e and Baf3), and demonstrated that one of the IR cell lines, P210Mo7eIR, had very high steady state levels of Nrf2 (>10-fold) compared with isogenic controls. In addition, real-time PCR studies of NRF2 anti-oxidant target transcripts including, NQO1, NQO2, HMO1, GCLM and GCLC in P210Mo7eIR cells showed that they significantly increased compared with control cells. Importantly, to determine if NRF2 plays a role in the sensitivity of P210Mo7eIR cells to imatinib, we performed siRNA knockdown of NRF2 followed by growth of these cells in the presence of imatinib, and find that downregulation of NRF2 resensitizes P210Mo7eIR cells to imatinib, dramatically increasing cell death, compared with that of siRNA scrambled controls. Furthermore, inhibition of NQO1/2 leads to sensitivity of P210Mo7eIR cells to imatinib. Conversely, overexpression of NRF2 cDNA constructs in imatinib sensitive P210Mo7e renders these cells resistant to imatinib, compared with mutated NRF2 and vehicle controls. To determine whether this mechanism is operative in primary samples from IR cells, we examined 5 IS and 5 IR CML BM samples for steady state levels of NRF2 protein and transcript levels of NRF2 target genes. One of 5 IR CML samples with no BCR-ABL mutations and no IS CML samples had significantly increased NRF2 levels and target anti-oxidant activity. These data suggest that significant upregulation of Nrf2 acute stress pathways may be an alternative mechanism for TKI resistance that does not involves mutations in BCR-ABL. Thus, inhibition of NRF2 itself or its downstream targets may be important in therapeutic strategies in this sub group of TKI resistant CML.