Abstract 647: NF-kB associates with tyrosine kinase inhibitor resistance in chronic myeloid leukemia
Authors: Rubio Andres J. , Olivas Idaly M. , Bencomo Alfonso E. , Gonzalez Mayra A. , Lara Joshua J. , Ellwood Rebecca , Ripoll-Fiol Carme , Nteliopoulos Georgios , Reid Alistair , Milojkovic Dragana , Apperley Jane , Sorouri-Khorashad Jamshid , Eiring Anna M. 
Published: September 2020

Abstract
Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are effective at eliminating most BCR-ABL1+ cells in chronic myeloid leukemia (CML), but do not target CML leukemic stem cells (LSCs), which are independent of BCR-ABL1 kinase activity. Our previous work showed that BCR-ABL1-independent resistance is largely driven by STAT3 (Eiring et al. Leukemia 2015). To understand how STAT3 contributes to TKI resistance, we performed RNA sequencing on TKI-sensitive K562-S cells versus TKI-resistant K562-R cells, which demonstrate kinase-independent resistance. Surprisingly, gene set enrichment analysis did not reveal STAT3-mediated transcription (p=1.0), but was reminiscent of TNFα signaling via NF-κB (p=0.024). Nucleocytoplasmic fractionation revealed higher levels of phospho-NF-κB in the nucleus of K562-R vs. K562-S controls, and in CD34+ progenitors from TKI-resistant CML patients (n=3) compared to TKI responders (n=2) and normal individuals (n=2). These data suggest NF-κB may be driving the transcriptional signature of TKI resistance, and implicate non-canonical functions for STAT3. To confirm increased NF-κB transcriptional activity in TKI resistance, K562-S and K562-R cells were transduced with an NF-κB luciferase reporter construct or a scrambled control. K562-R cells demonstrated increased NF-κB reporter activity compared to K562-S cells. To assess whether STAT3 activates NF-κB, reporter cells were co-infected with an inducible lentiviral shRNA targeting STAT3 (shSTAT3). STAT3 knockdown decreased NF-κB reporter activity in TKI-resistant cells, but increased reporter activity in TKI-sensitive controls. These data suggest that STAT3 may cooperate with NF-κB in the setting of TKI resistance. NF-κB and STAT3 have been shown to cooperatively bind to gene promoters of cytokines, specifically IL-6. ELISA assays demonstrated that K562-R cells produce autonomous IL-6, but not TNFα. Since IL-6 is a potent activator of STAT3, we treated TKI-resistant cells with the IL-6 receptor inhibitor, tocilizumab (100 and 1000 ng/ml). As expected, tocilizumab treatment reduced STAT3 Y705 phosphorylation as assessed by immunoblot analysis, but surprisingly had no effect on survival or NF-κB reporter activity in K562-R cells. These data suggest that STAT3 phosphorylation is not required for survival or NF-κB activation in TKI resistance. The role of unphosphorylated STAT3 in TKI resistance is currently being explored. We correlated our RNA sequencing data in TKI-resistant cell lines with microarray data in TKI-resistant patient samples (McWeeney et al. Blood 2010), identifying upregulation of the proteasome components, PSMD1 and PSMD3. qRT-PCR confirmed upregulation of PSMD3 but not PSMD1 in TKI resistance, and only the PSMD3 promoter was bound by NF-κB as assessed by ChIP. Lentiviral shRNAs targeting PSMD3 (shPSMD3) reduced colony formation of K562-S and K562-R cells, which correlated with induction of apoptosis, an increase of global protein ubiquitination, and decreased NF-κB activation, with little effects observed in normal cord blood. Altogether, our data implicate a positive feedback loop involving NF-κB and PSMD3 in TKI resistance and suggest that PSMD3 may be a novel target for treatment of TKI-resistant CML.
Citation Format: Andres J. Rubio, Idaly M. Olivas, Alfonso E. Bencomo, Mayra A. Gonzalez, Joshua J. Lara, Rebecca Ellwood, Carme Ripoll-Fiol, Georgios Nteliopoulos, Alistair Reid, Dragana Milojkovic, Jane Apperley, Jamshid Sorouri-Khorashad, Anna M. Eiring. NF-kB associates with tyrosine kinase inhibitor resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 647.