Rxra Overexpression Improves the Sensitivity to Imatinib In-Vitro/in-Vivo By Disrupting the Oxidative Capacity of Chronic Myeloid Leukemia Cells

Most patients who receive BCR-ABL1 kinase inhibitor therapy for CML see profound molecular responses. However, due to drug resistance clones and leukemic stem cells, not all patients benefit from TKI therapy. We have previously reported the dysregulated expression of nuclear hormone receptors in CML cell lines based on the sensitivity to Imatinib (IM) (Karathedath et al., ASH annual meeting abstracts 2013). We observed Retinoid X receptor alpha (RXRA) expression to be markedly down-regulated in CML cell lines (KCL22, LAMA84) that were inherently resistant to IM compared to IM-sensitive cell lines (EM2, KU812).

Here, we undertook a series of studies to decipher the role of RXRA in mediating sensitivity to IM. Initially, we stably overexpressed RXRA in the LAMA84 cell line with the lowest expression of RXRA. Post-RXRA overexpression (OE), we observed a significant reduction in cell proliferation in a time-dependent manner compared to empty vector (EV) transduced cells as assessed by the trypan blue exclusion (35.7 Vs. 25.9 hours). Next, we evaluated the sensitivity to IM by apoptosis assay, and RXRA OE cells were significantly sensitized to IM (58% vs. 21%. P<0.01). We then assessed the sensitivity to other TKIs nilotinib and dasatinib in the RXRA OE cells and found increased sensitivity. We evaluated the expression of major pro-survival down streaming kinase signaling pathways of BCR-ABL (AKT, CRKL, and STAT5) in the RXRA OE cells post IM exposure.

Interestingly, we observed diminished expression of p-CRKL in RXRA OE cells compared to EV, and further treatment with IM completely abolished the expression of p-AKT, p-STAT5, and p-CRKL in the RXRA OE cells vs. EV. It is well established that the leukemic cells show a metabolic shift towards oxidative phosphorylation to elicit therapy resistance; therefore, we performed extracellular flux analysis (Seahorse) to identify metabolic alteration post-RXRA OE. Interestingly, RXRA OE caused a significant reduction in the basal OCR rates, spare respiratory capacity, and ATP production compared to EV. We also measured the mitochondrial membrane potential (MMP) using JC-1 dye and found that reduced MMP in RXRA OE cells compared to EV. This suggests RXRA OE reduced the oxidative capacity of the LAMA84 cell line and thereby increased the sensitivity to TKI. We performed xenograft transplantation of the RXRA OE/EV cells and assessed the engraftment capacity and sensitivity to IM in-vivo. RXRA OE leads to reduced engraftment in the bone marrow and improved survival compared to EV transplanted mice (Median survival 50 vs 37 days). Further RXRA OE also led to an increased response to IM in-vivo, which resulted in better survival than IM-treated EV mice (Median survival 57 vs 48 days).

We probed for RXRA expression between CML CD34⁺ cells and health donor CD34⁺ cells and found significantly reduced expression. Next we assessed the effect of clinically available ligands specific to RXRA (Acitretin,Bexarotene and 9-cis-Retinoic acid) on CML CD34⁺ cells in combination with IM. Intriguingly, pretreament of CML CD34⁺cells with RXRA ligands followed by IM treatment, markedly reduced the viability and colongenic capacity of these cells compared to IM treatment alone.

In conclusion, we have identified that enforced expression of RXRA in CML cells improved sensitivity to TKI in-vitro and in-vivo. The profound sensitivity to TKI was found to be RXRA's disruptive effect on the OXPHOS metabolism. Interestingly RXRA OE reduced the engraftment potential of CML cells in-vivo, indicating a mechanism for altering the stemness capacity of CML cells. Further work is ongoing to elucidate the molecular mechanisms orchestrated by RXRA in CML cells.

No relevant conflicts of interest to declare.