Cholesterol esterification inhibition and imatinib treatment synergistically inhibit growth of BCR-ABL mutation-independent resistant chronic myelogenous leukemia

Abstract

Since the advent of tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, and dasatinib, chronic myelogenous leukemia (CML) prognosis has improved greatly. However, ~30-40% of patients develop resistance to imatinib therapy. Although most resistance is caused by mutations in the BCR-ABL kinase domain, 50-85% of these patients develop resistance in the absence of new mutations. In these cases, targeting other pathways may be needed to regain clinical response. Using label-free Raman spectromicroscopy, we evaluated a number of leukemia cell lines and discovered an aberrant accumulation of cholesteryl ester (CE) in CML, which was found to be a result of BCR-ABL kinase activity. CE accumulation in CML was found to be a cancer-specific phenomenon as untransformed cells did not accumulate CE. Blocking cholesterol esterification with avasimibe, a potent inhibitor of acyl-CoA cholesterol acyltransferase 1 (ACAT-1), significantly suppressed CML cell proliferation in Ba/F3 cells with the BCR-ABLT315I mutation and in K562 cells rendered imatinib resistant without mutations in the BCR-ABL kinase domain (K562R cells). Furthermore, the combination of avasimibe and imatinib caused a profound synergistic inhibition of cell proliferation in K562R cells, but not in Ba/F3T315I. This synergistic effect was confirmed in a K562R xenograft mouse model. Analysis of primary cells from a BCR-ABL mutation-independent imatinib resistant patient by mass cytometry suggested that the synergy may be due to downregulation of the MAPK pathway by avasimibe, which sensitized the CML cells to imatinib treatment. Collectively, these data demonstrate a novel strategy for overcoming BCR-ABL mutation-independent TKI resistance in CML.

Fig 1. CE accumulation in CML.

(a) Raman Spectra acquired from LDs of four leukemia cell lines, including RCH-ACV (ALL), K562 (CML), Kasumi-2 (ALL), and MOLM14 (AML).(b) Quantification of CE% out of total lipids in LDs in four leukemia cell lines. (c) Representative SRS images of Ba/F3 Cells overexpressing empty vector treated with or without IL-3, BCR-ABL^WT, or BCR-ABL^T315I cells. (d) Quantification of LD amount by area fraction analysis from SRS images. (e) Raman spectra of LDs in 32D cells overexpressing empty vector, BCR-ABL, BCR-ABL^T315I, or BCR-ABL^kinase-dead. (f) Quantification of CE% in LDs from 32D cells. For quantitative analysis, all the results are shown as means + SEM, n = 4~6. Two-way student t test was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 2. Imatinib and avasimibe show a significant synergy in inhibiting viability of K562R cells.

(a) Representative SRS images of K562 and K562R cells. (b) Quantification of CE% in LDs of K562 and K562R cells. The results are shown as means + SEM, n = 6. Two-way student t test was used for statistical analysis; n.s. indicates no significance. (c) 3D contour plot with colormap (d) linear plot of K562R cells treated with imatinib, avasimibe, or combination of imatinib and avasimibe at a molar concentration ratio of 1: 10 (IM: Ava) for 72 hours. (e) 3D contour plot with colormap (f) linear plot of K562 cells treated with imatinib, avasimibe, or combination of imatinib and avasimibe at a molar concentration ratio of 1: 10 (IM: Ava) for 72 hours.Viability was measured using the Cell Titer Glo assay, with all viabilities normalized to no inhibitor treatment group. The results are shown as means + SEM, n = 3.

Fig 3. Imatinib and avasimibe synergistically suppress K562R xenograft tumor growth in athymic nude mice.

(a) Tumor volume (mm³) measured by a caliper over the course of treatment for the four treatment groups. (b) Body weight (g) of the mice throughout the course of treatment. The results are shown as means + SEM, n = 8. One-way student t test was used for statistical analysis, * p < 0.05, *** p < 0.001

Fig 4. Avasimibe downregulates the MAPK pathway.

(a) Contour biaxials of pS6 (y-axis) and pCREB (x-axis) gated on pCRKL+ cells collected by CyTOF in K562R cells. Cells were treated for 0 or 4 hours with10μM avasimibe. (b) Effect of 30 minute 5μM imatinib treatment on sensitive and resistant patients normalized to the basal condition on the pooled MAPK pathway proteins together (All) and individually. Error bars represent standard deviation of fold change in each group of patients. T-tests were conducted comparing fold change in resistant patients to sensitive patients (p-values shown) and for general reduction in phosphorylation (*- p<0.05) (c-d) Bar graphs showing fold change of median protein expression after 10μM avasimibe and combination therapy normalized to 5μM imatinib in resistant and sensitive CML patients (n = 4 for all groups except SCML3 was omitted in the pERK group because zero pERK signal was observed). Imatinib treatment was for thirty minutes while avasimibe treatment was for four hours. (e) Heatmaps of CyTOF screens of non-lymphoid CD34⁺ CD38⁻ cells from cryopreserved bone marrow from a normal patient (top), cryopreserved bulk PBMCs from an imatinib-sensitive patient (middle), and cryopreserved bulk PBMCs from an imatinib-resistant patient without a BCR-ABL kinase domain mutation (bottom). Cells were treated with no inhibitor, 1μM imatinib, 10μM avasimibe, or imatinib plus avasimibe at the same concentrations. Imatinib stimulation was done for 30 minutes, while avasimibe stimulation was done for four hours. Heatmap tile color represents arcsinh ratio of medians normalized to the basal condition for each patient, see Bendall et al. 2011[23] for details. (f) Biaxials of p-p65/NFκB on the x-axis versus p-p38/MAPK on the y-axis in Lin^- CD34⁺ CD38⁻ collected by CyTOF from the resistant patient. Each plot represents one of the four stimulation conditions: basal (top left), imatinib (top right), avasimibe (bottom left), and imatinib + avasimibe (bottom right). The contour represents cell density.