Fig 1.
(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-ABLWT, or BCR-ABLT315I 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-ABLT315I, or BCR-ABLkinase-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.