Retinoic Acid Therapy Resistance Progresses from Unilineage to Bilineage in HL-60 Leukemic Blasts

<div><p>Emergent resistance can be progressive and driven by global signaling aberrations. All-<i>trans</i> retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10–20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D₃ (D₃). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D₃-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or “precommitment” phase) or subsequently (the late or “lineage-commitment” phase). HL-60 were treated with RA or D₃ for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D₃ was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D₃ responsiveness (unilineage maturation block), followed by bilineage maturation block and progressive signaling defects, notably the reduced expression of Vav1, Fgr, and c-Raf.</p></div

Percentage of cells expressing CD11b for WT HL-60 and R38+ and R38- RA-resistant HL-60 cells.

<p>D₃ increases the differentiation marker CD11b in RA-resistant HL-60 cell lines. (A) 48 h CD11b expression after sequential treatment with two inducing agents during the precommitment and lineage-commitment phases (RA/RA, RA/D₃, RA/-, D₃/D₃, D₃/RA, and D₃/-). (B) 72 h CD11b expression (continuation of treatment with second inducing agent). CD11b expression was assessed by flow cytometry (with APC-conjugated antibody) at 48 and 72 h after first treatment initialization. Gates to determine percent increase of expression with treatment were set to exclude 95% of the control population. For clarity, p-values are not indicated above bars due to the existence of multivariate comparison between cell lines, treatments, and time. However, p-values of interest are mentioned specifically in the main text.</p

6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells

<div><p>6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.</p></div

Percentage of cells in the G1/G0 phase for WT HL-60 and R38+ and R38- RA-resistant HL-60 cells.

<p>D₃ rescued G1/G0 arrest in R38+, and to a lesser degree in R38-, when added in the lineage-commitment stage. (A) 48 h G1/G0 arrest after sequential treatment with two inducing agents during the precommitment and lineage-commitment phases (RA/RA, RA/D₃, RA/-, D₃/D₃, D₃/RA, and D₃/-). (B) 72 h G1/G0 arrest (continuation of treatment with a second inducing agent). Untreated control gates were set to 45% G1/G0, 35% S and 20% G2/M. For clarity, p-values are not indicated above bars due to the existence of multivariate comparison between cell lines, treatments, and time. However, p-values of interest are mentioned specifically in the main text.</p

Quantified 48-60 and R38+ and R38− RA-resistant HL-60 cells.

<p>Repeat 48<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098929#pone-0098929-g006" target="_blank">Figure 6</a> were quantified using ImageJ and average fold change from control was graphed in GraphPad. Error bars represent standard error. Note that the fold change axis scale may differ for each bar graph.</p

Signaling protein expression for WT HL-60 and R38+ and R38− RA-resistant HL-60 cells.

<p>Individual Western blots of whole cell lysates are representative of at least three repeats. GAPDH loading controls were also performed on each individual blot to ensure even loading (not shown). WT HL-60 samples are indicated by WT, R38+ samples are indicated by + and R38− samples are indicated by ---. (A) 24 h protein expression after treatment with a single inducer (RA or D₃) during the precommitment phase. (B) 48 h protein expression after sequential treatment with two inducing agents during the precommitment and lineage-commitment phases (RA/RA, RA/D₃, RA/-, D₃/D₃, D₃/RA, and D₃/-). Quantified blot data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098929#pone-0098929-g007" target="_blank">Figure 7</a>.</p

Clustering analysis.

<p>(A) Agglomerative hierarchical clustering analysis was performed on average quantified Western blot data (signaling protein data only) using Cluster 3.0 and visualized with TreeView. The distance metric was Pearson's correlation coefficient. In the diagram, RR  =  RA/RA, RD  =  RA/D₃, R-  =  RA/-, DD  =  D₃/D₃, DR  =  D₃/RA, and D-  =  D₃/-. (B-E). Clustering analysis across all treatment cases and all results (cytometry phenotyping data and Western blot signaling protein data) was performed using SYSTAT 8.0. In B-E, CTD refers to pS289/296/301c-Raf. (B) Clustering for WT HL-60. (C) Clustering for R38+. (D) Clustering for R38−. (E) WT and R38+ cluster more closely to each other than to R38− HL-60 cells.</p

Percentage of cells exhibiting inducible respiratory burst for WT HL-60 and R38+ and R38- RA-resistant HL-60 cells.

<p>Oxidative metabolism (respiratory burst) at 72 h after sequential treatment with two inducing agents during the precommitment (24 h) and subsequent lineage-commitment phase (RA/RA, RA/D₃, RA/-, D₃/D₃, D₃/RA, and D₃/-). Respiratory burst activity in RA-resistant HL-60 cells was only marginally rescued by D₃ only when present both in precommitment and commitment stages of differentiation. WT HL-60 cells exhibit significant respiratory burst when RA or D₃ is present during the lineage-commitment phase. Gates to determine percent increase of expression with treatment were set to exclude 95% of the DMSO control population.</p

Hierarchical clustering between phenotypic markers (A) and treatments (B).

<p>The phenotype data was subject to hierarchical clustering analysis using the Pearson correlation coefficient as a distance metric and the average linkage method. Distances between clusters (1-Pearson correlation coefficient) are indicated on the x-axis.</p

Correlation analysis of the FICZ + RA-elicited signalsome.

<p>(A) The dendrogram resultant from the hierarchical clustering analysis of the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135668#pone.0135668.g005" target="_blank">Fig 5</a> using the Pearson correlation coefficient as the distance metric and the average linkage method. Distances between clusters (1-Pearson correlation coefficient) are indicated on the x-axis. (B) Pearson correlation coefficient matrix between signaling molecules and phenotypic markers for the control, RA, FICZ+RA, α-NF +RA and β-NF +RA.</p