Comparison between the compound ETP-39010 and other pan-PIMi.

<p>(A) Selectivity profile showing the IC₅₀ values of each of the compounds for the kinase activity of the indicated enzymes. (B) Percentage of inhibition of a panel of unrelated kinases by ETP-39010 and ETP-47551. A similar profile was found for ETP-47551, ETP-47652 and ETP-46638 compounds. (C) Sensitivity of PTCL cell lines to all pan-PIMi. (D) The newly developed pan-PIMi ETP-47551 reduced cell viability in all studied PTCL cell lines (IC₅₀ values calculated after 72 h of treatment are shown). (E) The pan-PIMi ETP-47551 strongly induced apoptosis in a time-dependent manner in all studied PTCL cell lines (*, p<0.05, from comparison with DMSO-treated cells). The percentage of non-viable cells was calculated as Annexin V+/7AAD− plus Annexin V+/7AAD+ cells in the PIMi-treated condition minus the DMSO-treated control. (F) The combination of ALKi + ETP-39010 was highly synergistic only in ALK+ ALCL cell lines, as was (G) the combination of ALKi + ETP-47551 (Combination Index, CI, <1 indicates synergism between the two drugs; CI ≈1 indicates an additive effect; CI>1 indicates antagonism).</p

PIM kinases as potential therapeutic targets in PTCL.

<p>(A) Gene expression profiling of tumoral samples from 38 human PTCL patients compared with 6 reactive lymph nodes (LN) by microarrays revealed a significantly increased expression of <i>PIM1</i> and <i>PIM2</i> genes (FDR<0.05), but not <i>PIM3</i>. The heatmap is shown in the upper panel, and the relative quantification (Log₂ ratio) comparing PIM expression in PTCL <i>versus</i> LN is shown in the lower panel. (B) GSEA ranked all significantly altered genes between PTCL and LN according to its correlation with <i>PIM1</i> or <i>PIM2</i> expression and displayed them in the red-to-blue bar. Each gene belonging to every pathway was interrogated whether it appeared positively (in the red region of the bar) or negatively (in the blue side) correlated. Using this approach GSEA identified a positive and significant correlation between <i>PIM1</i> and <i>PIM2</i> expression and Jak/STAT, NF-κB and IL-2 signaling pathways in the PTCL molecular signature (FDR<0.25). (C) PIM family genes mRNA level was measured by RT-qPCR in eight PTCL cell lines and (D) primary tumoral T cells from 5 Sézary Syndrome patients (SS #1–5), and compared with normal T cells isolated from 3 healthy donors (Control #1–3). The relative RNA amount of PIM has been calculated as a relative quantification, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112148#s2" target="_blank">Methods</a> section (RQ = 2^−ΔCt), normalized with non-tumoral cells: RQ in PTCL/RQ in healthy #3. In both settings, <i>PIM1</i>, and especially <i>PIM2</i>, but not <i>PIM3</i> expression was found to be increased in PTCL. (E) PIM kinase protein basal levels in PTCL cell lines measured by Western blot. PIM1 and PIM2 isoforms are also shown. (F) Distribution of PIM2 protein in a series of tumoral samples from 136 PTCL patients measured by immunohistochemistry. Negative, weakly positive and strongly positive samples were defined by the presence of <5%, 5–20% and >20% positive cells. (G) Distribution of PIM2 protein in the most common PTCL subtypes measured by immunohistochemistry.</p

Synergism between ALK and PIM inhibition in ALCL.

<p>(A) ALK expression was explored by Western blot in 4 PTCL cell lines. (B) IC₅₀ values were measured upon 72 h treatment with the ALKi Crizotinib: ALK+ ALCL cell lines were around 10 times as sensitive to the ALKi as the ALK− cells. (C) Cells were treated for 24 h with IC₅₀ of ALKi and pan-PIMi, alone and combined. The combination of ALKi + PIMi was highly synergistic (Combination Index, CI<1) and strongly enhanced apoptosis in ALK+ ALCL cell lines after 24 h, while this combination was antagonistic in ALK− PTCL cell lines (CI>1) (*, p<0.05 in comparison with DMSO). Data represent Annexin V+/PI− and Annexin V+/PI+ cells in each treatment. Black columns highlight the combined treatment.</p

PIM genetic silencing in PTCL by shRNA.

<p>(A) MyLa cells were infected with lentiviral particles containing the pLKO.1-puro vector with a non-targeting shRNA, the Turbo-GFP gene, or shRNAs for each of the PIM family genes. Cells were maintained under Puromycin selection for 3 weeks. Images were obtained 8 days post-infection with a Nikon Ti Epi-Fluorescence microscope (10X magnification). Green fluorescence was also assessed in the negative (NT sh) and the positive controls by flow cytometry, and percentages of green cells are indicated in the histograms. (B) Triple knockdown efficiency was measured by RT-qPCR and compared with the Non-Targeting shRNA (NTsh). Graphs show the silencing at the mRNA level 15 days after the lentiviral infection. RQ, relative quantification, has been calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112148#s2" target="_blank">Methods</a> section (RQ = 2^−ΔΔCt). (C) Western blots showing the effect of the triple lentiviral infection on PIM protein levels 15 days post-infection (NI, non-infected cells; NTsh, cells infected with the non-targeting shRNA; GFP, cells infected with Turbo-GFP; Triple sh, cells infected with shPIM1+ shPIM2+ shPIM3). (D) These knockdown conditions did not affect cell viability. The percentage of non-viable cells was calculated as Annexin V+/7AAD− plus Annexin V+/7AAD+ cells: (NI, non-infected cells; NTsh, cells infected with the non-targeting shRNA; GFP, cells infected with Turbo-GFP; Triple sh, cells infected with shPIM1+ shPIM2+ shPIM3).</p

Molecular response to the pan-PIMi in PTCL.

<p>(A) The pan-PIMi ETP-39010 reduced phosphorylation of 4E-BP1 in PTCL cell lines. D: DMSO; 5: 5 µM pan-PIMi; 10: 10 µM pan-PIMi. (B) Key effectors of apoptosis, such as Caspase-3 and BCL2, were affected by the pan-PIMi in PTCL cell lines, in a time and dose-dependent manner. D: DMSO; 5: 5 µM pan-PIMi; 10: 10 µM pan-PIMi. (C) Heat-map showing the commonly differentially expressed genes (FDR<0.05) in all PIMi-treated cell lines (10 µM) compared with DMSO-treated cells at each time point. STEM program was used to identify significant genes, and FatiGO recognized the pathways in which they were involved (adjusted p-value<0.05). (D) Amount and pattern of distribution of γH2A.X was tested by Western blot and immunofluorescence, respectively, in MyLa cells treated with 10 µM pan-PIMi for 24 h. Arrows show γH2A.X foci (40X magnification). Images were obtained by a fluorescence microscope (Axio Imager Z1, Zeiss, Oberkochen, Germany). (E) PTCL cell lines were treated with a range of doses of the combination pan-PIMi + Cisplatin for 72 h. Combination Index (<1) showed synergism between both drugs in all studied PTCL cell lines.</p

Simultaneous <i>PIM1+ PIM2+ PIM3</i> genetic silencing in PTCL cell lines by siRNA.

<p>(A) The triple knockdown was carried out in the HH, SU-DHL-1 and MyLa cell lines, using 33 nM siPIM1+33 nM siPIM2+33 nM siPIM3 for 24–72 h. As an average for the 3 PIM genes, the knockdown efficiency, measured by RT-qPCR, was around 70% at the mRNA level 24 h after the microporation (RQ, relative quantification, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112148#pone-0112148-g002" target="_blank">Figure 2</a>), and (B) around 50% at the protein level after 48 h, compared with the Non-Template Control (NTC). Numbers presented are PIM/tubulin ratios. (C) These knockdown conditions did not induce apoptosis in any PTCL cell lines. The percentage of non-viable cells was calculated as Annexin V+/PI− plus Annexin V+/PI+ cells.</p