PIM kinases as therapeutic targets against advanced melanoma

Abstract

Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients.

Keywords: PIM kinases; SGI-1776; melanoma; organometallics; therapy.

Figure 1. SM200 inhibits proliferation and invasion of 2D and 3D melanoma cells

(A) Melanoma cells and normal fibroblasts were treated with increasing doses of SM200 for 72 h, then were assessed using the alamarBlue assay. Results were normalized to the DMSO control. Data are represented as mean +/− SEM. A significant response to SM200 was detected for all melanoma cell lines compared to the fibroblasts (p < 0.0001). (B) Melanoma cell lines and normal fibroblasts were treated with SM200 [10 μM] for 72 h before staining with propidium iodide. All treated melanoma cell lines show a significant increase in cytotoxicity, but not fibroblasts. Data are represented as mean +/− SEM, and p-values are provided in the table below. (C) The AlamarBlue assay was conducted on collagen-embedded melanoma spheroids treated with SM200 for 72 h to assess spheroid growth. Data are represented as mean +/− SEM, from triplicate experiments and p-values are provided. (D) Collagen-embedded melanoma spheroids were treated for 72 h with SM200 [10 μM] and stained for live and dead cells. Green fluorescence indicates metabolically active (live) cells, red fluorescence indicates membrane compromised (dead) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 150 microns.

Figure 2. PIM kinases as targets for melanoma

(A) Chemical structure of SM200. The compound was used as a racemic mixture but only one enantiomer is shown. (B) Human kinome phylogenetic tree (TREEspot, DiscoveRx) displaying SM200 selectivity as assessed by an active-site-directed affinity screening approach against 451 human protein kinases (KINOMEScan). The human kinase dendogram shows the protein kinase families and the evolutionary relationships between the individual kinases. Compound hits are shown as red dots (%ctrl = percent of control: 0% = highest affinity drug binding, 100% = no affinity drug binding); PIM1 is indicated with an arrow. SM200 was profiled for protein kinase binding at a concentration of 1 μM and the top four inhibited kinases are shown in the right hand table. (C) Images of PIM1, PIM2, and PIM3 staining (IHC) of human metastatic melanoma tissue is shown. The images on the right correspond to a higher magnification of the images on left (boxed area). Scale bars represent 50 μM (left images) and 1 μM (right images). PIM1, PIM2, PIM3 expression was interpreted for fourteen different samples using the H-score method. Images for sample #4 (PIM1) and #17 (PIM2 and PIM3) are provided. The intensity and extent of staining on the entire tissue sections were assessed according to a four-tiered (0 to 3) scale.

Figure 3. Effects of PIM1 knockdown on melanoma cells

(A) Left panel: PIM1 was knocked down in 1205Lu and WM983B melanoma cells using two different shRNAs; expression of PIM isoforms was then assessed by western blot. Right panel: MTS assay shows decreased proliferation of melanoma cells with PIM1 knockdown. Data are represented as mean +/− SEM, from triplicate experiments. (B) Left panel: Phase contrast images of 3D spheroids generated with PIM1 knockdown melanoma cells. Scale bar represents 150 microns. Right panel: Quantitation of invasion from spheroids generated with PIM1 knockdown cells. Data are represented as mean +/− SEM, from three separate spheroids. (C) NSG mice were xenotransplanted using 1205Lu melanoma cells with PIM1 knocked down using 3 different shRNAs. Tumor volumes were measured at the indicated time points until day 15. Statistical analyses of tumor growth rates used ANOVA with groups defined by each shRNA. Three comparisons are shown for each shRNA group versus controls (p < 0.001 for all comparisons). Error bars represent SEM, n = 8 mice/group. (D) Western blot analyses of tumor lysates from PIM1 knockdown 1205Lu xenotransplanted mice (2 different tumors/group). Levels of all three PIM kinases are shown (below the 50 kDa marker) as well as p27 (below 35 kDa marker). Cleaved PARP and the loading control Hsp90 are also shown (below 100 kDa marker).

Figure 4. SGI-1776 inhibits proliferation and invasion of melanoma cells

(A) Melanoma cells and normal fibroblasts were treated with increasing doses of SGI-1776 for 72 h and were assessed using the alamarBlue assay. Results were normalized to the DMSO control. Data are represented as mean +/− SEM. A significant response to SGI-1776 was detected for all melanoma cell lines compared to the fibroblasts (p < 0.0001). (B) Three melanoma cell lines and normal fibroblasts were treated with SGI-1776 [10 μM] for 72 h before staining with propidium iodide. All treated melanoma cell lines show a significant increase in cytotoxicity, but not fibroblasts. Data are represented as mean +/− SEM. (C) Western blot analyses of adherent (2D) melanoma cells treated with SGI-1776 [10 μM] for 12 h. Levels of all PIM isoforms were investigated as well as multiple effectors involved in PIM signaling. Histone H3 was used as a loading control.

Figure 5. SGI-1776 displays anti-tumor activity in combination with a BRAF inhibitor in 3D melanoma models

(A) Western blot analysis showing endogenous levels of PIM-1, −2, and −3 in human melanoma cell lines, normal fibroblasts, and cell lines rendered resistant to BRAF inhibitors (BR). Hsp90 served as loading control. (B) Collagen-embedded melanoma spheroids were treated for 72 h with SGI-1776 [10 μM] or PLX4720 [5 μM] as single agents or in combination. Spheroids were then stained for live (green) and dead (red) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 150 microns. (C) PIM1 gene expression in melanoma patient samples pre-treatment and upon treatment progression on dabrafenib or vemurafenib. Total RNA was isolated from fresh frozen melanoma tumors in 21 patients (GSE50509). The graph shows data for 12 patients with greater PIM1 gene expression in progressing tumor samples (Prog) than in pretreated samples (Pre). The red and blue bars represent the normalized microarray expression of PIM1 (on a log scale) in Pre and Prog samples, respectively.

Figure 6. SGI-1776 displays anti-tumor activity in combination with a BRAF inhibitor in vivo

(A) NSG mice were xenotransplanted with 1205Lu melanoma cells and tumors were allowed to grow above 200 mm³. Mice were then treated with the single agents SGI-1776 (100 mg/kg 3×/week), or PLX4720 (200 mg/kg diet), or with the combination of both drugs. Tumor volumes were measured at the indicated time points. Statistical analyses of tumor volumes used one-way ANOVA with groups defined by treatment (p < 0.001 for all comparisons shown). Error bars represent SEM, n = 10 mice. (B) PIM kinase expression (all three isoforms) in mouse tumor lysates (n = 2/group); Histone H3 serves as a loading control. Lysates were collected 3 days after treatment initiation. (C) Western blot analyses of mouse tumor lysates (n = 3/group) isolated on day 17 of the experiment shown in (A). Effectors of the PI3K, MAPK, and STAT3 signaling pathways, as well as pBAD and the tumor suppressor p27, were evaluated. Histone H3 served as loading control.

Figure 7. SGI-1776 displays anti-tumor activity in combination with a PI3K inhibitor in 3D melanoma models

(A) Melanoma cells were treated with increasing doses of SGI-1776 or AZD6482 for 72 h and were assessed using the alamarBlue assay. The mean % of proliferation compared to the DMSO control is shown (from three experiments). Blue indicates no inhibition, red indicates high inhibition. Synergy was calculated for combination experiments using the Bliss formula. The Bliss number gives the difference between predicted and observed inhibition values (excess over Bliss); a positive value indicates synergy, a negative value indicates antagonism and values near zero indicate an overlap of predicted and observed combination effects. (B) 1205Lu spheroid lysates were interrogated for effectors of AKT and STAT3 signaling, as well as cleaved PARP by western blots. Vinculin was used as a loading control. (C) Collagen-embedded melanoma spheroids were treated for 72 h with SGI-1776 [5 μM] or AZD6482 [10 μM] as single agents or in combination before staining for live (green) and dead (red) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 150 microns.