Transformation of BaF3 cells by LTK-F568L requires JAK family kinase activity.

<p>(<b>A</b>) BaF3 cells transformed to cytokine independence by LTK-F568L were cultured in the presence of 0.5 µM (green squares) or 1 µM (blue triangles) JAK Inhibitor I (JI) or 0.1% DMSO (red circles) on Day 0. The total number of viable cells was determined by trypan blue exclusion. Dashed lines indicate viable cell counts going below the limit of detection of the hemacytometer towards zero. Similar results were obtained in two independent experiments. (<b>B</b>) IL-3-independent LTK-F568L BaF3 cells were treated with 1 µM (lane 2) JI or DMSO (lane 1) for three hours and immunoblotted for the indicated proteins (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031733#pone-0031733-g003" target="_blank">Figure 3</a> legend). Phosphotyrosine (pTyr) on LTK-F568L was detected by immunoblotting following immunoprecipitating LTK-F568L with HA antibodies.</p

LTK-F568L and LTK-R669Q induce PC12 cell neurite outgrowth.

<p>(<b>A</b>) PC12 cells were co-transfected with GFP and either vector, wildtype LTK, LTK-R669Q, or LTK-F568L. Cells were observed for neurite outgrowth. PC12 cells treated with 1 uM NGF are shown as control for neurite extensions. Photographs of representative cells were taken four days after transfection. Arrows indicate neurite outgrowth. Two photographs for both LTK-F568L and LTK-R669Q are shown. (<b>B</b>) GFP-positive PC12 cells transiently expressing either vector control (v, red bars), wildtype LTK (wt, black bars), LTK-R669Q (RQ, blue bars), or LTK-F568L (FL, green bars) were counted daily for ten days and the percent exhibiting neurite outgrowth is shown. GFP was strongly expressed through Day 7, after which expression began to diminish and became undetectable after Day 10. Data shown is the average of two independent experiments done in duplicate with error bars representing standard error of mean. At least 500 GFP-positive cells were assessed per plate.</p

LTK-F568L transforms BaF3 cells to cytokine-independence.

<p>(<b>A</b>) BaF3 cells stably expressing LTK-F568L (red inverted triangles), LTK-R669Q (green triangles), or wildtype LTK (blue squares) and parental control cells (black circles) were cultured in the absence of IL-3 at Day 0. The total number of viable cells was determined at each timepoint by trypan blue exclusion. Similar results were obtained in three independent experiments. The dashed line indicates cell numbers going below the detection of the hemacytometer towards zero. (<b>B</b>) Lysates from cells stably expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were cultured in the absence of IL-3 for six hours and immunoprecipitated with HA antibodies, followed by immunoblotting with HA and pTyr antibodies (first two blots). The same lysates were immunoblotted with antibodies to the indicated cell signaling proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031733#pone-0031733-g002" target="_blank">Figure 2</a> legend plus pShc-Y317, STAT3, pSTAT3-Y705, STAT5 and pSTAT5-Y694). Cell lysate from IL-3-independent LTK-F568L-expressing BaF3 cells was also analyzed (lane 5).</p

LTK-F568L and LTK-R669Q induce anchorage independent growth, which is inhibited by PF-2341066.

<p>(<b>A</b>) RIE cells stably expressing either wildtype LTK, LTK-F568L, or LTK-R669Q were plated in soft agar and assessed for anchorage-independent growth. LTK-F568L-expressing cells formed large colonies in soft agar while LTK-R669Q-expressing cells formed fewer colonies which were also significantly smaller. Including PF2341066 (PF) in the medium inhibited anchorage independent growth in soft agar. Photographs show representative fields after two weeks of culture. Results shown for are representative of similar findings of three independent experiments. (<b>B</b>) Quantitation of anchorage independent growth of RIE cells expressing wildtype LTK, LTK-F568L, and LTK-R669Q fourteen days after plating in the absence (blue bars) and presence of 0.5 µM (green bars) or 1.0 µM (black bars) PF2341066 (PF). (<b>C</b>) Quantitation of anchorage independent growth of RIE cells expressing wildtype LTK, LTK-F568L, and LTK-R669Q fourteen days after plating in the absence (blue bars) and presence of 0.5 µM (yellow bars) or 1 µM JAK Inhibitor (red bars). Data represents average colonies per field view per plate. Error bars represent standard error of the mean from two or three independent experiments performed in duplicate.</p

LTK-F568L transforms 32D cells to cytokine-independence.

<p>(<b>A</b>) 32D cells stably expressing LTK-F568L (red inverted triangles), LTK-R669Q (green triangles), or wildtype LTK (blue squares) and parental control cells (black circles) were cultured in the absence of IL-3 at Day 0. The total number of viable cells was determined at each timepoint by trypan blue exclusion. Similar results were obtained in three independent experiments. The dashed line indicates cell numbers going below the detection of the hemacytometer towards zero. (<b>B</b>) Lysates from cells stably expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were cultured in the absence of IL-3 for six hours and immunoprecipitated with HA antibodies, followed by immunoblotting with HA and pTyr antibodies (first two blots). The same lysates were immunoblotted with antibodies to the indicated cell signaling proteins (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031733#pone-0031733-g003" target="_blank">Figure 3</a> legend). Cell lysate from IL-3-independent LTK-F568L-expressing 32D cells was also analyzed (lane 5).</p

Comparison of ALK and LTK structure and sequence.

<p>(<b>A</b>) Schematic of ALK and LTK. ALK and LTK are RTKs, each containing an extracellular domain, a transmembrane domain (TMD, purple), and an intraceullar kinase domain (KD, green). ALK is a 1620 amino acid protein containing two MAM domains (blue) and an LDLa motif in its extracellular region. LTK is composed of 864 amino acids and has a much smaller extracellular domain that lacks these structures. The positions of the F1174L and R1275Q mutations of ALK and the corresponding mutations in LTK, F568L and R669Q, are indicated. (<b>B</b>) Alignment of ALK and LTK kinase domains. ALK and LTK proteins share 80% sequence identity in their kinase domains. Identical residues are indicated by an asterisk (*). Position of the F→L and R→Q residues of interest are shown in bold designated by arrows. Boundaries of the kinase domains were determined using the Simple Modular Architecture Research Tool, <a href="http://smart.embl-heidelberg.de/and" target="_blank">http://smart.embl-heidelberg.de/and</a> the alignment was done using BLAST.</p

LTK-F568L and LTK-R669Q transform epithelial cells.

<p>(<b>A</b>) Cell lysates from RIE cells stably expressing either empty vector (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were analyzed by immunoprecipitation and immunoblotting, as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031733#pone-0031733-g003" target="_blank">Figure 3</a> legend). (<b>B</b>) RIE cells stably expressing either wildtype LTK (wt), LTK-F568L (F568L), or LTK-R669Q (R669Q) were plated and reached confluency after six days. Cells were cultured further and growth was grossly assessed for the loss of contact inhibition. Cells expressing wildtype LTK did not grow beyond confluency, continuing to exhibit contact inhibition. However, cells expressing LTK-F568L continued to proliferate, forming swirling patterns of cells growing on top of the monolayer. Cells expressing LTK-R669Q formed distinct secondary morphological structures as these cells formed compact clusters of cells that grew on top of the monolayer of cells. Representative photographs of cells are shown: plates were stained with crystal violet (top row) after photographs were taken at 10× with a digital camera (middle) and 50× with a Zeiss Automated Fluorescent Microscope using AxioVision (bottom row). Results shown are representative of similar findings of three independent experiments.</p

Expression of LTK constructs in 293T cells.

<p>(<b>A</b>) Relative protein expression of wildtype and mutant LTK constructs in 293T cells. Lysates of 293T cells transiently expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were immunoblotted with an anti-HA antibody as well as HSP90 as a loading control. (<b>B</b>) 293T cell lysates containing the LTK proteins were treated with PNGase F (lanes 3, 5, and 7) or left untreated (lanes 2, 4, and 6) and immunoblotted with HA antibodies and HSP90 as a loading control. (<b>C</b>) HA-tagged LTK proteins expressed in 293T cells were immunoprecipitated and immunoblotted with HA antibodies as well as anti-phosphotyrosine (pTyr) antibodies (top). Antibodies that recognize the indicated proteins (Shc, pShC-Y239/Y240, ERK, pERK-T202/Y204, JAK1, pJAK1-Y1022/Y1023, JAK2, pJAK2-Y1007/Y1008, AKT, and pAKT-S473) were used to immunoblot cell lysates of 293T cells expressing LTK proteins (bottom). The phosphorylated form of the protein is designated with a “p” preceding the protein name.</p

Transformed LTK-F568L BaF3 cells are sensitive to the ALK inhibitor PF-2341066.

<p>(<b>A</b>) BaF3 cells transformed to cytokine independence by LTK-F568L were left untreated (0.1% DMSO, black circles) or cultured in the presence of 0.5 µM (blue squares), 1 µM (green triangles), or 2 µM (red inverted triangles) of the cMET/ALK inhibitor PF2341066 (PF) on Day 0. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. (<b>B</b>) IL-3-dependent (+IL-3) BaF3 cells harboring ALK-F1174L were cultured without (black circles) or with (blue squares) 0.5 µM PF2341066. BaF3 cells transformed to IL-3-independence by ALK-F1174L were cultured without (green triangles) or with (red inverted triangles) 0.5 µM PF2341066. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. (<b>C</b>) Parental BaF3 cells were cultured without (black circles) or with (blue squares) 0.5 µM PF2341066. BaF3 cells expressing wildtype LTK were cultured without (green triangles) or with (red inverted triangles) 0.5 µM PF2341066. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. (<b>D</b>) IL-3-dependent BaF3 cells expressing LTK-F568L were cultured without (black inverted triangles) or with (blue circles) 0.5 µM PF2341066. BaF3 cells transformed to IL-3 independence by LTK-F568L were cultured without (green squares) or with (red diamonds) 0.5 µM PF2341066. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. In (A) thru (D), the dashed lines indicate viable cell counts going below the limit of detection of the hemacytometer towards zero. For each of the experiments in (A) thru (D), similar results were obtained in each independent (at least two) experiment performed. (<b>E</b>) IL-3-independent BaF3 LTK-F568L-expressing cells were treated with 1 µM PF-2341066 (lane 2) or DMSO (lane 1) for 2 hours. Cell lysates were collected and immunoprecipitated and/or immunoblotted, as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031733#pone-0031733-g003" target="_blank">Figure 3</a> legend).</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies

<div><p>The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase–signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. <i>In vitro</i>, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated <i>ex vivo</i> and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. <i>In vivo</i>, single-agent INCB053914 inhibited Bcl-2–associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.</p></div