Pim-1 Kinase Phosphorylates and Stabilizes 130 kDa FLT3 and Promotes Aberrant STAT5 Signaling in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication

<div><p>The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic stem cells and acute myeloid leukemia (AML) cells and regulates their proliferation. Internal tandem duplication (ITD) mutation of FLT3 is present in a third of AML cases, results in constitutive activation and aberrant signaling of FLT3, and is associated with adverse treatment outcomes. While wild-type (WT) FLT3 is predominantly a 150 kDa complex glycosylated cell surface protein, FLT3-ITD is partially retained in the endoplasmic reticulum as a 130 kDa underglycosylated species associated with the chaperones calnexin and heat shock protein (HSP) 90, and mediates aberrant STAT5 signaling, which upregulates the oncogenic serine/threonine kinase Pim-1. FLT3 contains a Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that it might be a Pim-1 substrate. Pim-1 was indeed found to directly interact with and serine-phosphorylate FLT3. Pim-1 inhibition decreased the expression and half-life of 130 kDa FLT3, with partial abrogation by proteasome inhibition, in association with decreased FLT3 binding to calnexin and HSP90, and increased 150 kDa FLT3 expression and half-life, with abrogation by inhibition of glycosylation. These findings were consistent with Pim-1 stabilizing FLT3-ITD as a 130 kDa species associated with calnexin and HSP90 and inhibiting its glycosylation to form the 150 kDa species. Pim-1 knockdown effects were similar. Pim-1 inhibition also decreased phosphorylation of FLT3 at tyrosine 591 and of STAT5, and expression of Pim-1 itself, consistent with inhibition of the FLT3-ITD-STAT5 signaling pathway. Finally, Pim-1 inhibition synergized with FLT3 inhibition in inducing apoptosis of FLT3-ITD cells. This is, to our knowledge, the first demonstration of a role of Pim-1 in a positive feedback loop promoting aberrant signaling in malignant cells.</p></div

Pim-1 directly interacts with and serine phosphorylates FLT3.

<p><b>A.</b> Pim-1 interacts with FLT3 in cells. Lysates of MV4-11 and MOLM-14 FLT3-ITD cells, and of EOL-1 FLT3-WT cells, all of which express Pim-1, were immunoprecipitated with anti-Pim-1 and immunoblotted with anti-FLT3, and, reciprocally, immunoprecipitated with anti-FLT3 and immunoblotted with anti-Pim-1. 10% of the total cell lysate (TCL) used for immunoprecipitation is shown as input control. Interaction between Pim-1 and FLT3 was detected in MV4-11 and MOLM-14, and also in EOL-1, indicating that Pim-1 interacts with both FLT3-ITD and FLT3-WT. <b>B.</b> Pim-1 interacts with FLT3 in vitro. Lysates from Ba/F3-ITD and MV4-11 FLT3-ITD cells were incubated with immobilized GST-tagged Pim-1 fusion protein or control GST, followed by immunoblotting with anti-FLT3 antibody. Total cell lysate is shown as input control. GST-Pim-1 recombinant protein, but not GST control, bound to FLT3-ITD, confirming direct binding of Pim-1 kinase to FLT3-ITD <i>in vitro</i>. <b>C.</b> Pim-1 interacts with and phosphorylates FLT3 on serine residues between amino acids 571 and 993. An in <i>vitro</i> kinase assay was performed with purified GST-FLT3 peptide (571-993) containing the Pim-1 consensus phosphorylation site at amino acid 935 (RKRPS) as the substrate and immunoprecipitates from MV4-11 cells with anti-Pim-1 antibody or IgG control on Sepharose A beads as the kinase, in the absence and presence of 10 µM quercetagetin, followed by immunoblotting with the antibodies indicated. Immunoprecipitates with IgG and with beads alone are shown as negative controls. Direct interaction between Pim-1 and FLT3 resulted in serine phosphorylation of FLT3, which w<i>a</i>s completely inhibited by 10 µM quercetagetin. <b>D.</b> Quercetagetin inhibits FLT3 serine phosphorylation by Pim-1. After treatment with 10 µM quercetagetin for three hours, FLT3-ITD was immunoprecipitated from BaF3-ITD and MOLM-14 cell lines and immunoblotted for phoshoserine and FLT3. Quercetagetin decreased FLT3 serine phosphorylation in both cell lines. <b>E.</b> shRNA knockdown of Pim-1 inhibits serine phosphorylation of FLT3. MOLM-14 cells were transiently transfected with control shRNA or Pim-1 shRNA for 72 hours. FLT3-ITD was immunoprecipitated and then immunoblotted for phosphoserine. The total cell lysates were also immunoblotted with anti-Pim-1 to confirm Pim-1 knockdown. GAPDH is shown as a loading control. Pim-1 knockdown decreased FLT3 serine phosphorylation, consistent with serine phosphorylation of FLT3 by Pim-1.</p

Pim-1 knockdown also destabilizes FLT3-ITD.

<p><b>A.</b> Silencing Pim-1 with shRNA decreases FLT3-ITD expression. Pim-1 knockdown was performed as described in Materials and Methods, and expression of FLT3, Pim-1 and GAPDH control in Ba/F3-ITD, MOLM-14 and MV4-11 cells was measured by immunoblotting. Deceased expression of FLT3 was seen. (<b>B, C</b>). Pim-1 knockdown decreases half-life of 130 kDa FLT3. FLT3-ITD-expressing cells were treated with cycloheximide (CHX) combined with either 10 µM quercetagetin or DMSO for the times indicated. Expression of FLT3 and GAPDH control in MOLM-14 (<b>B</b>) and MV4-11 (<b>C</b>) cells was measured by immunoblotting. Decreased half-life of 130 kDa FLT3 was seen in both cell lines.</p

Pim-1 inhibition decreases FLT3 Y591 and STAT5 phosphorylation and Pim-1 expression in FLT3-ITD cells.

<p>Ba/F3-ITD and MV4-11 cells were treated with 10 µM quercetagetin or DMSO control for the times indicated, followed by immunoblotting (IB) with the antibodies indicated.</p

Pim-1 stabilizes 130 kDa FLT3-ITD A.

<p>Pim-1 inhibition with quercetagetin destabilizes 130 kDa and stabilizes 150 kDa FLT3. MV4-11 cells were treated with cycloheximide (CHX) in the presence or absence of 10 µM quercetagetin for the indicated time periods and immunoblotted for FLT3 and the loading control GAPDH. The expression ratio of each FLT3 isoform to GAPDH was quantitated by densitometric analysis, then normalized to “0” time point control and the normalized values are shown graphically as the percent of the FLT3 isoform remaining at various time points. <b>B.</b> Pim-1 inhibition with AR339 destabilizes 130 kDa and stabilizes 150 kDa FLT3. Cells were studied as in A, with 500 nM AR339. <b>C.</b> Pim-1 inhibition increases FLT3-ITD ubiquitination. MV4-11 and MOLM-14 cells were incubated with and without 10 µM quercetagetin for three hours and the protein lysates were immunoprecipitated with FLT3 antibody or control IgG and immunoblotted for ubiquitin. Total FLT3 in the protein lysates is also shown as control. A similar effect was seen in MV4-11 cells incubated with AR339 at a range of concentrations. <b>D.</b> Pim-1 inhibition enables glycosylation of 130 kDa FLT3, forming 150 kDa FLT3. MV4-11 cells were treated with 10 uM quercetagetin or DMSO control with and without the glycosylation inhibitor 2-deoxy-D-glucose (2-DG) for 24 hours, and immunoblotted with the indicated antibodies. Expression of 150 kDa FLT3 increased in the presence of quercetagetin, but this increase was abrogated by co-incubation with 2-DG, consistent with Pim-1 inhibition by quercetagetin enabling glycosylation of 130 kDa FLT3 to form 150 kDa FLT3.</p

Pim-1 inhibition decreases FLT3-ITD binding to its chaperone proteins calnexin and HSP90.

<p>Ba/F3-ITD, MV4-11 and MOLM-14 FLT3-ITD cells were incubated with the Pim-1 inhibitor 10 µM quercetagetin or DMSO control for 3 hours, and protein-protein interaction was studied by immunoprecipitation (IP) followed by immunoblotting (IB) with the antibodies indicated. Treatment with quercetagetin disrupted FLT3-ITD binding to calnexin (<b>A</b>) and decreased its binding to HSP90 (<b>B</b>).</p

Molecular analysis and favorable clinical outcomes in real-world patients with metastatic renal cell carcinoma

Prior biomarker studies have mainly been restricted to advanced RCC patients treated in clinical trials or have had limited integration of immunotherapy features such as programmed death ligand (PD-L)-1 with gene expression signatures intended to capture other canonical pathways to confirm their prognostic value. PD-L1 and PD-L2 by immunohistochemistry (IHC), PD-L2 messenger RNA (mRNA), and 10 gene expression profile (GEP) signatures targeting immune, angiogenesis and canonical pathways were analyzed in nephrectomy specimens from 227 advanced clear cell RCC (ccRCC) and 42 non-clear cell RCC (nccRCC) patients treated with targeted therapies including VEGF and mTOR inhibitors. Biomarker association with best overall response (BOR), progression-free survival (PFS), and overall survival (OS) were evaluated using multivariable modeling. Except for PD-L1 IHC and angiogenesis, tested with a nominal p-value of .05, multiplicity control was applied with a 0.1 significance level given limited experience in this setting. The strongest biomarker correlations were observed for hypoxia inducible factor (HIF)-2a and angiogenesis signatures (rho = 0.860 [ccRCC], 0.819 [nccRCC]); hypoxia and glycolysis signatures (rho = 0.943 [ccRCC], 0.973 [nccRCC]); PD-L2 mRNA and T-cell-inflamed GEP signatures (rho = 0.764 [ccRCC], 0.897 [nccRCC]); and PD-L2 mRNA and monocytic myeloid-derived suppressor cell signature (rho = 0.787 [ccRCC], 0.815 [nccRCC]). For ccRCC, higher angiogenesis expression was associated with improved BOR (OR:2.85 [95%CI:1.37, 5.93]), longer PFS (HR:0.61 [95%CI:0.45, 0.82]) and OS (HR:0.74 [95%CI:0.54, 1.00]); higher PD-L1 expression with shorter OS (HR:1.44 [95%CI:1.01, 2.07]). For nccRCC, there was more than a two-fold increased risk with longer OS associated with lower angiogenesis (HR:2.43 [95%CI:1.04, 5.68]), glycolysis (HR:7.03 [95%CI:1.51, 32.76]) and hypoxia (HR:8.83 [95%CI:1.69, 46.05]) gene signature expression. Data pointed at PD-L1 IHC and angiogenesis expression in ccRCC and hypoxia, glycolysis, and angiogenesis expression in nccRCC as potential prognostic factors. These findings may have implications for the design and interpretation of advanced RCC trials and to identify potential targets for combination therapy strategies.</p