A MCP1 fusokine with CCR2-specific tumoricidal activity

<p>Abstract</p> <p>Background</p> <p>The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development.</p> <p>Methods</p> <p>We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent.</p> <p>Results</p> <p>We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients.</p> <p>Conclusion</p> <p>Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2⁺malignancies including lymphoid and central nervous system malignancies.</p

A fusion cytokine coupling GMCSF to IL9 induces heterologous receptor clustering and STAT1 hyperactivation through JAK2 promiscuity.

Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes

GIFT9 stimulates the growth of BMMCs.

<p>(<b>A</b>) Flow cytometry analysis of BMMCs. (<b>B</b>) MTT assay of BMMCs after 5 days of culture with GMCSF/IL9 or GIFT9. *: P<0.05. (<b>C</b>) Model of GMCSF and IL9 receptor clustering and downstream signaling after GIFT9 stimulation. See text for further details.</p

GIFT9 stimulation did not increase JAK kinase activity and receptor polarization to lipid rafts is not the major mechanism of GIFT9-induced hyperphosphorylation of STAT1.

<p>(<b>A</b>) JAK1, JAK2, and JAK3 phosphorylation levels remain same after GIFT9 stimulation. Total JAK protein was used as a loading control. Normalized JAK phosphorylation levels in GMCSF+IL9 and GIFT9 treatments are shown. (<b>B</b>) Inhibition of lipid rafts did not affect the hyperphosphorylation of STAT1 induced by GIFT9. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown.</p

Mouse GIFT9 protein expression and biochemical analyses.

<p>(<b>A</b>) GIFT9 amino acid sequence. The signal peptides of each part are underscored, IL9 signal peptide serves as the linker of the two parts. (<b>B</b>) Western blot of GIFT9 protein in the condition media from 293T cells retrovirally transduced to express GIFT9. Recombinant mouse GMCSF and IL9 were used as controls. (<b>C</b>) Western Blot of phospho-STAT1, STAT3, and STAT5 in JawsII cells after GIFT9 stimulation. Total STAT protein was used as a loading control. (<b>D</b>) Western Blot of phospho-STAT1, STAT3, and STAT5 in MC/9 cells after GIFT9 stimulation without or with GMCSF-Rα antibody blocking. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.</p

JAK2 transphosphorylates STAT1 after GIFT9 treatment.

<p>(<b>A</b>) JAK2 inhibitor TG101348 treatment abolished hyperphosphorylation of STAT1 after GIFT9 stimulation. Total STAT protein was used as a loading control. (<b>B</b>) JAK3 inhibitor CP690550 treatment had a minor effect of STAT1 hyperphosphorylation induced by JAK2. Total STAT protein was used as a loading control. (<b>C</b>) Double inhibition of JAK1 and JAK2 by INCB018424 inhibited STAT5 phosphorylation after GMCSF/IL9 but not GIFT9 stimulation. Total STAT protein was used as a loading control. (<b>D</b>) Inhibition of all JAKs by INCB018424 and CP690550 depleted STATs phosphorylation. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.</p