Granulocyte-macrophage Colony-stimulating Factor Ligand Research Articles

The divergent role of the GM-CSF/GM-CSFR signaling pathway in cholangiocarcinoma and pancreatic ductal adenocarcinoma.

736 Background: The granulocyte-macrophage colony-stimulating factor ligand and receptor (GM-CSF/GM-CSFR) pathway plays a multifaceted role in the setting of cancer, exerting an anti-tumorigenic or pro-tumorigenic effect depending on GM-CSF expression. In previous work, we and others have shown increased GM-CSF expression to correlate with poor prognosis in patients with cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC). We also demonstrated how GM-CSF neutralization leads to reduced tumor size in murine CCA models, in part due to inhibition of alternative macrophage polarization. Here, we explore the impact of blocking GM-CSFR signaling in CCA and PDAC, two immunologically cold tumors with poor prognoses. Methods: Immunohistochemistry (IHC) and digital quantification of staining was performed on tissue from a human PDAC and CCA databank. Murine F4/80+ bone marrow-derived macrophages were cultured in tumor-conditioned media (TCM) derived from PDAC (KP2) or CCA (URCCA4.3) cell lines, with and without anti-GM-CSFR antibody. Quantitative real time PCR (qRT-PCR) analysis was performed for gene expression. URCCA4.3 and a luciferase-labeled PDAC (KCKO) cell line were orthotopically implanted into Bc/BIL3 knockout (KO) mice which lack GM-CSFR and C57BL/6 mice. Mice implanted with URCCA4.3 were enrolled into vehicle or gemcitabine treatment groups. Mice implanted with KCKO were untreated. Tumor growth was monitored by ultrasonography or bioluminescence imaging. Results: Digital analysis of IHC staining for GM-CSFR in human PDAC tumors revealed low expression to be associated with improved overall survival (OS) and greater number of days to recurrence, while high levels were associated with worse OS and fewer days to recurrence (p<0.01). In addition, human CCA specimens had greater GM-CSFR expression compared to normal uninvolved liver (p<0.01). qRT-PCR analysis of RNA from bone marrow-derived macrophages exposed to either CCA or PDAC TCM demonstrated lower expression of genes associated with alternative macrophage polarization ( Arg1, Cd274, Pdcd1lg2) and chronic inflammation ( Ccl2, Ccl17, Irf4) (all p<0.01) in response to GM-CSFR blockade. GM-CSFR KO mice implanted with URCCA4.3 demonstrated no differences in tumor growth with or without chemotherapy, compared to wild-type controls. Similarly, KO mice implanted with an immunogenic PDAC cell line (KCKO) had no differences in tumor size compared to wild-type controls. Conclusions: GM-CSFR expression is prognostic in the setting of PDAC and elevated in human CCA compared to normal liver. In vitro, GM-CSFR blockade downregulated pro-tumor genes associated with immunosuppression and inflammation. However, complete disruption of GM-CSFR signaling reversed the efficacy generated with GM-CSF ligand neutralization, suggesting some signaling is required for inducing anti-tumor immune responses.

Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

Discovery and characterization of a novel splice variant of the GM-CSF receptor α subunit

To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils. We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay. The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space. This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.

Granulocyte macrophage colony-stimulating factor in adenomyosis and autologous endometrium.

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been related to macrophage recruitment and activation and has been identified in the human endometrium. We determined whether adenomyosis expresses GM-CSF, and if present, compared GM-CSF protein expression in adenomyosis with that in autologous endometrium. We examined ectopic and eutopic endometrium from 16 premenopausal women who had hysterectomies for abnormal uterine bleeding, pelvic pain, or uterine prolapse. Serial sections of premenopausal uteri containing endometrium and adenomyosis were analyzed by immunohistochemistry for GM-CSF ligand and receptor and CD68 macrophages. We analyzed the intensity of staining for GM-CSF ligand and receptor and macrophages in the glandular epithelium and stroma of adenomyosis and autologous endometrium. The GM-CSF ligand localized primarily in the glandular epithelium and myometrium with only light stromal staining. Staining for GM-CSF ligand was significantly higher in adenomyotic glands compared with autologous endometrial glands (P = .002), especially during the secretory phase of the menstrual cycle. There were no statistical differences in the amount and intensity of staining of the GM-CSF receptor in adenomyosis and autologous endometrium. Adenomyotic tissue contained significantly more macrophages than matched autologous endometrium (P = .0004). Adenomyotic glandular epithelium had greater expression of the GM-CSF ligand compared with autologous endometrium from premenopausal women, which indicates that GM-CSF may play a role in increasing the levels of activated macrophages in women with adenomyosis.

Granulocyte-macrophage colony-stimulating factor promotes glucose transport and blastomere viability in murine preimplantation embryos.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion from epithelial cells lining the female reproductive tract is induced during early pregnancy by ovarian steroid hormones and constituents of seminal plasma. In this study we have investigated the influence of GM-CSF on development of preimplantation mouse embryos. Blastocyst-stage embryos were found to specifically bind (125)I-GM-CSF and analysis of GM-CSF mRNA receptor expression by reverse transcriptase-polymerase chain reaction indicated expression of the low-affinity alpha subunit of the GM-CSF receptor, but not the affinity-converting beta subunit (beta(c)), or GM-CSF ligand. GM-CSF receptor mRNA was present in the fertilized oocyte and all subsequent stages of development, and in blastocysts it was expressed in both inner cell mass and trophectoderm cells. In vitro culture of eight-cell embryos in recombinant GM-CSF accelerated development of blastocysts to hatching and implantation stages, with a maximum response at a concentration of 2 ng/ml (77 pM). Blastocysts recovered from GM-CSF-null mutant (GM-/-) mice on Day 4 of natural pregnancy or after superovulation showed retarded development, with the total cell number reduced by 14% and 18%, respectively, compared with GM+/+ embryos. Blastocysts generated in vitro from two-cell GM-/- and GM+/+ embryos were larger when recombinant GM-CSF was added to the culture medium (20% and 24% increases in total cell numbers in GM+/+ and GM-/- blastocysts, respectively). Incubation of blastocysts with recombinant GM-CSF elicited a 50% increase in the uptake of the nonmetabolizable glucose analogue, 3-O-methyl glucose. In conclusion, these data indicate that GM-CSF signaling through the low-affinity GM-CSF receptor in blastocysts is associated with increased glucose uptake and enhanced proliferation and/or viability of blastomeres. Together, the findings implicate a physiological role for maternal tract-derived GM-CSF in targeting the preimplantation embryo, and suggest that defective blastocyst development contributes to compromised pregnancy outcome in GM-CSF-null mutant mice.

Clinical studies of new "biologic" approaches to therapy of acute myeloid leukemia with monoclonal antibodies and immunoconjugates.

Conventional chemotherapeutic treatments of acute leukemias are often associated with life-threatening toxic effects due to a lack of specificity for hematopoietic cells. Monoclonal antibodies and fusion proteins that target antigens on leukemic blasts are being explored for their antileukemic effects and as a means of delivering chemotherapy or radiation directly to malignant cells. This approach might be safer and more effective than current non-specific chemotherapeutic agents. The cell surface antigens CD33 and CD45 are attractive targets. Although CD33 is expressed on acute myelocytic leukemic blast cells from about 90% of patients, normal hematopoietic stem cells lack this antigen, as do essentially all non-hematopoietic tissues. Anti-CD33 antibodies have been engineered to selectively target malignant myeloid and immature normal cells while sparing normal stem cells. Recently, anti-CD33 antibodies have also been used to deliver radiation or a cytotoxic agent directly to leukemic cells. The strategy for using CD45 as a target differs from CD33 in that it is expressed not only by the vast majority of leukemias, but also by normal stem cells. Therefore, 131I-labeled anti-CD45 antibody has been used in combination with conventional preparative regimens for patients receiving marrow transplantation for acute leukemia. Because the receptor for granulocyte-macrophage colony-stimulating factor is expressed by most myeloid leukemias, fusion proteins consisting of granulocyte-macrophage colony-stimulating factor ligand associated with diphtheria toxin have been proposed as a means of delivering a toxic agent directly to leukemic cells. Both unconjugated and conjugated antibodies show significant promise in the treatment of acute myelocytic leukemia.

Modulation of the Apoptotic Response of Human Myeloid Leukemia Cells to a Diphtheria Toxin Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractIt has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor–bearing cells. We now report that DT388–GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF–receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL ). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I–GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388–GM-CSF at 4 × 10−9 mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 × 10−9 mol/L to 3 × 10−13 mol/L. GM-CSF–receptor occupancy at 0.7 nmol/L GM-CSF–ligand concentration correlated with the protein synthesis IC50 . Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL , in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388–GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.