TGFβR1 Blockade with Galunisertib (LY2157299) Enhances Anti-Neuroblastoma Activity of the Anti-GD2 Antibody Dinutuximab (ch14.18) with Natural Killer Cells

Abstract

Purpose Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFβR1, was examined for its ability to enhance the efficacy of dinutuximab in combination with human ex vivo activated NK (aNK) cells against neuroblastoma. Experimental Design TGFB1 and TGFBR1 mRNA expression was determined for 249 primary neuroblastoma tumors by microarray analysis. The ability of galunisertib to inhibit SMAD activity induced by neuroblastoma patient blood and bone marrow plasmas in neuroblastoma cells was tested. The impact of galunisertib on TGFβ1-induced inhibition of aNK cytotoxicity and ADCC in vitro and on anti-neuroblastoma activity in NSG mice was determined. Results Neuroblastomas express TGFB1 and TGFBR1 mRNA. Galunisertib suppressed SMAD activation in neuroblastoma cells induced by exogenous TGFβ1 or by patient blood and bone marrow plasma, and suppressed SMAD2 phosphorylation in human neuroblastoma cells growing in NSG mice. In NK cells treated in vitro with exogenous TGFβ1, galunisertib suppressed SMAD2 phosphorylation and restored the expression of DNAM-1, NKp30, and NKG2D cytotoxicity receptors, TRAIL death ligand, the release of perforin and granzyme A, and the direct cytotoxicity and ADCC of aNK cells against NB cells. Addition of galunisertib to adoptive cell therapy with aNK cells plus dinutuximab reduced tumor growth and increased survival of mice injected with two neuroblastoma cell lines or a patient-derived xenograft. Conclusion Galunisertib suppresses activation of SMAD2 in neuroblastomas and aNK cells, restores NK cytotoxic mechanisms, and increases the efficacy of dinutuximab with aNK cells against neuroblastoma tumors.

NK Cell Function Regulation by TGF-β-Induced Epigenetic Mechanisms

“…Several strategies have been recently elaborated for the therapeutic inhibition of the TGFβ pathway, particularly to improve the antitumor immune responses. Combination therapy with an inhibitor of TGFβ type I receptor (TGFβR1) kinase, galunisertib, anti-GD2 antibody and ex vivo activated NK cells, has been used by Tran et al (55) in a mouse model of neuroblastoma with positive results. Genetically modified SMAD3-silenced human NK-92 cells inhibited cancer progression in two xenograft mouse models with human hepatoma and melanoma (56).…”

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes

“…CHLA-136 29–31 and CHLA-255 31–33 human NB cell lines and human NB patient-derived xenograft (PDX) COG-N-415x cells 34 were derived from patients with progressive disease and were provided by the Children’s Oncology Group (COG) Cell Culture and Xenograft Repository (www.COGcell.org). CHLA-136 cells express a medium level of GD2, are chemoresistant, and have genomic amplification of MYCN as previously described.…”

“…We transduced the firefly luciferase (Fluc) gene into CHLA-136 (CHLA-136-Fluc) cells, CHLA-255 (CHLA-255-Fluc) cells, and freshly isolated human monocytes using a lentivirus vector, as previously described. 31, 37 For specified experiments, CHLA-255 cells were transduced with the renilla luciferase gene (CHLA-255-hRL). Cells were tested for mycoplasma using MycoAlert (Lonza, Allendale, NJ, USA) and for correct identity using AmpFLSTR Identifiler PCR Amplification Kits (Applied Biosystems, Foster City, CA, USA), and were last tested at study completion.…”

Multifocal primary neuroblastoma tumor heterogeneity in siblings with co‐occurring PHOX2B and NF1 genetic aberrations

“…Despite the common somatic 17q gain, we found significant divergence of somatic CN and mutational variants across samples, similar to what has been described in multifocal lung cancer, prostate cancer, and glioblastoma, suggesting true multifocal primary sites of disease with independent development of tumors rather than oligometastatic spread. All somatic mutations identified were present in no more than two samples, although several have been described to have oncogenic potential and relevance in neuroblastoma, including FBN1 , FLT4/VEGFR3 , and N4BP1 (Table S1). Additional functional study of the listed genes may provide novel insight into neuroblastoma pathogenesis.…”