{
  "abstract": "Background The NF-κB family of transcription factors plays a key role in inflammation. NF-κB p50 homodimers repress proinflammatory genes. Multiple cancers grow slower in syngeneic p50(-/-) or p50(f/f);Lys-Cre mice. Murine tumor macrophages lacking p50 display increased expression of a subset of proinflammatory genes, which is associated with increased numbers of both total and activated tumor-infiltrating T cells. 1 2 We find that adoptive transfer of immature myeloid cells lacking p50 (p50-IMC) slows the growth of syngeneic murine prostate cancer, pancreatic ductal carcinoma, neuroblastoma, and glioblastoma tumors, when given after a dose of 5-fluorouracil.2–4 5-FU provides myelo-depletion and releases cancer cell neoantigens for myeloid cell presentation to T cells. Immature rather than mature cells are utilized to favor tumor localization and retain dendritic cell potential. Stat6 is another crucial transcription factor that modulates transcription of murine proinflammatory and anti-inflammatory genes downstream of IL-4. STAT6-IMC or p50/STAT6-IMC might be more effective than p50-IMC as human cancer immunotherapy. To help validate this idea, we developed human macrophages lacking p50, STAT6, or both and compared their proinflammatory gene expression patterns.Methods Human marrow CD34+ cells were nucleofected with Cas9:sgRNAs targeting the NFKB1 gene encoding p50, STAT6, or NFKB1 and STAT6. Cells were expanded in serum-free media containing SCF, TPO, FL, and IL-6, followed by transfer to serum-free media with M-CSF alone to produce monocytes, and finally to IMDM with human AB serum to generate macrophages. IL-4 was added for 24 hr, followed by RNA isolation and qRT-PCR and RNA-Seq. Supernatants collected 24-72 hr after IL-4 addition were analyzed for proinflammatory cytokines by Luminex assay.Results Highly efficient NFKB1 and STAT6 gene editing was confirmed by DNA analysis and Western blotting. Upon transfer of gene-edited cells to M-CSF, CD11b+CD14+ myeloid cells gradually accumulate and represent ~66% of the cell population after five days. Macrophages lacking STAT6 or p50 and STAT6, but not p50 alone, manifested increased expression of multiple proinflammatory pathways upon analysis of RNA-Seq data from three human donors. IL-6, TNFα, and CXCL10 proteins were each increased in the absence of p50 or STAT6, and IL-6 levels were increased an additional 3-fold in the absence of both gene-regulatory proteins.Conclusions Findings presented identify a means to generate human p50-IMC, STAT6-IMC, and p50/STAT6-IMC for cancer immunotherapy and demonstrate that absence of STAT6 contributes strongly to a proinflammatory human macrophage phenotype in an immune-suppressive context. Addition of CARs might further increase efficacy. 5 References Barberi T, Martin A, Suresh R, Barakat DJ, Harris-Bookman S, Drake CG, Lim M, Friedman AD. Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T cell induction of tumor-associated macrophage M2 polarization. Cancer Immunol. Immunoth. 2018;67(10):1491-1503.Cui C, Barberi T, Suresh R, Friedman AD. Adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC) impedes the growth of MHC-matched high-risk neuroblastoma. Mol. Oncol. 2021;15:1783-1796.Suresh R, Barakat DJ, Barberi T, Zheng L, Jaffee EM, Pienta KJ, Friedman AD. NF-κB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma. J. Immunother. Cancer 2020;8:e000244.Beck PJ, Barberi T, Friedman AD. Adoptive transfer of NF-κB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model. Cancer Res. 2022;83(Suppl. 7):4063.Alzubi MA, Barberi T, Friedman AD. PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny. Cancer Immunol. Immunoth. 2025;74:95.Ethics Approval This study was approved by the Johns Hopkins IRB (#00127577). Informed consent was obtained from marrow donors.",
  "authors": [
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA"
      ],
      "name": "Theresa Barberi"
    },
    {
      "affiliations": [
        "Johns Hopkins University School of Medicine, Baltimore, MD, USA"
      ],
      "name": "Alan D Friedman"
    }
  ],
  "title": "324 Investigating a potential human myeloid cell immunotherapy by targeting transcription factors that increase the proinflammatory phenotype of human macrophages in the presence of suppressive IL-4",
  "uid": "da6a7d8e-b747-5ce9-b9eb-bfb65b3eebe6"
}