A Stat1 bound enhancer promotes Nampt expression and function within tumor associated macrophages

Tumor associated macrophage responses are regulated by distinct metabolic states that affect their function. However, the ability of specific signals in the local tumor microenvironment to program macrophage metabolism remains under investigation. Here, we identify NAMPT, the rate limiting enzyme in NAD salvage synthesis, as a target of STAT1 during cellular activation by interferon gamma, an important driver of macrophage polarization and antitumor responses. We demonstrate that STAT1 occupies a conserved element within the first intron of Nampt, termed Nampt-Regulatory Element-1 (NRE1). Through disruption of NRE1 or pharmacological inhibition, a subset of M1 genes is sensitive to NAMPT activity through its impact on glycolytic processes. scRNAseq is used to profile in vivo responses by NRE1-deficient, tumor-associated leukocytes in melanoma tumors through the creation of a unique mouse strain. Reduced Nampt and inflammatory gene expression are present in specific myeloid and APC populations; moreover, targeted ablation of NRE1 in macrophage lineages results in greater tumor burden. Finally, elevated NAMPT expression correlates with IFNγ responses and melanoma patient survival. This study identifies IFN and STAT1-inducible Nampt as an important factor that shapes the metabolic program and function of tumor associated macrophages.


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Sample sizes were determined by pilot experiments to evaluate amplitude and variation of differences, and were increased if needed for replicated experiments presented in the manuscript, or were evaluated by the University of Utah Biostatistics core facility.
No datapoints were excluded from the experimental data presented in this manuscript.
Reproducibility of experimental findings was verified by independently repeating experiments in their entirety at least two times, as described in the manuscript. With the incidental exception of some pilot experiments not reported in this manuscript, presented experiments all showed the same trends and in most cases, similar levels of significance. Lack of replication of pilot experiments was due to issues such as inadequate sample sizes, or the need for procedural improvements.
Organisms and samples were randomly allocated into different experimental groups based on segregation of mouse and cell lines and genotyping to confirm their differences. Animals of the appropriate genotypes were then allocated into experimental groups described , as in the manuscript, as WT vs IFNgR deficient, or NREfl/fl vs. NREfl/fl bearing the appropriate Cre driver. These groups were also age and sex matched. Any possible covariates were controlled for by using mice from separate parallel crosses, by using littermate controls, controls bearing each of the genetic manipulations of the test animals or samples tested individually, and by testing for the desired genetic deletions in the final experimental samples that were analyzed.
No interventional or observational treatment studies were performed. Some experimental measurements were blinded by using separate researchers to analyze samples that were constructed and prepared by different experimenters and assigned non-descriptive numbers for the samples. Tumor mass measurements were accomplished by blinded researchers performing the dissection and weight measurements without information as to the experimental animal groups from which the samples were sourced. Otherwise, Blinding was not relevant to other aspects of this study because the samples were intrinsically defined and identified during construction, preparation, and analysis. Mouse B16-F10 cells were purchased from American Type Culture Collection (Manassas, VA) in 2014 and grown in DMEM with 10% FBS, penicillin and streptomycin at 37C with 5% CO2, then frozen in liquid nitrogen. For all experiments, cells were recovered from frozen aliquots and cultured for 1-2 weeks prior to inoculation of mice. 293-T: The 293T (ATCC CRL-3216) parental cell line has not been re-authenticated or subjected to mycoplasma testing in the past year. Cells were grown in DMEM with 10% FBS, penicillin and streptomycin at 37C with 5% CO2, then frozen in liquid nitrogen. For all experiments, cells were recovered from frozen aliquots before experimental manipulation and analysis. Cells lines were authenticated by gene expression under experimental conditions, the expression of fluorescent markers, and morphology.
The cell lines have not been subjected to mycoplasma testing in the past year.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
Mus musculus Strain C57BL/6J, male or female, ages 8-11 weeks Mus musculus Strain C57BL/6J NRE1 fl/fl, Biocytogen, male or female, ages 8-11 weeks none none All animal laboratory work was approved by the University of Utah Health IACUC and the Comparative Medicine Center.