Raffaella Morotti

NAPRT-deficient RMS models are sensitive to FK866 and cannot be rescued with NA

Functional validation of single-cell clones of CRISPR/Cas9 ribonucleoprotein (RNP) based NAPRT knock-out model

Supplementary Figure 2. NAPRT-deficient RMS models are not able to utilize NA to rescue cell viability

Supplementary Figure 3. Generation of plasmid based NAPRT overexpressing isogenic model.

Supplementary Figure 7. Clinical Correlates of NAPRT Expression in RMS patient samples.

Supplementary Tables with list of antibodies and description of TMAs

Supplementary Figure 5. Induction of apoptosis in RMS models with OT-82 treatment

Supplementary Figure 6. Tumor volumes in RMS isogenic models after OT-82 treatment.

New treatments are needed to improve survival in children with rhabdomyosarcoma (RMS). Nicotinamide adenine dinucleotide (NAD+) biosynthesis, regulated by the enzymes nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), represents a metabolic vulnerability due to high NAD+ turnover in cancers. Although NAMPT inhibitors (NAMPTi) show preclinical promise, clinical translation has been limited by toxicity and the lack of predictive biomarkers. In this study, we evaluated NAPRT expression in RMS and its potential as an actionable biomarker to guide NAMPTi therapy. NAPRT promoter methylation, transcript levels, and protein expression were assessed in RMS cells, patient-derived xenografts (PDX), and primary tumors (n = 109) from the Children’s Oncology Group. In vitro sensitivity to NAMPTi was tested in isogenic RMS cell lines, examining the role of NAPRT expression in mediating cytotoxicity and the ability of nicotinic acid (NA) to rescue viability. In vivo efficacy was assessed using cell-derived, NAPRT-isogenic, and orthotopic PDX models. NAPRT promoter hypermethylation was found in a subset of RMS models and patient samples. Immunohistochemistry showed loss of NAPRT protein in 30% to 40% of tumors, defined as <1% tumor cell staining. Methylation modestly correlated with protein expression. NAPRT-deficient cells were highly sensitive to NAMPTi, driven by NAD+ depletion and not reversible with NA. In vivo, NAMPTi induced significant tumor regression, which was not abrogated with NA administration in NAPRT-deficient models. NAPRT loss occurs in a subset of RMS and may offer a biomarker-driven strategy to expand the therapeutic window of NAMPTi. Further research is needed to understand NAPRT regulation and optimize biomarker assays in future clinical trials.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2 in vitro . Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (∼13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.