Chimeric antigen receptor (CAR) T cell expansion and persistence represent key factors to achieve complete responses and prevent relapses. These features are typical of early memory T cells, which can be highly enriched through optimized manufacturing protocols. Here, we investigated the efficacy and safety profiles of CAR T cell products generated from preselected naive/stem memory T cells (T N/SCM ), as compared with unselected T cells (T BULK ). Notwithstanding their reduced effector signature in vitro, limiting CAR T N/SCM doses showed superior antitumor activity and the unique ability to counteract leukemia rechallenge in hematopoietic stem/precursor cell–humanized mice, featuring increased expansion rates and persistence together with an ameliorated exhaustion and memory phenotype. Most relevantly, CAR T N/SCM proved to be intrinsically less prone to inducing severe cytokine release syndrome, independently of the costimulatory endodomain employed. This safer profile was associated with milder T cell activation, which translated into reduced monocyte activation and cytokine release. These data suggest that CAR T N/SCM are endowed with a wider therapeutic index compared with CAR T BULK .

The trimeric transcription factor (TF) NF-Y regulates the CCAAT box, a DNA element enriched in promoters of genes overexpressed in many types of cancer. The regulatory NF-YA is present in two major isoforms, NF-YAl (“long”) and NF-YAs (“short”). There is growing indication that NF-YA levels are increased in tumors. Here, we report interrogation of RNA-Seq TCGA (The Cancer Genome Atlas)—all 576 samples—and GEO (Gene Expression Ominibus) datasets of lung adenocarcinoma (LUAD). NF-YAs is overexpressed in the three subtypes, proliferative, inflammatory, and TRU (terminal respiratory unit). CCAAT is enriched in promoters of tumor differently expressed genes (DEG) and in the proliferative/inflammatory intersection, matching with KEGG (Kyoto Encyclopedia of Genes and Genomes) terms cell-cycle and signaling. Increasing levels of NF-YAs are observed from low to high CpG island methylator phenotypes (CIMP). We identified 166 genes overexpressed in LUAD cell lines with low NF-YAs/NF-YAl ratios: applying this centroid to TCGA samples faithfully predicted tumors’ isoform ratio. This signature lacks CCAAT in promoters. Finally, progression-free intervals and hazard ratios concurred with the worst prognosis of patients with either a low or high NF-YAs/NF-YAl ratio. In conclusion, global overexpression of NF-YAs is documented in LUAD and is associated with aggressive tumor behavior; however, a similar prognosis is recorded in tumors with high levels of NF-YAl and overexpressed CCAAT-less genes.

The CCAAT box is recognized by the trimeric transcription factor NF-Y, whose NF-YA subunit is present in two major splicing isoforms, NF-YAl (“long”) and NF-YAs (“short”). Little is known about the expression levels of NF-Y subunits in tumors, and nothing in lung cancer. By interrogating RNA-seq TCGA and GEO datasets, we found that, unlike NF-YB/NF-YC, NF-YAs is overexpressed in lung squamous cell carcinomas (LUSC). The ratio of the two isoforms changes from normal to cancer cells, with NF-YAs becoming predominant in the latter. NF-YA increased expression correlates with common proliferation markers. We partitioned all 501 TCGA LUSC tumors in the four molecular cohorts and verified that NF-YAs is similarly overexpressed. We analyzed global and subtype-specific RNA-seq data and found that CCAAT is the most abundant DNA matrix in promoters of genes overexpressed in all subtypes. Enriched Gene Ontology terms are cell-cycle and signaling. Survival curves indicate a worse clinical outcome for patients with increasing global amounts of NF-YA; same with hazard ratios with very high and, surprisingly, very low NF-YAs/NF-YAl ratios. We then analyzed gene expression in this latter cohort and identified a different, pro-migration signature devoid of CCAAT. We conclude that overexpression of the NF-Y regulatory subunit in LUSC has the scope of increasing CCAAT-dependent, proliferative (NF-YAshigh) or CCAAT-less, pro-migration (NF-YAlhigh) genes. The data further reinstate the importance of analysis of single isoforms of TFs involved in tumor development.

NF-Y is a pioneer trimeric transcription factor formed by the Histone Fold Domain (HFD) NF-YB/NF-YC subunits and NF-YA. Three subunits are required for DNA binding. CCAAT-specificity resides in NF-YA and transactivation resides in Q-rich domains of NF-YA and NF-YC. They are involved in alternative splicing (AS). We recently showed that NF-YA is overexpressed in breast and lung carcinomas. We report here on the overexpression of all subunits in the liver hepatocellular carcinoma (HCC) TCGA database, specifically the short NF-YAs and NF-YC2 (37 kDa) isoforms. This is observed at all tumor stages, in viral-infected samples and independently from the inflammatory status. Up-regulation of NF-YAs and NF-YC, but not NF-YB, is associated to tumors with mutant p53. We used a deep-learning-based method (DeepCC) to extend the partitioning of the three molecular clusters to all HCC TCGA tumors. In iCluster3, CCAAT is a primary matrix found in promoters of up-regulated genes, and cell-cycle pathways are enriched. Finally, clinical data indicate that, globally, only NF-YAs, but not HFD subunits, correlate with the worst prognosis; in iCluster1 patients, however, all subunits correlate. The data show a difference with other epithelial cancers, in that global overexpression of the three subunits is reported and clinically relevant in a subset of patients; yet, they further reinstate the regulatory role of the sequence-specific subunit.

NF-Y is the CCAAT-binding trimer formed by the histone fold domain (HFD), NF-YB/NF-YC and NF-YA. The CCAAT box is generally prevalent in promoters of “cancer” genes. We reported the overexpression of NF-YA in BRCA, LUAD and LUSC, and of all subunits in HCC. Altered splicing of NF-YA was found in breast and lung cancer. We analyzed RNA-seq datasets of TCGA and cell lines of head and neck squamous cell carcinomas (HNSCC). We partitioned all TCGA data into four subtypes, deconvoluted single-cell RNA-seq of tumors and derived survival curves. The CCAAT box was enriched in the promoters of overexpressed genes. The “short” NF-YAs was overexpressed in all subtypes and the “long” NF-YAl in Mesenchymal. The HFD subunits are overexpressed, except Basal (NF-YB) and Atypical (NF-YC); NF-YAl is increased in p53 mutated tumors. In HPV-positive tumors, high levels of NF-YAs, p16 and ΔNp63 correlate with better prognosis. Deconvolution of single cell RNA-seq (scRNA-seq) found a correlation of NF-YAl with Cancer Associated Fibroblasts (CAFs) and p-EMT cells, a population endowed with metastatic potential. We conclude that overexpression of HFD subunits and NF-YAs is protective in HPV-positive tumors; expression of NF-YAl is largely confined to mutp53 tumors and malignant p-EMT cells.