Citations

Abstract Natural killer (NK) cells represent key effectors of antitumor immunity, yet emerging evidence highlights populations with distinct roles in cancer. Despite such expanded diversity within the NK cell repertoire, we lack an understanding of how this heterogeneity affects immune responses and downstream clinical outcomes. Using single-cell RNA sequencing, we systematically profiled NK cells across cancer and uncovered a dichotomous phenotypic and functional landscape of tumor-infiltrating NK cells shaped by opposing intrinsic signaling programs that drive the expression of IFNG or TGFB1. These divergent programs are associated with distinct transcription factor circuits that integrate cues within the tumor microenvironment and skew NK cells toward proinflammatory or suppressive functions. We found that the capacity for NK cells to engage in either functional direction is intrinsically linked to their phenotypic identity. Canonical NK cells recruited from circulation predominantly directed suppressive TGFB1 signals toward effector CD8+ T cells in tumors. Of note, these subsets exhibited higher TGFB1 expression than intratumoral myeloid cells across tumor types. In contrast, a tissue-resident (TR) adaptive subset exhibited exclusively proinflammatory IFNG-driven profiles and was associated with prolonged survival in both primary and metastatic tumor settings. Moreover, these TR adaptive NK cells, but not other subsets, were linked to response to immune checkpoint blockade. Collectively, our study reveals a previously unrecognized regulatory axis in NK cells that shapes NK cell diversity and augments broader antitumor immune responses.

e14555 Background: Natural killer (NK) cells are innate lymphocytes that engage in direct cytotoxic responses against malignant cells and promote pro-inflammatory tumor microenvironments. While studies have predominantly focused on conventional NK cell subsets, recent findings uncovered adaptive and tissue-resident (TR) subsets with unique functional properties. The influence of distinct NK cell subsets on tumor immunobiology and clinical outcomes remains unknown. Methods: Single-cell RNA-sequencing (scRNA-seq) of primary, therapy-naïve tumors from 276 patients across 14 cancer types, as well as 39 patients treated with immune checkpoint blockade (ICB), was accessed through publicly available datasets. We employed systematic computational analyses to phenotype and examine intra-tumoral NK cells. Gene signatures for NK cell subsets were applied to bulk RNA-sequencing from 212,105 tumor samples to delineate their associations with clinical outcomes using the Caris CODEai database. Results: Six main subsets of NK cells were identified in solid tumors, including conventional CD56 dim and CD56 bright NK cells, alongside adaptive, TR adaptive, TR CD56 bright , and TR CD56 bright /adaptive subsets. Across cancer, TR adaptive NK cells emerged as the most prognostic subset, with high infiltration conferring significantly longer overall survival (HR[95% CI] = 0.68[0.66-0.69], p < 0.001) than conventional CD56 dim and CD56 bright subsets. This was also observed in cohorts of head and neck, non-small cell lung, and esophageal cancers, particularly those treated with ICB. Notably, elevated TR adaptive NK cell infiltration was more prognostic than high tumor mutational burden in these settings. This was most apparent in ICB-treated head and neck cancers (HR = 0.48[0.41-0.57], p < 0.001 vs HR = 0.90[0.76-1.04], p = 0.16). Additionally, TR adaptive NK cells were strongly correlated to the infiltration of CD8 + T cells and B cells in tumors. Underlying these observations, our scRNA-seq analyses demonstrated that TR adaptive NK cells exhibit the highest immunomodulatory activity of all subsets, expressing high levels of pro-inflammatory cytokines ( CCL3 , CXCL13 , and IFNG ) and low levels of immunosuppressive cytokines ( TGFB1 ). Mechanistically, this activity was linked to the upregulation of transcription factors associated with NK cell activation, function, and persistence and downregulation of genes related to stress response and exhaustion. Interestingly, scRNA-seq from paired pre- and post-ICB-treated tumors revealed a unique expansion of cytotoxic GZMB + TR adaptive NK cells after treatment. Conclusions: TR adaptive NK cells represent a novel subset of NK cells that may drive anti-tumor immune responses, particularly in the context of ICB treatment. Features of this unique population may be harnessed to design more effective NK cell-based therapies against solid tumors.

Abstract Purpose: Germline alterations in homologous recombination repair (gHRR) genes affect the pathogenesis, treatment options, and survival of patients with cancer. However, distinct gHRR gene alterations may differentially affect treatment response and oncogenic signaling. In this study, we interrogated genomic and transcriptomic data and assessed clinical outcomes of patients with gHRR mutations across four BRCA-associated cancers (breast, ovarian, pancreatic, and prostate cancers) to identify therapeutic vulnerabilities. Experimental Design: We assessed 24,309 patients undergoing matched tumor/normal next-generation DNA and RNA sequencing. Annotated gHRR gene variants [germline BRCA1, germline BRCA2, germline PALB2, germline ATM (gATM), and germline CHEK2] were analyzed. HRs were used to assess survival outcomes comparing germline versus sporadic groups. Somatic alterations and their frequencies were compared across gHRR-altered groups. Differential gene expression and gene set enrichment analysis were used to compare transcriptomic profiles. Results: Somatic TP53 mutations were depleted in gATM carriers (P &lt; 0.05) across all four BRCA-associated cancers by up to 2.5-fold. Tumors with germline BRCA1/2 mutations were associated with improved survival in patients with ovarian cancer and had consistent enrichment of TP53 mutations in all four cancers. gATM mutations displayed elevated p53 transcriptional activity in all four cancers, with significance reached in breast and prostate cancers (P &lt; 0.01). In breast, ovarian, and prostate cancers, gATM tumors demonstrated significantly increased inflammatory pathways (P &lt; 0.001). Finally, using gene dependency data, we found that cell lines that were highly dependent on ATM were co-dependent on canonical p53 function. Conclusions: gATM-associated cancers seem to require intact p53 activity and this synthetic essentiality may be used to guide targeted therapies that perturb canonical TP53 function.

Abstract Purpose: Around 10% to 15% of prostate cancers harbor recurrent aberrations in the Forkhead Box A1 gene, FOXA1, whereby the alteration type and the effect on the forkhead (FKH) domain affect protein function. We developed a FOXA1 classification system to inform clinical management. Experimental Design: A total of 5,014 prostate cancer samples were examined using whole-exome and -transcriptome sequencing from the Caris Life Sciences database. We denoted class 1 FOXA1 alterations as missense and in-frame insertions/deletions with subclasses oriented with respect to the FKH domain. These were in the first part of the FKH domain [class 1A: amino acids (AA) 168–246], within the Wing2 region of FKH (class 1B: AA 247–269), or outside FKH (class 1C: AA 1–167, 270+). Two hotspot missense mutations at R219 were denoted class 2. Class 3 included predicted truncating mutations with subclasses partitioned based on the FKH domain (class 3A: AA 1–269 and class 3B: AA 270+). Class 4 represented FOXA1 amplifications. Real-world overall survival and therapy outcomes were determined from insurance claims. Results: FOXA1 alterations did not influence survival when considered in aggregate but had distinct prognostic effects when stratified by class. In primary prostate samples, class 1A alterations were associated with overall improved survival (HR, 0.57; P = 0.03); a similar trend was seen in metastatic biopsies with class 1B (HR, 0.84; P = 0.09). Conversely, in primary specimens, class 1C exhibited worse survival upon second-generation androgen receptor signaling inhibitor treatment (HR, 1.93; P &lt; 0.001). Class 2 mutations (R219C/S) were enriched in neuroendocrine prostate cancers and were associated with overall poor survival (HR, 2.05; P &lt; 0.001) and worse outcomes to first-line androgen-deprivation therapies (HR, 2.5; P &lt; 0.001). Class 3A alterations indicated improved survival (HR, 0.70; P = 0.01), whereas class 3B alterations portended poor outcomes (HR, 1.50; P &lt; 0.001). Amplifications (class 4) indicated poor outcomes in metastatic samples (HR, 1.48; P = 0.02). Molecularly, different FOXA1 alteration classes harbored distinct mutational and immunologic features as well as unique transcriptional programs. Finally, relative to European Americans, African Americans had increased class 1C alterations, whereas Asian/Pacific Islander patients had increased class 1B alterations. Conclusions: FOXA1 alterations should not be interpreted in aggregate, as different classes are associated with divergent molecular features and clinical outcomes. Our revised classification schema facilitates clinical decision-making for patients with prostate cancer and uncovers important racial differences.