Christin E. Burd

Introduction: Depression and anxiety are common in adults with colorectal cancer (CRC), especially during chemotherapy, yet underlying biosocial mechanisms remain unclear. We examined whether biological age predicts these symptoms and whether neighborhood deprivation amplifies risk. Methods: This secondary deidentified electronic health record (EHR) analysis (2017 to 2023) included adults (≥18) with stage II–III CRC receiving chemotherapy (N = 958). Biological age was measured using 9 blood biomarkers (ie, Levine Clinical Biological Age). Depression and anxiety were assessed using PHQ-9 and GAD-7 tools. The Area Deprivation Index (ADI) was used to quantify neighborhood deprivation. Multivariable linear regression examined associations between biological aging and psychological symptoms over 6 months, with Benjamini–Hochberg adjustment (q-values) for multiple testing. Results: Mean biological age was 65.9 years at baseline and increased by 3 years during chemotherapy. Participants reported moderate depression and anxiety symptoms at baseline. In adjusted analyses, increases in biological age were associated with worsening depressive symptoms (B = 0.18, q = 0.048), whereas higher baseline biological age was associated with greater baseline depressive symptom severity (B = 0.31, q = 0.008). Neighborhood deprivation strengthened associations between biological aging and depressive symptoms at baseline and over time, including changes in biological age and age acceleration (all q ≤ 0.049). Conclusions: Blood biomarker–based biological age may indicate elevated risk of psychological distress in CRC, particularly in socioeconomically deprived neighborhoods. Integrating biological age and geospatial context may improve high-risk patient identification and guide targeted mental health care.

Immune checkpoint inhibitors (ICIs) have improved outcomes for patients with melanoma and are now the standard of care for high-risk and advanced disease. However, long-term benefits are observed in only around 25% of patients, with significant risk for immune-related adverse events, highlighting the need for predictive biomarkers. To develop a minimally invasive, pre-treatment biomarker strategy, we profiled functional and subset-specific transcripts in peripheral blood T lymphocytes (PBTLs) and applied machine learning to identify predictive signatures. Patients were enrolled prior to receiving ICI monotherapy in the adjuvant (Exploratory n=61, Validation=78) or metastatic (Exploratory n=48, Validation=46) settings. Following feature selection, random forest models were trained and benchmarked against empirical null models. In the adjuvant setting, CD160 and GZMB predicted recurrence (95th percentile), while treatment-limiting toxicity was predicted by a signature comprising TNFRSF18, VTCN1, TIGIT, CCR4, and AHR (97th percentile). In the metastatic setting, baseline CD45RB, a marker of T-cell differentiation, most strongly predicted progression within one year (93.9th percentile). Distinct signatures in the adjuvant and metastatic settings suggest differences in T-cell programs associated with patient outcomes. These findings support further evaluation of pre-treatment circulating T-cell transcriptional profiles as predictors of ICI response and toxicity in melanoma.

Blood-based DNA methylation is widely used as a molecular surrogate for traits ranging from circulating protein and metabolite levels to biological aging, but whether the CpGs driving these models reflect genuine gene regulation or statistical convenience is unresolved. Training elastic-net models using the expression of 77 T cell genes across 333 matched T-cell enriched blood samples, we show that predictability is governed by regulatory mode: genes associated with differentiation and lineage-identity are strongly predictable, whereas dynamically regulated genes encoding cytokines and immune checkpoint ligands are not. The predictive CpGs are neither at the gene promoter nor at master-regulator binding sites; instead, the signal is carried by gene-specific sets of distal CpGs enriched within T-cell enhancer chromatin states distributed throughout the genome. Methylation-predicted gene expression validated against directly measured NanoString values in an independent 228-sample cancer cohort, remaining highly concordant for lineage genes but only weakly concordant for stimulus-responsive checkpoint ligands, confirming the lineage-versus-stimulus divide independently of the training cohort. On-gene retraining and chromatin-state annotation provide complementary tests of the genomic context of predictive CpGs.

Supplementary Table 1 shows the list of antibodies used in this study. Supplementary Table 2 shows the input data and variables used for computational modeling. Supplementary Table 3 shows parameter estimates and confidence intervals for modeled cell populations. Supplementary Table 4 shows patient demographics, tumor characteristics, and treatment regimens.

Supplementary Figure S1 shows PD-1 blockade therapy combined with NK cell depletion improves tumor control and increases CD8⁺ T cell infiltration. Supplementary Figure S2 shows the combination of NK cell depletion and ICB is most effective when NK cells are depleted at an early stage. Supplementary Figure S3 shows NKRT accumulation is tumor-site specific. Supplementary Figure S4 shows phenotypic changes in CD4⁺ T cells upon NK cell depletion. Supplementary Figure S5 shows NKRTs are antigen-specific and exhibit enhanced tumor-controlling efficacy when combined with ICB. Supplementary Figure S6 shows NKRT formation depends on the timing of NK cell depletion. Supplementary Figure S7 shows NK cell depletion delays CD8⁺ T cell exhaustion in day 15 MC38 tumors. Supplementary Figure S8 shows NK cell depletion delays CD8⁺ T cell exhaustion in day 20 MC38 tumors. Supplementary Figure S9 shows NK cell depletion improves tumor control in the ACT setting. Supplementary Figure S10 shows distinct gene expression profiles in CD8⁺ T cells following NK cell depletion. Supplementary Figure S11 shows volcano plots of differentially expressed genes under specific conditions. Supplementary Figure S12 shows elevated p-STAT3 and p-STAT5 levels in CD8⁺ T cells from NK cell-depleted tumors. Supplementary Figure S13 shows cytokines play a critical role in NKRT formation and anti-tumor activity. Supplementary Figure S14 shows computational model fits of tumor growth curves and CD8⁺ T cell dynamics. Supplementary Figure S15 shows phenotypic variation in tumor-infiltrating NK cells across tumor models. Supplementary Figure S16 shows expression patterns of tissue-resident markers in CD8⁺ T cells and NK cell proportions in responders versus non-responders to immunotherapy.

Immune checkpoint blockers (ICB) targeting the PD-1/PD-L1 axis represent established therapies for many cancers. However, resistance occurs in most patients due to complex immune-suppressive mechanisms in the tumor microenvironment. NK cells can play effector roles in tumor control, but their impact on T-cell dysfunction and ICB efficacy remains controversial. Through genetic and antibody-mediated NK cell depletion, we found that a subset of tumor-associated NK cells plays a negative role in ICB sensitivity; they further impede CD8+ T-cell differentiation toward a CD69+ BCL2+ EOMES+ GZMB+ TIM3− GITR− phenotype. Mechanistically, the retinoic acid receptor α–dependent differentiation program in CD8+ T cells is hindered by tumor-infiltrating NK cells via competition for IFNα and IL-2. Finally, we observed that lower frequencies of NK cells correlate with better clinical responses to ICBs in patients with cancer. These findings suggest potential avenues for enhancing CD8+ T cell–centered immunotherapy by targeting regulatory NK cells. Significance: Although NK cells are traditionally viewed as antitumor effectors, our study uncovers their unexpected suppressive role in CD8+ T cell–based immunotherapy. By competing for cytokines, they disrupt retinoic acid receptor α–driven CD8+ T-cell differentiation and limit ICB efficacy. Clinically, reduced NK cell presence is associated with an enhanced immunotherapy response. See related article by Pozniak et al. p. XX

Aside from the canonical role of PDL1 as a tumour surface-expressed immune checkpoint molecule, tumour-intrinsic PDL1 signals regulate non-canonical immunopathological pathways mediating treatment resistance whose significance, mechanisms, and therapeutic targeting remain incompletely understood. Recent reports implicate tumour-intrinsic PDL1 signals in the DNA damage response (DDR), including promoting homologous recombination DNA damage repair and mRNA stability of DDR proteins, but many mechanistic details remain undefined. We genetically depleted PDL1 from transplantable mouse and human cancer cell lines to understand consequences of tumour-intrinsic PDL1 signals in the DNA damage response. We complemented this work with studies of primary human tumours and inducible mouse tumours. We developed novel approaches to show tumour-intrinsic PDL1 signals in specific subcellular locations. We pharmacologically depleted tumour PDL1 in vivo in mouse models with repurposed FDA-approved drugs for proof-of-concept clinical translation studies. We show that tumour-intrinsic PDL1 promotes the checkpoint kinase-2 (Chk2)-mediated DNA damage response. Intracellular but not surface-expressed PDL1 controlled Chk2 protein content post-translationally and independently of PD1 by antagonising PIRH2 E3 ligase-mediated Chk2 polyubiquitination and protein degradation. Genetic tumour PDL1 depletion specifically reduced tumour Chk2 content but not ATM, ATR, or Chk1 DDR proteins, enhanced Chk1 inhibitor (Chk1i) synthetic lethality in vitro in diverse human and murine tumour models, and improved Chk1i efficacy in vivo. Pharmacologic tumour PDL1 depletion with cefepime or ceftazidime replicated genetic tumour PDL1 depletion by reducing tumour Chk2, inducing Chk1i synthetic lethality in a tumour PDL1-dependent manner, and reducing in vivo tumour growth when combined with Chk1i. Our data challenge the prevailing surface PDL1 paradigm, elucidate important and previously unappreciated roles for tumour-intrinsic PDL1 in regulating the ATM/Chk2 DNA damage response axis and E3 ligase-mediated protein degradation, suggest tumour PDL1 as a biomarker for Chk1i efficacy, and support the rapid clinical potential of pharmacologic tumour PDL1 depletion to treat selected cancers.

This study examined how gut microbiota diversity and richness relate to T cell aging among 96 healthy adults of all ages. It also explored whether these links differed throughout the lifespan. Peripheral blood was obtained from 96 study participants (N=96, aged 21-72) to assess mRNA markers of T cell aging (p16^ink4a, p14^ARF, B3gat1, Klrg1) and DNA methylation. T cell aging mRNA markers were combined into an aging index and the Horvath epigenetic clock algorithm was used to calculate epigenetic age based on DNA methylation status of over 500 loci. Participants also collected a stool sample from which the V4 region of the 16S rRNA gene was sequenced to derive the Shannon and Simpson diversity indices, and the total count of observed operational taxonomic units (richness). Models controlled for BMI, comorbidities, sex, dietary quality, smoking status, physical activity, and sleep quality. Lower microbiota richness was associated with higher T cell age based on mRNA markers, but when probing the region of significance, this relationship was only significant among adults 45 years and older (p=.03). Lower Shannon diversity (p=.05) and richness (p=.07) marginally correlated with higher epigenetic age (i.e., greater T cell DNA methylation). Gut microbiota complexity may correspond with the rate of T cell aging, especially in mid-to-late life. These results suggest an interplay between the gut microbiome and immunological aging that warrants further experimental work.

An increasing number of cancer subtypes are treated with front‐line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University‐Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte‐specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non‐burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune‐based therapies.

Overall, some 30% of human cancers are driven by mutant RAS proteins, which have proven relatively difficult to target. While immunotherapy is effective for NRAS-mutant melanoma, no options exist for resistant disease, and mutant KRAS-driven lung and pancreatic carcinomas are much less responsive overall. Oncogenic pathways are often key targets for inhibition. However, pathway agonism has not been systematically explored as a therapeutic approach. BRAF-mutant melanomas respond to BRAF inhibitors (BRAFi) due to decreased ERK signaling. They can recover signaling by acquiring activating RAS/MEK mutations, but exhibit decreased proliferation in the absence of inhibitor, suggesting that supraphysiologic ERK signaling also compromises fitness. In line with this, we observed that increasing ERK hyperactivation in RAS-mutant cancers, particularly in those with a high ERK activity, might elevate ERK signaling to a degree to induce senescence and also create an inflammatory tumor microenvironment. We have shown that a majority of RAS-mutant cancer cell lines we tested undergo senescence-like arrest following exposure to all FDA-approved BRAF inhibitors at clinically relevant doses. Simultaneous MEK or ERK inhibition allows the cells to recover proliferation, and a BRAFi which does not induce ERK activation fails to have any effect, confirming that ERK hyperactivation is the key driver of this response. Importantly, these findings generalize across NRAS-mutant and KRAS-mutant cancer cell lines including in fresh human pancreatic adenocarcinoma explants. Finally, our data in two novel genomically-characterized, immunocompetent models of NRAS-mutant melanoma and KRAS-mutant pancreatic adenocarcinoma support our in-vitro findings. Both RAS-mutant murine models demonstrate that anti-PD1 therapy is substantially more efficacious when mice are additionally treated with BRAFi. Molecular profiling of the tumor microenvironment reveals the expression of cytokines related to senescence and marked infiltration of activated CD8+ T-cells. In order to implement more potentially effective immunotherapies, we have analyzed the expression of different immune-checkpoint molecules and we have observed a high expression of LAG3 and TIM3 in the melanoma NRAS-mutant model. We will further analyze the effect of those checkpoints in combination with BRAFi to potentiate the anti-tumoral responses and tumor regression. We propose that oncogenic pathway agonism is a novel, effective, and untested strategy to induce proliferation arrest and sensitization to immunotherapy. The impact of this data is in the ability to broadly sensitize RAS-mutant cancers to immunotherapy in the settings of de-novo and acquired resistance. The direct connection between a tumor cell signaling vulnerability to immunotherapy response is a major point of novelty and provides a clear molecular rationale for pursuing combined targeted and immune-based therapy. Citation Format: Alvaro de Mingo Pulido, Charles Adelmann, Brittney Sell, Karol Prieto, Kimberly Nguyen, Cynthia Dixey, Ivannie Ortiz-Rivera, Marco Napoli, Elsa Flores, Jason Fleming, Karen Mann, Christin Burd, Kenneth Y. Tsai. Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A010.