Dana-Farber Cancer Institute

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs optimized for therapeutic efficacy. Designed Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from any natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. An IL-2-based-Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without toxicity. Unexpectedly, an IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing striking efficacy in small cell lung cancer and pancreatic cancer models. Trikines obviate the need for cell engineering to customize STAT signatures for immunotherapy.

The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.

Categorical variants, or sets of genomic alterations constrained by shared properties, are pervasive across clinical, regulatory, and research domains in the biomedical ecosystem, yet their inconsistent and non-computable representation hinders data interoperability and clinical interpretation. We surveyed genomic knowledgebases spanning regulatory approvals and the biomedical literature and found that categorical variants underpin a substantial proportion of clinical genomics knowledge, but are largely described using incompatible bespoke models. To address this, we developed the GA4GH Categorical Variation Representation Specification (Cat-VRS), a constraint-based framework that provides a unified computable representation for both precise and intentionally broad categories across molecular and systemic variant domains. Cat-VRS enables harmonization of genomic knowledgebases, computable category-based search, and automated matching between assayed variants and categorical entities in clinical and research contexts. By providing a principled, extensible model for categorical variation, Cat-VRS enables computable reasoning over genomic variant categories and establishes a foundation for the standardized representation and exchange of genomic knowledge.

Although validated prognostic models exist for men with metastatic castration-resistant prostate cancer (mCRPC), current tools do not incorporate genomic biomarkers such as circulating tumor DNA (ctDNA) aneuploidy or pathogenic genetic alterations (PGAs). This study aimed to estimate the prevalence of PGAs in ctDNA, assess their correlation with ctDNA aneuploidy fraction, and evaluate their association with overall survival (OS). Additionally, we developed and validated a clinical-genetic (CG) model to predict OS. We analyzed ctDNA from 776 patients enrolled in the Alliance phase 3 trial (A031201). PGAs were derived using the AR-ctDETECT assay. The net reclassification improvement (NRI) evaluated the added value of the CG model, and the time-dependent area under the receiver operating characteristic curve (tAUC) assessed the accuracy of the OS model. Feature selection using random survival forest identified gains in androgen receptor (AR), AR enhancer, MYC, RSPO2, CCND1, BRAF, and MET; losses or pathogenic variants in ZBTB16, PTEN, MSH6, PPP2R2A, NKX3-1, ZFHX3, TP53, ZNFR3, RB1, FANCA, CHECK1, APC, and CHD1; and a pathogenic AR variant. The CG model significantly outperformed the clinical model, with an average tAUC of 0.77 (95% confidence interval [CI]: 0.73-0.79) for the CG model compared with the tAUC of 0.72 (95% CI: 0.69-0.75, p=0.01) for the clinical model, and NRI of 0.29 between the models. Patients were categorized by the predicted risk scores into poor-, intermediate-, and low-risk groups with median OS of 19.6, 33.6, and 60.8mo, respectively. Incorporation of PGAs from ctDNA into a CG model improved OS prediction by nearly 30% over a clinical model. This model can classify patients into risk groups and is useful for selecting patients in future mCRPC trials.

Prostate cancer progression is characterized by dysregulated lipid metabolism, with fatty acid synthase (FASN), the rate-limiting step in de novo lipogenesis (DNL), resulting in significant accumulation of saturated lipids. Here, we investigate whether pharmacologic FASN inhibition creates a metabolic state that increases reliance on exogenous polyunsaturated fatty acids (PUFAs). Inhibition of FASN profoundly alters membrane phospholipid composition, driving compensatory incorporation of PUFAs into membrane phospholipids, thus increasing susceptibility to lipid peroxidation and oxidative damage. Combined FASN inhibition and PUFA exposure increased reactive oxygen species, induced mitochondrial hyperpolarization, and enhanced lipid peroxidation in both hormone-sensitive and castration-resistant prostate cancer models. Marked inhibition of human and murine prostate cancer organoids is achieved ex vivo . In genetically engineered, DNL-reliant Hi-Myc mice, a diet enriched in PUFAs significantly inhibited invasive carcinoma compared to a saturated fat-enriched diet. Environmental PUFAs modulate and enhance the therapeutic efficacy of FASN-targeted strategies. These findings set the stage for pharmacologic and dietary intervention in prostate cancer patients.