Andrew J. Aguirre

Purpose: Preclinical data suggest that the anti-cancer efficacy of ATR inhibitors can be synergistically enhanced by low-dose gemcitabine. To test the clinical feasibility of this regimen, we conducted a phase 1 trial of low-dose gemcitabine combined with the ATR inhibitor elimusertib (BAY1895344). Patients and Methods: This phase 1 study evaluated the combination of gemcitabine and elimusertib in patients with previously treated, incurable solid tumors. The primary objective was to assess the safety and tolerability of this regimen and to determine the maximum tolerated dose (MTD). Secondary objectives included assessment of objective response rate (ORR) per RECIST v.1.1 and characterization of the pharmacokinetic (PK) profile. Results: Fourteen patients were enrolled; most patients had pancreatic cancer (n=6) or ovarian cancer (n=6). The median age was 63.5 years of age, and the median number of prior therapies was 2. The regimen caused significant toxicity, and four patients (28%) discontinued the trial due to treatment-related adverse events. The trial was stopped prior to establishing an MTD due to the pharmaceutical company's decision to halt development of the agent. A confirmed partial response was observed in one patient with platinum-refractory ovarian cancer harboring a gain in CCNE1. Another patient with pancreatic adenocarcinoma harboring biallelic ATM loss remained on trial for 9.5 months with stable disease, with an 85% decrease in CA19-9. Conclusions: The combination of low-dose gemcitabine combined with elimusertib was not tolerable. Although evidence of anti-tumor activity was observed, significant improvements in tolerability are required for future ATR inhibitor combinations to be clinically feasible.

Purpose: Clonal hematopoiesis (CH) is increasingly recognized as a proinflammatory driver of adverse outcomes in solid tumors. Tumor-infiltrating immune cells with CH (TI-CH) actively promote tumor growth and those harboring TI-CH experience significantly worse survival. We investigated the prevalence and impact of CH on outcomes in patients with treatment-naïve metastatic pancreatic ductal adenocarcinoma (PDAC). Methods: Peripheral blood from 125 patients with metastatic PDAC enrolled in the PASS-01 trial (ClinicalTrials.gov identifier: NCT04469556 ) was evaluated. CH was defined as the presence of pathogenic or likely pathogenic variants with a variant allele frequency (VAF) of ≥1% in a 23 gene panel. Survival was estimated using the Kaplan-Meier method and compared using the log-rank test. Hazard ratios (HRs) were estimated using multivariable Cox proportional hazards regression. Results: CH was detected in 36.8% (46 of 125) of patients, and DNMT3A (60.9%) and TET2 (17.4%) were the most frequently mutated genes. There was no significant difference in progression-free survival (PFS; median, 4.9 v 5.2 months; HR, 1.01 [95% CI, 0.68 to 1.49]; P = .95) and overall survival (OS; median, 9.4 v 9.6 months; HR, 0.99 [95% CI, 0.63 to 1.53]; P = .95) between CH-positive and CH-negative patients, respectively. Among patients with basal-like subtype (n = 23), the presence of CH was associated with a numerically shorter PFS (median, 2.4 v 4.7 months; HR, 2.50 [95% CI, 0.93 to 6.84]; P = .06) and OS (median, 6.2 v 10.2 months; HR, 1.37 [95% CI, 0.51 to 3.69]; P = .52). In contrast, classical subtype showed no significant differences in survival outcomes by CH. Conclusion: CH is prevalent in metastatic PDAC but not independently prognostic. Further examination of CH in basal subtype tumors and the role of TI-CH in PDAC is needed.

Tumor phenotype and therapeutic vulnerability are shaped by dynamic transcriptional states that are not captured by genetic profiling alone and are difficult to monitor longitudinally. Existing plasma-based approaches have largely been developed using samples with high circulating tumor DNA fractions, evaluated across expression quantiles rather than individual genes, and rarely benchmarked head-to-head. Using timepoint-matched plasma epigenomes and tumor transcriptomes, we identify biologically grounded relationships between circulating chromatin, fragmentation, and transcription. H3K4me3 and H3K36me3 encode distinct positional information reflecting promoter activity and transcriptional elongation, while gene-body fragment periodicity predicts expression. We integrate these relationships into APEX, a machine-learning framework for genome-wide single-gene expression inference. Across 559 plasma samples spanning 24 cancer types and clinically relevant tumor fractions, APEX outperforms existing approaches for individual-gene inference, resolves pancreatic cancer transcriptional subtypes, tracks therapeutic targets in small-cell lung cancer, and identifies NECTIN4 expression as a biomarker of response to enfortumab vedotin in urothelial cancer.

KRAS G12C (Gly ¹² →Cys) inhibitors (G12Cis) have improved outcomes for patients with KRAS G12C–mutated non–small cell lung cancer (NSCLC), but their clinical benefit is limited by the emergence of resistance mechanisms. Here, we demonstrate that combining the RAF/MEK clamp avutometinib and the focal adhesion kinase (FAK) inhibitor defactinib with a G12Ci enhanced antitumor activity by deepening mitogen-activated protein kinase (MAPK) pathway suppression and simultaneously inhibiting adaptive resistance pathways induced by G12Ci and avutometinib treatment, including FAK and PI3K signaling. In KRAS G12C NSCLC mouse models, the triplet combination produced greater tumor growth inhibition than either sotorasib alone or the sotorasib plus avutometinib doublet. Moreover, in KRAS G12C NSCLC mouse models resistant to sotorasib, the addition of avutometinib and FAK inhibition restored sensitivity to sotorasib and increased both the depth and durability of antitumor responses. Collectively, these findings demonstrate that concurrent targeting of KRAS G12C, RAF/MEK, and FAK signaling can overcome multiple resistance mechanisms and provide a strong rationale for the clinical evaluation of sotorasib, avutometinib, and defactinib in patients with KRAS G12C–mutated NSCLC (RAMP 203; NCT05074810).

Solid tumors evade immunotherapy because of immunosuppressive microenvironments that limit immune cell persistence and function. Although interleukin-12 (IL-12) potently activates antitumor immunity, its clinical use has been constrained by systemic toxicity. Here we engineered a CAR natural killer (NK) cell platform that integrated IL-12 signaling to enhance antitumor immunity. IL-12 signaling synergized with CAR activation to sustain mTORC1 activity through convergent Ras-ERK and PI3K pathways, promoting metabolic fitness, autonomous expansion, and sustained effector function. IL-12 also activated bystander NK cells, T cells, and macrophages to remodel the tumor microenvironment. To improve safety, IL-12 was tethered to a collagen-binding A3-domain, restricting its activity to the extracellular matrix and limiting systemic exposure. In ovarian and pancreatic cancer models, matrix-anchored IL-12 CAR NK cells expanded without exogenous cytokines and achieved durable tumor control. These findings suggest that mTORC1-mediated integration of CAR and cytokine signaling can enhance immune fitness and overcome immunosuppressive tumor microenvironments.

Daraxonrasib is an orally bioavailable RAS(ON) multi-selective tri-complex inhibitor of the oncogenic mutant and wild-type variants of N, H and KRAS. We previously reported encouraging efficacy in a phase 1/2 clinical trial evaluating daraxonrasib monotherapy at clinically active dose levels in patients with previously treated, RAS mutant metastatic pancreatic adenocarcinoma (PDAC), providing the basis for confirmatory evaluation in the randomized phase 3 RASolute 302 clinical trial. Here we report mechanisms of acquired resistance to daraxonrasib monotherapy observed through targeted sequencing of over 800 genes in paired pretreatment and end of treatment circulating tumor DNA samples from 44 patients in the phase 1/2 clinical trial. Treatment-emergent genomic alterations in the RAS signaling pathway were observed in more than half (26 of 44; 59%) of these patients, including, most notably, mutant KRAS amplifications in one-third (16 of 44; 36%), as well as alterations in receptor tyrosine kinase (RTK) (4 of 44; 9%), MAPK (11 of 44; 25%) and PI3K (4 of 44; 9%) pathways. Notably, no acquired secondary KRAS mutations were observed, distinct from resistance profiles of mutant-selective KRAS G12C(OFF) inhibitors. To corroborate these clinical findings, we found, or mechanistically established, concordant mechanisms of daraxonrasib resistance in human and murine preclinical models of PDAC, including mutant KRAS and MYC amplification and RTK upregulation, with these alterations guiding various combination therapy concepts. Notably, daraxonrasib combined with agents targeting DNA damage response, RTKs or the mutant-selective RAS(ON) G12D inhibitor zoldonrasib averted resistance in preclinical models. Collectively, these results show that most daraxonrasib genomic resistance mechanisms drive reactivation of RAS pathway signaling and guide potential combination strategies in PDAC for further investigation.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer that frequently presents with disseminated disease. The PDA metastatic microenvironment imposes distinct metabolic stressors, potentially generating context-dependent vulnerabilities. Therefore, we employed CRISPR-based genetic screening in a model of PDA liver metastasis to identify novel and possibly targetable liabilities. Remarkably, ferritin heavy chain (FTH1) emerged as the most prominent liver-specific dependency – loss of FTH1 suppressed tumor growth specifically in the liver microenvironment. FTH1 deletion and subsequent disruption of iron handling triggers mitochondrial dysfunction and ionic imbalance, including cytosolic calcium overload. These perturbations result in the activation of a transcriptional program that triggers anti-tumor immunity mediated by immunostimulatory cytokine IL36G. Mechanistically, FTH1 deletion and subsequent ionic imbalance causes decreased protein levels of the tumor suppressor Stk11 (LKB1) which we propose to be mediated by an RNA G-quadruplex located in the 5′-UTR of LKB1. The loss of LKB1 protein levels alters signaling cascades resulting in reduced SIK signaling and inhibition of nonsense mediated decay, ultimately leading to Il36g mRNA stabilization. Taken together, this work elucidates novel ionic disruptions that regulate the translation of LKB1 through a previously undescribed quadruplex in the 5′UTR, altering signaling axes that can be targeted to generate an anti-tumor immune response in PDA.

Identification of 4 groups within TCGA PAAD data.

Phenotypes in acinar cell of origin Arf knockout and double Arf and Trp53 knockout mouse cohorts