Mechanisms of resistance to oncogenic KRAS inhibition in pancreatic cancer.

KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.

Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-Specific Transcriptome and Molecular Subtype.

Longitudinal monitoring of patients with advanced cancers is crucial to evaluate both disease burden and treatment response. Current liquid biopsy approaches mostly rely on the detection of DNA-based biomarkers. However, plasma RNA analysis can unleash tremendous opportunities for tumor state interrogation and molecular subtyping. Through the application of deep learning algorithms to the deconvolved transcriptomes of RNA within plasma extracellular vesicles (evRNA), we successfully predicted consensus molecular subtypes in patients with metastatic colorectal cancer. Analysis of plasma evRNA also enabled monitoring of changes in transcriptomic subtype under treatment selection pressure and identification of molecular pathways associated with recurrence. This approach also revealed expressed gene fusions and neoepitopes from evRNA. These results demonstrate the feasibility of using transcriptomic-based liquid biopsy platforms for precision oncology approaches, spanning from the longitudinal monitoring of tumor subtype changes to the identification of expressed fusions and neoantigens as cancer-specific therapeutic targets, sans the need for tissue-based sampling. SIGNIFICANCE: The development of an approach to interrogate molecular subtypes, cancer-associated pathways, and differentially expressed genes through RNA sequencing of plasma extracellular vesicles lays the foundation for liquid biopsy-based longitudinal monitoring of patient tumor transcriptomes.

Integrated Molecular Characterization of Intraductal Papillary Mucinous Neoplasms: An NCI Cancer Moonshot Precancer Atlas Pilot Project.

Intraductal papillary mucinous neoplasms (IPMN) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low-grade (LG) to high-grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multiregion sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy-number alterations (CNA) and transcriptional changes that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histologic dysplasia obtained from 23 patients, followed by whole-exome and whole-transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification being associated with HG progression and with cases that harbored co-occurring PDAC. Furthermore, the combined assessment of single-nucleotide variants (SNV) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery as well as pathways related to glycosylation were a common feature of progression to HG. In addition, the established PDAC transcriptional subtypes (basal-like and classical) were readily apparent within IPMNs. Taken together, this work emphasizes the role of 1q copy-number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in noninvasive precursor lesions, and reinforces that evolutionary pathways in IPMNs are heterogenous, comprised of both SNV and CNA-driven events. SIGNIFICANCE: Integrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of IPMNs, which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.

Integrated spatial transcriptomics and lipidomics of precursor lesions of pancreatic cancer identifies enrichment of long chain sulfatide biosynthesis as an early metabolic alteration.

Background: The development of diverse spatial profiling technologies has provided an unprecedented insight into molecular mechanisms driving cancer pathogenesis. Here, we conducted the first integrated cross-species assessment of spatial transcriptomics and spatial metabolomics alterations associated with progression of intraductal papillary mucinous neoplasms (IPMN), bona fide cystic precursors of pancreatic ductal adenocarcinoma (PDAC). Methods: Matrix Assisted Laster Desorption/Ionization (MALDI) mass spectrometry (MS)-based spatial imaging and Visium spatial transcriptomics (ST) (10X Genomics) was performed on human resected IPMN tissues (N= 23) as well as pancreata from a mutant Kras;Gnas mouse model of IPMN. Findings were further compared with lipidomic analyses of cystic fluid from 89 patients with histologically confirmed IPMNs, as well as single-cell and bulk transcriptomic data of PDAC and normal tissues. Results: MALDI-MS analyses of IPMN tissues revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Integrated ST analyses confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1 , and FA2H , were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid identified several sulfatide species, including the C24:0(OH) and C24:1(OH) species, to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Targeting of sulfatide metabolism via the selective galactosylceramide synthase inhibitor, UGT8-IN-1, resulted in ceramide-induced lethal mitophagy and subsequent cancer cell death in vitro , and attenuated tumor growth of mutant Kras;Gnas allografts. Transcript levels of UGT8 and FA2H were also selectively enriched in PDAC transcriptomic datasets compared to non-cancerous areas, and elevated tumoral UGT8 was prognostic for poor overall survival. Conclusion: Enhanced sulfatide metabolism is an early metabolic alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception.

Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential.

Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross-species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while reexpression of Nkx6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis. SIGNIFICANCE: Identification of the molecular features driving IPMN development and differentiation is critical to prevent cancer progression and enhance risk stratification. We used spatial profiling to characterize the epithelium and microenvironment of IPMN, which revealed a previously unknown link between NKX6-2 and gastric differentiation, the latter associated with indolent biological potential. See related commentary by Ben-Shmuel and Scherz-Shouval, p. 1768. This article is highlighted in the In This Issue feature, p. 1749.

Integrative transcriptomic analysis identifies a novel gene signature to predict prognosis of pancreatic cancer in different subtypes.

BACKGROUND: Recent advances on pancreatic cancer molecular classifications have identified several subtypes with distinct characteristics, treatment response, and prognosis. We aim to identify the consensus gene signature that could predict the prognosis of pancreatic cancer. METHODS: Transcriptomic data was acquired from TCGA database. Differentially expressed genes (DEGs) were identified by comparing the Basal-like, Quasi-mesenchymal and Squamous subtype to other subtypes. A new model was constructed by the least absolute shrinkage and selection operator to stratify patients into high and low-risk groups. The prognosis, transcriptomic profiles, and immune infiltration were examined between these groups. RESULTS: We constructed a signature consisting of nine genes, and the GSEA analysis showed that the genomic profile of high-risk tumors is associated with the basal-like and squamous gene set enrichment. Patients with high-risk tumors had worse overall survival (P < 0.001) and progression free survival (P = 0.033), and are associated with a higher expression of KRAS downstream targets such as SDC1, ITGB4 and SLC2A1, which are involved in KRAS mediated macropinocytosis and tumor invasion. Meanwhile, several recurrence-associated genes increased in the high-risk tumors, including ITGA3 and TP63, which have been shown to mediate enhancer-dependent genomic reprogramming towards the squamous phenotype. The tumor immune infiltration profile analysis showed that high-risk tumors are characterized with an immune suppressive microenvironment. CONCLUSION: The integrative transcriptomic analysis identifies a consensus gene signature that can discriminate pancreatic cancer subtypes and determine patient prognosis by evaluating the genomic reprogramming and the level of immune infiltration profile in pancreatic cancer.

Elucidation of Tumor-Stromal Heterogeneity and the Ligand-Receptor Interactome by Single-Cell Transcriptomics in Real-world Pancreatic Cancer Biopsies.

PURPOSE: Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. With molecular subtypes of PDAC gaining relevance in the context of therapeutic stratification, the ability to characterize heterogeneity of cancer-specific gene expression patterns is of great interest. In addition, understanding patterns of immune evasion within PDAC is of importance as novel immunotherapeutic strategies are developed. EXPERIMENTAL DESIGN: Single-cell RNA sequencing (scRNA-seq) is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. RESULTS: Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast subclasses, and predicts for a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. CONCLUSIONS: Our analysis demonstrates that the use of de novo biopsies from patients with PDAC paired with scRNA-seq may facilitate therapeutic prediction from limited biopsy samples.

APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP.

MicroRNA (miRNA) deregulation in prostate cancer (PCa) contributes to PCa initiation and metastatic progression. To comprehensively define the cancer-associated changes in miRNA targeting and function in commonly studied models of PCa, we performed photoactivatable ribonucleoside-enhanced cross-linking immunoprecipitation of the Argonaute protein in a panel of PCa cell lines modeling different stages of PCa progression. Using this comprehensive catalogue of miRNA targets, we analyzed miRNA targeting on known drivers of PCa and examined tissue-specific and stage-specific pathway targeting by miRNAs. We found that androgen receptor is the most frequently targeted PCa oncogene and that miR-148a targets the largest number of known PCa drivers. Globally, tissue-specific and stage-specific changes in miRNA targeting are driven by homeostatic response to active oncogenic pathways. Our findings indicate that, even in advanced PCa, the miRNA pool adapts to regulate continuing alterations in the cancer genome to balance oncogenic molecular changes. These findings are important because they are the first to globally characterize miRNA changes in PCa and demonstrate how the miRNA target spectrum responds to staged tumorigenesis.

SRC-2 orchestrates polygenic inputs for fine-tuning glucose homeostasis.

Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.

Steroid receptor coactivator 1 is an integrator of glucose and NAD+/NADH homeostasis.

Steroid receptor coactivator 1 (SRC-1) drives diverse gene expression programs necessary for the dynamic regulation of cancer metastasis, inflammation and gluconeogenesis, pointing to its overlapping roles as an oncoprotein and integrator of cell metabolic programs. Nutrient utilization has been intensely studied with regard to cellular adaptation in both cancer and noncancerous cells. Nonproliferating cells consume glucose through the citric acid cycle to generate NADH to fuel ATP generation via mitochondrial oxidative phosphorylation. In contrast, cancer cells undergo metabolic reprogramming to support rapid proliferation. To generate lipids, nucleotides, and proteins necessary for cell division, most tumors switch from oxidative phosphorylation to glycolysis, a phenomenon known as the Warburg Effect. Because SRC-1 is a key coactivator responsible for driving a hepatic gluconeogenic program under fasting conditions, we asked whether SRC-1 responds to alterations in nutrient availability to allow for adaptive metabolism. Here we show SRC-1 is stabilized by the 26S proteasome in the absence of glucose. RNA profiling was used to examine the effects of SRC-1 perturbation on gene expression in the absence or presence of glucose, revealing that SRC-1 affects the expression of complex I of the mitochondrial electron transport chain, a set of enzymes responsible for the conversion of NADH to NAD(+). NAD(+) and NADH were subsequently identified as metabolites that underlie SRC-1's response to glucose deprivation. Knockdown of SRC-1 in glycolytic cancer cells abrogated their ability to grow in the absence of glucose consistent with SRC-1's role in promoting cellular adaptation to reduced glucose availability.

Electric field driven torque in ATP synthase.

FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.