Cancer Research

ABSTRACTOverexpression of PHGDH, the rate-limiting enzyme in the serine synthesis pathway, promotes melanomagenesis, melanoma cell proliferation, and survival of metastases in serine-low environments such as the brain. While PHGDH amplification explains PHGDH overexpression in a subset of melanomas, we find that PHGDH levels are universally increased in melanoma cells due to oncogenic BRAFV600E promoting PHGDH transcription through mTORC1-mediated translation of ATF4. Importantly, PHGDH expression was critical for melanomagenesis as depletion of PHGDH in genetic mouse models blocked melanoma formation. Despite BRAFV600E- mediated upregulation, PHGDH was further induced by exogenous serine restriction. Surprisingly, BRAFV600E inhibition diminished serine restriction-mediated PHGDH expression by preventing ATF4 induction, creating a potential vulnerability whereby melanoma cells could be specifically starved of serine by combining BRAFV600E inhibition with exogenous serine restriction. Indeed, we show that this combination promoted cell death in vitro and attenuated melanoma growth in vivo. This study identified a melanoma cell-specific PHGDH-dependent vulnerability.

KRAS is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC), where hotspot alterations in codons 12, 13, and 61 drive tumor initiation and progression. Although distinct biochemical properties have been described for individual KRAS mutants, whether they generate unique allele-specific signaling programs in PDAC cells remains unresolved. Here, we systematically interrogated the molecular consequences of seven common KRAS mutant variants in reconstituted isogenic, KRAS-deficient PDAC cell lines by integrated transcriptomic, proteomic, and phosphoproteomic profiling. We found that baseline cellular state, rather than allele identity, was the predominant driver of molecular variation. Comparisons with established KRAS reference signatures revealed significant but moderate overlap at the mRNA level and less so at the proteome level. Pathway analyses highlighted interferon response and mitochondrial translation as recurrently altered across alleles, while phosphoproteomic data confirmed robust ERK1/2 activity and suppression of DYRK kinase substrates by mutant KRAS expression. Importantly, no robust allele-specific molecular programs were identified. Together, our study establishes a comprehensive multi-omics resource for KRAS signaling in PDAC and demonstrates that cellular context exerts a stronger influence than allele identity in shaping molecular profiles, with implications for interpreting putative allele-specific signaling dependencies and therapeutic vulnerabilities.

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

RAP guanine exchange factors (RAPGEF3/4) also known as EPAC1/2 (Exchange Protein Activated by cyclic AMP) are important signaling proteins. In cutaneous melanoma, we reported that loss of dependency on RAPGEF3/4 is associated with metastatic progression. Here, we investigated the molecular mechanisms underlying EPAC1/2 signaling in melanoma. Using transformed human melanocytes, chemical inhibition and genetic deletion of EPAC in Braf/Pten mice, we show that EPAC activation is an early event in melanomagenesis and is required for the growth of transformed melanocytes in vitro and melanomagenesis in vivo. Query of the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of melanoma tumors showed that low EPAC mRNA and RAP1-GTP protein correlate with better diseases free survival of patients with primary melanoma. RNAseq analysis of patient-matched primary and metastatic melanoma cells treated with EPAC inhibitor ESI-09 revealed that TXNIP, an important regulator of redox homeostasis, is a downstream effector of EPAC-RAP1 signaling. Our data also show that EPACs promote melanoma growth by regulation of redox homeostasis and mitochondrial reactive oxygen species through activation of mechanistic target of rapamycin complex 1 (mTORC1) that stabilizes hypoxia-inducible factor 1-alpha (HIF-1), a transcriptional activator of TXNIP and glycolytic enzymes. Our data suggest that targeting mechanisms that metastatic melanoma cells employ to bypass EPAC dependency as a potential therapeutic approach for melanoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/696903v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1b0bc1eorg.highwire.dtl.DTLVardef@e8499org.highwire.dtl.DTLVardef@1237175org.highwire.dtl.DTLVardef@1edbd02_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pancreatic ductal adenocarcinoma (PDAC) arises in a nutrient-deprived microenvironment through progressive stages from pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma. While serine metabolism supports tumor growth across multiple cancer types, the stage-specific role of de novo serine synthesis in PDAC evolution remains undefined. Here, we show that expression of phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of serine biosynthesis, increases progressively from PanIN to invasive PDAC in human and mouse specimens. Using genetically engineered mouse models with inducible PHGDH knockdown, we found that PHGDH loss delayed PDAC development. Unexpectedly, PHGDH-deficient tumors did not increase reliance on exogenous serine, and dietary serine/glycine manipulation had no effect on tumor development. Instead, stable isotope tracing and metabolomic profiling revealed that PHGDH loss suppressed mTOR signaling, reduced expression of the glutamine transporter ASCT2, and impaired glutamine uptake and utilization. Leveraging this metabolic liability, we demonstrated that PHGDH-deficient tumors exhibited selective sensitivity to the glutamine antagonist DRP-104, whereas PHGDH-intact tumors were resistant. These findings reveal an unanticipated connection between serine biosynthesis and glutamine metabolism in PDAC and identify a therapeutic vulnerability that may be exploited through combined metabolic targeting. Statement of significancePHGDH supports PDAC progression not primarily through serine provision, but by maintaining glutamine metabolism and mTOR signaling. This unanticipated metabolic crosstalk creates a synthetic lethal vulnerability to glutamine antagonism in PHGDH-deficient tumors, providing a rationale for combining serine synthesis pathway inhibitors with glutamine-targeting therapies in pancreatic cancer.

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. Our in vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, we observed treatment-independent enhancement of tumor cell proliferation by fibroblast-produced secreted factors. Using spatial statistics analyses, we found that proximity to stroma is often associated with enhanced tumor cell proliferation in vivo. Based on these observations, we hypothesized an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate the feasibility of this hypothesis, we developed a spatial agent-based model of stroma impact on proliferation/death dynamics. The model was quantitatively parameterized using inferences from histological analyses and experimental studies. We found that the observed enhancement of tumor cell proliferation within stroma-proximal niches can enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, our study supports the existence of a novel, indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes.

High-grade serous ovarian carcinoma (HGSC) is an aggressive malignancy for which bulk transcriptomic subtypes are used to stratify tumors, interpret biology, and guide biomarker development. The four TCGA-derived subtypes, mesenchymal (C1.MES), immunoreactive (C2.IMM), proliferative (C5.PRO), and differentiated (C4.DIF), are consistently observed across cohorts. However, despite their prominence, these subtypes have not translated into therapeutic utility, and their biological basis remains unresolved. Here, we show that HGSC transcriptomic subtypes are largely determined by tumor cellular composition rather than intrinsic malignant transcriptional programs. By integrating controlled single-cell-derived pseudobulk simulations with deconvolution-based analysis of 1,834 primary HGSC tumors across RNA-seq and microarray cohorts, we demonstrate that subtype probabilities align along a composition-driven axis of stromal and immune variation. Cellular composition alone predicted subtype labels with high accuracy (ROC-AUC = 0.81-0.95) and explained a substantial fraction of subtype-associated transcriptomic variation, with the mesenchymal (C1.MES) subtype representing the most robust and reproducible example of composition-driven signal. Although a secondary, composition-independent expression signal is detectable, it does not define the dominant structure of subtype classification. These findings redefine HGSC transcriptomic subtypes as features of the tumor ecosystem rather than discrete malignant states. This reinterpretation has immediate implications for studies that use subtype labels to infer tumor-intrinsic biology and provides a generalizable framework for separating composition-driven and intrinsic signals in bulk tumor data. Significance StatementHGSC transcriptomic subtypes lack consistent clinical utility and remain biologically ambiguous. We show subtype assignments are largely driven by tumor cellular composition, and less so by distinct intrinsic tumor states.

BRCA1 promoter methylation predicts sensitivity to PARP inhibitors in high-grade serous ovarian cancer, yet therapeutic resistance is common and mechanistically unresolved. Using long-read direct DNA sequencing of patient-derived xenografts and cell lines, we resolved BRCA1 methylation at single-molecule resolution with structural and transcriptomic analyses. We revealed two convergent PARP inhibitor and platinum resistance mechanisms, validated in patient tumors. First, focal, allele-specific loss of BRCA1 methylation arose through local cis-acting genomic alterations, instead of global epigenetic reprogramming. Engineered in cis sequence alterations near the methylated BRCA1 promoter were sufficient to induce methylation loss, restore homologous recombination, and confer resistance. Similar associations were observed across the genome, suggesting this mechanism extends beyond BRCA1. Second, BRCA1 expression was restored despite intact promoter methylation via structural variant-mediated promoter bypass or alternative transcription initiation. Together, these findings redefine BRCA1 methylation loss as a locus-restricted process and reveal multiple routes by which tumors escape PARP inhibitor therapy. Statement of SignificanceWe show that high-grade serous ovarian cancers can restore BRCA1 expression after therapy through multiple genomic mechanisms, including local methylation loss and promoter bypass, thereby re-establishing homologous recombination and driving PARP inhibitor resistance. These findings challenge reliance on BRCA1 methylation alone as a predictive biomarker and support rational combination therapies for more durable responses.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the HER2V777L; PIK3CAH1047Rtransgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers show accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancers have resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by neratinib plus trastuzumab deruxtecan. Proteomic and RNA-Seq analysis of HP breast cancers showed increased gene expression of Cyclin D1 and p21WAF1/Cip1 and changes in cell cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has very similar HER2 and PIK3CA mutations. Both of these drug combinations are being tested in phase 1 clinical trials and this study provides valuable preclinical evidence for them.

We previously reported that the phosphate exporter XPR1 is required to prevent toxic phosphate accumulation in ovarian cancer cells. To guide therapeutic development, we sought to systematically compare potential strategies to inhibit XPR1: directly targeting the phosphate efflux channel, targeting its partner protein KIDINS220, or inhibiting the synthesis of inositol pyrophosphates (PP-InsPs), metabolites which activate XPR1. We evaluated functional domains in XPR1 and KIDINS220 using mutational scanning and found that loss of function mutations in XPR1 clustered in distinct regions throughout the protein, with the most deleterious mutations in the PP-InsP-binding domain. In contrast, loss of function mutations in KIDINS220 were infrequent and altered the localization of XPR1, consistent with a scaffolding role for KIDINS220. These data highlight the functional relevance of PP-InsPs, which we confirmed by inhibiting their synthesis using IP6K inhibitors. We demonstrate that IP6K inhibition phenocopies XPR1 inhibition across hundreds of cancer cell lines, with the mechanism of sensitivity solely due to inhibition of cellular phosphate efflux. Finally, we show that IP6K inhibitors decrease tumor burden in xenograft models of ovarian cancer, but that the rapid resynthesis of PP-InsPs requires high exposures to achieve efficacy. This study comprehensively evaluates the XPR1-dependent phosphate efflux network and reinforces the concept of directly targeting XPR1 as a precision medicine strategy to benefit patients with ovarian cancer.

The diversity of structural variants (SVs) in melanoma and how they impact oncogenesis are incompletely known. We performed harmonized analysis of SVs across melanoma histological and genomic subtypes, and we identified distinct global properties between subtypes. These included the frequency and size of SVs and SV classes, their relation to chromothripsis events, and the role of topologically associated domain (TAD) boundary altering SVs on cancer-related genes. Following our prior identification of double-stranded break repair deficiency in a subset of triple wild-type cutaneous melanoma, we identified MRE11 and NBN loss-of-function SVs in melanomas with this mutational signature. Experimental knockouts of MRE11 and NBN, followed by olaparib cell viability assays in melanoma cells, indicated that dysregulation of each of these genes may cause sensitivity to PARPi in cutaneous melanomas. Broadly, harmonized analysis of melanoma SVs revealed distinct global genomic properties and molecular drivers, which may have biological and therapeutic impact. Statement of SignificanceThe diversity of SVs in melanoma, and how they directly or indirectly impact oncogenesis, are incompletely known. Here we present analysis of melanoma SVs that reveal distinct global genomic properties and molecular drivers, some of which point to opportunities for further biological and therapeutic investigation.

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient with BRAFV600E/K melanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository of patient-derived xenograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these R-PDXs, we executed in vivo compound repurposing screens using 11 FDA-approved agents from an NCI-portfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in [~]70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phospho-AKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial. SINGLE SENTENCE SUMMARYBroad target inhibition effective as a salvage strategy in BRAF/MEK inhibitor-acquired resistance PDX

Background & AimsPancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma, which contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. MethodsWe used a dual recombinase mouse model to delete Hif1 or Hif2 in -smooth muscle actin (SMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2 expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. ResultsCAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. ConclusionsTogether, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Here, we show that hypoxia drives especially long-lasting epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) primarily through a positive-feedback histone methylation-MAPK signaling axis. We find that transformed cells preferentially undergo EMT in hypoxic tumor regions in multiple model systems and that hypoxia drives a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, can last for weeks. We further demonstrate that hypoxia reduces histone demethylase KDM2A activity, suppresses PP2 family phosphatase expression, and activates MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors play only a supporting role. This mechanism can be antagonized in vivo by combinations of MAPK inhibitors that may be effective in multi-drug therapies designed to target EMT.

Adaptive therapy is an evolution-based treatment approach that aims to maintain tumor volume by employing minimum effective drug doses or timed drug holidays. For successful adaptive therapy outcomes, it is critical to find the optimal timing of treatment switch points. Mathematical models are ideal tools to facilitate adaptive therapy dosing and switch time points. We developed two different mathematical models to examine interactions between drug-sensitive and resistant cells in a tumor. The first model assumes genetically fixed drug-sensitive and resistant populations that compete for limited resources. Resistant cell growth is inhibited by sensitive cells. The second model considers phenotypic switching between drug-sensitive and resistant cells. We calibrated each model to fit melanoma patient biomarker changes over time and predicted patient-specific adaptive therapy schedules. Overall, the models predict that adaptive therapy would have delayed time to progression by 6-25 months compared to continuous therapy with dose rates of 6%-74% relative to continuous therapy. We identified predictive factors driving the clinical time gained by adaptive therapy. The first model predicts 6-20 months gained from continuous therapy when the initial population of sensitive cells is large enough, and when the sensitive cells have a large competitive effect on resistant cells. The second model predicts 20-25 months gained from continuous therapy when the switching rate from resistant to sensitive cells is high and the growth rate of sensitive cells is low. This study highlights that there is a range of potential patient specific benefits of adaptive therapy, depending on the underlying mechanism of resistance, and identifies tumor specific parameters that modulate this benefit.

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To test this, we mutated the substrate binding site of Glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative branch of the pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors but when these tumors spontaneously metastasized the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species (ROS), decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing the production of NADPH through glutaminolysis. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutamine as compared to control melanomas. The oxidative pentose phosphate pathway and compensatory glutaminolysis thus confer layered protection against oxidative stress during metastasis. SignificanceMelanoma metastasis is limited by oxidative stress. Cells that enter the blood experience high levels of ROS and usually die of ferroptosis. We found that melanoma cells become more dependent upon the oxidative branch of the pentose phosphate pathway to manage oxidative stress during metastasis. When pentose phosphate pathway function was disabled by G6PD mutation, the melanoma cells increased their utilization of malic enzyme, fueled by increased consumption of glutamine in the tricarboxylic acid cycle. Melanoma cells thus have redundant and layered protection against oxidative stress.

The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment contains hypoxic tissue subdomains and cancer-associated fibroblasts (CAFs) of multiple subtypes that play tumor-promoting and -restraining roles. Here, we demonstrate that hypoxia promotes an inflammatory-like CAF phenotype and that hypoxic CAFs selectively promote epithelial-mesenchymal transition (EMT) in PDAC cancer cells through growth factor-mediated cell crosstalk. By analyzing patient tumor single-cell transcriptomics and conducting an inhibitor screen, we identified IGF-2 and HGF as specific EMT-inducing growth factors produced by hypoxic CAFs. We further found that reactive oxygen species-activated NF-{kappa}B cooperates with hypoxia-dependent histone methylation to promote IGF-2 and HGF expression in hypoxic CAFs. In lineage-traced autochthonous PDAC mouse tumors, hypoxic CAFs resided preferentially near hypoxic, mesenchymal cancer cells. However, in subcutaneous tumors engineered with hypoxia fate-mapped CAFs, once-hypoxic re-oxygenated CAFs lacked a spatial correlation with mesenchymal cancer cells. Thus, hypoxia promotes reversible CAF-malignant cell interactions that drive EMT through druggable signaling pathways. One-sentence summaryWe show that hypoxic fibroblasts in pancreas cancer leverage histone methylation and ROS-mediated NF-{kappa}B activation to produce growth factors that drive epithelial-mesenchymal transition in malignant cells, demonstrating how tumor stromal features cooperate to initiate a signaling process for disease progression.

The molecular pathways involved in the response to radiation therapy in pancreatic ductal adenocarcinoma (PDAC) remain poorly understood. We aimed to elucidate the adaptive mechanisms and cellular interactions within PDAC to radiation therapy (RT). We constructed a transcriptomic landscape of the cellular subtypes and spatially resolved neighborhoods from 50 patient samples, including 16 longitudinally matched single cell RNA sequencing and 34 spatial transcriptomics specimens. To resolve shortcomings of cell-type mixtures in spatial data, we developed a novel statistical method called SpaCCI (spatially aware analysis of cell-cell interactions) to profile cell-cell interactions and ligand-receptor enrichment. This revealed CXCL12/TGF{beta}-driven persister cell niches where activated fibroblasts reprogram tumor- associated macrophages and spatially exclude stress-response CD8 T cells after RT. Persister cancer cells displayed transcriptional evidence of recalcitrance to metal-induced cell death pathways of ferroptosis and cuproptosis which were recapitulated in preclinical models. Our study reveals the selective pressures experienced by PDAC following RT that may help provide insight for future multimodal therapeutic strategies.

PurposeImmune checkpoint inhibitors (ICI) have demonstrated promising therapeutic benefit although a majority will not respond. Here we identify and validate predictive biomarkers from plasma-derived exosomes that allow non-invasive monitoring of tumor intrinsic and host immune status and prediction of ICI success.\n\nExperimental DesignTranscriptomic profiling of peripheral blood bulk exosomes and tumors from a discovery cohort of 50 patients with metastatic melanoma treated with ICI was undertaken; a further validation cohort of 30 patients was utilized to validate findings from the discovery cohort. We designed a Bayesian probabilistic model to partition bulk exosomes into tumor-specific and non-tumor-specific proportions.\n\nResultsExosomal RNA signatures exhibit significant correlations with tumor transcriptomes. Exosomal profiles reflect several key biological drivers of ICI resistance or melanoma progression, exhibit significantly differentially expressed genes and pathways, and correlate with and are predictive of clinical response to therapy. Our deconvolution model estimates contributions from tumor and non-tumor sources, enabling more precise interpretation of differentially-expressed genes and pathways. Exosomal RNA-seq mutational information can be used to segregate responders and non-responders.\n\nConclusionsPeripheral blood-derived exosomes can serve as a non-invasive biomarker to jointly probe tumor-intrinsic and immune changes to ICI, and can potentially function as predictive markers of ICI responsiveness and a monitoring tool for tumor persistence and immune activation.\n\nStatement of SignificanceWe use transcriptomic analysis of bulk, non-selected, peripheral blood derived exosomes to reveal both tumor-intrinsic and immune-derived signatures predictive of early response to immune checkpoint inhibitor therapy. We develop a novel computational model to classify exosomal transcripts into tumor and non-tumor components and establish relevance in immune checkpoint blockade therapy. We show that tumor driver load from RNA-seq mutational calls are significantly different between responders and non-responders.