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.

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

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.

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.

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.

Despite the availability of several FDA-approved therapies, metastatic melanoma remains a significant clinical challenge, particularly for patients with brain metastases, which frequently represent the site of treatment failure and a major cause of melanoma-related mortality. Melanoma exhibits a strong propensity to metastasize to the brain, yet the molecular mechanisms driving this lethal progression remain incompletely understood, limiting the development of effective treatment options. Building on our prior discovery that focal adhesion kinase (FAK) is a key mediator of AKT1-driven brain metastasis, we sought to validate the role of FAK in melanoma progression and metastatic dissemination. Using complementary autochthonous and syngeneic mouse models of BRAF-mutant melanoma, we evaluated the impact of FAK expression on overall survival, primary tumor growth, and metastasis. Through the generation of targeted FAK mutants, we distinguished kinase-dependent from kinase-independent functions and demonstrate that FAK promotes melanoma metastasis in a kinase-dependent manner. Furthermore, we establish that FAK functions downstream of PTEN to drive metastatic progression. Collectively, these findings support the therapeutic potential of FAK inhibition, either alone or in combination with existing treatments, to more effectively combat metastatic melanoma and inform the development of emerging FAK-targeted therapies.

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 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.

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.

Melanoma progression and resistance to targeted therapies remain major clinical challenges, driven in part by the remarkable phenotypic plasticity of melanoma cells. Identifying molecular mechanisms that couple tumor survival to adaptative drug responses is therefore essential. Here, we identify PSMD14, a proteasome-associated deubiquitinase, as an essential regulator of melanoma plasticity, growth, survival, and therapeutic resistance with strong prognostic significance in metastatic disease. An unbiased siRNA screen targeting the human deubiquitinase family revealed PSMD14 (proteasome 26S subunit, non-ATPase 14) as a top regulator of melanoma cell proliferation. Integrative analyses of DepMap, TCGA, and patient-derived datasets revealed that PSMD14 is frequently upregulated in melanoma, enriched in metastatic lesions, and significantly associated with poor patient outcome. Functional and pharmacological studies demonstrated that genetic depletion or inhibition of PSMD14 suppresses proliferation, clonogenic and long-term growth, and viability of melanoma cells across BRAF-, NRAS-, and NF1-driven genotypes, while inducing DNA damage and apoptosis. Consistently, PSMD14 inhibition markedly reduced tumor growth in Nras and Braf syngeneic mouse models. Mechanistically, we uncover a non-proteolytic role for PSMD14 as an epigenetic regulator of chromatin state. Proteomic and biochemical analyses identified histone H2A as a direct interactor and substrate of PSMD14. PSMD14 deubiquitinates H2A at lysine 119 independently of the proteasome, antagonizing the Polycomb E3 ligase RING1B. Loss of PSMD14 allows the increment of H2AK119 ubiquitination, transcriptional repression of pro-survival genes, including MCL1 and BCL2, and apoptotic cell death, effects rescued by RING1B depletion. Importantly, we demonstrate that the PSMD14-H2A axis governs melanoma adaptation to MAPK pathway inhibition. PSMD14 expression and H2AK119 ubiquitination dynamically correlate with therapeutic response, drug-tolerant persistence, and acquired resistance. Targeting PSMD14 genetically or pharmacologically enhances the efficacy of BRAF and MEK inhibitors, suppresses the emergence of drug-tolerant persister cells, and prevents tumor relapse in vivo. Together, this findings establish PSMD14 as a chromatin-rewiring enzyme that links proteostasis to epigenetic control of melanoma plasticity and therapy resistance, highlighting PSMD14 as a promising biomarker and therapeutic target in aggressive and drug-resistant melanoma.

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.

Prostate cancer is the second most common malignancy among men, with androgen deprivation therapy (ADT) serving as the standard treatment due to the hormone sensitivity of prostate tumors. However, therapeutic resistance frequently develops, leading to castration-resistant prostate cancer (CRPC), an aggressive and lethal disease. A recently defined subtype, stem cell-like CRPC (CRPC-SCL), accounts for approximately 25% of CRPC cases and demonstrates poor responsiveness to ADT. CRPC-SCL is characterized by the expression of Cluster of Differentiation 44 (CD44), a glycoprotein that promotes hyaluronic acid binding and uptake. Within CRPC-SCL patient-derived xenograft (PDX) model, CD44 high (CD44hi) cells exhibit enhanced tumorigenicity and proliferative capacity. Importantly, iron metabolism emerges as a critical regulator of this population: CD44hi cells maintain elevated intracellular iron, which sustains CD44 expression and stem cell-like properties by modulating H3K9me2 modification. Leveraging this vulnerability, inhibition of the iron-regulatory factor NRF2 was shown to increase intracellular free iron and selectively induce ferroptosis in CD44hi cells. These findings highlight the therapeutic potential of targeting iron metabolism to induce ferroptosis as a novel treatment strategy for CRPC-SCL.

E3 ubiquitin ligases mediating turnover of proteins engaged in cancer progression point to key regulatory nodes. To uncover modifiers of metastatic competency, we conducted an in vivo genome-wide CRISPR-inactivation screen using cultured breast circulating tumor cells, following intravascular seeding and lung colonization. We identified HECTD4, a previously uncharacterized gene encoding a conserved potential HECT domain-containing ubiquitin transferase, as a potent tumor and metastasis suppressor. We show that purified HECTD4 mediates ubiquitin conjugation in vitro, and proteomic studies combined with ubiquitin remnant profiling identify a major degradation target as the prostaglandin synthetic enzyme cyclooxygenase-2 (COX-2; PTGS2). In addition to COX-2 itself, HECTD4 targets its regulatory kinase MKK7. In breast cancer models, HECTD4 expression is induced as cells lose adherence to the matrix, and its depletion massively increases COX-2 expression, enhancing anchorage-independent proliferation and tumorigenesis. Genetic or pharmacologic suppression of COX-2 reverses the pro-tumorigenic and pro-metastatic phenotype of HECTD4-depleted cells. Thus, HECTD4 encodes an E3 ubiquitin ligase that downregulates COX-2 suppressing anchorage-independence in epithelial cancer cells. Significance StatementA genome-wide CRISPR-inactivation screen identified the previously uncharacterized E3 ubiquitin ligase HECTD4, as a tumor and metastasis suppressor, with COX-2 as its major degradation target. The pro-tumorigenic and pro-metastatic effect of HECTD4 suppression depends on COX-2 stabilization, which is critical for anchorage-independent growth, providing a basis for investigating COX-2 inhibition to prevent metastatic recurrence.

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we show that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, non-natural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.