AP-1 Mediates Cellular Adaptation and Memory Formation During Therapy Resistance.

Cellular responses to environmental stimuli are typically thought to be governed by genetically encoded programs. We demonstrate that melanoma cells can form and maintain cellular memories during the acquisition of therapy resistance that exhibit characteristics of cellular learning and are dependent on the transcription factor AP-1. We show that cells exposed to a low dose of therapy adapt to become resistant to a high dose, demonstrating that resistance was not purely selective. The application of therapy itself results in the encoding of transient gene expression into cellular memory and that this encoding occurs for both transiently induced and probabilistically arising expression. Chromatin accessibility showed concomitant persistence. A two-color AP-1 reporter system showed that these memories are encoded in cis , constituting an example of activating cis epigenetics. Our findings establish the formation and maintenance of cellular memories as a critical aspect of gene regulation during the development of therapy resistance.

A Pan-Cancer Patient-Derived Xenograft Histology Image Repository with Genomic and Pathologic Annotations Enables Deep Learning Analysis.

Patient-derived xenografts (PDX) model human intra- and intertumoral heterogeneity in the context of the intact tissue of immunocompromised mice. Histologic imaging via hematoxylin and eosin (H&E) staining is routinely performed on PDX samples, which could be harnessed for computational analysis. Prior studies of large clinical H&E image repositories have shown that deep learning analysis can identify intercellular and morphologic signals correlated with disease phenotype and therapeutic response. In this study, we developed an extensive, pan-cancer repository of >1,000 PDX and paired parental tumor H&E images. These images, curated from the PDX Development and Trial Centers Research Network Consortium, had a range of associated genomic and transcriptomic data, clinical metadata, pathologic assessments of cell composition, and, in several cases, detailed pathologic annotations of neoplastic, stromal, and necrotic regions. The amenability of these images to deep learning was highlighted through three applications: (i) development of a classifier for neoplastic, stromal, and necrotic regions; (ii) development of a predictor of xenograft-transplant lymphoproliferative disorder; and (iii) application of a published predictor of microsatellite instability. Together, this PDX Development and Trial Centers Research Network image repository provides a valuable resource for controlled digital pathology analysis, both for the evaluation of technical issues and for the development of computational image-based methods that make clinical predictions based on PDX treatment studies. Significance: A pan-cancer repository of >1,000 patient-derived xenograft hematoxylin and eosin-stained images will facilitate cancer biology investigations through histopathologic analysis and contributes important model system data that expand existing human histology repositories.

Nextflow pipeline for Visium and H&E data from patient-derived xenograft samples.

We designed a Nextflow DSL2-based pipeline, Spatial Transcriptomics Quantification (STQ), for simultaneous processing of 10x Genomics Visium spatial transcriptomics data and a matched hematoxylin and eosin (H&E)-stained whole-slide image (WSI), optimized for patient-derived xenograft (PDX) cancer specimens. Our pipeline enables the classification of sequenced transcripts for deconvolving the mouse and human species and mapping the transcripts to reference transcriptomes. We align the H&E WSI with the spatial layout of the Visium slide and generate imaging and quantitative morphology features for each Visium spot. The pipeline design enables multiple analysis workflows, including single or dual reference genome input and stand-alone image analysis. We show the utility of our pipeline on a dataset from Visium profiling of four melanoma PDX samples. The clustering of Visium spots and clustering of H&E imaging features reveal similar patterns arising from the two data modalities.

Spatiotemporal profiling defines persistence and resistance dynamics during targeted treatment of melanoma.

Resistance of BRAF-mutant melanomas to targeted therapy arises from the ability of cells to enter a persister state, evade treatment with relative dormancy, and repopulate the tumor when reactivated. Using spatial transcriptomics in patient derived xenograft models, we capture clonal lineage evolution during treatment, finding the persister state to show increased oxidative phosphorylation, decreased proliferation, and increased invasive capacity, with central-to-peripheral gradients. Phylogenetic tracing identifies intrinsic- and acquired-resistance mechanisms (e.g. dual specific phosphatases, Reticulon-4, CDK2) and suggests specific temporal windows of potential therapeutic efficacy. Using deep learning to analyze histopathological slides, we find morphological features of specific cell states, demonstrating that juxtaposition of transcriptomics and histology data enables identification of phenotypically-distinct populations using imaging data alone. In summary, we define state change and lineage selection during melanoma treatment with spatiotemporal resolution, elucidating how choice and timing of therapeutic agents will impact the ability to eradicate resistant clones. Statement of Significance: Tumor evolution is accelerated by application of anti-cancer therapy, resulting in clonal expansions leading to dormancy and subsequently resistance, but the dynamics of this process are incompletely understood. Tracking clonal progression during treatment, we identify conserved, global transcriptional changes and local clone-clone and spatial patterns underlying the emergence of resistance.

Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity.

The circadian clock regulator Bmal1 modulates tumorigenesis, but its reported effects are inconsistent. Here, we show that Bmal1 has a context-dependent role in mouse melanoma tumor growth. Loss of Bmal1 in YUMM2.1 or B16-F10 melanoma cells eliminates clock function and diminishes hypoxic gene expression and tumorigenesis, which could be rescued by ectopic expression of HIF1α in YUMM2.1 cells. By contrast, over-expressed wild-type or a transcriptionally inactive mutant Bmal1 non-canonically sequester myosin heavy chain 9 (Myh9) to increase MRTF-SRF activity and AP-1 transcriptional signature, and shift YUMM2.1 cells from a Sox10high to a Sox9high immune resistant, mesenchymal cell state that is found in human melanomas. Our work describes a link between Bmal1, Myh9, mouse melanoma cell plasticity, and tumor immunity. This connection may underlie cancer therapeutic resistance and underpin the link between the circadian clock, MRTF-SRF and the cytoskeleton.

ERK hyperactivation serves as a unified mechanism of escape in intrinsic and acquired CDK4/6 inhibitor resistance in acral lentiginous melanoma.

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.

HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation.

By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ T cell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity.

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8+ T cells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8+ T cell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8+ T cell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better T cell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 T cells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.

Nextflow Pipeline for Visium and H&E Data from Patient-Derived Xenograft Samples.

Highlights: We have developed an automated data processing pipeline to quantify mouse and human data from patient-derived xenograft samples assayed by Visium spatial transcriptomics with matched hematoxylin and eosin (H&E) stained image. We enable deconvolution of reads with Xenome, quantification of spatial gene expression from host and graft species with Space Ranger, extraction of B-allele frequencies, and splicing quantification with Velocyto. In the H&E image processing sub-workflow, we generate morphometric and deep learning-derived feature quantifications complementary to the Visium spots, enabling multi-modal H&E/expression comparisons. We have wrapped the pipeline into Nextflow DSL2 in a scalable, portable, and easy-to-use framework. Summary: We designed a Nextflow DSL2-based pipeline, Spatial Transcriptomics Quantification (STQ), for simultaneous processing of 10x Genomics Visium spatial transcriptomics data and a matched hematoxylin and eosin (H&E)-stained whole slide image (WSI), optimized for Patient-Derived Xenograft (PDX) cancer specimens. Our pipeline enables the classification of sequenced transcripts for deconvolving the mouse and human species and mapping the transcripts to reference transcriptomes. We align the H&E WSI with the spatial layout of the Visium slide and generate imaging and quantitative morphology features for each Visium spot. The pipeline design enables multiple analysis workflows, including single or dual reference genomes input and stand-alone image analysis. We showed the utility of our pipeline on a dataset from Visium profiling of four melanoma PDX samples. The clustering of Visium spots and clustering of imaging features of H&E data reveal similar patterns arising from the two data modalities.

H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Melanoma-intrinsic NR2F6 activity regulates antitumor immunity.

Nuclear receptors (NRs) are implicated in the regulation of tumors and immune cells. We identify a tumor-intrinsic function of the orphan NR, NR2F6, regulating antitumor immunity. NR2F6 was selected from 48 candidate NRs based on an expression pattern in melanoma patient specimens (i.e., IFN-γ signature) associated with positive responses to immunotherapy and favorable patient outcomes. Correspondingly, genetic ablation of NR2F6 in a mouse melanoma model conferred a more effective response to PD-1 therapy. NR2F6 loss in B16F10 and YUMM1.7 melanoma cells attenuated tumor development in immune-competent but not -incompetent mice via the increased abundance of effector and progenitor-exhausted CD8+ T cells. Inhibition of NACC1 and FKBP10, identified as NR2F6 effectors, phenocopied NR2F6 loss. Inoculation of NR2F6 KO mice with NR2F6 KD melanoma cells further decreased tumor growth compared with NR2F6 WT mice. Tumor-intrinsic NR2F6 function complements its tumor-extrinsic role and justifies the development of effective anticancer therapies.

Diverse clonal fates emerge upon drug treatment of homogeneous cancer cells.

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells1-7. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy7-9; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.

Targeting Fatty Acid Reprogramming Suppresses CARM1-expressing Ovarian Cancer.

The arginine methyltransferase CARM1 exhibits high expression levels in several human cancers, with the trend also observed in ovarian cancer. However, therapeutic approaches targeting tumors that overexpress CARM1 have not been explored. Cancer cells exploit metabolic reprogramming such as fatty acids for their survival. Here we report that CARM1 promotes monounsaturated fatty acid synthesis and fatty acid reprogramming represents a metabolic vulnerability for CARM1-expressing ovarian cancer. CARM1 promotes the expression of genes encoding rate-limiting enzymes of de novo fatty acid metabolism such as acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN). In addition, CARM1 upregulates stearoyl-CoA desaturase 1 (SCD1) that produces monounsaturated fatty acid by desaturation. Thus, CARM1 enhances de novo fatty acids synthesis which was subsequently utilized for synthesis of monounsaturated fatty acids. Consequently, inhibition of SCD1 suppresses the growth of ovarian cancer cells in a CARM1 status-dependent manner, which was rescued by the addition of monounsaturated fatty acids. Consistently, CARM1-expressing cells were more tolerant to the addition of saturated fatty acids. Indeed, SCD1 inhibition demonstrated efficacy against ovarian cancer in both orthotopic xenograft and syngeneic mouse models in a CARM1-dependent manner. In summary, our data show that CARM1 reprograms fatty acid metabolism and targeting SCD1 through pharmacological inhibition can serve as a potent therapeutic approach for CARM1-expressing ovarian cancers. Significance: CARM1 reprograms fatty acid metabolism transcriptionally to support ovarian cancer growth by producing monounsaturated fatty acids, supporting SCD1 inhibition as a rational strategy for treating CARM1-expressing ovarian cancer.

Targeting Upregulated cIAP2 in SOX10-Deficient Drug Tolerant Melanoma.

Drug tolerance and minimal residual disease (MRD) are likely to prelude acquired resistance to targeted therapy. Mechanisms that allow persister cells to survive in the presence of targeted therapy are being characterized but selective vulnerabilities for these subpopulations remain uncertain. We identified cellular inhibitor of apoptosis protein 2 (cIAP2) as being highly expressed in SOX10-deficient drug tolerant persister (DTP) melanoma cells. Here, we show that cIAP2 is sufficient to induce tolerance to MEK inhibitors, likely by decreasing the levels of cell death. Mechanistically, cIAP2 is upregulated at the transcript level in SOX10-deficient cells and the AP-1 complex protein, JUND, is required for its expression. Using a patient-derived xenograft model, we demonstrate that treatment with the cIAP1/2 inhibitor, birinapant, during the MRD phase delays the onset of resistance to BRAF inhibitor and MEK inhibitor combination therapy. Together, our data suggest that cIAP2 upregulation in SOX10-deficient subpopulations of melanoma cells induces drug tolerance to MAPK targeting agents and provides a rationale to test a novel therapeutical approach to target MRD.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

BACKGROUND: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. METHODS: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. FINDING: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. INTERPRETATION: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. FUNDING: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

ERK Hyperactivation Serves as a Unified Mechanism of Escape in Intrinsic and Acquired CDK4/6 Inhibitor Resistance in Acral Lentiginous Melanoma.

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in > 60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression are a unified mechanism of both intrinsic and acquired CDK4i/6i resistance. MEK and/or ERK inhibition increases CDK4i/6i efficacy in a patient-derived xenograft (PDX) model of ALM and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach to improve outcomes for patients with advanced ALM.

Tumor-Derived Small Extracellular Vesicles Inhibit the Efficacy of CAR T Cells against Solid Tumors.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable success in the treatment of hematologic malignancies. Unfortunately, it has limited efficacy against solid tumors, even when the targeted antigens are well expressed. A better understanding of the underlying mechanisms of CAR T-cell therapy resistance in solid tumors is necessary to develop strategies to improve efficacy. Here we report that solid tumors release small extracellular vesicles (sEV) that carry both targeted tumor antigens and the immune checkpoint protein PD-L1. These sEVs acted as cell-free functional units to preferentially interact with cognate CAR T cells and efficiently inhibited their proliferation, migration, and function. In syngeneic mouse tumor models, blocking tumor sEV secretion not only boosted the infiltration and antitumor activity of CAR T cells but also improved endogenous antitumor immunity. These results suggest that solid tumors use sEVs as an active defense mechanism to resist CAR T cells and implicate tumor sEVs as a potential therapeutic target to optimize CAR T-cell therapy against solid tumors. SIGNIFICANCE: Small extracellular vesicles secreted by solid tumors inhibit CAR T cells, which provide a molecular explanation for CAR T-cell resistance and suggests that strategies targeting exosome secretion may enhance CAR T-cell efficacy. See related commentary by Ortiz-Espinosa and Srivastava, p. 2637.

Autophagy Paradox: Strategizing Treatment Modality in Melanoma.

OPINION STATEMENT: The primordial autophagy process, originally identified as a starvation response in baker's yeast, has since been shown to have a wide spectrum of functions other than survival. In many cases, it is accepted that autophagy operates as a key tumor suppressor mechanism that protects cells from adverse environmental cues by enforcing homeostasis and maintaining the functional and structural integrity of organelles. Paradoxically, heightened states of autophagy are also seen in some cancers, leading to the prevailing view that the pro-survival aspect of autophagy might be hijacked by some tumors to promote their fitness and pathogenesis. Notably, recent studies have revealed a broad range of cell-autonomous autophagy in reshaping tumor microenvironment and maintaining lineage integrity and immune homeostasis, calling for a renewed understanding of autophagy beyond its classical roles in cell survival. Here, we evaluate the increasing body of literature that argues the "double-edged" consequences of autophagy manipulation in cancer therapy, with a particular focus on highly plastic and mutagenic melanoma. We also discuss the caveats that must be considered when evaluating whether autophagy blockade is the effector mechanism of some anti-cancer therapy particularly associated with lysosomotropic agents. If autophagy proteins are to be properly exploited as targets for anticancer drugs, their diverse and complex roles should also be considered.