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Although alterations in xenobiotic metabolism are considered causal in the development of bladder cancer, the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in bladder cancer. This metabolic signature distinguished both normal and benign bladder from bladder cancer. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing bladder cancer from controls and also nonmuscle from muscle-invasive bladder cancer. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in bladder cancer. In particular, we validated tumor-associated hypermethylation in the cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) promoters of bladder cancer tissues by bisulfite sequence analysis and methylation-specific PCR and also by in vitro treatment of T-24 bladder cancer cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Furthermore, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of bladder cancer specimens compared with matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of bladder cancer, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression. Cancer Res; 71(24); 7376–86. ©2011 AACR.

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Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224–35. ©2016 AACR.

Supplementary Figure 2 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes. [Cancer Res 2007;67(18):8511–8]

This study examined DNA methylation associated with acute lymphoblastic leukemia (ALL) and showed that selected molecular targets can be pharmacologically modulated to reverse gene silencing. A CpG island (CGI) microarray containing more than 3,400 unique clones that span all human chromosomes was used for large-scale discovery experiments and led to 262 unique CGI loci being statistically identified as methylated in ALL lymphoblasts. The methylation status of 10 clones encompassing 11 genes (DCC, DLC-1, DDX51, KCNK2, LRP1B, NKX6-1, NOPE, PCDHGA12, RPIB9, ABCB1, and SLC2A14) identified as differentially methylated between ALL patients and controls was independently verified. Consequently, the methylation status of DDX51 was found to differentiate patients with B- and T-ALL subtypes (P = 0.011, Fisher's exact test). Next, the relationship between methylation and expression of these genes was examined in ALL cell lines (NALM-6 and Jurkat) before and after treatments with 5-aza-2-deoxycytidine and trichostatin A. More than a 10-fold increase in mRNA expression was observed for two previously identified tumor suppressor genes (DLC-1 and DCC) and also for RPIB9 and PCDHGA12. Although the mechanisms that lead to the CGI methylation of these genes are unknown, bisulfite sequencing of the promoter of RPIB9 suggests that expression is inhibited by methylation within SP1 and AP2 transcription factor binding motifs. Finally, specific chromosomal methylation hotspots were found to be associated with ALL. This study sets the stage for acquiring a better biological understanding of ALL and for the identification of epigenetic biomarkers useful for differential diagnosis, therapeutic monitoring, and the detection of leukemic relapse. [Cancer Res 2007;67(6):2617–25]

Supplementary Table 2 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Supplementary Table 1 from Large-Scale CpG Methylation Analysis Identifies Novel Candidate Genes and Reveals Methylation Hotspots in Acute Lymphoblastic Leukemia

Supplementary Figure 1 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

The Supplemental materials and method file has methods for the Generation of mammospheres and tumorospheres, Preparation of single cell suspension from mammary gland, methods for RNA Isolation and Real-time PCR, and Clonogenic assay.

Supplementary Figure legends provide clear description for the Supplementary Figures S1-S7.

The Supplemental figure file contains severn additional figures. Supplementary figure 1 shows that the Lin-CD49f+CD24+ cells are tumorigenic, Supplementary Figure 2 provide additional evidence for myoepithelial stem and luminal progenitor cells are the targets of genetic mutations/epigenetic modifications that lead to tumor cell of origin, Supplementary Figure 3, shows that the treatment with DNMT and HDAC inhibitors reduces colony formation, Supplementary Figure 4l provide a Flow chart for RNA sequencing analysis, Supplementary Figure 5 shows a heat map for differential expression of genes in normal and in breast cancer, Supplementary Figure 6 shows RAD51AP1 expression in basal breast cancer, and Supplementary Figure 7 shows SPC25 expression in basal breast cancer

Chimeric antigen receptor engineered T cells (CARTs) for HCC and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAbs) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects.New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained three hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with approximately 17-fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects. In vitro, low-avidity 8F8 CARTs killed both hGPC3The low-avidity 8F8-BBz CART resists exhaustion and apoptosis inside tumor lesions, demonstrating a greater therapeutic potential than high-avidity CARTs.

Brown adipocytes share the same developmental origin with skeletal muscle. Here we find that a brown adipocyte-to-myocyte remodeling also exists in mature brown adipocytes, and is induced by prolonged high fat diet (HFD) feeding, leading to brown fat dysfunction. This process is regulated by the interaction of epigenetic pathways involving histone and DNA methylation. In mature brown adipocytes, the histone demethylase UTX maintains persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high Prdm16 expression. PRDM16 then recruits DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation and suppressing its expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity., Brown adipocytes contribute to energy balance, and adipocyte development and brown adipocyte thermogenesis are in part regulated by epigenetic modifications. Here the authors report that the histone demethylase Utx maintains brown adipocyte identity via demethylation of PRDM16, which in turn represses myogenic remodelling via DNMT1-mediated Myod1 promoter hypermethylation in mice.

Activation of brown fat thermogenesis increases energy expenditure and alleviates obesity. Sympathetic nervous system (SNS) is important in brown/beige adipocyte thermogenesis. Here we discover a fat-derived “adipokine” neurotrophic factor neurotrophin 3 (NT-3) and its receptor Tropomyosin receptor kinase C (TRKC) as key regulators of SNS growth and innervation in adipose tissue. NT-3 is highly expressed in brown/beige adipocytes, and potently stimulates sympathetic neuron neurite growth. NT-3/TRKC regulates a plethora of pathways in neuronal axonal growth and elongation. Adipose tissue sympathetic innervation is significantly increased in mice with adipocyte-specific NT-3 overexpression, but profoundly reduced in mice with TRKC haploinsufficiency (TRKC +/−). Increasing NT-3 via pharmacological or genetic approach promotes beige adipocyte development, enhances cold-induced thermogenesis and protects against diet-induced obesity (DIO); whereas TRKC + /− or SNS TRKC deficient mice are cold intolerant and prone to DIO. Thus, NT-3 is a fat-derived neurotrophic factor that regulates SNS innervation, energy metabolism and obesity. Activation of brown adipose tissue thermogenesis increases energy expenditure and promotes weight loss in mice. Here the authors identify neurotrophic factor neurotrophin 3 (NT-3) as an adipokine that regulates sympathetic nervous system growth and innervation in adipose tissue and increases white adipose beiging.

Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-T cell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to T cell cytotoxicity. We observed that DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas(+) tumor cells and suppresses established melanoma growth in immune competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein level on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted T cells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data determine that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.

Tumor cell resistance to ferroptosis, which is a recently discovered CTL induced cell death pathway, remains incompletely understood. We report here that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells are resistant to intrinsic ferroptosis induction and also exhibit decreased ferroptosis in response to tumor specific CTL killing. Loss of IRF8 increases p53 express in tumor cells and knocking out Tp53 in IRF8.KO cells restored tumor cell sensitivity to intrinsic ferroptosis induction. We were able to show that treating tumor-bearing mice with IRF8-encoding plasmid NTC9385R-mIRF8 encapsulated in a DOTAP-cholesterol lipid nanoparticle significantly reduced tumor growth and increased the percentage of dead cells in the tumor. Our data therefore determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. Citation Format: Dakota Poschel, Mercy Kehinde-Ige, John Klement, Dafeng Yang, Alyssa Merting, Natasha Savage, Huidong Shi, Kebin Liu. IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB036.

Adoptive cell therapy has revolutionized the landscape of cancer immunotherapy, exhibiting unprecedented success in treating various types of cancer. Substantial investigative efforts have been devoted to gaining a deeper understanding of the ways in which adoptively transferred T cells eliminate tumors and to discovering strategies that enhance T cell functionality in the tumor microenvironment. We have established an experimental mouse model in which adoptive transfer of tumor-specific CD4+ T cells leads to eradication of established tumors. Using this model, we previously reported on the profound disruption of tumor metabolism induced by adoptive T cell therapy. However, the detailed mechanisms that enable tumor-reactive CD4+ T cells to alter tumor metabolism remain largely undefined. In the current study, we aimed to identify the key metabolic enzymes and pathways targeted by antitumor CD4+ T cells that lead to progressive tumor regression. Utilizing next-generation sequencing strategies, including bulk and single-cell RNA sequencing, we examined the metabolic changes cancer cells undergo in response to treatment, and the immune cell compositions in the tumor microenvironment. Additionally, we performed an in vivo CRISPR knockout screen to identify the metabolic genes that either sensitize tumor cells to adoptive cell therapy or contribute to tumor resistance. Results from our study may also illuminate potential targets in cancer metabolism that can be exploited using small-molecule inhibitors when adoptive cell therapy is not feasible. Citation Format: Caitlin Brandle, Ogacheko D. Okoko, Md Yeashin Gazi, Xin Wang, Mercy Kehinde-Ige, Huidong Shi, Gang Zhou. Antitumor CD4+ T cells reprogram tumor metabolism to drive progressive tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 268.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

Adoptive Cell Therapy (ACT) holds great promise to transform cancer treatments. However, applying ACT to a broad patient population still faces major challenges. The significant barriers to the success of ACT have been attributed to multiple factors, including functional exhaustion, poor persistence, and the inability of T cells to efficiently compete against tumor cells for metabolic resources in the tumor microenvironment (TME). These functional deficiencies highlight the importance of targeting T cell metabolism to improve ACT treatment outcomes. In the TME, tumor cells preferentially utilize glycolysis to rapidly generate ATP, converting pyruvate into lactate and creating a hypoxic environment that is inconducive for effector T cells due to a lack of access to the metabolites needed for sustenance, leading to T cell exhaustion, and declined proliferation. Consequently, T cells lose their function and are unable to persist against tumor cells. The expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells has been shown to improve their persistence, polyfunctionality, and anti-tumor effects. Our hypothesis is that the improved polyfunctionality and persistence of CASTAT5-CD4+ T cells are partly due to the reprogramming of their metabolic landscape by CASTAT5, resulting in improved metabolic fitness. Single-cell metabolic pathway enrichment analysis of the identified polyfunctional CASTAT5-CD4+ T cells revealed increased capacity for glycolysis, electron transport chain, oxidative phosphorylation, and amino acid metabolism. Single-cell Flux Estimation Analysis (scFEA) further revealed increased metabolic fluxes in amino acid uptake and synthesis, glycolysis, tricarboxylic acid (TCA) cycle, and polyamine synthesis. Interestingly, CASTAT5-CD4+ T cells showed increased polyamine (spermine and spermidine) synthesis, with the upregulation of the metabolic fluxes from putrescine to spermine, catalyzed by spermidine synthase (Srm) and spermine synthase (Srs), both of which are also upregulated in the polyfunctional CD4+ T cell populations. Spermidine and spermine have been shown to enhance mitochondrial metabolism in T cells, increasing their proliferation and survival. Here, we show that there is increased synthesis of spermine in CASTAT5-CD4+ T cells, which may contribute to their increased capacity for oxidative phosphorylation and TCA cycle, leading to the observed improvements in their persistence and anti-tumor function. These findings suggest a role for the polyamine synthesis pathway in the improvement of T cell function. Further studies modulating targets within the polyamine synthesis pathway will help provide an improved understanding of the underlying mechanisms affecting T cell function, thus, unveiling potential metabolic targets to improve the efficacy of tumor-specific T cells. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Zhi-Chun Ding, Gang Zhou, Huidong Shi. Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3693.

3D printing technology is considered as a highly flexible method which can achieve a various customized end products. The employment of bio-based materials can significantly decrease the environmental footprint of the end 3D printing products. This study presents the preparation of thermoplastic starch (TPS)/poly(lactic acid) (PLA)/poly(butyleneadipate-co-terephthalate) (PBAT) composite that dedicated for the FDM 3D printing technology, the ratio of TPS:PLA:PBAT was fixed at 50:40:10 wt%. In addition, the chain extender ADR4468 (CE) was added to improve the brittleness of the blends to obtain better 3D printing filament. The mechanical properties of blends were improved by the addition of CE with 113 % increase in elongation at break and the 190 % raise in impact strength. Dynamic rheological analysis showed the maximum degree of complex viscosity and melt strength when the content of CE reached 1 wt%. The successful printability of TPS-based filament was demonstrated by accurate and complex printing samples. This paper provided a method to prepare highly renewable filaments for 3D printing.

As the high throughput technologies rapidly develop, multiple types of genomic data become available within and across different studies. It has become a challenging task in modern statistical research to use all types of genomic data to infer some disease-prone genetic information. In this work, we propose an integrative analysis of multiple and different types of genomic data, clinical covariates and survival data under a framework of an accelerated failure time with frailty model. The proposed integrative approach aims to answer some aspects of the complex problem in genomic data analysis by finding relevant genomic features and inferring patients’ survival time using identified features. The proposed integrative approach is developed using a weighted least-squares with a sparse group LASSO penalty as the objective function to simultaneously estimate and select the relevant features. Extensive simulation studies are conducted to assess the performance of the proposed method with two types of genomic data, DNA methylation data and copy number variation data, on 600 genes and three clinical covariates. The simulation results show promises of the proposed method. The proposed method is applied to the analysis of the Cancer Genome Atlas data on Glioblastoma, a lethal brain cancer, and biologically interpretable results are obtained.

Hematological neoplasms are predominantly defined by chromosomal aberrations that include structural variations (SVs) and copy number variations (CNVs). The current standard-of-care (SOC) genetic testing for the detection of SVs and CNVs relies on a combination of traditional cytogenetic techniques that include karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarrays (CMA). These techniques are labor-intensive, time and cost-prohibitive, and often do not reveal the genetic complexity of the tumor. Optical genome mapping (OGM) is an emerging technology that can detect all classes of SVs in a single assay. We report the results from our clinical validation (in a CLIA setting) of the OGM technique for hematological neoplasms. The study included 92 sample runs (including replicates) using 69 well-characterized unique samples (59 hematological neoplasms and 10 controls). The technical (QC metrics and first-pass rate) and analytical performance [sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV)] were evaluated using the clinical samples. The reproducibility was evaluated by performing inter-run, intra-run, and inter-instrument comparisons using six samples run in triplicates. The limit of detection (LoD) for aneuploidy, translocation, interstitial deletion, and duplication was assessed. To confirm the LoD, samples at 12.5%, 10%, and 5% allele fractions (theoretical LoD range) were run in triplicates. The technical performance resulted in a 100% first-pass rate with all samples meeting the minimum QC metrics. The analytical performance showed a sensitivity of 98.7%, specificity of 100%, accuracy of 99.2%, PPV of 100%, and NPV of 98%, which included the detection of 61 aneuploidies, 34 deletions, 28 translocations, 11 duplications/amplifications, 15 insertions/additional material not identified with karyotyping, 12 marker chromosomes, and one each of ring chromosome, inversion and isochromosome. OGM demonstrated robust technical and analytical inter-run, intra-run, and inter-instrument reproducibility. The LoD was determined to be at 5% allele fraction for all the variant classes evaluated in the study. In addition, OGM demonstrated higher resolution to refine breakpoints, identify the additional genomic material, marker, and ring chromosomes. OGM identified several additional SVs, revealing the genomic architecture in these neoplasms that provides an opportunity for better tumor classification, prognostication, risk stratification, and therapy selection. This study is the first CLIA validation report for OGM for genome-wide structural variation detection in hematological neoplasms. Considering the technical and analytical advantages of OGM compared to the current SOC methods used for chromosomal characterization, we highly recommend OGM as a potential first-tier cytogenetic test for the evaluation of hematological neoplasms.

Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα–RIP1–mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3–DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. Significance: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.

CD73, an ecto-5′-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73hi population in human colorectal cancers (CRCs) and two CD73− murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A2A and A2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A2B-mediated ADO-CAF-CD73 feedforward circuit and A2A-mediated immune suppression is crucial for improving therapeutic outcomes.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathways, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

Obesity results from a chronic energy imbalance due to energy intake exceeding energy expenditure. Activation of brown fat thermogenesis has been shown to combat obesity. Epigenetic regulation, including DNA methylation, has emerged as a key regulator of brown fat thermogenic function. Here we aimed to study the role of Dnmt3b, a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat thermogenesis and obesity. We found that the specific deletion of Dnmt3b in brown fat promotes the thermogenic and mitochondrial program in brown fat, enhances energy expenditure, and decreases adiposity in female mice fed a regular chow diet. With a lean phenotype, the female knockout mice also exhibit increased insulin sensitivity. In addition, Dnmt3b deficiency in brown fat also prevents diet-induced obesity and insulin resistance in female mice. Interestingly, our RNA-seq analysis revealed an upregulation of the PI3K-Akt pathway in the brown fat of female Dnmt3b knockout mice. However, male Dnmt3b knockout mice have no change in their body weight, suggesting the existence of sexual dimorphism in the brown fat Dnmt3b knockout model. Our data demonstrate that Dnmt3b plays an important role in the regulation of brown fat function, energy metabolism and obesity in female mice.

Increasing energy expenditure through activation of brown fat thermogenesis is a promising therapeutic strategy for the treatment of obesity. Epigenetic regulation has emerged as a key player in regulating brown fat development and thermogenic program. Here, we aimed to study the role of DNA methyltransferase 3b (Dnmt3b), a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat function and energy homeostasis. We generated a genetic model with Dnmt3b deletion in brown fat-skeletal lineage precursor cells (3bKO mice) by crossing Dnmt3b-floxed (fl/fl) mice with Myf5-Cre mice. Female 3bKO mice are prone to diet-induced obesity, which is associated with decreased energy expenditure. Dnmt3b deficiency also impairs cold-induced thermogenic program in brown fat. Surprisingly, further RNA-seq analysis reveals a profound up-regulation of myogenic markers in brown fat of 3bKO mice, suggesting a myocyte-like remodeling in brown fat. Further motif enrichment and pyrosequencing analysis suggests myocyte enhancer factor 2C (Mef2c) as a mediator for the myogenic alteration in Dnmt3b-deficient brown fat, as indicated by decreased methylation at its promoter. Our data demonstrate that brown fat Dnmt3b is a key regulator of brown fat development, energy metabolism and obesity in female mice.

Obesity is accompanied by low-grade systemic inflammation that etiologically contributes to obesity-induced cardiovascular disease (CVD). Growing evidence supports that neutrophil, the most abundant type of leukocytes in human, is most likely to be the target peripheral leukocyte subtype initiating the inflammatory cascade in obesity. However, few studies have systematically assessed the genome wide changes in neutrophils associated with obesity. In this study, a hypothesis-free OMIC approach (i.e. the discovery phase) and a target approach (i.e. the validation phase) were used to identify obesity related neutrophil activation markers and their roles on CVD risks. In the discovery phase, genome wide DNA methylation, RNA-sequencing and quantitative proteomics were obtained from purified neutrophils (12 obese vs. 12 lean). In the validation phase, gene expression levels of the promising genes from the OMIC platforms were measured in 81 obese cases vs. 83 lean controls, and the association between the expression levels and CVD risks were evaluated. Significant difference was found for one gene, alkaline phosphatase, liver/bone/kidney (ALPL), across 3 OMIC platforms. In the validation phase, the gene expression levels of ALPL in leukocytes were significantly higher in obese compared with lean subjects (p ALPL expression level showed significantly positive association with CVD risk factors (p ALPL of neutrophil activation in response to obesity and provided evidence that obesity induced change in ALPL expression was associated with CVD risk factors.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2223.

Background & Aims Chimeric antigen receptor engineered T cells (CARTs) for hepatocellular carcinoma (HCC) and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAb) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects. Methods and results New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained 3 hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with ~17 fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects. In vitro, low-avidity 8F8 CARTs killed both hGPC3high and hGPC3low HCC tumor cells to the same extent as high-avidity GC33 CARTs. 8F8 CARTs expanded and persisted to a greater extent than GC33 CARTs, resulting in durable responses against HCC xenografts. Importantly, compared to GC33 CARTs, there were 5 folds more of 8F8-BBz CARTs in the tumor mass for a longer period of time. Remarkably, the tumor infiltrating 8F8 CARTs were less exhausted and apoptotic, and more functional than GC33 CARTs. Conclusion The novel low-avidity 8F8-BBz CART resists exhaustion and apoptosis inside tumor lesions, demonstrating a greater therapeutic potential than high-avidity CARTs.

Cancer cells always require more nutrients, energy, and biosynthetic activity to sustain their rapid proliferation than normal cells. Previous studies have shown the impact of THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), on transcription regulation and cell-cycle arrest in numerous cancers, but its effects on cellular metabolism in cancer cells remain unknown. In this study we elucidated the anticancer mechanism of THZ1 in human non-small-cell lung cancer (NSCLC) cells. We showed that treatment with THZ1 (10−1000 nM) dose-dependently suppressed the proliferation of human NSCLC cell lines H1299, A549, H292, and H23, and markedly inhibited the migration of these NSCLC cells. Furthermore, treatment with THZ1 (50 nM) arrested cell cycle at G(2)/M phase and induced apoptosis in these NSCLC cell lines. More importantly, we revealed that treatment with THZ1 (50 nM) blocked the glycolysis pathway but had no effect on glutamine metabolism. We further demonstrated that THZ1 treatment altered the expression pattern of glutaminase 1 (GLS1) isoforms through promoting the ubiquitination and degradation of NUDT21. Combined treatment of THZ1 with a glutaminase inhibitor CB-839 (500 nM) exerted a more potent anti-proliferative effect in these NSCLC cell lines than treatment with THZ1 or CB-839 alone. Our results demonstrate that the inhibitory effect of THZ1 on the growth of human NSCLC cells is partially attributed to interfering with cancer metabolism. Thus, we provide a new potential therapeutic strategy for NSCLC treatment by combining THZ1 with the inhibitors of glutamine metabolism.

The presence of polyfunctional CD4(+) T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4(+) T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4(+) T cells to undergo robust expansion, infiltrate tumors vigorously and elicit antitumor CD8(+) T cell responses in a CD4(+) T-cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4(+) T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4(+) T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to co-express CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4(+) CAR T cells in a mouse B-cell lymphoma model. Notably, the optimal therapeutic outcome was obtained when both CD4(+) and CD8(+) CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8(+) T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4(+) T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4(+) T cells to potentiate adoptive T-cell therapy for cancer.

NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications. Impact statement Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Despite evidences linking methylation changes in the cancer tissues, little is known about the methylation modification in the peripheral blood. With the current study, we identified differential methylation regions (DMRs) across human genome by collecting the blood samples of colorectal cancer (CRC) patients compared to that of their blood-related family who shared genetic inheritance and environmental influences, and unrelated obese and non-obese controls by accessing publicly available Gene Expression Omnibus data. We performed genome-wide analyses using the reduced representation bisulfite sequencing (RRBS) method covering about 25% of CpGs for whole human genome of the four groups (n = 5 each). In comparison to the non-obese controls, we observed significant DMRs in CRC for genes involved in tumorigenesis including MLH3, MSH2, MSH6, SEPT9, GNAS; and glucose transporter genes associated with obesity and diabetes including SLC2A1/GLUT1, and SLC2A3/GLUT3 that were reported on methylation being modified in cancer tissues. In addition, we observed significant DMRs in CRC for genes involved in the methylation pathways including PEMT, ALDH1L1, and DNMT3A. CRC and family members shared significant DMRs for genes of tumorigenesis including MSH2, SEPT9, GNAS, SLC2A1/GLUT1 and SLC2A3/GLUT3); and CAMK1, GLUT1/SLC2A1 and GLUT3/SLC2A3 genes involved in glucose and insulin metabolism that played vital role in development of obesity and diabetes. Our study provided evidences that these differentially methylated genes in the blood could potentially serve as candidate biomarkers for CRC diagnostic and may provide further understanding on CRC progression. Further studies are warranted to validate these methylation changes for diagnostic and prevention of CRC.

We previously observed that disruption of FK506‐binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)‐induced cardiac hypertrophy in mice, whereas the adenovirus‐mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)‐induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6−/−) mice and cardiac‐specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini‐pump. The results showed that FKBP12.6 deficiency aggravated AngII‐induced cardiac hypertrophy, while cardiac‐specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII‐induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII‐induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+]i), in which the protein significantly inhibited the key Ca2+/calmodulin‐dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF‐2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII‐induced cardiac hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways.

Chinese rice wine is a popular traditional alcoholic beverage in China, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome sequence of the yeast strain Saccharomyces cerevisiae N85 for Chinese rice wine production. Several assembly methods were used to integrate Pacific Bioscience (PacBio) and Illumina sequencing data to achieve high-quality genome sequencing of the strain. The genome encodes more than 6,000 predicted proteins, and 238 long non-coding RNAs, which are validated by RNA-sequencing data. Moreover, our annotation predicts 171 novel genes that are not present in the reference S288c genome. We also identified 65,902 single nucleotide polymorphisms and small indels, many of which are located within genic regions. Dozens of larger copy-number variations and translocations were detected, mainly enriched in the subtelomeres, suggesting these regions may be related to genomic evolution. This study will serve as a milestone in studying of Chinese rice wine and related beverages in China and in other countries. It will help to develop more scientific and modern fermentation processes of Chinese rice wine, and explore metabolism pathways of desired and harmful components in Chinese rice wine to improve its taste and nutritional value.

Summary Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton’s tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.