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Introduction T cell recognition of cancer antigens represents the end effector mechanism of successful cancer immunotherapy (1). Advances in the areas of T cell biology, gene engineering, and antigen identification [...], BACKGROUND. Neoantigens derived from [KRAS.sup.MUT] have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore [KRAS.sup.MUT] immunogenicity and the properties of 4 T cell receptors (TCRs) specific for [KRAS.sup.G12V] restricted to the HLA-A3 superfamily of class I alleles. METHODS. A phase 1 clinical vaccine trial targeting [KRAS.sup.MUT] was conducted. TCRs targeting [KRAS.sup.G12V] restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics. RESULTS. Vaccination against [KRAS.sup.MUT] resulted in the priming of [CD8.sup.+] and [CD4.sup.+] T cell responses. [KRAS.sup.G12V] -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against [KRAS.sup.WT]. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected [CD8.sup.+] T cells exhibited potent lytic activity against [KRAS.sup.G12V] cancers, while only HLA-A*11:01-restricted TCR-T [CD4.sup.+] T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All [KRAS.sup.G12V]-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell. CONCLUSION. This study identifies [KRAS.sup.G12V]-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies. TRIAL REGISTRATION. ClinicalTrials.gov NCT03592888. FUNDING. AACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

Introduction Many cancers arise from a single mutation in a proto-oncogene that drives tumor growth. These cancers are often treated with genotype-directed 'targeted therapies,' small-molecule inhibitors that specifically disable growth [...], Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found that the combination of genotype- directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized antitumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations--including EGFR mutations, ALK fusions, or [KRAS.sup.G12C] mutations. We observed downregulation of [beta]2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Introduction Immunotherapy based on blockade of the programmed cell death protein-1/programmed cell death ligand 1 (PD-1/PD-L1) pathway has demonstrated pronounced efficacy in restoring antitumor activity in some patients with solid [...], Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD- 1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive [CD8.sup.+] T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Author(s): King L. Hung [sup.1] , Kathryn E. Yost [sup.1] , Liangqi Xie [sup.2] [sup.3] [sup.4] , Quanming Shi [sup.1] , Konstantin Helmsauer [sup.5] , Jens Luebeck [sup.6] [sup.7] , [...], Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation.sup.1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome.sup.2,3. Here we show that ecDNA hubs--clusters of around 10-100 ecDNAs within the nucleus--enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy. Extrachromosomal DNA (ecDNA) congregates in clusters called ecDNA hubs that promote intermolecular interactions between gene-regulatory regions and thereby amplify the expression of oncogenes such as MYC in cancer cell lines.

Introduction Protein ubiquitylation is a posttranslational modification that couples with the proteasome to form the ubiquitin-proteasome system (UPS). By controlling the ubiquitylation and degradation of more than 80% of normal [...], Ubiquitin-conjugating enzyme E2C (UBE2C) mediates ubiquitylation chain formation via the K11 linkage. While previous in vitro studies showed that UBE2C plays a growth-promoting role in cancer cell lines, the underlying mechanism remains elusive. Still unknown is whether and how UBE2C plays a promoting role in vivo. Here we report that UBE2C was indeed essential for growth and survival of lung cancer cells harboring Kras mutations, and UBE2C was required for [Kras.sup.G12D]-induced lung tumorigenesis, since Ube2c deletion significantly inhibited tumor formation and extended the lifespan of mice. Mechanistically, [Kras.sup.G12D] induced expression of UBE2C, which coupled with [APC/C.sup.CDH1] E3 ligase to promote ubiquitylation and degradation of DEPTOR, leading to activation of mTORC signaling. Importantly, DEPTOR levels fluctuated during cell cycle progression in a manner dependent on UBE2C and CDH1, indicating their physiological connection. Finally, Deptor deletion fully rescued the tumor inhibitory effect of Ube2c deletion in the [Kras.sup.G12D] lung tumor model, indicating a causal role of Deptor. Taken together, our study shows that the UBE2C/CDH1/DEPTOR axis forms an oncogene and tumor suppressor cascade that regulates cell cycle progression and autophagy and validates UBE2C an attractive target for lung cancer associated with Kras mutations.

Introduction Mutations of the KRAS gene are the most common drivers of tumor development across a spectrum of human cancers, such as cancers of the lung, colon, and pancreas (1-3). [...], KRAS is one of the most frequently activated oncogenes in human cancers. Although the role of KRAS mutation in tumorigenesis and tumor maintenance has been extensively studied, the relationship between KRAS and the tumor immune microenvironment is not fully understood. Here, we identified a role of KRAS in driving tumor evasion from innate immune surveillance. In samples of lung adenocarcinoma from patients and Kras- driven genetic mouse models of lung cancer, mutant KRAS activated the expression of cluster of differentiation 47 (CD47), an antiphagocytic signal in cancer cells, leading to decreased phagocytosis of cancer cells by macrophages. Mechanistically, mutant KRAS activated PI3K/STAT3 signaling, which restrained miR-34a expression and relieved the posttranscriptional repression of miR-34a on CD47. In 3 independent cohorts of patients with lung cancer, the KRAS mutation status positively correlated with CD47 expression. Therapeutically, disruption of the KRAS/CD47 signaling axis with KRAS siRNA, the [KRAS.sup.G12C] inhibitor AMG 510, or a miR-34a mimic suppressed CD47 expression, enhanced the phagocytic capacity of macrophages, and restored innate immune surveillance. Our results reveal a direct mechanistic link between active KRAS and innate immune evasion and identify CD47 as a major effector underlying the KRAS-mediated immunosuppressive tumor microenvironment.

Introduction High mammographie density (MD) associates with elevated lifetime risk of malignancy (1-4). Every 3% to 6% increase in MD corresponds to a 10% increase in relative risk for breast [...], Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that isstifferand correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographie density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographie density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Alct activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographie density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographie density.

Author(s): John P. Morris IV [sup.1] , Jossie J. Yashinskie [sup.2] [sup.3] [sup.4] , Richard Koche [sup.3] , Rohit Chandwani [sup.5] [sup.6] [sup.7] [sup.8] , Sha Tian [sup.1] , Chi-Chao [...], The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC).sup.1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development.sup.2. Wild-type p53 is also known to modulate cellular metabolic pathways.sup.3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of [alpha]-ketoglutarate ([alpha]KG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable [alpha]KG. Increased levels of the [alpha]KG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of [alpha]KG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase--an enzyme of the tricarboxylic acid cycle--specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of [alpha]KG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that [alpha]KG is an effector of p53-mediated tumour suppression, and that the accumulation of [alpha]KG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression. Restoring the function of p53 in a mouse model of pancreatic ductal adenocarcinoma leads to the accumulation of [alpha]-ketoglutarate, which increases levels of the 5-hydroxymethylcytosine chromatin modification and results in reduced tumour-cell fitness.

Introduction Nucleophosmin (NPM1) is one of the most frequently mutated genes in de novo acute myeloid leukemia (AML) (~30% of cases) (1). Only approximately 50% of patients with NPM1-mutated AML [...], Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.

Introduction Cancer remains a deadly disease since patients often relapse after surgery and adjuvant therapy, progressing to advanced metastatic stages (1). Recent studies have shown that cancer cells, beyond their [...], Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell- autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

Assessment of the response of acute myeloid leukemia (AML) to intensive therapy is central to therapeutic decision making. It is well-established that detection of measurable residual disease (MRD) after remission-induction [...], * Context.--Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective.--To investigate the feasibility of using next-generation sequencing (NgS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000X-500 000X) sequencing (NGS-qNPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design.--A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-housedesigned bioinformatic pipeline. NGS-qNPM1 was com pared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results.--The NGS-qNPM1 assay had a sensitivity of between [10.sup.-4] and [10.sup.-5] and showed high concordance and correlation with reference methodologies. Moreover, the NGS-qNPM1 assay was able to be integrated into the laboratory's existing, targeted amplicon-based sequencing workflow. Conclusions.--An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities. (Arch Pathol Lab Med. 2018;142:606-612; doi: 10.5858/arpa.2017-0229-0A)