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Subject demographics in the University of Michigan sample set

Normalized spectra counts of AK1, CLIC1, and SOD1 in eight CRC cell lines

Case/control ratios of 22 proteins selected for antibody microarrays in pre-diagnostic CRC plasma samples from the Women's Health Initiative cohort

Supplementary Methods

Normalized intensity of high density antibody array and CEA levels in the University of Michigan sample set

MAPRE1 protein expression in tissue microarray

Simple Summary Although pathologic complete response (pCR) to neoadjuvant chemoradiation (nCRT) in locally advanced rectal cancer (LARC) is associated with better outcomes, a subset of tumors exhibit resistance to nCRT. Therefore, there is a need of biomarkers to predict the nCRT response and increment efforts for personalized therapeutic options. To this end, we analyzed pretreatment plasma proteome of a mouse model of rectal cancer treated with concurrent chemoradiation, resulting in identification and validation of plasma VEGFR3 as a potential predicting biomarker. In addition, plasma levels of EGFR and COX2, previously validated tissue-based predicting biomarkers, were significantly higher in non-pCR than pCR LARC patients, indicating that EGFR and COX2 can also serve as blood-based biomarkers. The performance of the biomarker panel combining VEGFR3, EGFR, and COX2 were significantly improved compared to that of each marker alone, providing a rationale for further integration and refinement of the biomarker panel and validation in the larger sample sets. Abstract The current standard of care for patients with locally advanced rectal cancer (LARC) is neoadjuvant chemoradiation (nCRT) followed by total mesorectal excision surgery. However, the response to nCRT varies among patients and only about 20% of LARC patients achieve a pathologic complete response (pCR) at the time of surgery. Therefore, there is an unmet need for biomarkers that could predict the response to nCRT at an early time point, allowing for the selection of LARC patients who would or would not benefit from nCRT. To identify blood-based biomarkers for prediction of nCRT response, we performed in-depth quantitative proteomic analysis of pretreatment plasma from mice bearing rectal tumors treated with concurrent chemoradiation, resulting in the quantification of 567 proteins. Among the plasma proteins that increased in mice with residual rectal tumor after chemoradiation compared to mice that achieved regression, we selected three proteins (Vascular endothelial growth factor receptor 3 [VEGFR3], Insulin like growth factor binding protein 4 [IGFBP4], and Cathepsin B [CTSB]) for validation in human plasma samples. In addition, we explored whether four tissue protein biomarkers previously shown to predict response to nCRT (Epidermal growth factor receptor [EGFR], Ki-67, E-cadherin, and Prostaglandin G/H synthase 2 [COX2]) also act as potential blood biomarkers. Using immunoassays for these seven biomarker candidates as well as Carcinoembryonic antigen [CEA] levels on plasma collected before nCRT from 34 patients with LARC (6 pCR and 28 non-pCR), we observed that levels of VEGFR3 (p = 0.0451, AUC = 0.720), EGFR (p = 0.0128, AUC = 0.679), and COX2 (p = 0.0397, AUC = 0.679) were significantly increased in the plasma of non-pCR LARC patients compared to those of pCR LARC patients. The performance of the logistic regression model combining VEGFR3, EGFR, and COX2 was significantly improved compared with the performance of each biomarker, yielding an AUC of 0.869 (sensitivity 43% at 95% specificity). Levels of VEGFR3 and EGFR were significantly decreased 5 to 7 months after tumor resection in plasma from 18 surgically resected rectal cancer patients, suggesting that VEGFR3 and EGFR may emanate from tumors. These findings suggest that circulating VEGFR3 can contribute to the prediction of the nCRT response in LARC patients together with circulating EGFR and COX2.

The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen–specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.

Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the KRAS gene. To understand the role of oncogenic KRAS in CRC, we engineered a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele (Krasmut) and conditional null alleles of Apc and Trp53 (iKAP). The iKAP model recapitulates tumor progression from adenoma through metastases. Whole-exome sequencing revealed that the Krasmut allele was heterogenous in primary tumors yet homogenous in metastases, a pattern consistent with activated Krasmut signaling being a driver of progression to metastasis. System-level and functional analyses revealed the TGF-β pathway as a key mediator of Krasmut-driven invasiveness. Genetic extinction of Krasmut resulted in specific elimination of the Krasmut subpopulation in primary and metastatic tumors, leading to apoptotic elimination of advanced invasive and metastatic disease. This faithful CRC model provides genetic evidence that Krasmut drives CRC invasion and maintenance of metastases.

This presentation will highlight our use of mouse models to dissect molecular and cellular interactions in the tumor microenvironment, including (i) the illumination of the role of myeloid cells in driving immune suppression in prostate cancer, and the impact of combined MDSC-targeted and immune checkpoint blockade therapies in the treatment of metastatic castration-resistant prostate cancer; (ii) the generation and analysis of oncogenic Kras in an inducible colorectal cancer mouse model, revealing the role of IRF2 in the recruitment of myeloid cells into the tumor microenvironment and informing patient selection for more effective ICB therapy in CRC; (iii) the concept of “synthetic essentiality” (a means by which to target specific tumor-suppressor gene deficiencies in cancer), exemplified by the identification of the role of CHD1 in modulation of the tumor microenvironment in Pten-deficient prostate cancer; (iv) the discovery of a symbiotic interaction between glioma cell (LOX) and macrophage (SPP1) in PTEN-null glioma, as well as macrophage reprogramming in immune suppression in glioblastoma; and (v) the role of KRAS in cancer metabolism, in particular how the tumor microenvironment may support KRAS-directed cancer cell metabolism and provide a nonautonomous mechanism enabling escape from Kras-dependent tumor growth. These illustrative examples provide translational opportunities to improve cancer patient treatment and survival. Citation Format: Adam Boutin, Peiwen Chen, Prasenjit Dey, Pingping Hou, Wen-Ting Liao, Xin Lu, Haoqiang Ying, Di Zhao, Ronald A. DePinho. Modeling and understanding tumor biologic mechanisms [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA01.

The precise biologic functions and mechanisms of oncogenic KrasG12D (Kras*) in therapeutic resistance, including immune therapy, are not understood. Here, utilizing a colorectal cancer (CRC) mouse model engineered with an inducible Kras* transgene and conditional null APC and Trp53 alleles (iKAP) that we recently established (Boutin et al., Gene & Development 2017), we observed that KRAS*-expressing invasive tumors possess a prominent immune-suppressive profile characterized by a preponderance of myeloid-derived suppressor cells (MDSCs). Integrated transcriptomic and genomic analyses identified the Interferon Regulatory Factor 2 (IRF2) gene as a key downstream target of KRAS* mediating tumor progression and immune suppression. Mechanistically, integrated transcriptomics, cytokine profiling and ChIP-seq analysis identified CXCL3 as a key direct target of IRF2, and pharmacologic inhibition of CXCL3 or its receptor CXCR2 resulted in suppression of MDSC migration and infiltration in KRAS*-expressing CRC tumors. Translational studies established that de novo anti-PD1 resistance of KRAS*-expressing tumors can be overcome by enforced IRF2 expression or by treatment with small-molecule CXCR2 antagonist SX-682. Correspondingly, CRC patient tumors with higher IRF2 expression show increased response to anti-PD1 therapy. This KRAS*-mediated immune suppressive mechanism provides a framework for patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced CRC. Citation Format: Wenting Liao, Michael Overman, Adam Boutin, Scott Kopetz, Ron DePinho, Alan Wang. KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B52.

Oncogenic Kras (Kras*) plays a key role in tumor maintenance and therapeutic resistance in several cancer types, although the precise biologic functions and mechanisms are not completely understood. We had previously shown in pancreatic ductal adenocarcinoma that some tumors underwent spontaneous relapse and were devoid of Kras* expression and downstream canonical MAPK signaling, instead acquiring amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras*-independent tumor maintenance. In a second previous study, we developed a faithful colorectal cancer (CRC) mouse model and showed that Kras* drove invasion and maintenance in CRC. Here, utilizing this CRC mouse model, we demonstrate that KRAS* promotes a pronounced immune-suppressive profile distinguished by a predominance of myeloid-derived suppressor cells (MDSCs). We performed integrated transcriptomic and genomic analyses that identified IRF2 (Interferon Regulatory Factor 2) as a key KRAS* effector mediating immune suppression. IRF2 suppresses MDSC migration and infiltration in KRAS*-expressing CRC tumors by targeting the CXCL3/CXCR2 axis. We also observed that de novo resistance of KRAS*-expressing tumors to anti-PD1 therapy could be overcome by enforced IRF2 expression or by treatment with SX-682, a small-molecule CXCR2 antagonist. In addition, CRC patient tumors with higher IRF2 expression showed increased response to anti-PD1 therapy. This KRAS*-mediated immune-suppressive mechanism provides a framework for CRC patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced disease. Citation Format: Wen-Ting Liao, Michael J. Overman, Adam Boutin, Prasenjit Dey, Di Zhao, Guocan Wang, Jiexi Li, Zhengdao Lan, Jun Li, Xiaoying Shang, Ming Tang, Shan Jiang, Xingdi Ma, Peiwen Chen, Riham Katkhuda, Krittiya Korphaisarn, Deepavali Chakravarti, Qing Chang, Jianhua Zhang, Dipen M. Maru, Dean Y. Maeda, John A. Zebala, Scott Kopetz, Y. Alan Wang, Ronald A. DePinho. Context-dependent role of KRAS in GI malignancies [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA27.

Tumor-associated macrophages (TAMs) are potent drivers of an immunosuppressive tumor microenvironment and may be an attractive therapeutic target. Experimental therapies tested thus far block myeloid cell differentiation, leading to ineffective antigen presentation and minimal antitumor activity. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. We have developed novel, engineered exosome therapeutic candidates that selectively deliver antisense oligos (ASOs) to TAMs, targeting key transcription factors that control the immunosuppressive program, such as STAT6 and C/EBPβ. These transcription factors, which are otherwise “undruggable” with current therapeutic strategies, are activated through various signaling molecules such as IL-4 and IL-10, which results in a protumorigenic macrophage phenotype (M2). Exosomes loaded with STAT6 or C/EBPβ ASOs (exoASO) induced dose-dependent knockdown (KD) of target genes in primary human M2 macrophages with greater potency than the free ASO. Gene expression analysis and cytokine assays showed that exoASO-mediated KD resulted in a marked reprogramming to a proinflammatory (M1) phenotype, as measured by a significant decrease in CD163 and TGF-β1 expression, and a concomitant induction of TNFα and IL-12p40 production. Intratumorally dosed exoASO preferentially associated with tumor myeloid cells, resulting in a 50% KD of STAT6 and C/EBPβ mRNA transcripts in tumor-associated CD11b+ cells, compared to a 20% KD with an equivalent dose of free ASO. Modulation of mRNAs for CD206, Csf1r, and iNos was also observed, and is consistent with M2 to M1 conversion. ExoASO-STAT6 treatment of mice with CT26 tumors showed significant antitumor activity as a monotherapy (80% tumor growth inhibition, 30% complete responses). Similar monotherapy activity was also observed in vivo with exoASO C/EBPβ therapy. In contrast, equivalent doses of free ASO or anti-CSF1R monotherapy did not have any significant effects on tumor growth. Thus, exoASO therapeutics can specifically target and effectively reduce the expression of transcription factors in TAMs, inducing effective reprogramming and resulting in potent antitumor activity. Collectively, exoASOs against STAT6 and C/EBPβ represent a first-in-class strategy to target tumor-associated myeloid cells in a highly selective manner. Citation Format: Suhrut Kamerkar, Dalia Burzyn, Charan Leng, Olga Burenkova, Su Chul Jang, Raymond Yang, Adam Boutin, Katherine Kirwin, Tong Zi, William Dahlberg, Eric Zhang, Kelvin Zhang, Scott Estes, Kyriakos Economides, Timothy Soos, Sriram Sathyanarayanan. Reprogramming of tumor-associated M2 macrophages with antisense oligonucleotide-loaded exosomes results in potent single-agent antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A50.

Blood-based biomarkers for early detection of colorectal cancer could complement current approaches to colorectal cancer screening. We previously identified the APC-binding protein MAPRE1 as a potential colorectal cancer biomarker. Here, we undertook a case–control validation study to determine the performance of MAPRE1 in detecting early colorectal cancer and colon adenoma and to assess the potential relevance of additional biomarker candidates. We analyzed plasma samples from 60 patients with adenomas, 30 with early colorectal cancer, 30 with advanced colorectal cancer, and 60 healthy controls. MAPRE1 and a set of 21 proteins with potential biomarker utility were assayed using high-density antibody arrays, and carcinoembryonic antigen (CEA) was assayed using ELISA. The biologic significance of the candidate biomarkers was also assessed in colorectal cancer mouse models. Plasma MAPRE1 levels were significantly elevated in both patients with adenomas and patients with colorectal cancer compared with controls (P < 0.0001). MAPRE1 and CEA together yielded an area under the curve of 0.793 and a sensitivity of 0.400 at 95% specificity for differentiating early colorectal cancer from controls. Three other biomarkers (AK1, CLIC1, and SOD1) were significantly increased in both adenoma and early colorectal cancer patient plasma samples and in plasma from colorectal cancer mouse models at preclinical stages compared with controls. The combination of MAPRE1, CEA, and AK1 yielded sensitivities of 0.483 and 0.533 at 90% specificity and sensitivities of 0.350 and 0.467 at 95% specificity for differentiating adenoma and early colorectal cancer, respectively, from healthy controls. These findings suggest that MAPRE1 can contribute to the detection of early-stage colorectal cancer and adenomas together with other biomarkers. Cancer Prev Res; 8(11); 1112–9. ©2015 AACR.

Background: KRAS mutations have been associated with lung metastases at diagnosis of metastatic colorectal cancer (mCRC), but the impact of this mutation on subsequent development of lung metastasis is unknown. We investigated KRAS mutation as a predictor of lung metastasis development. Methods: We retrospectively evaluated data from patients with mCRC whose tumour was tested for KRAS mutation from 2008 to 2010. The relationships of KRAS mutational status with time-to-lung metastasis (TTLM) and overall survival (OS) were analysed. Results: Of the 494 patients identified, 202 (41%) had tumours with KRAS mutation. KRAS mutations were associated with a shorter TTLM (median 15.2 vs 22.4 months; hazard ratio=1.40; P=0.002) and a two-fold greater odds of developing lung metastases during the disease course in patients with liver-limited mCRC at diagnosis (72 vs 56%, P=0.007). Overall survival did not differ by KRAS status. Conclusions: Lung metastasis was more likely to develop during the disease course in patients whose tumour had a KRAS mutation than in those whose tumour did not have a KRAS mutation. This finding may have an impact on decision making for surgical resection of metastatic disease.

Exosomes are natural and abundant nanoscale vesicles for intercellular communication, capable of transferring biological instructions between neighboring and distant cell types. Translational research efforts have focused on exploiting this communication mechanism to deliver exogenous pharmacologic payloads to treat a variety of diseases including cancer. Functionalization of the exosome surface with proteins and peptides is an important strategy to maximize the potential of exosomes as therapeutics. Comparative proteomic analysis (LC/MS) of stringently purified exosomes led to the identification of several highly enriched and unique proteins, including a transmembrane glycoprotein (Protein X, PrX), belonging to the immunoglobulin superfamily. Stable expression of PrX in a producer cell line resulted in a 200-fold increase of PrX on the secreted exosomes. Protein X was extensively characterized and the minimum structural requirements for exosome enrichment were determined. With our engExTM platform, we developed precision engineered exosome therapeutics using PrX as a scaffold to enable high-density exosome surface display of an array of structurally and biologically diverse proteins, including enzymes, antibodies, type I cytokines, and TNF superfamily members. These proteins were genetically fused to PrX and overexpressed in a producer cell. Significantly higher transgene expression on secreted exosomes was achieved compared to conventional scaffolds, including the tetraspanins CD9/CD63/CD81 and LAMP2B. Oligomerization of PrX coupled with avidity effects inherent in exosome surface display resulted in a clear activity advantage compared to free protein. Protein X-mediated display of CD40L on exosomes resulted in a 20-fold potency increase in B cell activation over recombinant CD40L. Furthermore, expression of CD40L redirected exosome uptake from phagocytic antigen presenting cells (APCs) to B cells, demonstrating exosome surface modifications can alter cellular tropism. We also evaluated the functionality of IL-12 tethered to the exosome surface and demonstrated superior tumor retention compared to free cytokine, resulting in robust anti-tumor activity in anti-PD-1 refractory B16F10 tumor models. These results demonstrate the potential of the engExTM platform to generate novel exosome therapeutics. Citation Format: Kevin Dooley, Ke Xu, Sonya Haupt, Nuruddeen Lewis, Rane Harrison, Shelly Martin, Christine McCoy, Chang Ling Sia, Su Chul Jang, Katherine Kirwin, Russell McConnell, Bryan Choi, Adam T. Boutin, Damian Houde, Jorge Sanchez-Salazar, Agata Villiger-Oberbek, Kyriakos D. Economides, John D. Kulman, Sriram Sathyanarayanan. engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2150.

The biological functions and mechanisms of oncogenic KRASG12D (KRAS∗) in resistance to immune checkpoint blockade (ICB) therapy are not fully understood. We demonstrate that KRAS∗ represses the expression of interferon regulatory factor 2 (IRF2), which in turn directly represses CXCL3 expression. KRAS∗-mediated repression of IRF2 results in high expression of CXCL3, which binds to CXCR2 on myeloid-derived suppressor cells and promotes their migration to the tumor microenvironment. Anti-PD-1 resistance of KRAS∗-expressing tumors can be overcome by enforced IRF2 expression or by inhibition of CXCR2. Colorectal cancer (CRC) showing higher IRF2 expression exhibited increased responsiveness to anti-PD-1 therapy. The KRAS∗-IRF2-CXCL3-CXCR2 axis provides a framework for patient selection and combination therapies to enhance the effectiveness of ICB therapy in CRC.

Here, Boutin et al. investigated the role of oncogenic KRAS in colorectal cancer (CRC). They generated a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele (Krasmut) and conditional null alleles of Apc and Trp53 (iKAP), which provides genetic evidence that Krasmut drives CRC invasion and maintenance of metastases.

SummaryTo assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm−/− mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1β, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1β or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma1 and have surveyed exons of protein-coding genes for mutations in 11 affected individuals2,3. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with β-catenin4 in colorectal carcinogenesis5,6, the fusion lacks the TCF4 β-catenin–binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.

Skin, the first barrier against invading microorganisms, is hypoxic, even under baseline conditions. The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha, the principal regulator of cellular adaptation to low oxygen, is strongly expressed in skin epithelium. HIF-1alpha is now understood to play a key role in the bactericidal capacity of phagocytic cells such as macrophages and neutrophils. In the skin, keratinocytes provide a direct antibacterial activity through production of antimicrobial peptides, including cathelicidin. Here, we generate mice with a keratinocyte-specific deletion of HIF-1alpha and examine effects on intrinsic skin immunity. Keratinocyte HIF-1alpha is seen to provide protection against necrotic skin lesions produced by the pathogen group A Streptococcus. RNA interference studies reveal that HIF-1alpha regulation of keratinocyte cathelicidin production is critical to their antibacterial function.

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per . Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. IS Ec13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.

Vascular flow through tissues is regulated via a number of homeostatic mechanisms. Localized control of tissue blood flow, or autoregulation, is a key factor in regulating tissue perfusion and oxygenation. We show here that the net balance between two hypoxia-inducible factor (HIF) transcription factor isoforms, HIF-1α and HIF-2α, is an essential mechanism regulating both local and systemic blood flow in the skin of mice. We also show that balance of HIF isoforms in keratinocyte-specific mutant mice affects thermal adaptation, exercise capacity, and systemic arterial pressure. The two primary HIF isoforms achieve these effects in opposing ways that are associated with HIF isoform regulation of nitric oxide production. We also show that a correlation exists between altered levels of HIF isoforms in the skin and the degree of idiopathic hypertension in human subjects. Thus, the balance between HIF-1α and HIF-2α expression in keratinocytes is a control element of both tissue perfusion and systemic arterial pressure, with potential implications in human hypertension.

A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha and their negative regulator von Hippel-Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis.

SummarySkin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1α inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen characterized by aggregative adherence (AA) to cultured human mucosal epithelium cells. We have recently characterized a 10.2-kDa protein, called dispersin, which is exported from the bacteria and which promotes dispersal of EAEC across the intestinal mucosa. Here, we present evidence that dispersin is exported by a putative ABC transporter complex, which is encoded by a genetic locus of the EAEC virulence plasmid pAA2. We demonstrate that the locus comprises a cluster of five genes (designated aat-PABCD), including homologs of an inner-membrane permease (AatP), an ATP-binding cassette protein (AatC) and the outer membrane protein TolC (AatA). We show that, like TolC, AatA localizes to the outer membrane independently of its ABC partner. Dispersin appears to require the Aat complex for outer membrane translocation but not for secretion across the inner membrane. We also show that, like the dispersin gene, transcription of the aat cluster is dependent on AggR, a regulator of virulence genes in EAEC. We propose that the aat cluster encodes a specialized ABC transporter, which plays a role in the pathogenesis of EAEC by transporting dispersin out of the bacterial cell.

We present the complete genome sequence of uropathogenic Escherichia coli , strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli . The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear “backbone,” or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Because telomerase is activated in 85–90% of all human cancers, inhibition of telomerase activity has been proposed as an anti-cancer strategy. However, telomere erosion resulting from telomerase inhibition can generate rampant genomic instability which may fuel adaptive mechanisms. Here, we determined the impact of telomerase activation and its subsequent extinction in T-cell lymphomas arising in Atm−/− mice engineered with an inducible telomerase reverse transcriptase-estrogen receptor (TERT-ER) knock-in allele. Endogenous telomerase reactivation in late generation TERTER/ER Atm−/− mice with telomere dysfunction enabled full malignant progression with accompanying alleviation of telomere checkpoint signaling and acquisition of copy number alterations targeting genes known to be important in human T-cell lymphomas/leukemia. Upon telomerase extinction, tumors eventually showed impaired growth due to reinstatement of telomere dysfunction-induced checkpoint signaling, yet subsequently resumed growth with accompanying alternative lengthening of telomeres (ALT) features. Transcriptomic analysis of ALT+ tumors showed altered transcriptional networks centering on mitochondrial biology and oxidative defense, and genomic analysis showed amplification of PGC-1β, a master regulator of mitochondrial biogenesis and oxidative defense. Relative to telomerase+ tumors, ALT+ tumors showed marked sensitivity to PGC-1β or SOD2 knockdown which compromised mitochondrial function and/or caused increased reactive oxygen species (ROS). Together, these studies provide in vivo genetic evidence that naturally arising tumors adapt to telomerase extinction by acquisition of both ALT and mitochondrial maintenance mechanisms, and show that targeting mitochondrial function and oxidative defense may enhance the effectiveness of anti-telomerase therapy in cancer.