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Objectives Daily stress and cardiovascular reactivity may be important mechanisms linking cumulative life event stress with cardiovascular health and may help to explain racial health disparities. However, studies have yet to examine links between exposure to life event stress, daily stress exposure, and cardiovascular reactivity. This study assessed links between trajectories of life event stress exposure, daily stressors, and cardiovascular reactivity among Black and White individuals. Methods Participants are from the Stress and Well-being in Everyday Life Study in which 238 individuals (109 Black 129 White; ages 33–93), drawn from the longitudinal Social Relations Study, reported life event stress in 1992, 2005, 2015, and 2018. Of those individuals, 169 completed an ecological momentary assessment study in which they reported stress exposure every 3 hr, and 164 wore a heart rate monitor for up to 5 days. Results Latent class growth curve models revealed 2 longitudinal trajectories of life event stress: moderate-increasing and low-decreasing. Individuals in the moderate-increasing stress trajectory reported greater daily stress exposure and links did not vary by race. Black individuals in the low-decreasing trajectory and White individuals in the moderate-increasing trajectory showed positive associations between daily stress and heart rate (i.e., were reactive to daily stress exposure). The link between daily stress and heart rate was not significant among Black individuals in the moderate-increasing trajectory and White individuals in the low-decreasing trajectory. Discussion Individuals who experience more life events across the adult life course report greater daily stress exposure which has important implications for daily cardiovascular health. Black individuals with moderate-increasing life event stress show evidence of blunted daily stress reactivity (nonsignificant association between daily stress and heart rate) whereas Black individuals with low-decreasing life event stress show evidence of stress reactivity (positive association between daily stress and heart rate). White individuals showed the opposite pattern (albeit marginally). These findings expand the weathering hypothesis and indicate that chronic life event stress may be associated with blunted stress reactivity among Black individuals.

Supplemental Figure 2: STING agonists stimulate cytokines in tumor, plasma and peripheral tissues following IT dosing.

Figure S3. Illustration of modeling and correcting for dependence of each marker through the expression level of housekeeping genes.

Figure S1A-C - Dot plots and histograms from flow cytometry analysis of SK-MEL-5-educated M-MDSCs and PBMCs cultured in medium alone.Figure S1D - FACS result of cell surface staining of cytokine-induced M-MDSCs and PBMCs cultured in medium alone.Figure S1E - Cell surface staining of monocytes cultured in the presence of M-CSF and IL4 and SK-MEL-5-educated M-MDSCs by staining with HLA-DR.Figure S2 A-B - ILT3 is highly expressed on CD45+CD33+CD14+ myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Figure S3 - Single-cell RNAseq (scRNAseq) of human immune cells in non-small cell lung cancer (NSCLC) public dataset.Figure S4 - Association of LILRB4 expression with deconvoluted cell fractions for human tumor types in the TCGA database.Figure S5 - Suppression of autologous CD8+ T cell proliferation by cytokine-induced M-MDSCs.Figure S6 - CD33+ cells from PBMCs cultured in medium alone are not suppressive.Figure S7 - Differentially expressed genes (DEG) between SK-MEL-5 educated M-MDSCs and monocytes.

Table S1 - Antibodies used for flow cytometry-based assessment of binding of anti-ILT3 and anti-ILT4 antibody to human primary cells and SK-MEL-5-educated monocytic myeloid-derived suppressor cells.Table S2 - Antibodies used for flow cytometry-based assessment of ILT3 expression on myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Table S3 - Differential gene expression in SK-MEL-5 educated MDSCs treated with anti-ILT3 vs. isotype control using RNAseq.Table S4 - Pantherdb.org analysis for GO term enrichment of the 623 signatures.

Supplementary Methods and Figures

Supplementary Table S1. Genes tested for Prostate CTC digital PCR Assay; Supplementary Table S2. Clinical information for prostate cancer patients and healthy donor subjects; Supplementary Table S3. Clinical information for patients enrolled on prospective trial of first-line abiraterone for mCRPC; Supplementary Table S4. Clinical information for patients with localized prostate cancer in radical prostatectomy study; Supplementary Table S5. Droplet digital PCR primers used in this study.

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell–inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell–inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFβ biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Figure S1A-C - Dot plots and histograms from flow cytometry analysis of SK-MEL-5-educated M-MDSCs and PBMCs cultured in medium alone.Figure S1D - FACS result of cell surface staining of cytokine-induced M-MDSCs and PBMCs cultured in medium alone.Figure S1E - Cell surface staining of monocytes cultured in the presence of M-CSF and IL4 and SK-MEL-5-educated M-MDSCs by staining with HLA-DR.Figure S2 A-B - ILT3 is highly expressed on CD45+CD33+CD14+ myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Figure S3 - Single-cell RNAseq (scRNAseq) of human immune cells in non-small cell lung cancer (NSCLC) public dataset.Figure S4 - Association of LILRB4 expression with deconvoluted cell fractions for human tumor types in the TCGA database.Figure S5 - Suppression of autologous CD8+ T cell proliferation by cytokine-induced M-MDSCs.Figure S6 - CD33+ cells from PBMCs cultured in medium alone are not suppressive.Figure S7 - Differentially expressed genes (DEG) between SK-MEL-5 educated M-MDSCs and monocytes.

Supplementary Figure S1. (A) Schematic of d-CTC assay. Whole blood is processed using the CTC-iChip, resulting in a bulk product highly enriched for intact CTCs (labeled green) but also contains WBCs (labeled red) and RBCs (unlabeled); Supplementary Figure S2. Graphs showing droplet digital PCR signal for each gene in 12 metastatic prostate cancer patients compared to 34 healthy male control subjects; Supplementary Figure S3. (A, B) Graphs of relationships between Prostate CTCM Score and serum PSA, and CTC droplet digital PCR KLK3 signal and serum PSA at the pre-treatment time point; Supplementary Figure S4. (A) Kaplan-Meier curves for radiographic progression-free survival (R-PFS) by CTC HOXB13 status at pretreatment; Supplementary Figure S5. (A) Heatmap of droplet digital PCR CTC signal after whole transcriptome amplification for blood samples from healthy donor controls arranged by sex and age, and from patients with clinically localized prostate cancer, arranged by D'Amico Risk Group; Supplementary Figure S6. (A) Box plots showing pre-operative leave-one-out cross validated (LOOCV) CTCL Score in clinically localized prostate cancer patients according to microscopic SVI or pelvic LN involvement identified at the time of radical prostatectomy

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion.Implications:These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Supplementary Data from Reverse Translating Molecular Determinants of Anti–Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models

Supplemental Methods, Tables S1-2, Table Legends and Figure Legends Table S1.List of qRT-PCR primers used for epithelial, mesenchymal, inducer and housekeeping genes. Table S2. RNA-ISH quantitation of expression scores used in Figure 5.

Supplemental Table 1: Log2Ratio and p values for all 310 genes shown in Figure 6

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation.Significance:This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160

Supplemental Figures and Tables

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell–inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell–inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFβ biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. Significance: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms. See related commentary by Spasevska and Myklebust, p. 3160

Objective: Smartphone industry is growing day by day.According to studies, maximum smartphone users watch videos on their smartphones.With the time, android operating system is becoming popular due to the applications which are based on android SDKsthat attract the attention of the mobile phone users. Methods: However, with the increase of its popularity video editing in android devices is becoming a complex task for many reasons as well like poor video quality after editing, slow editing process, editing tool crashes while editing etc. There is a need of an attractivetool and an efficient environmentto deal with such complex tasks. Findings: This study discusses the novel and easy-to-use interface of video editing in android devices. The developed tool can perform video encoding which generates number of frames per second. We have applied image processing techniques to add images or text to any frame of video and can apply different effects like covers, sepia, gamma, rotation, snow. Then the tool can regenerate video from the frames produced after editing and application of various effects. Application:The resultant video will be quality conscious and can be generated in multiple formats like MP4, 3GP, and MOV reliably. Keywords: Android Phones, Digital Video, Image Processing, Smartphones, Video Processing, Video Editing

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2′3′-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti–PD-1–resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti–PD-1 treatment by enhancing the anti–PD-1 immune profile.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. Implications: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Blood-based biomarkers are critical in metastatic prostate cancer, where characteristic bone metastases are not readily sampled, and they may enable risk stratification in localized disease. We established a sensitive and high-throughput strategy for analyzing prostate circulating tumor cells (CTC) using microfluidic cell enrichment followed by digital quantitation of prostate-derived transcripts. In a prospective study of 27 patients with metastatic castration-resistant prostate cancer treated with first-line abiraterone, pretreatment elevation of the digital CTCM score identifies a high-risk population with poor overall survival (HR = 6.0; P = 0.01) and short radiographic progression-free survival (HR = 3.2; P = 0.046). Expression of HOXB13 in CTCs identifies 6 of 6 patients with ≤12-month survival, with a subset also expressing the ARV7 splice variant. In a second cohort of 34 men with localized prostate cancer, an elevated preoperative CTCL score predicts microscopic dissemination to seminal vesicles and/or lymph nodes (P < 0.001). Thus, digital quantitation of CTC-specific transcripts enables noninvasive monitoring that may guide treatment selection in both metastatic and localized prostate cancer. Significance: There is an unmet need for biomarkers to guide prostate cancer therapies, for curative treatment of localized cancer and for application of molecularly targeted agents in metastatic disease. Digital quantitation of prostate CTC-derived transcripts in blood specimens is predictive of abiraterone response in metastatic cancer and of early dissemination in localized cancer. Cancer Discov; 8(3); 288–303. ©2018 AACR. See related commentary by Heitzer and Speicher, p. 269. This article is highlighted in the In This Issue feature, p. 253

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

Objective: To explore if and how women perceived their prenatal care to have changed as a result of COVID-19 and the impact of those changes on pregnant women. Design: Qualitative analysis of open-ended prompts included as part of an anonymous, online, cross-sectional survey of pregnant women in the United States. Setting: Online survey with participants from 47 states within the U.S. Participants: Self-identified pregnant women recruited through Facebook, Twitter, and other online sources. Measurements and findings: An anonymous, online survey of pregnant women (distributed April 3 - 24, 2020) included an open-ended prompt asking women to tell us how COVID-19 had affected their prenatal care. Open-ended narrative responses were downloaded into Excel and coded using the Attride-Sterling Framework. 2519 pregnant women from 47 states responded to the survey, 88.4% of whom had at least one previous birth. Mean age was 32.7 years, mean weeks pregnant was 24.3 weeks, and mean number of prenatal visits at the point of the survey was 6.5. Predominant themes of the open narratives included COVID-19’s role in creating structural changes within the healthcare system (reported spontaneously by 2075 respondents), behavioral changes among both pregnant women and their providers (reported by 429 respondents), and emotional consequences for women who were pregnant (reported by 503 respondents) during the pandemic. Changes resulting from COVID-19 varied widely by provider, and women's perceptions of the impact on quality of care ranged from perceiving care as extremely compromised to perceiving it to be improved as a result of the pandemic. Key conclusions and Implications for practice: Women who are pregnant during the COVID-19 pandemic have faced enormous upheaval as hospitals and healthcare providers have struggled to meet the simultaneous and often competing demands of infection prevention, pandemic preparedness, high patient volumes of extremely sick patients, and the needs of ‘non-urgent’ pregnant patients. In some settings, women described very few changes, whereas others reported radical changes implemented seemingly overnight. While infection rates may drive variable responses, these inconsistencies raise important questions regarding the need for local, state, national, or even global recommendations for the care of pregnant women during a global pandemic such as COVID-19.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription., The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

The internet of things (IOT) is becoming growing field with the time which connects many people to many things. Electoral voting systems allow voters to vote at any location without geographical restrictions all over the world which considers mobility, privacy, security, and easiness. Current systems adopted all over the world have peculiar problems in each of them. The main idea of this paper is to design a voting system that uses smart phone's fingerprint sensors in order to provide high accuracy, security, and mobility to make voting system more practical and location-free.

Due to the rapid growth of data in the field of big data and bioinformatics, the analysis and management of the data is a very difficult task for the scientist and the researchers. Data exists in many formats like in the form of groups and clusters. The data that exist in the group form and have some repetition patterns called Motifs. A lot of tools and techniques are available in the literature to detect the motifs in different fields like neural networks, antigen/antibody protein, metabolic pathways, DNA/RNA sequences and Protein-Protein Interactions (PPI). In this paper, motif detection is done in tumor antigen protein, namely, cellular tumor antigen p53 (Guardian of the protein and genome) that regulate the cell cycle and suppress the tumor growth in the human body. As tumor is a death causing disease and creates a lot of other diseases in human beings like brain stroke, brain hemorrhage, etc. So there needs to investigate the relation of the tumor protein that prevents the human from not only brain tumor but also from a lot of other diseases that is created from it. To find out the gap between the motifs in the tumor antigen the GLAM2 is used that detects the distance between the motifs very efficiently. Same tumor antigen protein is evaluated at different tools like MEME, TOMTOM, Motif Finder and DREME to analyze the results critically. As tumor protein exists in multiple species, so comparison of homo tumor antigen protein is also done in different species to check the diversity level of this protein. Our purposed approach gives better results and less computational time than other approaches for different types of user characteristics.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.

During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial-mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells. Individual cells with few neighbours dispersed with fast, straight trajectories, whereas cells that encountered many neighbours migrated collectively with epithelial biomarkers. We modelled these emergent dynamics using a physical analogy to phase transitions during binary-mixture solidification, and validated it using drug perturbations, which revealed that individually migrating cells exhibit diminished chemosensitivity. Our measurements also indicate a degree of phenotypic plasticity as cells interconvert between individual and collective migration. The study of multicellular behaviours with single-cell resolution should enable further quantitative insights into heterogeneous tumour invasion.

Acute myeloid leukemia (AML) is a malignancy with complex genetics, poor survival rates and high rates of chemotherapy resistance. This study reports the establishment of a novel bone marrow mononuclear cell 3D-culture model and the application of this model to define sensitivity and resistance biomarkers as evident by DNA and RNA expression signatures in response to standard Cytarabine (Ara-C) chemotherapy. By simulating the bone marrow microenvironment, we optimized the growth conditions using frozen bone marrow mononuclear cells (BMMC) derived from healthy donors. We validated our platform by demonstrating that healthy BMMCs are capable of differentiating into all hematopoietic lineages and various types of stromal cells. Using this platform, we then characterized BMMC samples from 47 AML patients. Sixty percent of AML samples were capable of proliferating and 23 samples demonstrated robust growth. RNA sequencing was performed for 3D and 2D cultures to determine which conditions are superior at maintaining original gene expression signatures as compared to the uncultured bone marrow, To elucidate specific genetic mutations and/or gene expression signatures associated with the ability of ex vivo growth of these AML donors, whole exosome sequencing and RNA Sequencing were performed. The model identified unique gene expression signatures that correlated with sensitive as well as resistant donors in response to Ara-C treatment. These data demonstrate the translational value of our platform which should be widely applicable to evaluate other therapies in AML. Citation Format: Haiyan Xu, Eric S. Muise, Sarah Javaid, Lan Chen, Razvan Cristescu, My Sam Mansueto, Nicole Follmer, Jennifer Cho, Kimberley Kerr, Rachel Altura, Michelle Machacek, Benjamin Nicholson, George Addona, Ilona Kariv, Hongmin Chen. Identification of predictive genetic signatures of Cytarabine responsiveness in acute myeloid leukemia using a novel 3D translational culture model of primary bone marrow [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 LB-029.

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.

Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.

The expression, replication and repair of eukaryotic genomes require the fundamental organizing unit of chromatin, the nucleosome, to be unwrapped and disassembled. We have developed a quantitative model of nucleosome dynamics which provides a fundamental understanding of these DNA processes. We calibrated this model using results from high precision single molecule nucleosome unzipping experiments, and then tested its predictions for experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex hMSH2-hMSH6. We found that this calibrated model quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate DNA sequences and four distinct histone modification states. In addition, this model provides mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone modifications on this disassembly reaction. This model's precise agreement with current experiments suggests that it can be applied more generally to provide important mechanistic understanding of the numerous nucleosome alterations that occur during DNA processing.

Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA-histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2-hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.

DNA nucleotide mismatches and lesions arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains approximately 146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling whereby hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlight unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ~19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters, and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer., The p53-inducible antiproliferative gene BTG2 is suppressed in many cancers, in the absence of inactivating gene mutations. Here the authors show that the histone lysine methyltransferase SETD1A suppresses the expression of several p53 target genes including BTG2 by inducing a network of microRNAs.

Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. Implications: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases. Mol Cancer Res; 13(5); 934–43. ©2015 AACR.

Examination of circulating tumor cells (CTCs) holds the potential of offering a real-time non-invasive window into tumor biology. Information gleaned from CTC detection and characterization can be used for early cancer detection, choice of therapy decisions and long-term monitoring for disease recurrence and the emergence of drug resistance mechanisms. However, technical difficulties with CTC isolation and the inherent limitations of imaging-based analysis have hindered the broad clinical use of CTCs as biomarkers. To overcome these concerns, we have combined our unbiased microfluidic CTC enrichment technology, the iChip, with a highly sensitive and specific multi-gene RNA-based biomarker panel to develop an assay that detects CTC signatures in patient blood samples in a high throughput and quantitative fashion. This assay was applied to a cohort of breast cancer patients, including women with both localized and metastatic disease. It successfully identified CTC signal in 50-70% of metastatic and 20-40% of localized pretreatment patient samples. Patient CTC scores, assigned based on a multi-marker prediction algorithm, correlated with cancer stage and grade, but not with hormone receptor status, suggesting the applicability of the assay to a wide range of breast cancer subtypes. To determine if CTC scoring was useful for disease monitoring, we performed monthly blood collection from metastatic patients starting a new line of treatment. CTC score as early as one month after initiation of therapy, but not at pretreatment, was predictive of progression-free survival and treatment outcome, suggesting that real-time kinetic changes in CTC signatures are more clinically informative that one-time static evaluation of their presence in a given sample. In addition, a subset of high-risk genes showed expression patterns over the course of treatment that highly correlated with disease recurrence. This observation indicates that a multi-marker panel can parse out dynamic changes in cancer gene expression programs and provide a molecular insight into treatment responses that are not achievable by assays built around one or few markers. In conclusion, our novel RNA-based method of identifying CTC signatures in liquid biopsies provides a sensitive platform for breast cancer detection and monitoring that goes beyond CTC enumeration. It offers a non-invasive quantitative molecular characterization of tumor gene expression that can be used to guide informed clinical decisions in both standard course of care and clinical trial settings. Citation Format: Tanya T. Kwan, Aditya Bardia, Tilak Sundaresan, Laura Spring, Mark Kalinich, David Miyamoto, Xin Hong, Joseph LiCausi, Uyen Ho, Sarah Javaid, Erin Silva, Lecia Sequist, Shyamala Maheswaran, Daniel Haber. A novel RNA-based assay for the detection and monitoring of circulating tumor cell signatures in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1736. doi:10.1158/1538-7445.AM2017-1736

MK-4280 is a humanized IgG4 monoclonal antibody (mAb) that binds to the immune checkpoint receptor Lymphocyte Activation Gene-3 (LAG-3) to block the interaction with its ligand, Major Histocompatibility Complex (MHC) Class II. LAG-3 is frequently co-expressed with other immune checkpoint receptors, most notably programmed cell death protein-1 (PD-1), on T cells with an exhausted phenotype. LAG-3 and PD-1 cooperate to regulate peripheral immune tolerance in healthy individuals, and, conversely, play critical roles in several diseases, including autoimmunity, graft rejection, viral infections, and cancer. Co-blockade of LAG-3 and PD-1 in immunocompetent mouse tumor models have demonstrated augmented anti-tumor activity over single agents. However, the molecular mechanisms behind these combination effects have not been fully investigated. Here, preclinical proof-of-biology studies are presented for co-targeting LAG-3 and PD-1 in cancer. c28G10-mG1-[D265A] (abbreviated 28G10-mG1) is a rat:mouse chimera that mimics MK-4280 by its ability to directly block the mouse LAG-3:MHC Class II interaction without initiating Fc-mediated effector functions. As a single agent, 28G10-mG1 demonstrated modest anti-tumor activity across several syngeneic mouse tumor models, despite evidence of systemic drug exposure and target engagement (as assessed by sLAG-3-mAb complex accumulation). The combination of 28G10-mG1 and the anti-mouse PD-1 blocking antibody mDX400 resulted in greater tumor growth inhibition and increased numbers of complete responses (CR) over mDX400 alone in the MBT-2 tumor model. Furthermore, animals that had achieved CR to combination therapy were subsequently protected from MBT-2 re-challenge, suggesting the establishment of immune memory. RT-qPCR analyses revealed up-regulation of immune-related genes, primarily at Day 4 in the blood and Day 8 in the tumor with mDX400, but not 28G10-mG1, treatment. However, when combined with mDX400, 28G10-mG1 further altered the expression of many immune-related genes that were perturbed by mDX400 single agent therapy. Genes unique to combination treatment were also observed. Significantly, immune pathway signatures associated with clinical efficacy to Keytruda were upregulated with combination therapy. Tumor transcriptome and network analysis by RNAseq revealed enrichment in several immune- and cytokine-related pathways with combination therapy compared to mDX400 single agent therapy. Taken together, these preclinical oncology studies support the concept of co-targeting LAG-3 to increase the therapeutic efficacy of PD-1 blockade. Clinical investigation of MK-4280 in combination with anti-PD-1 therapy (pembrolizumab/Keytruda) is ongoing. Citation Format: Brian B. Haines, Sarah Javaid, Long Cui, Heather Hirsch, Saso Cemerski, Terri McClanahan, Manjiri Sathe, Shuli Zhang, Michael Rosenzweig, Brian Long, Rene de Waal Malefyt. Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4714. doi:10.1158/1538-7445.AM2017-4714

Summary Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6–48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.