Understanding the complex tumor microenvironment is key to the development of personalized therapies for the treatment of cancer including colorectal cancer (CRC). In the past decade, significant ...advances in the field of immunotherapy have changed the paradigm of cancer treatment. Despite significant improvements, tumor heterogeneity and lack of appropriate classification tools for CRC have prevented accurate risk stratification and identification of a wider patient population that may potentially benefit from targeted therapies. To identify novel signatures for accurate prognostication of CRC, we quantified gene expression of 12 immune‐related genes using a medium‐throughput NanoString quantification platform in 93 CRC patients. Multivariate prognostic analysis identified a combined four‐gene prognostic signature (TGFB1, PTK2, RORC, and SOCS1) (HR: 1.76, 95% CI: 1.05–2.95, *p < 0.02). The survival trend was captured in an independent gene expression data set: GSE17536 (177 patients; HR: 3.31, 95% CI: 1.99–5.55, *p < 0.01) and GSE14333 (226 patients; HR: 2.47, 95% CI: 1.35–4.53, *p < 0.01). Further, gene set enrichment analysis of the TCGA data set associated higher prognostic scores with epithelial–mesenchymal transition (EMT) and inflammatory pathways. Comparatively, a lower prognostic score was correlated with oxidative phosphorylation and MYC and E2F targets. Analysis of immune parameters identified infiltration of T‐reg cells, CD8+ T cells, M2 macrophages, and B cells in high‐risk patient groups along with upregulation of immune exhaustion genes. This molecular study has identified a novel prognostic gene signature with clinical utility in CRC. Therefore, along with prognostic features, characterization of immune cell infiltrates and immunosuppression provides actionable information that should be considered while employing personalized medicine.
Colorectal cancer has a complex tumor microenvironment that also provides a unique opportunity for the molecular classification of these patients. In this study, we have used NanoString technology to quantify potentially prognostic mRNAs in the Augusta University cohort. Further, a similar prognostic pattern was observed in the TCGA data set.
Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies ...with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.
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•Enniatin A targets the chaperone Hsp90 without inducing a heat shock response•Enniatin A induces immunogenic cell death in aggressive TNBC models•Enniatin A reduces immunosuppression by reducing PD-L1 protein and activating CX3CR1•Enniatin A promotes CD8+ T cell-mediated anti-tumor immunity against TNBC
Microenvironment; Biological sciences; Molecular biology; Cancer
Abstract
Molecular chaperones are guardians of the proteome and of cellular homeostasis. Dysregulation in the function of the Hsp90 chaperoning machine in particular has been implicated in metabolic, ...oncological, neurodegenerative, and cardiovascular diseases. Targeting Hsp90 has been shown to have a combinatorial impact on dysfunctional circuitries that underlie human cancers. Thus, several inhibitors targeting the N-terminal ATP-binding pocket of Hsp90 are undergoing clinical trials as anticancer agents. They inactivate the ATPase activity of the chaperone, causing proteasomal degradation of its “client” oncogenic proteins. Although these inhibitors provided a proof-of-principal that Hsp90 is a valuable therapeutic target, they exhibit modest activity in the clinic, in part because they concomitantly induce a heat shock response (HSR), which turns on pro-survival pathways. Consequently, new mechanisms to inhibit the Hsp90 chaperone machine that do not induce the heat shock response are needed.
To address this, we have developed a unique high throughput screen (HTS) based on the progesterone receptor (PR) and the five purified chaperones required for folding steroid receptors, i.e., Hsp90, Hsp70, Hsp40, Hsp70/Hsp90 organizing protein (Hop), and p23. During our preliminary screening of natural products, we discovered that the compound AD07 kills cancer cells through inhibition of the Hsp90 machine without a significant induction of HSR. In addition, AD07 induces a powerful T cell-mediated immune response, resulting in highly efficient tumor killing in a syngeneic mouse model and long-term memory against the primary tumor. At the molecular level, AD07 reduces the protein levels of two key mediators of tumor-induced immune tolerance: programmed cell death ligand-1 (PDL-1) and indolamine 2,3 dioxygenase (IDO). We therefore propose that AD07 is a promising anti-tumor agent targeting the Hsp90 machine through a novel mechanism of action involving cancer cell toxicity, which increases its immunogenicity, and modulation of the tumor microenvironment to reduce immunotolerance.
Citation Format: Ahmed Chadli, Nada Eisa, Vincent Crowley, Sumin Lu, Yasmeen Jilani, Hasan Korkaya, Brian Blagg. A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy 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 12.
Abstract
Colorectal cancer (CRC) affects nearly 1.4 million individuals every year and is the third most common cause of deaths due to cancer. There is a need to identify novel prognostic biomarkers ...with accurate prognostic and therapeutic implications. In this study, we sought to identify prognostic gene expression signatures and explore their relationship with the tumor microenvironment. Under the IRB approved protocol, FFPE blocks of CRC patients were acquired from the Medical College of Georgia, Augusta, USA. Tumor regions and adjacent normal tissue regions were identified and marked by a board-certified pathologist. RNA isolation was performed from tumor sections (n = 88) and adjacent normal regions (n = 16). A panel of 17 genes with the highest perturbation in TCGA dataset was investigated on the FFPE tissues. The quantification of the mRNA molecules was performed using total RNA (300 ng) on the NanoString platform. It uses a specific molecular barcoding system to tag target nucleic acid molecules with very high sensitivity and specificity. The immunological dynamics of the prognostic genes were analyzed through TIMER (Tumor-Infiltrating Immune Cells) web portal using the TCGA-COAD dataset. In the clinical dataset, MCM4, YWHAB, LRRC59 and DPP7 were highly expressed in tumor tissue compared to adjacent control and were associated with worse prognosis (Log-rank test, p < 0.001*). However, PI4K2B, PCMT1 and PBX1P1 showed higher expression in adjacent normal tissue compared to tumor regions (Student's t-test, p < 0.001*). Further analysis using TIMER portal revealed that copy number alterations in YWHAB were significantly associated with decreased tumor infiltration of B cells, CD8+ T cells, neutrophils and dendritic cells (Wilcoxon test, p < 0.001*). The predominant alterations observed were arm-level gain and high amplification. Further, arm-level gain of MCM4 was associated with lower infiltration of B cells and CD8+ T cells (Wilcoxon test, p < 0.001*) and FBXO46 was associated with lower infiltration of B cells only (Wilcoxon test, p < 0.05*). Additional comparison between CRC tumor and adjacent normal tissue was explored using “DiffExp” module of TIMER web portal. The comparison showed that YWHAB, DPP7 and MCM4 were expressed at higher amounts in tumor tissues compared to adjacent normal tissue. These preliminary analyses, therefore reveal the significance of prognostic genes and their association with the infiltration of immune cells that in turn can be of therapeutic value. A comprehensive network of prognostic gene signatures and its perturbations in immune cell functioning can aid in efficient prognostic classification and design of new therapies.
Citation Format: Pankaj Kumar Ahluwalia, Ashis Mondal, Meenakshi Ahluwalia, Nikhil Sahajpal, Kimya Jones, Yasmeen Jilani, Allan Njau, Ravindra Kolhe. Exploring prognostic and immunological impact of a novel 4 gene signature in colorectal cancer patients abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4334.
Abstract
A clinical next generation sequencing (NGS) assay comprises various factors taken into consideration including selection of appropriate markers thereby panel size, nucleic acid input, ...assessment and interpretation, and workflow. The downstream benefits of the development of such panels therefore involve improvement in prognosis, patients' outcome, and benefit from targeted therapy. Myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN, acute myeloid leukemia (AML) are associated with somatic mutations in around 25 to 50 genes. Herein, we describe clinical validation of a DNA based targeted NGS assay utilizing QIAact Myeloid DNA UMI panel in combination with QIAGEN GeneReader NGS system. The assay employs unique molecular index technology (UMI) into a gene specific, primer based target enrichment technology enabling sequencing of specific regions of interest providing an integrated solution by simultaneously assessing many candidate genes for actionable mutations. The QIAact Myeloid DNA UMI panel is a 25 gene panel for markers of known clinical significance, including single nucleotide variants (SNV), and large insertion/deletion mutations. At the lab, 40 individual AML specimens with known mutation profiles were tested. The limit of detection (LOD) was assessed by sequencing Seraseq myeloid mutation DNA mix as positive control. For accuracy, inter-run and intra-run controls were also implemented. All samples were previously characterized by using another 54 gene NGS panel. The samples were prepared using QIAact Myeloid DNA UMI panel kits, sequenced with QIAGEN GeneReader NGS system, and mutations identified using the QIAGEN Clinical Insight (QCI) Analyze software suite, adjusted specifically for variant calling. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection (LOD) studies also determined that input of 40ng DNA was optimal for high analytical sensitivity. High positive and negative percentage agreement with prior results was observed across all variant categories. The assay was also able to identify 52bp deletion CALR type 1 variant, and CEBPα amplicons mutations which require separate bi-directional Sanger sequencing for identification. Also, the assay was able to identify FLT3 ITDs up to 101bp insertion which are detected by performing PCR separately. Hence, the study presents the data establishing the QIAact Myeloid DNA UMI panel suitable for implementation as a routine clinical NGS test for myeloid malignancies. The optimized chemistry allows high-throughput analytical sensitivity for detection of highly relevant mutations including large Insertion/Deletion (InDel).
Citation Format: Meenakshi Ahluwalia, Ashis Mondal, Nikhil Sahajpal, Allan Njau, Kimya Jones, Pankaj Kumar Ahluwalia, Yasmeen Jilani, Ravindra Kolhe. Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 816.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the ...feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4). The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply.
Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates ...with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.
Abstract
Recent findings have shown that the Heat Shock Protein 90 (Hsp90) co-chaperone UNC45A is overexpressed in ovarian and breast cancers. Previously, we have shown that UNC45A is a centrosomal ...protein essential for cervical tumor cell growth through activation of the checkpoint kinase 1 (ChK1). In this report, we further examined the role of UNC45A in breast tumorigenesis using a variety of biochemical and cell biology techniques and animal models. We confirmed that UNC45A is highly overexpressed in human breast-infiltrating ductal carcinomas as compared to adjacent normal tissues. Silencing UNC45A in vitro blocked the proliferation of all breast cancer subtypes and drastically reduced tumor growth of the triple negative MDA-MB-231 cell line implanted in mammary fat pads of NOD/SCID mice. However, loss of UNC45A did not affect the proliferation of normal mammary cells. Remarkably, UNC45A becomes more nuclear in human cancer tissues and cancer cell lines as compared to normal tissues and non-transformed Hs578Bst and HME mammary cell lines, respectively. This suggests an important nuclear function for UNC45A during tumorigenesis. Microarray analysis of mRNA from Hs578T cells showed that loss of UNC45A alters the expression of 121 genes, involved in cancer and cellular development and growth networks. Relevant to cell proliferation, we found that Nek7 gene was significantly repressed upon silencing UNC45A, which was validated by RTqPCR and Western blot analyses in multiple breast cancer cell lines. Nek7 is a member of the NIMA (never in mitosis, gene A) family of serine/threonine kinases. It plays a key role in centrosomal separation during mitosis. This correlates neatly with our observation that loss of UNC45A causes a centrosomal separation defect, cell proliferation arrest and death of breast cancer cell lines. ChIP experiments showed that UNC45A binds to the promoter of the Nek7 gene, suggesting direct transcriptional regulation. Interestingly, the UNC45A sequence contains four LxxLL motifs, which are thought to be signatures for co-activator binding to nuclear receptors. Furthermore, computational analysis identified two glucocorticoid response elements (GRE) consensus sequences in the Nek7 promoter, suggesting its transcriptional regulation by the glucocorticoid receptor (GR). This hypothesis was further strengthened by a significant decrease in the mRNA and protein levels of Nek7 upon silencing GR. Thus, our data suggest that UNC45A functions as a GR co-activator to control Nek7 gene transcription. Consistent with this, immunoprecipitation experiments confirmed that UNC45A and GR form endogenous complexes, and treatment of Hs578T and MCF7 cell lines with dexamethasone upregulates Nek7 mRNA and protein levels. In conclusion our data strongly support the premise that UNC45A promotes Nek7 transcription through activation of GR, and thus controls centrosomal separation and cancer cell proliferation.
Citation Format: Yasmeen Jilani, Nada H. Eisa, Kashish Kainth, Sumin Lu, Nehal M. Elsherbiny, Laila A. Eissa, Mamdouh M. Elshishtawy, Hasan Korkaya, Abdeljabar El Andaloussi, Ahmed Chadli. The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation 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 4493. doi:10.1158/1538-7445.AM2017-4493
Seaweeds have been used as nutraceuticals because of certain metabolites present in some species including polyphenols. Seven species of seaweeds collected from the Karachi coast were screened for ...the first time both for phycochemical analysis and pharmacological activities. The phycochemicals quantified included phenols, flavonoids, tannins and saponins. Melanothamnus afaqhusainii showed the highest content of phenols (2.16mg GAE/g) while highest flavonoids were observed in Coelarthrum muelleri (4.59mg RE/g). Tannins were found in low amounts while saponins were observed as major constituents among the seaweeds ranging from 1.34-3.47% in Sargassum swartzii and Codium flabellatum, respectively. Saponins were not analysed in Rhodophyta due to gel formation. Pharmacological screening revealed analgesic and anti-inflammatory effects. Both the methods of analgesic activities have shown significant increase in reaction time when methanol extracts were used. The reason for delayed anti-inflammatory activity of Solieria robusta and C. muelleri was found correlating with its gel forming ability. While Cystoseira indica and C. flabellatum exhibited highly significant effect from 1st to 5th h. Results suggested that selected seaweeds had potential in combating both acute and chronic inflammation and pain and hence can be used for therapeutic efficacy.
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