Inactivation Of Tumor Suppressor Genes Research Articles

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

Recurrent noncoding U1snRNA mutations drive cryptic splicing in SHH medulloblastoma.

Summary ParagraphRecurrent somatic single nucleotide variants (SNVs) in cancer are largely confined to protein coding genes, and are rare in most pediatric cancers1–3. We report highly recurrent hotspot mutations of U1 spliceosomal small nuclear RNAs (snRNAs) in ~50% of Sonic Hedgehog medulloblastomas (Shh-MB), which were not present across other medulloblastoma subgroups. This U1-snRNA hotspot mutation (r.3a>g), was identified in <0.1% of 2,442 cancers across 36 other tumor types. Largely absent from infant Shh-MB, the mutation occurs in 97% of adults (Shhδ), and 25% of adolescents (Shhα). The U1-snRNA mutation occurs in the 5′ splice site binding region, and snRNA mutant tumors have significantly disrupted RNA splicing with an excess of 5′ cryptic splicing events. Mutant U1-snRNA mediated alternative splicing inactivates tumor suppressor genes (PTCH1), and activates oncogenes (GLI2, CCND2), represents a novel target for therapy, and constitutes a highly recurrent and tissue-specific mutation of a non-protein coding gene in cancer.

1170P - A parent-of-origin effect of the RB1 mutations in retinoblastoma with low penetrance and variable expressivity

BackgroundRetinoblastoma (RB) is the most common pediatric intraocular neoplasm caused by the biallelic inactivation of the RB1 tumor suppressor gene. In 40% of cases, the development of RB is mediated by a germline mutation in one of the alleles of RB1. Patients with germline mutation develop bilateral tumor with penetrance of more than 90%. However, some families demonstrate cases of RB with low penetrance (unaffected carries) and variable expressivity (carries develop either bi- or uni- RB).It is believed that the phenotypic manifestation of hereditary retinoblastoma depends on the functional type of the germinal mutation in the RB1. The molecular mechanisms underlying the variable phenotypic manifestation of the same mutation in different family members are currently explained by the parent-of-origin effect of RB1 mutation. MethodsUsing NGS of the RB1 we have analyzed DNA from blood of 331 unrelated patients with RB (226 patients with uni- RB and 105 with bi- RB). DNA samples of available family members were also examined for the presence of an identified mutation using Sanger sequencing. ResultsWe identified 11 germline mutations in the RB1 that led to the RB with low penetrance and/or variable expressivity in 12 families. Among the identified mutations:, 25.0% - are missense mutations, 58.3% - are splice mutations and 16.7% - are frame shift mutations. In 91,7% of cases, probands inherited the mutant allele from their fathers, who were either clinically healthy carriers (7 families) or had the uni-/bilateral form of RB (3 families).Table: 1170PTable: 1170PMutationsCarrier (Proband, P)Form (uni, U; bi-lateral, B; no symptoms, -)c.1364G>C; c.1573G>A; c.1981C>T; с.607+1G-T (2 families); c.45_76del; с.83delP ♂U -c.861G>CP ♀U -c.939G>AP ♂ Grandfather UncleU - - Uc.380+1G-A; с.1695+5G-T; c.1696-2A-GP ♂B U ConclusionsThe identification of mutations in the RB1 leading to the development of RB with low penetrance and variable expressivity, is necessary for adequate treatment and competent determination of the risk of developing the disease in other family members. Legal entity responsible for the studyState assignment of Ministry of Science and Higher Education of the Russian Federation. FundingThe state assignment of Ministry of Science and Higher Education of the Russian Federation. DisclosureAll authors have declared no conflicts of interest.

Mathematical modelling the pathway of genomic instability in lung cancer

Genomic instability plays a significant role in lung cancer. Although substantial research has been conducted using both clinical and theoretical studies, it is still a hotly debated issue to whether genomic instability is necessary or whether genomic instability precedes oncogenes activation and tumor suppressor genes inactivation for lung cancer. In response to this issue, we come up with a mathematical model incorporating effects of genomic instability to investigate the genomic instability pathway of human lung cancer. The presented model are applied to match the incidence rate data of lung cancer from the Life Span Study cohort of the atomic bomb survivors in Nagasaki and Hiroshima and the Surveillance Epidemiology and End Results registry in the United States. Model results suggest that genomic instability is necessary in the tumorigenesis of lung cancer, and genomic instability has no significant impact on the net proliferation rate of cells by statistical criteria. By comparing the results of the LSS data to those of the SEER data, we conclude that the genomic instability pathway exhibits a sensitivity to radiation exposure, more intensive in male patients.

PTEN Mouse Models of Cancer Initiation and Progression

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated, deleted, and functionally inactivated tumor suppressor genes in human cancer. PTEN is found mutated both somatically and in the germline of patients with PTEN hamartoma tumor syndrome (PHTS). PTEN encodes a dual lipid and protein phosphatase that dephosphorylates the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), in turn negatively regulating the oncogenic PI3K-AKT pathway, a key proto-oncogenic player in cancer development and progression. Because of importance of PTEN in tumorigenesis, a large number of sophisticated genetically engineered mouse models (GEMMs) has been designed to elucidate the underlying mechanisms by which the "PTEN pathway" promotes tumorigenesis, while simultaneously providing a well-tailored system for the identification of novel therapies and offering platforms for new drug discoveries. This review summarizes the major cancer mouse models through which the PTEN pathway has been genetically deconstructed, and outlines the rapid development of GEMMs toward more detailed functional and tissue-specific analysis.

Targeting mRNA processing as an anticancer strategy.

Discoveries in the past decade have highlighted the potential of mRNA as a therapeutic target for cancer. Specifically, RNA sequencing revealed that, in addition to gene mutations, alterations in mRNA can contribute to the initiation and progression of cancer. Indeed, precursor mRNA processing, which includes the removal of introns by splicing and the formation of 3' ends by cleavage and polyadenylation, is frequently altered in tumours. These alterations result in numerous cancer-specific mRNAs that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumour-suppressor genes. Abnormally spliced and polyadenylated mRNAs are also associated with resistance to cancer treatment and, unexpectedly, certain cancers are highly sensitive to the pharmacological inhibition of splicing. This Review summarizes recent progress in our understanding of how splicing and polyadenylation are altered in cancer and highlights how this knowledge has been translated for drug discovery, resulting in the production of small molecules and oligonucleotides that modulate the spliceosome and are in clinical trials for the treatment of cancer.

Friend or foe-IDH1 mutations in glioma 10 years on.

The identification of recurrent point mutations in the isocitrate dehydrogenase 1 (IDH1) gene, albeit in only a small percentage of glioblastomas a decade ago, has transformed our understanding of glioma biology, genomics and metabolism. More than 1000 scientific papers have been published since, propelling bench-to-bedside investigations that have led to drug development and clinical trials. The rapid biomedical advancement has been driven primarily by the realization of a neomorphic activity of IDH1 mutation that produces high levels of (d)-2-hydroxyglutarate, a metabolite believed to promote glioma initiation and progression through epigenetic and metabolic reprogramming. Thus, novel inhibitors of mutant IDH1 have been developed for therapeutic targeting. However, numerous clinical and experimental findings are at odds with this simple concept. By taking into consideration a large body of findings in the literature, this article analyzes how different approaches have led to opposing conclusions and proffers a counterintuitive hypothesis that IDH1 mutation is intrinsically tumor suppressive in glioma but functionally undermined by the glutamate-rich cerebral environment, inactivation of tumor-suppressor genes and IDH1 copy-number alterations. This theory also provides an explanation for some of the most perplexing observations, including the scarcity of proper model systems and the prevalence of IDH1 mutation in glioma.

Modulation of all-trans retinoic acid-induced MiRNA expression in neoplastic cell lines: a systematic review

BackgroundCancer is a genetic and epigenetic disease that involves inactivation of tumor suppressor genes and activation of proto-oncogenes. All-trans retinoic acid (ATRA) is an isomer of retinoic acid involved in the onset of differentiation and apoptosis of a number of normal and cancer cells, functioning as an anti-cancer agent in several neoplasms. Ectopic changes in the expression of certain microRNAs (miRNAs) occur in response to ATRA, leading to phenotypic alterations in neoplastic cell lines. Moreover, the modulation of miRNA patterns upon ATRA-treatment may represent an effective chemopreventive and anti-cancer therapy strategy. The present systematic review was performed to provide an overview of the modulation of ATRA-induced miRNA expression in different types of neoplastic cells and identify the efficacy of intervention factors (i.e., concentration and duration of treatment) and how they influence expression profiles of oncogenesis-targeting miRNAs.MethodsA systematic search was conducted according to the PRISMA statement via the US National Library of Medicine MEDLINE/PubMed bibliographic search engine.ResultsThe search identified 31 experimental studies involving human cell lines from nine different cancer types (neuroblastoma, acute myeloid leukemia, breast cancer, lung cancer, pancreatic cancer, glioma, glioblastoma, embryonal carcinoma, and colorectal cancer) treated with ATRA at concentrations ranging from 10− 3 μmol/L to 102 μmol mol/L for 24 h to 21 days.ConclusionThe concentrations used and the duration of treatment of cancer cells with ATRA varied widely. The presence of ATRA in the culture medium of cancer cells was able to modulate the expression of more than 300 miRNAs, and inhibit invasive behavior and deregulated growth of cancer cells, resulting in total tumor remission in some cases. ATRA may thus be broadly effective for neoplasm treatment and prevention, although these studies may not accurately represent in vivo conditions. Additional studies are required to elucidate ATRA-induced miRNA modulation during neoplasm treatment.

Hepatocellular Carcinoma: Molecular Mechanisms and Targeted Therapies.

Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumors worldwide. HCC is a complex process that is associated with several etiological factors, which in turn result in aberrant activation of different cellular and molecular pathways and the disruption of balance between activation and inactivation of protooncogenes and tumor suppressor genes, respectively. Since HCC most often occurs in the setting of a diseased or cirrhotic liver and most of the patients are diagnosed at the late stage of disease, prognosis is generally poor. At present, limited treatment options with marginal clinical benefits are available. Systemic therapy, particularly in the form of conventional cytotoxic drugs, are generally ineffective. In recent years, molecular-targeted therapies have been clinically used to treat various cancers, including liver cancer. This approach inhibits the growth of tumor cells by interfering with molecules that are involved in carcinogenesis, which makes it more selective and specific than cytotoxic chemotherapy. Many clinical trials have been carried out while using molecular targeted drugs in advanced HCC with many more in progress. The clinical trials in HCC to date have evaluated a single-targeted therapy alone, or two or more targeted therapies in parallel. The aim of this review is to provide insight of various molecular mechanisms, leading to HCC development and progression, and also the range of experimental therapeutics for patients with advanced HCC. The review will summarize different clinical trials data the successes and failures of these treatments, as well as the most effective and approved drugs designed against HCC.

Understanding Cancer Through the Lens of Epigenetic Inheritance, Allele-Specific Gene Expression, and High-Throughput Technology.

Epigenetic information is characterized by its stable transmission during mitotic cell divisions and plasticity during development and differentiation. This duality is in contrast to genetic information, which is stable and identical in all cells in an organism with exception of immunoglobulin gene rearrangements in lymphocytes and somatic mutations in cancer cells. Allele-specific analysis of gene expression and epigenetic modifications provides a unique approach to studying epigenetic regulation in normal and cancer cells. Extension of Knudson's two-hits theory to include epigenetic alteration as a means to inactivate tumor suppressor genes provides better understanding of how genetic mutations and epigenetic alterations jointly contribute to cancer development. High-throughput technology has greatly accelerated cancer discovery. Large initiatives such as TCGA have shown that epigenetic components are frequent targets of mutations in cancer and these discoveries provide new insights into understanding cancer etiology and generate new opportunities for cancer therapeutics.

Genetic alterations in anaplastic thyroid carcinoma and targeted therapies.

Thyroid cancer is the most common type of endocrine malignancy, and its incidence is increasing. Anaplastic thyroid cancer (ATC), referring to undifferentiated subtypes, is considered to be aggressive and associated with poor prognosis. Conventional therapies, including surgery, chemotherapy and radioiodine therapy, have been used for ATC, but these do not provide any significant reduction of the overall mortality rate. The tumorigenesis, development, dedifferentiation and metastasis of ATC are closely associated with the activation of various tyrosine cascades and inactivation of tumor suppressor genes, including B-Raf proto-oncogene, serine/threonine kinaseV600E, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α,tumor protein 53 mutations and telomerase reverse transcriptase mutation. These pathways exert their functions individually or through a complex network. Identification of these mutations may provide a deeper understanding of ATC. A variety of tyrosine kinase inhibitors have been successfully employed for controlling ATC growth in vitro and in xenografts. Certain novel compounds are still in clinical trials. Multi-kinase inhibitors provide a novel approach with great potential. This systematic review determined the prevalence of the major genetic alterations and their inhibitors in ATC.

The Relationship between the Methylation of Promoter Regions of Tumor Suppressor Genes PTEN and APC with Endometrial Cancer.

Background:Endometrial neoplasms is one of the most typical gynecologic diseases with harmful effects. Promoter hypermethylation is an important mechanism of the inactivation of tumor suppressor genes in endometrial neoplasms. Epigenetic changes of the PTEN and APC genes have shown to be present in various cancers. Therefore, in this study, we have investigated the association between the promoter hypermethylation of PTEN and APC genes with endometrial neoplasms. Methods:For this study, 28 patients with endometrial neoplasms as well as 22 controls were studied. Analysis of the promoter methylation regions of PTEN and APC genes were performed by Methylation-Specific PCR. Results:The frequency of PTEN and APC genes promoter methylation was 28.57% and 17.86% in tumor tissues, and 11.54% and 3.85% in blood samples, respectively. We found a significant relationship between blood and tissue in PTEN methylation (p = 0.0353). Additionally, we determined a closely significant difference between normal tissue and tumor tissue of the PTEN gene (p = 0.0787) and blood and tissue samples of the APC gene in methylated promoter regions (p=0.0623). Furthermore, these results suggest that there is no significant relationship between the promoter methylation of PTEN and APC with clinical characteristics.Conclusion:DNA methylation deficiency is a well known highlighted factor in tumorigenesis, therefore the promoter hypermethylation of PTEN and APC can be indicated as a risk factor in endometrial neoplasms.

PTEN Tumor-Suppressor: The Dam of Stemness in Cancer.

PTEN is one of the most frequently inactivated tumor suppressor genes in cancer. Loss or variation in PTEN gene/protein levels is commonly observed in a broad spectrum of human cancers, while germline PTEN mutations cause inherited syndromes that lead to increased risk of tumors. PTEN restrains tumorigenesis through different mechanisms ranging from phosphatase-dependent and independent activities, subcellular localization and protein interaction, modulating a broad array of cellular functions including growth, proliferation, survival, DNA repair, and cell motility. The main target of PTEN phosphatase activity is one of the most significant cell growth and pro-survival signaling pathway in cancer: PI3K/AKT/mTOR. Several shreds of evidence shed light on the critical role of PTEN in normal and cancer stem cells (CSCs) homeostasis, with its loss fostering the CSC compartment in both solid and hematologic malignancies. CSCs are responsible for tumor propagation, metastatic spread, resistance to therapy, and relapse. Thus, understanding how alterations of PTEN levels affect CSC hallmarks could be crucial for the development of successful therapeutic approaches. Here, we discuss the most significant findings on PTEN-mediated control of CSC state. We aim to unravel the role of PTEN in the regulation of key mechanisms specific for CSCs, such as self-renewal, quiescence/cell cycle, Epithelial-to-Mesenchymal-Transition (EMT), with a particular focus on PTEN-based therapy resistance mechanisms and their exploitation for novel therapeutic approaches in cancer treatment.

Generation of two H1 hESC sublines carrying deletions of RB1 exon 1/promoter in heterozygous or compound heterozygous state

Biallelic inactivation of the retinoblastoma tumor suppressor gene (RB1) causes formation of retinoblastoma, a retinal eye tumor occurring in early childhood. Using the CRISPR/Cas9 nickase system, exon 1 of RB1 was deleted, including the RB1 promoter. As a result, sublines were generated carrying deletions of RB1 exon 1/promoter on one or both alleles.

The essence of the new blood vessels formation process in cancers based on the example of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is still one of the most enigmatic breast cancer types and no firm, efficient therapy has been established yet. Regarding its molecular profile and lack of specific receptors, angiogenesis, as an inseparable part of tumor biology, was taken into consideration as one of the leading causes of tumor progression. Taking into account cancerous neovascularization stages and recent scientific researches, it appears probable to understand the process of tumor progression and separate several ways of inhibiting this crucial phenomenon determining neoplasm invasiveness. Combination of traditional therapy and new antiangiogenic agents is a chance for patients to receive treatment as effective as patients with other types of breast cancer or cancer at all. New therapeutic methods may impact the genome level of carcinogenesis. There are many studies aimed at blocking transformed protooncogenes or restore the activity of tumor suppressor genes. Dealing with the process of cancer cells hypoxia and understanding the role of hypoxia inducible factor (HIF) and vascular endothelial growth factor (VEGF) – main angiogenesis inducers – let us introduce proper drugs blocking their activity (e.g. indenopyrazole 21, bevacizumab). Monoclonal antibodies, genetic therapies and newly synthesized drugs replace previously used therapeutic methods as poly (ADP-ribose) polymerase (PARP) inhibitors, mTOR kinase inhibitors, or Ras inhibitors. These new treatment methods include sunitinib, Aurora A kinase/tyrosine kinase inhibitor ENMD-2076, tested due to their multi-target actions, oncolytic viral therapy or small interfering RNA (siRNA). Despite numerous scientific and clinical studies, triple-negative breast cancer remains difficult to treat and is characterized by significantly worse prognosis, statistically shorter five-year survival rate and tendency to relapse.

Abstract 3490: Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population

Abstract Background: CRC is the 3rd most commonly diagnosed malignancy and 4th leading cause of cancer related deaths worldwide. Over two decades of extensive research from the West has revealed three major tumorigenesis pathways responsible for CRC viz. a) Chromosomal Instability (CIN) arising from aberrant activation of Wnt signalling, b) Microsatellite Instability (MSI) caused by inactivation of MisMatch Repair (MMR) genes and c) CpG Island Methylator Phenotype resulting in promoter DNA methylation induced inactivation of tumour suppressor genes. Previous results from our laboratory showed a significant proportion of Early Onset Sporadic Rectal Cancer (EOSRC) to be driven neither by aberrant Wnt signalling nor MSI. We also identified a significant proportion of MSI+ familial CRC not exhibiting loss either of the four major MMR genes indicating involvement of addition MMR or non-MMR genes. We therefore endeavoured to determine status of MSI in sporadic CRC samples from the Indian population. We are performing analysis of the status of other molecular features including CIMP, BRAF/KRAS/p53 mutation status, Wnt, etc. to obtain a wholesome molecular profile of sporadic CRC from the Indian population. Methodology: The microsatellite pattern in CRC tumours was determined using the five microsatellite marker panel as per the Bethesda guidelines. Expression status of MMR proteins (MLH1, MSH2, MSH6 &amp; PMS2) was evaluated using immunohistochemistry. Mutations in BRAF (V600E) and KRAS (Codon 12 and 13) were identified by Sanger sequencing. CIMP status is being determined using the MethyLight technology as well as methylation specific PCR. Results: A comprehensive screen of close to 200 CRC samples revealed a significantly higher proportion of sporadic CRC tumors (41%; 50% of colonic and 31% of rectal cancer samples) exhibiting MSI as compared to 14% reported in The Cancer Genome Atlas (TCGA) data. Among MSI-high tumours, only 27% and 15% showed loss of MLH1 or MSH2 respectively, a significantly lower frequency compared to Caucasian data. Instability in the BAT25 microsatellite was significantly high in tumors harboring MLH1 loss. More importantly, a significant proportion of MSI+ CRC tumors were devoid of loss of either of the four major MMR genes. In addition, the BRAF V600E mutation, a known cause of MLH1 silencing, is absent in all but one MSI+ tumors. Work on determining status of CIMP and Wnt signalling is currently underway. Conclusions: Significantly higher proportion of sporadic CRCs from Indian population exhibit MSI but the underlying mechanism appears to be novel and distinct from those identified from the Caucasian population. Deep sequencing based studies are currently underway to determine molecular aberrations driving MSI in the Indian sporadic CRC. Note: This abstract was not presented at the meeting. Citation Format: Ashmala Naz, Vijaya Tourani, Satish Rao, Murali Dharan Bashyam. Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population [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 3490.

Abstract 1660: Identification of allelic imbalance utilizing heterozygous genotype allele frequencies and intensities

Abstract Allelic imbalance (AI) events, such as amplification, deletion or copy-neutral loss-of-heterozygosity (cn-LOH) can result in the activations of oncogenes or inactivations of tumor suppressor genes that are critical to the process of carcinogenesis and metastasis. However, tumor samples often have low tumor-cellularity and small subclones that require sensitive and robust algorithm for AI detections. To overcome this challenge, our lab has developed an original software application hapLOH (Vattathil and Scheet, 2013) that identifies AI with high sensitivity in SNP array data by utilizing heterozygous genotype allele frequencies in SNP array data using a Hidden Markov Model. Motivated by the plethora of next-generation sequencing (NGS) data, we carried this logic and functionality and developed hapLOHseq (San Lucas et al, 2016) for detection of subtle AI in NGS data using reference and alternate allele read depths and a set of haplotype estimates based on 1000 genome data. Recently, we sought to improve AI detection and provide support for different data types by developing haploh-cn. Haploh-cn extends our previous tools by supporting both SNP array and NGS data, and by incorporating intensity data and Log R ratios (LRR) into the underlying Hidden Markov Model for SNP array data, and incorporating sequencing depth for NGS data. In order to evaluate haploh-cn’s performances, we have downloaded The Cancer Genome Atlas Lung Adenocarcinoma whole exome sequencing data and corresponding Affymetrix SNP6 array data. Then, we simulated tumor cellularity by performing in-silico dilution of NGS and SNP array data. For NGS data, we down sampled tumor sequencing BAM files and mixed in matched-normal sequencing reads. For SNP6 array data, we combined the adjusted B allele frequencies of heterozygous sites in the tumor sample and matched-normal sample. Using the AI events detected in the undiluted tumor samples as the gold standard, we assessed the performance of haploh-cn in the computational diluted samples. Our results showed that haploh-cn had high sensitivity without sacrificing specificity in diluted tumor samples. In summary, haploh-cn is a robust and powerful method for profiling subtle AI in NGS and SNP array data. Citation Format: Kyle Chang, Francis A. San Lucas, Zuhal Ozcan, Smruthy Sivakumar, Yasminka A. Jakubek, Richard G. Fowler, Paul Scheet. Identification of allelic imbalance utilizing heterozygous genotype allele frequencies and intensities [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 1660.

Abstract 4513: Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors

Abstract Although cancer usually arises from the combined effects of oncogene activation and tumor suppressor gene inactivation, most targeted cancer treatments are focused on the inhibition of oncoproteins, with less consideration given to the possible concomitant effects on tumor suppressors. Here we describe a critical role of the DLC1 tumor suppressor in the growth regulation of cancer and have identified two kinases that directly phosphorylate DLC1 and attenuate its tumor suppressor activity, leading to the hypothesis that reactivation of DLC1 by the relevant kinase inhibitors should be evaluated as a candidate biomarker of clinical response. We determined that the SRC and AKT kinases directly phosphorylate and attenuate the Rho-GAP and tumor suppressor activities of DLC1 by distinct, cooperative mechanisms and that inhibition of the SRC and/or AKT kinases has strong antitumor activity in two DLC1-positive tumor models that have high SRC and AKT activities, the MMTV-PyMT breast cancer model and tumor xenografts from a NSCLC line. In both models, treatment with the kinase inhibitors results in cooperative DLC1 reactivation, which was monitored by the reactivation of the Rho-GAP and tumor suppressor activities of DLC1. Reactivation of DLC1 makes a critical contribution to the antitumor activity of the AKT or the SRC inhibitors in both models, and the combined treatment with inhibitors of both kinases has even greater Rho-GAP and antitumor activities, which is correlated with greater reductions in RhoA-GTP and its downstream signaling. Furthermore, the combined treatment with AKT and SRC inhibitors induced cellular senescence and apoptosis to a greater degree than single agent treatment, as measured by beta-galactosidase and annexin V expression after short-term tumor treatment. Their antitumor activity is much weaker in isogenic tumors that are DLC1-negative or express DLC1 mutants that are not activatable by the AKT and the SRC inhibitors. A limitation of this therapeutic approach is that it is likely to benefit only those tumors that express moderate to high levels of DLC1 protein. However, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein expression through the epigenetic destabilization of the protein. We hypothesize that it could be beneficial to treat this class of solid tumors with a combination of drugs that can stabilize the DLC1 protein and concurrently inhibit the AKT and SRC activities. Our results indicate the feasibility of this approach, as treatment with proteasome inhibitors in several tumor cell lines that lack detectable DLC1 protein but express its mRNA increased the steady state level of DLC1 protein, which enabled SRC and AKT inhibitors to more potently inhibit tumor cell growth and reduce RhoA-GTP and its signaling. Thus, reactivation of the DLC1 tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors. Citation Format: Brajendra K. Tripathi, Meghan F. Anderman, James H. Doroshow, Douglas R. Lowy. Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors [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 4513.

Abstract 5136: Adapting long read sequencing technologies for targeted and single cell applications

Abstract Structural variations (SV), a hallmark of genomic instability in cancer, include insertions, deletions, duplications, inversions, or translocations that can either activate oncogenes or inactivate tumor suppressor genes. These variants contribute to the complexity of the cancer genome but have not been successfully resolved yet. While short-read sequencing has aided cancer genomics, it has performed poorly in SV detection, with false positive and false negative rates of 50% or more. Long-read sequencing generates reads that are &amp;gt;10 kb long and has helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. Long reads can span SV with a single continuous read, giving a clearer idea of the variation, its position, and size. Currently, long-read sequencing methods are greatly limited by the DNA preparation step, with fragment lengths and yield often compromised by standard extraction methods. The potential to generate reads at the megabase level relies greatly on library preps using large amounts (several μg) of DNA input, which prevents its application to samples like patient tumors that are often limited in DNA. Our overarching goal is to develop a targeted approach that generates large contiguous reads from moderate to low input DNA masses. To this end, we use different extraction, amplification, fragmentation, and enrichment strategies to efficiently target and resolve complex regions of the genome that are often associated with disease conditions. Development of a targeted long-read sequencing strategy using low DNA input would give us the potential to explore the landscape of genetic variation across several individuals and heterogeneous cell populations. We are currently employing and adapting CRISPR-based targeting techniques to enrich for specific regions of the genome from intact cells and/or high-molecular-weight DNA. Citation Format: Shruti Iyer, Sara Goodwin, W. Richard McCombie. Adapting long read sequencing technologies for targeted and single cell applications [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 5136.

Abstract 5182: Chromatin reader machinery as target for overcoming resistance to DNA-demethylating epi-drug decitabine

Abstract Based on the current understanding of the epigenetic landscape, cancer methylome is highly disrupted, which makes DNA methylation an excellent target for anti-cancer cures. To date, azacytidine and its congener, decitabine (DAC) are the most successful DNA demethylating epigenetic drugs. However, scientists are yet to find the reversal of clinical resistance to these drugs. Yet another challenge remains to be the decreased efficacy of these promising therapies in the cure of solid tumors. We used a soild tumor, HCT116 colorectal cancer cell line and developed resistance against DAC. DAC-resistant HCT116 cells were used to study the epigenetic cross-talk between DNA methylation and chromatin modifications. The screening of parental and DAC-resistant HCT116 cells against inhibitors of epigenetic writers-erasers-readers unveiled increased sensitivity of resistant cells to BET inhibitor (inhibitor of reader enzymes), (+)-JQ1. BET inhibitor further mediated augmented response on cell cycle phases of resistant cells, increased anti-proliferative effects in xenograft models of resistant cells, and synergystic effects in combination with DAC in HCT116 parental cells, both in vitro and in vivo. We then sequenced the transcriptome of DAC-sensitive and -resistant HCT116 cells, and (+)-JQ1 treated-(DAC-resistant) cells, using RNA-seq. The RNA-seq data revealed the overexpression of critical oncogenes, and their binding inactivation of key tumor suppressor genes (TSGs) in resistant cells. The most significant and biologically relevant transcriptional changes were further validated by qRT-PCR, and in addition their methylation status was determined by bisulphite sequencing. We discovered that the expressions of down-regulated TSGs were driven by promoter methylation, exposing these tumor-suppressive signatures as biomarkers which might differentiate between DAC-resistance and sensitivity, whereas, overexpression of oncogenes was independent of promoter methylation. Interestingly, the expressions of up-regulated oncogenes which define cell identity, mainly those involved in signaling of inflammatory pathways (including some bromodomain-specific genes) were reversed on treatment with (+)-JQ1. Further, siRNA-mediated genetic inhibition of bromodomains in resistant cells phenocopied therapeutic inhibition by (+)-JQ1. These data unveil the chromatin “reader proteins”, as regulators of dysregulated oncogenic expressions in DAC-resistant cells. The present study provides novel insights into the epigenomic landscape of DAC-resistant colorectal cancer cells, and put forward, the alternative therapeutic regimen for DAC-resistant patients. This study was supported by Czech Ministry of Education, Youth and Sports (LO1304, LM2015091, LM2015064), Technology Agency of the Czech Republic (TE01020028) and Internal Grant Agency of Palacky University (IGA_LF_2016_019). Citation Format: Khushboo Agrawal, Petr Vojta, Rastislav Slavkosky, Ivo Frydrych, Petr Dzubak, Marian Hajduch. Chromatin reader machinery as target for overcoming resistance to DNA-demethylating epi-drug decitabine [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 5182.