Ahmed, Atique

Adult-type diffusely infiltrating gliomas, of which glioblastoma is the most common and aggressive, almost always recur after treatment and are fatal. Improved understanding of therapy-driven tumor evolution and acquired therapy resistance in gliomas is essential for improving patient outcomes, yet the majority of the models currently used in preclinical research are of therapy-naïve tumors. Here, we describe the development of therapy-resistant IDH-wildtype glioblastoma patient-derived xenografts (PDX) through orthotopic engraftment of therapy naïve PDX in athymic nude mice, and repeated in vivo exposure to the therapeutic modalities most often used in treating glioblastoma patients: radiotherapy and temozolomide chemotherapy. Post-temozolomide PDX became enriched for C>T transition mutations, acquired inactivating mutations in DNA mismatch repair genes (especially MSH6), and developed hypermutation. Such post-temozolomide PDX were resistant to additional temozolomide (median survival decrease from 80 days in parental PDX to 42 days in a temozolomide-resistant derivative). However, temozolomide-resistant PDX were sensitive to lomustine (also known as CCNU), a nitrosourea which induces tumor cell apoptosis by a different mechanism than temozolomide. These PDX models mimic changes observed in recurrent GBM in patients, including critical features of therapy-driven tumor evolution. These models can therefore serve as valuable tools for improving our understanding and treatment of recurrent glioma.

Abstract Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Tumor recurrence in glioblastoma multiforme (GBM) is often attributed to acquired resistance to the standard chemotherapeutic agent, temozolomide (TMZ). Promoter methylation of the DNA repair gene MGMT (O6-methylguanine–DNA methyltransferase) has been associated with sensitivity to TMZ, whereas increased expression of MGMT has been associated with TMZ resistance. Clinical studies have observed a downward shift in MGMT methylation percentage from primary to recurrent stage tumors; however, the evolutionary processes that drive this shift and more generally the emergence and growth of TMZ-resistant tumor subpopulations are still poorly understood. Here, we develop a mathematical model, parameterized using clinical and experimental data, to investigate the role of MGMT methylation in TMZ resistance during the standard treatment regimen for GBM—surgery, chemotherapy, and radiation. We first found that the observed downward shift in MGMT promoter methylation status between detection and recurrence cannot be explained solely by evolutionary selection. Next, our model suggests that TMZ has an inhibitory effect on maintenance methylation of MGMT after cell division. Finally, incorporating this inhibitory effect, we study the optimal number of TMZ doses per adjuvant cycle for patients with GBM with high and low levels of MGMT methylation at diagnosis.

Abstract Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) have global genomic hypermethylation, are less aggressive than IDH1 wild-type (IDH1wt) gliomas, and generally grow poorly in vitro and in vivo. Yet little data exist that connect specific hypermethylation events to this unique phenotype. We previously reported that the gene encoding Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas relative to IDH1wt gliomas. TF was originally described in blood coagulation, but also can enhance tumor malignancy via protease-activated receptor 2 (PAR2) signaling, though its role in gliomas is not as well understood. In this study, we further explored the significance of TF on malignancy in IDH1wt and IDH1mut gliomas, and showed that receptor tyrosine kinases (RTKs) and β-catenin are downstream effectors of TF-PAR2 signaling. Studies were performed using 6 patient derived glioma cell lines, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was assessed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using conditioned medium. Markers of malignancy included BrdU incorporation for cell proliferation, transwell invasion, and soft agar colony formation. Nude mice (N≥6/group) were transplanted with glioma cells and monitored for growth using bioluminescence imaging. The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, with corresponding lower TF protein in IDH1mut cells. Treatment of IDH1mut glioma cells with a demethylating agent, decitabine, increased F3 transcription 5-fold and PCA 19-fold in IDH1mut cells. TF increased the in vivo "take rate" of IDH1mut GBM164 from 0% to 100% (P=0.0001, Fisher's exact test). Conversely, TF knockdown in IDH1wt cells greatly reduced cell proliferation, invasion, colony formation, and extended median survival of engrafted mice by 86% (P=0.001). In both IDH1wt and IDH1mut cells, TF-PAR2 activated β-catenin, ERK1/2, and Akt, and was associated with multiple pathways that increase high-grade behavior. Disrupting TF-PAR2 signaling had the greatest impact on tumor growth in IDH1wt gliomas driven by epidermal growth factor receptor (EGFR), a major receptor tyrosine kinase (RTK) involved in glioma malignancy. TF-PAR2 transactivated EGFR through a Src-dependent intracellular pathway, even when extracellular stimulation of EGFR was blocked, and further enhanced phosphorylation of the constitutively activated EGFRvIII. In IDH1mut cells, TF-PAR2 signaled through another RTK, PDGFR. In contrast, TF-PAR2 stimulated invasion in all glioma cell lines through an RTK-independent, β-catenin-dependent mechanism. These results demonstrate the importance of TF-PAR2 in gliomas, and show that its suppression is a critical component of the IDH1mut phenotype. Citation Format: Dusten J. Unruh, Snezana Mirkov, Seamus Caragher, Jann Sarkaria, Atique Ahmed, C. David James, Craig Horbinski. Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5443.

Abstract Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signal transduction cascade. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung and breast cancer. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and well-established Drosophila development system, we are able to study RAS activation and SINA function under normal physiological conditions. In the developing Drosophila eye, photoreceptor cells are recruited sequentially and acquire their distinctive cell fates through a series of local inductive events. The 800x cell arrays allowed us to dissect the role of SINA/SIAH downstream of RAS activation in photoreceptor cell development. To delineate SINA function, we performed an F1 modifier screen using ethyl methanesulfonate (EMS) and X-ray radiation, isolating 28 novel sina mutant alleles. These mutant alleles exhibit much stronger mutant phenotypes than those of the previously published sina2 and sina3 alleles, suggesting that the sina2 and sina3 alleles are hypomorphic alleles. Sequencing analysis of these sinamutant alleles reveals the functional roles of mutated residues and protein domains. To define SINA/SIAH functional conservation, we have generated a complete panel of transgenic fly models that express either wild-type (WT) or dominant negative (DN) SINA/SIAH. The corresponding UAS-sina/siahGOF/LOF phenotypes have been characterized using sev-, GMR-, dpp- and salivary gland-GAL4 drivers to elucidate the developmental outcomes of altered SINA/SIAH expression upon RAS activation. Ectopic expression of sinaWT/DN/siahWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum, causing PNS neurodegenerative phenotypes. Our results show that the biological functions of fly SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Mechanistic insights and regulatory principles learned from Drosophila can be directly applied to cancer biology to develop and validate next-generation anti-SIAH-based anti-K-RAS and anticancer therapy in the future. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development [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 4824. doi:10.1158/1538-7445.AM2017-4824

Abstract Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/RAS signaling pathway has been well established in promoting tumor growth, invasion and metastasis of human breast cancer. Even though oncogenic K-RAS mutations are rare in breast cancer, activation of the growth-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated HER2/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the ERBB/RAS pathway – the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These results suggest that SIAH2 may be an attractive and new drug target for novel breast cancer therapy. Developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. By attacking the growth-promoting HER2/EGFR/RAS pathway at multiple signaling modules simultaneously – a multi-pronged synergistic inhibition at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel, we may be in a position to halt the genesis, progression and metastasis of human breast cancer. Thus, SIAH-dependent proteolysis may be an Achilles heel for breast cancer. SIAH inhibition may represent an innovative and logical way to inhibit HER2/EGFR/K-RAS activation, alleviate tumor burdens, halt cancer metastasis, and improve the efficacy for the future treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2011-1659

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive tumor growth and metastasis in human cancer. Currently, there are no effective ways to treat human cancers that have oncogenic K-RAS mutations that confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS is an urgent goal and the major challenge in cancer therapy and treatment. Instead of targeting an upstream signaling module such as EGFR/HER2/KRAS/B-RAF/MEK/MAPK/AKT/mT0R, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of seven-in-absentia (SINA), an evolutionarily conserved RING E3 ligase, an essential downstream signaling module, and a critical gatekeeper required for proper ERBB/RAS/RAF signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we conducted preclinical studies to dissect SIAH function in the context of ERBB/K-RAS-mediated tumorigenesis and metastasis in human cancer cells and tumor specimens. We found that: 1) SIAH is a novel biomarker for human cancer, i.e., SIAH specifically decorates proliferating tumor cells (not stromal or immune cells in the tumor microenvironment). SIAH expression is progressively and markedly upregulated as human cancer progresses to the advanced stages in human pancreatic, lung, breast, and prostate cancers; and 2) inhibiting SIAH function is highly effective to abolish tumorigenesis and metastasis in multiple of the most aggressive human cancer cell lines known in the literature that include pancreatic cancer cells (MiaPaCa and Panc-1), lung cancer cells (A549, H727, and UMC11), invasive breast cancer cells (MDA-MB-231, MDA-MB-468, and MDA-MB-435), colorectal cancer cells (SW620 and HCT116) and hormonal-refractory prostate cancer cells (PC-3 and DU-145) in our preclinical studies. These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS activation in human cancer cells. Using anti-SIAH molecules to block K-RAS signaling in human cancer is an excellent example of science going from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials). As a highly evolutionarily conserved E3 ligase and the most downstream gatekeeper of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is uniquely and logically positioned to become a next-generation anti-K-RAS drug target. Our preclinical studies have demonstrated that SIAH-dependent proteolysis is an Achilles' heel for human cancer cells. Albeit at an early preclinical stage, knowledge gained from our study has great promise that has generated many excitements with immediate translational values. Anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies. By attacking the oncogenic ERBB/K-RAS/B-RAF pathway using multipronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR), and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a great position to halt the genesis, progression, and metastasis of the most aggressive and the deadliest forms of human cancers. If successful, we hope to translate our findings to the cancer clinics to treat many cancer patients with metastatic diseases in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B14.

Abstract Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer. [Cancer Res 2007;67(24):11798–810]

AbstractWe have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.