Ahmed, Atique U.

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

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.

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. 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 hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina 2 and sina 3 alleles, providing us with an opportunity to study sina loss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) Dm SINA and human SIAH. The altered Dm SINA/hSIAH expression under the control of sev ‐, GMR ‐, and dpp ‐ and salivary gland ‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of Dm SINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINA WT/DN /SIAH WT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future. Support or Funding Information Extramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Abstract There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA’s importance is its high degree of 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 cancers. 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 Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer [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 83.

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