Citations

Abstract Glioblastoma multiforme (GBM) is a particularly aggressive primary brain tumor that is relatively common in adults. The disease comes with a dismal prognosis, often with expected survival just over a year when treated with surgery, chemotherapy, and radiation. A promising addition to our GBM treatment arsenal are oncolytic viruses (OVs), a class of immunotherapy now in clinical trials. The most effective OVs home in on tumor cells, killing them and releasing tumor-associated antigens that elicit antitumor immunity. This effectiveness, however, varies depending on the cell state—a property that may be influenced by the composition of the extracellular matrix (ECM) of GBM. Cellular communication network factor 1 (CCN1) is found in the ECM of the majority of GBMs and is predictive of resistance to OVs, particularly those derived from herpes simplex virus type 1 (HSV-1). In this study, our aim is to understand how extracellular CCN1 alters the GBM intracellular state in a manner that confers resistance to OVs. We began by using our NetDecoder platform to build CCN1-specific prioritized protein-protein interaction (PPI) networks from published LN229 human GBM transcriptomic datasets. From this network, we identified 12 PPI edges that dominate the network flow in CCN1-high LN229 cells when compared to CCN1-low controls. The 21 unique nodes that comprise these binary PPI edges were extracted and subjected to downstream biological process overrepresentation analyses using Gene Ontology. We found that the cytokine-mediated signaling pathway (FDR = 4.2 x 10-11; enrichment ratio = 15.8) and the cellular response to cytokine stimulus (FDR = 3.1 x 10-9; enrichment ratio = 10.9) were highly enriched when mapped to these nodes. We conclude that CCN1, acting as a paracrine signal in the GBM tumor microenvironment, primes the cell to inhibit HSV-1 and other candidate OV replication prior to any virus interactions, thus contributing to immunovirotherapy resistance. While CCN1 and its cell surface integrin binding partners are ostensible drug targets for overcoming this resistance, pharmacologic interventions that target CCN1-specific high flow edges in our prioritized PPI network may be more effective. Citation Format: Dileep D. Monie, Cristina Correia, Cheng Zhang, Choong Ung, Richard G. Vile, Hu Li. Innate immunity and cytokine signaling pathways drive resistance to HSV-1 oncolytic immunovirotherapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1679.

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

Abstract Purpose: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs. Experimental Design: Matched case–control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays. Results: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1−/− T47D breast cancer cell line. Conclusions: This matched case–control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

Abstract BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Abstract Introduction: Topoisomerase I is a nuclear enzyme that relaxes torsional strain in DNA by nicking one DNA strand, allowing rotation, then resealing the DNA backbone. This is an important enzyme in replication and transcription, and therefore has been targeted by anti-cancer agents such as camptothecins. These agents intercalate into DNA at the topoisomerase I active site and inhibit the religation step catalyzed by the enzyme, causing increased topoisomerase I-DNA complexes that impede advancing replication forks or transcription complexes, leading to DNA damage and cell death. Platinating agents also stabilize topoisomerase I-DNA covalent complexes. The topotecan/carboplatin combination has recently shown promise in combination with the PARP inhibitor veliparib in aggressive and transformed myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (K Pratz et al., Clin Cancer Res. 23:899, 2017). Given that both classes of agents have a narrow therapeutic window, it is important to try to predict the sensitivity/resistance of these agents in malignancies such as acute myeloid leukemia (AML) and transformed MPNs. Some of mechanisms of drug resistance that have been described are decreased numbers of drug-stabilized topoisomerase I-DNA complexes due to drug efflux, diminished topoisomerase I expression or topoisomerase I mutation. Thus, being able to detect and quantify these DNA-protein complexes may provide insight into drug sensitivity. Methods: Using a unique monoclonal antibody that detects topoisomerase I-DNA covalent complexes (A. Patel et al., Nucleic Acids Res. 44:2816, 2016), immunofluorescence techniques were used to quantify these topoisomerase I-DNA complexes in AML cell lines (SET2, HEL, K562, U937, ML2, MV-4-11, THP1, HL60, ML1, and Molm13) and clinical AML samples. To verify the fidelity of the anti-topoisomerase I-DNA covalent complex antibody (TopIcc), siRNA knockdown methods were utilized with confirmatory immunofluorescence and Western blotting. Slot blots were additionally used to assess the presence of these complexes in AML cell lines. Western blots were performed to assess topoisomerase I levels in AML cell lines. Topotecan drug sensitivity analysis was performed by using an MTS assay. Results: With treatment of the topoisomerase I inhibitor, topotecan, there was an increase in the percentage of leukemia cells positive for the topoisomerase I-DNA covalent complexes. The degree of increase in complexes varied across cell lines and patient samples. To ensure we were detecting TopIcc by immunofluorescence, we performed a topoisomerase-I siRNA knockdown to confirm the specificity of the antibody for TopIcc's. Slot blots on multiple AML cell lines confirmed the presence of topoisomerase-DNA covalent complexes. The Western blot of all the AML cell lines, ordered from low to high TopIcc revealed increasing amounts of topoisomerase I. The MTS assay revealed lower topotecan IC50 values in three of the cell lines with higher complex formation, including Molm13, ML1 and ML2 (IC50 6-8 nM compared to 13-46 nM in the other cell lines). Conclusions: Based on recent clinical results, there has been renewed interested in topoisomerase I inhibitors, especially for aggressive and transformed MPNs. Topoisomerase I-DNA complex repair/downregulation has been a hallmark of drug resistance to topoisomerase I inhibitors. Therefore, detecting and quantifying these complexes in treated samples has been hypothesized to provide insight into the drug sensitivity, which is particularly important in camptothecins and platinating agents given their narrow therapeutic windows. Immunodetection techniques using an anti-TopIcc antibody may help predict sensitivity of AML to topoisomerase I inhibitors and will be used as a correlative biomarker study in an upcoming phase II randomized study of topotecan/carboplatin with or without veliparib or placebo in advanced myeloproliferative disorders and chronic myelomonocytic leukemia (ClinicalTrials.gov NCT03289910). Disclosures Pratz: AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Kaufmann:Mayo Clinic: Patents & Royalties: Co-inventor on a patent held by Mayo Clinic for the use of the anti-TOP1cc antibody as a diagnostic reagent.

Abstract With the emergence of genome editing technologies and synthetic biology, it is now possible to engineer genetic circuits driving a cell's phenotypic response to a stressor. However, capturing a continuous response, rather than simply a binary ‘on’ or ‘off’ response, remains a bioengineering challenge. No tools currently exist to identify gene candidates responsible for predetermining and fine-tuning cell response phenotypes. To address this gap, we devised a novel Regulostat Inferelator (RSI) algorithm to decipher intrinsic molecular devices or networks that predetermine cellular phenotypic responses. The RSI algorithm is designed to extract gene expression patterns from basal transcriptomic data in order to identify ‘regulostat’ constituent gene pairs, which exhibit rheostat-like mode-of-cooperation capable of fine-tuning cellular response. Our proof-of-concept study provides computational evidence for the existence of regulostats and that these networks predetermine cellular response prior to exposure to a stressor or drug. In addition, our work, for the first time, provides evidence of context-specific, drug–regulostat interactions in predetermining drug response phenotypes in cancer cells. Given RSI-inferred regulostat networks offer insights for prioritizing gene candidates capable of rendering a resistant phenotype sensitive to a given drug, we envision that this tool will be of great value in bioengineering and medicine.

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [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 5885.

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (T CM ) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected T CM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo . Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV + memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

Significance There is a need for a robust and efficient eukaryotic polypeptide display platform for the discovery and affinity maturation of antibodies and other scaffolds, offering a powerful addition to the existing display technologies that use microorganisms. This study is significant in that it demonstrates just such a technology, avian leukosis virus (ALV) polypeptide display, supplying an innovative and substantive departure from the status quo of antibody display that uses platforms based on microorganisms. The use of eukaryotic cells provides the assurance of maximizing the proper folding, posttranslational modifications, and display of the single-chain Fv antibodies and the virus fusion proteins in virions and on the cell surface. ALV display offers virus and cell display platforms in a eukaryotic expression system.