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Co-culture model of endogenous Vstat120 impact on microglia response to oHSV-infected glioma.

Ewing sarcoma subcutaneous tumors stained for CD68+ macrophages, with hematoxylin counterstain.

List of genes differentially induced ({greater than or equal to} 1.5 fold) in macrophages cultured with rHSVQ1 (Q) vs. RAMBO (R) infected glioma cells (UI, uninfected).

List of genes significantly up-regulated (>1.5 fold) in macrophages upon culture with glioma cells infected with RAMBO (R) or rHSVQ1 (Q) relative to uninfected (UI) cells.

Mutated and dysregulated protein kinases, such as EGFR and PI3K, have become major targets in cancer therapy due to the growth advantage they confer and the frequency of alterations. In glioblastoma (GBM), EGFR and PI3K are two of the most mutated genes and result in hyperactivation of these kinases. However, single agent inhibition has offered minimal success, largely due to the development of resistance from compensatory downstream signaling pathways. One strategy to circumvent resistance and improve efficacy in GBM is to use combination therapy, such as concurrent inhibition of EGFR or PI3K with inhibition of relevant epigenetic modulators. In GBM, lysine-specific demethylase 1 (LSD1) is an important epigenetic regulator that has shown to promote kinase signaling in cancer models. Therefore, the present study sought to define the relationship between the EGFR/PI3K signaling pathway and LSD1 and to evaluate the efficacy of co-inhibition of EGFR/PI3K and LSD1 in GBM.Transcriptional changes were examined following knockdown of LSD1 in isogenic human GBM cells using RNA-seq. These data were analyzed by GSEA to evaluate biological processes associated with the LSD1 expression. We identified several kinase signaling processes that were enriched in GBM cells with wild type LSD1 such as gene sets for regulation of PI3K activity, RTK binding, and transmembrane RTK activity. The effect of kinase inhibition on LSD1 expression was assessed using western blot analysis. We also evaluated the effects of LSD1 inhibition on expression of downstream kinase signaling proteins. Three kinase inhibitors directed against either EGFR or PI3K were evaluated in GSCs as single agents and in combination with LSD1 inhibitors. The three kinase inhibitors, osimertinib, erlotinib, and BKM120, all have evidence of some brain penetrance. We also evaluated a novel dual kinase inhibitor, MTX-241, which targets both EGFR and PI3K simultaneously. Five LSD1 inhibitors were assessed for their ability to enhance efficacy of EGFR/PI3K inhibitors.Our results demonstrate that LSD1 protein expression can be modulated by stimulation of EGFR in patient-derived GSCs. We observed an increase in LSD1 protein expression following the addition of EGF in GSC 17 cells. The concurrent inhibition of EGFR/PI3K and LSD1 enhanced the in vitro efficacy compared to single agent, supporting convergence of kinase signaling and LSD1 dependent pathways. In fact, several treatment combinations of EGFR/PI3K inhibitors and LSD1 inhibitors resulted in synergistic effects in multiple GSC lines.In summary, our results highlight the need for effective therapy combinations that can reduce the population of GSCs and avoid the adaptive resistance that is typical of kinase inhibitors. Future studies will focus on evaluating the efficacy and tolerability of the most promising treatment combinations in vivo using orthotopic xenograft models of the GSCs. Citation Format: Lea Stitzlein, Matthew Luetzen, Caitlin McCabe, Maninder Khosla, Melissa Singh, Xiaoping Su, Yue Lu, Joy Gumin, Frederick Lang, Christopher Whitehead, Judith Sebolt-Leopold, Joya Chandra. Efficacy of EGFR/PI3K signaling inhibition is enhanced with LSD1 inhibition in glioblastoma stem cell (GSC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3329.

Vascularization is a common pathology for many solid tumors, and therefore anti-angiogenic strategies are being investigated as a therapeutic target for treatment. Numerous studies are also being conducted regarding the effects of oncolytic viruses, including ImlygicTM, an FDA approved oncolytic herpes simplex virus-1 (oHSV) for the treatment of highly vascularized tumors such as Kaposi sarcoma (NCT04065152), and brain tumors. To our knowledge, the effects of combining oncolytic HSV with angiogenesis inhibition on endothelial cell activation has not been previously described. Here, we tested the effects of Rapid Antiangiogenesis Mediated By Oncolytic Virus (RAMBO), an oHSV which expresses a potent anti-angiogenic gene Vasculostatin on endothelial cell activation in heavily vascularized solid tumors. oHSV treatment induces endothelial cell activation, which inhibits virus propagation and oncolysis in adjacent tumor cells in vitro. Consistently, this was also observed in intravital imaging of intracranial tumor-bearing mice in vivo where infected tumor endothelial cells could efficiently clear the virus without cell lysis. Quantitative real-time PCR (Q-PCR), leukocyte adhesion assay, and fluorescent microscopy imaging data, however, revealed that RAMBO virus significantly decreased expression of endothelial cell activation markers and leukocyte adhesion, which in turn increased virus replication and cytotoxicity in endothelial cells. In vivo RAMBO treatment of subcutaneously implanted sarcoma tumors significantly reduced tumor growth in mice bearing sarcoma compared to rHSVQ. In addition, histological analysis of RAMBO-treated tumor tissues revealed large areas of necrosis and a statistically significant reduction in microvessel density (MVD). This study provides strong preclinical evidence of the therapeutic benefit for the use of RAMBO virus as a treatment option for highly vascularized tumors.

Current and former smoker AML patients have worse survival outcomes compared to never smokers. This worsened prognosis of smokers with AML can also be seen in patients who carry activating mutations of the Fms-like tyrosine kinase 3 (FLT3) and are treated with regimens that include newly approved kinase inhibitors. While the impact of genetic mutations on survival in AML have been studied, and some have been therapeutically targeted, the role of cigarette smoking or cigarette smoke exposure (which is potentially modifiable) on leukemia progression or treatment response is understudied. In order to elucidate molecular effects of cigarette smoke exposure (CSE) that contribute to the poor prognosis of AML patients, we developed a cigarette smoke exposure model for mice to mimic the current and former smoking habits of AML patients. NOD-SCID mice were exposed to CSE in a smoking robot for 2 hours, 5 days/week, for 2 weeks or to air alone as a control. Mice were then injected with luciferase-tagged human AML cell lines, and leukemic burden was monitored through non-invasive bioluminescent imaging. Control "non-smoking" mice were only subject to AML cell injection. Enhanced early leukemic-burden was observed two distinct FLT3-ITD AML models, MOLM13 and MOLM14, within one week post AML introduction (p-value Cigarette smoke exposure globally alters DNA methylation in blood cells and these changes can persist for decades. Independent of mutations, DNA methylation patterns in AML patients have prognostic significance. To understand how CSE accelerated leukemic growth in vivo, DNA methylation was evaluated using reduced representative bisulfite sequencing. More than two hundred significant alterations in DNA methylation across the promoter region of genes were found AML cells from spleen samples of CSE MOLM13-bearing mice as compared to non-smoking mice. Among the genes with the most significantly altered DNA methylation were GATA-2, an important protein for hematopoietic differentiation, and aryl-hydrocarbon receptor repressor (AHRR), a gene whose hypomethylation is a hallmark of cigarette smoke exposure. To identify the impact of cigarette smoke exposure on the leukemia cells in the absence of the tumor microenvironment we treated AML cells directly using a cigarette smoke condensate (CSC) that contains the chemicals in cigarette smoke used in the previously described CSE model. MOLM13 cells either treated with DMSO or 10ug/ml CSC every passage for two weeks were injected into NOD-SCID mice. This model resulted in enhanced leukemic burden 3, 10, and 17 days after leukemic introduction (p-value Evaluation of in vitro CSC treated AML cells was conducted to identify causes for the enhanced leukemic burden. While CSC treatment yielded no changes in proliferation or survival of the cells over the course of two months, within one week there was increased expression of DNMT1 in several cells lines. Increased basal and maximal oxygen consumption, and modulation of the antioxidant gene, HO-1, was also observed along with modulation of AHRR and GATA-2, reinforcing roles for methylation data gained from in vivo CSE experiments. Discovering the mechanisms promoting AML progression from cigarette smoke exposure will lead to improved, tailored treatment for AML patients with smoking histories and our further studies of these gene changes will aid in that endeavor. Disclosures Jabbour: Takeda: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding. Konopleva:Genentech: Consultancy, Research Funding; Ascentage: Research Funding; Forty-Seven: Consultancy, Research Funding; Calithera: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Agios: Research Funding; Amgen: Consultancy; AstraZeneca: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; AbbVie: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Sanofi: Research Funding. DiNardo:Agios: Consultancy, Honoraria, Research Funding; Syros: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Calithera: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; MedImmune: Honoraria; ImmuneOnc: Honoraria; Jazz: Honoraria.

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV). Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1−/− mice were used. Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1−/− or wild-type non–tumor-bearing mice revealed the safety of this approach. Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809–19. ©2016 AACR.

Soft Tissue Sarcoma (STS), a cancer which forms in the connective tissue, requires aggressive treatment. Metastatic soft tissue sarcomas are associated with a median prognosis of about twelve months accompanied frequently with many signs and symptoms leading to a poor quality of life. These tumors frequently have angiogenic properties. Angiogenesis is responsible for the formation of new blood vessels and is associated with local progression and increased metastatic disease. Strategies that work to block angiogenesis are currently being investigated as a treatment for STS. Oncolytic virus therapy is a novel treatment strategy that utilizes modified viruses to specifically replicate and lyse cancer cells. The combination of angiogenesis inhibitors with oncolytic virus treatment has not been tested for STS. Here we tested the effect of combining oncolytic viral therapy with antiangiogenic gene therapy. RAMBO is a HSV-1 derived oncolytic virus that codes for the production of Vstat120, a gene with potent antiangiogenic effects. We have tested a panel of sarcoma cells and shown effective infection and lysis of these cells by RAMBO and HSVQ-1 in vitro. In vivo, we compared the antitumor efficacy of RAMBO and HSVQ-1 against sarcoma tumors grown in mice. Briefly, mice were implanted with 150-300mm3 A673 sarcoma tumors were injected with a PBS, HSVQ-1, or RAMBO (5.5×106 pfu). Mice were followed for tumor size over a period of time. Tumor volumes over time were logarithm (base 2) transformed for variance stabilization. The linear mixed model was used to compare the three groups while accounting for the variance-covariance structure due to repeated measures at different days from the same mouse. Statistically significant improvement in antitumor efficacy was observed in RAMBO treated tumors, compared to HSVQ-1 and control. H&E staining of tumor tissue reveals large areas of necrosis in RAMBO treated tumors. RAMBO and its inherent antiangiogenic gene therapy demonstrated significant efficacy in these sarcoma models and further investigations are underway with combination therapeutic strategies.