Citations

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

Abstract Introduction: Lung cancer is the leading cause of cancer death in the U.S. and in the world. In 2018, lung cancer alone resulted in 25.3% of all cancer deaths evaluated worldwide. Lung Adenocarcinoma (LUAD) is a major subtype of lung cancer and accounts for 40% of these tumors. Our group has recently discovered that about 20% of LUAD are characterized by abnormally high expression of the neuroendocrine transcription factor, ASCL1. In these tumors, ASCL1 orchestrates a network of genomic changes that create opportunities for treatment. We have shown that LUAD tumors expressing high ASCL1 have RET mRNA levels that are predictive of patient survival (p < 0.004), and, these tumors were susceptible to EGFR inhibitors, with having a functional interaction between RET and EGFR. We found EGFR inhibitor, Lapatinib, to have highest efficacy, indicating it to be a primary drug treatment for ASCL1+ (A+) LUAD. However, we and others have observed Lapatinib induces resistance to EGFR therapy over time, and, Lapatinib therapy associated by-pass signaling pathways have recently gained attention. Therefore, we hypothesize that for a long lasting therapy, it is important to identify additional drugs that in combination with EGFR inhibitor Lapatinib will eliminate A+ LUAD tumors. Methodology and Results: An in vitro preliminary screen of approx. 2000 FDA approved small molecule inhibitors done with A+ and A- LUAD cell lines in collaboration at NIH revealed potential candidates for alternative pathways, 80 of which showed a differential sensitivity to A+ LUAD as single agent. We identified best 28 drugs in total: 23 selected from this pre-screen (based on their effectiveness to A+LUAD lines) and 5 additional chosen based on their biological relevance to the disease, which may act upon putative compensatory pathways in EGFR therapy resistance. Our in vitro drug screening in HCC1833 ASCL1+ and ASCL1 knockdown lines has revealed 8 out of 28 drugs to synergize as per their IC50 and fixed ratios, with Lapatinib. From these 8 leads, we have confirmed a definite synergy of Lapatinib with DDR-1 7rh, an inhibitor of ECM receptor DDR-1, using Mac-Synergy-II software. DDR-1, Discoidin domain receptor-1, is a collagen receptor highly upregulated in NSCLC, and is associated with poor prognosis in LUAD. Our microarray data on lapatinib treated tumors from mice PDX has shown ECM signatures to be among top upregulated. Additionally, TM4SF1, a signaling molecule associated with DDR-1, was found to associate with poor outcome in A+LUAD (P=0.0001). Other synergistic drugs identified include, a K+ channel opener (Retigabine), Wnt signaling and Lyn kinase inhibitors (PRI-724, and Bafetinib), Tubulin inhibitor (Docetaxel) and 3 multi-tyrosine kinase inhibitors. Interestingly, Wnt pathway was found to be elevated in Lapatinib treated ASCL1+LUAD in our microarray data. Moreover, Wnt signaling in known to crosstalk with ECM and DDR-1 in other tumors. K+ channel dysfunction is associated with neuronal/neuroendocrine disorders and Lyn kinases are the predominant SRC kinases in brain tumors, indicating their possible involvement in ASCL1+ neuroendocrine tumor pathophysiology. Conclusion: Taken together this data reveals a potential involvement of ECM (DDR-1 receptor) and Wnt signaling in effect of Lapatinib, and the identified leads as path to novel combination therapies for A+ LUAD. Citation Format: Kshama Gupta, Farhad Kosari. Identification of the synergistic drugs in combination with EGFR therapy that can target ASCL1+ Lung Adenocarcinoma (A+ LUAD) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A143. doi:10.1158/1535-7163.TARG-19-A143