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Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family which has been extensively studied for its roles in neural development, long-term memory, brain injury, and neurodegenerative diseases. BDNF signaling through tropomyosin receptor kinase B (TrkB) stimulates neuronal cell survival. For this reason, small molecule TrkB agonists are under pre-clinical develoment for the treatment of a range of neurodegenerative diseases and injuries. Our laboratory recently reported BDNF is secreted by pro-regenerative endothelial progenitor cells (EPCs) which support hematopoietic reconstitution following total body irradiation (TBI). Here we report BDNF-TrkB signaling plays a novel regenerative role in bone marrow and thymic regeneration following radiation injury. Exogenous administration of BDNF or TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following myelosuppressive radiation injury promoted faster recovery of mature blood cells and hematopoietic stem cells capable of multi-lineage reconstitution. BDNF promotes hematopoietic regeneration via activation of PDGFRα+bone marrow mesenchymal stem cells (MSCs) which increase secretion of hematopoietic cytokines interleukin 6 (IL-6) and leukemia inhibitory factor (LIF) in response to TrkB activation. These data suggest pharmacologic activation of the BDNF pathway with either BDNF or 7,8-DHF may be beneficial for treatment of radiation or chemotherapy induced myelosuppression.

Purpose/Objectives: Radiation therapy is used by more than 50% of breast cancer patients, but radiation doses can be limited by normal tissue side effects. For example, breast cancer radiation therapy can improve breast cancer-specific survival, but increase cardiac deaths in those with left-sided cancers. Identifying genetic factors that can enhance tumor radiation sensitivity while decreasing normal tissue toxicities has the potential to improve the therapeutic ratio of radiation therapy – leading to more cures and less long-term toxicities. The use of animal models with differing genetic backgrounds to assess radiation toxicity, followed by genetic mapping of radiosensitivity phenotypes, has the potential to identify new targets that can predict cardiac toxicity from radiation therapy. This project examines how genetic host factors alter normal tissue toxicity risks from breast cancer radiation. Materials/Methods: Inbred female SS rats and SS.BN3 consomic rats, that are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. For this study, adult female SS and SS.BN3 rats received image-guided whole heart radiation to a dose of 21 Gy (3 fields, AP and 2 laterals). Cardiac troponin was serially measured at 2, 6, and 12 weeks, and echocardiograms with strain analysis were performed at baseline and 3 months. The Student's t-test was used to compare values. Results: The SS female rats exhibited enhanced cardiac toxicity compared to SS.BN3 rats, with cardiac troponin levels elevated at 12 weeks (0.32 ng/ml vs.0.08 ng/ml for SS vs. SS.BN3, p=0.01), and moderate to severe pericardial effusions seen in 6 of 9 SS rats vs. 2 of 7 SS.BN3 rats. At 3 months post-radiation, echocardiograms revealed increased left ventricular posterior wall thickness at end diastole (LVPWd) in SS vs. SS.BN3 rats (0.25 vs. 0.20 cm, p=0.002) and increased left ventricular mass (LVM) in SS vs. SS.BN3 rats (1.54 vs. 1.28 g, p Conclusions: These results demonstrate that genetic variant on rat chromosome 3 alter the radiosensitivity to single fraction cardiac radiation therapy. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). These models will also be expanded to test whether similar results are seen with fractionated cardiac radiation therapy. This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer. Citation Format: Bergom C, Schlaak R, Frei A, Fish BL, Harmann L, Gasperetti T, Schottstaedt AM, Flister MJ, Medhora M, Strande JL. The use of consomic animal models to identify genetic factors that modulate radiation-induced cardiac toxicity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-11-18.

Objectives: The tumor microenvironment (TME) can impact breast cancer tumor growth, progression, and treatment responses. Data suggests that genetic variants in not only breast cancer cells, but also in the TME, can also alter these processes. We have utilized a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel to identify genetic variants in the TME that affect radiation sensitivity. Materials/Methods: Human triple negative breast cancer MDA-MD-231 cells were implanted into immunodeficient (IL2RG KO) consomic rat strains that are genetically identical except for chromosome 3 is inherited from a separate strain (SS and SS.BN3 strains). On day 10, tumors were treated with 3 daily ionizing radiation (IR) treatments of 4 Gy or sham, and tumor growth was monitored. Tumors were also harvested for hypoxia staining using pimonidazole or for RNA-seq. RNA-Seq was performed and a custom SSRS protocol was used to align both rat and human transcripts. This yielded transcript and gene level estimated fold-change and adjusted p-values for human- and rat-derived transcripts separately. E077 mammary tumor cells were implanted into adult female immune competent C57/Bl6 mice. On day 5, tumors were treated with 5 daily IR treatments of 5 Gy or sham. Either vehicle or a mAb to the Notch ligand Dll4 (Genentech) was given twice weekly. Chi-square, Fisher’s exact, and Kolmogorov-Smirnov tests and empirical cumulative distribution plots for differential expression significance values were performed. Results: Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor IR sensitivity, as human breast cancer xenografts in the consomic strain (SS.BN3) were significantly more IR sensitive than SS rat strain tumors (supra-additive). Vascular gene pathways were differentially expressed, and tumor vascular phenotypes were distinct, with SS.BN3 tumors with increased but poorly functioning blood vessels. Hypoxia was similar at baseline, but increased in SS.BN3 tumors following IR. These results were consistent with less Dll4 expression in the SS.BN3 TME. The use of a Dll4-targeted mAb in mice demonstrated that targeting Dll4 enhanced mammary tumor IR responses. Conclusion: CXM demonstrated TME genetic variants can affect IR sensitivity of genetically identical tumor cells. Using SSRS, we identified candidate genes on rat chromosome 3 that may potentially influence IR sensitivity, and our studies ultimately led to identification of the Notch ligand Dll4 as a target to enhance breast cancer IR responses. Future studies will investigate the possibility of the Dll4 pathway as a therapeutic target, as well as interrogate other pathways responsible for changes in IR sensitivity seen in the CXM model. Determining TME factors that affect the IR sensitivity will allow more tailored and effective treatments. Citation Format: Carmen Bergom, Michael W. Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Rachel A. Schlaak, Shirng-Wern Tsaih, Michael J. Flister. Genetic variants in the tumor microenvironment alter radiation responses in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA004.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG&minus, /&minus, )) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p &lt, 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Purpose/Objectives: Over 50% of breast cancer patients receive radiation therapy, but radiation doses can be limited by normal tissue toxicity. Radiation therapy can improve breast cancer-specific survival, but cardiac morbidity can be increased in patients with left-sided tumors. We used rat genetic models to identify targets to improve the therapeutic ratio of radiation. We assessed the radiation responsiveness of mammary tumors and the heart in genetically similar consomic rats conducive to genetic mapping. Materials/Methods: Female SS rats and SS.BN3 consomic rats, which are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. Human MDA-MD-231 cells or syngeneic mammary tumor cells developed from DMBA-induced mammary tumors were implanted orthotopically into immunodeficient or immunocompetent SS and SS.BN3 rats, respectively, and tumors were treated locally with mock or 5x4 Gy. To examine cardiac toxicity, adult female SS and SS.BN3 rats received image-guided localized whole-heart radiation to a dose of 24 Gy or 9 Gy x 5 (AP and 2 lateral fields, weighted 1:1:1). Echocardiograms with strain analysis were performed at baseline, 3 months and 5 months. The Student's t-test was used to compare values. Results: The BN strain-derived genetic variant(s) on rat chromosome 3 is important for tumor radiation sensitivity. Tumors in SS.BN3 rats were significantly more radiosensitive than tumors in the parental SS strain. A supra-additive effect was seen with both tumor cell lines, with recurrence-free survival of 30% vs. 67% at 137 days in SS vs SS.BN3 rats (p=0.02) in xenografts, and recurrence-free survival of 9% vs. 100% at 141 days in SS vs. SS.BN3 rats (p Conclusions: These results demonstrate that genetic variants on rat chromosome 3 alter the sensitivity to radiation therapy, enhancing tumor responses to radiation and protecting the heart, thus improving the therapeutic ratio. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer. Citation Format: Rachel A. Schlaak, Anne Frei, Aronne M. Schottstaedt, Brian L. Fish, Leanne Harmann, Tracy Gasperetti, Michael J. Flister, Meetha Medhora, Jennifer L. Strande, Carmen R. Bergom. Novel genetic rat models to identify factors that modulate cardiac and tumor radiation sensitivity [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 4165.

Background: The tumor microenvironment (TME) is known to impact tumor growth, metastatic potential, and treatment response. Nearly all studies of head and neck cancer (HNC) have focused on somatic mutations in the malignant cells. We hypothesized that genetic determinants limited to the microenvironment would influence HNC growth and metastatic potential. Approach: To demonstrate the impact of genetic differences in the TME on HNC cell line in vivo growth we utilized a novel tool, the consomic xenograft model (CXM). A consomic rat has an entire chromosome substituted into the isogenic background of another inbred strain by selective breeding. Use of immunodeficient (IL2Rγ-/-) consomic rats allows one to study the influence of stromal genetics on tumor biology without the confounding effect of differences in the immune system through the orthotopic implantation of cancer cells into different consomic rat strains. In this system, any differences in tumor growth or metastases are due to differences in the TME rather than cancer cells or immune response. We utilized SS and SS.BN3 consomic rat strains, previously shown to affect the growth of breast tumors, to study the effects of the TME on HNC tumor growth using two well-characterized HPV negative HNC cell lines, SCC-6 (base of tongue derived) and SCC-22b (derived from a hypopharyngeal cancer that had metastasized to lymph nodes). Both cell lines were modified to stably express luciferase. HNC cells were inoculated into the tongue of SS and SS.BN3 animals and tumor growth was monitored by biophotonic imaging after luciferin injection. Results: A significant difference in the tumor growth was seen between rat strains for both cell lines, with the SS.BN3 rats exhibiting less tumor growth and metastasis. Median luciferase activity from baseline increased by 4.1-fold vs. 1.1 fold in SCC-6 tumors in SS vs SS.BN3 rats, respectively (p Conclusions: The use of the CXM model demonstrates an important role for the TME in the growth and metastatic spread of HNC cell lines. This model allows for future congenic mapping to identify the causative genetic variants in the TME mediating the HNC changes in tumor growth and metastasis. Citation Format: Michael W. Straza, Amy Rymaszewski, Kwangok P. Nickel, Anne Frei, Anirban Chatterjee, Rachel Schlaak, Amit Joshi, Michael Flister, Randy J. Kimple, Carmen Bergom. The consomic xenograft model identifies genetic changes in the tumor microenvironment that alter the growth and metastasis of head and neck cancers [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 5898. doi:10.1158/1538-7445.AM2017-5898

Progress in elucidating the molecular basis of breast cancer has allowed for treatment breakthroughs such as anti-estrogen and Her2-targeted therapy. It has also shaped the approaches to both surgical and systemic therapy. However, no similar use of molecular information has been utilized to better direct the use of radiation therapy. The development of predictive tools for the radiosensitivity of tumors could allow for personally tailored radiation doses, with treatment de-escalation for radiosensitive tumors, or dose escalation or the use of adjunct treatments in the case of radioresistant tumors. Communication between malignant tumor cells and the tumor microenvironment (TME) underlies most aspects of tumor biology, including chemotherapy and radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cell-sorting. Here we utilize these unique techniques to identify genetic variants in the TME that can affect radiation sensitivity. In CXM, human triple negative breast cancer MDA-MD-231 cells are orthotopically implanted into immunodeficient (IL2Rγ-/-) consomic rat strains, which are rat strains in which an entire chromosome is introgressed into the isogenic background of another inbred strain by selective breeding. Because the strain backgrounds are different but the tumor cells are not varied, the observed changes in tumor progression are due to genetic differences in the non-malignant TME. We hypothesized that the tumors in SS.BN3 rats (identical to SS rats but with BN strain chromosome 3) would be more sensitive to radiation due to increased tumor vascularity via CD31 staining, and increased tumor blood volume capacity, as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Our studies demonstrate differential responses to radiation in the CXM model comparing parental SS (IL2Rγ) rats to SS.BN3 (IL2Rγ) rats treated with fractionated radiation therapy (4 Gray x 3), with altered tumor growth kinetics and tumor recurrence rates. A difference was seen in time to 5-fold increase in tumor growth, with 44 vs. >130 days for SS versus SS.BN3 rats (supra-additive, p130 days) in the SS versus SS.BN3 rats (p=0.02). These results suggest that genetic determinants in the TME affect the radiation sensitivity of genetically identical tumor cells. Using SSRS, we identified a number of candidates on rat chromosome 3 that may potentially influence radiation sensitivity by altering the tumor vasculature. Future studies will further dissect the pathways responsible for the changes in radiation sensitivity. Determining TME factors that affect the radiation sensitivity of tumors has the potential to allow for more tailored and effective radiation treatments in breast cancer. Citation Format: Carmen Bergom, Michael Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Shirng-Wern Tsaih, Howard Jacob, Michael J. Flister. Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B07.

Glioblastoma (GB) is the most aggressive malignancy affecting the central nervous system (CNS) with a median survival of 12 to 15 months even with surgery, radiation and chemotherapy. Previous research demonstrates that increased activation of NF-κB is critical for GB growth, proliferation and the up regulation of genes involved in cytokine production, cell cycle regulation, apoptosis and cell adhesion. Understanding the molecular targets that regulate NF-κB may provide more effective therapeutic targets for GB. DiRas family small GTPases, which are homologous to pro-oncogenic Ras GTPases, are tumor suppressive rather than tumor promoting and include DiRas1, DiRas2 and DiRas3 (ARHI). DiRas1 and DiRas2 have been suggested to be tumor suppressive in CNS malignancies, but the role of DiRas3 in CNS malignancies remains unknown. Here we demonstrate that expression of DiRas3 protein in GB cell lines is absent, although DiRas3 is expressed in non-malignant glial cells. Re-expression of DiRas3 in U-87 cells reduces cell proliferation by 20%. Using a NF-κB transcriptional activity luciferase reporter assay demonstrates that DiRas3 expression reduces NF-κB transcriptional activity by 70% compared to vector control. Further experiments demonstrate that decreased NF- κB activity occurs via reduced phosphorylation of the NF-κB inhibitor IκBα. The reduced phosphorylation of IκBα could be a result of decreased AKT and ERK activity, as increased ERK and AKT activity can stimulate NF-κB pathways. Our lab has previously demonstrated that the most common binding partner for DiRas1 and DiRas2 was the small GTPase binding protein SmgGDS, and DiRas1 and DiRas2 also reduce NF- κB activation. However, DiRas3 does not interact with SmgGDS, suggesting that DiRas3 can reduce NF-κB in a SmgGDS-independent manner. Understanding the role of DiRas3 and its binding partners in mediating NF- κB activation may lead to novel therapeutics for glioblastoma. Citation Format: Amy Rymaszewski, Michael Straza, Anne Frei, Carmen Bergom. The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3678.

The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

A number of malignancies are promoted by the activation of Rho GTPases. Regulators of RhoA activity, including the RhoGEF, SmgGDS, can also promote cancer development. Both RhoA and SmgGDS have been shown to enhance NF-ĸB activation in a number of tumors. Utilizing mass spectrometry, we identified the tumor suppressive small GTPase DiRas1 as a novel binding partner for SmgGDS. DiRas1 is a tumor suppressor in malignant gliomas and esophageal squamous cell carcinomas. Examining DiRas gene expression using the ONCOMINE mRNA microarray database (www.oncomine.org) revealed that DIRAS1 and DIRAS2 are markedly decreased in central nervous system tumors, including anaplastic astrocytomas and GBMs, when compared to normal tissue. The role of DiRas1 in other cancers is largely undetermined, and the ways in which DiRas1 may antagonize pro-oncogenic small GTPase signaling is largely unknown. To define how DiRas1 expression may affect SmgGDS functions, we tested the association of DiRas family proteins with SmgGDS. SmgGDS robustly bound wildtype DiRas1, and its closely-related family member DiRas2, but only weakly bound DiRas3 in vitro and in cell lines. In addition, DiRas1, DiRas2, or DiRas3 protein expression decreased proliferation of HEK293T cells. DiRas1 expression could promote less activated RhoA, as a low level of DiRas1 expression caused markedly decreased RhoA-SmgGDS binding in a number of cell lines. DiRas1 expression also abrogated SmgGDS- and RhoA-mediated NF-ĸB activation in a concentration-dependent manner. SmgGDS may exhibit GEF activity toward DiRas1, as a dominant negative DiRas1 (S21N) binds to SmgGDS more than the wildtype protein. However, DiRas1 is predominantly GTP-bound in cells, likely due to a high rate of guanine nucleotide dissociation and low intrinsic GTPase activity. Thus, DiRas1 expression may in part promote tumor suppression by non-productively binding SmgGDS, resulting in less RhoA activity. Taken together, these results suggest that the tumor suppressive small GTPase DiRas1 can antagonize RhoA signaling by competing for binding of the RhoGEF SmgGDS. Further characterizing these actions may lead to novel therapeutic targeting of RhoA activation in cancer malignancies. Citation Format: Andrew D. Hauser, Kristen M. Barr, Anne C. Frei, Patrick Gonyo, Ellen L. Lorimer, Carol L. Williams, Carmen Bergom. The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4443. doi:10.1158/1538-7445.AM2014-4443