Research

Our goal is to improve the therapeutic ratio of radiation by understanding and minimizing radiation-induced cardiac dysfunction and improving tumor radiation responses.

We focus on utilizing unique genetic models to identify new targets for improving tumor responses to radiation and to minimize damage to the heart from radiation exposure.

More than 50% of cancer patients receive radiation as part of their therapy. Patients who have tumor locations in the thorax often receive unavoidable incidental radiation exposure to the heart, which can lead to cardiac dysfunction months to years after treatment. We currently lack biomarkers or therapies to identify those patients at highest risk of cardiac dysfunction before or after treatments. Our laboratory aims to identify novel therapeutic targets and biomarkers of radiation-induced cardiac toxicity in order to better prevent, minimize, and treat this long-term toxicity.

Consomic Rat Model to Determine Genetic Variants that Influence Cardiac Radiation Dysfunction

We have used chromosome substitution models to demonstrate that rat chromosome 3 harbors genetic variants that can influence radiation sensitivity of mammary tumors and the heart. These studies have led to identification of therapeutic targets that may protect the heart and/or improve tumor responses to radiation therapy.

These studies stemmed from our discovery that inbred SS and BN rat strains have differences in the severity of heart dysfunction after localized heart irradiation. The SS rats are much more sensitive to cardiac radiation than the BN rats, as demonstrated in Figure 1.

**Figure 1. The SS rat strain is more sensitive to radiation than BN rat strain. A.** Adult female rats received 24 Gy of image-guided radiation therapy (RT) using 3 equally-weighted fields. B. The SS rats had increased heart to body weight ratios (an indicator of cardiac hypertrophy) 5 months after RT, while the BN rats did not (*Schlaak et al., AJP – Heart Circ Physiol 2019*).

These findings led us to utilize a chromosome substitution model, called a consomic rat panel (Figure 2), in order to determine the chromosome(s) harboring genetic variants influencing the severity of radiation-induced cardiac dysfunction. Consomic models allow genetic mapping of complex traits. Rats have 21 chromosomes, and thus each consomic rat has ~5% difference in genetic variants from the SS strain. Due to data available on rgd.mcw.edu on the SS-BN consomic animals (not shown), we hypothesized that chromosome 3 harbors genetic variants influencing cardiac radiation injury. Therefore, we started our consomic studies comparing cardiac dysfunction after localized radiation in SS versus SS.BN3 consomic rats (Figure 3).

**Figure 2. The consomic rat panel.** A schematic representation of the SS and SS.BN3 consomic rat genomes.The numbered bars represent chromosomes that are derived from SS (white) or BN (black) rat strains. The only genetic difference between SS and SS.BN3 is the inheritance of chromosome 3 from the SS or BN rats

**Figure 3. Testing whether rat chromosome 3 harbors genetic variants influencing cardiac radiation sensitivity.** The SS and SS.BN3 rats received localized cardiac radiation, as in Figure 1, and received echocardiograms at 0, 3, and 5 months after radiation.

These studies revealed that the SS rats are more sensitive to cardiac radiation, as measured by mortality (Figure 4A), pleural effusions (Figure 4B), pericardial effusions (not shown), and other echocardiogram parameters, such as radial strain (Figure 5). In addition, the SS rats had significantly more myocardial necrosis at 5 months post-radiation than the SS.BN3 rats (Schlaak et al., AJP – Heart Circ Physiol 2019). We saw similar results in male rats, as well as rats administered 9 Gy x 5 daily fractions of localized cardiac radiation, with the SS rats being more sensitive than the SS.BN3 rats.

**Figure 4. The SS rat strain is more sensitive to radiation than BN rat strain. A.** Adult female rats received 24 Gy of image-guided radiation therapy. 5/11 SS rats died by 5 months post-radiation, while 0/7 BN rats died. B. The SS rats had increased pleural fluid at 2.5 and 5 months post-radiation than the SS.BN3 rats, indicating more severe inflammation and/or heart failure (*Schlaak et al., AJP – Heart Circ Physiol 2019*).

**Figure 5. The SS rat strain is more sensitive to radiation than BN rat strain. A.** Adult female rats received 24 Gy of image-guided radiation therapy. Echocardiograms at 5 months post-radiation revealed significantly lower radial strain (and circumferential, not shown) in the SS versus SS.BN3 rats. **B,C.** The SS rats had significantly more necrosis in the myocardium than SS.BN3 rats (*Schlaak et al., AJP – Heart Circ Physiol 2019*).

We are genetically mapping the cardiac radiosensitivity phenotype on rat chromosome 3. However, we have also utilized RNA sequencing to identify potentially important pathways that are differentially expressed and regulated after radiation in the SS and SS.BN3 rat hearts. One week after 24 Gy of radiation to the heart, we see significant differences in gene expression between SS and SS.BN3 rats in a number of pathways, including cardiac hypertrophy, mitochondrial dysfunction, sirtuin signaling, and ubiquitin pathways (Figure 6). These studies have led to additional studies exploring how these pathways contribute to radiation-induced cardiac dysfunction.

**Figure 6. RNA-seq analysis of irradiated SS and SS.BN3 hearts reveal a number of differentially expressed pathways and genes.** Total RNA was extracted and RNA-seq was performed on RNA from the left ventricle of female SS and SS.BN3 rats 1 week after 24 Gy of localized heart radiation. Differential expression analysis was performed, and IPA gene network analysis identified significantly enriched gene ontologies including cardiac hypertrophy, mitochondrial dysfunction, sirtuin signaling, and ubiquitin signaling, which included multiple candidate genes that reside on RNO3 (inset), *Schlaak et al., AJP – Heart Circ Physiol 2019.*

Genetic mapping has started using chromosome 3 congenic rats (Figure 7). Preliminary results from these studies have suggested an ~25 MB region in rat chromosome 3 contains variants influencing the severity of radiation-induced cardiac dysfunction. Further genetic studies are being performed to better characterize the genes involved in cardiac radiation sensitivity.

**Figure 7. Schematic of congenic mapping. A.** Consomic studies revealed that the presence of variants on the BN strain rat chromosome 3 confers resistances to radiation-induced cardiac damage. B. Schematic of congenic rat chromosome 3 to test which area(s) of rat chromosome 3 are important for radiation sensitivity. C. These congenic rats are treated similarly to the studies in Figure 4. Studies examine whether the congenics have similar phenotypes to the SS or the SS.BN3 rats.

Consomic Tumor Model to Determine Genetic Variants in the Tumor Microenvironment that Influence Responses to Radiation

Another focus of our research is using genetic chromosome substitution models to identify factors in the tumor microenvironment that improve breast cancer responses to radiation therapy (Figure 8). This model utilizes chromosome substitution of rat chromosome 3, similar to the cardiac studies above, in immunocompetent or immunocompromised rats (with IL-2 receptor gamma knocked out). Using this model, we found that mammary tumors treated with localized tumor radiation (4 Gy x 3) had significantly enhanced tumor responses in the SS.BN3 rats, compared to the SS rats (Figure 9). The factors in the tumor microenvironment responsible for these changes are currently being explored, using species specific RNA-seq, optical imaging of tumor vasculature, and pharmacologic targeting of pathways differentially regulated in the microenvironment.