Hello, my name is Alfredo Pallerina, and I'm a second year student at the West Virginia School of Osteopathic Medicine. Today, I will be presenting my project entitled, Investigation of Dual Effects of Compound 968 Analogues in Treating Breast Cancer and Cancer-Related Pain. Breast cancer is the second leading cause of cancer with the highest mortality rate in females in the United States. A subtype of breast cancer is triple negative breast cancer, which has the worst prognosis and even lower survival rates, especially with brain metastasis, shown in this figure right here. Currently, the treatment for brain metastasis of breast cancer is largely ineffective, leading us to seek new treatments. In this project, we studied glutaminase inhibitors as potential drugs to treat brain metastasis of breast cancer. Glutaminases are rate-limiting enzymes important in glutamine metabolism, and it plays a critical role in cancer. Therefore, glutaminase inhibition has become a potential strategy for cancer treatment. There are a number of glutaminase inhibitors available, such as compound 968, also known as C968, as shown in this left compound right here. These inhibitors showed anti-cancer activities, however, due to their poor in vivo efficacy and toxicity, they did not pass clinical trials. To find better glutaminase inhibitors, our collaborators designed about 100 C968 analogs, all of which we screened. Eventually, we found that analog 15 had good anti-cancer activity, as shown over here. In addition to anti-cancer, the compounds also have anti-pain activity. There is a transcription factor called NRF2, which can be activated during pain, ultimately leading to pain reduction. Other pain signaling pathways, such as ERK, JNK, and AKT, are also activated during pain. To study pain, we use CIPN, or chemotherapy-induced peripheral neuropathy model, in our project. Our preliminary studies showed that glutaminase inhibitors C968 and analog 15 can activate NRF2, indicating that they can be promising treatment options for both cancer and pain. To study the anti-cancer effects of C968 and analog 15, we use the triple negative breast cancer cell line and its brain metastasis counterpart. The 231 is the parental regular breast cancer cell line, as shown here. And 231BR cells is the brain metastasized cell line, as shown over here. To study the anti-pain activities and effects, we use CIPN mouse model, which is indicated in the figure below, right here. First, we performed an NTT assay and found that both compounds can inhibit the growth of 231 and 231BR cells. We also measured the IC50 value of both compounds in cell lines. Here we can see that analog 15 has lower IC50 values in both cell lines compared to C968, indicating that analog 15 may have better efficacy, as shown over here. Here in this figure, we also measured the expression of glutaminase 1 and NRF2 in both cell types, and we found that glutaminase has higher expression and NRF2 has lower expression in 231BR cells compared to 231 cells. This indicates our compound may be a good option to treat brain metastasis of breast cancer. In this figure, we use a kit to measure glutaminase 1 inhibition. Compared to C968, analog 15 showed better inhibition of glutaminase 1 activity. In this figure here, we show the WNT signal transaction pathway, which is important in triple negative breast cancer oncogenesis. We measured some important proteins in the WNT pathway, which is something that we are still working on. In the bottom figure here, we show that the analog 15 has a better inhibition on GSK3 alpha beta and beta-catenin in 231BR cells compared to the C968. So far, we investigated the anti-cancer activity of the two compounds. Next, we will demonstrate their effects in pain reduction. In this part of the study, we use the CIPN model to study the anti-pain activities and effects of these drugs. As shown here in figures A and B, this represents the effect of C968 in males and female mice, respectively. We can see that compared to the VHOCO control, C968 accentuated the development of CIPN in both sexes. Over here in figures C and D, we represent the effects of analog 15 in male and female mice, respectively. We can see that analog 15 accentuated the development of CIPN in males, but not in females. So explore the reason why analog 15 only showed effectiveness in pain relief in male mice. We did some mechanism studies. We measured the activation of ERK, JNK, and AKT pathways in male and female mice, treated with VHOCO, C968, and analog 15. As we can see here in figures B and D, we see that analog 15 increased the phosphorylation of ERK and AKT only in the female mice, as shown here. This particularly explained why analog 15 showed no anti-pain activity effects in the female mice. In conclusion, based on our findings, the glutaminase inhibitors C968 and analog 15 could inhibit both regular and brain metastasized breast cancer cells in vitro. But overall, analog 15 may have better anti-cancer and anti-metastasis activity than C968. For pain treatment, C968 accentuated the development of CIPN in both male and female mice, while analog 15 accentuated the development of CIPN mainly in the male mice. Our studies may lead to finding new drugs to treat cancer and cancer-related pain at the same time. Additionally, our project demonstrates osteopathic significance, as it will be helpful in bringing the body back to homeostasis, eventually integrating the behavioral model of osteopathic care. For future plans, we will further study the compound's anti-cancer and anti-pain activities and effects and explore their mechanism. That is all I have for today, and thank you so much for listening.

glutaminase inhibitors

triple-negative breast cancer

analog 15

brain metastasis

pain management