Elucidating Glutamine Metabolism in Breast to Bone Metastasis

Project Summary Several aggressive types of breast cancer (BCa) are nutritionally reliant on glutamine metabolism, and glutamine uptake has been shown to be enhanced in BCa bone-homing clonal cell populations. This is especially significant as bone metastasis will occur in approximately 70% of women with metastatic BCa and remain clinically

incurable. The enzyme glutaminase (GLS) catalyzes the conversion of glutamine to glutamate, and I have shown that its loss in bone tropic BCa cell lines results in reduced osteolysis. During outgrowth of bone metastases, bone destruction is driven by changes in bone-forming osteoblasts and bone-degrading osteoclasts, known as

the “vicious cycle.” As the metabolic conversion catalyzed by GLS is also essential to osteoblast lineage commitment, a genetically modified mouse model was used to delete Gls in osteoblast progenitors. Surprisingly, loss of GLS function in osteoblast progenitors led to reduced bone destruction and osteoclast numbers. This

proposal seeks to identify the mechanisms by which glutamine metabolism in the bone niche influences bone destruction and how they could be exploited therapeutically. I hypothesize that loss of functional GLS in bone- tropic BCa cells induces a senescence-like phenotype that reduces tumor burden and confers sensitivity to

senolytic therapy while GLS loss in osteoblast progenitors limits release of osteoclastogenic signals, reducing bone destruction by cutting off the “vicious cycle.” In Specific Aim 1, I will follow up on data from an RNA-seq analysis, which indicated upregulation of Ypel2 (Yippee-like protein 2) in GLS KO cells, a gene that has been

linked to cell cycle arrest and senescence in endothelial cells. I will use CRISPR/Cas9 to KO and overexpress Ypel2 in various GLS WT and KO breast cancer cell lines and test for cell cycle activity and detect markers of senescence. I will also test for the presence and levels of osteolytic factors such as PTHrP (in vitro) and RANKL

(in vivo). Finally, I will test the effect of genetic deletion and pharmacologic inhibition of GLS (CB-839) on sensitivity of aggressive breast cancer cell lines E0771, 4T1-2, and MDA-MB-231-BoM to senolytic treatment in vitro and in vivo. In Specific Aim 2, to determine the effect of osx-driven GLS KO (GLSOBKO) on osteoblast

differentiation and function, I will co-culture osteoblasts from mice with GLSOBKO with bone-tropic BCa cells ex vivo and test for osteoblast differentiation and function, including production of osteoblast-specific proteins (RUNX2, OPN, OCN, etc.). This will allow me to determine the effect of tumor cell signaling on the compromised

osteoblast progenitors. To determine the effect of GLS loss in osteoblast progenitors on osteoclastogenesis and osteoclast function in tumor-bearing bones, I will perform RNA-scope analysis and Tartrate-Resistant Acid Phosphatase (TRAP) staining on tissue sections from GLSOBKO mice and wild type controls. Findings from this

research have the potential to provide new information about a highly morbid, yet incurable manifestation of metastatic breast cancer and could even raise the possibility of using pre-existing cancer therapies targeting glutamine metabolism to ameliorate bone metastatic burden in breast cancer.