Bone Stromal Glutamine Renders Tumor cell PARP Inhibitor Resistance

The majority of patients with late-stage breast cancer develop distal bone metastases. The bone microenvironment can affect response to therapy, and uncovering the underlying mechanisms could help identify improved strategies for treating bone metastatic breast cancer. Here, we observed that osteoclasts reduced the sensitivity of breast cancer cells to DNA damaging agents, including cisplatin and the PARP inhibitor (PARPi) olaparib. Metabolic profiling identified elevated glutamine production by osteoclasts. Glutamine supplementation enhanced the survival of breast cancer cells treated with DNA damaging agents, while blocking glutamine uptake increased sensitivity and suppressed bone metastasis. GPX4, the critical enzyme responsible for glutathione oxidation, was upregulated in cancer cells following PARPi treatment through stress-induced ATF4-dependent transcriptional programming. Increased glutamine uptake and GPX4 upregulation concertedly enhanced glutathione metabolism in cancer cells to help neutralize oxidative stress and generate PARPi resistance. Analysis of paired patient samples of primary breast tumors and bone metastases revealed significant induction of GPX4 in bone metastases. Combination therapy utilizing PARPi and zoledronate, which blocks osteoclast activity and thereby reduces the microenvironmental glutamine supply, generated a synergistic effect in reducing bone metastasis. These results identify a role for glutamine production by bone-resident cells in supporting metastatic cancer cells to overcome oxidative stress and develop resistance to DNA-damaging therapies. Overall design: 5×10^5 MDA-MB-231 cells were plated on 10 cm dishes and cultured for 24 hours. Cells were then treated with either DMSO or 40 µM Olaparib for 3 days. Cells were lysed for RNA extraction and cDNA library construction and RNA-seq analysis; Macrophages were isolated from bone marrow cells flushed from the tibia of 6-week-old C57/BL6 mice and filtered through a 70 µm cell-strainer before over-night culture in a-MEM with 10% FBS. The following day non-adherent cells were plated and supplemented with 50 ng/ml M-CSF for 2 days. 2×106 Cells were then re-plated in 12 well plates at the presence of 100 ng/ml RANKL in DMEM plus 10% FBS, with media changed every 2 days (media was changed twice a day from Day 5 and on). Matured osteoclasts and macrophages were lysed for RNA extraction and cDNA library construction and RNA-seq analysis.