DkkMo, a vivo morpholino antisense inhibitor of Dkk-1 targets tumor growth, bone lesions, metastatic potential and immune evasive host stroma in a patient-derived xenograft model of osteosarcoma
Introduction
Osteosarcoma (OS) is the most common primary bone malignancy and, like many forms of malignant bone disease (MBD), progression is accompanied by tumor growth, metastases, and destruction of bone. The current standard for treating OS is with adjuvant chemotherapy followed by surgical removal of the primary tumor. Chemotherapy has the capacity to increase 5-year survival from 20–40% (surgery alone) to 55–90%. Survival rates within the chemotherapeutic responder category can be stratified into those with greater than 90% tumor necrosis after adjuvant therapy versus the remainder, who have 5-year survival rates of 90% and 55%, respectively. The effectiveness of chemotherapy can be negatively influenced by tumor-induced osteolytic bone lesions (OLs) that provide a protective environment for tumor propagation and survival. The canonical Wnt (cWnt) inhibitor Dickkopf-1 (Dkk-1) has been shown to drive bone destruction and tumor survival in several forms of MBD, including OS.
Methods
A Dkk-1 blocking vivo morpholino (referred to as DkkMo) was custom designed and manufactured by Genetools LLC (Philomath, OR) to target the 5’ end of the human Dkk-1 transcript (Pan et al. 2022, PMID: 35277659). Animal studies were performed in accordance with a protocol approved by Texas A&M University IACUC. A patient-derived osteosarcoma xenograft (Goldstein et al. 2016, PMID: 27049730) was implanted next to the medial femur of 2-month-old NSG mice. Some of the mice received an intraperitoneal administration of 12.5 mg per kg DkkMo and untreated controls received vehicle. Over three weeks, primary tumor volume was measured by ultrasound imaging. At the conclusion of the experiment, final tumor volume, tumor necrosis, and bone destruction was measured by micro-computed tomography (Pan et al. 2022). Pulmonary metastases were measured by quantitative reverse transcriptase PCR. Single cell resolution transcriptomic sequencing (scSeq) was performed using 10x technology by the Texas A&M Genome Sciences and Society genomics core.
Results
As a single agent, DkkMo, exhibited the capacity to slow tumor expansion, increase tumor necrosis, inhibit metastases and preserve bone in a patient derived xenograft based murine model of orthotopic OS. With scSeq, DkkMo was demonstrated to substantially reduce the frequency of dividing tumor cells and reinitiate an osteogenic phenotype in most of the tumor cells analyzed. DkkMo also inhibited the infiltration of myeloid suppressor-like cells in the tumor stroma, supporting recent recognition of the role of Dkk-1 in immune modulation. Unlike many traditional chemotherapeutics, DkkMo did not exhibit signs of toxicity and did not cause weight loss.
Conclusion
These preclinical findings indicate that DkkMo has the potential to safely target tumor growth, survival, metastases and local bone tissue destruction, and therefore may represent a powerful means to improve treatment of OS and other MBDs.