Pacritinib mitigates doxorubicin resistance in osteosarcoma by inhibiting WNT/beta-catenin, efflux proteins and receptor tyrosine kinase network

Chemoresistance remains the major obstacle to desirable responses in osteosarcoma (OS). We have previously identified pacritinib (PCT), an FDA-approved JAK2 inhibitor, as a modulator of the AXL/beta-catenin axis. In parental osteosarcoma cells, pacritinib treatment reduced proliferation, induced apoptosis, and suppressed beta-catenin-dependent transcription. Our docking studies, using Seam Dock server, supported direct engagement of both AXL and beta-catenin with pacritinib. In vivo experiments showed growth inhibition of K7M3 tumors by pacritinib treatment in mice without any toxicity. Here, we evaluated the effect of pacritinib in sensitizing doxorubicin-resistant (DXR) osteosarcoma cells to doxorubicin.To determine key players in causing resistance, we first performed RNASeq analysis in doxorubicin-resistant (DXR) MG63 osteosarcoma cells and compared with parental MG63 sensitive -cells, with or without 1.5 µM pacritinib treatment for 24 hours. Gene-set enrichment from transcriptome data revealed significant activation of WNT/beta-catenin programs in DXR cells compared to sensitive counterparts, with increased expression of canonical targets and receptors (e.g., WNT ligands, FZD receptors, TCF/LEF targets) and reciprocal loss of antagonists (DKK/SFRP/WIF1). Notably, drug-efflux machinery mainly ABCB1/MDR1 was elevated in MG63-DXR cells. In addition, receptor tyrosine kinase (RTK) network including AXL was also enhanced in DXR cells. The signature proteins identified by RNASeq analysis were further confirmed in MG63-DXR cells. Western blots confirmed the overexpression of AXL, beta-catenin, and MDR1 in MG63-DXR cells relative to sensitive MG63 cells. Sulforhodamine-B cell viability assay showed that pacritinib reduced the growth of MG63-DXR cells in a dose-dependent manner. Growth suppressive effects were more pronounced in MG63-DXR cells when treated with pacrinitib in combination with doxorubicin, as compared to any individual treatments, showing synergism. Pacritinib treatment decreased AXL and beta-catenin protein levels, attenuated downstream beta-catenin targets, as well as reduced MDR1 in MG63-DXR cells. Notably, RNA-seq data of pacritinib-treated MG63-DXR cells showed a “reversal” trend: transcripts that were elevated in DXR (including beta-catenin and efflux genes) were reduced by pacritinib treatment, whereas WNT antagonists increased, supporting pathway-level inhibition. An in vivo experiment is currently underway to validate that pacritinib exhibits synergistic effect with doxorubicin in suppressing MG63-DXR tumors in mice.Taken together, our data indicates that pacritinib synergistically enhances the effects of doxorubicin in suppressing the growth of MG63-DXR osteosarcoma tumors through a well-defined mechanism.