Targeting iRhom to Improve Colon Cancer Immunochemotherapy - Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Approximately 80%- 90% metastatic CRC (mCRC) are treated with chemotherapy or molecular-targeted agents with unsatisfactory overall survivals. Immunotherapy, especially immune checkpoint blockade, has demonstrated promise in CRC with deficient mismatch repair (dMMR) or high level of microsatellite instability (MSI-H). However, most patients (95% of mCRC cases) are of proficient mismatch repair (pMMR) or microsatellite stable (MSS) phenotype and often do not benefit from current immunotherapy approaches. There is still a need to develop novel mechanism- based immunochemotherapy for mCRC. IRhom proteins (iRhom1 and iRhom2) are catalytically inactive relatives of rhomboid intramembrane proteases. iRhom proteins are distributed in many tissues and play an important role in regulating the stability and trafficking of other membrane proteins. IRhom1 was reported to be overexpressed in breast cancer (BCa) and head and neck cancer, and knockdown (KD) of iRhom1 caused apoptosis or autophagy to epithelial cancer cells and inhibited the xenograft tumor growth. Our TCGA data analysis shows that iRhom1 is also significantly upregulated in CRC and pancreatic cancer. The levels of iRhom1 expression are negatively correlated with the clinical prognosis of CRC patients and drug response. KEGG pathway analysis of RNAseq data from iRhom1 knockout (KO) murine CRC cells (CT26) shows that various oncogenic pathways are negatively affected by iRhom1 KO while the apoptosis pathway is upregulated. IRhom1 expression is also negatively correlated with CD8+ T cell infiltration in several types of cancers including CRC. Our preliminary data show that iRhom1 negatively regulates antigen processing and presentation, suggesting a likely role of iRhom1 overexpression in CRC immunoresistance. This application is focused on exploring the therapeutic potential of the combination of RNAi-mediated iRhom1 KD and chemotherapy using nanoparticles (NPs). We have developed a new nanocarrier (PCL-CP) that is highly effective in codelivery of siRNA and lipophilic/hydrophobic small molecule anticancer agents. Importantly, this nanocarrier is highly effective in targeting various types of tumors through both tumor endothelium targeting (likely through transcytosis) and EPR effect. Codelivery of iRhom1 siRNA and CPT-SAHA (a prodrug conjugate of camptothecin and SAHA) led to significantly enhanced tumor growth inhibition and an improvement in tumor immune microenvironment. In this application, three (3) aims will be pursued to further characterize the nanocarrier and elucidate the mechanism of synergistic action of the codelivery. Aim 1 will develop and characterize the biophysical and biological properties of PCL-CP NPs coloaded with siiRhom1 and CPT-SAHA. Aim 2 will investigate the tumor-targeting efficiency of the nanocarrier, and the PK and tissue biodistribution of CPT-SAHA and siRNA coloaded into the nanocarrier. Aim 3 will investigate the in vivo therapeutic efficacy, the underlying mechanism, and the toxicity profile of the CPT- SAHA/iRhom1 siRNA-coloaded NPs in murine and human CRC models. Successful completion of this study will lead to the development of a new and improved immunochemotherapy for the treatment of mCRC.
