Pancreatic cancer is one of the most aggressive malignancies with poor prognosis. The effectiveness of currently available conventional therapies is limited by chemoresistance and systemic toxicity. Thus, there is an urgent need to develop novel and effective anticancer agents by targeting critical pathways that drive tumorigenesis in pancreatic cancer. Pancreatic cancer cells rely on cholesterol for their growth. Cholesterol plays an important role in the cellular metabolism that is reprogrammed in cancer cells to meet the increased energy and biosynthetic demands associated with rapid tumor growth. Targeting cholesterol metabolism is an attractive strategy for inhibiting tumor growth. The sigma-2 receptor (S2R), also known as TMEM97, is an endoplasmic reticulum (ER) protein involved in the regulation of cellular cholesterol levels and is overexpressed in pancreatic cancer. Therefore, targeting S2R is a promising therapeutic approach for pancreatic cancer. We synthesized a series of quinolinyl pyrazinamides showing cytotoxicity in pancreatic cancer cells and selected JR235 for mechanistic studies. JR235 kills the cancer cells through induction of ROS, autophagy, and cell cycle arrest. Bru-seq and proteomics analyses of JR235-treated cells showed the deregulation of genes and proteins involved in cholesterol/lipid biosynthesis and GPCR signaling. Screening of JR235 for its binding affinity against a panel of GPCRs and membrane proteins revealed selectivity and nano-molar affinity to S2R. JR235 binds to cellular S2R and a BODIPY-labeled derivative localizes in the ER. Furthermore, treatment with JR235 results in accumulation of lipid droplets. TMEM97 knockdown resulted in only partial reduction in JR235’s cytotoxicity suggesting the involvement of additional targets. Importantly, JR235 is synergistic with cholesterol and lipid biosynthesis inhibitors. This is the first in-depth study that uses bioinformatics analysis for the investigation of the mechanistic signature of sigma-2 ligands (S2Ls), like JR235, providing unprecedented insights into their role in cholesterol metabolism. In addition, our findings provide a strong rationale for the development of S2Ls as combination therapies with other anticancer agents including statins. In the second part of the dissertation, we focused on validating the target of napabucasin, a reported signal transducer and transcription factor 3 (STAT3) inhibitor being evaluated in multiple clinical trials for the treatment of pancreatic cancer. To better elucidate its mechanism of action, we designed a napabucasin-based proteolysis targeting chimera (PROTAC), XD2-149, that resulted in inhibition of STAT3 signaling in pancreatic cancer cells without inducing proteasome-dependent degradation of STAT3. Proteomics analysis of XD2-149 treated cells revealed the downregulation of the E3 ubiquitin-protein ligase ZFP91. XD2-149 induces degradation of ZFP91 with DC50 values in the nanomolar range. The cytotoxicity of XD2-149 was significantly, but not fully, reduced with ZFP91 knockdown providing evidence for its multi-targeted mechanism of action. The NQO1 inhibitor, dicoumarol rescued the cytotoxicity of XD2-149 but not ZFP91 degradation suggesting that the NQO1-induced cell death is independent of ZFP91. Our findings provide a rationale for the development of ZFP91-targeted therapeutics for the treatment of pancreatic cancer. In summary, this dissertation validates S2R and ZFP91 as important therapeutic targets in pancreatic cancer and provides new tool compounds to elucidate their role in tumor progression. Importantly, our work provides the foundation for the development of novel and promising S2L combination therapies for the treatment of pancreatic cancer.