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The Notch pathway is highly conserved and involved in both cell proliferation and apoptosis. There are five ligands involved: Delta-like ligand 1 (DLL1), DLL2, DLL4, Jagged 1 (JAG1), and JAG2. When the ligands bind to the Notch receptor, the intracellular domain (NICD) is cleaved by ADAM, a disintegrin and metalloproteinase as well as γ-secretase (Wong et al. 2019). After NICD is released, it is able to bind to a translational complex and initiate transcription of proteins that either influence proliferation or apoptosis. Vascular endothelial growth factor (VEGF) signaling also contributes to tumor angiogenesis and works in collaboration with Notch ligand DLL4 to further the cancerous phenotype. More specifically, overexpression of ligands DLL4 and JAG2 have been found in pancreatic adenocarcinoma (Gao et al. 2017). Targeting of both these ligands, as well as VEGF, is therefore crucial in halting self-renewal of PaCSCs. It has been shown that elevated expression of DLL4 leads to therapeutic resistance against VEGF receptors, making a synergistic approach to targeting these two more appealing (Takebe et al. 2015). The bispecific antibody targeting DLL4 and VEGF receptors, Navicixizumab, is currently under clinical trial for colorectal and ovarian cancer, and the PancreAss Kickers plan to incorporate it into their treatment as well (OncoMed Pharmaceuticals 2018). A similar bispecific antibody to target overexpressed JAG2 and VEGF receptors can also be developed and incorporated into the treatment to stop the self-renewal PaCSC process.

The sonic hedgehog pathway is a third highly conserved pathway involved in tissue growth and repair as well as PaCSC self-renewal. In pancreatic adenocarcinoma, KRAS promotes sonic hedgehog expression via the inflammatory transcription factor NF-kB (Gu et al. 2016). The sonic hedgehog protein inhibits the receptor patched, which releases its inhibition of the protein smoothened. Smoothened leads to the activation and nuclear localization of GLI transcription factors, which regulates expression of target genes. An appealing target for this pathway is smoothened, and inhibitors BMS-833923 and PF-0444913 serve as effective treatments for pancreatic adenocarcinoma (Jia et al. 2019). These two inhibitors work most efficiently in combination with gemcitabine, but also have shown beneficial effects on their own (Jia et al. 2019). The researchers plan to incorporate these two smoothened inhibitors, BMS-833923 and PF-0444913, into their treatment as well to block the self-renewal of PaCSCs.

Although resistance can develop in PaCSCs in response to treatment with OXPHOS inhibitors, it can be combated through the inhibition of the protein Myc. PaCSCs’ dependence on OXPHOS is mediated by Peroxisome proliferator-activated receptor γ co-activator 1 α (PGC-1α), a transcription factor that is inversely regulated by the expression of the c-Myc oncogene. The Myc protein is expressed at low to undetectable levels in PaCSCs normally, thus indicating a high expression of PGC-1α (Sancho et al. 2015). When resistance in PaCSCs arises, c-Myc expression increases as well. This resistance has been reversed with inhibition of c-Myc (Sancho et al. 2015). Therefore, the researchers will include a c-Myc inhibitor in all phases of treatment to continually combat this resistance. RRx-001 is a minimally toxic c-Myc inhibitor that has been found to effectively target CD133+/CD44+ cancer stem cells from colon cancer (Oronsky et al. 2018). It also inhibits Wnt signaling as a result of this c-Myc downregulation. The researchers plan to incorporate RRx-001 into their treatment to further suppress PaCSC self-renewal and resistance.

CPI-613 is a lipoate derivative that inhibits pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Anderson et al. 2018). These enzymes in the Krebs cycle allow for carbons from either glucose or glutamine to enter cellular respiration, thereby preventing oxidative phosphorylation. CPI-613 activates pyruvate dehydrogenase kinase, which phosphorylates and inactivates pyruvate dehydrogenase. CPI-613 activates a redox process through a burst of reactive oxygen species that blocks α-ketoglutarate dehydrogenase’s activity (Stuart et al. 2014). A maximum tolerated dose of 500 mg/m² of CPI-613 in combination with the chemotherapy FOLFIRINOX approximately doubled the response rate of patients with metastatic pancreatic adenocarcinoma and showed promising effects for median survival rates (Alistar et al. 2017). The researchers imagine similar effects to be possible in combination with phenformin. By eliminating mitochondrial OXPHOS in PaCSCs, these cells will have no energy to survive and will undergo apoptosis. Since these PaCSCs are the main drivers of metastasis, eradicating them also exterminates their EMT, metastatic niche, chemoresistant, and plastic functions, resulting in an overall greater prognosis for the patient.

Phenformin is a potent biguanic OXPHOS complex I inhibitor that has shown greater than 30% tumor growth inhibition in 5 out of 12 pancreatic adenocarcinoma xenograft models (Rajeshkumar et al. 2017). It is more potent than metformin, another OXPHOS complex I inhibitor, which only suppressed tumor growth via mitochondrial membrane inhibition to the same degree in 3 out of 12 pancreatic adenocarcinoma xenograft models with a five-fold higher dose (Rajeshkumar et al. 2017). Additionally, phenformin does not require a cellular transporter, unlike metformin (Iversen et al. 2017). OXPHOS inhibitors, such as phenformin, have also been shown to resensitize previously therapy-resistant cancer cells (Matassa et al. 2016). Due to its high efficacy, the Pancreass Kickers plan to incorporate phenformin into their treatment in conjunction with a Krebs cycle inhibitor, devimistat (CPI-613).

Cancer stem cells (CSCs) are believed to be the main drivers of metastasis, chemoresistance, and relapse of pancreatic adenocarcinoma due to their plasticity and cooperation with the tumor microenvironment (Sancho et al. 2016). Due to these diverse functions, complete eradication of CSCs poses a challenge. CSCs are also able to undergo metabolic reprogramming depending on stressors in the tumor microenvironment, and current literature suggests that it is the metabolic plasticity of CSCs themselves that allows for survival in different environmental stressors, leading to further metastasis (Peiris-Pagès et al. 2016). Pancreatic cancer stem cells (PaCSCs) in particular are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) to survive. This serves as their preferred mechanism for energy production (Sancho et al. 2015). Another hallmark of CSC survival and proliferation is the notion of self-renewal, most commonly driven in PaCSCs through the Notch, Wnt/β-catenin, and Hedgehog signaling pathways (Wong et al. 2019). Therefore, the researchers plan to target PaCSCs through OXPHOS as well as self-renewal signaling in order to most effectively eradicate this metastatic driver.

Cancer stem cells (CSCs) are believed to be the main drivers of metastasis, chemoresistance, and relapse of pancreatic adenocarcinoma due to their plasticity and cooperation with the tumor microenvironment (TME) to further the disease (Sancho et al. 2016). Due to these diverse effects, eradication of CSCs poses a challenge. CSCs are also able to undergo metabolic reprogramming depending on stressors in the TME, and current literature suggests that it is the metabolic plasticity of CSCs themselves that allows for survival in different environmental stressors, leading to further metastasis (Peiris-Pagès et al. 2016). Pancreatic cancer stem cells (PaCSCs) in particular are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) to survive, which serves as their preferred mechanism for energy production (Sancho et al. 2015). Another hallmark of CSCs is the notion of self-renewal, most commonly driven in PaCSCs through the Notch, Wnt/β-catenin, and Hedgehog signaling pathways (Wong et al. 2019). Therefore, the researchers plan to target PaCSCs through OXPHOS as well as self-renewal signaling in order to most effectively eradicate this metastatic driver.

Another therapy approach described to target the survival function of the metastatic niche are gap junction inhibitors. In the brain metastatic niche, astrocytes were shown to interact with disseminated tumor cells (DTCs) through gap junctions in order to further their survival through the second messenger cGAMP and the activation of the STING pathway as well as interferon-alpha and tumor necrosis factor (Chen et al. 2016). Therefore, by inhibiting these gap junctions, cancerous cells are not able to utilize surrounding cells to further their survival. Some of these gap junction inhibitors include the FDA-approved tonaberset and meclofenamate. This therapy is useful in halting cell survival but is systemic as many cells throughout the body interact through gap junctions, including those that are healthy. Instead, these gap junction inhibitors may be better utilized in synergy with a more specific treatment to target only cancerous cells.

Another therapy approach described to target the protection functions of the metastatic niche are STAT3 inhibitors. STAT3 is a transcription factor involved in cellular immunity, proliferation, apoptosis, and differentiation. In brain metastasis, the STAT3 inhibitor Legasil (Silibinin) reduced the immunosuppressive effect of astrocytes (Priego et al. 2018). Legasil prevents the activation these astrocytes which allows for CD8+ T cells to kill cancerous cells, a function previously suppressed by these cells allowing them to evade the immune response. With this STAT3 inhibitor activity, the protective function of the metastatic niche is eliminated. This therapy is systemic as STAT3 is a transcription factor prevalent throughout the body and inhibiting this transcription factor may affect healthy cells as well as cancerous ones. This treatment may also be better functioning within a more specific treatment targeted at solely tumor cells.  

One therapy approach described to target both the anchorage and proliferation functions in the metastatic niche are anti-VEGF treatments. This anti-anchorage treatment is helpful in combating metastatic dormancy as it disrupts the niche’s function to enhance mobilization of tumor cells. In breast cancer cells, the anti-VEGF antibody as well as VEGF receptor inhibitor SU1498 were shown to significantly reduce cell adhesion to blood vessels (Shen et al. 2010). As a result, this also inhibits the proliferation of cells in the metastatic niche as blocking cell adhesion to blood vessels does not allow the tumor cells to utilize resources to grow. Although this therapy is appealing, VEGF is a common growth factor in cells throughout the body, making this treatment nonspecific and harmful to other healthy cells as well as cancerous ones.