Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6–AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5′-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5′-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6–AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

Background: Not merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and Ecadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis. Methods: Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed. Results: Elevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients. Conclusions: Together with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK.

Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.

Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating β1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations.

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