PEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. While aberrant expression of PEAK1 has been linked to cancer progression, its normal physiological role in vertebrate biology is not known. Here we provide evidence that PEAK1 plays a central role in orchestrating new vessel formation in vertebrates. Deletion of the PEAK1 gene in zebrafish, mice, and human endothelial cells (ECs) induced severe defects in new blood vessel formation due to deficiencies in EC proliferation, survival, and migration. Gene transcriptional and proteomic analyses of PEAK1-deficient ECs revealed a significant loss of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression, as well as downstream signaling to its effectors, ERK, Akt, and Src kinase. PEAK1 regulates VEGFR2 expression by binding to and increasing the protein stability of the transcription factor GATA-binding protein 2 (GATA2), which controls VEGFR2 transcription. Importantly, PEAK1-GATA2-dependent VEGFR2 expression is mediated by EC adhesion to the ECM and is required for breast cancer-induced new vessel formation in mice. Also, elevated expression of PEAK1 and VEGFR2 mRNA are highly correlated in many human cancers including breast cancer. Together, our findings reveal a novel PEAK1-GATA2-VEGFR2 signaling axis that integrates cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer.

There remains intense interest in tractable approaches to target or silence the KRAS oncoprotein as a rational therapeutic strategy to attack pancreatic ductal adenocarcinoma (PDAC) and other cancers that overexpress it. Here we provide evidence that accumulation of the KRAS oncoprotein is controlled by a self-regulating feed-forward regulatory loop that utilizes a unique hypusinated isoform of the translation elongation factor eIF5A and the tyrosine kinase PEAK1. Oncogenic activation of KRAS increased eIF5A–PEAK1 translational signaling, which in turn facilitated increased KRAS protein synthesis. Mechanistic investigations show that this feed-forward positive regulatory pathway was controlled by oncogenic KRAS-driven metabolic demands, operated independently of canonical mTOR signaling, and did not involve new KRAS gene transcription. Perturbing eIF5A–PEAK1 signaling, by genetic or pharmacologic strategies or by blocking glutamine synthesis, was sufficient to inhibit expression of KRAS, eIF5A, and PEAK1, to attenuate cancer cell growth and migration, and to block tumor formation in established preclinical mouse models of PDAC. Levels of KRAS, eIF5A, and PEAK1 protein increased during cancer progression with the highest levels of expression observed in metastatic cell populations. Combinatorial targeting of eIF5A hypusination and the RAS–ERK signaling pathway cooperated to attenuate KRAS expression and its downstream signaling along with cell growth in vitro and tumor formation in vivo. Collectively, our findings highlight a new mechanistic strategy to attenuate KRAS expression as a therapeutic strategy to target PDAC and other human cancers driven by KRAS activation. Significance: These findings highlight a new mechanistic strategy to attenuate KRAS expression as a therapeutic strategy to target human cancers driven by KRAS activation. Cancer Res; 78(6); 1444–56. ©2018 AACR.

: Repeated intravenous (IV) administration of radiation-attenuated sporozoite (RAS) vaccines induces Plasmodium-specific CD8⁺ liver-resident memory T (Trm) cells in mice and achieves sterile protection against challenge. Our heterologous “prime-and-trap” vaccine strategy was previously shown to simplify and improve upon RAS vaccination. Prime-and-trap vaccination combines epidermal priming by DNA-encoded circumsporozoite protein (CSP) antigen followed by a single IV dose of freshly dissected RAS (fresh-RAS) to direct and trap activated and expanding CD8⁺ T cells in the liver. Prime-and-trap vaccination protects mice against wild-type sporozoite (spz) challenge. Assessment of prime-and-trap vaccines in nonhuman primate (NHP) models and/or humans would be greatly enabled if fresh-RAS could be replaced by cryopreserved RAS (cryo-RAS). Here, we investigated if fresh-RAS could be replaced with cryo-RAS for prime-and-trap vaccination in BALB/cj mice. Despite a reduction in spz vaccine liver burden following cryo-RAS administration compared with fresh-RAS, cryo-RAS induced a similar level of Plasmodium yoelii (Py) CSP-specific CD8⁺ liver Trm cells and completely protected mice against Py spz challenge 112 days after vaccination. Additionally, when the glycolipid adjuvant 7DW8-5 was co-administered with cryo-RAS, 7DW8-5 permitted the dose of cryo-RAS to be reduced four-fold while still achieving high rates of sterile protection. In summary, cryo-RAS with and without 7DW8-5 were compatible with prime-and-trap malaria vaccination in a mouse model, which may accelerate the pathway for this vaccine strategy to move to NHPs and humans.

Attenuated whole organism vaccines targeting the malaria liver stage reliably confer sterile immunity. These vaccines completely protect female mice from infection, but protection in male mice remains unproven. We discover that male mice vaccinated with prime-and-trap, a whole organism-based vaccine strategy, exhibit poorer protection against Plasmodium sporozoite challenge than females. We investigate this sex difference, and identify vaccinated males have fewer hepatic memory CD8⁺ T cells than females when scaling for liver biomass, and reduced inflammatory responses post-vaccination. Surgical hormone manipulation clarifies that the presence of testicular hormones hinders protection in male mice. The presence of androgens does not affect memory CD8⁺ T cell quantity nor quality, but reduces recruitment of CD8⁺ T cells in male liver tissues via a restricted inflammatory response. Here, we show both males and females form functional memory responses following prime-and-trap vaccination, but the presence of androgens during sporozoite challenge impair protection in male mice.

7DW8-5 is a potent glycolipid adjuvant that improves malaria vaccine efficacy in mice by inducing IFN-γ and increasing protective CD8⁺ T cell responses. The addition of 7DW8-5 was previously shown to improve the efficacy of a CD8⁺ T cell-mediated heterologous ‘prime-and-trap’ malaria vaccine against Plasmodium yoelii sporozoite challenge in inbred female mice. Here, we report significant differential sex-specific responses to 7DW8-5 in inbred and outbred mice. Male mice express significantly less IFN-γ and IL-4 compared to females following intravenous 7DW8-5 administration. Additionally, unlike in female mice, 7DW8-5 did not improve the vaccine efficacy against sporozoite challenge in prime-and-trap vaccinated male mice. Our findings highlight the importance of including both female and male sexes in experimental adjuvant studies.