Mechanisms that maintain cancer stem cells are crucial to tumor progression. The ID2 protein underpins cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. We report that DYRK1A and DYRK1B kinases phosphorylate ID2 on Threonine-27 (T27). Hypoxia down regulates this phosphorylation via inactivation of DYRK1, whose activity is stimulated in normoxia by the oxygen sensing prolyl hydroxylase PHD1. ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin-2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

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The l‐type amino acid transporter 1 (LAT1, SLC7A5) imports dietary amino acids and amino acid drugs (e. g., l‐DOPA) into the brain, and plays a role in cancer metabolism. Though there have been numerous reports of LAT1‐targeted amino acid‐drug conjugates (prodrugs), identifying the structural determinants to enhance substrate activity has been challenging. In this work, we investigated the position and orientation of a carbonyl group in linking hydrophobic moieties including the anti‐inflammatory drug ketoprofen to l‐tyrosine and l‐phenylalanine. We found that esters of meta‐carboxyl l‐phenylalanine had better LAT1 transport rates than the corresponding acylated l‐tyrosine analogues. However, as the size of the hydrophobic moiety increased, we observed a decrease in LAT1 transport rate with a concomitant increase in potency of inhibition. Our results have important implications for designing amino acid prodrugs that target LAT1 at the blood‐brain barrier or on cancer cells.

T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical TCR-induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8 + T-cells undergo activation but also simultaneous homeostatic inhibition manifest in upregulation of immune checkpoint molecules and functional suppression. T-cell transferred into BMT recipients were protected from homeostatic inhibition by PD1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT-'immunotransplant'increased T-cell homeostatic activation and anti-tumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL-7/IL-15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T-cell in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies.

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers . It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 mM), much closer to the EAAT1 value of 0.6 mM. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.