Discovery and Targeting of a Noncanonical Mechanism of Sarcoma Resistance to ADI-PEG20 Mediated by the Microenvironment.

PURPOSE: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 reexpression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20. EXPERIMENTAL DESIGN: Tumor initiation and growth rates were measured for a spontaneous Ass1 knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied in vitro and in vivo. RESULTS: Conditional Ass1 KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. Ass1 KO cells grew robustly through arginine starvation in vivo, while ADI-PEG20 remained completely lethal in vitro, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Coculture with Ass1-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect in vitro and in vivo. CONCLUSIONS: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes.

TDP-43 Oligomerization and Phase Separation Properties Are Necessary for Autoregulation.

Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3' untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3' UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling.

High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules.

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.