DON inhibits FGAMS, a de novo purine biosynthesis enzyme, with the lowest Ki.

Inhibiting MTAP sensitizes tumor cells to DRP-104 treatments.

Prostate cancer cells develop adaptation to purine shortage inducer, such as DRP-104, and employ microtubules to assemble purinosome to enhance purine biosynthesis efficiency.

Treating tumor-bearing mice with DRP-104 in combination with MTDIA does not affect animal’s body weight but alters the tumor’s immune microenvironment.

Gene expression and functional assays implicate purine biosynthesis as a driver of prostate cancer pathogenesis.

The MTAP-dependent purine salvage is a source of purine supply for cancer cells.

A graphical abstract summarizing tumor cells’ strategies for adapting to and their vulnerabilities under purine shortage stress.

PURPOSE:Purine metabolism is a promising therapeutic target in cancer; however, how cancer cells respond to purine shortage, particularly their adaptation and vulnerabilities, remains unclear. EXPERIMENTAL DESIGN:Using the recently developed purine shortage-inducing prodrug DRP-104 and genetic approaches, we investigated the responses in prostate, lung, and glioma cancer models. RESULTS:We demonstrate that when de novo purine biosynthesis is compromised, cancer cells employ microtubules to assemble purinosomes, multiprotein complexes of de novo purine biosynthesis enzymes that enhance purine biosynthesis efficiency. Although this process enables tumor cells to adapt to purine shortage stress, it also renders them more susceptible to the microtubule-stabilizing chemotherapeutic drug docetaxel. Furthermore, we show that although cancer cells primarily rely on de novo purine biosynthesis, they also exploit methylthioadenosine phosphorylase (MTAP)-mediated purine salvage as a crucial alternative source of purine supply, especially under purine shortage stress. In support of this finding, combining DRP-104 with an MTAP inhibitor significantly enhances tumor suppression in prostate cancer models in vivo. Finally, despite the resilience of the purine supply machinery, purine shortage-stressed tumor cells exhibit increased DNA damage and activation of the cGAS-STING pathway, which may contribute to impaired immunoevasion and provide a molecular basis of the previously observed DRP-104-induced antitumor immunity. CONCLUSIONS:Together, these findings reveal purinosome assembly and purine salvage as key mechanisms of cancer cell adaptation and resilience to purine shortage while identifying microtubules, MTAP, and immunoevasion deficits as therapeutic vulnerabilities.

Glutamine is a conditionally essential amino acid for the growth and survival of rapidly proliferating cancer cells. Many cancers are addicted to glutamine, and as a result, targeting glutamine metabolism has been explored clinically as a therapeutic approach. Glutamine-catalyzing enzymes are highly expressed in primary and metastatic head and neck squamous cell carcinoma (HNSCC). However, the nature of the glutamine-associated pathways in this aggressive cancer type has not been elucidated. Here, we explored the therapeutic potential of a broad glutamine antagonist, DRP-104 (sirpiglenastat), in HNSCC tumors and aimed at shedding light on glutamine-dependent pathways in this disease. We observed a potent antitumoral effect of sirpiglenastat in HPVand HPV + HNSCC xenografts. We conducted a whole-genome CRISPR screen and metabolomics analyses to identify mechanisms of sensitivity and resistance to glutamine metabolism blockade. These approaches revealed that glutamine metabolism blockade results in the rapid buildup of polyunsaturated fatty acids (PUFAs) via autophagy nutrient-sensing pathways. Finally, our analysis demonstrated that GPX4 mediates the protection of HNSCC cells from accumulating toxic lipid peroxides; hence, glutamine blockade sensitizes HNSCC cells to ferroptosis cell death upon GPX4 inhibition. These findings demonstrate the therapeutic potential of sirpiglenastat in HNSCC and establish a novel link between glutamine metabolism and ferroptosis, which may be uniquely translated into targeted glutamine-ferroptosis combination therapies.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases T regs , and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

Pancreatic ductal adenocarcinoma (PDAC) cells use glutamine (Gln) to support proliferation and redox balance. Early attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. Here, we demonstrated that treating PDAC cells with a Gln antagonist, 6-diazo-5-oxo- l -norleucine (DON), led to a metabolic crisis in vitro. In addition, we observed a profound decrease in tumor growth in several in vivo models using sirpiglenastat (DRP-104), a pro-drug version of DON that was designed to circumvent DON-associated toxicity. We found that extracellular signal-regulated kinase (ERK) signaling is increased as a compensatory mechanism. Combinatorial treatment with DRP-104 and trametinib led to a significant increase in survival in a syngeneic model of PDAC. These proof-of-concept studies suggested that broadly targeting Gln metabolism could provide a therapeutic avenue for PDAC. The combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome.

Our recently published K1N2-score robustly predicts KEAP1/NFE2L2-mutations and pathway activation status, while its accessibility might be limited. We tested if the RNA expression data of six pathway-related genes and NQO1-IHC might be a reliable alternative using 348 KEAP1/NFE2L2 mutation-enriched NSCLC. While TXNRD1 RNA testing was the best-performing single-gene test, the combination of single-gene screening and validation with the K1N2-score achieved the highest performance when predicting mutation status or pathway activation.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. A factor that contributes to the poor prognosis of the disease is the complex tumor microenvironment (TME). The PDAC TME is composed of excessive fibrosis and desmoplasia, which creates a harsh environment resulting in hypoxia and altered nutrient availability. To promote survival and proliferation in this environment, PDAC cells can reprogram glutamine (Gln) metabolism. Previous studies have demonstrated that PDAC cells use Gln to support proliferation and redox balance. However, earlier attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. We hypothesized that a Gln analogue, such as 6-Diazo-5-oxo-L-norleucine (DON), could broadly target Gln metabolism in PDAC and prevent rapid adaptation. Indeed, DON treatment led to a significant decrease in PDAC proliferation and a profound reduction of various metabolites involved in central carbon and nucleotide metabolism, suggesting that DON creates a metabolic crisis. In addition, we observed a significant decrease in tumor growth in various in vivo models (syngeneic, immunodeficient and PDXs) using DRP-104 (sirpiglenastat), a novel pro-drug version of DON that was designed to circumvent DON associated GI toxicity and allow the therapeutic exploration of broad Gln antagonism. Mechanistically, we found that ERK signaling is increased as a compensatory mechanism through the increased activity of receptor tyrosine kinases (RTKs). Combinatorial treatment of DRP-104 and Trametinib (MEK1/2) inhibitor led to a significant increase in survival in a syngeneic model PDAC. Taken together, these pre-clinical results suggest that broadly targeting Gln metabolism could provide a new therapeutic avenue for PDAC and that the combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome. Citation Format: Joel Encarnacion-Rosado, Alec C. Kimmelman, Robert Wild. Targeting pancreatic cancer metabolic dependencies through glutamine antagonism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A059.

KEAP1 is mutated in approximately 20% of non-small cell lung cancer (NSCLC) and is associated with poor response rates to checkpoint blockade. Given the therapeutic challenge presented by KEAP1 mutant tumors and the great potential held by immunotherapeutic approaches, there is an urgent need to identify the mechanisms that mediate immune evasion in patients with KEAP1 mutant lung adenocarcinoma. We previously demonstrated that metabolic rewiring in KEAP1 mutant tumors leads to increased dependency on glutamine as a substrate for multiple anabolic pathways. DRP-104 (Sirpiglenastat) is a novel glutamine antagonist which inhibits all glutamine consuming reactions. Using pre-clinical mouse models and patient derived xenograft models we demonstrate that DRP-104 robustly impairs the growth of KEAP1 mutant tumors. Furthermore, using an immunocompetent orthotopic lung adenocarcinoma mouse model we find that treatment with DRP-104 increases survival of Keap1 mutant tumor bearing mice treated with anti-PD1 therapy. We have dissected the mechanism by which DRP-104 impacts KEAP1 mutant tumors and the surrounding immune microenvironment. Notably, DRP-104 impairs nucleotide synthesis in KEAP1 mutant tumors and thereby inhibits cancer cell growth. In addition, DRP-104 reduces exhausted T cell and T regulatory populations within the tumor microenvironment as well as increases effector T cell function. Overall, we find that DRP-104 impairs the growth of KEAP1 mutant tumors through both targeting of tumor intrinsic metabolic vulnerabilities and through tumor extrinsic immunostimulatory mechanisms such as promoting the expansion of functional anti-tumor T cell populations. This data provides additional rationale for the ongoing phase I/II clinical trial (NCT04471415) using DRP-104 in NSCLC with mutations in KEAP1 as well as for combining DRP-104 with checkpoint blockade. Citation Format: Ray Pillai, Sarah LeBoeuf, Ali Rashidfarrokhi, Shih Ming Huang, Triantafyllia Karakousi, Anastasia-Maria Zavitsanou, Warren Wu, Volkan Sayin, Robert Wild, Sergei Koralov, Thales Papagiannakopoulos. Glutamine antagonist DRP-104 enhances anti-tumor responses in KEAP1 mutant lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4904.

Supplementary Figure S1. LMNA-NTRK1 genetic rearrangement detected in patient's plasma and xeno. Supplementary Figure S2. Acquisition of mutations in the TRKA kinase domain drives secondary resistance to entrectinib in Ba/F3 TRKA WT cells. Supplementary Figure S3. NTRK1 mutations confer resistance to TRKA inhibition. Supplementary Figure S4. Homology alignment of NTRK1 p.G595 and p.G667 variants. Supplementary Table S1. NGS analysis of NTRK1 gene in patient's plasma and xeno. Supplementary Table S2. NGS analysis of patient's derived samples. Supplementary Table S3. Summary of serial ctDNA analyses. Supplementary Table S4. The p.G595R and p.G667C mutations confer resistance to multiple TRK inhibitors. Supplementary Table S5. NTRK1 p. G595R, p.G667C and LMNA-NTRK1 fusion probes for ddPCR.

BACKGROUND:Prostate cancer (PCa) continues to be one of the leading causes of cancer deaths in men. While androgen deprivation therapy is initially effective, castration-resistant PCa (CRPC) often recurs and has limited treatment options. Our previous study identified glutamine metabolism to be critical for CRPC growth. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) blocks both carbon and nitrogen pathways but has dose-limiting toxicity. The prodrug DRP-104 is expected to be preferentially converted to DON in tumor cells to inhibit glutamine utilization with minimal toxicity. However, CRPC cells' susceptibility to DRP-104 remains unclear. METHODS:Human PCa cell lines (LNCaP, LAPC4, C4-2/MDVR, PC-3, 22RV1, NCI-H660) were treated with DRP-104, and effects on proliferation and cell death were assessed. Unbiased metabolic profiling and isotope tracing evaluated the effects of DRP-104 on glutamine pathways. Efficacy of DRP-104 in vivo was evaluated in a mouse xenograft model of neuroendocrine PCa, NCI-H660. RESULTS:DRP-104 inhibited proliferation and induced apoptosis in CRPC cell lines. Metabolite profiling showed decreases in the tricarboxylic acid cycle and nucleotide synthesis metabolites. Glutamine isotope tracing confirmed the blockade of both carbon pathway and nitrogen pathways. DRP-104 treated CRPC cells were rescued by the addition of nucleosides. DRP-104 inhibited neuroendocrine PCa xenograft growth without detectable toxicity. CONCLUSIONS:The prodrug DRP-104 blocks glutamine carbon and nitrogen utilization, thereby inhibiting CRPC growth and inducing apoptosis. Targeting glutamine metabolism pathways with DRP-104 represents a promising therapeutic strategy for CRPC.

Supplementary Data from Compensatory Insulin Receptor (IR) Activation on Inhibition of Insulin-Like Growth Factor-1 Receptor (IGF-1R): Rationale for Cotargeting IGF-1R and IR in Cancer

Supplementary Figures 1-9 and Tables 1-3 from Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2: Distinct from Rapamycin

Supplementary Figure from Sirpiglenastat (DRP-104) Induces Antitumor Efficacy through Direct, Broad Antagonism of Glutamine Metabolism and Stimulation of the Innate and Adaptive Immune Systems