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Research ArticleHematologyMetabolismOncology
Open Access | 
10.1172/JCI199709
1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
Find articles by Yang, H. in: PubMed | Google Scholar
1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
Find articles by Zingaro, V. in: PubMed | Google Scholar
1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
Find articles by Vu, H. in: PubMed | Google Scholar
1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
Find articles by Nguyen, H. in: PubMed | Google Scholar
1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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1Helen Diller Family Comprehensive Cancer Center, and
2Department of Urology, School of Medicine, UCSF, San Francisco, California, USA.
3Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, New York, USA.
4Department of Pathology,
5Department of Neurology, and
6Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.
7Arc Institute, Palo Alto, California, USA.
8Department of Pathology and Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
9Eugene McDermott Center for Human Growth and Development, Children’s Research Institute, Department of Pediatrics, and
10Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
11Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Address correspondence to: Davide Ruggero, 1450 3rd St., San Francisco, California, 94158, USA. Email: Davide.Ruggero@ucsf.edu. Or to: Haojun Yang, 101 Nicolls Road, Stony Brook, New York, 11794, USA. Email: Haojun.Yang@stonybrook.edu.
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Published July 15, 2026 - More info
Published in Volume 136, Issue 14 on July 15, 2026
Related article:
Abstract
Malignant cells must rapidly synthesize nucleotides to grow and proliferate. Antimetabolite chemotherapies throw a wrench in this process by administering decoy molecules resembling nucleotide precursors that cells cannot use, such as 6-mercaptopurine (6MP) and methotrexate. While this approach remains an essential tool in the treatment of lymphoblastic leukemias and B cell non-Hodgkin lymphomas, approximately 1 in 3 patients will eventually develop therapy-resistant malignancies. In this issue of the JCI, Yang et al. investigated the metabolic adaptations that enable therapy-resistant tumors to grow in the presence of these drugs. Using their previously described mouse model of MYC-driven large B cell lymphoma, they identified that increased expression of the vesicular oligopeptide and histidine transporter SLC15A3 drives dipeptide accumulation in therapy-resistant cells. In lieu of finding other ways to make more nucleotides, these adaptations force cell growth by boosting mTOR signaling. This cunning adaptation, however, is also a vulnerability that can be targeted clinically.
Authors
× AbstractAntimetabolites, chemotherapy targeting nucleotide biosynthesis, are among the oldest and most widely used cancer treatments, yet resistance remains a daunting barrier, especially in the fight against B cell lymphomas. However, the underlying mechanisms of this resistance have long remained elusive. Using an innovative, integrated omics approach, we unexpectedly identified that the accumulation of dipeptides and upregulation of the dipeptide transporter SLC15A3 underlie resistance to nucleotide deficiency in a Myc-driven large B cell lymphoma mouse model. A similar mechanism occurred after long treatment of human B cell lymphoma cells with the chemotherapeutic purine synthesis inhibitor 6-mercaptopurine (6MP). Mechanistically, we demonstrated that dipeptides containing essential amino acids activated the growth and survival mTOR complex 1 (mTORC1) signaling pathway. Notably, SLC15A3 specifically interacted with mTOR on the lysosome, boosting mTORC1 activity selectively in resistant lymphoma cells but not in parental cancer cells. Silencing SLC15A3 diminished mTORC1 activity and restored resistant lymphoma sensitivity to 6MP. Strikingly, resistant lymphomas, but not primary tumors, exhibited heightened sensitivity to the clinical mTOR inhibitor, rapamycin, in culture and in vivo. We extended these findings in human lymphoma biopsies, which revealed increased SLC15A3 expression following antimetabolite therapy. Together, our study uncovered a metabolic adaptation that fuels cancer resistance to nucleotide deficiency and positions the mTORC1 inhibitor, rapamycin, as a potential therapeutic strategy for transforming the management of chemotherapy-resistant lymphomas.
IntroductionAntimetabolites that target nucleotide biosynthesis, such as 6-mercaptopurine (6MP) and methotrexate, remain a cornerstone in chemotherapy treatment of acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (1–4). However, resistance to these therapies remains a major obstacle to curative outcomes (5). Understanding how cancer cells adapt to survive nucleotide deficiency is essential for developing new therapeutic strategies and overcoming drug resistance.
Cancer cells monopolize nucleotide biosynthesis for their uncontrolled growth. Our previous work pinpointed the molecular mechanisms by which this was achieved downstream of Myc-driven lymphoma, which is implicated in more than 80% of Burkitt’s lymphoma cases (6). Specifically, Myc regulates the expression of phosphoribosyl pyrophosphate synthetase 2 (PRPS2), a rate-limiting enzyme in nucleotide biosynthesis. By generating what we believe is the first genetic loss-of-function mouse model for PRPS2 (Prps2null), we demonstrated that inhibition of PRPS2-dependent nucleotide production results in a profound block in Myc-driven tumorigenesis, while PRPS2 deficiency has no effects on normal development and physiology (7). However, despite the initial protection against lymphoma, approximately 40% of mice eventually develop tumors at later stages and become resistant to nucleotide deficiency (7). This model not only mirrors the therapeutic effects of antinucleotide drugs but also provides a powerful system to investigate the metabolic adaptations that underlie resistance to nucleotide-targeted therapies. Cancer cells are known to reprogram their metabolic networks to sustain growth under stress, including nutrient deprivation and chemotherapy-induced metabolic bottlenecks. While much attention has focused on how tumors adapt through changes in de novo nucleotide biosynthesis or salvage pathways (8), less is known about the metabolic rewiring that reprograms cellular signaling pathways to support survival under conditions of nucleotide deficiency.
In this study, employing an integrated approach combining metabolomics, RNA sequencing (RNA-seq), and ribosome profiling in the context of both genetically engineered mouse models (Eμ-Myc/+Prps2null) and pharmacologically resistant human lymphoma cells, we uncovered a potentially novel dipeptide/mTOR complex 1 (mTORC1) signaling axis that enables lymphoma cells to bypass nucleotide stress. We found that resistance to nucleotide deficiency is associated with increased intracellular uptake of dipeptides enriched in essential amino acids, accompanied by an upregulation of the dipeptide transporter SLC15A3. Mechanistically, we found a selective interaction between SLC15A3 and mTOR proteins on the lysosome in resistant cancer cells, resulting in enhanced SLC15A3-dependent mTORC1 activity. We functionally show that genetically and pharmacologically derived resistant lymphomas, but not primary tumors, become sensitive to the mTORC1 inhibitor rapamycin, in culture and mouse models. In addition, we found that the vitamin D pathway acts as an upstream repressor of SLC15A3 transcription, and upregulation of SLC15A3 renders resistant cells vulnerable to active vitamin D3. Importantly, we observed an increase in SLC15A3 levels in lymphoma patient samples following antimetabolite chemotherapy. These findings uncover a crosstalk between dipeptide metabolism and the mTORC1 pathway that is exploited by cancer cells to adapt and grow under conditions of nucleotide deficiency. Most excitingly, we identify rapamycin, an already FDA-approved clinical compound, as a powerful new strategy to combat lymphoma resistance to antimetabolite chemotherapy, opening promising new avenues for targeted treatment.
ResultsNucleotide deficiency–resistant large B cell lymphoma upregulates dipeptide metabolism. The Myc oncogene relies on the PRPS2-dependent nucleotide pathway for lymphoma initiation and progression. However, a percentage of Myc-transgenic mice eventually bypass nucleotide deficiency to form tumors (7). To investigate how large B cell lymphoma cancer cells rewire specific metabolic pathways to acquire resistance to PRPS2-mediated nucleotide deficiency, we performed an unbiased ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) metabolomic profiling of B cells isolated from wild-type, Prps2null, Eμ-Myc/+, and Eμ-Myc/+ Prps2null mice as premalignant samples, as well as lymphoma cells from Eμ-Myc/+ and Eμ-Myc/+ Prps2null mice as tumor samples. This profiling identified a total of 529 known metabolites, and their normalized levels (adjusted to Bradford protein concentration) across all mouse genetic backgrounds comparing premalignant cells and tumors, which are presented in the heatmap (Figure 1A and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI199709DS1). To provide functional context, metabolites were grouped into 8 superpathways based on broad biochemical domains: amino acid, nucleotide, lipid, peptide, carbohydrate, xenobiotic, energy, and cofactors/vitamins. Within tumors, a distinct cluster emerged that clearly distinguished Eμ-Myc/+ from Eμ-Myc/+Prps2null lymphomas and was enriched in metabolites belonging to the peptide superpathway (Figure 1A). To further quantify metabolic rewiring in response to Prps2 deletion during Myc-driven tumorigenesis, we calculated the differential abundance (DA) score, a summary statistic commonly used in metabolomics enrichment analysis (Figure 1B). The DA score captures the overall direction of change within each pathway by comparing the number of significantly increased versus decreased metabolites. In Eμ-Myc/+Prps2null tumors, the DA score revealed an overall decrease in the nucleotide superpathway compared with Eμ-Myc/+ lymphomas (Figure 1B). Specifically, Eμ-Myc/+Prps2null tumors exhibited significant decreases in adenine, cytidine, and thymidine compared with Eμ-Myc/+ lymphomas, along with nonsignificant reductions in AICAR, inosine, and hypoxanthine, as well as in the nucleosides/bases adenosine, guanosine, and guanine (Supplemental Figure 1A and Supplemental Table 1). These data suggest that Myc-overexpressing tumors can circumvent the effects of nucleotide depletion independently of restoring nucleotide levels. Interestingly, the peptide superpathway exhibited the greatest increase among all superpathways in Eμ-Myc/+Prps2null tumors (Figure 1B). Particularly, we observed an augmentation in dipeptides — peptides composed of 2 amino acids (Supplemental Figure 1B). The significantly upregulated dipeptides predominantly contained glutamine or glycerol paired with essential amino acids, including leucine, isoleucine, and valine (Figure 1C). These findings suggest a specific metabolic reprogramming of dipeptide metabolism in nucleotide-deficient Eμ-Myc/+ Prps2null lymphomas.
Figure 1Genetically antinucleotide-resistant lymphoma specifically upregulates dipeptides and their transporter SLC15A3. (A) Heatmap showing relative metabolite levels (compared with WT B cells) of premalignant (PM) B cells from WT, Prps2null, Eμ-Myc/+, and Eμ-Myc/+ Prps2null mice and lymphomas (T) from Eμ-Myc/+, and Eμ-Myc/+ Prps2null mice (n = 5). (B) Differential abundance (DA) score for each superpathway (n = 5). A DA score approaching 1 indicates a coordinated increase in metabolite abundance within the pathway, whereas a score near –1 indicates widespread depletion. (C) Relative levels of significantly altered dipeptides between lymphoma from Eμ-Myc/+ Prps2null and Eμ-Myc/+ mice. (D) Top enriched pathways enriched in genes that are upregulated at both transcriptional and translational levels using BioPlanet pathway enrichment analysis. (E) Relative mRNA expression of indicated genes comparing lymphoma between Eμ-Myc/+ Prps2null and Eμ-Myc/+ mice. (F) Immunoblot analysis of indicated proteins in lymphoma from Eμ-Myc/+ Prps2null and Eμ-Myc/+ mice. β-Actin serves as the loading control. Individual data and mean ± SEM were presented in C and E and analyzed using 2-way ANOVA; *P < 0.05, **P < 0.01; ****P < 0.0001.
As Myc directly regulates both transcription and translation, to gain a deeper understanding of gene expression changes underlying the activation of specific metabolic pathways associated with resistance to nucleotide deficiency, we conducted RNA-seq and Ribo-seq analyses to profile the transcriptome and translatome of Eμ-Myc/+Prps2null lymphomas in comparison with Eμ-Myc/+ lymphomas. The analysis of these omics approaches revealed that the top enriched pathway in Eμ-Myc/+ Prps2null lymphomas corresponds to Transport of amino acids/oligopeptides, which was upregulated at both the transcriptional and translational levels (Figure 1D, Supplemental Figure 1C, and Supplemental Table 2). This finding is consistent with our metabolomics results showing an increase in the peptide superpathway. Among the proteins belonging to this pathway, we identified SLC15A3, a transporter that is specific for dipeptides (9). B cells express 2 dipeptide transporters, SLC15A3 and SLC15A4 (10, 11). We further validated that only Slc15a3, but not Slc15a4, was upregulated in Eμ-Myc/+ Prps2null lymphomas at the mRNA (Figure 1E) and protein levels (Figure 1F), while there was no increase of SLC15A3 or SLC15A4 in premalignant B cells (Supplemental Figure 1D). In addition to dipeptides, SLC15A3 also transports histidine (9). Consistent with increased Slc15a3 expression, metabolites involved in histidine metabolism were also elevated in Eμ-Myc/+ Prps2null lymphomas (Supplemental Figure 1E). Together, these findings demonstrate a distinct upregulation of dipeptide metabolism and its transporter, SLC15A3, in Myc-driven lymphoma upon nucleotide deprivation.
Pharmacologically nucleotide-deficient B cell lymphomas upregulate SLC15A3 and dipeptide metabolism. To strengthen the clinical significance of our data, we next sought to investigate whether the dipeptide pathway may also contribute to chemotherapy-induced resistance to nucleotide deficiency in human cancer cells. To this end, we generated an antimetabolite-resistant lymphoma cell line. We utilized Ramos cells, a human B lymphocyte cell line derived from a patient with Burkitt lymphoma commonly used as a model for B cell biology and Burkitt lymphoma. We treated Ramos cells with 6MP, a chemotherapy drug that targets the second step after PRPS2 within the purine biosynthesis pathway (Figure 2A) and is widely used in the treatment of lymphoma and leukemia (4, 12, 13). By subjecting the cells to a low-dose, long-term 6MP treatment followed by gradual dose escalation, we established a 6MP-resistant Ramos cell line (6MPR) capable of tolerating up to 10 μM 6MP (Figure 2B). Similar to Eμ-Myc/+ Prps2null lymphomas, SLC15A3 levels were significantly elevated in 6MPR cells at both mRNA levels and protein levels (Figure 2, C and D), while SLC15A4 remained unchanged. In addition, 6MPR Ramos cells exhibited an increase of dipeptide uptake, as demonstrated by treatment with an AMCA-labeled dipeptide (Figure 2E and Supplemental Figure 2A). Knockdown of SLC15A3 in resistant cells significantly decreased the AMCA-labeled dipeptide uptake (Figure 2F and Supplemental Figure 2, B and C).
Figure 26MP-resistant human lymphomas upregulate SLC15A3 and dipeptide uptake. (A) Schematic of the de novo purine biosynthesis pathway starting from ribose-5-phosphate. (B) Relative cell survival of parental Ramos cells and 6MP-resistant (6MPR) Ramos cells treated with varying concentrations of 6MP for 2 days. (C) Relative mRNA expression levels of SLC15A3 and SLC15A4 in parental and 6MPR Ramos cells. (D) Immunoblot analysis of SLC15A3 protein levels in parental and 6MPR Ramos cells. β-Actin serves as the loading control. (E) Quantification of dipeptide-AMCA (7-amino-4-methylcoumarin-3-acetic acid, a fluorescent reference) levels in cell lysates from parental or 6MPR Ramos cells treated with increasing concentrations of dipeptide-AMCA. (F) Relative dipeptide-AMCA in parental and 6MPR Ramos cells or 6MPR with SLC15A3 shRNA in basal condition. (G) Cell viability of parental and 6MPR Ramos cells treated with varying concentrations of Gly-Leu or Gly-Sar in amino acid–free HBSS medium. (H) Cell viability of parental and 6MPR Ramos cells treated with varying concentrations of 1 mM different dipeptides in amino acid–free HBSS medium. (I) Cell viability of parental and 6MPR Ramos cells treated with 1 mM Gly-Leu or histidine in amino acid–free HBSS medium. Individual data and mean ± SEM were presented in B, C, and E–I, and E–I were analyzed using 2-way ANOVA; *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001.
To investigate the functional role of upregulated dipeptides in 6MP-resistant cells, we assessed cell viability in amino acid–free HBSS supplemented with Gly-Leu, a dipeptide enriched in Eμ-Myc/+ Prps2null lymphomas. Supplementation with Gly-Leu showed a dose-dependent effect on resistant cell survival but not with a nonhydrolysable dipeptide (Gly-Sar) (Figure 2G). We further examined the dipeptides with another 2 essential amino acids, Gly-Ile and Gly-Val, and found that all dipeptides with essential amino acids selectively enhanced the survival of 6MPR cells but not parental cells. Importantly, downregulation of SLC15A3 attenuated this survival advantage (Figure 2H). In contrast, Gly-Ala, the dipeptide with a nonessential amino acid, failed to enhance resistant cells’ survival, as did supplementation with free histidine (Figure 2I). These findings suggest that antimetabolite-resistant lymphomas upregulate dipeptide metabolism and adopt a novel mechanism to utilize dipeptides as an alternative nutrient source to support survival.
Elevated dipeptides enhance mTORC1 activation in resistant lymphoma through SLC15A3. To investigate how dipeptides contribute to cell survival and resistance, we examined the utilization of dipeptides in antimetabolite-resistant lymphoma. We focused on the significantly upregulated dipeptides, which consist of glycine/glutamine and essential amino acids (Figure 1D). Glycine and glutamine are important for de novo nucleotide biosynthesis; essential amino acids such as leucine, isoleucine, and valine are substrates in branched-chain amino acid (BCAA) metabolism and potent activators of mTORC1 activation, a central regulator of cell proliferation and survival (Figure 3A). One hypothesis is that glycine and glutamine from dipeptides may contribute carbon (from glycine) and nitrogen (from glutamine) atoms, respectively, to fuel nucleotide biosynthesis. To test this, we performed isotope tracing experiments using (13C)-labeled glycine with leucine and (15N)-labeled glutamine with leucine. We detected significant increases in GMP and XMP levels in 6MPR cells derived from the dipeptide of (13C)-labeled glycine with leucine but no changes in pyrimidines from the (AMIDE-15N)-glutamine–containing dipeptide (Figure 3, B and C). Next, we investigated whether the essential amino acid component of the dipeptides supports BCAA metabolism by tracing (13C)-labeled leucine with glutamine. However, no significant differences were found in BCAA pathway metabolites between parental and resistant cells (Figure 3D).
Figure 3Elevated dipeptides are associated with enhanced mTORC1 activation in resistant lymphomas. (A) Schematic of pathways derived from dipeptide consisting of Gly/Gln with branched-chain amino acids (BCAA). (B) Percentage of 13C within different metabolites from parental or 6MP-resistant Ramos cells treated with (13C) Gly-Leu. (C) Percentage of 15N within different metabolites from parental or 6MP-resistant Ramos cells treated with (15N) Gln-Leu. (D) Percentage of 13C within different metabolites from parental or 6MP-resistant Ramos cells treated with Gln-(13C) Leu. (E) Immunoblot analysis of indicated proteins in Ramos cells treated with different concentrations of Gly-Leu in serum-free medium. (F) Immunoblot analysis of indicated proteins in parental or 6MP-resistant Ramos cells in full medium condition. (Internal control β-actin was the same one from Figure 2D.) (G) Immunoblot analysis of indicated proteins in Eμ-Myc/+Prps2null and Eμ-Myc/+ lymphomas. (H) Immunoblot analysis of the indicated proteins in parental and 6MP-resistant Ramos cells with or without shRNA-mediated knockdown of SLC15A3 under basal conditions. (I) Relative cell survival of parental and 6MP-resistant Ramos cells with or without
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