Targeting Asparagine Metabolism as a Therapeutic Vulnerability in Pediatric H3-Mutant High-Grade Gliomas
Presenter Status
Post-Doctorial Research
Abstract Type
Basic Research
Primary Mentor or Principal Investigator
Vivek Nand Yadav
Presentation Type
Poster
Start Date
19-5-2026 12:00 PM
End Date
19-5-2026 1:00 PM
Abstract Text
Problem Statement/Question:
Diffuse hemispheric glioma (DHG, H3G34R-mutant) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive pediatric brain tumors with limited treatment options and extremely poor prognosis. Effective targeted metabolic therapies for these tumors remain largely undefined. It is unknown whether asparagine metabolism serves as a specific therapeutic vulnerability in pediatric high-grade gliomas.
Background/Project Intent (Aim Statement):
Pediatric high-grade gliomas (pHGGs) are among the most lethal childhood cancers, with no effective therapies and poor survival. Asparagine (ASN), essential for protein synthesis and metabolic balance, supports tumor growth through its biosynthetic enzyme asparagine synthetase (ASNS), often upregulated in aggressive cancers to promote resistance. While L-asparaginase has transformed leukemia therapy, ASN metabolism in gliomas remains unexplored. Our data show elevated ASNS expression in pediatric glioma tissues compared to normal brain, suggesting metabolic dependency. This study aims to determine whether pharmacologic (ASX-173) or genetic inhibition of ASNS can suppress tumor growth in H3G34R-mutant pHGG, identifying asparagine metabolism as a novel, targetable vulnerability for this fatal pediatric cancer.
Methods (include PDSA cycles):
Glioma cell lines were cultured in complete or asparagine-free media and treated with L-asparaginase, the ASNS inhibitor ASX-173, or combination therapies. Stable ASNS shRNA knockdown models were generated for mechanistic studies. Cell proliferation, viability, clonogenic survival, and IC₅₀ values were assessed, while qPCR, immunoblotting, and immunohistochemistry evaluated ASNS expression and pathway activity. RNA sequencing compared transcriptomic responses under control, ASN-deprived, and ASX-173–treated conditions, complemented by public single-cell RNA-seq analysis. Experimental parameters were optimized for dosing and combination effects. Future work will assess the in vivo efficacy of ASX-173 in a DHG tumor model to validate ASNS inhibition as a therapeutic strategy.
Results:
Single-cell RNA-sequencing and immunohistochemical analysis of pHGG patients and murine tumors showed increased ASNS expression in tumors compared to the normal brain, indicating higher in situ asparagine biosynthesis. In vitro, deprivation of ASN or NEAA alone had minimal impact on cell growth, suggesting limited reliance on extracellular ASN uptake. Conversely, pharmacologic inhibition of ASNS with ASX-173 decreased proliferation in a dose-dependent manner, with tumor cells showing lower nanomolar IC50 values than normal human astrocytes, indicating greater sensitivity to blocking de novo ASN synthesis. Treatment with L-asparaginase alone resulted in little growth inhibition, further supporting reduced reliance on extracellular ASN. Importantly, combined treatment with ASX-173 and L-asparaginase caused significant synergistic suppression of viability across multiple glioma models. ASN deprivation or ASNS inhibition caused a compensatory increase in ASNS protein expression in multiple glioma models. Overall, these findings suggest that pediatric glioma cells mainly rely on ASNS-mediated synthesis rather than uptake of ASN, revealing a new metabolic vulnerability that can be targeted.
Conclusions:
PHGGs depend on ASNS-driven asparagine synthesis for survival. ASX-173–mediated ASNS inhibition markedly reduces tumor growth and heightens metabolic stress sensitivity. Combined ASX-173 and L-asparaginase treatment shows potent synergy, further suppressing tumor viability. These results identify asparagine metabolism as a key therapeutic vulnerability in H3-mutant pediatric gliomas and highlight ASX-173–based strategies as a promising avenue to improve outcomes in this lethal cancer.
Targeting Asparagine Metabolism as a Therapeutic Vulnerability in Pediatric H3-Mutant High-Grade Gliomas
Problem Statement/Question:
Diffuse hemispheric glioma (DHG, H3G34R-mutant) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive pediatric brain tumors with limited treatment options and extremely poor prognosis. Effective targeted metabolic therapies for these tumors remain largely undefined. It is unknown whether asparagine metabolism serves as a specific therapeutic vulnerability in pediatric high-grade gliomas.
Background/Project Intent (Aim Statement):
Pediatric high-grade gliomas (pHGGs) are among the most lethal childhood cancers, with no effective therapies and poor survival. Asparagine (ASN), essential for protein synthesis and metabolic balance, supports tumor growth through its biosynthetic enzyme asparagine synthetase (ASNS), often upregulated in aggressive cancers to promote resistance. While L-asparaginase has transformed leukemia therapy, ASN metabolism in gliomas remains unexplored. Our data show elevated ASNS expression in pediatric glioma tissues compared to normal brain, suggesting metabolic dependency. This study aims to determine whether pharmacologic (ASX-173) or genetic inhibition of ASNS can suppress tumor growth in H3G34R-mutant pHGG, identifying asparagine metabolism as a novel, targetable vulnerability for this fatal pediatric cancer.
Methods (include PDSA cycles):
Glioma cell lines were cultured in complete or asparagine-free media and treated with L-asparaginase, the ASNS inhibitor ASX-173, or combination therapies. Stable ASNS shRNA knockdown models were generated for mechanistic studies. Cell proliferation, viability, clonogenic survival, and IC₅₀ values were assessed, while qPCR, immunoblotting, and immunohistochemistry evaluated ASNS expression and pathway activity. RNA sequencing compared transcriptomic responses under control, ASN-deprived, and ASX-173–treated conditions, complemented by public single-cell RNA-seq analysis. Experimental parameters were optimized for dosing and combination effects. Future work will assess the in vivo efficacy of ASX-173 in a DHG tumor model to validate ASNS inhibition as a therapeutic strategy.
Results:
Single-cell RNA-sequencing and immunohistochemical analysis of pHGG patients and murine tumors showed increased ASNS expression in tumors compared to the normal brain, indicating higher in situ asparagine biosynthesis. In vitro, deprivation of ASN or NEAA alone had minimal impact on cell growth, suggesting limited reliance on extracellular ASN uptake. Conversely, pharmacologic inhibition of ASNS with ASX-173 decreased proliferation in a dose-dependent manner, with tumor cells showing lower nanomolar IC50 values than normal human astrocytes, indicating greater sensitivity to blocking de novo ASN synthesis. Treatment with L-asparaginase alone resulted in little growth inhibition, further supporting reduced reliance on extracellular ASN. Importantly, combined treatment with ASX-173 and L-asparaginase caused significant synergistic suppression of viability across multiple glioma models. ASN deprivation or ASNS inhibition caused a compensatory increase in ASNS protein expression in multiple glioma models. Overall, these findings suggest that pediatric glioma cells mainly rely on ASNS-mediated synthesis rather than uptake of ASN, revealing a new metabolic vulnerability that can be targeted.
Conclusions:
PHGGs depend on ASNS-driven asparagine synthesis for survival. ASX-173–mediated ASNS inhibition markedly reduces tumor growth and heightens metabolic stress sensitivity. Combined ASX-173 and L-asparaginase treatment shows potent synergy, further suppressing tumor viability. These results identify asparagine metabolism as a key therapeutic vulnerability in H3-mutant pediatric gliomas and highlight ASX-173–based strategies as a promising avenue to improve outcomes in this lethal cancer.
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Poster Board Number: 40