Biology subjects

Sundaram, R. K.

Publications and source records attributed to Sundaram, R. K..

4 recordsLinked to original sources

bioRxiv2026

Biomarker-Targeted O6-Guanine Alkylation Potentiates ATR Inhibitor Response in De Novo and Relapsed/Refractory AML

Acute myeloid leukemia (AML) remains limited by high relapse rates and a scarcity of biomarker-directed therapies, underscoring the need to identify actionable vulnerabilities and mechanisms of therapeutic resistance. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that directly reverses mutagenic O6-alkylguanine lesions, is epigenetically silenced in multiple cancers. Although MGMT silencing is predictive of temozolomide (TMZ) response in glioblastoma with significant survival advantage, prior clinical trials of TMZ in AML have shown only modest responses. Therefore, the prevalence and therapeutic relevance of MGMT silencing, as well as genetic factors that modify its therapeutic response in AML, remain to be comprehensively investigated. Here, we first profiled MGMT status across a diverse cohort of 23 de novo and 16 relapsed/refractory (R/R) primary AMLs using an integrated analysis of MGMT mRNA expression, promoter methylation, and protein expression. We found that approximately 25-30% of AMLs harbor MGMT silencing, in contrast to consistent MGMT expression in healthy CD34+ hematopoietic stem and progenitor cells. We further identified frequent loss of mismatch repair (MMR) proteins in these AML cohorts, a known resistance mechanism against TMZ in glioblastoma. Using CRISPR knockout screening and genetically engineered AML models, we found that MMR loss also drives both upfront and acquired TMZ resistance in MGMT-silenced AML. These findings identify MMR deficiency as an important and previously underappreciated genetic contributor to the modest responses observed in prior trials of TMZ in AML. To therapeutically exploit MGMT silencing while bypassing genetic determinants of resistance, including MMR deficiency, we evaluated clinically and preclinically characterized MGMT-dependent DNA-alkylating agents and identified a fluoroethylating analog of TMZ, N3-(2-fluoroethyl) imidazotetrazine (KL50), with pronounced and selective activity in MGMT-silenced AML. KL50 retained antileukemic activity irrespective of MMR status, significantly prolonging survival in humanized MISTRG6 mice harboring primary AML patient-derived xenografts. Mechanistically, KL50 induced DNA damage through the time-dependent formation of interstrand DNA crosslinks, bypassing MMR dependence and triggering a replication stress response dominated by ATR signaling. Pharmacologic ATR inhibition synergized with KL50, producing marked antileukemic activity and significantly extending survival across AML models without compromising hematologic safety. Together, these findings establish MGMT silencing as a prevalent and therapeutically actionable biomarker in AML, define MMR status as a key determinant of TMZ response in AML but not KL50 sensitivity, and provide a translational rationale for combining low-dose O6-fluoroethylating imidazotetrazines with ATR inhibitors to target both de novo and R/R AML across diverse genetic backgrounds.

cancer biology↗

bioRxiv2026

Leveraging Homologous Recombination Deficiency via the Repositioned Prodrug CB1954

AO_SCPLOWBSTRACTC_SCPLOWHomologous recombination deficiency (HRD) is an actionable vulnerability found in a substantial fraction of human cancers, yet current HRD-directed therapies are limited by toxicity, incomplete responses, and acquired resistance. Many DNA-damaging agents were developed before DNA repair biomarkers were available, suggesting that abandoned agents may harbor previously unrecognized genotype-selective activity. Here, through a focused screen of DNA-damaging agents in isogenic homologous recombination-proficient and -deficient models, we identify CB1954, a decades-old nitrobenzamide aziridine prodrug, as highly selective for BRCA2-deficient tumor cells. CB1954 forms DNA interstrand crosslinks independent of HR status, but selectively induces DNA-damage signaling, apoptosis, and loss of clonogenic survival in HR-deficient cells. Targeted DDR CRISPR screening and isogenic validation define a distinct repair dependency for the Fanconi anemia and homologous recombination pathways, with limited dependence on mismatch repair or nucleotide excision repair. Genetic and pharmacologic perturbation of NQO2, the bioactivating enzyme for CB1954, reveals a bifurcated mechanism in which NQO2-dependent activation selectively contributes to HRD cytotoxicity, while aziridine-dependent lesions likely account for residual activity in HR-proficient cells. CB1954 exhibits favorable preclinical pharmacokinetic properties and genotype-dependent antitumor activity in BRCA2-deficient xenografts. These findings reposition CB1954 as a historically overlooked HRD-selective agent and demonstrate that biomarker-guided profiling of DNA-damaging agents can uncover new opportunities for precision oncology.

cancer biology↗

bioRxiv2025

NAPRT expression and epigenetic regulation in pediatric rhabdomyosarcoma as a potential biomarker for NAMPT inhibition

PurposeNew treatments are needed to improve survival in children with rhabdomyosarcoma (RMS). NAD biosynthesis, regulated by the enzymes NAPRT and NAMPT, represents a metabolic vulnerability due to high NAD turnover in cancers. Although NAMPT inhibitors (NAMPTi) show preclinical promise, clinical translation has been limited by toxicity and the lack of predictive biomarkers. Here, we evaluated NAPRT expression in RMS and its potential as an actionable biomarker to guide NAMPTi therapy. Experimental DesignNAPRT promoter methylation, transcript levels, and protein expression were assessed in RMS cells, PDXs, and primary tumors (n=109) from the Childrens Oncology Group. In vitro sensitivity to NAMPTi was tested in molecularly diverse and isogenic RMS cell lines, examining the role of NAPRT expression in mediating cytotoxicity and the ability of nicotinic acid (NA) to rescue viability. In vivo efficacy was assessed using NAPRT-isogenic orthotopic xenograft models. ResultsNAPRT promoter hypermethylation was found in a subset of RMS models and patient samples. Immunohistochemistry showed loss of NAPRT protein in 30-40% of tumors, defined as <1% tumor cell staining. Methylation modestly correlated with protein expression. NAPRT-silenced cells were highly sensitive to NAMPTi, driven by NAD depletion and not reversible with NA. In vivo, NAMPTi induced significant tumor regression, which was not abrogated with NA administration in NAPRT-silenced models. ConclusionsNAPRT loss occurs in a subset of RMS, offering a potential strategy to expand the therapeutic window of NAMPTi. Further research is needed to understand NAPRT regulation and optimize biomarker assay strategies for use in future clinical trials.

cancer biology↗

bioRxiv2020

Targeted DNA Damage Repair CRISPR/Cas9 Knockout Screen Identifies Novel Classification of Poly-ADP Ribose Polymerase Inhibitors Based on Key Base Excision Repair Proteins

DNA repair deficiencies have become an increasingly promising target for novel therapeutics within the realm of clinical oncology. Recently, several inhibitors of Poly(ADP-ribose) Polymerases (PARPs) have received approval for the treatment of cancers primarily with deleterious mutations in the homologous recombination (HR) proteins, BRCA1 and BRCA2. Despite numerous clinical trials which have been completed or are currently ongoing, the mechanism of action by which PARP inhibitors selectively kill tumor cells is poorly understood. While many believe "trapping" of PARP proteins to DNA at sites of damage is the most important determinant driving cytotoxicity by these drugs, clinically effective inhibitors exist with a diverse range of PARP-trapping qualities. These findings suggest that characterization of inhibitors as strong versus weak trappers does not properly capture the intra-class characteristics of these drugs. Here, we use a novel, targeted DNA damage response (DDR) CRISPR/Cas9 screening library to reveal heterogenous genetic dependencies on the base excision repair (BER) pathway for PARP inhibitors, which is not correlated with PARP trapping ability or catalytic inhibition of PARP. These findings demonstrate that inhibition of PARylation and induction of PARP trapping are not the only factors contributing to distinct biological activity for different PARP inhibitors, and they provide insight into the optimal choice of PARP inhibitors for use in the setting of specific DDR defects. AUTHOR SUMMARYTargeted cancer therapies rely on our general understanding of which genetic mutations are involved in both sensitivity and resistance to such anticancer agents. In this study, we describe the use of functional genetic screening to evaluate the role of various DNA repair proteins in response to inhibitors of PARP, a quintessential example of targeted therapy. While PARP inhibitors are best known for their utility in cancers with homologous recombination defects, we show that some inhibitors within this class may have additional functionality in cancers with deficient base excision repair. These findings highlight not only the importance of PARP inhibitor selection in the appropriate context, but also the mechanistic differences that exist within this class of inhibitors. It is our hope that our findings will inspire future work evaluating the use of specific PARP inhibitor selection in designing clinical trials to further expand the use of PARP inhibitors beyond tumors with homologous recombination deficiencies.

cancer biology↗