The effect of DNA-PKi and etoposide combinations on non-malignant epithelial cell lines

The DNA-PKi enhancement of EWS cells sensitivity to etoposide DNA-PK inhibition is a class effect

TOP2 poisons synergize with DNA-PK inhibition in EWS

The synergistic combination of the DNA-PK inhibitor M3814 and etoposide extends to multiple Ewing sarcoma cell lines

Assessment of cell confluency confirms DNA-PK inhibition selectively synergizes with etoposide

A screen of multiple drug combinations demonstrates the selectivity of the synergistic activity of the DNA-PK clinical inhibitor M3814

In vivo compound preparation and administration information

Etoposide combined with DNA-PK inhibition induces apoptosis in TP53 wild-type and mutant Ewing sarcoma cell lines

Abstract Although improvements in local therapy have increased the 5-year survival rate for localized Ewing sarcoma (EWS) from less than 20% to 70%, little progress has been made in the treatment of metastatic disease, which has a 5-year survival rate of less than 30%. A major challenge in the field is a lack of preclinical models that can recapitulate spontaneous metastatic disease and can be used to better understand the disease and identify new vulnerabilities. In this study, we present and characterize a murine model of spontaneous distant EWS metastasis derived from human patient-derived xenografts (PDX) in two different mouse backgrounds that mimics the clinical progression of the human disease. A panel of seven molecularly diverse patient-derived xenograft (PDX) models (SJ18, SJ17, S049, NCH1, NCH4, PDMR-098, PDMR-077) were injected orthotopically into the gastrocnemius muscle in the left hind leg of athymic nude and NOD SCID gamma (NSG) mice. Once primary tumors reached 1500 mm3, hind limb amputation survival surgery was performed, and we observed animals for the development of distant spontaneous metastases. EWS PDXs formed spontaneous macrometastases in multiple sites including lymph nodes, lung, liver, and kidney in both NSG and nude mice. Each PDX model exhibited a distinct pattern of macrometastasis formation, based on site, metastasis frequency, and mouse strain. Immunohistochemical analysis for CD99 positivity revealed the presence of micrometastases in some locations where no macrometases were evident. The highest frequency of distant metastases (macro- plus micro-) was seen in PDMR-098 and SJ18 models in NSG mice (75% and 73.3% respectively); SJ18 most frequently metastasized to lung, whereas PDMR-098 most frequently metastasized to liver and mesenteric lymph nodes. Comparing results in NSG versus nude mice, we observed variations in metastasis frequency and site preference, with NSG mice demonstrating a higher rate of metastasis overall. Here we describe a preclinical model of spontaneous distant EWS metastasis that recapitulates the characteristics of human disease. The site and frequency of metastases vary based on the specific PDX model as well as the mouse background, highlighting that modeling metastasis formation is a multifactorial process driven by complex interactions between the tumor and host. Metabolomic profiling conducted to compare primary and macrometastatic tumors between models and metastatic sites revealed differences in multiple metabolic pathways. Future studies will focus on identifying potentially targetable metabolic vulnerabilities that could be used in the development of treatments for metastatic EWS. Citation Format: Ali Mokhtar Mahmoud, Unsun Lee, Arnulfo Mendoza, Christine M. Heske, . Establishment and analysis of spontaneous metastatic PDX models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6178.

Defining parameters for the evaluation of effects of DNA-PK inhibitors plus TOP2 poisons in preclinical models of EWS

Concomitant AZD-7648 and etoposide treatments impair tumor growth

Evaluation of the DNA-PK inhibitors plus TOP2 poisons in preclinical models of EWS

Abstract RMS is a common pediatric soft tissue sarcoma for which new therapies are critically needed. We previously demonstrated that RMS is highly sensitive to inhibitors of NAMPT, which catalyzes the rate-limiting step of the NAD+ salvage pathway and is the only pharmacologically targetable NAD+ production enzyme. Treatment with the NAMPT inhibitor OT-82 results in complete tumor regressions in vivo, however, upon intermittent treatment, acquired resistance develops in some models. As acquired drug resistance is a known impediment to the clinical efficacy of targeted agents, we sought to elucidate potential mechanisms of OT-82 resistance in RMS. Mice with orthotopic fusion-positive (FP) and fusion-negative (FN) RMS xenograft tumors were treated with OT-82 for 8 weeks on the clinical dosing schedule. After stopping treatment, mice were observed for recurrence and retreated when tumors regrew to >900 mm3. Tumors that progressed on treatment were harvested and converted to cell lines. Two resistant cell lines (1 FP - Rh30-mRes and 1 FN - RD-mRes) were selected for further study. Incucyte live cell analysis confirmed retention of OT-82 resistance in vitro with resistant cells maintaining proliferation at doses of OT-82 up to 30X above the IC50 of parental cells. Resistant cells exposed to OT-82 maintained ATP levels consistent with that of untreated controls. After 24h of OT-82 treatment, NAD+ loss was observed in both parental and resistant cells, however resistant cells recovered NAD+ levels within 48-96h. Effects on glucose metabolism, measured using extracellular flux and metabolomic analyses demonstrated that in the presence of OT-82, only resistant cells maintained glycolytic function. Specifically, metabolites downstream of the NAD+-dependent enzyme glyceraldehyde-3-phosphase dehydrogenase were reduced in parental cells but maintained in resistant cells. Analysis of protein expression of NAD+ synthesis enzymes NAMPT, NAPRT, and QPRT revealed that Rh30-mRes expresses more QPRT, however, genetic silencing of QPRT did not reverse resistance, suggesting upregulation of compensatory NAD production enzymes is not a primary mechanism of resistance. Whole exome sequencing revealed that each resistant cell line has a distinct, previously unreported mutation in NAMPT. Protein modeling suggests each mutation affects the drug binding pocket of NAMPT, with the S17F variant in Rh30-mRes resulting in a collapse of the pocket and the S241C variant in RD-mRes reducing binding affinity of OT-82. These findings are consistent with functional studies demonstrating that Rh30-mRes is resistant to multiple other NAMPT inhibitors whereas RD-mRes is resistant only to OT-82. Together, these data suggest that acquired resistance to NAMPT inhibitors in RMS models involves the development of mutations in the target protein affecting drug binding and affinity. Citation Format: Ariana Elizabeth Nelson, Abantika Chakraborty, David Bell, Victor J. Collins, Ali Mokhtar Mahmoud, Ying Wu, SOPHIA VARRIANO, Arnulfo Mendoza, Sameer Issaq, Parthav Jailwala, Jack F. Shern, Ernesto Suárez, Joseph Ivanic, Christine M. Heske. Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor-resistant rhabdomyosarcoma (RMS) models exhibit alterations in metabolic and genomic profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1798.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and represents a tumor of high unmet clinical need as standard systemic therapy consists of cytotoxic agents only. We have previously demonstrated that RMS models are highly sensitive to inhibition of nicotinamide phosphoribosyltransferase (NAMPT). NAMPT catalyzes the rate-limiting step of the NAD+ salvage pathway and is currently the only pharmacologically targetable NAD+ production enzyme. Treatment with the clinical NAMPT inhibitor OT-82 results in complete tumor regressions in vivo, however, upon intermittent treatment, acquired resistance develops in some in vivo models. As acquired drug resistance is a known impediment to the clinical efficacy of targeted agents, we sought to elucidate potential mechanisms of acquired resistance to OT-82 in RMS. Mice with orthotopic fusion-positive (FP) and fusion-negative (FN) RMS xenograft tumors were treated with OT-82 for 8 weeks on the clinical dosing schedule. Upon cessation of treatment, mice were observed for recurrence and retreated when tumors regrew to a volume of >900 mm3. Tumors that progressed on treatment were converted to cell lines. Two resistant cell lines (1 FP and 1 FN) were selected for further study. Incucyte live cell analysis confirmed retention of OT-82 resistance in vitro with resistant cell lines maintaining proliferation at doses of OT-82 up to 30 times above the IC50 of parental lines. Resistance to multiple other NAMPT inhibitors (daporinad and KPT-9274) was observed in one of the resistant cell lines. Resistant cells exposed to OT-82 maintained ATP levels consistent with that of untreated controls. After 24 hours of OT-82 treatment, NAD+ loss was observed in both parental and resistant cell lines, however resistant cell lines were able to recover NAD+ levels within 48-96 hours. Downstream effects on glucose metabolism, measured using extracellular flux and metabolomic analyses, demonstrated that in the presence of OT-82 resistant cells maintained their glycolytic function, whereas parental cells did not. Specifically, the products downstream of the NAD+-dependent enzyme glyceraldehyde-3-phosphase dehydrogenase were reduced in parental cell lines but maintained in resistant cell lines. Analysis of gene and protein expression of NAD+ synthesis enzymes NAMPT, NAPRT, and QPRT demonstrated changes in NAPRT and QPRT expression at the transcriptional level and at the protein level in resistant cell lines. Whole exome sequencing revealed that each resistant cell line has a distinct, previously unreported mutation in NAMPT. Together, these data suggest that acquired resistance to NAMPT inhibitors in RMS models involves cellular alterations in the biochemical, metabolomic, and genomic profiles of cells. Ariana E. Nelson, Victor J. Collins, Ali Mokhtar Mahmoud, Ying Wu, Sophia Varriano, Arnulfo Mendoza, Sameer Issaq, Parthav Jailwala, Jack F. Shern, Christine M. Heske. NAMPT inhibitor-resistant rhabdomyosarcoma models exhibit alterations in metabolic and genomic profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3945.

Cancer cell metabolism is altered to meet increased metabolic demands of proliferating cancer cells. Reprogrammed metabolic pathways represent potential cell type and context specific therapeutic opportunities. Targeting NAD+ production via inhibition of the NAD+ salvage pathway presents one such potential opportunity, as NAD+ is essential for energy metabolism and other downstream processes. Presently, nicotinamide phosphoribosyltransferase (NAMPT) is the only clinically targetable enzyme in this pathway. We conducted a high-throughput cancer cell line screen and identified that neuroblastoma (NB) cells are significantly more sensitive to NAMPT inhibitors (NAMPTis) than most other solid tumors, with IC50 values >20-fold less than the cell line panel average. NB is the most common extracranial solid tumor in children and a tumor of high unmet need, accounting for 15% of pediatric cancer deaths. We used two clinical NAMPTis under early phase study (OT-82 and KPT-9274) to validate our drug screen results in 10 molecularly diverse NB cell lines including 2 NB PDX-derived cell lines. We investigated the mechanistic effects of NAMPT inhibition on NAD+-dependent pathways and analyzed the in vivo effects of OT-82 in 3 orthotopic NB models. Treatment of NB cell lines with NAMPTis resulted in non-apoptotic cell death within 48 hours and failure of cells to proliferate even following replacement with drug-free media. NAMPTis reduced intracellular NAD+ levels and co-treatment with NMN, the product of NAMPT, fully rescued cell viability, verifying NAD+-dependence and on-target activity of each NAMPTi. Moreover, reductions in ATP of 50% and >90% were observed after 24 hours and 72 hours of treatment, respectively. Examination of drug effects on glucose metabolism using extracellular flux and metabolomics analyses demonstrated cell line-specific effects, including reduction in oxidative phosphorylation and/or glycolysis with depletion of metabolites produced via NAD+-consuming enzymes. Investigation of the effects of NAMPTis on other key NAD+ consuming enzymes including sirtuin 1 (SIRT1) and poly (ADP-ribose) polymerase (PARP), demonstrated significant loss of activity of both enzymes in a time-dependent manner. As loss of PARP and SIRT1 activity can compromise DNA repair, comet assays were used to assess the extent of DNA damage and revealed an increase in DNA damage upon NAMPTi treatment in all models. In vivo studies testing OT-82 in orthotopic xenografts demonstrated significant tumor shrinkage. Across models, 23/26 mouse tumors had average volume reductions of 67% (range 10%-99%). Tissue studies from these experiments are ongoing and will be reported. Together, these data demonstrate that in NB, multiple critical pathways are impacted by the loss of NAD+ mediated by NAMPT inhibition and suggest NAMPTis may have translational potential as a novel agent against NB. Amy Yu, Sophia Varriano, Amy James, Kristine Isanogle, Nimit Patel, Caleb Kim, Unsun Lee, Victor J. Collins, Ariana E. Nelson, Ming Sun, Ye Yang, Bhushan L. Thakur, Arnulfo Mendoza, Sameer H. Issaq, John F. Shern, Mirit I. Aladjem, Daniel R. Crooks, Joseph D. Kalen, Simone Difilippantonio, Craig J. Thomas, Rosa Nguyen, Carol J. Thiele, Christine M. Heske. NAMPT inhibition impacts energy metabolism, induces DNA damage, and mediates tumor regression in preclinical neuroblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6921.

This abstract has been removed: please see Elsevier policy on article withdrawal (https://www.elsevier.com/about/policies-and-standards/article-withdrawal). This article has been removed at the request of the author. This abstract has been removed because it was not presented at the 2025 BMT Tandem Meeting.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Supplementary Materials and Methods

5' UTR structures of BONCAT identified proteins

Supplementary Figure 3 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis