Disruption of dNTP homeostasis by ribonucleotide reductase hyperactivation overcomes AML differentiation blockade.

Differentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop-mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.

EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.

Symmetry of bilateral hallux valgus deformity: A radiographic study.

Hallux valgus (HV) is a common anatomical deformity leading to pain and difficulty with footwear and mobility. Bilateral HV deformity is much more common than unilateral although it remains unclear whether the severity of deformity is equal between feet. The objective was to investigate the severity and symmetry of HV in patients with bilateral symptomatic deformity presenting for surgery. Weight-bearing radiographs of patients presenting with symptomatic bilateral HV were reviewed. The hallux valgus angle (HVA) and intermetatarsal angle (IMA) were measured and classified as mild, moderate, or severe. Left-to-right comparison was undertaken to assess whether the degree of deformity was similar for each foot. The relationship between age, HVA, and IMA was also assessed. Between July 2014 and June 2020, 322 ft (161 patients with bilateral deformity) underwent corrective HV surgery. Of those, 6.8%, 64.6%, and 28.4% were classified as mild, moderate, and severe, respectively on the left side, and on the right 6.2%, 67.7%, and 26.1% were classified as mild, moderate, and severe respectively. There was no statistically significant difference between feet for either IMA (p = 0.06) or HVA (p = 0.85). There was a moderate correlation (R = 0.41, p ≤ 0.001) between HVA and IMA. There was only a 'weak' or 'very weak' correlation between age and HVA or IMA. Patients presenting for surgery with symptomatic bilateral HV have symmetrical moderate radiographic deformity at the time they present for consideration of surgical intervention.

Eltrombopag inhibits the proliferation of Ewing sarcoma cells via iron chelation and impaired DNA replication.

BACKGROUND: The treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities. METHODS: We analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug. RESULTS: We found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known "off-target" effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress. CONCLUSIONS: Ewing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.

CDKs in Sarcoma: Mediators of Disease and Emerging Therapeutic Targets.

Sarcomas represent one of the most challenging tumor types to treat due to their diverse nature and our incomplete understanding of their underlying biology. Recent work suggests cyclin-dependent kinase (CDK) pathway activation is a powerful driver of sarcomagenesis. CDK proteins participate in numerous cellular processes required for normal cell function, but their dysregulation is a hallmark of many pathologies including cancer. The contributions and significance of aberrant CDK activity to sarcoma development, however, is only partly understood. Here, we describe what is known about CDK-related alterations in the most common subtypes of sarcoma and highlight areas that warrant further investigation. As disruptions in CDK pathways appear in most, if not all, subtypes of sarcoma, we discuss the history and value of pharmacologically targeting CDKs to combat these tumors. The goals of this review are to (1) assess the prevalence and importance of CDK pathway alterations in sarcomas, (2) highlight the gap in knowledge for certain CDKs in these tumors, and (3) provide insight into studies focused on CDK inhibition for sarcoma treatment. Overall, growing evidence demonstrates a crucial role for activated CDKs in sarcoma development and as important targets for sarcoma therapy.

SN-38 Conjugated Gold Nanoparticles Activated by Ewing Sarcoma Specific mRNAs Exhibit In Vitro and In Vivo Efficacy.

The limited delivery of chemotherapy agents to cancer cells and the nonspecific action of these agents are significant challenges in oncology. We have previously developed a customizable drug delivery and activation system in which a nucleic acid functionalized gold nanoparticle (Au-NP) delivers a drug that is selectively activated within a cancer cell by the presence of an mRNA unique to the cancer cell. The amount of drug released from sequestration to the Au-NP is determined by both the presence and the abundance of the cancer cell specific mRNA in a cell. We have now developed this technology for the potent, but difficult to deliver, topoisomerase I inhibitor SN-38. Herein, we demonstrate both the efficient delivery and selective release of SN-38 from gold nanoparticles in Ewing sarcoma cells with resulting efficacy in vitro and in vivo. These results provide further preclinical validation for this novel cancer therapy and may be extendable to other cancers that exhibit sensitivity to topoisomerase I inhibitors.

Causes and consequences of aneuploidy in cancer.

Genetic instability, which includes both numerical and structural chromosomal abnormalities, is a hallmark of cancer. Whereas the structural chromosome rearrangements have received substantial attention, the role of whole-chromosome aneuploidy in cancer is much less well-understood. Here we review recent progress in understanding the roles of whole-chromosome aneuploidy in cancer, including the mechanistic causes of aneuploidy, the cellular responses to chromosome gains or losses and how cells might adapt to tolerate these usually detrimental alterations. We also explore the role of aneuploidy in cellular transformation and discuss the possibility of developing aneuploidy-specific therapies.

Performance and predictors of recruitment success in National Heart, Lung, and Blood Institute's cardiovascular clinical trials.

Background/Aims Identifying predictors of recruitment success in clinical trials, particularly prior to study launch, could contribute to higher study completion rates and improved scientific return on investment. This article evaluates the performance of clinical trials funded by the National Heart, Lung, and Blood Institute that began recruitment before and after implementation of National Heart, Lung, and Blood Institute's 2009 Accrual Policy and identifies study-related factors that predict recruitment success. Methods A retrospective analysis of National Heart, Lung, and Blood Institute's cardiovascular clinical trials with initial funding from 1996 to 2012 was performed to assess recruitment success. Success was defined as ≥100% enrollment of the proposed sample size within the duration initially proposed by investigators. Trials were assigned to categories (pre-policy vs post-policy) based on whether the first patient was enrolled before or after the 2009 Accrual Policy implementation. Potential determinants of successful recruitment were evaluated using multivariable logistic regression. Results Of 167 trials analyzed, 26.3% met the definition of success. Twenty-four trials (14.4%) were terminated early and 15 (62.5%) for insufficient recruitment. Trials failed due to <100% enrollment (22.8%), longer duration (19.8%), or both (31.1%). Trials testing behavioral interventions, those conducted within a National Heart, Lung, and Blood Institute-funded network, and those with normal controls were predictive of success. The proportion of successful clinical trials increased from 23% in the pre-policy era to 30% post-policy, although the difference was not statistically significant ( p = 0.29). Conclusion Enrollment success rates for National Heart, Lung, and Blood Institute's clinical trials are concerning. The 2009 National Heart, Lung, and Blood Institute Accrual Policy did not significantly improve trial success. Clinical trials testing behavioral interventions, those conducted within networks, and those with normal controls were predictive of recruitment success. Components of networks may provide model practices to help other trials attain success, including close attention to oversight activities such as recruitment plans, real-time enrollment monitoring, corrective action plans to address shortfalls, and close sponsor-investigator collaborations.

Development of Secondary Acute Myeloid Leukemia in a Pediatric Patient Concurrently Receiving Primary Therapy for Ewing Sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue sarcoma that requires intensive therapy, which can cause secondary malignancies. We present a rare case of early, treatment-related AML in a pediatric patient concurrently receiving primary therapy for Ewing sarcoma. Despite AML-directed therapy, our patient died secondary to complications of hyperleukocytosis. Cytogenetic and mutation profiling of the leukemia cells revealed the DNA-topoisomerase-II-inhibitor-associated t(9;11)(p22;q23) translocation and clonal KRAS and BRAF mutations. This report highlights the importance of monitoring for treatment-related effects in cancer therapy, as well as the need for novel, less toxic approaches in Ewing sarcoma therapy.

Data monitoring committees: Promoting best practices to address emerging challenges.

BACKGROUND AND PURPOSE: Data monitoring committees are responsible for safeguarding the interests of study participants and assuring the integrity and credibility of clinical trials. The independence of data monitoring committees from sponsors and investigators is essential in achieving this mission. Creative approaches are needed to address ongoing and emerging challenges that potentially threaten data monitoring committees' independence and effectiveness. METHODS: An expert panel of representatives from academia, industry and government sponsors, and regulatory agencies discussed these challenges and proposed best practices and operating principles for effective functioning of contemporary data monitoring committees. RESULTS AND CONCLUSIONS: Prospective data monitoring committee members need better training. Options could include didactic instruction as well as apprenticeships to provide real-world experience. Data monitoring committee members should be protected against legal liability arising from their service. While avoiding breaches in confidentiality of interim data remains a high priority, data monitoring committees should have access to unblinded efficacy and safety data throughout the trial to enable informed judgments about risks and benefits. Because overly rigid procedures can compromise their independence, data monitoring committees should have the flexibility necessary to best fulfill their responsibilities. Data monitoring committee charters should articulate principles that guide the data monitoring committee process rather than list a rigid set of requirements. Data monitoring committees should develop their recommendations by consensus rather than through voting processes. The format for the meetings of the data monitoring committee should maintain the committee's independence and clearly establish the leadership of the data monitoring committee chair. The independent statistical group at the Statistical Data Analysis Center should have sufficient depth of knowledge about the study at hand and experience with trials in general to ensure that the data monitoring committee has access to timely, reliable, and readily interpretable insights about emerging evidence in the clinical trial. Contracts engaging data monitoring committee members for industry-sponsored trials should have language customized to the unique responsibilities of data monitoring committee members rather than use language appropriate to consultants for product development. Regulatory scientists would benefit from experiencing data monitoring committee service that does not conflict with their regulatory responsibilities.

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.

Effect of the use and timing of bone marrow mononuclear cell delivery on left ventricular function after acute myocardial infarction: the TIME randomized trial.

CONTEXT: While the delivery of cell therapy after ST-segment elevation myocardial infarction (STEMI) has been evaluated in previous clinical trials, the influence of the timing of cell delivery on the effect on left ventricular function has not been analyzed. OBJECTIVES: To determine the effect of intracoronary autologous bone marrow mononuclear cell (BMC) delivery after STEMI on recovery of global and regional left ventricular function and whether timing of BMC delivery (3 days vs 7 days after reperfusion) influences this effect. DESIGN, SETTING, AND PATIENTS: A randomized, 2 × 2 factorial, double-blind, placebo-controlled trial, Timing In Myocardial infarction Evaluation (TIME) enrolled 120 patients with left ventricular dysfunction (left ventricular ejection fraction [LVEF] ≤ 45%) after successful primary percutaneous coronary intervention (PCI) of anterior STEMI between July 17, 2008, and November 15, 2011, as part of the Cardiovascular Cell Therapy Research Network sponsored by the National Heart, Lung, and Blood Institute. INTERVENTIONS: Intracoronary infusion of 150 × 106 BMCs or placebo (randomized 2:1) within 12 hours of aspiration and cell processing administered at day 3 or day 7 (randomized 1:1) after treatment with PCI. MAIN OUTCOME MEASURES: The primary end points were change in global (LVEF) and regional (wall motion) left ventricular function in infarct and border zones at 6 months measured by cardiac magnetic resonance imaging and change in left ventricular function as affected by timing of treatment on day 3 vs day 7. The secondary end points included major adverse cardiovascular events as well as changes in left ventricular volumes and infarct size. RESULTS: The mean (SD) patient age was 56.9 (10.9) years and 87.5% of participants were male. At 6 months, there was no significant increase in LVEF for the BMC group (45.2% [95% CI, 42.8% to 47.6%] to 48.3% [95% CI, 45.3% to 51.3%) vs the placebo group (44.5% [95% CI, 41.0% to 48.0%] to 47.8% [95% CI, 43.4% to 52.2%]) (P = .96). There was no significant treatment effect on regional left ventricular function observed in either infarct or border zones. There were no significant differences in change in global left ventricular function for patients treated at day 3 (−0.9% [95% CI, −6.6% to 4.9%], P = .76) or day 7 (1.1% [95% CI, −4.7% to 6.9%], P = .70). The timing of treatment had no significant effect on regional left ventricular function recovery. Major adverse events were rare among all treatment groups. CONCLUSION: Among patients with STEMI treated with primary PCI, the administration of intracoronary BMCs at either 3 days or 7 days after the event had no significant effect on recovery of global or regional left ventricular function compared with placebo. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00684021.

Effect of transendocardial delivery of autologous bone marrow mononuclear cells on functional capacity, left ventricular function, and perfusion in chronic heart failure: the FOCUS-CCTRN trial.

CONTEXT: Previous studies using autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy. OBJECTIVE: To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular end-systolic volume (LVESV), or enhances maximal oxygen consumption in patients with coronary artery disease or LV dysfunction, and limiting heart failure or angina. DESIGN, SETTING, AND PATIENTS: A phase 2 randomized double-blind, placebo-controlled trial of symptomatic patients (New York Heart Association classification II-III or Canadian Cardiovascular Society classification II-IV) with a left ventricular ejection fraction of 45% or less, a perfusion defect by single-photon emission tomography (SPECT), and coronary artery disease not amenable to revascularization who were receiving maximal medical therapy at 5 National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) sites between April 29, 2009, and April 18, 2011. INTERVENTION: Bone marrow aspiration (isolation of BMCs using a standardized automated system performed locally) and transendocardial injection of 100 million BMCs or placebo (ratio of 2 for BMC group to 1 for placebo group). MAIN OUTCOME MEASURES: Co-primary end points assessed at 6 months: changes in LVESV assessed by echocardiography, maximal oxygen consumption, and reversibility on SPECT. Phenotypic and functional analyses of the cell product were performed by the CCTRN biorepository core laboratory. RESULTS: Of 153 patients who provided consent, a total of 92 (82 men; average age: 63 years) were randomized (n = 61 in BMC group and n = 31 in placebo group). Changes in LVESV index (-0.9 mL/m(2) [95% CI, -6.1 to 4.3]; P = .73), maximal oxygen consumption (1.0 [95% CI, -0.42 to 2.34]; P = .17), and reversible defect (-1.2 [95% CI, -12.50 to 10.12]; P = .84) were not statistically significant. There were no differences found in any of the secondary outcomes, including percent myocardial defect, total defect size, fixed defect size, regional wall motion, and clinical improvement. CONCLUSION: Among patients with chronic ischemic heart failure, transendocardial injection of autologous BMCs compared with placebo did not improve LVESV, maximal oxygen consumption, or reversibility on SPECT. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00824005.

Inhibitor of DNA binding 2 (ID2) regulates the expression of developmental genes and tumorigenesis in ewing sarcoma.

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.

Augmenting chemotherapy with low-dose decitabine through an immune-independent mechanism.

The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.