Targeted Inhibition of Snail Activity in Breast Cancer Cells by Using a Co(III) -Ebox Conjugate.

The transition from a non-invasive to an invasive phenotype is an essential step in tumor metastasis. The Snail family of transcription factors (TFs) is known to play a significant role in this transition. These TFs are zinc fingers that bind to the CAGGTG Ebox consensus sequence. Co(III) -Ebox is a cobalt(III) complex attached to an Ebox oligonucleotide that confers specificity towards Snail TFs. Co(III) -Ebox has been shown to inhibit Snail-mediated embryonic neural crest development in Xenopus laevis, but its efficacy in inhibiting Snail-induced cancer cell invasiveness has not been explored. Here, we describe the efficacy of Co(III) -Ebox in inhibiting the invasive aspects of heregulin-ß1(HRG)-treated breast cancer cells. Co(III) -Ebox was found to inhibit the capacity of Snail to repress target genes after HRG induction. Snail inhibition by Co(III) -Ebox reduced the invasive propensity of cells in 2D and 3D, thereby demonstrating promise in inhibiting metastasis.

Feasibility of personalized circulating tumor DNA detection in stage II and III melanoma.

The objective of this study was to evaluate the feasibility of developing personalized, tumor-informed assays for patients with high-risk resectable melanoma and examine circulating tumor DNA (ctDNA) levels in relation to clinical status. Pilot prospective study of clinical stage IIB/C and resectable stage III melanoma patients. Tumor tissue was used to design bespoke somatic assays for interrogating ctDNA in patients' plasma using a multiplex PCR (mPCR) next-generation sequencing (NGS)-based approach. Plasma samples for ctDNA analysis were collected pre-/post-surgery and during surveillance. Out of 28 patients (mean 65 years, 50% male), 13 (46%) had detectable ctDNA prior to definitive surgery and 96% (27/28) tested ctDNA-negative within 4 weeks post-surgery. Pre-surgical detection of ctDNA was significantly associated with the later-stage ( P  = 0.02) and clinically evident stage III disease ( P  = 0.007). Twenty patients continue in surveillance with serial ctDNA testing every 3-6 months. With a median follow-up of 443 days, six out of 20 (30%) patients developed detectable ctDNA levels during surveillance. All six of these patients recurred with a mean time to recurrence of 280 days. Detection of ctDNA in surveillance preceded the diagnosis of clinical recurrence in three patients, was detected concurrent with clinical recurrence in two patients and followed clinical recurrence in one patient. One additional patient developed brain metastases without detection of ctDNA during surveillance but had positive pre-surgical ctDNA. Our results demonstrate the feasibility of obtaining a personalized, tumor-informed mPCR NGS-based ctDNA assay for patients with melanoma, particularly in resectable stage III disease.

Feasibility of Longitudinal ctDNA Assessment in Patients with Uterine and Extra-Uterine Leiomyosarcoma.

Background: Leiomyosarcomas (LMS) are aggressive malignancies with a propensity for early relapse. Current surveillance modalities include physical exam and imaging; however, radiological response to therapy may only manifest after 4-6 cycles of treatment. Herein, we evaluated the feasibility of longitudinal circulating tumor DNA (ctDNA) assessment in LMS patients to identify disease progression. Methods: We performed a retrospective review of patients with LMS who underwent treatment at Stanford Cancer Center between September 2019 and May 2022. ctDNA detection was performed using a personalized, tumor-informed ctDNA assay. Genomic analysis was conducted to characterize tumor mutation burden (TMB) and known driver mutations. Results: A total of 148 plasma samples were obtained from 34 patients with uterine (N = 21) and extrauterine (N = 13) LMS (median follow-up: 67.2 (19-346.3) weeks] and analyzed for ctDNA presence. Nineteen patients had metastatic disease. The most frequently mutated driver genes across sub-cohorts were TP53, RB1, and PTEN. Patients were stratified into four sub-cohorts (A-D) based on ctDNA kinetics. ctDNA levels tracked longitudinally with progression of disease and response to therapy. Conclusion: Our results indicate that while undetectable ctDNA may suggest a lower likelihood of relapse, ctDNA positivity may indicate progressive disease, enabling closer monitoring of patients for early clinical intervention.

Malic Enzyme 1 Absence in Synovial Sarcoma Shifts Antioxidant System Dependence and Increases Sensitivity to Ferroptosis Induction with ACXT-3102.

PURPOSE: To investigate the metabolism of synovial sarcoma (SS) and elucidate the effect of malic enzyme 1 absence on SS redox homeostasis. EXPERIMENTAL DESIGN: ME1 expression was measured in SS clinical samples, SS cell lines, and tumors from an SS mouse model. The effect of ME1 absence on glucose metabolism was evaluated utilizing Seahorse assays, metabolomics, and C13 tracings. The impact of ME1 absence on SS redox homeostasis was evaluated by metabolomics, cell death assays with inhibitors of antioxidant systems, and measurements of intracellular reactive oxygen species (ROS). The susceptibility of ME1-null SS to ferroptosis induction was interrogated in vitro and in vivo. RESULTS: ME1 absence in SS was confirmed in clinical samples, SS cell lines, and an SS tumor model. Investigation of SS glucose metabolism revealed that ME1-null cells exhibit higher rates of glycolysis and higher flux of glucose into the pentose phosphate pathway (PPP), which is necessary to produce NADPH. Evaluation of cellular redox homeostasis demonstrated that ME1 absence shifts dependence from the glutathione system to the thioredoxin system. Concomitantly, ME1 absence drives the accumulation of ROS and labile iron. ROS and iron accumulation enhances the susceptibility of ME1-null cells to ferroptosis induction with inhibitors of xCT (erastin and ACXT-3102). In vivo xenograft models of ME1-null SS demonstrate significantly increased tumor response to ACXT-3102 compared with ME1-expressing controls. CONCLUSIONS: These findings demonstrate the translational potential of targeting redox homeostasis in ME1-null cancers and establish the preclinical rationale for a phase I trial of ACXT-3102 in SS patients. See related commentary by Subbiah and Gan, p. 3408.

Targeting one-carbon metabolism requires mTOR inhibition: a new therapeutic approach in osteosarcoma.

The rate-limiting enzyme of serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH), contributes to rapid growth and proliferation when it is overexpressed in cancer. We recently described the metabolic adaptations that occur upon PHGDH inhibition in osteosarcoma. PHGDH inhibition causes metabolite accumulation that activates the mechanistic target of rapamycin (mTOR) signaling, sensitizing osteosarcoma to non-rapalog mTOR inhibition.

Pathogenesis and Current Treatment of Osteosarcoma: Perspectives for Future Therapies.

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. The standard-of-care curative treatment for osteosarcoma utilizes doxorubicin, cisplatin, and high-dose methotrexate, a standard that has not changed in more than 40 years. The development of patient-specific therapies requires an in-depth understanding of the unique genetics and biology of the tumor. Here, we discuss the role of normal bone biology in osteosarcomagenesis, highlighting the factors that drive normal osteoblast production, as well as abnormal osteosarcoma development. We then describe the pathology and current standard of care of osteosarcoma. Given the complex heterogeneity of osteosarcoma tumors, we explore the development of novel therapeutics for osteosarcoma that encompass a series of molecular targets. This analysis of pathogenic mechanisms will shed light on promising avenues for future therapeutic research in osteosarcoma.

Interim Analysis of the Phase II Study: Noninferiority Study of Doxorubicin with Upfront Dexrazoxane plus Olaratumab for Advanced or Metastatic Soft-Tissue Sarcoma.

PURPOSE: To report the interim analysis of the phase II single-arm noninferiority trial, testing the upfront use of dexrazoxane with doxorubicin on progression-free survival (PFS) and cardiac function in soft-tissue sarcoma (STS). PATIENTS AND METHODS: Patients with metastatic or unresectable STS who were candidates for first-line treatment with doxorubicin were deemed eligible. An interim analysis was initiated after 33 of 65 patients were enrolled. Using the historical control of 4.6 months PFS for doxorubicin in the front-line setting, we tested whether the addition of dexrazoxane affected the efficacy of doxorubicin in STS. The study was powered so that a decrease of PFS to 3.7 months would be considered noninferior. Secondary aims included cardiac-related mortality, incidence of heart failure/cardiomyopathy, and expansion of cardiac monitoring parameters including three-dimensional echocardiography. Patients were allowed to continue on doxorubicin beyond 600 mg/m2 if they were deriving benefit and were not demonstrating evidence of symptomatic cardiac dysfunction. RESULTS: At interim analysis, upfront use of dexrazoxane with doxorubicin demonstrated a PFS of 8.4 months (95% confidence interval: 5.1-11.2 months). Only 3 patients were removed from study for cardiotoxicity, all on > 600 mg/m2 doxorubicin. No patients required cardiac hospitalization or had new, persistent cardiac dysfunction with left ventricular ejection fraction remaining below 50%. The median administered doxorubicin dose was 450 mg/m2 (interquartile range, 300-750 mg/m2). CONCLUSIONS: At interim analysis, dexrazoxane did not reduce PFS in patients with STS treated with doxorubicin. Involvement of cardio-oncologists is beneficial for the monitoring and safe use of high-dose anthracyclines in STS.See related commentary by Benjamin and Minotti, p. 3809.

Metabolic compensation activates pro-survival mTORC1 signaling upon 3-phosphoglycerate dehydrogenase inhibition in osteosarcoma.

Osteosarcoma is the most common pediatric and adult primary malignant bone cancer. Curative regimens target the folate pathway, downstream of serine metabolism, with high-dose methotrexate. Here, the rate-limiting enzyme in the biosynthesis of serine from glucose, 3-phosphoglycerate dehydrogenase (PHGDH), is examined, and an inverse correlation between PHGDH expression and relapse-free and overall survival in osteosarcoma patients is found. PHGDH inhibition in osteosarcoma cell lines attenuated cellular proliferation without causing cell death, prompting a robust metabolic analysis to characterize pro-survival compensation. Using metabolomic and lipidomic profiling, cellular response to PHGDH inhibition is identified as accumulation of unsaturated lipids, branched chain amino acids, and methionine cycle intermediates, leading to activation of pro-survival mammalian target of rapamycin complex 1 (mTORC1) signaling. Increased mTORC1 activation sensitizes cells to mTORC1 pathway inhibition, resulting in significant, synergistic cell death in vitro and in vivo. Identifying a therapeutic combination for PHGDH-high cancers offers preclinical justification for a dual metabolism-based combination therapy for osteosarcoma.

PHGDH as a mechanism for resistance in metabolically-driven cancers.

At the forefront of cancer research is the rapidly evolving understanding of metabolic reprogramming within cancer cells. The expeditious adaptation to metabolic inhibition allows cells to evolve and acquire resistance to targeted treatments, which makes therapeutic exploitation complex but achievable. 3-phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme of de novo serine biosynthesis and is highly expressed in a variety of cancers, including breast cancer, melanoma, and Ewing's sarcoma. This review will investigate the role of PHGDH in normal biological processes, leading to the role of PHGDH in the progression of cancer. With an understanding of the molecular mechanisms by which PHGDH expression advances cancer growth, we will highlight the known mechanisms of resistance to cancer therapeutics facilitated by PHGDH biology and identify avenues for combatting PHGDH-driven resistance with inhibitors of PHGDH to allow for the development of effective metabolic therapies.

Diagnosis and management of malignant peripheral nerve sheath tumors: Current practice and future perspectives.

One of the most common malignancies affecting adults with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the malignant peripheral nerve sheath tumor (MPNST), a highly aggressive sarcoma that typically develops from benign plexiform neurofibromas. Approximately 8-13% of individuals with NF1 will develop MPNST during young adulthood. There are few therapeutic options, and the vast majority of people with these cancers will die within 5 years of diagnosis. Despite efforts to understand the pathogenesis of these aggressive tumors, the overall prognosis remains dismal. This manuscript will review the current understanding of the cellular and molecular progression of MPNST, diagnostic workup of patients with these tumors, current treatment paradigms, and investigational treatment options. Additionally, we highlight novel areas of preclinical research, which may lead to future clinical trials. In summary, MPNST remains a diagnostic and therapeutic challenge, and future work is needed to develop novel and rational combinational therapy for these tumors.

Systems level profiling of arginine starvation reveals MYC and ERK adaptive metabolic reprogramming.

Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many carcinomas and in the majority of sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing ASS1. As arginine-based multiagent therapies are being developed, further characterization of the changes induced by arginine starvation is needed. In order to develop a systems-level understanding of these changes, activity-based proteomic profiling (ABPP) and phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-PEG20-sensitive and resistant sarcoma cells. When integrated with metabolomic profiling, this multi-omic analysis reveals that cellular response to arginine starvation is mediated by adaptive ERK signaling and activation of the Myc-Max transcriptional network. Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by enhancing glutamine and pyruvate anaplerosis and altering lipid metabolism to recycle citrate for oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, these multi-omic approaches could provide valuable tools for evaluating response to metabolically targeted therapies.

Cisplatin exposure causes c-Myc-dependent resistance to CDK4/6 inhibition in HPV-negative head and neck squamous cell carcinoma.

The loss of p16 is a signature event in Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) that leads to increased Cyclin Dependent Kinase 4/6 (CDK) signaling. Palbociclib, a CDK4/6 inhibitor, is active for the treatment of a subset of HNSCC. In this study, we analyzed patient response data from a phase I clinical trial of palbociclib in HNSCC and observed an association between prior cisplatin exposure and CDK inhibitor resistance. We studied the effects of palbociclib on cisplatin-sensitive and -resistant HNSCC cell lines. We found that while palbociclib is highly effective against chemo-naive HNSCC cell lines and tumor xenografts, prior cisplatin exposure induces intrinsic resistance to palbociclib in vivo, a relationship that was not observed in vitro. Mechanistically, in the course of provoking a DNA damage-resistance phenotype, cisplatin exposure upregulates both c-Myc and cyclin E, and combination treatment with palbociclib and the c-Myc bromodomain inhibitor JQ1 exerts a synergistic anti-growth effect in cisplatin-resistant cells. These data show the benefit of exploiting the inherent resistance mechanisms of HNSCC to overcome cisplatin- and palbociclib resistance through the use of c-Myc inhibition.

Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors.

PURPOSE: The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1- tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. EXPERIMENTAL DESIGN: ASS1- tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1+) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. RESULTS: In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. In vivo studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. CONCLUSIONS: The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM.

Targeted delivery of gold nanoparticle contrast agents for reporting gene detection by magnetic resonance imaging.

Detection of protein expression by MRI requires a high payload of Gd(III) per protein binding event. Presented here is a targeted AuDNA nanoparticle capable of delivering several hundred Gd(III) chelates to the HaloTag reporter protein. Incubating this particle with HaloTag-expressing cells produced a 9.4 contrast-to-noise ratio compared to non-expressing cells.