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Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K–Akt–mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K–Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K–Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599–609. ©2018 AACR.

Figures S1-3 show regulation of Par-4 by PI3K-Akt-Foxo pathway. Table 1 shows primer sequences.

Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS–FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS–FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

Collateral lethality occurs when loss of one paralog renders cancer cells dependent on the remaining paralog. Combining genome scale CRISPR/Cas9 screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout (KO) sensitized cells to VRK1 suppression, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 suppression. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced Barrier-to-Autointegration Factor (BAF) phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.Statement of SignificanceWe credential VRK1 as a target in adult and pediatric gliomas, and neuroblastomas with VRK2 promoter methylation. This demonstrates the utility of paralog-driven synthetic lethal interactions for biomarker-linked, targeted therapeutics.

Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.

People who are exposed to life-threatening trauma are at risk of developing posttraumatic stress disorder (PTSD). In addition to psychological manifestations, PTSD is associated with an increased risk of myocardial infarction, arrhythmias, hypertension, and other cardiovascular problems. We previously reported that rats exposed to a predator-based model of PTSD develop myocardial hypersensitivity to ischemic injury. The present study characterized cardiac changes in histology and gene expression in rats exposed this model. Male rats were subjected to two cat exposures (separated by a period of 10 days) and daily cage-mate changes for 31 days. Control rats were not exposed to the cat or cage-mate changes. Ventricular tissue was analyzed by RNA sequencing, western blotting, histology, and immunohistochemistry. Multifocal lesions characterized by necrosis, mononuclear cell infiltration, and collagen deposition were observed in hearts from all stressed rats but none of the control rats. Gene expression analysis identified clusters of upregulated genes associated with endothelial to mesenchymal transition, endothelial migration, mesenchyme differentiation, and extracellular matrix remodeling in hearts from stressed rats. Consistent with endothelial to mesenchymal transition, rats from stressed hearts exhibited increased expression of α-smooth muscle actin (a myofibroblast marker) and a decrease in the number of CD31 positive endothelial cells. These data provide evidence that predator-based stress induces myocardial lesions and reprogramming of cardiac gene expression. These changes may underlie the myocardial hypersensitivity to ischemia observed in these animals. This rat model may provide a useful tool for investigating the cardiac impact of PTSD and other forms of chronic psychological stress.

Background: Neuroblastoma is an extracranial solid tumor of childhood that arises from the developing sympathetic nervous system. Half of patients with high-risk disease do not survive despite multimodal therapy, and survival after relapse is unlikely. Recent studies have characterized two distinct neuroblastoma cell subtypes: the adrenergic (ADRN) subtype of sympathetic noradrenergic-like identity and the mesenchymal (MES) subtype of neural crest-like identity. ADRN-like cells are more aggressive and proliferative, while the MES-like cells are chemotherapy resistant. Neuroblastoma tumors are heterogenous and comprised of both ADRN and MES cell subtypes, which likely leads to differential responses to therapy. Thus, to eliminate cells that may lead to tumor relapse, it is critical to develop therapies against both neuroblastoma subtypes. Methods: To identify candidate subtype-specific therapeutic targets, we used ssGSEA to classify human neuroblastoma tumors into ADRN- or MES-dominant and performed differential expression. We validated subtype-specific targets in additional datasets and models, including transdifferentiated neuroblastoma cell lines (ADRN-to-MES) after inducible overexpression of the MES transcription factor PRRX1. Results: We show ADRN-specific expression of known preclinical and clinical neuroblastoma targets with limited or no expression in MES-dominant tumors. We propose CD276 (B7-H3) and L1CAM as pan-subtype targets with similar expression in both subtypes and stable expression after ADRN-to-MES transdifferentiation. In MES-specific neuroblastoma patient samples and cell lines, we identify the receptor tyrosine kinase AXL as a candidate MES target. MES neuroblastoma cell lines are more sensitive to small molecule AXL inhibitors (Cabozantinib, NPS-1034, and ONO-7475) compared to ADRN cell lines. We also observe higher expression of PDL1 and immunosuppressive chemokines in MES-dominant tumors and cell lines. Finally, we detect AXL and Gas6 transcripts in tumor-resident macrophages and fibroblasts, which may further promote immune evasion. Conclusion: Here we have identified and prioritized ADRN-specific, MES-specific, and pan-subtype neuroblastoma therapeutic targets and suggest AXL-targeted therapy may eliminate both MES-dominant neuroblastoma cells and immune cell populations that contribute to an immunosuppressive microenvironment. Citation Format: Nathan M. Kendsersky, Nathaniel W. Mabe, Alvin Farrel, Liron D. Grossmann, Michal Odrobina, Kimberly Stegmaier, John M. Maris. Identification of ADRN-specific, MES-specific, and pan-subtype therapeutic targets in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3889.

SummaryThe survival of residual tumor cells following therapy and their eventual recurrence constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during tumor relapse. We used cellular barcoding to directly monitor clonal dynamics during tumor recurrence in a genetically engineered mouse model. We found that the clonal diversity of tumors progressively decreased during tumor regression, residual disease, and recurrence. Only a fraction of subclones survived oncogene withdrawal and persisted in residual tumors. The minimal residual disease phase itself was accompanied by a continued attrition of clones, suggesting an ongoing process of selection during dormancy. The reactivation of dormant residual cells into recurrent tumors followed several distinct evolutionary routes. Approximately half of the recurrent tumors exhibited a striking clonal dominance in which one or two subclones comprised the vast majority of the tumor. The majority of these clonal recurrent tumors exhibited evidence of de novo acquisition of Met amplification, and were sensitive to small-molecule Met inhibitors. A second group of recurrent tumors exhibited marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors were not sensitive to Met inhibitors, but were instead dependent upon an autocrine IL-6 – Stat3 pathway. These results suggest that the survival and reactivation of dormant tumors can proceed via multiple independent routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically reexpressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo necroptosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

Epigenetic dysregulation is a common feature of cancer, and is thought to underlie many aspects of tumor progression. Using a genetically engineered mouse model of breast cancer recurrence, we show that recurrent mammary tumors undergo widespread epigenomic and transcriptional alterations, and acquire dependence on the G9a histone methyltransferase. Genetic ablation of G9a delays tumor recurrence, and pharmacologic inhibition of G9a slows the growth of recurrent tumors. Mechanistically, G9a activity is required to silence pro-inflammatory cytokines, including TNF, through H3K9 methylation at gene promoters. G9a inhibition induces re-expression of these cytokines, leading to p53 activation and necroptosis. Recurrent tumors upregulate receptor interacting protein kinase-3 (RIPK3) expression and are dependent upon RIPK3 activity. High RIPK3 expression renders recurrent tumors sensitive to necroptosis following G9a inhibition. These findings demonstrate that epigenetic rewiring – specifically G9a-mediated silencing of pro-necroptotic proteins – is a critical step in tumor recurrence and suggest that G9a is a targetable dependency in recurrent breast cancer.

Tumor relapse is the leading cause of death in breast cancer, largely due to the fact that recurrent tumors are frequently resistant to chemotherapy. We previously reported that downregulation of the proapoptotic protein Par-4 promotes tumor recurrence in genetically engineered mouse models of breast cancer recurrence. In the present study, we examined the mechanism and functional significance of Par-4 downregulation in recurrent tumors. We found that epithelial-to-mesenchymal transition (EMT) promotes epigenetic silencing of Par-4 in recurrent tumors. Par-4 silencing proceeded through binding of the EMT transcription factor Twist to the Par-4 promoter, where Twist induced a unique bivalent chromatin domain. This bivalent configuration conferred plasticity at the Par-4 promoter, and Par-4 silencing could be reversed with pharmacologic inhibitors of Ezh2 and HDAC1/2. Using an epigenome editing approach to reexpress Par-4 by specifically reversing the histone modifications found in recurrent tumors, we found that Par-4 reexpression sensitized recurrent tumors to chemotherapy in vitro and in vivo. Upon reexpression, Par-4 bound to the protein phosphatase PP1, caused widespread changes in phosphorylation of cytoskeletal proteins, and cooperated with microtubule-targeting drugs to induce mitotic defects. These results identify Twist-induced epigenetic silencing of Par-4 as a targetable axis that promotes chemoresistance in recurrent breast cancer.

Gαi-coupled receptors play important roles in protecting the heart from ischemic injury. Regulator of G protein signaling (RGS) proteins suppress Gαi signaling by accelerating the GTPase activity of Gαi subunits. However, the roles of individual RGS proteins in modulating ischemic injury are unknown. In this study, we investigated the effect of RGS6 deletion on myocardial sensitivity to ischemic injury. Hearts from RGS6 knockout (RGS6−/−) and RGS6 wild-type (RGS6+/+) mice were subjected to 30 minutes of ischemia and 2 hours of reperfusion on a Langendorff heart apparatus. Infarcts in RGS6−/− hearts were significantly larger than infarcts in RGS6+/+ hearts. RGS6−/− hearts also exhibited increased phosphorylation of β2-adrenergic receptors and G protein–coupled receptor kinase 2 (GRK2). Mitochondrial GRK2 as well as caspase-3 cleavage were increased significantly in RGS6−/− hearts compared with RGS6+/+ hearts after ischemia. Chronic propranolol treatment of mice prevented the observed increases in ischemic injury and the GRK2 phosphorylation observed in RGS6−/− hearts. Our findings suggest that loss of RGS6 predisposes the ventricle to prodeath signaling through a β2AR-GRK2–dependent signaling mechanism, and they provide evidence for a protective role of RGS6 in the ischemic heart. Individuals expressing genetic polymorphisms that suppress the activity of RGS6 may be at increased risk of cardiac ischemic injury. Furthermore, the development of agents that increase RGS6 expression or activity might provide a novel strategy for the treatment of ischemic heart disease.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities., The cellular composition of recurrent tumors can provide insight into resistance to therapy and inform on second line therapies. Here, using a genetically modified mouse, the authors perform barcoding experiments of the primary tumors to allow them to study the clonal dynamics of tumor recurrence.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically re-expressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo programmed necrosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

People who are exposed to life-threatening trauma are at risk of developing posttraumatic stress disorder (PTSD). In addition to psychological manifestations, PTSD is associated with increased risk...

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K–Akt–mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K–Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K–Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599–609. ©2018 AACR.

Aims Regulator of G protein signaling (RGS) proteins act as molecular “off switches” that terminate G protein signaling by catalyzing the hydrolysis of Gα-bound GTP to GDP. Many different Gαi-coupled receptors have been implicated in the cardioprotective effects of ischemic preconditioning. However, the role of RGS proteins in modulating cardioprotection has not been previously investigated. We used mice that were homozygous (GS/GS) or heterozygous (GS/+) for a mutation in Gαi2 rendering it RGS-insensitive (G184S) to determine whether interactions between endogenous RGS proteins and Gαi2 modulate Gαi-mediated protection from ischemic injury Methods and Results Langendorff-perfused mouse hearts were subjected to 30 min global ischemia and 2 hours reperfusion. Infarcts in GS/GS (14.5 % of area at risk) and GS/+ (22.6 % of AAR) hearts were significantly smaller than those of +/+ hearts (37.2 % of AAR) and recovery of contractile function was significantly enhanced in GS/GS and GS/+ hearts compared to +/+ hearts. The cardioprotective phenotype was not reversed by wortmannin or U0126 but was reversed by 5-hydroxydecanoic acid and HMR 1098, indicating that RGS insensitive-Gαi2 protects the heart through a mechanism that requires functional ATP-dependent potassium channels but does not require acute activation of extracellular regulated kinase or Akt signaling pathways. Conclusions This is the first study to demonstrate that Gαi2 mediated cardioprotection is suppressed by RGS proteins. These data suggest that RGS proteins may provide novel therapeutic targets to protect the heart from ischemic injury.