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Neuroblastoma (NB) is an aggressive pediatric cancer that originates from neural crest tissues of the sympathetic nervous system. NB is highly heterogeneous both from a clinical and a molecular perspective. Clinically, this cancer represents a wide range of phenotypes ranging from spontaneous regression of 4S disease to unremitting treatment-refractory progression and death of high-risk metastatic disease. At a cellular level, the heterogeneous behavior of NB likely arises from an arrest and deregulation of normal neural crest development. In the present review, we summarize our current knowledge of neural crest development as it relates to pathways promoting 'stemness' and how deregulation may contribute to the development of tumor-initiating CSCs. There is an emerging consensus that such tumor subpopulations contribute to the evolution of drug resistance, metastasis and relapse in other equally aggressive malignancies. As relapsed, refractory disease remains the primary cause of death for neuroblastoma, the identification and targeting of CSCs or other primary drivers of tumor progression remains a critical, clinically significant goal for neuroblastoma. We will critically review recent and past evidence in the literature supporting the concept of CSCs as drivers of neuroblastoma pathogenesis.

Several glomerular pathologies resulting from podocyte injury are linked to genetic variation involving the MYH9 gene, which encodes the heavy chain of non-muscle myosin-IIA (NM-IIA). However, the functional role of NM-IIA has not been studied extensively in podocytes. We hypothesized that NM-IIA is critical for maintenance of podocyte structure and mechanical function. To test this hypothesis, we studied murine podocytes in vitro subjected to blebbistatin inhibition of NM-II activity, or RNA interference-mediated, isoform-specific ablation of Myh9 gene and protein (NM-IIA) or its paralog Myh10 gene and protein (NM-IIB). Using quantitative immunofluorescence microscopy, traction force microscopy, and attachment and "wound healing" assays, we found that NM-IIA ablation altered podocyte actin cytoskeletal structure and focal adhesion distribution, decreased cell attachment and contractility, and increased cell motility. Blebbistatin treatment had similar effects. NM-IIB ablation produced cells that exhibited poor attachment, but cytoskeletal structural organization, contractility and motility were maintained. These findings indicate that NM-IIA is essential for maintenance of podocyte cytoskeletal structure and mechanical function in vitro, and NM-IIB does not replace it in this role when NM-IIA expression is altered. We conclude that critical podocyte functions may be affected by MYH9 mutations or disease-associated haplotypes. © 2016 Wiley Periodicals, Inc.

Background Blood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis. Methods An Enzyme-Linked Lectin-Sorbent Assay (ELLecSA) that uses magnetic microbeads coated with an engineered version of the human opsonin, Mannose Binding Lectin, containing the Fc immunoglobulin domain linked to its carbohydrate recognition domain (FcMBL) was developed to quantify pathogen-associated molecular patterns (PAMPs) in whole blood. This assay was tested in rats and pigs to explore whether it can detect infections and monitor disease progression, and in prospectively enrolled, emergency room patients with suspected sepsis. These results were also compared with data obtained from non-infected patients with or without traumatic injuries. Results The FcMBL ELLecSA was able to detect PAMPS present on, or released by, 85% of clinical isolates representing 47 of 55 different pathogen species, including the most common causes of sepsis. The PAMP assay rapidly ( 81%), specificity (> 89%), and diagnostic accuracy of 0·87. It also distinguished infection from trauma-related inflammation in the same patient cohorts with a higher specificity than the clinical sepsis biomarker, C-reactive Protein. Conclusion The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections, even when blood cultures are negative and antibiotic therapy has been initiated. It may help to triage patients with suspected systemic infections, and serve as a companion diagnostic to guide administration of emerging dialysis-like sepsis therapies., Highlights • The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections. • The FcMBL ELLecSA distinguished infection from trauma-related inflammation. • It can detect infection even when blood cultures are negative and antibiotic therapy has been initiated. Current diagnostics of sepsis using blood cultures and molecular diagnostic tests fail to detect bloodstream infections in most infected patients, whereas the inflammatory biomarkers of infection that have a higher sensitivity of detection, lack specificity in distinguishing infection from trauma-related inflammation. Therefore we have leveraged a broad-spectrum pathogen binding opsonin and developed a rapid test to directly diagnose the presence of infection in the blood to triage patients and guide antibiotic therapy.

Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Kruppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

Neuroblastoma (NB) is a poorly differentiated, aggressive pediatric solid tumor for which half of high-risk cases have no identified genetic alteration. While such high-risk tumors are often infiltrated by macrophages that generate an inflammatory gene signature predictive of poor outcome, the initiation mechanism of this pro-tumorigenic inflammation remains unknown. In our analysis of clinical outcome-linked NB gene expression data, high tumor expression of TNFR1 was strongly correlated with poor event-free survival (5-year EFS 15% vs 70%, bonf p=9.3e-07), highlighting the potential importance of TNFα signaling in NB. Using TNFα knockout human NB cell lines, we determined that monocytes, but not NB cells, produce TNFα during in vitro co-culture. Furthermore, using a TNFα converting enzyme inhibitor to prevent secretion of soluble TNFα, we found that the membrane-bound form of TNFα (mTNFα) expressed on monocytes was critical for NF-κB pathway activation and downstream production of IL-6, a known NB growth factor. Neutralization of TNFα or its receptor TNFR1, but not TNFR2, in a monocyte-NB co-culture prevented NF-κB activation in monocytes, reducing IL-6 production by 50% compared to controls (p Our results demonstrate that TNFR1-expressing NB cells activate monocytes via contact-dependent mTNFα signaling, leading to NF-κB activation and production of IL-6, a known driver of NB progression and therapy resistance. These findings reveal a novel, druggable mechanism of tumor-promoting inflammation in NB.

High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite significant dose escalation of intense therapies, long-term survival for NB patients remains poor ( Citation Format: Saurabh Agarwal, Julie Tomolonis, Sanjeev Vasudevan, Jason Shohet. Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B21.

Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically. Citation Format: Chun Shik Park, Ye Shen, Andrew Lewis, Koramit Suppipat, Monica Puppi, Julie Tomolonis, Taylor Chen, Paul Pang, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Rachel Rau, Daniel Lacorazza. Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 145.

Several glomerular pathologies resulting from podocyte injury are linked to genetic variation involving the MYH9 gene, which encodes the heavy chain of non-muscle myosin-IIA (NM-IIA). However, the functional role of NM-IIA has not been studied extensively in podocytes. We hypothesized that NM-IIA is critical for maintenance of podocyte structure and mechanical function. To test this hypothesis, we studied murine podocytes in vitro subjected to blebbistatin inhibition of NM-II activity, or RNA interference-mediated, isoform-specific ablation of Myh9 gene and protein (NM-IIA) or its paralog Myh10 gene and protein (NM-IIB). Using quantitative immunofluorescence microscopy, traction force microscopy, and attachment and "wound healing" assays, we found that NM-IIA ablation altered podocyte actin cytoskeletal structure and focal adhesion distribution, decreased cell attachment and contractility, and increased cell motility. Blebbistatin treatment had similar effects. NM-IIB ablation produced cells that exhibited poor attachment, but cytoskeletal structural organization, contractility and motility were maintained. These findings indicate that NM-IIA is essential for maintenance of podocyte cytoskeletal structure and mechanical function in vitro, and NM-IIB does not replace it in this role when NM-IIA expression is altered. We conclude that critical podocyte functions may be affected by MYH9 mutations or disease-associated haplotypes. © 2016 Wiley Periodicals, Inc.

Introduction: Increasing evidence suggest that epigenetic regulators play a critical role in cancer cell heterogeneity and in maintaining tumor sub-populations with enhanced tumor-initiating and drug-resistant capacities. Recently, we discovered a drug-resistant, highly tumorigenic, self-renewing cell sub-population with features of cancer stem cells (CSCs) in pediatric neuroblastoma (NB). This sub-population, characterized by surface expression of G-CSF receptor (CD114), can escape initial therapy and cause refractory and aggressively invasive relapsed disease. Recently, we found that the CSF3R gene that code for CD114 is expressed specifically in CD114+ sub-population ( Methods: Chromatin immunoprecipitation (ChIP)-qPCR for the histone marks H3K4me3 and H3K27me3 was performed in sorted NB sub-populations. Pathway qPCR arrays and orthotopic xenografts mouse model were used to determine the effects of epigenetic inhibitors in vitro and in vivo. Results: We found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are overexpressed in CD114+ NB cells. These regulators maintain a reversible epigenetic switch of high H3K4me3 and low H3K27me3 histone marks at the CSF3R locus in CD114+ cells for active transcription. Targeting these epigenetic regulators with specific small-molecule inhibitors MM-102 (inhibit MLL1) and GSK-J4 (inhibit JMJD3) in NB, reverses the histone patterns at CSF3R promoter locus and block gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. These inhibitors also inhibit the expression of cancer stem cell specific genes as determined by pathway arrays. Inhibiting MLL1 and JMJD3 in NB orthotopic xenografts significantly decreases tumor size (p Conclusion: Overall, we demonstrate that epigenetic regulators MLL1 and JMJD3 regulate the expression of critical genes and maintain NB CSC sub-population. Targeting these epigenetic regulators reduce NB tumorigenicity, metastasis and increase drug sensitivity. Further developing these strategies will pave the way for incorporating epigenetic inhibitors in current therapies for effectivity improving long-term cure rates. Citation Format: Saurabh Agarwal, Zaowen Chen, Julie A. Tomolonis, Sanjeev A. Vasudevan, Jason M. Shohet. Epigenetic regulation of neuroblastoma tumorigenicity through MLL1 and JMJD3 modulation in cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4781. doi:10.1158/1538-7445.AM2017-4781

Chronic myeloid leukemia (CML) is the first blood cancer known to originate from a single hematopoietic stem cell (HSC) by expression of BCR-ABL, a product of the chromosomal translocation t(9;22), that slowly progress to a lethal fast-growing leukemia caused by malignant reprogramming of progenitor cells (blast crisis). Although CML can be successfully managed with targeted therapy by suppressing BCR-ABL kinase activity with tyrosine kinase inhibitors (TKI), patients remain in remission as long as they adhere to a lifelong treatment. The leukemic stem cell (LSC) population emerges as a key ‘CML reservoir’ that escapes TKI therapy by developing BCR-ABL-independent mechanisms of self-renewal and survival. LSC still remains an elusive target because of our poor understanding of specific self-renewal mechanisms and inability to selectively eliminate LSC without damaging normal hematopoiesis. Thus, there is a need for alternative drugs for relapse patients to prevent reactivation of BCR-ABL-positive LSC after stopping chemotherapy or emergence of chemoresistance and as frontline therapy to achieve treatment-free remission. We found that somatic deletion of the transcriptional factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. This data suggest that KLF4 promotes self-renewal of LSCs whereas serial transplantation indicates that KLF4 restricts stress self-renewal of normal HSCs and thus inhibition of KLF4 function would impair LSC self-renewal without altering blood production. Analyses of global gene expression in purified LSCs and genome-wide binding of KLF4 in a murine CML cell line revealed that KLF4 represses the gene encoding for the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). Immunoblots revealed elevated levels of DYRK2 protein in KLF4-deficient LSCs purified from the bone marrow of CML mice. Because phosphorylation of c-Myc and p53 by DYRK2 induces proteosomal degradation and cell death, respectively, we found that DYRK2 upregulation in KLF4-deficient LSCs was associated with a reduction of c-Myc protein and increased cleavage of PARP. As a proof-of-principle of the therapeutic potential of this finding, we explored the efficacy of vitamin K3 to eradicate LSCs by inhibiting the ubiquitin E3 ligase SIAH2 in charge of DYRK2 degradation. Vitamin K3 efficiently reduced cell viability in a panel of human-derived CML cell lines by inducing Dyrk2 expression and apoptosis. The identification of Dyrk2 as a critical mediator of LSC downfall is an innovative paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in CML patients. Citation Format: Chun Shik Park, Ye Shen, Koramit Suppipat, Julie Tomolonis, Monica Puppi, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Daniel Lacorazza. KLF4 promotes self-renewal by repressing DYRK2-mediated degradation of c-Myc in leukemic stem cells: development of targeted therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3334.