Single-nucleus expression characterization of non-enhancing region of recurrent high-grade glioma.

Background: Non-enhancing (NE) infiltrating tumor cells beyond the contrast-enhancing (CE) bulk of tumor are potential propagators of recurrence after gross total resection of high-grade glioma. Methods: We leveraged single-nucleus RNA sequencing on 15 specimens from recurrent high-grade gliomas (n = 5) to compare prospectively identified biopsy specimens acquired from CE and NE regions. Additionally, 24 CE and 22 NE biopsies had immunohistochemical staining to validate RNA findings. Results: Tumor cells in NE regions are enriched in neural progenitor cell-like cellular states, while CE regions are enriched in mesenchymal-like states. NE glioma cells have similar proportions of proliferative and putative glioma stem cells relative to CE regions, without significant differences in % Ki-67 staining. Tumor cells in NE regions exhibit upregulation of genes previously associated with lower grade gliomas. Our findings in recurrent GBM paralleled some of the findings in a re-analysis of a dataset from primary GBM. Cell-, gene-, and pathway-level analyses of the tumor microenvironment in the NE region reveal relative downregulation of tumor-mediated neovascularization and cell-mediated immune response, but increased glioma-to-nonpathological cell interactions. Conclusions: This comprehensive analysis illustrates differing tumor and nontumor landscapes of CE and NE regions in high-grade gliomas, highlighting the NE region as an area harboring likely initiators of recurrence in a pro-tumor microenvironment and identifying possible targets for future design of NE-specific adjuvant therapy. These findings also support the aggressive approach to resection of tumor-bearing NE regions.

Low- and high-grade glioma-associated vascular cells differentially regulate tumor growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of pro-tumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express pro-tumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wildtype (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to anti-angiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and anti-angiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo co-transplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wildtype HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFß1-GPM6A signaling.

Low- and high-grade glioma endothelial cells differentially regulate tumor growth.

Background: A key feature distinguishing high-grade glioma (HGG) from low-grade glioma (LGG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor endothelial cells (TEC) from HGG are molecularly and functionally distinct from normal brain EC and secrete higher levels of pro-tumorigenic factors that promote glioma growth and progression. However, it remains unclear whether TEC from LGG also express pro-tumorigenic factors, and to what extent they functionally contribute to glioma growth. Methods: Transcriptomic profiling was conducted on tumor endothelial cells (TEC) from grade II/III (LGG, IDH-mutant) and grade IV HGG (IDH-wildtype). Functional differences between LGG- and HGG-TEC were evaluated using growth assays, resistance to anti-angiogenic drugs and radiation therapy. Conditioned media and specific factors from LGG- and HGG-TEC were tested on patient-derived gliomasphere lines using growth assays in vitro and in co-transplantation studies in vivo in orthotopic xenograft models. Results: LGG-TEC showed enrichment of extracellular matrix and cell cycle-related gene sets and sensitivity to anti-angiogenic therapy whereas HGG-TEC displayed an increase in immune response-related gene sets and anti-angiogenic resistance. LGG- and HGG-TEC displayed opposing effects on growth and proliferation of IDH-wildtype and mutant tumor cells. Asporin (ASPN), a small leucine rich proteoglycan enriched in LGG-TEC was identified as a growth suppressor of IDH-wildtype GBM by modulating TGFΒ1-GPM6A signaling. Conclusions: Our findings indicate that TEC from LGG and HGG are molecularly and functionally heterogeneous and differentially regulate the growth of IDH-wildtype and mutant tumors.

Interfer(on)ing with Zika virus.

In this issue of Neuron, Bulstrode et al.1 demonstrate that glioblastoma slice cultures, unlike neural progenitors, are refractory to Zika virus infection. The anti-infective mechanism is myeloid-lineage cell-secreted interferon beta. These studies have implications for therapeutics in both glioblastoma and Zika virus infections.

Pathway-based approach reveals differential sensitivity to E2F1 inhibition in glioblastoma.

Analysis of tumor gene expression is an important approach for the classification and identification of therapeutic vulnerabilities. However, targeting glioblastoma (GBM) based on molecular subtyping has not yet translated into successful therapies. Here, we present an integrative approach based on molecular pathways to expose new potentially actionable targets. We used gene set enrichment analysis (GSEA) to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene signatures. We identified key targets that are predicted to be differentially activated between tumors and were functionally validated in a library of gliomasphere cultures. Resultant cluster-specific gene signatures associated not only with hallmarks of cell cycle and stemness gene expression, but also with cell-type specific markers and different cellular states of GBM. Several upstream regulators, such as PIK3R1 and EBF1 were differentially enriched in cells bearing stem cell like signatures and bear further investigation. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only a subset of gliomasphere cultures predicted to be E2F1 signaling dependent. Our in vivo work also validated the functional significance of E2F1 in tumor formation capacity in the predicted samples. E2F1 inhibition also differentially sensitized E2F1-dependent gliomasphere cultures to radiation treatment. Our findings indicate that this novel approach exploring cancer pathways highlights key therapeutic vulnerabilities for targeting GBM.

P300 promotes tumor recurrence by regulating radiation-induced conversion of glioma stem cells to vascular-like cells.

Glioma stem cells (GSC) exhibit plasticity in response to environmental and therapeutic stress leading to tumor recurrence, but the underlying mechanisms remain largely unknown. Here, we employ single-cell and whole transcriptomic analyses to uncover that radiation induces a dynamic shift in functional states of glioma cells allowing for acquisition of vascular endothelial-like and pericyte-like cell phenotypes. These vascular-like cells provide trophic support to promote proliferation of tumor cells, and their selective depletion results in reduced tumor growth post-treatment in vivo. Mechanistically, the acquisition of vascular-like phenotype is driven by increased chromatin accessibility and H3K27 acetylation in specific vascular genes allowing for their increased expression post-treatment. Blocking P300 histone acetyltransferase activity reverses the epigenetic changes induced by radiation and inhibits the adaptive conversion of GSC into vascular-like cells and tumor growth. Our findings highlight a role for P300 in radiation-induced stress response, suggesting a therapeutic approach to prevent glioma recurrence.

Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.

High-Dimensional Analysis of Circulating and Tissue-Derived Myeloid-Derived Suppressor Cells from Patients with Glioblastoma.

We will first describe analysis of MDSC subsets from patient tumors with multicolor flow cytometry. The key components of this methodology are to obtain viable single cell suspensions and eliminate red blood cell contamination.

Discovery of a dual inhibitor of NQO1 and GSTP1 for treating glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a universally lethal tumor with frequently overexpressed or mutated epidermal growth factor receptor (EGFR). NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation. METHODS: High-throughput screen was used for agents selectively active against GBM cells with EGFRvIII mutations. Co-crystal structures were revealed molecular details of target recognition. Pharmacological and gene knockdown/overexpression approaches were used to investigate the oxidative stress in vitro and in vivo. RESULTS: We identified a small molecular inhibitor, "MNPC," that binds to both NQO1 and GSTP1 with high affinity and selectivity. MNPC inhibits NQO1 and GSTP1 enzymes and induces apoptosis in GBM, specifically inhibiting the growth of cell lines and primary GBM bearing the EGFRvIII mutation. Co-crystal structures between MNPC and NQO1, and molecular docking of MNPC with GSTP1 reveal that it binds the active sites and acts as a potent dual inhibitor. Inactivation of both NQO1 and GSTP1 with siRNA or MNPC results in imbalanced redox homeostasis, leading to apoptosis and mitigated cancer proliferation in vitro and in vivo. CONCLUSIONS: Thus, MNPC, a dual inhibitor for both NQO1 and GSTP1, provides a novel lead compound for treating GBM via the exploitation of specific vulnerabilities created by mutant EGFR.

Metronomic capecitabine as an immune modulator in glioblastoma patients reduces myeloid-derived suppressor cells.

BACKGROUNDMyeloid-derived suppressor cells (MDSCs) are elevated in the circulation of patients with glioblastoma (GBM), present in tumor tissue, and associated with poor prognosis. While low-dose chemotherapy reduces MDSCs in preclinical models, the use of this strategy to reduce MDSCs in GBM patients has yet to be evaluated.METHODSA phase 0/I dose-escalation clinical trial was conducted in patients with recurrent GBM treated 5-7 days before surgery with low-dose chemotherapy via capecitabine, followed by concomitant low-dose capecitabine and bevacizumab. Clinical outcomes, including progression-free and overall survival, were measured, along with safety and toxicity profiles. Over the treatment time course, circulating MDSC levels were measured by multiparameter flow cytometry, and tumor tissue immune profiles were assessed via time-of-flight mass cytometry.RESULTSEleven patients total were enrolled across escalating dose cohorts of 150, 300, and 450 mg bid. No serious adverse events related to the drug combination were observed. Compared with pretreatment baseline, circulating MDSCs were found to be higher after surgery in the 150-mg treatment arm and lower in the 300-mg and 450-mg treatment arms. Increased cytotoxic immune infiltration was observed after low-dose capecitabine compared with untreated GBM patients in the 300-mg and 450-mg treatment arms.CONCLUSIONSLow-dose, metronomic capecitabine in combination with bevacizumab was well tolerated in GBM patients and was associated with a reduction in circulating MDSC levels and an increase in cytotoxic immune infiltration into the tumor microenvironment.TRIAL REGISTRATIONClinicalTrials.gov NCT02669173.FUNDINGThis research was funded by the Cleveland Clinic, Case Comprehensive Cancer Center, the Musella Foundation, B*CURED, the NIH, the National Cancer Institute, the Sontag Foundation, Blast GBM, the James B. Pendleton Charitable Trust, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. Capecitabine was provided in kind by Mylan Pharmaceuticals.

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis.

Building Bonds: Cancer Stem Cells Depend on Their Progeny to Drive Tumor Progression.

Little is currently known about how cancer stem-like cells (CSCs) interact with their more restricted progeny. In this issue of Cell Stem Cell, Wang et al. (2018) demonstrate a novel bidirectional signaling axis between CSCs and their progeny that is mediated by brain-derived neurotrophic factor and VGF accelerating glioma progression.

Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase.

Tumors adapt their phenotypes during growth and in response to therapies through dynamic changes in cellular processes. Connexin proteins enable such dynamic changes during development, and their dysregulation leads to disease states. The gap junction communication channels formed by connexins have been reported to exhibit tumor-suppressive functions, including in triple-negative breast cancer (TNBC). However, we find that connexin 26 (Cx26) is elevated in self-renewing cancer stem cells (CSCs) and is necessary and sufficient for their maintenance. Cx26 promotes CSC self-renewal by forming a signaling complex with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK), resulting in NANOG stabilization and FAK activation. This FAK/NANOG-containing complex is not formed in mammary epithelial or luminal breast cancer cells. These findings challenge the paradigm that connexins are tumor suppressors in TNBC and reveal a unique function for Cx26 in regulating the core self-renewal signaling that controls CSC maintenance.

A molecular cascade modulates MAP1B and confers resistance to mTOR inhibition in human glioblastoma.

Background: Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. Methods: To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. Results: An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. Conclusions: These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

A 4-miRNA signature to predict survival in glioblastomas.

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06-23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93-33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45-0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression.

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.

Taking a Toll on Self-Renewal: TLR-Mediated Innate Immune Signaling in Stem Cells.

Innate immunity has evolved as the front-line cellular defense mechanism to acutely sense and decisively respond to microenvironmental alterations. The Toll-like receptor (TLR) family activates signaling pathways in response to stimuli and is well-characterized in both resident and infiltrating immune cells during neural inflammation, injury, and degeneration. Innate immune signaling has also been observed in neural cells during development and disease, including in the stem and progenitor cells that build the brain and are responsible for its homeostasis. Recently, the activation of developmental programs in malignant brain tumors has emerged as a driver for growth via cancer stem cells. In this review we discuss how innate immune signaling interfaces with stem cell maintenance in the normal and neoplastic brain.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling.

BACKGROUND: Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. METHODS: MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. RESULTS: Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. CONCLUSIONS: Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.

Cx25 contributes to leukemia cell communication and chemosensitivity.

Leukemia encompasses several hematological malignancies with shared phenotypes that include rapid proliferation, abnormal leukocyte self-renewal, and subsequent disruption of normal hematopoiesis. While communication between leukemia cells and the surrounding stroma supports tumor survival and expansion, the mechanisms underlying direct leukemia cell-cell communication and its contribution to tumor growth are undefined. Gap junctions are specialized intercellular connections composed of connexin proteins that allow free diffusion of small molecules and ions directly between the cytoplasm of adjacent cells. To characterize homotypic leukemia cell communication, we employed in vitro models for both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and measured gap junction function through dye transfer assays. Additionally, clinically relevant gap junction inhibitors, carbenoxolone (CBX) and 1-octanol, were utilized to uncouple the communicative capability of leukemia cells. Furthermore, a qRT-PCR screen revealed several connexins with higher expression in leukemia cells compared with normal hematopoietic stem cells. Cx25 was identified as a promising adjuvant therapeutic target, and Cx25 but not Cx43 reduction via RNA interference reduced intercellular communication and sensitized cells to chemotherapy. Taken together, our data demonstrate the presence of homotypic communication in leukemia through a Cx25-dependent gap junction mechanism that can be exploited for the development of anti-leukemia therapies.