Targeting Wnt signaling for improved glioma immunotherapy.

Introduction: Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic Wnt/ß-catenin signaling is associated with a poor prognosis and the proliferation of glioma stem-like cells, leading to malignant transformation and tumor progression. Impressive results in a subset of cancers have been obtained using immunotherapies including anti-CTLA4, anti-PD-1, and anti-PD-L1 or chimeric antigen receptor (CAR) T cell therapies. However, the heterogeneity of tumors, low mutational burden, single antigen targeting, and associated antigen escape contribute to non-responsiveness and potential tumor recurrence despite these therapeutic efforts. In the current study, we determined the effects of the small molecule, highly specific Wnt/CBP (CREB Binding Protein)/ß-catenin antagonist ICG-001, on glioma tumor cells and the tumor microenvironment (TME)-including its effect on immune cell infiltration, blood vessel decompression, and metabolic changes. Methods: Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated in vitro cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001. Results: In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/ß-catenin target gene Survivin/BIRC5-a hallmark of Wnt/CBP/ß-catenin inhibition. We found that in a syngeneic mouse model of glioma (K-luc), ICG-001 treatment enhanced tumor infiltration by CD3+ and CD8+ cells with increased expression of the vascular endothelial marker CD31 (PECAM-1). We also observed differential gene expression and induced immune cell infiltration in tumors pretreated with ICG-001 and then treated with CAR T cells as compared with single treatment groups or when ICG-001 treatment was administered after CAR T cell therapy. Discussion: We conclude that specific Wnt/CBP/ß-catenin antagonism results in pleotropic changes in the glioma TME, including glioma stem cell differentiation, modulation of the stroma, and immune cell activation and recruitment, thereby suggesting a possible role for enhancing immunotherapy in glioma patients.

Depletion of kinesin motor KIF20A to target cell fate control suppresses medulloblastoma tumour growth.

During mammalian brain development, neural progenitor cells proliferate extensively but can ensure the production of correct numbers of various types of mature cells by balancing symmetric proliferative versus asymmetric differentiative cell divisions. This process of cell fate determination may be harnessed for developing cancer therapy. Here, we test this idea by targeting KIF20A, a mitotic kinesin crucial for the control of cell division modes, in a genetic model of medulloblastoma (MB) and human MB cells. Inducible Kif20a knockout in both normal and MB-initiating granule neuron progenitors (GNPs) causes early cell cycle exit and precocious neuronal differentiation without causing cytokinesis failure and suppresses the development of Sonic Hedgehog (SHH)-activated MB. Inducible KIF20A knockdown in human MB cells inhibits proliferation both in cultures and in growing tumors. Our results indicate that targeting the fate specification process of nascent daughter cells presents a novel avenue for developing anti-proliferation treatment for malignant brain tumors.

Spindle Orientation-Independent Control of Cell Fate Determination by RGS3 and KIF20A.

It was proposed that similar to its role in the invertebrate nervous system, mitotic spindle orientation (or cell cleavage plane orientation) of a dividing neural progenitor cell specifies the fate of daughter cells in the mammalian brain, modulating the production of neurons via symmetric versus asymmetric cell divisions during the course of neurogenesis. Experimental tests of the sufficiency of spindle/cleavage plane orientation in mammalian cell fate determination have yielded conflicting results. On the other hand, the necessity of spindle/cleavage plane orientation in mammalian cell fate determination has not yet been addressed. Here we examined the necessity of spindle/cleavage plane orientation during cortical neurogenesis in mice with loss-of-function of the RGS3-KIF20A interaction axis. We present evidence that while inactivation of RGS3 or KIF20A was linked to a shift in neural progenitor cells from proliferative to differentiative divisions in the developing cortex, these genetic mutations did not lead to anticipated alteration in the orientation of spindle/cleavage plane. Our results indicate that the RGS3-KIF20A axis regulates the balance between proliferation and differentiation in the mammalian cortex employing a mechanism independent of spindle/cleavage plane orientation. These data also caution against using spindle/cleavage plane orientation as the synonym for cell fate determination.

SEPT7 Interacts with KIF20A and Regulates the Proliferative State of Neural Progenitor Cells During Cortical Development.

Balanced proliferation and differentiation of neural progenitor cells (NPCs) are critical for brain development, but how the process is regulated and what components of the cell division machinery is involved are not well understood. Here we report that SEPT7, a cell division regulator originally identified in Saccharomyces cerevisiae, interacts with KIF20A in the intercellular bridge of dividing NPCs and plays an essential role in maintaining the proliferative state of NPCs during cortical development. Knockdown of SEPT7 in NPCs results in displacement of KIF20A from the midbody and early neuronal differentiation. NPC-specific inducible knockout of Sept7 causes early cell cycle exit, precocious neuronal differentiation, and ventriculomegaly in the cortex, but surprisingly does not lead to noticeable cytokinesis defect. Our data uncover an interaction of SEPT7 and KIF20A during NPC divisions and demonstrate a crucial role of SEPT7 in cell fate determination. In addition, this study presents a functional approach for identifying additional cell fate regulators of the mammalian brain.

KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development.

Balanced symmetric and asymmetric divisions of neural progenitor cells (NPCs) are crucial for brain development, but the underlying mechanisms are not fully understood. Here we report that mitotic kinesin KIF20A/MKLP2 interacts with RGS3 and plays a crucial role in controlling the division modes of NPCs during cortical neurogenesis. Knockdown of KIF20A in NPCs causes dislocation of RGS3 from the intercellular bridge (ICB), impairs the function of Ephrin-B-RGS cell fate signaling complex, and leads to a transition from proliferative to differentiative divisions. Germline and inducible knockout of KIF20A causes a loss of progenitor cells and neurons and results in thinner cortex and ventriculomegaly. Interestingly, loss of function of KIF20A induces early cell cycle exit and precocious neuronal differentiation without causing substantial cytokinesis defect or apoptosis. Our results identify a RGS-KIF20A axis in the regulation of cell division and suggest a potential link of the ICB to regulation of cell fate determination.

Prospective separation and transcriptome analyses of cortical projection neurons and interneurons based on lineage tracing by Tbr2 (Eomes)-GFP/Dcx-mRFP reporters.

In the cerebral cortex, projection neurons and interneurons work coordinately to establish neural networks for normal cortical functions. While the specific mechanisms that control productions of projection neurons and interneurons are beginning to be revealed, a global characterization of the molecular differences between these two neuron types is crucial for a more comprehensive understanding of their developmental specifications and functions. In this study, using lineage tracing power of combining Tbr2(Eomes)-GFP and Dcx-mRFP reporter mice, we prospectively separated intermediate progenitor cell (IPC)-derived neurons (IPNs) from non-IPC-derived neurons (non-IPNs) of the embryonic cerebral cortex. Molecular characterizations revealed that IPNs and non-IPNs were enriched with projection neurons and interneurons, respectively. Expression profiling documented cell-specific genes including differentially expressed transcriptional regulators that might be involved in cellular specifications, for instance, our data found that SOX1 and SOX2, which were known for important functions in neural stem/progenitor cells, continued to be expressed by interneurons but not by projection neurons. Transcriptome analyses of cortical neurons isolated at different stages of neurogenesis revealed distinct temporal patterns of expression of genes involved in early-born or late-born neuron specification. These data present a resource useful for further investigation of the molecular regulations and functions of projection neurons and interneurons.

Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis.

DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.

5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations.

The base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) and 5-methyl-2'-deoxycytidine (5mdC) in genomic DNA, we investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC were depleted substantially with up to 5-fold reduction compared with normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to more than 30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1. Furthermore, immunohistochemical analysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. The data therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Transcriptome analysis of neural progenitor cells by a genetic dual reporter strategy.

Global analysis of stem/progenitor cells promises new insight into mechanisms that govern self-renewal and cellular potential, an unresolved question of stem/progenitor cell biology. Despite rapid advance of genome-wide profiling methods, the difficulty in cell purification remains a major challenge for global analysis of somatic stem/progenitor cells. Genetic tagging with a reporter provides a powerful tool for identification and isolation of a specific mature cell type; however, for stem/progenitor cells, reporter retention by progeny may be a concern for impurity. Here, we describe a genetic system combining a progenitor cell specific label with a second tag for marking differentiation. We present evidence that differential labeling of neural progenitor cells and their progeny enables prospective purification of these two cell types, whereas isolation based on a single marker compromises the purity of the intended progenitor population. Comparative expression profiling between the purified progenitors and progeny documents a neural progenitor cell transcriptome and uncovers an important role of Tyro3/Axl/Mer receptor tyrosine kinases in the maintenance of neural progenitor cells. This study establishes a general strategy for isolation of somatic stem/progenitor cells and provides a transcriptome database of neural progenitor cells useful for identification of causal factors of neural progenitor cell state, global dissection of epigenetic control of cellular potential, as well as for developing biomarkers or targets of brain cancer stem/initiating cells for therapeutic interventions.

Gα subunit coordinates with ephrin-B to balance self-renewal and differentiation in neural progenitor cells.

Proper development of the mammalian brain requires that neural progenitor cells balance self-renewal and differentiation under precise temporal and spatial regulation, but the underlying mechanisms are not well understood. In this study, we identify Gα subunit as a positive regulator of mammalian neurogenesis, working with the regulator of G protein signaling (RGS)-mediated ephrin-B signaling pathway as two opposing forces to maintain a balance between self-renewal and differentiation in the developing mouse cerebral cortex. Multiple Gα(i) subunits are expressed by cortical neural progenitor cells during the course of cortical neurogenesis. Activation of Gα(i) signaling, through in utero electroporation-mediated expression of wild-type and constitutively active Gα(i) subunits, counteracts the function of ephrin-B in cortical neural progenitors to induce differentiation. Genetic knock-in of an RGS-insensitive G184SGα(i2) causes early cell cycle exit and a reduction of cortical neural progenitor cells and leads to a defect in the production of late born cortical neurons, similar to what is observed in mutant mice with deficiency in ephrin-B reverse signaling pathway. This study reveals a role of Gα subunit in mammalian neurogenesis and uncovers a developmental mechanism, coordinated by the Gα and ephrin-B signaling pathways, for control of the balance between self-renewal and differentiation in neural progenitor cells.

Essential role of PDZ-RGS3 in the maintenance of neural progenitor cells.

Ephrin-B plays an important role in neural progenitor cells to regulate self-renewal and differentiation. Cellular and embryological evidence suggest this function of ephrin-B is mediated through a PDZ-dependent reverse signaling mechanism. Here, we have genetically investigated the function of PDZ-RGS3, a proposed downstream signaling mediator of ephrin-B function, and found that knockout of PDZ-RGS3 caused early cell cycle exit and precocious differentiation in neural progenitor cells of the developing cerebral cortex, reminiscent of the phenotype observed in ephrin-B1 knockout mice. This resulted in a loss of cortical neural progenitor cells during cortical neurogenesis and led to impairment in the production of late born cortical neurons. These results reveal an essential role of PDZ-RGS3 in maintaining the balance between self-renewal and differentiation of neural progenitor cells and provide genetic evidence linking PDZ-RGS3 to ephrin-B reverse signaling. As ephrin-B molecules are often differentially expressed in different types of neural progenitor/stem cells during development or in adult life, deletion of PDZ-RGS3 can achieve a uniform loss of function of the ephrin-B/regulator of G protein-signaling (RGS) pathway, thereby providing a genetic tool useful for dissecting the mechanisms and functions of the ephrin-B/RGS reverse signaling pathway in neural progenitor/stem cell regulation.

ZHX2 Interacts with Ephrin-B and regulates neural progenitor maintenance in the developing cerebral cortex.

Neural progenitor cells in the ventricular zone of the developing mammalian cerebral cortex give rise to specialized cortical cell types via consecutive rounds of proliferation and differentiation, but the mechanisms by which progenitor cell self-renewal and differentiation are regulated during cortical development are not well understood. Here, we show that zinc-finger and homeodomain protein 2 (ZHX2) is specifically expressed in neural progenitor cells during cortical neurogenesis. ZHX2 binds to the cytoplasmic domain of ephrin-B1, which is expressed in cortical neural progenitors and plays a role in neural progenitor cell maintenance. ZHX2 acts as a transcriptional repressor in cell, and its repressor activity is enhanced by coexpression of an ephrin-B1 intracellular domain. Blocking ZHX2 function in cultured neural progenitor cells or in the embryonic cortex causes neuronal differentiation, whereas overexpression of ZHX2 and an ephrin-B1 intracellular domain disrupts the normal differentiation of cortical neural progenitor cells. This study identifies ZHX2 as a novel regulator of neural progenitor cell maintenance and suggests a potential nuclear mechanism of the ephrin-B function in the cortex.

Regulation of neural progenitor cell state by ephrin-B.

Maintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B-PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifically seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells. Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.

Rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-DsRed-express.

Characterization of neural promoter/enhancers is essential for understanding gene regulation during brain development and provides useful genetic tools. However, it relies on the use of transgenic mice. We report a method for the rapid in vivo analysis of neural promoter/enhancers in the developing mouse brain and its application in the isolation of the doublecortin (DCX) promoter/enhancer for genetic labeling of young neurons. In the present study, we demonstrated that reporter genes introduced into the developing mouse cerebral cortex by in utero electroporation can achieve promoter/enhancer-specific patterns of expression. We used the in utero electroporation system to isolate a genomic fragment of the doublecortin gene that can direct reporter expression faithful to doublecortin in young neurons of the cerebral cortex. Finally, we showed that the DCX promoter identified via electroporation could reproduce doublecortin expression in the entire central nervous system in DCX-DsRed-express transgenic mice. The results of our study provide a convenient, reliable, and rapid method for in vivo analysis of neural promoter/enhancers in the developing mouse brain.

The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription.

EMSA and footprinting analyses have revealed that the -489-- -414 bp and the -390-- -345 bp (designated DC and PC respectively) upstream of the Aspergillus nigerT21 glaA gene were bound by one protein factor in the A. nigerT21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. nigerT21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA revealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. nigerT21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.

Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression.

Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.

Detection of a protein, AngCP, which binds specifically to the three upstream regions of glaA gene in A. niger T21.

Electromobility shift assay (EMSA) was used to scan 600 bp of 5' cis regulatory sequence of Aspergillus niger (A. niger) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starchinduced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNase I footprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.

[Construction of recipient strain of expression-secretion system in filamentous fungi].

Glucoamylase overproducing A. niger T21 was mutated by UV mutagensis. An extracellular acid protease-deficient mutant, A. niger T21-201, which produced only 0.76% extracellular acid protease activity of the parent strain, was screened by casein-degradating plate and determination of protease activity. Moreover, the growth properties and the ability to secrete glucoamylase of A. niger T21-201 are identical to these of starting strain T21. The comparison of expression-secretion levels of heterologous gene in A. niger T21-201 and T21 was carried out with bacterial vhb as reporter, the level of expression-secretion of VHb in A. niger T21-201 was 6-7 times higher than that in T21, but the transcriptional levels of vhb gene in both strains were similar revealed by Northern blot. Therefore, it was demonstrated that the deficiency of acid protease of recipient T21-201 has significant effect on the protection of heterologous protein.