CREB: A multifaceted transcriptional regulator of neural and immune function in CNS tumors

in the CNS. CREB is a kinase inducible transcription factor which regulates many CNS functions, including neurogenesis, neuronal survival, neuronal activation and long-term memory. Here, we discuss how CREB-regulated mechanisms operating in diverse cell types, which control development and function of the CNS, are co-opted in CNS tumors.


Introduction
The cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a widely expressed, kinase inducible transcription factor, activated by growth factors, peptide hormones and calcium ions, and has a prominent role in the development and function of the CNS (Lonze and Ginty, 2002).By regulating the transcription of genes involved in long-term potentiation (LTP), a process by which the strength of synaptic connections between neurons is increased, CREB has been linked to mechanisms underpinning synaptic plasticity, learning and memory (Bourtchuladze et al., 1994).CREB dysfunction is also associated with a range of neurological and psychiatric disorders (Mertz et al., 2020); (Xiao et al., 2018).Neurophysiological functions regulated by CREB are co-opted in CNS cancers, including the most common CNS cancer, glioblastoma (GBM).The first direct evidence that CREB can impart oncogenic behaviors upon cells was the observation that CREB is elevated in blast cells from patients with acute lymphoid and myeloid leukemia, accompanied by amplification of the CREB1 gene (Shankar et al., 2005).Since this discovery, CREB-dependent transcriptional activation has been linked to oncogenesis via the regulation of cancer hallmark cellular functions, including cell proliferation and cell survival.Accumulating evidence suggests that CREB regulates malignancy in many solid cancers, including the CNS (Daniel et al., 2018).Cancers affecting the CNS are particularly fascinating with respect to CREB's role due to the co-option of several CREB-dependent functions regulating normal cell, neural and immune physiology, which converge Abbreviations: CREB, cAMP response element binding protein; cAMP, cyclic adenosine monophosphate; GBM, glioblastoma; LTP, long-term potentiation; bZIP, basic leucine zipper; CREM, cAMP responsive modulator; ATF1, activating transcription factor 1; PI3K, phosphatidylinositol-3-kinase; MAPK, mitogen-activated protein kinase; KID, kinase inducible domain; GHRH, growth hormone releasing hormone; BDNF, brain derived neurotrophic factor; AD, Alzheimer's disease; PKA, protein kinase A; GFP, green fluorescent protein; TAM, tumor associated macrophage; CBP, CREB Binding Protein.
to regulate oncogenesis.Here, we discuss CREB-dependent functions operating during CNS development and postnatal function in different cell types of the CNS tumor microenvironment.We highlight key discoveries which demonstrate that co-option of CREB-dependent neurophysiological and immune functions cooperate to promote malignancy, tumor cell invasion and protection of CNS cancer cells from immune attack.

CREB structure and function
CREB is a ubiquitous basic leucine zipper (bZIP) transcription factor with pleiotropic roles and is encoded by the CREB1 gene (Montminy and Bilezikjian, 1987).Two closely related genes, CREM and ATF1, encode for the proteins, cAMP responsive modulator (CREM) and activating transcription factor 1 (ATF1), respectively, which together with CREB, comprise the kinase inducible CREB/ATF transcription factor family (Fig. 1).CREB1 is the ancestral gene and only member of the CREB/ATF family described in several invertebrate species, including the freshwater coelenterate, Hydra (Galliot et al., 1995), sea slug, Aplysia (Dash et al., 1990) and fruit fly, Drosophila (Smolik et al., 1992).Unlike CREB, both CREM and ATF1 exhibit cell specific and developmentally restricted expression patterns.Genetic deletion experiments in mice have demonstrated a level of redundancy, so that when expressed in the same cell types, CREM and ATF1 can compensate for the loss of CREB with respect to fundamental cellular functions, including cell survival (Mantamadiotis et al., 2002); (Bleckmann et al., 2002).Importantly, while CREB protein levels may remain static, it is the phosphorylation of Ser 133 that dictates activity allowing for highly dynamic transcriptional regulation of many genes, which regulate neural and tumor cell functions.CREB homodimers or heterodimers with CREM or ATF1 bind to cAMP responsive elements (CRE), which are eight base pair sequences, where the consensus sequence is 5′ TGACGT(C/A)(G/A) 3′.CREB contributes to the transcriptional regulation of at least 4000 target genes (Zhang et al., 2005).

CREB signaling
Activation of the cAMP, phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways lead to the stimulation and nuclear translocation of several serine-threonine kinases which phosphorylate CREB on key serine residues, including serine at position 133 (Ser133), which is in the kinase inducible domain (KID) (Figs. 1 and 2) (Steven et al., 2020).Phosphorylation of Ser133 results in recruitment of CREB-binding protein (CBP)/p300 transcriptional coactivators and binding of the CREB KID to the CBP/p300 KIX domain.The CREB-CBP interaction activates transcription by acetylating nucleosomal histones and interaction with proteins of the transcription preinitiation complex, including RNA polymerase II (Shaywitz and Greenberg, 1999).Besides CBP/p300, other CREB transcriptional co-factors have also been identified, including transducer of CREB/ CREB-regulated transcription coactivator (TORC/CRTC) and DREAM (Conkright et al., 2003); (Ledo et al., 2002).

Cell-specific CREB-mediated gene expression
As CREB is a major hub of activity-driven cell signaling programs in many cell types, the question about how CREB orchestrates distinct functional behaviors, which converge to facilitate pro-oncogenic outcomes arises.Some of the answers are provided by data derived from several studies investigating cell specific CREB target genes.Comparison of gene promoter CREB binding and gene expression in three cell types: pheochromocytoma PC12 cell line, which exhibit neuronal characteristics, H4IIE hepatoma cells and primary cortical neurons determined that the different cell types, including neurons and glia, exhibit distinct CREB-dependent transcriptional programs (Cha-Molstad et al., 2004).By using chromatin immunoprecipitation and genomic footprinting assays to determine gene promoter occupancy by CREB, combined with mRNA expression analysis, this study showed that CREB exhibits cellspecific target gene promoter binding which correlates with mRNA expression levels.The study determined that CREB promoter binding correlates with DNA methylation.Another study reveals that even ontogenetically related cell types exhibit distinct CREB transcriptomes.Pardo and colleagues examined and compared the expression of CREB target genes in rat neurons and astrocytes (Pardo et al., 2017) and showed that there was only a minimal overlap between CREB target gene expression between astrocytes and neurons.Examination of 108 bona-fide target genes that are positively regulated by CREB in neurons and astrocytes, revealed that only two were common to both cell types.These studies provide insight into how CREB contributes to the regulation of diverse functions in tissues, which exhibit extensive cellular heterogeneity.

CREB in CNS development
In studies investigating the role of CREB in the development of multicellular organisms, it was demonstrated that CREB is required for Fig. 1.CREB family polypeptide domain structure.The major CREB, CREM and ATF1 isoforms are shown with the functional polypeptide domains aligned.Q1 and Q2 are the glutamine rich domains, which contribute to the strength of transcriptional activation.The α-domain is unique to the longest CREB isoform, CREBα, which also exhibits the highest transcriptional activation, compared to CREM, AFT1 and the other CREB isoforms.The kinase-inducible domain (KID) harbors the core conserved amino acid sequence (RRPSYRKIL), and includes a key serine residue (S), which is the substrate for upstream serine-threonine kinases.When the serine residue in the KID is phosphorylated, the KID forms a stable interaction with the transcriptional co-factors CBP and/or p300, to initiate the assembly of the transcription machinery.The bZip domain harbors the leucine-zipper domain, which is involved in protein dimerization, and harbors the DNA-binding domain.Created with BioRender.com.head regeneration in the freshwater polyp species, Hydra (Galliot et al., 1995).Further studies in several species contributed to the consensus view that CREB is essential for the normal development of the head and brain.For example, disruption of CREB translation in the clawed frog species Xenopus, led to aberrant head development, with microcephaly being the dominant phenotype (Lutz et al., 1999).Further insights into the role of CREB in regulating brain development come from experiments showing that inhibition of CREB in zebrafish embryos lead to reduced neural progenitor cell proliferation and aberrant midbrainhindbrain organization (Dworkin et al., 2007).
A series of mouse models generated in the laboratory of Günther Schütz, targeting all CREB-CREM-ATF1 family members in mice, provided unprecedented insight on the role of all the closely related CREB transcription factors (Fig. 3).The specific roles of specific CREB isoforms, CREM and ATF1 were investigated in mice at various stages of development, including cell-specific targeting of CREB in the brain.Several of the mutant mouse models are discussed below.
In the mouse olfactory bulb, which is a brain region exhibiting robust lifelong neurogenesis and neuronal migration, CREB regulates the differentiation and survival of immature neurons (Giachino et al., 2005).Targeted neural progenitor cell-specific deletion of CREB, using a nestin-Cre transgene, showed that CREB regulates hypothalamic development, where CREB mutant mice exhibit dwarfism due to the reduced expression of growth hormone releasing hormone (GHRH), due to attenuation of the hypothalamic-pituitary-growth hormone axis, resulting in dwarfism (Mantamadiotis et al., 2006).In vivo and in vitro experiments demonstrate that CREB regulates both neural precursor cell survival and proliferation, as well as stem cell functions in the subventricular zone (Dworkin et al., 2007); (Dworkin et al., 2009); (Herold et al., 2011).
Deletion of CREB and ATF1 in mouse embryonic stem cells, resulted in early preimplantation blastula stage embryonic death due to apoptosis of the pluripotent stem cells in the inner cell mass (Bleckmann et al., 2002).Deletion of only CREB in mouse embryonic stem cells results in multi-organ developmental delay and the mice died at birth due to severe lung underdevelopment (Rudolph et al., 1998).Upon closer examination of the brain in newborn mice, it was observed that they exhibited enlarged lateral ventricles and reduced thickness of the corpus callosum and the anterior commissure, suggesting a reduction in cellularity and/or neurogenesis during prenatal development.Many studies have since investigated the role of CREB in neurogenesis, all showing that CREB regulates neural progenitor cell expansion in the healthy mammalian brain and during post-injury regeneration (Zhu et al., 2004); (Herold et al., 2011); (Gundersen et al., 2013); (Xia et al., 2015).
A study investigating cerebellar development in chickens and rats showed that in granule cell precursors, CREB acts as an intermediary signaling-transcription factor, linking upstream activation by extracellular matrix proteins and downstream activation of sonic hedgehog (SHH), to promote granule cell differentiation (Pons et al., 2001).Transient activation of CREB in the thalamus during early postnatal life of the mouse contributes to the remodeling of retinogeniculate projections (Pham et al., 2001).This highlights the involvement of CREB in coordinating exquisite neurodevelopmental processes, including neural circuitry remodeling in the thalamus, which is exemplified by the development of the visual cortex during the first few weeks of life (Godement et al., 1984).Although the majority of studies investigating the role of CREB in brain relate to functions requiring neuronal activation, CREB-mediated signaling has also been shown to regulate cortical neuron differentiation, prior to the establishment of fully functional synaptic contacts, demonstrating that CREB also contributes to activityindependent neuronal functions (Landeira et al., 2018).

CREB regulates diverse neural functions
A landmark study conducted in Dr Michael Greenberg's laboratory demonstrated that transcriptional regulation of FOS was directly linked to CREB activation, resulting from membrane depolarization by calcium influx leading to neuronal stimulation or pharmacological induction of cAMP activity (Sheng et al., 1990).This observation coincided with the identification of an invertebrate CREB ortholog, where Nobel Laureate, Eric Kandel's laboratory, identified a DNA-binding phospho-protein in the sea slug Aplysia, the expression of which enhanced and stabilized neuronal synaptic strength (Dash et al., 1990).This work provided a molecular mechanism which explained prior observations by Kandel and colleagues on how short-term gill-withdrawal reflex memory was converted into a long-term, gill-withdrawal reflex memory in Aplysia (Castellucci and Kandel, 1974).Further studies demonstrated that CREB's role in promoting neuronal synaptic stability correlated with the stabilization of long term reflex memory in Aplysia (Bartsch et al., 1995), long-term memory in other invertebrates, including Drosophila (Yin et al., 1994) and hippocampal-dependent memory in mice (Bourtchuladze et al., 1994); (Kogan et al., 1997).Investigations using additional experimental animal models have supported a role for CREB in the regulation of memory-dependent behaviors, although there are reports downplaying CREB as a key transcriptional activator of hippocampaldependent memory (Perazzona et al., 2004); (Balschun et al., 2003).Genetic manipulation of CREB led to the discovery that this transcription factor regulates various types of memory and neural circuit function, with prominent roles in non-hippocampal memory, involving drug reward and addiction, anxiety, depression and fear conditioning and the regulation of key immediate early genes, including FOS (Valverde et al., 2004); (Balschun et al., 2003); (Josselyn et al., 2001); (Lamprecht et al., 1997); (Carlezon et al., 1998); (Chen et al., 2001).Genetic targeting of specific serine residues in CREB, which are phosphorylated, have revealed fascinating insights into more subtle CREB functions in the brain and how manipulation of the CREB signaling circuitry affects the brain and behavior.Substitution of serine at position 142 to alanine, showed that CREB Ser142 phosphorylation is involved in the entrainment of the mammalian circadian clock and the circadian locomotor activity, which was attenuated in mice lacking CREB Ser142 (Gau et al., 2002).A similar phenotype was observed when Ser133 was mutated to alanine (Wheaton et al., 2018), suggesting that phosphorylation of single serine residues in CREB leads to subtle stimulus-induced behavioral changes, and that there is likely an inbuilt compensatory mechanism(s), where multiple serine phosphorylation events regulate overlapping Fig. 3. CNS and non-CNS phenotypes in CREB knockout mice.CREB KO mice lack all CREB isoforms (Rudolph et al., 1998), die within 15 min of birth due to respiratory distress caused by severely delayed lung maturation.These mice also exhibit delayed brain development observed as hypocellularity and increased ventricular space and reduced thickness of the corpus callosum.Double knockout CREB and ATF1 mice, which bypass compensatory redundancy of these factors (Bleckmann et al., 2002), die at E3.5, prior to implantation stage and exhibit developmental arrest, as well as severe apoptosis of the inner mass cells.CREBαΔ mice are hypomorphs, lacking the two major CREB isoforms, CREBα and CREBΔ, resulting in upregulated expression of CREBβ (Hummler et al., 1994).CREBαΔ mice exhibit a deficiency in hippocampal-dependent long-term memory and an associated deficit in hippocampal long-term potentiation (LTP).Conditional mutant CREB mice, in which the CREB gene was specifically deleted in the CNS during early embryonic development, exhibit pituitary hypoplasia, dwarfism, and hormone dependent sex-specific deficits (Mantamadiotis et al., 2006).CREM, CREB double mutant mice, where CREB was deleted specifically in the brain in a CREM knockout background (Mantamadiotis et al., 2002), exhibited progressive neuronal loss in the hippocampus and striatum.Mice with pro-oncogenic brain-specific Pik3ca-Pten mutations targeted to neural progenitor cells and targeted CREB deletion in the same cells showed delayed CNS tumor growth (P.M. Daniel, Filiz, Brown, et al. 2018).Created with BioRender.com.functions.This is further highlighted by data demonstrating that mice with the serine to alanine mutation at position 133, express normal levels of several CREB target genes implicated in learning and memory, and showed no deficits in learning (Briand et al., 2015).

Neurodevelopmental disorders
Altered neuronal plasticity and cognitive impairments are common pathophysiological features of many psychiatric disorders (Arguello and Gogos, 2012).Chronic stress strongly contributes to the development of psychiatric illnesses by impacting neuroplasticity at several levels (Pittenger and Duman, 2008).Notably, CREB and BDNF (brain derived neurotrophic factor) are critically involved in these processes (Carlezon et al., 2005); (Aguilera and Liu, 2012).However, a causal link between CREB, its transcriptional activity and psychiatric disorders remains elusive.A correlation between CREB1 mutations and expression has been reported in studies examining genomic data from approximately 100,000 people, where approximately half were patients with either bipolar disorder, schizophrenia or unipolar major depressive disorder, and half were controls with no diagnosed neurological or psychological disorders (Xiao et al., 2018); (Li et al., 2014).Two single-nucleotide polymorphisms (SNPs) in the CREB-1 gene were discovered which were associated with an increased risk of bipolar disorder and abnormal hippocampal activation (Li et al., 2014).Importantly, the study by Xiao et al., examined CREB1 DNA sequence and mRNA expression data from a subset of patients across all psychiatric disorders.This identified that CREB1 mRNA expression was downregulated in psychiatric patients, compared with healthy controls.Protein-protein interaction analysis showed that CREB directly interacted with several psychiatric disorder risk genes, including kinases, AKT3 (AKT serine/threonine kinase 3) and MAPK3 (mitogen-activated protein kinase 3), which converge to directly or indirectly activate CREB, and CREB transcriptional co-factors, MEF2C (myocyte enhancer factor 2C) and EP300 (E1A binding protein p300) (Xiao et al., 2018).These genes have roles in brain development and neurodevelopmental disorders, including microcephaly and callosal hypoplasia, further supporting the role of CREB in signaling and transcriptional dysregulation that underpins these disorders (Boland et al., 2007); (Cosgrove et al., 2021); (Pucilowska et al., 2015); (Lipinski et al., 2022).
CREB regulates the expression of many genes associated with epileptogenesis, including BDNF (Esvald et al., 2020) and the calcium channel gene, CATSPER1 (Oviedo et al., 2018).CREB is also implicated in the regulation of glutamate homeostasis and signaling, with glutamate acting as a mediator of epileptogenesis in specific neuropathological conditions or following brain surgery (Navon et al., 2012); (Grove-Strawser et al., 2010); (Yuen et al., 2012).In transient global ischemia, pCREB expression levels increase during reperfusion.Ischemia-resistant dentate granule cells exhibit higher levels of CREB phosphorylation compared with ischemia-sensitive CA1 hippocampal neurons, suggesting a protective role of CREB phosphorylation (Hu et al., 1999).
Disruption of a signaling-transcription axis involving CREB and the numerous proteins directly interacting with CREB suggests molecular dysfunction, manifested as an aberration of gene expression affecting many genes regulating all stages of brain development and multiple brain functions.This type of transcriptional dysregulation is reminiscent of other diseases affecting the central nervous system, including Huntington's Disease, in which the mutant huntingtin protein harboring long stretches of glutamine residues, encoded by mutant CAG trinucleotide repeats in the Huntingtin gene, interferes with neuronal gene expression (Zhai et al., 2005).

CREB-dependent neuronal survival & neurodegeneration
Although diverse in etiology and neuroanatomy, neurodegenerative diseases are characterized by progressive cognitive and/or motor decline that directly associate with progressive neuronal loss.Alterations in neuron specific CREB function is one of the key molecular mechanisms regulating neuronal health and survival.A study showing okadaic acid induced apoptosis could be prevented in cultured neurons expressing high levels of pCREB provided the first direct evidence that CREB promotes neuronal survival (Walton et al., 1999).This was confirmed in a mouse model where conditional CNS-specific postnatal loss of CREB, combined with deletion of the CREM gene, to bypass transcriptional compensation by CREM, led to both hippocampal CA1 and striatal dopaminergic neuronal death (Mantamadiotis et al., 2002).The mechanisms underpinning CREB's role in regulating neuronal survival is complex but upregulation of activity-regulated inhibitor of death (AID) genes by CREB is one of the main mechanisms that have been identified (Tan et al., 2012).Another mechanism by which CREB may promote neuroprotection is via regulation of autophagy (Carloni et al., 2010).
CREB regulates the expression of key autophagy genes in liver cells (Seok et al., 2014) and appears to do the same in many cell types, including in neurons.Autophagy, like apoptosis, is a regulated cell death process which promotes the destruction of organelles that have accumulated toxic metabolites and damaged macromolecules, thus facilitating the maintenance of tissue and organ health (Mizushima and Levine, 2020).Since mature neurons are long-lived, they accumulate toxic metabolites and damaged macromolecules over time, and are particularly vulnerable to the consequences of autophagy deregulation.This was shown in neurons using rapamycin inhibition of mTOR, which led to an increase in CREB activation, resulting in enhanced autophagy and neuroprotection in mice with hypoxic brain injury (Balduini et al., 2012).
Impaired CREB expression and activation has been long recognized in Alzheimer's disease (AD) patient brains and rodent models of AD (Yamamoto-Sasaki et al., 1999); (Pugazhenthi et al., 2011).CREB activation is associated with neuroprotection, thought to occur via CREB regulated expression of the brain-derived neurotrophic factor gene (BDNF) (Shieh and Ghosh, 1999).AD is characterized by the loss of neurons and degeneration of synapses, compounded by the accumulation of β-amyloid (Aβ) peptides and phosphorylated tau protein.CREB is also implicated in AD pathology, where Aβ accumulation-induced memory deficits are, in part, caused by a disruption of CREB transactivation, resulting in the downregulation of the expression of neuroplasticity, memory genes, and neuroprotective genes, thus accounting for reduced BDNF expression in AD neurons (España et al., 2010); (Caccamo et al., 2010).

CREB regulates diverse glioma cell functions
The role of CREB in regulating cellular functions, which are also shared as hallmarks of cancer, has been investigated for over two decades and discussed in detail previously (Conkright and Montminy, 2005); (Sapio et al., 2020).Despite having a prominent role in brain functions and a growing body of research investigating CREB in non-CNS cancers, information about CREB in brain cancer has been slow to emerge.The first study reporting a role for CREB in glioma cell function, showed that the combined targeting of MAPK signaling and CREB, inhibited glioma cell proliferation (Cuevas et al., 2003).A comparison of glioma tissues from 64 patients representing low-grade glioma, oligodendroglioma, high-grade glioma and anaplastic astrocytoma, demonstrated that pCREB expression was present in 100% of astrocytoma samples and 46 % of oligodendroglioma samples, consistent with the view that CREB has a role in high grade cancer cell functions, including cell proliferation and cell invasion (Barresi et al., 2015).A correlation between increasing glioma tumor grade and pCREB expression was also seen in other studies, examining independent glioma tissue (Daniel et al., 2014).
Given the large number of genes whose mRNA expression is potentially regulated or co-regulated by CREB, it is unsurprising that CREB is involved in the control of many pro-oncogenic cellular functions in glioma cells.For instance, CREB activation via the PKA (protein kinase A)/cAMP pathway has been linked to glioma differentiation (Li et al., 2007).In cells derived from the pediatric primary brain cancer, medulloblastoma, CREB signaling activity correlates with cell differentiation and cell survival (Armandari et al., 2021).Activation of the PKA/ cAMP pathway also promotes apoptosis in glioma cells, but this appears to operate independently of CREB (Daniel et al., 2016).The diversity of stimuli which trigger similar outcomes via CREB in glioma cells, is highlighted by a study showing that nitric oxide stimulation of the inositol-requiring enzyme 1, requires downstream CREB activation to trigger cell death (Kim et al., 2010).These studies demonstrate the complexity of cell signaling and transcriptional control of oncogenesis in glioma cells and likely, in cancer cells, more broadly.Moreover, these studies demonstrate that although CREB is involved in regulating fundamental cell biology of cancer cells, some CREB-dependent functions are cancer cell specific and depend on the coincident co-expression and coactivation of cell signaling-transcription programs.
Spatial analysis of GBM tissue has demonstrated the existence of regionally heterogeneous PI3K and MAPK cell signaling and CREB activation (Daniel et al., 2018).This cell signaling heterogeneity reflects and underpins tumor heterogeneity, suggesting that, not only is there a diverse cellular heterogeneity but also the establishment of a regional and dynamic flux of cell activation and gene expression patterns, defining cell neighborhood-specific communication and function.The specific cell signal transduction pathways activated and co-activated in GBM tissue also correlate with PCNA and CD44 expression.Specifically, GBM regions where MAPK and CREB, but not PI3K signaling are activated, exhibit robust PCNA expression, suggesting that cells in these regions are proliferating, consistent with previous data demonstrating that CREB is required for efficient GBM cell proliferation, in vitro and in vivo (Daniel et al., 2018); (Daniel et al., 2014).Regions where activation of MAPK and CREB were high, also co-expressed high levels of CD44, suggesting that this activation axis regulates GBM cancer stem cell function and GBM cell invasion.This is consistent with previous work showing constitutive CREB activation in brain tumor cells, in which PI3K is also constitutively activated, and that CREB loss disrupts cancer cell invasion (Daniel et al., 2018).Independent experiments showed that CREB also regulates GBM stem cell function.Activation of CREB via CDK5, stimulated glioma stem cell expansion and conversely, inhibition of the CDK5-CREB axis had the opposite effect, and that reduction in glioma stem cell number was due to attenuation of glioma stem cell selfrenewal (Mukherjee et al., 2018).
Spatial analysis of cell signaling activation in GBM shows that pCREB + cell clusters are present in GBM, suggesting that dynamic and heterogeneous CREB activation, regulates cell-specific functions (Dinevska et al., 2022).Co-activation of pERK1/2 and pCREB was observed in dense neoplastic cell and tumor-associated endothelial cell clusters, where rapid cell expansion and migration occurs.Although most of the cells expressing pCREB in CNS tumors are neoplastic cells, tumor infiltrating immune cells also exhibited a grade-dependent increase in pCREB expression, with the most prominent immune cells being tumor associated microglia and macrophages (Dinevska et al., 2022).
CREB is also involved in a fascinating GBM cell-cell communication network, where GBM cell survival and cell proliferation is regulated in physically networked GBM cell clusters which communicate via the calcium-activated potassium channel, KCa3.1.The networked GBM cells establish rhythmic activation of intracellular MAPK, NF-ϰB and CREB (Hausmann et al., 2022).This rhythmic GBM cell activation resembles a novel cellular communication mechanism recently discovered in GBM, which involves cell-to-cell communication between neurons and glioma cells, which is discussed in detail in section 3.2.These novel observations highlight co-option of a neurophysiological process in malignant brain cancer.
Compelling evidence that CREB directly regulates brain cancer cell malignancy has been demonstrated in studies investigating the biology and pathology of glioma using a genetically engineered mouse model.Mice with neural progenitor cell-specific oncogenic mutations with mutations targeting the PI3K-PTEN pathway, develop high-grade malignant glioma (Daniel et al., 2018).Mice with concomitant targeted deletion of the Creb1 gene exhibit extended symptom-free survival, almost double that of the Creb1 wild-type mice.The Creb1-deleted tumors displayed a phenotype resembling a lower grade tumor, with no evidence of tumor cell invasion into the healthy brain parenchyma, low cell density and large neoplastic cells, unlike the Creb1 wild-type tumors which showed high tumor cell density, extensive invasion into the healthy brain parenchyma and along white matter tracts.Creb1-deleted tumor cells derived from the mice exhibited a lower proliferation rate, due to an aberration in the cell cycle, evidenced by an increase in cells in G0/G1-phase and fewer cells in S-phase, consistent with CREBs role in human GBM cell lines, where CREB was shown to directly regulate the expression of several cell cycle proteins, including cyclin D1 (Daniel et al., 2014).

CREB in neural-cancer crosstalk
A role for neural regulation of tumor biology has been considered since the first report describing the presence of nerves in tumors (Young, 1897).The role of nerve cells in specific cancer types remains a relatively new area of research, with significant gaps in knowledge.It has been demonstrated that cancer-neuronal crosstalk regulates both cancer cell intrinsic functions, including neoplastic cell proliferation and migration, as well as indirect effects, mediated via regulation of the tumor immune microenvironment, comprehensively reviewed by Reavis and colleagues (Reavis et al., 2020).Most tumor innervation research to date has focused on non-CNS cancers, and tumor innervation by the peripheral nervous system.As the brain is unique with respect to innervation, it is only recently that an understanding of the functional interaction of brain tumor cells and nerve cells in the CNS, has come to light.A series of studies have led the path toward understanding the mechanisms underpinning neural-brain cancer crosstalk and how this interaction contributes to oncogenesis.The first ground-breaking studies demonstrated that glioma cells can form electrically and chemically active synapses with surrounding neurons, where the glioma cells are the postsynaptic cells, and that excitatory calcium-stimulated glutamatergic activation of the presynaptic neurons stimulates glioma cell proliferation (Venkataramani et al., 2019); (Venkatesh et al., 2019).Extending these findings, Venkataramani and colleagues showed that upon pharmacological stimulation of cells in glioma-neuronal co-cultures, pCREB expression increased in glioma cells (Venkataramani et al., 2022).The functional consequences of this stimulus were investigated in mice, where green fluorescent protein (GFP)-tagged glioma cells were transplanted into brains and monitored by intravital imaging.Glioma cells exhibited enhanced migratory speed, migratory branching and invasive activity.Administration of a small molecule CREB inhibitor, inhibited glioma cell invasion, an effect attributed to the inhibition of calcium-dependent neuronal activation and reduction in the formation of tumor microtubes, which are thought to be important for cell-to-cell communication and facilitating cell invasion.The mechanism regulating neural-glioma synapse generation may be a pathological mimic of the physiological role which CREB plays in synapse formation.CREB activation in neurons enhances structural plasticity, where there is an increase in the formation of naive synapses, which are then available for the generation of mature functional synapses, formed by future experience-dependent stimulation (Marie et al., 2005).CREB is also required for spine density proliferation in stimulated neurons (Murphy and Segal, 1997), which, in neural-glioma interactions, likely promotes stabilization and enhanced pro-oncogenic communication between the interacting cells.

CREB-regulated tumor immunity
The GBM tumor microenvironment is highly immunosuppressive, with high levels of anti-inflammatory cytokines, many of which are secreted by pro-tumor, immunosuppressive immune cells, including monocyte-derived macrophages (Klemm et al., 2020).Tumor associated macrophages (TAMs), which include macrophages and microglia, are considered to be the primary immune cells that establish an immunosuppressive microenvironment in brain tumors, and can represent up to half of all cells in the tumor microenvironment (Andersen et al., 2021); (Grabowski et al., 2021); (Darmanis et al., 2017).The immunosuppressive milieu in the brain tumor microenvironment is complex and composed of many co-opted cytokines and chemokines, including arginase-1 (ARG1), transforming growth factor beta (TGFβ) and interleukin-10 (IL-10) (Klemm et al., 2020).IL-10 is an archetypal antiinflammatory cytokine which has emerged as an important immunosuppressive factor in brain cancer, with high IL-10 receptor alpha (IL10RA) and IL-10 receptor beta (IL10RB) mRNA expression, associated with poor outcome in patients with GBM (Widodo et al., 2021).Spatial transcriptomics has also been pivotal in identifying IL-10 secreting macrophages as major determinants of T-cell dysfunction and tumor immunosuppression in GBM (Ravi et al., 2022).CREB is one of the key transcription factors regulating IL-10 transcription, with deletion of CREB binding sites in the IL10 promoter, diminishing IL-10 expression in monocytes (Platzer et al., 1999).In breast cancer, CREB activation is important for the polarization of TAMs, from an anti-tumor M1-like phenotype, to an immunosuppressive pro-tumor M2-like state (Na et al., 2020); TAMs with high pCREB expression were immunosuppressive M2-like cells.Moreover, in vitro, and in vivo, mouse experiments have demonstrated that pharmacological inhibition of the upstream CREB kinase PKA, blocked pCREB activation and M2-like TAM polarization led to reduced tumor growth.In another study investigating breast cancer immuno-oncology, the CREB-specific small molecule inhibitor, 666-15, blocked the expression of immunosuppressive factor, ARG1 in macrophages (Su et al., 2021).In a pancreatic ductal adenocarcinoma (PDAC) mouse, CREB inhibition reversed tumor immunosuppression.Specifically, this study reported that administration of the CREB inhibitor, 666-15 in the PDAC mice, reduced the number of tumor infiltrating F4/80-positive immunosuppressive TAMs and FoxP3-positive regulatory T cells (Tregs), which was accompanied by increased infiltration of CD8 + T cells (Srinivasan et al., 2018).
A fascinating macrophage-neural communication mechanism, in which attenuation of CREB activity occurs, has been proposed in AD pathology (Nancy (Bartolotti and Lazarov, 2019).Studies suggest that peripheral blood monocytes (PMBC), which under pathological conditions, differentiate into macrophages, regulate neural function in the AD brain, either directly, by crossing the blood brain barrier or indirectly, by supplying macrophage-derived, secreted factors which disrupt neural cell function (N.(Bartolotti et al., 2016).Attenuation of CREB function in PBMC would lead to reduced expression of key anti-inflammatory cytokines, such as IL-10, thereby shifting the balance of the brain tissue toward a pro-inflammatory milieu.Attenuation of CREB activation in neurons would lead to reduced expression of pro-survival signals and subsequent neuronal loss, contributing to the overt phenotype associated with AD.The observations with respect to macrophage biology in the AD brain are consistent with the biology of TAMs in brain tumors, which contribute to tumor immunosuppression.
In summary, multiple independent studies show that CREB is a key transcriptional regulator of cytokines, chemokines and growth factors produced by TAMs and other immune cells, which promote immunosuppression (Fig. 4).Although experiments have not been conducted in brain tumors, similar CREB-dependent tumor immune mechanisms likely operate in cancers of the CNS.

Conclusion
Based on the well-defined biological functions regulated by CREB in the healthy CNS, in cancer cells, and the innate immune system, targeting CREB in CNS cancers would interfere with at least three key prooncogenic hallmarks, which together, are unique to the CNS (Fig. 4).The three hallmarks include cancer cell intrinsic functions, as well as CNS tumor microenvironment functions.Two hallmarks are common to all cancers, namely cancer cell proliferation and macrophage-mediated immunosuppression.The third hallmark, neural-cancer crosstalk, although not unique to the CNS, likely plays a more prominent role in the CNS, compared to non-CNS tumors.As CREB is a prominent molecular regulator of neurophysiological functions in the healthy brain, malignant cells in the CNS co-opt CREB-regulated neural functions to propagate neural-cancer crosstalk, which act in concert with CREBregulated cancer proliferation and CREB-regulated immunosuppression, to promote aggressive cancer growth and spread.
Compared to targeting more tractable protein structures, such as kinase active sites, transcription factors were historically viewed as 'undruggable'.This view persisted due to challenges associated with the nature of the molecular structure of many transcription factors and the inability to efficiently target protein-DNA interactions.The lack of inhibitors targeting protein-DNA binding is likely due to the highly positively charged DNA binding interfaces being difficult targets for small-molecule inhibitors.However, protein-protein interactions between transcription factors and transcriptional coactivators are more easily targeted.A series of naphthol-based small molecules have been synthesized which target the CREB-CBP interaction, which is required for CREB's transcriptional activity.Several naphthol-based small molecule inhibitors of CREB have been developed (Best et al., 2004); (Xie et al., 2015); (Mitton et al., 2016).Experiments in mice have demonstrated that these CREB inhibitors disrupt tumor growth in several mouse models by disrupting cancer cell proliferation and reducing the number of immunosuppressive tumor infiltrating immune cells (Kim et al., 2021); (Xie et al., 2015); (Qin et al., 2020); (Srinivasan et al., 2018); (Kim et al., 2022).
As CREB is widely expressed, there is potential for substantial offtarget effects, which must be considered when assessing the safety of pharmacological inhibition.Although studies suggest that pharmacological inhibition of CREB would be safe in adults, CREB inhibition is likely to disrupt organ development during embryogenesis.CREB is critical for efficient neurogenesis during pre-and postnatal brain development, and that constitutive CREB activity is required for neural progenitor cell expansion and survival (Dworkin et al., 2007); (Dworkin et al., 2009); (Mantamadiotis et al., 2012).However, in adult mice, sustained administration of the naphthol-based small molecule CREB inhibitor 666-15 is safe, with no observed deleterious effects in liver, heart and kidney, following administration of 10 mg/kg five days a week, for three weeks (Li et al., 2016).Given the poor outlook for adult patients with GBM and the limited treatment options, the benefit in disease control using a CREB inhibitor to control tumor development will likely outweigh short-term undesirable off-target effects.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 2 .
Fig. 2. The canonical CREB signaling axis.Activation of cell surface receptors by ligands activates diverse downstream cytoplasmic proteins which eventually activate diverse serine-threonine kinases, which act as CREB-kinases, including protein kinase A, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP2) and AKT1 (also known as protein kinase B).CREB, which is constitutively expressed and present in the cell nucleus, is phosphorylated at several serine residues, with Ser133, located in the Kinase-Inducible-Domain (KID) being the critical phosphorylated residue.Phosphorylation of Ser133 stabilizes the binding of CREB homo-or hetero-dimers to cAMP Response Elements (CRE) in gene promoters.Phosphorylation also triggers the recruitment of transcriptional co-factors, including CREB Binding Protein (CBP) or the closely related p300.The recruitment of the CREB-binding co-factors lead to recruitment of a repertoire of transcriptional cofactors to assemble the transcriptional machinery, including activation of RNA polymerase II and initiation of transcription of the target genes at the transcription start site (TSS).Created with BioRender.com.

Fig. 4 .
Fig. 4. CREB hallmarks of cancer in the central nervous system.CREB regulates intrinsic oncogenic functions in neoplastic cells, including cell proliferation by direct transcriptional regulation of cell cycle factors, including cyclin D1 (CCND1).CREB also regulates the brain tumor microenvironment by direct transcriptional regulation of immunosuppressive factors in tumor associated macrophages (M2 macrophages), including IL-10, ARG1 and VEGFA.CREB also regulates neural-glioma communication, where neuronal stimulation results in CREB activation which triggers the expression of immediate early genes, including FOS and neural growth factors, such as brain derived neurotrophic factor (BDNF), which lead to the formation and stabilization of neuronal-glioma cell synapses, which regulate migration and invasion of glioma cells into the healthy brain parenchyma.Created with BioRender.com.