3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

Single base-pair resolution analysis of DNA binding motif with MoMotif reveals an oncogenic function of CTCF zinc-finger 1 mutation.

Defining the impact of missense mutations on the recognition of DNA motifs is highly dependent on bioinformatic tools that define DNA binding elements. However, classical motif analysis tools remain limited in their capacity to identify subtle changes in complex binding motifs between distinct conditions. To overcome this limitation, we developed a new tool, MoMotif, that facilitates a sensitive identification, at the single base-pair resolution, of complex, or subtle, alterations to core binding motifs, discerned from ChIP-seq data. We employed MoMotif to define the previously uncharacterized recognition motif of CTCF zinc-finger 1 (ZF1), and to further define the impact of CTCF ZF1 mutation on its association with chromatin. Mutations of CTCF ZF1 are exclusive to breast cancer and are associated with metastasis and therapeutic resistance, but the underlying mechanisms are unclear. Using MoMotif, we identified an extension of the CTCF core binding motif, necessitating a functional ZF1 to bind appropriately. Using a combination of ChIP-Seq and RNA-Seq, we discover that the inability to bind this extended motif drives an altered transcriptional program associated with the oncogenic phenotypes observed clinically. Our study demonstrates that MoMotif is a powerful new tool for comparative ChIP-seq analysis and characterising DNA-protein contacts.

Achieving clinical success with BET inhibitors as anti-cancer agents.

The transcriptional upregulation of oncogenes is a driving force behind the progression of many tumours. However, until a decade ago, the concept of 'switching off' these oncogenic pathways represented a formidable challenge. Research has revealed that members of the bromo- and extra-terminal domain (BET) motif family are key activators of oncogenic networks in a spectrum of cancers; their function depends on their recruitment to chromatin through two bromodomains (BD1 and BD2). The advent of potent inhibitors of BET proteins (BETi), which target either one or both bromodomains, represents an important step towards the goal of suppressing oncogenic networks within tumours. Here, we discuss the biology of BET proteins, advances in BETi design and highlight potential biomarkers predicting their activity. We also outline the logic of incorporating BETi into combination therapies to enhance its efficacy. We suggest that understanding mechanisms of activity, defining predictive biomarkers and identifying potent synergies represents a roadmap for clinical success using BETi.

HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site.

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lys in each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCE The results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lys during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I.

CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.

Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes.

Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary.

The p16(INK4a) tumor suppressor gene is a frequent target of epigenetic inactivation in human cancers, which is an early event in breast carcinogenesis. We describe the existence of a chromatin boundary upstream of the p16 gene that is lost when this gene is aberrantly silenced. We show that the multifunctional protein CTCF associates in the vicinity of this boundary and absence of binding strongly coincides with p16 silencing in multiple types of cancer cells. CTCF binding also correlates with RASSF1A and CDH1 gene activation, and CTCF interaction is absent when these genes are methylated and silenced. Interestingly, defective poly(ADP-ribosyl)ation of CTCF and dissociation from the molecular chaperone Nucleolin occur in p16-silenced cells, abrogating its proper function. Thus, destabilization of specific chromosomal boundaries through aberrant crosstalk between CTCF, poly(ADP-ribosyl)ation, and DNA methylation may be a general mechanism to inactivate tumor suppressor genes and initiate tumorigenesis in numerous forms of human cancers.

Chemotherapy reduces PARP1 in cancers of the ovary: implications for future clinical trials involving PARP inhibitors.

BACKGROUND: PARP inhibitors have shown promising clinical results in cancer patients carrying BRCA1/2 mutations. Their clinical efficacy could logically be influenced by PARP1 protein levels in patient tumors. METHODS: We screened three cohorts of patients with ovarian cancer, totaling 313 samples, and evaluated PARP1 protein expression by immunohistochemistry with further validation by western blotting. RESULTS: We observed that up to 60 % of tumors showed little PARP1 protein expression. In serous ovarian tumors, comparing intratumoral PARP1 expression between chemo-naïve and post-chemotherapy patients revealed a decrease in intratumoral PARP1 following chemotherapy in all three cohorts (immunohistochemistry: p < 0.001, n = 239; western blot: p = 0.012, n = 74). The findings were further confirmed in a selection of matched samples from the same patients before and after chemotherapy. CONCLUSION: Our data suggest that patients should be screened for PARP1 expression prior to therapy with PARP inhibitors. Further, the observed reduction of intratumoral PARP1 post-chemotherapy suggests that treating chemo-naïve patients with PARP inhibitors prior to the administration of chemotherapy, or concurrently, might increase the responsiveness to PARP1 inhibition. Thus, a change in the timing of PARP inhibitor administration may be warranted for future clinical trials.

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors.

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.

p66ShcA potentiates the cytotoxic response of triple-negative breast cancers to PARP inhibitors.

Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.

Reprogramming of Nucleotide Metabolism Mediates Synergy between Epigenetic Therapy and MAP Kinase Inhibition.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Retinoic acid modulates chromatin to potentiate tumor necrosis factor alpha signaling on the DIF2 promoter.

Transcriptional activation by nuclear hormone receptors is well characterized, but their cooperation with other signaling pathways to activate transcription remains poorly understood. Tumor necrosis factor alpha (TNFalpha) and all-trans retinoic acid (RA) induce monocytic differentiation of acute promyelocytic leukemia (APL) cells in a synergistic manner. We used the promoter of DIF2, a gene involved in monocytic differentiation, to model the mechanism underlying the cooperative induction of target genes by RA and TNFalpha. We show a functional RA response element in the DIF2 promoter, which is constitutively bound by PML/RARalpha in APL cells. RA stimulates release of corepressors and recruitment of chromatin modifying proteins and additional transcription factors to the promoter, but these changes cause only a modest induction of DIF2 mRNA. Co-stimulation with RA plus TNFalpha facilitates binding of NF-kappaB to the promoter, which is crucial for full induction of transcription. Furthermore, RA plus TNFalpha greatly enhanced the level of RNA Pol II phosphorylation on the DIF2 promoter, via synergistic recruitment of TFIIH. We propose that RA mediates remodeling of chromatin to facilitate binding of transcription factors, which cooperate to enhance Pol II phosphorylation, providing a mechanism whereby nuclear receptors interact with other signaling pathways on the level of transcription.

Antitumor activity of the dual BET and CBP/EP300 inhibitor NEO2734.

Bromodomain and extra-terminal domain (BET) proteins, cyclic adenosine monophosphate response element-binding protein (CBP), and the E1A-binding protein of p300 (EP300) are important players in histone acetylation. Preclinical evidence supports the notion that small molecules targeting these proteins individually or in combination can elicit antitumor activity. Here, we characterize the antitumor activity of the pan BET/CBP/EP300 inhibitor NEO2734 and provide insights into its mechanism of action through bromodomain-binding assays, in vitro and in vivo treatments of cancer cell lines, immunoblotting, and transcriptome analyses. In a panel of 60 models derived from different tumor types, NEO2734 exhibited antiproliferative activity in multiple cell lines, with the most potent activity observed in hematologic and prostate cancers. Focusing on lymphoma cell lines, NEO2374 exhibited a pattern of response and transcriptional changes similar to lymphoma cells exposed to either BET or CBP/EP300 inhibitors alone. However, NEO2734 was more potent than single-agent BET or CBP/EP300 inhibitors alone. In conclusion, NEO2734 is a novel antitumor compound that shows preferential activity in lymphomas, leukemias, and prostate cancers.

Small-Cell Carcinoma of the Ovary, Hypercalcemic Type-Genetics, New Treatment Targets, and Current Management Guidelines.

Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and highly aggressive ovarian malignancy. In almost all cases, it is associated with somatic and often germline pathogenic variants in SMARCA4, which encodes for the SMARCA4 protein (BRG1), a subunit of the SWI/SNF chromatin remodeling complex. Approximately 20% of human cancers possess pathogenic variants in at least one SWI/SNF subunit. Because of their role in regulating many important cellular processes including transcriptional control, DNA repair, differentiation, cell division, and DNA replication, SWI/SNF complexes with mutant subunits are thought to contribute to cancer initiation and progression. Fewer than 500 cases of SCCOHT have been reported in the literature and approximately 60% are associated with hypercalcemia. SCCOHT primarily affects females under 40 years of age who usually present with symptoms related to a pelvic mass. SCCOHT is an aggressive cancer, with long-term survival rates of 30% in early-stage cases. Although various treatment approaches have been proposed, there is no consensus on surveillance and therapeutic strategy. An international group of multidisciplinary clinicians and researchers recently formed the International SCCOHT Consortium to evaluate current knowledge and propose consensus surveillance and therapeutic recommendations, with the aim of improving outcomes. Here, we present an overview of the genetics of this cancer, provide updates on new treatment targets, and propose management guidelines for this challenging cancer.

Methionine Metabolism Shapes T Helper Cell Responses through Regulation of Epigenetic Reprogramming.

Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.

Beyond EZH2: is the polycomb protein CBX2 an emerging target for anti-cancer therapy?

Introduction: Epigenetic modifications are important regulators of transcription and appropriate gene expression answering an environmental stimulus. In cancer, these epigenetic modifications are altered, which impact the transcriptome, promoting initiation and cancer progression. Thus, targeting epigenetic machinery has proven to be an efficient cancer therapy. Areas covered: We review CBX2 as a therapeutic target. CBX2 is a polycomb protein, responsible for polycomb-repressive complex 1 (PRC1) targeting to chromatin via recognition of the repressive mark H3K27me3. Mechanistically, CBX2 overexpression may be implicated in poor survival by maintaining cancer stem cells in an undifferentiated state and via repression of tumor suppressors. We discuss strategies used to target CBX proteins and provide insights into biomarker considerations that may be important when targeting CBX family members for anti-cancer therapy. Expert opinion: CBX2 inhibition is a promising approach for the targeting of polycomb complexes in the cancer stem cell niche. However, extensive optimization of the current field of small molecules targeting CBX family proteins will be critical to reach in vivo, or clinical, utility.