Epigenetic Therapy for Epithelioid Sarcoma.

Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer.

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/ß-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.

Acetylation Enhances TET2 Function in Protecting against Abnormal DNA Methylation during Oxidative Stress.

TET proteins, by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are hypothesized, but not directly shown, to protect promoter CpG islands (CGIs) against abnormal DNA methylation (DNAm) in cancer. We define such a protective role linked to DNA damage from oxidative stress (OS) known to induce this abnormality. TET2 removes aberrant DNAm during OS through interacting with DNA methyltransferases (DNMTs) in a "Yin-Yang" complex targeted to chromatin and enhanced by p300 mediated TET2 acetylation. Abnormal gains of DNAm and 5hmC occur simultaneously in OS, and knocking down TET2 dynamically alters this balance by enhancing 5mC and reducing 5hmC. TET2 reduction results in hypermethylation of promoter CGIs and enhancers in loci largely overlapping with those induced by OS. Thus, TET2 indeed may protect against abnormal, cancer DNAm in a manner linked to DNA damage.

Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia.

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

A phase II study of guadecitabine combined with irinotecan vs regorafenib or TAS-102 in irinotecan-refractory metastatic colorectal cancer patients.

DNA methyltransferase inhibitors (DNMTi) have demonstrated benefit in reversing resistance to systemic therapies for several cancer types. In a phase II trial of guadecitabine and irinotecan compared to regorafenib or TAS-102 in pts with advanced mCRC refractory to irinotecan. Patients with mCRC refractory to irinotecan were randomized 2:1 to guadecitabine and irinotecan (Arm A) vs standard of care regorafenib or TAS-102 (Arm B) on a 28-day cycle. Between January 15, 2016 and October 24, 2018, 104 pts were randomized at four international sites, with 96 pts undergoing treatment, 62 in Arm A and 34 in Arm B. Median overall survival was 7.15 months for Arm A and 7.66 months for Arm B (HR 0.93, 95% CI: 0.58-1.47, P = .75). The Kaplan-Meier rates of progression free survival at 4 months were 32% in Arm A and 26% in Arm B. Common ≥Grade 3 treatment related adverse events in Arm A were neutropenia (42%), anemia (18%), diarrhea (11%), compared to Arm B pts with neutropenia (12%), anemia (12%). Guadecitabine and irinotecan had similar OS compared to standard of care TAS-102 or regorafenib, with evidence of target modulation. Clinical trial information: NCT01896856.

Pharmacologic induction of innate immune signaling directly drives homologous recombination deficiency.

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.

Betacoronavirus-specific alternate splicing.

Viruses can subvert a number of cellular processes including splicing in order to block innate antiviral responses, and many viruses interact with cellular splicing machinery. SARS-CoV-2 infection was shown to suppress global mRNA splicing, and at least 10 SARS-CoV-2 proteins bind specifically to one or more human RNAs. Here, we investigate 17 published experimental and clinical datasets related to SARS-CoV-2 infection, datasets from the betacoronaviruses SARS-CoV and MERS, as well as Streptococcus pneumonia, HCV, Zika virus, Dengue virus, influenza H3N2, and RSV. We show that genes showing differential alternative splicing in SARS-CoV-2 have a similar functional profile to those of SARS-CoV and MERS and affect a diverse set of genes and biological functions, including many closely related to virus biology. Additionally, the differentially spliced transcripts of cells infected by coronaviruses were more likely to undergo intron-retention, contain a pseudouridine modification, and have a smaller number of exons as compared with differentially spliced transcripts in the control groups. Viral load in clinical COVID-19 samples was correlated with isoform distribution of differentially spliced genes. A significantly higher number of ribosomal genes are affected by differential alternative splicing and gene expression in betacoronavirus samples, and the betacoronavirus differentially spliced genes are depleted for binding sites of RNA-binding proteins. Our results demonstrate characteristic patterns of differential splicing in cells infected by SARS-CoV-2, SARS-CoV, and MERS. The alternative splicing changes observed in betacoronaviruses infection potentially modify a broad range of cellular functions, via changes in the functions of the products of a diverse set of genes involved in different biological processes.

Targeting the cancer epigenome for therapy.

Next-generation sequencing has revealed that more than 50% of human cancers harbour mutations in enzymes that are involved in chromatin organization. Tumour cells not only are activated by genetic and epigenetic alterations, but also routinely use epigenetic processes to ensure their escape from chemotherapy and host immune surveillance. Hence, a growing emphasis of recent drug discovery efforts has been on targeting the epigenome, including DNA methylation and histone modifications, with several new drugs being tested and some already approved by the US Food and Drug Administration (FDA). The future will see the increasing success of combining epigenetic drugs with other therapies. As epigenetic drugs target the epigenome as a whole, these true 'genomic medicines' lessen the need for precision approaches to individualized therapies.

Cancer epigenetics: tumor heterogeneity, plasticity of stem-like states, and drug resistance.

The existence of subpopulations of cells in cancers with increased tumor-initiating capacities and self-renewal potential, often termed "cancer stem cells," is a much discussed and key area of cancer biology. Such cellular heterogeneity is very important because of its impact on therapy and especially states of treatment resistance. A major question is whether there is plasticity for evolution of these cell states during tumorigenesis that can involve movement between cell populations in a reversible fashion. In this review, we discuss the possible role of epigenetic abnormalities as well as genetic alterations in such dynamics and in the creation of cellular heterogeneity in cancers of all types.

DNA methyltransferase inhibitors induce a BRCAness phenotype that sensitizes NSCLC to PARP inhibitor and ionizing radiation.

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.

Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential.

Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden.

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality.

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.