Gallbladder cancer (GBC) is a highly aggressive malignancy lacking clinically available targeted therapeutic agents. Super-enhancers (SEs) are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in cancers. To study SE in GBC, the genomic distribution of H3K27ac is profiled in multiple GBC tissue and cell line samples to establish the SE landscape and its associated core regulatory circuitry (CRC). The biliary lineage factor SOX9 and Wnt pathway effector TCF7L2, two master transcription factor (TF) candidates identified by CRC analysis, are verified to co-occupy each other's SE region, forming a mutually autoregulatory loop to drive oncogenic SE reprogramming in a subset of GBC. The SOX9/TCF7L2 double-high GBC cells are highly dependent on the two TFs and enriched of SE-associated gene signatures related to stemness, ErbB and Wnt pathways. Patients with more such GBC cells exhibited significantly worse prognosis. Furthermore, SOX9/TCF7L2 double-high GBC preclinical models are found to be susceptible to SE-targeted CDK7 inhibition therapy in vitro and in vivo. Together, this study provides novel insights into the epigenetic mechanisms underlying the oncogenesis of a subset of GBCs with poorer prognosis and illustrates promising prognostic stratification and therapeutic strategies for treating those GBC patients in future clinical trials.

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.

Supplementary Figs. S1 to S9 Supplementary table legends

Supplementary Table S1_Mouse_ sgRNA library_Sequences

Supplementary Table S2_Candidates_ sgRNA library screening results

Supplementary Table S3_Short Tandem Repeat (STR) analysis

Supplementary Table S5_PCR and qPCR primers

Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with MYC amplification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; significantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confirmed the therapeutic efficacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1 , two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.

Abstract Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. Significance: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.

PURPOSE:In medulloblastoma (MB), group 3 (G3) patients with MYC amplification tend to exhibit worse prognosis, thus creating a need for novel effective therapies. As the driver and crucial dependency for MYC-amplified G3-MB, MYC has been proven to be a prospective therapeutic target. Here, we aimed to identify novel effective therapeutic strategies against MYC-amplified G3-MB via targeting MYC translation.MATERIALS AND METHODS:Major components of translation initiation complex eIF4F were subjected to MB tumor dataset analysis, and EIF4A1 was identified to be a potential therapeutic target of MYC-amplified G3-MB. Validation was performed through genetic or pharmacological approaches with multiple patient-derived tumor models of MYC-amplified G3-MB in vitro and in vivo. Underlying mechanisms were further explored by Western blot, quantitative real-time PCR and mass spectrometry (MS) analyses.RESULTS:MB tumor datasets analyses showed that EIF4A1 was significantly up-regulated in G3-MB patients relative to normal cerebella, positively correlated with MYC in G3-MB at transcriptional level and a crucial cancer dependency in MYC-amplified G3-MB cells. Targeting EIF4A1 with a CRISPR/Cas9 approach or small-molecule inhibitor silvestrol effectively attenuated growth in multiple preclinical models of MYC-amplified G3-MB via blocking proliferation and inducing apoptosis. Mechanistically, EIF4A1 inhibition effectively impeded MYC expression at translational level, and its potency was positively associated with MYC level. Whole-proteome MS analysis of silvestrol-treated cells further unveiled other biological functions and pathways influenced by EIF4A1 inhibition.CONCLUSION:Our investigation shows that interrupting MYC translation by EIF4A1 inhibition could be a potential effective therapeutic approach when treating patients with MYC-amplified G3-MB.

5-Hydroxymethylcytosine (5hmC) is a DNA modification that is generated by the oxidation of 5-methylcytosine (5mC) in a reaction catalyzed by the ten-eleven translocation (TET) family enzymes. It tends to mark gene activation and affects a spectrum of developmental and disease-related biological processes. In this manuscript, we present a 5hmC selective chemical labelling technology (hmC-Seal) to capture and sequence 5hmC-containing DNA fragments with low input. We tested 10 tumour/adjacent colon cancer tissues and 10 tumour/healthy plasma samples. Furthermore, we tested if this methodology could generate the 5hmC differential genes among cancer patients, healthy controls and precancerous adenoma patients from plasma. Robust cancer-specific epigenetic signatures were identified for colon cancers. The results show that 5hmC is mainly distributed in gene active regions. The results also indicate the potential application of 5hmC change signals in early stage of colon cancer, even show potential in the diagnosis of precancerous adenoma. We demonstrated the robustness of the 5hmC-Seal method in tissue and cell-free DNA (cfDNA) as potential biomarkers. Moreover, this study provides the potential value and feasibility of 5hmC-Seal approach on colorectal cancer (CRC) early detection. We believe this strategy could be an effective liquid biopsy-based diagnosis and a potential prognosis method for colon cancer using cfDNA.

The aberrant hedgehog (Hh) pathway plays important roles in multiple cancer types, therefore serving as a promising drug target. Current clinically available hedgehog-targeted drugs act mostly by antagonizing the upstream component smoothened; however, both primary and acquired resistance to FDA-approved smoothened inhibitor (SMOi) drugs have been described. We have recently demonstrated that the BET inhibitor effectively suppresses SMOi-resistant Hh-driven cancers through antagonizing transcription of GLI1 and GLI2, the core transcriptional factors of Hh pathway, suggesting epigenetic or transcriptional targeted therapy represents an anti-Hh therapeutic strategy that can overcome SMOi resistance. Here we performed an unbiased screening of epigenetic or transcriptional targeted small molecules to test their inhibitory effects on GLI1 and GLI2 transcription or cell viability of Hh-driven tumor lines. THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), is identified as the top hit in our screening. We then confirmed that antagonizing CDK7 by either small-molecule inhibitors or the CRISPR-Cas9 approach causes substantial suppression of GLI1 and GLI2 transcription, resulting in effective inhibition of Hh-driven cancers in vitro and in vivo. More importantly, antagonizing CDK7 retains inhibitory activity against Hh-driven cancers with almost all so-far described primary or acquired SMOi resistance. Furthermore, we reveal a synergy between CDK7 inhibition and BET inhibition on antagonizing aberrant Hh pathway and Hh-driven cancers that are either responsive or resistant to SMOi. Our results illustrate transcriptional inhibition through targeting CDK7 as a promising therapeutic strategy for treating Hh-driven cancers, especially those with primary or acquired resistance to SMOi drugs.

Diffuse Intrinsic Pontine Glioma (DIPG) remains to be a lethal type of pediatric brain tumor to date, indicating an urgent need of finding novel therapeutic strategy. Through screening of a collection of anti-tumor agents against a patient-derived DIPG primary tumor cell line, we identified a few novel therapeutic candidates for treating DIPG, among which BIRC5 inhibitor YM155 was the top potent agent. Next, we confirmed that YM155 could selectively inhibit multiple DIPG primary cell lines with IC50 less than 10nM. Moreover, our gene expression analyses showed that BIRC5 was significantly upregulated in DIPG primary tumor tissues and paired normal cortex tissues from the same patient. Genetically targeting of BIRC5 with CRISPR-Cas9 or RNAi approach could also effectively disrupt the growth of multiple DIPG cell lines, confirming BIRC5 as a valid therapeutic target for treating DIPG. Mechanistically, BIRC5 inhibition could cause cell cycle arrest, shut off proliferation and induce massive apoptosis. Furthermore, we tested the combinatory inhibitory effects of YM155 plus chemotherapy or targeted therapy drugs with proven anti-DIPG activity, including THZ1, JQ1, Gemcitabine, Panobinostat, GSK-J4 and et al. The preliminary results demonstrated YM155 could work synergistically with a few of above-mentioned drugs in vitro. We are also testing the in vivo inhibitory effects of targeting BIRC5 against DIPG preclinical PDX models. Together, our study identified BIRC5 inhibition as a novel therapeutic strategy against DIPG.

Medulloblastoma is the most common type of malignant pediatric brain tumor. It can be categorized into 4 major subgroups with distinct biological and clinical signatures: WNT, SHH, Group 3 and Group 4. MYC-amplified Group 3 medulloblastoma tend to metastasis and have the worst prognosis, however, they have been recently shown to be effectively inhibited by epigenetic drugs such as BET inhibitor and HDAC inhibitor in preclinical studies, suggesting epigenetic drug could serve as novel and promising therapeutic strategy for them. Based on these previous findings, we performed epigenetic drug combination screening by using patient derived MYC-amplified Group 3 medulloblastoma primary cell lines based on BET inhibitor JQ1 and HDAC inhibitor panobinostat, and identified some drug combinations with potential synergistic inhibitory effects. Further tests on cell viability, proliferation and apoptosis have validated the synergy of some of our identified epigenetic drug combinations. Currently we are testing their combinatory treatment by using in vivo mouse and human MYC-amplified Group 3 medulloblastoma tumor models. RNA-seq and ChIP-seq analyses will also be performed to dissect the underlying molecular mechanism of our identified epigenetic drug combinations. Our study will provide preclinical efficacy and molecular mechanism of novel epigenetic drug combinations with synergistic inhibitory effects against MYC-amplified Group 3 medulloblastoma.

Atypical Teratoid/Rhabdoid Tumor (AT/RT) is one of the most common aggressive brain tumors in infants with very poor prognosis. Although epigenetic dysregulation caused by loss of functional SMARCB1 has been known to serve as its tumor hallmark, oncogenic driver as well as diagnostic biomarker, effective targeted therapeutic strategies against AT/RT are still needed. Our recent epigenetic drug screening identified that BET inhibitor (BETi) and HDAC inhibitor (HDACi) could dramatically inhibit cell viability of multiple AT/RT primary cell lines, which belong to different epigenetic subtypes. They exert their therapeutic effects through inhibiting proliferation and inducing apoptosis based on our data from EdU incorporation assay and Annexin V staining assay. Moreover, these two types of epigenetic drugs exhibited synergistic inhibitory effects against AT/RT when working together in vitro. Compared to single drug treatment, combination treatment resulted in significantly stronger inhibition of cell viability, proliferation and survival. Gene expression analyses of drug-treated AT/RT cell lines found that BETi+HDACi combinatory treatment cause more profound down-regulation of crucial oncogenic driver/marker genes of each AT/RT subtype. In summary, our study has begun to illustrate BETi+HDACi as a novel combinatory therapeutic strategy against multiple subtypes of AT/RT. Next, in vivo therapeutic effects against multiple subtypes of AT/RT patient-derived xenograft (PDX) models with these two types of epigenetic drugs individually or in combination, as well as the underlying molecular mechanism of their synergy for each subtype, will be further investigated to provide a solid rational for initiating BETi+HDACi trial of AT/RT in near future.

Small ubiquitin-like modifier (SUMO)/sentrin-specific protease 1 (SENP1), a member of the SENP family, is highly expressed in several neoplastic tissues. However, the effect of SENP1 in acute promyelocytic leukemia (APL) has not been elucidated. In the present study, it was observed that SENP1 deficiency had no effect on the spontaneous apoptosis or differentiation of NB4 cells. Arsenic trioxide (As2O3) could induce the upregulation of endoplasmic reticulum (ER) stress, resulting in the apoptosis of NB4 cells. Additionally, knockdown of SENP1 significantly increased As2O3-induced apoptosis in NB4 cells transfected with small interfering RNA targeting SENP1. SENP1 deficiency also increased the accumulation of SUMOylated X-box binding protein 1 (XBP1), which was accompanied by the downregulation of the messenger RNA expression and transcriptional activity of the XBP1 target genes endoplasmic reticulum-localized DnaJ 4 and Sec61a, which were involved in ER stress and closely linked to the apoptosis of NB4 cells. Taken together, these results revealed that the specific de-SUMOylation activity of SENP1 for XBP1 was involved in the ER stress-mediated apoptosis caused by As2O3 treatment in NB4 cells, thus providing insight into potential therapeutic targets for APL treatment via manipulating XBP1 signaling during ER stress by targeting SENP1.

Alzheimer's disease (AD) is a common neurodegenerative disease affecting cognitive function in the elderly, which is characterized by the presence of extracellular deposits of insoluble amyloid-β plaques and neuronal loss. Modern pharmacology and drug development usually follow a single-target principle, which might contribute to the failure of most compounds in clinical trials against AD. Considering AD is a multifactorial disease, a combination therapeutic strategy that applies drugs with different mechanisms would be an alternative way. Smart Soup (SS), a Traditional Chinese Medicine formula, is composed of three herbaceous plants and has been applied in the treatment of amnesia in China for hundreds of years. In this work, we studied the clinical potency of the combination of SS and Aricept in AD therapy. In the in vivo model, both longevity and locomotive activity of AD transgenic Drosophila were improved remarkably in the combined medicine treated group. We also observed less amyloid-β deposition and retarded neuronal loss following the combined drug treatment. In the retrospective cohort study, we found the combination therapy exerted better therapeutic effect on AD patients. Our study revealed that combination therapy with multiple drug targets did have a better therapeutic outcome. It provides a new strategy to develop an optimum pharmaceutical approach against AD.