A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma

Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection None.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability H3K27ac ChIP-seq and RNA-seq datasets performed in this study are deposited at the NCBI Gene Expression Omnibus under the accession GSE166254. H3K27ac ChIP-seq datasets previously performed in FAP adenomas and normal colon crypts were accessed from the Gene Expression Omnibus under the accession GSE7773717. Broad Institute Cancer Cell Line Encyclopedia (CCLE) RNA-seq datasets for CRC cell lines were accessed from the Gene Expression Omnibus under the April 2020 accession PRJNA52338023. COLO205 in vitro and orthotopic tumor H3K27ac ChIP-seq datasets presented in fig. S2C-E, 3A-B were accessed under the accession GSE12618843. RELA ChIP-seq datasets from adipocytes were accessed under the accession GSM156673577; RELA ChIP-seq datasets from A549 cells were accessed under the accession GSM84787678; STAT1 ChIP-seq datasets from HeLa cells were accessed under the accession GSM38550579; STAT1 ChIP-seq datasets from monocytes were accessed under the accession GSM105701180; STAT3 ChIP-seq datasets from MDA-MB-468 cells were accessed under the accession GSM227800381; CTCF ChIP-seq from normal colon tissue was accessed from ENCODE82. Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No statistical analyses were performed to pre-determine sample size. Power analyses performed retroactively validate our 15 patient cohort saturated and was sufficiently powered for novel super-enhancer discovery. For all other experiments, a minimum of 3 biological replicates were used for the determination of statistical analysis in accordance with standard rigor and reproducibility practice guidelines by the NIH.
Data exclusions No data were excluded in this study.

Replication
The majority of in vitro experiments were performed 3 or more times on independent samples, and all results were reproducible. Where experiments were performed once, the phenotypes were robust and validated using orthogonal methods and/or the same experiment performed in different cell lines. The exact n for each experiment is noted in the Figure Legends. Mouse studies were not replicated but included sufficient sample size to account for biological variability.
Randomization For mouse studies, cages were allocated randomly. Randomization of samples into experimental groups did not apply for all other experiments (cell culture).

Blinding
Investigators were not blinded for the analysis of experiments as it was not feasible.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Antibodies Antibodies used The following primary antibodies were used for IHC: anti-CD11c (CST #97585), anti-CD68 (CST #97778), anti-perforin (CST #31647), and anti-Ly-6G (CST #87048). The following primary antibodies were used for immunoblot: anti-PDZK1IP1 (Sigma #HPA014907), anti-GAPDH (Cell Signaling Technologies, CST #2118), anti-V5 (CST #13202), anti-phosphorylated STAT3 at Y705 (CST #9145). H3K27ac ChIP-seq antibodies were purchased from abcam (#ab177178). Horse radish peroxidase conjugated secondary antibodies: mouse (Thermo #31432) and rabbit (Thermo #31460) both used at 1:2000 concentrations).

Validation
The following antibodies were validated by the Ramon Parsons laboratory using knockout or overexpression studies: anti-CD68 (CST #97778), anti-PDZK1IP1 (Sigma #HPA014907), anti-V5 (CST #13202). The following antibodies were validated by the manufacturer using knockout or overexpression studies: anti-Ly-6G (CST #87048), anti-CD68 (CST #97778), anti-V5 (CST #13202), anti-PDZK1IP1 (Sigma #HPA014907). The anti-phosphorylated STAT3 at Y705 (CST #9145) was validated using an inhibitor of STAT3 C188-9, and by the manufacturer via interferon stimulation of cell culture. The H3K27ac antibody was validated by the manufacturer through the use of histone deacetylase inhibitor Trichostatin A as well as a peptide array to assess for binding to other histone modifications.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) The following cell lines were used in this study: HT29, COLO205, DLD1, and HEK293T all purchased from ATCC.

Authentication
HT29 was authenticated using short tandem repeat (STR) profiling at the Mount Sinai Oncological Sciences Sequencing Core Facility. HEK293T, COLO205, and DLD1 were not authenticated.

Mycoplasma contamination
All cell lines tested negative for mycoplasma.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Homozygous Nu/J mice (#002019, Jax), at 6-8 weeks of age were used for xenograft studies. Tumors were initiated 7 days after delivery to allow acclimation of animals. Mice were euthanized before tumors reached a maximum size of 2000mm^3. Male mice were used in this study to recapitulate the majority of CRC cases observed in humans.

Wild animals
No wild animals were used. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
No population characteristics--de-identified patient surgical samples, exempt.

Recruitment
No recruitment--de-identified patient surgical samples, exempt.

Ethics oversight
All human subjects work was approved by the Mount Sinai Hospital Institutional Review Board (IRB) under Protocol IRB-19-01860. All samples were de-identified by The Mount Sinai Biorepository.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.
Raw and processed sequencing data has been deposited at the Gene Omnibus under the accession GSE166254. The token: sfmteagypdghhyd may be used for reviewer access.