Inhibition of histone acetyltransferase function radiosensitizes CREBBP/EP300 mutants via repression of homologous recombination, potentially targeting a gain of function

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment.


Statistics
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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 All studies must disclose on these points even when the disclosure is negative.

Sample size
Generally, in vitro studies were performed in 3-8 separate samples. In vivo studies were performed in 7-14 tumors per treatment arm. No a priori sample size determination was performed as generally these samples sizes (3 minimum for in vitro, 7 minimum for in vivo) were felt to be sufficient to evaluate treatment effect based our previous similar work.
Data exclusions No data were excluded.

Replication
All attempts at replication were successful. Generally in vitro data (as well as the UMSCC47 in vivo study) was repeated twice at a minimum.
Randomization In animal studies, tumors were randomly allocated once they reached ~150 mm3. For in vitro experiments, randomization has no expected benefit, as there is nothing to randomize against, as the cells all originate from the same source immediately prior to treatment.

Blinding
Blinding was not possible due to delivery of radiation in animal tumors. Two investigators were assigned to measure tumor volume to minimize bias. In regard to in vitro studies, blinding on a volume needed for each experiment would require more resources than are available in most laboratories and would provide minimal, if any, benefit for reasons similar to the lack of randomization.

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.  Authentication STR genotyping was performed prior to use for each cell line.

Mycoplasma contamination
Cell lines were tested for mycoplasma prior to use, any found to be positive were treated with antibiotics prior to use. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Ethics oversight
This is a publicly available, de-identified database of information.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

March 2021
Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation TUNEL Assay Following experimental treatments, all cells were collected including floating cells and TUNEL staining was performed using the APO-DIRECT Kit (BD Pharmingen) according to the manufacturer's protocol. 500,000-1 million cells were fixed in 1% paraformaldehyde on ice for 30min. Cells were then washed in PBS and fixed in 70% ethanol overnight at -20C. Cells were washed twice with provided buffer then stained with 50μl of DNA labeling solution at 37°C for 45-60 min . Cells were then rinsed twice with provided buffer and resuspended in 300ul of rinse buffer. Cells were then analyzed by flow cytometry using the BD Accuri C6 Plus flow cytometer (BD Biosciences) with 488nm laser, 533/30 filter and FL1 detector. 10,000 events were measured per sample. Standard SSC and FSC gating were used to exclude debris. From the gated dot plot display, additional gating was applied at the edge of the unstained cell population (~4 log) and any events to the right of this population was gated as positive apoptosis (~5 log). 2μg/ml puromycin 24 and 48h, in addition to kit controls, were used as positive control samples to assist in proper delineation.
Cell cycle 24h after irradiation, all cells were collected including floating cells. Cells were then pelleted by centrifugation at 300 x g for 5min and washed with PBS twice, then fixed using 70% ethanol for at least 30min at 4oC or overnight at -20C. After fixation, cells were pelleted and washed once with PBS, then resuspended in propidium iodide 50ug/ml (Sigma Aldrich), 100ug/ml Rnase A (Sigma Aldrich) in PBS and incubated at room temperature for 30min. Samples were then analyzed by flow cytometry using the Accuri C6 Plus flow cytometer (BD Biosciences). Standard SSC and FSC gating were used to exclude debris. Standard gate was further gated by FL2-H and FL2-A, and additionally a third gating was applied, FL2-H by width, to remove doublets. A histogram was generated from these events and cell cycle distribution was quantified using FCS Express v7 using 1 cycle DNA fit analysis.
Stably selected cells were electroporated with 6μg pCBASceI (Addgene, Plasmid 26477) and 2μg mCherry (Addgene, Plasmid 41583) and incubated in 1.5μM A485/A486, 10μM ATMi (KU-55933) or 100nM ATRi (BAY-1895344) for a total of 72h. Flow cytometry was run using BD Accuri C6 Plus and standard SSC and FSC gating excluded debris. A dot plot display of FL1 (gfp) by FL2 (rfp) were gated at the edge of negative control groups DRGFP or EJ5GFP. Any events to the right and upward from this gate were considered positive for repair.

Instrument
BD Accuri C6 Plus flow cytometer (BD Biosciences) Software BD Accuri C6 Plus and FCS Express v7 Cell population abundance Tunel assay: Standard SSC and FSC gating were used to exclude debris with 10,000 events evaluated. From the gated dot plot display, additional gating was applied at the edge of the unstained cell population (~4 log) and any events to the right of this population was gated as positive apoptosis (~5 log). 2μg/ml puromycin 24 and 48h, in addition to kit controls, were used as positive control samples to assist in proper delineation.
Cell cycle: Standard SSC and FSC gating were used to exclude debris with 10,000 events evaluated. Standard gate was further gated by FL2-H and FL2-A, and additionally a third gating was applied, FL2-H by width, to remove doublets. A histogram was generated from these events and cell cycle distribution was quantified using FCS Express v7 using 1 cycle DNA fit analysis.
HR/NHEJ Assays: Flow cytometry was run using BD Accuri C6 Plus and standard SSC and FSC gating excluded debris with 5,000 events analyzed. A dot plot display of FL1 (gfp) by FL2 (rfp) were gated at the edge of negative control groups DRGFP or EJ5GFP. Any events to the right and upward from this gate were considered positive for repair.