Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1)

BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.


Variant calling
The sequencing quality of each sample was assessed using the NGS QC toolkit (2.3.3) and, where necessary, adapter and read end trimming were performed using cutadapt (1.8.3) and in-house scripting respectively.
Paired-end reads were then aligned to the hg19 reference using BWA-MEM (0.7.10).
Alignments are sorted and indexed by Picard (1 .138) and locally realigned using GATK  Then, the supernatants were collected and the cytokine array (ab133998, Abcam) carried out according to the manufacturer's instructions.

Cytokine-independent growth in 32D and SKNO-1 cells
To analyze the effect of BRCC3 or Brcc3 knock-out on cytokine-independent growth, 32D cells stably expressing the Cas9 enzyme were infected with multiple sgRNA vectors

Mass spectrometric analysis
To analyse interaction partners of BRCC3 wild type (WT) or mutants, we applied stable isotope labelling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry 1 . A coimmunoprecipitation was performed in OCI-AML5 stably over-expressing an empty control, FLAG-tagged BRCC3 WT, BRCC3 R81G, or BRCC3 D135E. The samples were then separated by SDS-PAGE on 12 % self-made Bis-Tris gels. The gel was stained by colloidal coomassie staining by pre-incubating in staining solution (34 % (v/v) methanol, 2 % (v/v) phosphoric acid, 17 % (w/v) ammonium sulfate) for 1 h at RT and subsequent addition of 0.066 % (w/v) Coomassie brilliant blue G-250. After incubation overnight, the gel was de-stained with ddH2O.
Following trypsin digestion, peptides eluted from de-stained gel slices were subjected to mass spectrometric analysis using an LTQ Orbitrap Velos Pro system (Thermo Fisher Scientific) online coupled to an U3000 RSLCnano (Thermo Fisher Scientific) as described previously 2 with the following exceptions: A binary solvent gradient consisting of solvent A (0.1 % FA) and solvent B (86 % ACN, 0.1 % FA) was used for separation. The column was initially equilibrated in 5 % B. In a first elution step, the percentage of B was raised from 5 % to 15 % in 5 min, followed by an increase from 15 % to 40 % B in 30 min. The column was washed with 95 % B for 4 min and re-equilibrated with 5 % B for 25 min.
Using the embedded Andromeda search engine 3 within MaxQuant Vers. 1.5.2.8 4 , MS/MS spectra were correlated with the UniProt human reference proteome set. The respective SILAC amino acids, Arg6 + Lys4 and Arg10+Lys8 were selected, while Carbamidomethylated cysteine was considered as a fixed modification along with oxidation (M), and acetylated protein N-termini as variable modifications. False discovery rates were set on both, peptide