P-054: TRIM33 loss in Multiple Myeloma is associated with defective DNA repair and sensitivity to PARP inhibition

Abstract

<h3>Background</h3> Chromosomal instability is a hallmark of Multiple Myeloma (MM) with most patients displaying cytogenetic abnormalities which can often act as prognostic indicators. Such abnormalities can arise due to defects in the DNA Damage Response (DDR). TRIM33 is an E3 ligase and transcription co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 is involved in PARP-dependent DDR and regulation of chromosomal stability. Here, we investigated the influence of TRIM33 loss in MM, focusing on its role in the DDR and whether this could be exploited therapeutically. <h3>Methods</h3> The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with survival, structural variants and common cytogenetic abnormalities. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells for in vitro studies. Protein expression and interactions were assessed by co-immunoprecipitation, western blotting and/or immunofluorescence. Clonogenic survival assays were used to assess response to Olaparib. <h3>Results</h3> Previously we identified a subset of MM patients with TRIM33 loss and identified that these patients exhibit significantly more chromosomal structural variants (deletions, inversions, duplications and translocations) (p<0.0001) and a significantly poorer overall survival (p<0.0001). Additionally, we have determined the frequency of common recurrent primary and secondary cytogenetic abnormalities for these patients. No recurrent primary cytogenetic abnormalities were associated with TRIM33 loss. However, high-risk secondary chromosome 1 aberrations were associated with loss of TRIM33, both del(1p) and gain(1q) (p<0.0001 and p=0.0474 respectively). In vitro, TRIM33 knockdown resulted in increased formation of 53BP1 foci and increased γH2AX expression (p<0.001) indicating unrepaired DNA damage typical of a DDR defect. Following induced DNA damage using 2Gy irradiation (IR), TRIM33 is recruited to chromatin within 5 minutes, with levels returning to basal by 30 minutes. The chromatin remodelling enzyme ALC1 is known to regulate sensitivity to PARP inhibition and TRIM33 is required for its timely removal from sites of damage. TRIM33 transiently interacts with ALC1 within 15 minutes of 2Gy IR. TRIM33 knockdown did not affect ALC1 expression. However, knockdown did sensitize MM cells to the PARP inhibitor Olaparib reducing the IC50 from 1.7µM to 780nM. <h3>Conclusion</h3> Here, we show that a subgroup of MM patients have TRIM33 loss and these patients have high-risk disease and poor outcome. We show that TRIM33 is recruited to damaged chromatin where it regulates ALC1 activity. Therefore, TRIM33 loss results in a DDR defect leading to chromosomal abnormalities. However, TRIM33 loss-associated DDR defects can be exploited therapeutically using Olaparib which is currently approved for the treatment of BRCA1/2 mutated breast and ovarian cancers.