P-188: Exploiting Transcription-Replication Conflicts As a Novel Therapeutic Intervention in Multiple Myeloma

Introduction: The evolution of Multiple Myeloma (MM), a plasma cell (PC) disorder, is driven by the accumulation of genomic abnormalities that lead to cell cycle dysregulation, and is therefore associated with replication stress. PCs are antibody-secreting cells associated with a high transcriptional stress. Transcription/Replication Conflicts (TRCs) arising from collisions between the replication and transcription machineries can promote tumor progression but can also represent an Achille’s heel to cancer cells. Here, we investigated the therapeutic interest of increasing TRCs to target specifically malignant PCs using a G-quadruplex-stabilizing small molecule.

Methods: The prognostic value of 13 genes involved in TRC management was evaluated in three independent cohorts of MM patients and used to build the TRC score.
The G-quadruplex (G4) stabilizer Pyridostatin (PDS) was used as a strategy to increase TRCs. The response of 18 Human Myeloma Cell Lines (HMCLs) to PDS was evaluated with proliferation assay. DNA damage response was investigated using western-blot and immunofluorescence detection of H2AX and 53BP1.
The cytotoxic effect of PDS on primary cells from MM patients was determined using the co-culture of MM cells with bone-marrow microenvironment cells and flow cytometry.

Results: A signature of genes involved in TRCs management was found overexpressed in malignant PCs compared to normal PCs, suggesting that they adapted to an elevated replication stress through the upregulation of a TRC-resolving machinery. Combining those genes into a TRC score identified high risk MM patients that could benefit from a TRCs-increasing therapy.
Treatment with PDS was associated with significant toxicity in 13 HMCLs with an IC50 ≤ 4 M whereas 5 cell lines demonstrated higher resistance to PDS. It was associated with cell cycle arrest in G2/M, DNA damage and apoptosis. Importantly, primary myeloma cells were more sensitive to PDS treatment than the normal cells of the bone marrow micro-environment.
PDS was able to improve the efficacy of current MM treatments. Firstly, PDS synergized with Melphalan leading to increased DNA damage, cell cycle arrest and apoptosis. Secondly, HMCLs and primary MM cells with a high TRC score were more sensitive to Panobinostat, and increasing R-loop formation with Panobinostat and stabilizing these R-loops with PDS had a synergistic effect in HMCLs as well as on primary MM cells. Thirdly, PDS-treated HMCLs were associated with cGAS-STING pathway activation, and a high TRC score was associated with early relapse in a cohort of MM patients treated with the monoclonal antibody Daratumumab, suggesting that these patients could benefit from a TRCs-increasing strategy.

Conclusions: TRCs represent an actionable Achille’s heel for malignant PCs that could be targeted by new therapies such as G4 stabilizers, to improve the efficacy of current MM treatments and the outcome of myeloma patients.