Exploiting Transcription-Replication Conflicts As a Novel Therapeutic Intervention in Multiple Myeloma
Authors: Dutrieux Laure , Lin Yea-Lih , Lutzmann Malik , Requirand Guilhem , Robert Nicolas , Vincent Laure , Cartron Guillaume , Herbaux Charles , Rodriguez Raphael , Cogne Michel , Pasero Philippe , Moreaux Jerome 
Published: November 2021

Abstract
Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained in malignant PCs meaning that these cells display an elevated transcriptional stress. Besides, malignant PCs face oncogene-induced replication stress concomitantly with cell cycle deregulation. Consequently, transcription and replication in malignant PCs need to be tightly coordinated to avoid too much interferences that would increase replication stress and genomic instability. A failure to cope with these transcription/replication conflicts (TRCs) could have a significant impact on mutagenesis involved in MM development. Importantly, these effects might open the therapeutic possibility of TRCs enhancement to specifically kill malignant PCs.
Based on these observations, we identified a signature of 13 TRCs resolution factors significantly overexpressed in MM patients. Considering the potent role of TRCs resolution in MM cell adaptation to replication stress, we sought to identify the TRCs resolution factors that are associated with a poor outcome in MM. High expression of 9 out of the 13 TRCs resolution factors significantly overexpressed in malignant PCs are associated with a poor outcome in MM (TT2 cohort, n = 345). We gathered the prognostic value of these 9 genes within a Gene Expression Profile (GEP)-based TRC resolution score (TRC score). High TRC score is associated with a poor outcome in two independent cohorts of newly diagnosed MM patients treated by high dose therapy and autologous stem cell transplantation (Arkansas, TT2 cohort, n = 345; CoMMpass cohort, n = 674) (Fig.1A). Interestingly, we investigated the link between the TRC score and the MM cells drug response using our collection of human myeloma cell lines (HMCLs), and identified that HMCLs with high TRC score values are significantly more sensitive to Panobinostat histone deacetylase inhibitor, currently used in MM treatment at relapse (n = 11, p value &lt; 0.05). Histone acetylation has been shown to promote R-loop formation that constitutes obstacles to replication fork progression. Using primary MM cells from patients (n = 12) co-cultured with their bone marrow microenvironment, we found that a high TRC score value is associated with a higher toxicity of Panobinostat (p value &lt; 0.01). Therefore, the TRC score allows the identification of a MM patients subgroup with a poor outcome that could benefit from Panobinostat treatment.
Interestingly, TRCs are promoted by R-loop formation and G-quadruplex (G4) stabilizers treatment. R-loops are formed by the reannealing of the nascent RNA with the template DNA (called an RNA:DNA hybrid). G4s are four-stranded secondary DNA structures, constituted of stacked guanine tetrads. Both structures are formed during transcription in G-rich DNA regions and can represent a barrier for replication fork progression if unscheduled. G4s can stabilize R-loops which have been shown to mediate DNA damage induced by G4 stabilizers. Interestingly, treatment with the G4 stabilizer Pyridostatin (PDS) was associated with significant toxicity on HMCLs (n = 15) (Fig.1B), and on primary MM cells of patients cocultured with their bone marrow microenvironment (n = 5, p value &lt; 0.05). Interestingly, the combination of PDS and Panobinostat has a synergistic effect in HMCLs. We also found a correlation between HMCLs TRC score and the response to two Bromodomain and Extra-Terminal motif (BET) proteins inhibitors, I-BET-762 and RVX-208. The synergistic effect of PDS combination with I-BET-762 was validated in vitro. BET proteins inhibition has been shown to increase R-loop formation and DNA damage. Furthermore, we used inducible RNase H expression in HMCLs to specifically degrade RNA:DNA hybrids. RNase H expression resulted in a significant reduction of DNA damage response after PDS treatment (Fig.1C). Our results underline that spontaneous replication stress and genomic instability are related to R-loop formation and TRCs in MM cells.
Altogether, these results emphasize the therapeutic potential of TRCs targeting in MM using G4 stabilizers alone or in combination with current treatments.
Figure 1 Figure 1.

Disclosures
Vincent: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Cartron: Roche, Celgene-BMS: Consultancy; Danofi, Gilead, Novartis, Jansen, Roche, Celgene-BMS, Abbvie, Takeda: Honoraria. Herbaux: Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Moreaux: Diag2Tec: Consultancy.