The Oncolytic Measles Virus Preferentially Infects p53 Abnormal Myeloma Cells
Authors: Lok Anne , Descamps Geraldine , Tessoulin Benoit , Moreau Philippe , Le Gouill Steven , Tangy Frederic , Amiot Martine , Pellat-Deceunynck Catherine 
Published: October 2019

Abstract
P53 plays a pleiotropic role in cell homeostasis by regulating stress-induced adaptive responses leading to survival or cell death. Although it is well demonstrated that the lack of a functional p53 pathway favors cancers, it remains still unclear whether it is related to its genome guardian or in apoptosis inducer role. Leonova and Gudkov demonstrated that, at least in mice, p53 and CpG methylation prevent expression of the so-called junk DNA, expression of which induces suicidal IFN responses (PNAS 2013;110:E89). Cancer cells mostly harbor hypo methylated DNA, reversible or irreversible silencing of p53 pathway and defects in suicidal IFN responses. Although the molecular bases are still not well understood, oncolytic viruses such as the Measles Virus (MV) preferentially infect and kill tumor cells. The aim of this study was to investigate whether del(17p)+/p53 mutant myeloma cells were preferentially sensitive to the Schwarz vaccine MV strain and whether p53 was involved in. Plasma cells from patients with multiple myeloma (MM) or MGUS over express CD46, the main receptor for the vaccine MV strain, as compared to normal plasma cells and to hematopoietic cells. To assess MV infection and replication, we used the GFP modified strain (MV-GFP) and measured GFP expression at day 4 after infection (MOI=1) using flow cytometry. We showed that p53 abnormal MM cell lines (n=25) over expressed CD46 (p=0.039) and were preferentially infected by MV (p=0.03) as compared with p53 wild type MM cell lines (n=12). Expressions of CD46 and GFP were directly correlated (p=0.014, r=0.505) and GFP expression correlated with cell death (n=37, p<0.0001, r=0.82). CD46 inhibition (using CD46 siRNA or anti-CD46 mAb) fully inhibited infection and death. CProduction of alpha IFN was not frequent (11 out of 37 cell lines), was irrespective of TP53 status and not related to MV resistance (no production of beta IFN was found). To assess whether p53 pathway regulates MV infection/replication, we treated cells 24 hours with the MDM2 inhibitor nutlin3a prior to MV infection. In all wild type and in none p53 abnormal cell lines, nutlin3a-pretreatment induced an increase in DR5 expression (Tessoulin B et al, Blood 2014;124:1626) and a decrease in CD46 expression: the mean of nutlin3a/control DR5 ratio of expression was 1.60 ± 0.27 and 1.04 ± 0.05 in wild type (n=10) and p53 abnormal cell lines (n=4), respectively (p=0.001) while that of nutlin3a/control CD46 ratio expression was 0.55 ± 0.08 and 0.97 ± 0.03, respectively, (p=0.0007). At 24 hours, cells were washed to remove nutlin3a and 9 cell lines sensitive to MV (5 wild type and 4 abnormal) were infected with MV (MOI=1). Decrease in CD46 expression was associated with decrease in MV replication in all p53 wild type (mean nutlin3a/control ratio 0.53 ± 0.05, n=5) but in none p53 abnormal cell lines (1.02 ± 0.18, n=4), p=0.016. We assessed infection of primary cells in unpurified mononuclear cells (MOI=1) from bone marrow or peripheral blood harboring various CD138+ infiltrate. Compared with CD138- cells, CD138+ cells over expressed CD46 (MFI ratio 100 versus 22, p=0.0003) and were preferentially infected by MV (GFP+ 62% versus 20%, p=0.0005, GFP MFI ratio 14 versus 5, p=0.0011, n=17). Assessment of TP53 status in primary cells was determined using FISH (del17p) and the functional assay of DR5 increase in response to nutlin3a. Although del(17p)+ myeloma cells over expressed CD46 and were highly infected by MV when compared to del(17p)- myeloma cells, differences didn't reach statistical significance. We further showed that nutlin3a induced decrease in CD46 expression and MV infection in 3 primary cells without del(17p) but did not in 2 primary CD138+ cells with del(17p): decrease in CD46 expression correlated with decrease in GFP expression (n=5, p=0.0167, r=1.00) and with increase in DR5 expression (p=0.033, r=-0.89). These data showed that p53 abnormal myeloma cells (cell lines and primary cells) were strongly sensitive to the oncolytic MV, which could be of interest for patients with del(17p) refractory to current therapy. The molecular mechanism of p53-induced decrease in CD46 expression and MV infection is under investigation.

Disclosures
Moreau: Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria.