ALBERT

Description: Although most multiple myeloma (MM) cases are characterized by the detection of a monoclonal immunoglobulin in the serum, about 15% of the patients present only immunoglobulin light chains, detected either in the urine or serum or both. These patients are designated as having light-chain (LC) MM. Using fiber-fluorescent in situ hybridization, and in contrast to patients and myeloma cell lines secreting heavy chains (who presented a legitimate functional IgH rearrangement in every case), LC MM never displayed a functional IgH recombination. Interestingly, most LC MM cases presented one IgH allele with a germline configuration (including the DJ region), the second allele being usually involved in an illegitimate recombination. Of note, most of these translocations occurred close to (or at) switch regions, even though in some cases, breakpoints involving nonswitch regions were observed. Thus, this study clearly showed that LC MM is due to the absence of legitimate IgH rearrangement at the DNA level, reflecting possible abnormalities in the IgH gene recombinations during B-cell maturation. Furthermore, it showed that this defect did not prevent the activation of the switch process because most of 14q32 translocations observed in LC MM occurred at switch regions.

Description: Abstract 4072 The development of novel treatments such as those based on human monoclonal antibodies (mAb) are currently being evaluated in preclinical and early clinical studies in cancers. There is convincing evidence supporting a key role for antibody-dependent cell-mediated cytotoxicity (ADCC) in the clinical response of several therapeutic monoclonal antibodies. In myeloma, CD38 and CD138, both highly expressed by all myeloma cells, represent the best targets for ADCC and humanized Abs are currently in development for both antigens. The aim of the present study was to assess the CD38 and CD138-ADCC sensitivity of a large panel of human myeloma cell lines (HMCLs) representative of the disease heterogeneity. To reproductively assess ADCC, we used mouse FcgRIII expressing human T lymphocytes as cytotoxic effector cells. We measured ADCC sensitivity of 20 HMCLs with two mouse IgG1 anti-CD38 (clones T16 and HIT2), with two mouse IgG1 anti-CD138 (clones BB4 and MI15) and with a mouse IgG2a anti-HLA class-I (clone W6.32) by a standard 51Cr release assays performed at an effector to target ratio of 10:1. Unexpectedly, despite high level of CD38, only 3 cells lines among 20 were sensitive to anti-CD38/ADCC. No correlation was observed between anti-CD38/ADCC sensibility and the CD38 expression level. No correlation was observed too between sensitivity and representative molecular heterogeneity of HMCLs. The absence anti-CD38/ADCC was not due to intrinsic resistance lysis through ADCC since these cell lines were lysed when using an anti-HLA class-I mAb (or anti-CD20 mAbs for one CD20+ HMCL). Furthermore, absence of ADCC was not due to antibody-induced antigenic modulation. Surprisingly too, none HMCL was lysed by anti-CD138 mAbs despite high expression of CD138. Moreover, primary myeloma cells purified from peripheral blood of a patient were also resistant to both anti-CD38 and anti-CD138 mAbs. These results show that ADCC activity mediated by a particular monoclonal antibody varies according to the target cell and independently of the antigen expression level, suggesting that the antigen “environment” might affect the interactions between antibody, Fc receptor and effector cells. Further studies will be required to determine the CD38 and CD138 “environment” factors on myeloma cell that can influence ADCC. Disclosures: No relevant conflicts of interest to declare.

Description: Patients with multiple myeloma (MM) carrying a del(17)(p13) (del17p) deletion and/or TP53 mutation at diagnosis have a shortened survival whatever the treatment received. By using a large and characterized collection of human myeloma cell lines (HMCLs), we have shown that TP53mutated/deleted cell lines are more resistant to melphalan and nutlin3a (MDM2 inhibitor) than TP53wild-type HMCLs (Surget S, Cancer Res 2012;72:4562). The absence of a functional p53 pathway prevents the induction of p53 target genes, including pro-apoptotic genes, and therefore impairs apoptosis. In cancer cells, p53 mutations are frequent and highly expressed mutant p53 proteins exert a dominant effect. Pharmacological drugs such as PRIMA-1Met/APR246, which has been recently evaluated in a phase I/II study (Lehmann S J Clin Oncol 2012;30:3633) offers an interesting opportunity to redirect the activity of mutant misfolded p53 proteins. With this aim, the efficiency of PRIMA-1Met was assessed in 22 HMCLs and 16 primary MM samples characterized for del17p. HMCLs were heterogeneously sensitive to PRIMA-1Met, with an LD50 median value of 35 µM (range 4-〉100 µM). We failed to observe any correlation with HMCL’s TP53 status since the LD50 median values of TP53wt, TP53mut and TP53del HMCLs were not significantly different (p=0.1, Kruskal Wallis test) at respectively 21 µM (range 4-70, n=7), 45 µM (21-〉100, n=13) and 21 µM (8-33, n=2). Primary MM samples were also heterogeneously sensitive, with a median cell death value induced by 10 µM PRIMA-1Met of 51% (n=16, range 5-100%). The 5 del17p+ samples showed no significant difference in their sensitivity to PRIMA-1Met from the 11 del17p- (p=0.31 Mann Whitney). Moreover, the sensitivity of 2 TP53wt and 2 TP53mut HMCLs was unchanged upon p53 silencing, and PRIMA-1Met failed to increase the expression of such p53 target genes as p21, Puma or DR5 (TRAIL-R2) in both TP53wt and TP53mut/del HMCLs. By contrast, nutlin3a induced cell death and increased DR5 expression in all TP53wt but in none of the TP53mut/del HMCLs (p

Description: IGF-1 is a well-known growth factor for human myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for the treatment of multiple myeloma. In this study, we investigated the effect of a humanized antagonistic monoclonal antibody to the IGF-1R (AVE1642). We showed that AVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines (HMCL). Since we had previously shown the functional relevance of CD45 expression in the growth of myeloma cells, and the association of CD45 negative (CD45neg) status with a less favorable clinical outcome, both CD45 positive (CD45pos) and CD45neg HMCL were studied. We found that AVE1642 strongly inhibited the growth of CD45neg HMCL, leading to a G1 growth arrest, whereas it had almost no effect on the growth of CD45pos HMCL. Indeed, by cell counting over four days in standard culture conditions, AVE1642 induced a significant growth inhibition of the CD45neg LP-1 HMCL of 90% whereas it had no effect on the growth of the CD45pos XG-1 HMCL. Furthermore, AVE1642 binding induced a significant reduction of IGF-1R expression. In western blot, a decrease of 60% the IGF-1R expression of the LP-1 CD45neg HMCL was observed. This decrease was also confirmed by flow cytometry analysis. Finally, a stable CD45 silencing XG-1 cell line was generated and became sensitive to AVE1642. By cell counting over four days in standard culture conditions, AVE1642 inhibited the growth of the shCD45neg XG-1 HMCL of 56% whereas it had almost no effect on the growth of the shLuci-XG-1 HMCL. Thus, for the first time, we provided direct evidence that the expression of CD45 renders myeloma cells resistant to IGF1R inhibition. Furthermore, AVE1642 in combination with Bortezomib strongly increased the apoptosis induced by Bortezomib alone on CD45negHMCL. The CD45neg LP-1 HMCL was considered as a modestly sensitive cell line since 10nM of Bortezomib, which is in the range of therapeutic concentrations, induced 40±16% of apoptosis. Bortezomib in combination with AVE1642 was found to induce 80±1% of apoptosis in the CD45neg LP-1 HMCL. Apoptosis induction by AVE1642/Bortezomib combination was associated with an important induction of Noxa, a BH3-only pro-apoptotic protein that was increased by the addition of AVE1642. We also found a strong increase of caspase-3, -8 activation under this combination compared to Bortezomib alone. Taken together, these results support that therapy directed against IGF-1R associated with Bortezomib, could be beneficial in treating CD45neg patients, a population known for poor clinical outcome.

Description: Targeting anti-apoptotic proteins of the BCL2 family by BH3 mimetics is a new promising therapeutic approach in multiple myeloma (MM). The specific BH3 mimetic targeting BCL2, BCLXL or MCL1 trigger apoptosis and exploit the dependency on these different anti-apoptotic proteins to kill tumor cells. Because MM is mostly considered as dependent on MCL1, the recent clinical availability of MCL1 BH3 mimetics underlines an urgent need to better define patients that would benefit from a MCL1 targeted therapy. In the present study, we used a BH3 mimetic toolkit that includes venetoclax, A1155463 and A1210477, which target BCL2, BCLXL and MCL1 respectively to define dependencies/co-dependencies in a large cohort of 60 myeloma patients (21 at diagnosis and 39 at relapse). Alternatively, MCL1 dependency was confirmed using the S63845 MCL1 inhibitor in MM patient samples. Mononuclear bone marrow/blood cells were treated overnight with the respective BH3 mimetic and cell death was specifically measured in the tumor cell population. Primary MM cells dependencies were stratified using PCA analysis in three groups as highly dependent, intermediately dependent or not dependent. Our study demonstrated that half of patients at diagnosis were BCL2 dependent while only 10% were BCLXL dependent. The dependence on BCL2 or BCLXL was not significantly different between samples at diagnosis and relapse. Strikingly, we found that the MCL1 dependency was 33% at diagnosis while it was 69% at relapse, suggesting a significant increase in MCL1 dependency during the disease progression (p=0.01). Besides, 36% of overall patients showed co-dependencies on BCL2/MCL1. We also identified primary MM cells that did not depend on any of the three pro-survival molecules, both at diagnosis and relapse. Among this cohort of MM patients, 47 samples were further analyzed for the presence of recurrent translocations (t(11;14), t(6;14), t(4;14) and t(14;16)) allowing the analysis of dependencies in the different subgroups; these recurrent translocations lead to the overexpression of CCND1, CCND3, MMSET and MAF oncogenes, respectively. We found that BCL2 dependency was significantly higher in CCND1 subgroup (83%) compared to all other subgroups (20%, p=0.008). We also confirmed the BCL2/BCLXL mRNA ratio as a valuable biomarker to define BCL2 dependence (p=0.0001). At diagnosis, MCL1 dependency was absent in patients not harboring the common recurrent translocations while at relapse 6 out 9 patients not harboring the recurrent translocations were MCL1 dependent, indicating an increase of MCL1 dependency at relapse in this subgroup (p=0.03). Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection of cell death observed by BAK knock-down and the complete disruption of MCL1/BAK complexes upon A1210477 treatment, observed in MM cell lines and also in a patient sample. Interestingly, this complex was also dissociated in A1210477 resistant cells but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. Thus, BCLXL may act as a sink to bind freed pro-apoptotic proteins from MCL1 and limits MM cell death triggered by the specific targeting of MCL1. In conclusion, our study highlights the potential clinical use of BH3 mimetics in MM treatment guided by the practical ex-vivo testing of myeloma cell dependencies using the BH3 toolkit. This strategy could be used to identify the respective and tailored use of venetoclax, MCL1 BH3 mimetics or their combination in myeloma treatment. Disclosures Moreau: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Key Points Myeloma cells are highly sensitive to PRIMA-1Met, independent of p53. PRIMA-1Met induces myeloma cell death by impairing GSH/ROS balance.

Description: 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

Description: Multiple Myeloma (MM) is a fatal plasma-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow. IL-6 and IGF-1 are known to be essential growth and survival factors in this malignancy. Beside these well characterized growth factors, other growth factors such as HGF, HB-EGF and FGF have been involved in this malignancy. Even though all of them seem to be involved in myeloma cell proliferation, the hierarchy of each growth factor remains to be established. In the present study, a serum-free cytokine-free and collagen-based assay which does not allow the generation of endogeneous myeloma colonies was used to identify the clonogenic factors of fourteen myeloma cell lines. We selected seven myeloma cell lines expressing CD45 on 100% of cells and seven other cell lines lacking CD45 expression. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 of 14 myeloma cell lines. For some cell lines (LP-1, L363 and XG-2) the percentage of clonogenic cells reached 40% to 50% indicating that clonogenic cells are CD138+ and do not represent a particular CD138-subpopulation. In contrast, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some CD45-myeloma cell lines and at a less extend than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from 5 of 8 CD45- myeloma cell lines. We searched for a correlation between myeloma-colony formation and the signaling pathway induced by IGF-1 and FGF in MM cells. It appears that activation of both the PI3Kinase and ERK pathway by IGF-1 and FGF fully correlates with their capacity to stimulate the clonogenicity of CD45-myeloma cell lines. In conclusion, the CD45 phenotype of myeloma cells discriminates their signaling and proliferative response to IL-6 and growth factors. These results give a strong rational to the stratification of prognostic value discriminating CD45+ from CD45- MM. Furthermore, treatment strategies for CD45- patients should combined the disruption of signaling induced by both IL-6 and IGF-1 (or other growth factors) whereas CD45+ patients could be targeted through IL-6 only.

Description: The coronavirus pandemic raging worldwide since December 2019 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which invades human cells via the angiotensin-converting enzyme 2 (ACE2) receptor. Although it has already been identified in many organs, ACE2 expression remains largely unknown in the head and neck (HN) sphere. Thus, this study aims to investigate its protein expression in several sites of the upper aerodigestive tract in order to highlight potential routes of infection. We compared ACE2 immunohistochemical expression between 70 paraffin-embedded specimens with two different antibodies and reported the quantified expression in each histological location. Surprisingly, we obtained different results depending on the antibody, an absence of labeling having been observed with a monoclonal antibody raised against the extracellular domain, whereas the polyclonal, against the cytoplasmic part of the protein, revealed enriched ACE2 expression, particularly in sinuses, vocal cords, salivary glands and oral cavity epithelial cells. The interpretation of these discordant results has brought several exciting lines of reflection. In conclusion, this study provides possible routes of entry for the SARS-CoV-2 in HN region and, above all, has led us to encourage caution when studying the ACE2 expression which is currently at the center of all attention.