An IFNγ-Mediated Immune Inflamed Microenvironment State in Multiple Myeloma with TP53 Loss That Can be Therapeutically Exploited
Authors: Larrayoz Marta , Arriazu Elena , Zabaleta Aintzane , Roncal Carmen , Tamayo Ibon , Llopiz Diana , Jimenez Maddalen , Castro Carla , Celay Jon , Vicente Carmen , Guerrero Camila , Sanz Sonia , Garcia-Barchino Maria J , Barrena Naroa , Puig Noemi , Cedena Romero Maria Teresa , Calasanz Maria Jose , Rodríguez-Otero Paula , San-Miguel Jesús F. , Hernaez Mikel , Agirre Xabier , Sarobe Pablo , Prosper Felipe , Paiva Bruno , Martinez-Climent Jose A. 
Published: December 2024

The impact of individual genetic lesions in the bone marrow (BM) immune microenvironment of high-risk multiple myeloma (MM) is uncertain. To experimentally address this issue, three genetically engineered mouse models carrying either standard-risk genetic lesions (BIcγ1 and CyclinD1-BIcγ1 mice) or a monoallelic TP53 deletion (TP53-BIcγ1 mice), considered a high-risk genetic lession, were generated. Sequential characterization of mice from early to late MM-like stages was carried out using multi-parametric flow cytometry, whole exome sequencing (WES) and single-cell RNA and T-cell receptor sequencing (scRNA/TCRseq) in BM tumor and immune cells.
Median overall survival (mOS) of TP53-BIcγ1 mice was shorter than in standard risk MM models (mOS, 258 vs 305 vs 301 days; respectively; p&lt;0,01). While CRAB signs, Ig secretion, and accumulation of GFP+CD138+B220-IgM- MM cells in BM with multi-focal growth pattern progressively increased in each model, TP53-BIcγ1 mice showed higher tumor burden and increased MM cell proliferation, while lower IgG/IgA secretion than non-TP53 models. WES revealed higher tumor mutation burden (TMB) and copy number variation (CNV) in the TP53 model (p=0,001 for both). Validating mouse data, among 598 patients with newly diagnosed MM in the CoMPass study, 62 cases with TP53 loss/mutation (10,4%) showed increased TMB and CNV with respect to those without TP53 alterations (p=0,001 and p=0,0001, respectively).
The impact of TP53-driven genomic instability on the immune microenvironment was evaluated by scRNA/TCRseq. An inflammatory BM microenvironment with clonally expanded effector-memory CD8+ T cells was observed in TP53-BIcγ1 mice vs standard-risk models, which showed immune exhaustion, proliferation, and IFN response transcriptional signatures. To validate mouse data, patients with smoldering MM (n=177), newly diagnosed active MM (n=579), and refractory/relapsed MM (n=279), carrying del(17p) that increased across stages (6,8%, 9,2% and 28%, respectively; p&lt;0,0001), were characterized with flow cytometry. Resembling the models, higher number of MM cells was observed in patients with del(17p) along with expansion of BM CD3+CD27- T lymphocytes that is a phenotypic surrogate of T cell clonality. These findings were particularly evident in refractory/relapsed cases. Functional assays found higher neoantigen load in MM cells from TP53-BIcγ1 mice, where neoantigen-containing peptides selectively bound to T-cell MHC-I/II molecules in T cells ex vivo. In vivo, prophylactic neoantigenic vaccination expanded reactive CD8+ T cell clones, delayed MM onset, and extended mouse survival.
Further analyses of BM innate immune cells in TP53-BIcγ1 mice revealed increased NK cells with mature CD11b+CD27- phenotype and impaired cytotoxicity, immune exhaustion, and IFN response signatures, and abundant M2-like monocytes and pro-inflammatory macrophages. Likewise, MM patients with del(17p) showed alterations in CD56+ NK cells and higher number of monocytes in the BM. Subsequent investigations revealed that while IFNα was modestly produced by mono/macrophages in TP53-BIcγ1 mice, clonal immune-exhausted CD8+ T cells and mature CD11b+CD27- NK cells highly secreted IFNγ, leading to type II IFN signalling response in MM cells and in virtually all examined BM immune cells. Among them, expanded subpopulations of CD4+ regulatory T cells and myeloid-derived suppressor cells with high IFN signalling were observed in TP53-BIcγ1 mice vs non-TP53 models, which highlights an IFNγ-mediated immunosuppressive microenvironment that promotes MM progression.
Finally, the clinical relevance of IFN signalling was evaluated in CoMMpass MM patients. MM cells with TP53 loss/mutation exhibited increased expression of type II IFNGR2 receptor and downstream IRF1, IRF3, IRF7 and IRF9 genes with respect to MM cells without TP53 alteration. Furthermore, a type II IFN gene transcription signature correlated with shorter PFS (26 vs 40 months; p=0,0004) and OS (67 vs 95 months; p=0,0038) in univariate analyses, and showed independent prognostic value in multivariate models with HR of 1.4 (1.2-1.8), p&lt;0,001 for PFS, and HR of 1.5 (1.1-1.9), p=0,008 for OS.
Taken together, our data reveal an IFNγ-mediated immune inflamed microenvironment state induced by MM cells with high neoantigen load, which could be leveraged to treat patients with high-risk MM defined by TP53 abnormalities.