An IFNγ-Mediated Immune Inflamed Microenvironment State in Multiple Myeloma with TP53 Loss That Can be Therapeutically Exploited

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<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<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<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.