Background:

Myeloma multiple (MM) disease is a type of blood cancer characterized by abnormal plasma cell growth in the bone marrow (BM) that leads to end organ damage (1).
MM cells share several characteristics with their normal counterparts, including the expression of pan-plasma cell antigens CD138, B cell maturation antigen (BCMA), and CD38. At the same time, MM cells also modify their phenotype by somatic mutations, chromosomal translocations, or copy number variations (2, 3) to induce aberrant surface antigens such as CD56 (4-6); promote compensatory mechanisms to overcome proteotoxic stress or lack of nutrients (7); and elude the immune system (8). 

CD56, also called neuronal cell adhesion molecule 1 (NCAM1), is aberrantly expressed on the surface of MM cells, while absent in normal plasma cells. We recently showed that CD56 controls transcription factor CREB1 (cAMP responsive element binding protein 1) by inducing its phosphorylation and therefore its binding to the DNA (9). 

Results:

In our new paper (10), we studied how IFN-gamma signaling and CREB1 regulate the function of HLA-E, a non-classical major histocompatibility complex (MHC) class I molecule involved in immune escape by preventing natural killer (NK) cell-mediated cytotoxicity (11). It is well known that the immune system of patients with MM is unbalanced towards an immunosuppressive phenotype, with an increase of exhausted or anergic T cells and inhibitory NK cells (8). These abnormalities reduce the responses to immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, T cell engagers, and chimeric antigen receptor (CAR) T-cell therapies (12).

We first demonstrated that HLA-E expression is present in pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance-MGUS and smoldering multiple myeloma-SMM) and in overt MM, but not in normal plasma cells, indicating it is an early myelomagenesis event. Moreover, HLA-E is also present in extracellular vesicles released by myeloma cells; therefore, targeting HLA-E expression could hence be a strategy to prevent MM progression or to reduce bloodstream dissemination, by removal of decoy particles in the circulation.

We then observed that patients with high CREB1 expression have activation of IFN-gamma-related pathways and greater HLA-E levels. We confirmed that CREB1 binds to the promoter of HLA-E by ChIP-sequencing analysis. We then established CREB1 gain-of and loss-of function models showing that CREB1 modulates the expression of immune checkpoint HLA-E on MM cells at both mRNA and protein levels. Since IFN-gamma also induces HLA-E by STAT1 signaling activation (13), we modulated STAT1 signaling by overexpression of STAT1 or treatment with the cytokine itself. We observed that IFN-gamma treatment or the combined overexpression of STAT1-CREB1 were additive in increasing HLA-E expression, while IFN-gamma treatment with CREB1 inhibition could not induce HLA-E and blocked STAT1 activation. In contrast, STAT1 limited CREB1 activation in a negative feedback manner.

            IMiDs activate the phosphorylation of STAT1, resulting in the transcription of IFN-gamma target genes (14). We showed that IMiDs also upregulate HLA-E expression, while proteasome inhibitors (PIs), such as bortezomib and carfilzomib, decrease the expression of HLA-E either as single agents or in combination with IMiDs.

            Finally, HLA-E expression on cancer cells delivers an inhibitory signal to CD56^bright NKG2A⁺ CD16^- NK cells preventing the lysis of the malignant cells. The inhibition of CREB1, especially in combination with pomalidomide, restored NK cell-mediated cytotoxicity toward MM cell lines and patient samples. Our findings emphasize the role of CREB1 in immune escape, leading to novel therapeutic approaches for patients with MM.

What’s next:

            In these studies, we identify that CREB1 inhibition is effective in killing MM cells by activating NK cell-mediated cytotoxicity. CD94/NKG2A-HLA-E axis is an important immune checkpoint which has not been fully explored clinically yet. Indeed, targeting NKG2A by monoclonal antibodies (15, 16) has been validated in preclinical models but showed variable activity in clinical trials (17). CREB1 inhibitors have never been tested in human studies, but we are currently working on bringing this new therapeutic approach to the clinic. Since CD56 activates CREB1, we envisage that combining CREB1 inhibition with IMiDs will be specifically effective in patients with CD56+ disease, as shown in our vitro models (9).

            Finally, CREB1 has several roles in normal cells, and we anticipate that other processes could be affected by this transcription factor. Ongoing studies are aimed at understanding the overall pathways induced by CREB1 in MM, with the potential to translate these studies to other CREB1-driven cancers (18), for the benefit of an even bigger number of patients.

References:

1. Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011;364(11):1046-60.
2. Carrasco DR, Tonon G, Huang Y, Zhang Y, Sinha R, Feng B, et al. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients. Cancer Cell. 2006;9(4):313-25.
3. Keats JJ, Reiman T, Maxwell CA, Taylor BJ, Larratt LM, Mant MJ, et al. In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression. Blood. 2003;101(4):1520-9.
4. Ferguson ID, Patino-Escobar B, Tuomivaara ST, Lin YT, Nix MA, Leung KK, et al. The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance. Nat Commun. 2022;13(1):4121.
5. Flores-Montero J, de Tute R, Paiva B, Perez JJ, Bottcher S, Wind H, et al. Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma. Cytometry B Clin Cytom. 2016;90(1):61-72.
6. Yao L, Wang JT, Jayasinghe RG, O'Neal J, Tsai CF, Rettig MP, et al. Single-Cell Discovery and Multiomic Characterization of Therapeutic Targets in Multiple Myeloma. Cancer Res. 2023;83(8):1214-33.
7. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7(8):585-98.
8. Cohen AD, Raje N, Fowler JA, Mezzi K, Scott EC, Dhodapkar MV. How to Train Your T Cells: Overcoming Immune Dysfunction in Multiple Myeloma. Clin Cancer Res. 2020;26(7):1541-54.
9. Cottini F, Rodriguez J, Hughes T, Sharma N, Guo L, Lozanski G, et al. Redefining CD56 as a Biomarker and Therapeutic Target in Multiple Myeloma. Mol Cancer Res. 2022;20(7):1083-95.
10. Ismael A, Robinette AJ, Huric L, Schuetz J, Dona K, Benson D, et al. CREB1 promotes expression of immune checkpoint HLA-E leading to immune escape in multiple myeloma. Leukemia. 2024.
11. Braud VM, Allan DS, O'Callaghan CA, Soderstrom K, D'Andrea A, Ogg GS, et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature. 1998;391(6669):795-9.
12. Friedrich MJ, Neri P, Kehl N, Michel J, Steiger S, Kilian M, et al. The pre-existing T cell landscape determines the response to bispecific T cell engagers in multiple myeloma patients. Cancer Cell. 2023;41(4):711-25 e6.
13. Gobin SJ, van den Elsen PJ. Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G. Hum Immunol. 2000;61(11):1102-7.
14. Fedele PL, Willis SN, Liao Y, Low MS, Rautela J, Segal DH, et al. IMiDs prime myeloma cells for daratumumab-mediated cytotoxicity through loss of Ikaros and Aiolos. Blood. 2018;132(20):2166-78.
15. Andre P, Denis C, Soulas C, Bourbon-Caillet C, Lopez J, Arnoux T, et al. Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells. Cell. 2018;175(7):1731-43 e13.
16. Kamiya T, Seow SV, Wong D, Robinson M, Campana D. Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells. J Clin Invest. 2019;129(5):2094-106.
17. Geurts VCM, Voorwerk L, Balduzzi S, Salgado R, Van de Vijver K, van Dongen MGJ, et al. Unleashing NK- and CD8 T cells by combining monalizumab and trastuzumab for metastatic HER2-positive breast cancer: Results of the MIMOSA trial. Breast. 2023;70:76-81.
18. Zheng T, Huang J, Xiang X, Li S, Yu J, Qu K, et al. Systematical analysis reveals a strong cancer relevance of CREB1-regulated genes. Cancer Cell Int. 2021;21(1):530.