1111 - Immune-profiling of ibrutinib-treated CLL patients revealed TMBIM6 as a potential target for CLL and its high expression as an independent variable associated with poor prognosis

CLL is distinguished by a marked T-cell disfunction that aggravates at disease progression. Ibrutinib treatment is commonly used in first-line and relapsed/refractory CLL. Discontinuations due to adverse effects, progression/Richter transformation and secondary resistances are current limitations. Identifying potential adaptive mechanisms to ibrutinib may help to overcome these limitations. Additionally, although there are some discrepancies between in-patient and in vitro studies, ibrutinib has been reported to improve T-cell function.
Our study aimed to longitudinally assess the immune profile of ibrutinib-treated CLL patients to evaluate its effect in T-cell function and to identify potential adaptive mechanisms. To do so, blood samples from 28 patients from GELLC7 trial (NCT03280160) were collected, at least, at two timepoints, including before treatment (BT), at 1, 3, 6 and 12 months during the first year of single-agent ibrutinib treatment. Peripheral blood mononuclear cells and plasma were isolated and stored to perform flow cytometry, RNA/DNAseq, migration assays, and cytokine determination.
T-cell immunophenotyping revealed a decrease of CD4+ and CD8+ T cells expressing PD1 and CD244 upon ibrutinib treatment. The immunosuppressive/supportive subpopulations of T-regulatory cells and T-follicular helper cells were also reduced. Bulk-RNAseq of purified T cells revealed 170 differential expressed genes (DEG) upon 6 months of ibrutinib treatment. These genes participate in biological processes (Gene Ontology) related to cell stimulation and regulation of cell activation. Gene set enrichment analysis (GSEA) showed a downregulation of oxidative metabolism, proliferation, and activation pathways, indicating a reduction of the CLL inflammatory burden (Figure 1). Furthermore, there was a significant decrease of several inflammatory/migration chemokines (CCL2/CCL3/CCL4/CXCL10/CXCL12/CXCL13/CCL19) in plasma at 6 months.
CLL-cell immunophenotyping showed a decreased expression of markers related to adhesion (CD44/CD62L), immunosuppression (CD200/BTLA), and migration (CXCR5/CCR7) at 6 months of ibrutinib treatment. However, CXCR4 expression increased upon ibrutinib treatment, and the proliferative CXCR4dimCD5br population was reduced.
We investigated CXCR4-mediated migration as a potential mechanism of CLL-cell adaptation to ibrutinib. To this end, we evaluated the CXCL12-migratory capacity of CLL cells from 14 patients at BT and upon 6 months of BTK inhibition. We observed a reduced CXCL12-migratory capacity, but still some CLL cells were able to migrate under BTK inhibition. To characterize CLL cells that retained migratory capacity under BTK inhibition, we compared the recurrent genetic alterations and gene expression profiling of migrated cells from BT and after 6 months of ibrutinib. Targeted-DNAseq of migrated cells from the two timepoints revealed that, in 6 out of 12 patients, there was an alteration of the subclonal composition of CLL cells that retained migratory capacity under BTK inhibition with a recurrent enrichment of subclones harboring mutations in ATM and a NF-kB gene (NFKBIE or IKBKB). BTK or PLCG2 mutations were not observed at any timepoint. GSEA showed an upregulation of mTORC1 and Myc pathways in cells that retained migratory capacity under BTK inhibition. Among the DEG, TMBIM6 was the most significantly upregulated gene (FDR < 0.05). TMBIM6 gene is related to apoptosis and mTOR-Akt axis and associated with poor prognosis in several solid tumors. We evaluated the prognostic impact of TMBIM6 expression using public clinical data from the CLL-map portal. TMBIM6 high expression was associated with poor overall survival in the univariable analysis (HR:2.23, p-value < 0.001) (Figure 2), and in the multivariate analysis including age, gender, IGHV status and recurrent genetic alterations. We targeted TMBIM6 with BIA, an TMBIM6 antagonist, to assess its impact in CLL viability (Kim et al., 2020). BIA induced CLL-cell apoptosis in suspension and co-cultured with a bone marrow stromal cell line plus CD40L and CpG ODN. Moreover, ibrutinib addition to BIA treatment enhanced the apoptotic effect of BIA. Collectively, these results indicate that TMBIM6 may be a potential novel target for CLL. Further experiments describing the molecular effects of BIA should be performed.
In conclusion, ibrutinib treatment impacted on T cells from CLL patients by reducing the expression of exhaustion markers and the frequency of T-regulatory/follicular helper subpopulations. In CLL cells, we observed a downregulation of markers related to adhesion, immunosuppression, and migration, and an overexpression of TMBIM6 in CLL cells that retained migrative capacity towards CXCL12 under ibrutinib. Furthermore, we identified TMBIM6 high expression as an independent poor prognostic variable and demonstrated that BIA induces apoptosis of CLL cells ex vivo revealing TMBIM6 as a potential novel target for CLL.