Multiomic Evaluation of Immunologic Changes in Chronic Lymphocytic Leukemia with Venetoclax Treatment

Introduction:

Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the US, is characterized by the proliferation of malignant B-cells that exert immunosuppressive effects through complex, partially understood mechanisms. Immunosuppression in CLL leads to increased risk of secondary malignancies, opportunistic infections, and treatment failure to T-cell based immunotherapies (e.g. chimeric antigen receptor T-cells [CART]). Venetoclax (Ven) is a highly effective BCL2 inhibitor that promotes apoptosis in CLL cells and may enhance T-cell function.

Methods:

Peripheral blood (plasma & peripheral blood mononuclear cells [PBMC]) specimens and clinicopathologic data from 13 consented patients (BDR-164122) with CLL initiating treatment with Ven were prospectively collected at baseline (D0) and after Ven ramp up at 30 days post-treatment (D30). Specimens were analyzed via flow cytometry (FC, including T-cell subsets and exhaustion markers), RNA sequencing (on both PBMC, [PB-RNAseq] and T-cells separated by negative selection [TcRNAseq]), Reduced Representation Bisulfite Sequencing (RRBS), and a Biocrates MxP Quant 500 XL metabolomic panel. Comparative multiomic analysis was performed using machine learning and bioinformatics tools (including R, CIBERSORTx, & NIH-DAVID) and differentially expressed genes (DEGs), methylated regions (DMRs), and regulated metabolites were analyzed.

CAR T-cells (anti-CD19 scFv, CD28 transmembrane/intracellular signaling domain, TCR ζ-chain signaling domain, lentiviral vector) were manufactured at both D0 and D30 (N=5) with transduction efficiency evaluated by FC. Cytotoxicity against Luciferase transfected Raji cells (Raji-Luc) was measured.

Results:

All but 1 patient had bulky disease prior to D0, and 12 patients were successfully de-bulked prior to D30 (7 with Ven alone, and 6 with Ven and an anti-CD20 antibody). Post-treatment D30 flow cytometry revealed an increase in CD8+ T-cell frequency, as well as CD8+ T_Stem Cell Memory (TSCM) and T_naïve (TN) memory cell subsets. D30 TcRNAseq correspondingly revealed significant upregulation of genes important for CD8+ and CD4+ memory T cell (TIMP2, CPEB3) and CD8+ memory development (PRKCZ). TcRNAseq DEG analysis revealed significant upregulation of genes involved in both T-cell activation/survival (TNFSF13B, ZDHHC21, VAV3) and negative regulators of T-regulatory cell (TReg) development and enhanced ferroptosis (ALOX5).

PB-RNAseq revealed significant post-treatment upregulation of DEGs governing M1 macrophage polarization (TLR4), monocyte/macrophage differentiation markers (CD68, CD163, CCR1, CSF1, IL32), neutrophil migration (CXCR2), and neutrophil chemoattraction (CXCL8), as well as T-cell inflammatory cytokine production and cytotoxic function (GZMB, GZMK, TNFAIP2). CIBERSORTx immune cell deconvolution clustering correlated with FC, and revealed 3 transcriptomic subgroups that mirrored treatment responses (% change in circulating CLL cells), with better responders having 1698 positively expressed DEGs associated with T-cell activation (ITK, CD4, CD8A, CD8B, CD28) and cytokine signaling (IL7, EREG, CSF1R, CCR2).

Metabolomic analyses revealed that lysophosphatidylcholine and lysophosphatidylethanolamine were significantly increased in D30 plasma, while spermine, spermidine (known to polarize CD4+ T-cells to TReg), taurine, and glutamate plasma concentrations decreased at D30. The most hypermethylated D30 DMRs by RRBS were GFI1B, ALOXE3, HLA-DOA, and PRDM16 while KCNB1 and TMEM275 were the most hypomethylated D30 DMRs.

D0 CART had noticeably impaired post-transduction CART expansion compared with D30 post-Ven CART. Enhanced CART cytotoxicity was observed at D30 post-Ven in 3 of 5 patients by Raji-Luc cytotoxicity assays, relative to intra-patient D0 CART.

Conclusions:

One month of Ven treatment and de-bulking of CLL cells was found to significantly enhance immune cell activation and function. Upregulation of genes and associated phenotypic changes that correlate with improved cytotoxicity with CART (e.g. TSCM and TN T-cell subsets) were observed, as were pro-inflammatory changes in innate immune cells (e.g. M1 macrophage polarization). Some of these changes appear to be metabolically regulated. Our findings suggest Ven enhances CART cytotoxicity and T-cell function, which warrants further translational validation.