CLL-532 Changes in Proteome of CLL (Chronic Lymphocytic Leukemia) Lymphocytes During Investigator-Initiated Trial of Ibrutinib Alone and in Combination With Venetoclax in Treatment-Naïve Patients With CLL

Context: Combined ibrutinib and venetoclax is an effective treatment for CLL. Objective: Our investigator-initiated-trial (NCT02756897) combining ibrutinib and venetoclax (Jain N. et al. NEJM. 380:2095,2018) demonstrated 72% achieving bone marrow u-MRD (undetectable minimal residual disease) status for patients with treatment-naïve (TN) CLL. These u-MRD status were durable (Jain N. et al. JAMA-Oncol. 7:1213,2021). These results were recapitulated in CAPTIVATE and GLOW trials. Our objective is to elucidate the proteomic landscape during therapy. Patients: Fourteen TN-CLL patients were selected from NCT02756897 trial. Methods: Ibrutinib was administered for 3 28-d-cycles; venetoclax was initiated starting at C4D1. Fifty-six peripheral blood samples from 14 patients were obtained at the baseline (C1D1), after one cycle of ibrutinib (C2D1), after 3 cycles of ibrutinib (C4D1) and in one week after venetoclax addition to ibrutinib (C4D8). Mass-spectrometry-based proteomic profiling was performed on these samples. Lysates of the cancer cells were trypsinized, and the peptides were fractionated by liquid chromatography and analyzed by untargeted mass spectrometry. Results: Analysis of proteomic data yielded identification of >1,000 proteins per time-point. K-mean clustering combined with Gap-Statistics identified 8 cluster patterns. Overall, 70 proteins were up-regulated, and 116 proteins were down-regulated. Early changes included upregulation of nuclear pore proteins NUP214, NUP133 and NUP155 at C2D1. PCK2 and PCYT1A were significantly downregulated after ibrutinib treatment. UIMC and SGSH proteins were consistently upregulated throughout all timepoints. Seven selected proteins (AIF1, ARHGAP25, PCK2, PCYT1A, PPBP, PTPN6, S100A11) were further validated using immunoblot analyses. After 1 cycle of ibrutinib, pathways that dominated maximum changes were energy metabolism, cell death, B- and T-cell signaling, and immune response; these were maintained after 3 cycles of ibrutinib. Impact on transcription and protein synthesis inhibition initiated after first cycle of ibrutinib but dominated after 3 cycles of ibrutinib. After one week of venetoclax, at C4D8 timepoint, the main changes included transcription pathway, signal transduction and genes responsible for energy metabolism. Cytoskeleton related pathways are initially downregulated and become upregulated after the addition of venetoclax. Conclusions: The CLL lymphocytes during ibrutinib and venetoclax therapy displayed significant proteome differences compared to baseline. Further validation studies are ongoing to evaluate the roles of these proteins in CLL pathophysiology and therapy response.