Ibrutinib Disrupts the Metabolic Program of CLL Cells in-Vivo
Authors: Rozovski Uri , Harris David M. , LI Ping , Liu Zhiming , Jain Preetesh , Ferrajoli Alessandra , Burger Jan A. , O'Brien Susan M. , Bose Prithviraj , Thompson Philip A. , Jain Nitin , Wierda William G. , Keating Michael , Estrov Zeev 
Published: October 2019

Abstract
Introduction: Unlike their normal resting B cell counterparts, chronic lymphocytic leukemia (CLL) cells proliferate. Approximately 1% of the total CLL cell clone expands daily. To adjust for the increase in energetic demands imposed by continuous proliferation, CLL cells undergo metabolic reprogramming and, as recently shown (Rozovski U, et al. Mol Cancer Res. 2015; 13:944-53), CLL cells utilize fat in a manner similar to that of adipocytes. The recent introduction of the oral Bruton tyrosine kinase inhibitor (BTK) ibrutinib revolutionized the treatment of CLL. Because the proliferation of CLL cells is driven by lipid metabolism and ibrutinib inhibits the B cell receptor-induced proliferation of CLL cells, we sought to determine whether ibrutinib also disrupts the metabolic program that provides CLL cells with their unique energy requirements.
Methods: We prospectively studied serial peripheral blood samples from 16 patients with CLL. The patients' peripheral blood CLL cells were analyzed prior to and during treatment with ibrutinib. All patients received a daily dose of 420 mg ibrutinib. In addition, we performed in vitro studies using CLL cells from 3 ibrutinib-naïve patients. CLL cells were analyzed for free-fatty acids (FFA) consumption and for the rate of cellular apoptosis using propiduim iodide (PI) and annexin V staining analyzed by flow cytometry.
Results: To study lipid metabolism of CLL cells we incubated peripheral blood CLL cells from 3 randomly selected ibrutinib-naïve patients in the presence or absence of FFA and measured the concentration of culture media-dissolved O2 (dO2). Like in our previous study (Rozovski U, et al. Mol Cancer Res. 2015; 13:944-53), we found that CLL cells metabolized FFA and, as a result, the levels of dO2 decreased. However when the cells were co-cultured with FFA and ibrutinib, the delta dO2 (dO2 with FFA minus dO2 without FFA) remained unchanged, suggesting that ibrutinib blocked FFA metabolism in CLL cells.Then, to determine whether ibrutinib also inhibited CLL-cell lipid metabolism in patients treated with ibrutinib, we collected 2 to 5 consecutive PB samples (median: 5) from 16 CLL patients prior to and during treatment with ibrutinib. Unlike the 12% reduction in delta dO2 detected in untreated patients' CLL cells incubated with FFA in vitro, a 6% reduction in delta dO2 was detected in CLL cells of patients treated with ibrutinib 4 days into treatment and after a median of 147 days of ibrutinib treatment a change in delta dO2 was no longer detected. These data suggest that ibrutinib-treated cells lost their capacity to utilize FFA or that the number of FFA consuming circulating CLL cells declined until they were no longer detected. In addition, whereas ibrutinib induced apoptosis of CLL cells in a dose-dependent manner in vitro, ibrutinib did not induce apoptosis at the same time points in vivo, suggesting that interruption of FFA metabolism does not lead to apoptotic cell death and that the metabolic and proapoptotic pathways are not linearly intertwined in CLL cells.
In conclusion: Treatment with ibrutinib changes the metabolic profile CLL cells. Even after short exposure to the drug the cells were less capable of utilizing FFA, and after longer exposure, the cells could no longer utilize FFA. Whether ibrutinib induced reduction in FFA metabolism decreases the proliferation capacity of CLL cells remains to be determined.

Disclosures
Burger: Pharmacyclics: Research Funding. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Novimmune: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; BMS: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria. Wierda:Novartis: Research Funding; Abbvie: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding.