Molecular Mechanisms of Ibrutinib Resistance: Defining a Logical Approach to Improving Targeted Therapy in CLL
Authors: Farag Sara MK , Newton Darren , Doody Gina , Mahmoud Lotfy Abdelnaby , Fouda Manal Ibrahim , El Ghannam Doaa Mahmoud , Hillmen Peter 
Published: October 2019

Abstract
Introduction
Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the western world. The importance of B cell receptor (BCR) signaling in CLL pathology has led to the emergence of a number of targeted therapies specific to kinases in this pathway. Ibrutinib, a potent irreversible Bruton's Tyrosine Kinase (BTK) inhibitor, is highly effective in CLL with minimal side effects. However, resistance to Ibrutinib is now emerging among patients in several clinical trials.
Aims/Methods
To understand the molecular mechanisms underlying ibrutinib resistance to aid in early detection of resistant clones and to inform further treatment choices, we have developed an in vitro ibrutinib resistant model using BCR signalling dependent B cell lymphoma lines (Ramos and Namalwa). These cells have been conditioned by exposure to escalating doses of ibrutinib - starting from 0.001μM and increased over time to a lethal dose of 0.5μM in order to select drug-resistant cells. Single cell clones have been isolated from both the naïve and the resistant cells. The growth kinetics of 7 Ramos and 5 Namalwa resistant clones has reached the same pattern as the parental cells. Additionally, 3 naïve clones were isolated from each sensitive parental cell line.
Dysregulation of the BCR signaling pathway was characterized by both flow cytometry, using specific anti-phosphokinase antibodies, and by Western blotting. In addition, RNA from these clones was isolated and analyzed on an Affymetrix HTA2.0 gene expression array (n=6 from two cell lines paralleled to one naïve clone from each cell line).
Results
In Ramos (n=3), gene expression profiling revealed significant upregulation of BCL2 and interferon response genes including interferon gamma (IFNG). However, in Namalwa (n=3), in addition to BCL2 upregulation, the expression arrays showed the overexpression of BCR signaling pathway genes including BTK itself, CD79B, LCK and GAB2 (Growth factor receptor-bound protein 2- associated-binder 2), suggesting hyper-activation of BCR pathway and constitutive activation of BTK and downstream kinases.
Moreover, initial screening showed that not all of the resistant clones behave similarly. In Ramos, one clone apparently escapes ibrutinib inhibition by modifying different pathways compared to the other analysed clones, suggesting the potential for multiple mechanisms by which the malignant B cell clones can evade the toxic activity of targeted therapy.
Results of this analysis were validated by qPCR and additional resistant clones were tested for BCL2 and IFNG expression. In Ramos, 7 resistant clones showed significant upregulation of BCL2 (mean 9.7-fold increase, range 2.1- 31.8, SEM 4.5) and IFNG(mean increase 8.3, range 2.6-19.1, SEM 2.2). IFNγ, is a known immune modulator linked to various stress signals indicating one possible resistance mechanism of malignant cells to anti-cancer therapies.
In Namalwa, qPCR of 5 resistant clones showed significant upregulation of BTK (mean increase 10.3, range 2- 31: SEM: 5.4) and confirmed at the protein expression level by Western blotting as well; where the resistant cells over-express BTK protein compared to their respective naïve cells (n=6, mean increase 1.6, range 1.1- 1.9, SEM 0.16). Moreover, CD79B was over-expressed (mean over-expression 12.4, range 1.5- 22.2, SEM: 4.3) and 6-fold overexpression of LCK (mean increase 6, range 3.1- 9.5, SEM: 1.4) compared to the analysed naïve clones. Furthermore, validation with qPCR showed 12-fold upregulation of the GAB2gene (mean upregulation 12.7, SEM: 4.8), an adaptor protein which transmits crucial signals to activate downstream PI3K signaling with links to leukemogenesis.
Conclusion
Our results demonstrate that common mechanisms of resistance to BCR inhibition include both the up-regulation of B-cell receptor signaling activity and the up-regulation of anti-apoptotic molecules, particularly BCL2. More potent and more target-specific BTK inhibitors may overcome the hyperactivity of the BCR pathway. In contrast, inhibition of BCL2 with venetoclax may be beneficial in overcoming the emergent resistance to ibrutinib as these cells become more sensitive to BCL2 inhibition. These initial results support the combination of ibrutinib with the pro-apoptotic BCL2 inhibitor, venetoclax, which we are currently studying in the Bloodwise TAP CLARITY study.

Disclosures
Hillmen: Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.