Ibrutinib is especially effective in CLL patients with unmutated IgHV (UM-CLL), although presence of unmutated IgHV is usually associated with poor clinical outcome and shorter PFS. Sustained BCR signaling is one major reason for this. The BCL2 protein BIM plays a pivotal role in regulating apoptosis and high expression of BIM has been associated with presence of unmutated IgHV. This led us to investigate if there is a causality between BIM regulation and ibrutinib sensitivity.


We used quantitative mass spectometry (MS) to analyze the (phospho) proteome of CLL patients with unmutated and mutated IgHV (n=3 respectively) upon BCR stimulation and ibrutinib treatment. BTKC481S overexpressing cells were used to validate our findings from MS.


Among all identified (phospho)peptides we found a striking phosphorylation pattern of BIM when comparing UM-CLL and M-CLL. Interestingly, we found that only phosphorylation of S77 was significantly altered, but not phosphorylation of S69 as previously reported. Phosphorylation of BIM S77 was upregulated upon BCR stimulation only in UM-CLL, but not in M-CLL. It is known, that BIM phosphorylation of both S69 and S77 leads to degradation of the protein by ubiquitination. In line with this and with the increase in BIM phosphorylation, total level of BIM decreased upon BCR stimulation.

Furthermore we identified BIM as a target of ibrutinib. The BTK inhibitor was able to reduce phosporylation of BIM S77 both in UM-CLL and M-CLL. In accord with the observations of decreased BIM expression upon BCR stimulation, the decrease in phosphorylation of BIM induced by ibrutinib led to higher total levels of BIM.

In order to investigate if phosphorylation of BIM is BTK dependent we generated an ibrutinib-resistant BTK mutant (C481S) that was overexpressed in Ramos cells. While ibrutinib treatment led to a decrease of phosphorylation of BIM S77 in wildtype cells, no change in phosphorylation could be observed in cells expressing the BTKC481S mutant, which suggests that BIM regulation by ibrutinib is indeed BTK dependent.

Similar to ibrutinib, SYK inhibitor entospletinib leads to reduction of BIM phoshorylation in wildtype Ramos. But in contrast to ibrutinib, both BTK and BIM phosphorlyation were also reduced upon entospletinib treatment in Ramos cells expressing the BTKC481S mutant, which indicates that entospletinib can overcome ibrutinib resistance caused by BTKC481S mutation.


For the first time we found that BIM S77 is a target of ibrutinib. Whereas BCR stimulation led to a differential response dependent on IgHV, ibrutinib was able to target BIM both in UM-CLL and M-CLL in a BTK dependent manner. Furthermore, ibrutinib can regulate total BIM levels by preventing protein degradation. Interestingly, the SYK inhibitor entospletinib can also target BIM, but is able to overcome ibrutinib resistance mediated by BTKC481S mutation.


Hallek:Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau. Frenzel:Gilead: Research Funding; Abbvie: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.

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