Ibrutinib In Chronic Lymphocytic Leukemia Research Articles

Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib

BackgroundIbrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance.MethodsWe performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis.ResultsWe identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell–receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib.ConclusionsResistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell–receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.)

P53 Pathway Alterations Influence Survival and Treatment Response In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy, with survival rates that range from months to decades. Numerous genetic markers have been identifying that predict for poor survival outcomes. One of the most significant prognostic risk factors is loss of integrity of the p53 pathway. It is well known that loss of the short arm of chromosome 17 (17p-), containing the p53 locus, is a major predictor poor outcome in CLL, however, there is a lack of clarity how specific p53 mutations influence cancer progression. It is our contention that understanding the impact of p53 mutations in CLL progression will facilitate the development of better treatments options. We are using a mouse model of B-CLL (Eµ-TCL1) that also carry a p53 hot-spot mutation p53R172H, corresponding to R175H in humans, to study the impact of p53 mutation on treatment response and survival. Cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ were treated with fludarabine at 35mg/kg per day for 5 days each month. We have observed significant changes in survival and selective pressure on the p53 locus following fludarabine treatment, leading to a refractory population of clones. Mice in the Eu-TCL1 cohort received a median of 1 treatment cycle (Range; 1-3), while the mice in the Eµ-TCL1;p53R172H/+ cohort received a median of 3 treatment cycles (Range;1-4), suggesting that the mutant cohort are likely to benefit initially with treatment only to succumb to a refractory clone of CLL. Mice in the Eµ-TCL1 cohort had an overall survival of 373 days (range; 341-464 days) in comparison to 322 days (range; 270-382days) in the Eµ-TCL1;p53R172H/+ cohort, indicating a trend toward inferior survival in the Eµ-TCL1;p53R172H/+ cohort. Interestingly, mice in the Eµ-TCL1 cohort had significant splenomegaly (40%) as compared to the Eµ-TCL1;p53R172H/+ cohort (0%). However, we observed that all Eµ-TCL1;p53R172H/+ had extensive extranodal disease with mice having abdominal and/or cervical mass, which is suggestive of Richter's transformation. Together, these data indicate that fludarabine based therapies may have a profound negative transformational impact on CLL progression when used as a treatment strategy in the context of p53 mutations. These results would suggest that less cytotoxic therapies may benefit patients harboring p53 mutations. To this end, we are evaluating the effect of Ibrutinib therapy in the context of p53 mutations. We are currently treating cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ mice with Ibrutinib 25mg/kg/day. Our preliminary results suggest Ibrutinib's innovative mechanism may lessen some of these selective pressures on the p53 pathway and may indicate Ibrutinib is a superior treatment option compared to fludarabine when p53 mutations are present. In conjunction with these mouse studies, we are also determining the impact of p53 mutation in human patients by examining changes in CLL patient samples and cell lines following fludaribine and Ibrutinib treatment. This dual mouse/human strategy represents an ideal system in which to study genetic anomalies in the p53 pathway and examine the dynamic molecular pressures exerted by both fludarabine and Ibrutinib in CLL. Disclosures:No relevant conflicts of interest to declare.

Effective Inhibition Of Tumor Microenvironment Interactions In CLL Patients Treated With The BTK Inhibitor Ibrutinib Results In Sustained Inhibition Of Tumor Proliferation and Survival Pathways

▪Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, tissue resident CLL cells show an increase in both B-cell receptor (BCR) and NF-κB signaling; pathways known to regulate survival, proliferation and migration of CLL cells. One key signaling molecule in this pathway is Bruton's tyrosine kinase (BTK) that is activated directly downstream of the BCR and known to be up-regulated in CLL cells (Herman et al., Blood 2011). Ibrutinib, a covalent BTK inhibitor currently in clinical trials for CLL, has been shown to induce apoptosis and inhibit proliferation and tumor burden both in vitro and in mouse models of CLL (Herman et al., Blood 2011; Ponader et al., Blood 2012, Herman et al., Leukemia 2013). Recently, in a multicenter study, ibrutinib has been shown to induce objective clinical responses and reduce lymphadenopathy in the majority of patients, regardless of the presence of adverse prognostic markers (Byrd et al., NEJM 2013).It has been shown that CLL cells in the lymph node and bone marrow microenvironments demonstrate higher levels of BCR and NF-κB signaling as well as increased cell activation and proliferation (Herishanu et al., Blood 2011). We therefore sought to determine the in vivo effect of ibrutinib on tumor cell activation and proliferation in these microenvironmental niches. We have previously demonstrated that ibrutinib inhibits BCR and NF-κB in the lymph node microenvironment within the first 24 hours after initiation of therapy (Herman et al., ASH 2012). We here expand our previous results by evaluating the long term effects of ibrutinib in the tissue compartment. Because repeat sampling of lymph node tissue on therapy is not practical, we assessed changes in the bone marrow compartment. We obtained bone marrow aspirates pre-treatment and after two cycles of ibrutinib (Day 56). We first evaluated the BCR gene signature using a previously validated set of BCR regulated genes (Herishanu et al., 2011). We found that BCR signaling was significantly inhibited in CLL cells sampled from the bone marrow in 8/8 patients evaluated (P = 0.01). Similarly we also found inhibition of the NF-κB gene signature in all patients evaluated (P = 0.01). In fact, every patient evaluated demonstrated a reduction in both signatures, but there was substantial variation among patients in the extent to which these pathways were inhibited. This variability did not appear to correlate with clinical and prognostic factors, such as IGHV mutation status, deletion of chromosome 17p, or prior treatment status. However, the degree of inhibition of NF-κB signaling was strongly correlated with the degree of inhibition of BCR signaling (r = 0.96, P < 0.001), suggesting that the BCR (and/or a second equally BTK dependent pathway) plays a major role in activating NF-κB also in bone marrow resident CLL cells. To confirm our results we next evaluated the phosphorylation of proteins activated downstream of the BCR. We found that CLL cells showed a significant reduction in both PLCγ2 and ERK phosphorylation (mean reduction 52.9% and 71.2%, respectively; P < 0.01). Next, using the proliferation marker Ki67 we found a significant reduction in tumor proliferation in the bone marrow on ibrutinib (from a median of 6.6% KI67+ CLL cells pre-treatment to 1.1% on Day 56, P = 0.003). Lastly, we also found a significant reduction in the cell surface expression of the activation markers CD69 and CD86 (mean reduction of 57% (P = 0.001) and 82% (P = 0.03), respectively).In conclusion, our data demonstrate that ibrutinib effectively inhibits BCR, NF-κB, and ERK signaling. This occurs very quickly as demonstrated in the lymph node and is sustained on treatment as shown in the bone marrow. The strong and sustained reduction in proliferation and activation of CLL cells in the tissue microenvironment suggests that BTK is indeed a central hub mediating the nourishing and protective effects of the tumor microenvironment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Kinetics Of Chronic Lymphocytic Leukemia Cells In Tissues and Blood During Therapy With The BTK Inhibitor Ibrutinib

Bruton's tyrosine kinase (BTK), an essential component of B-cell-receptor (BCR) signaling, is blocked by ibrutinib, a BTK inhibitor that is clinically active in patients with chronic lymphocytic leukemia (CLL). Characteristically, ibrutinib causes CLL cell redistribution from tissue sites into the peripheral blood during the initial weeks of therapy, resulting in a transient surge in lymphocytosis. This activity of ibrutinib is thought to be important for the anti-leukemia activity of ibrutinib in CLL, because it removes CLL cells from the tissue microenvironment and consequently deprives the leukemia cells from growth- and survival-promoting cellular and molecular signals that are present in the tissues. However, clinically it has been noted that the magnitude of lymphocytosis during ibrutinib therapy is lesser than what would be expected from the rapid reduction in overall tissue disease burden, seen during the early weeks of ibrutinib therapy, especially in patients with bulky lymph nodes.Therefore, we aimed to quantify the relative amount of tissue CLL cells redistributed into the peripheral blood, and to better characterize the dynamics of this redistribution phenomenon. Crucial kinetic parameters were estimated by fitting a two-compartment mathematical model to clinical data that document serial blood lymphocyte counts of CLL patients as well as changes in total tissue burden, calculated from radiological measurements of lymph node and spleen volumes during ibrutinib therapy. This was done in a cohort of ten relapsed CLL patients, treated with single-agent ibrutinib, 420 mg PO daily on the Phase 1b/2 ibrutinib study at MD Anderson Cancer Center. We found that during ibrutinib therapy, on average 1.7 ± 1.1% of the cells die per day in the blood, while in tissue 2.7% ± 0.99% of the cells die per day. These death rates during treatment are approximately 5 times higher than those estimated in previous studies in the absence of treatment. The average fraction of the tissue CLL cells that was redistributed into the blood during ibrutinib therapy accounted for 23.3 ± 17% (range: 1.9% to 52.6%) of the calculated total tissue disease burden, with an average time for achieving a 50% reduction in tissue CLL cells of 20.3 ± 3.6 days. These findings indicate that generally a relatively small fraction of the total tissue CLL cell burden is re-distributed into the blood during ibrutinib therapy, and that a significant amount of drug-induced cell death occurs in tissue compartments. Disclosures:Off Label Use: Ibrutinib for CLL therapy. James:Pharmacyclics: Employment, Equity Ownership. O'Brien:Pharmacyclics: Research Funding. Burger:Pharmacyclics: Research Funding.

Ibrutinib Inhibits B-Cell Adhesion and Causes An Efflux Of Chronic Lymphocytic Leukemia Cells From The Tissue Microenvironment Into The Blood Leading To a Transient Treatment-Induced Lymphocytosis

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, monoclonal B-cells in the peripheral blood (PB), bone marrow, and secondary lymphoid organs. Ibrutinib targets Bruton's tyrosine kinase (BTK); which is activated downstream of the B-cell receptor (BCR), a pathway known to regulate the survival, proliferation, and migration of B-cells. In vitro, ibrutinib has been shown to induce apoptosis and inhibit proliferation (Herman et al., Blood 2011). Recently, clinical trials demonstrated that ibrutinib is well tolerated and can induce objective clinical responses in CLL (Advani et al., JCO 2013; Byrd et al., NEJM 2013). Interestingly, ibrutinib leads to a transient lymphocytosis that resolves over the first 2-3 months on treatment. This lymphocytosis is thought to arise because of a redistribution of CLL cells due to the release of tumor cells from the tissue compartment into the peripheral blood and/or due to inhibition of re-entry of circulating cells into the tissue sites. In our single agent phase II trial of ibrutinib in CLL (NCT01500733) over 40% of patients reached their peak absolute lymphocyte count (ALC) already within 24 hours of starting treatment. We therefore hypothesized that the increase in ALC may be primarily due to efflux of cells from the lymph node, and that this would result in an increased number of cells in the blood having the immunophenotypic characteristics of tissue resident cells. Indeed, compared to the matching pre-treatment samples, circulating CLL cells on day 2 showed increased expression of the activation marker CD38 and of the proliferation marker Ki67 (P < 0.01). Consistent, with a treatment-induced release of already activated cells from tissue sites, by day 14 the circulating cells have lower expression of CD38 and Ki67 than at baseline. Next we sought to determine whether ibrutinib decreases the expression of the adhesion molecules CD62L and CD49d. By day 28 of treatment, we indeed found a statistically significant decrease in the expression of CD62L (P= .02; median reduction 38%) but only a minimal reduction in CD49d (median reduction 13%; P > 0.05).To evaluate the in vivo effect of ibrutinib on cell adhesion we chose a functional assay that models tumor-stromal interactions in the tissue microenvironment. We collected PB tumor cells from patients pre-treatment and on day 28 of treatment. In vitro, these cells were allowed to adhere to fibronectin-coated plates for 1 hour. After rigorous washing, we quantified the number of adherent cells by fluorescence microscopy. CLL cells from ibrutinib treated patients showed a dramatic reduction in their ability to adhere to extracellular matrix component fibronectin compared to matching pre-treatment cells (median inhibition 96%, range 75%-99%; P< 0.001). This reduction in adhesion was demonstrable both within 24 hours and after 4 weeks on therapy. Interestingly, the inhibitory effect of ibrutinib on cell adhesion was independent of IGHV mutational status.In conclusion, we present direct in vivoevidence that the ibrutinib induced lymphocytosis develops within hours and is, in significant part, due to the release of previously activated cells from the tissue microenvironment. The rapid onset of the lymphocytosis and the dramatic defect in adhesion, in the absence of major changes in the expression of adhesion molecules, suggests that ibrutinib directly interferes with an intracellular signaling network required for cell adhesion.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood samples to make this research possible. Also, we acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

In Vivo Effects Of Ibrutinib On The Migration Of Chronic Lymphocytic Leukemia Cells Differ Between Patients and Reduce The Ability Of The Bone Marrow Microenvironment To Attract The Tumor Cells

The Bruton's tyrosine kinase inhibitor ibrutinib has recently been shown to be well tolerated, and to induce objective clinical responses in the majority of patients, irrespective of adverse prognostic markers (Byrd et al., NEJM 2013). Despite the demonstrated clinical activity, ibrutinib also leads to a transient lymphocytosis that is thought to reflect a redistribution of cells from tissue compartments into the peripheral blood. The mechanisms contributing to this lymphocytosis are not well understood. To date, two groups have demonstrated that in vitro treatment with ibrutinib inhibits migration of CLL cells in chemokine gradients (de Rooji et al., Blood 2012 and Ponader et al., Blood 2012). Here we sought to assess the in vivo effect of ibrutinib on cellular migration. To validate our assay we first treated CLL cells with 1uM ibrutinib for 1 hour in vitro and measured migration of CLL cells to a mix of SDF-1 (at 200ng/mL) and CCL19 (at 100ng/mL), two chemo-attractants known to induce migration of CLL cells. Migration was assayed in a dual chamber system separated by a membrane with 5µm pores after 3 hours of incubation. Confirming published data we found a significant reduction in the migration index (ratio of migration to chemokines divided by migration to media alone) of ibrutinib treated cells compared to untreated cells (mean reduction 24%; P = 0.04). Next, we analyzed the migration of CLL cells obtained from patients (n = 9) enrolled on a clinical trial with single agent ibrutinib that were sampled pre-treatment and after 4 weeks on drug. We observed highly variable responses; in about half of the patients treated cells showed increased migration, while in the other half there was decreased migration to the SDF-1/CCL19 mix. Interestingly, patients showing a decrease in migration on treatment often had del17p but there was no difference in regards to IGHV mutation status and no correlation to the degree of lymphocytosis observed in the patient. T-cell migration was not affected by ibrutinib. In order to extend the analysis to a mix of chemo-attractants that the tumor cells may encounter in vivo we used the supernatant harvested from bone marrow aspirates and found that it efficiently induced migration of CLL cells in the dual chamber assay (mean fold increase 5.2 compared to control). Comparing CLL cells from patients sampled pre-treatment to those obtained on treatment day 28 we again found the same mixed effects of ibrutinib on the ability of CLL cells to migrate to bone marrow plasma as we had observed with the SDF-1/CCL19 mix. Thus, direct inhibition of CLL cell migration can account for only a subset of patients with treatment-induced lymphocytosis. Given reports that ibrutinib can inhibit cytokine and chemokine secretion from CLL cells and T-cells (Ponader et al., Blood, 2012; Herman et al., Blood, 2011), we hypothesized that ibrutinib treatment might change the content of chemo-attractants in the bone marrow We therefore compared the ability of the bone marrow plasma obtained pre-treatment and after 2 months on ibrutinib to attract CLL cells (these cells were obtained from the peripheral blood pre-treatment from the same patient donating the marrow). We found that in 4/4 patients evaluated there was a significant reduction in the migration of CLL cells to the on-treatment bone marrow plasma compared to the matching pre-treatment sample (mean decrease 20%; P < 0.05).In conclusion, migration of CLL cells from patients on ibrutinib can be inhibited or increased, with most del17p patients showing decreased migration. Intriguingly, these patients tend to have slower resolution of the treatment induced lymphocytosis, raising the question whether inhibition of homing to tissue sites could affect the time to resolution of the lymphocytosis. In addition, we provide evidence that bone marrow plasma on ibrutinib therapy has a reduced capacity to attract CLL cells, suggesting that ibrutinib may alter the composition of the bone marrow microenvironmentThis work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Cytokine and T-Cell Phenotypic Changes Upon In Vivo Ibrutinib Therapy For CLL – Targeting Both CLL Cells and The Tumor-Microenvironment

IntroductionProliferation of chronic lymphocytic leukemia (CLL) cells is highly dependent on the microenvironment. B-cell receptor (BCR) signaling and interactions of the tumor cells with elements of the tissue microenvironment including T cells and macrophages appear to be of particular importance (Burger et al, Blood 2009; Herishanu at al, Blood 2011; Bagnara at al, Blood 2011). The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is highly effective in blocking BCR signaling and leads to impressive clinical responses in CLL (Byrd et al, NEJM 2013). BTK is a member of the TEC kinase family that also includes TEC, IL2-inducible T cell kinase (ITK), and BMX/ETK. BTK is not expressed in T cells; however ITK, which is expressed in T cells, is directly inhibited by ibrutinib, and the drug reduces cytokine secretion from activated T cells without inducing apoptosis (Herman et al, Blood, 2011). Here, we sought to determine the in vivo effect of ibrutinib on T cells and cytokine levels in CLL patients treated with single agent ibrutinib. MethodsThe effect of ibrutinib on T-cell subsets, T-cell activation, and cytokine profiles was assessed in 10 CLL patients treated with 420mg ibrutinib daily in an ongoing phase II trial (NCT01500733). Matched samples of viably frozen peripheral blood mononuclear cells obtained from patients pre-treatment and after 6 months on ibrutinib were analyzed by flow cytometry. Cytokine levels pre-treatment and on days 1, 28, months 2, and 6 on ibrutinib were measured in the same patients using the Milliplex human cytokine assay. ResultsConsistent with inhibition of BCR signaling in CLL cells, CCL3 and CCL4 serum levels were rapidly and significantly decreased by ibrutinib as described previously (Ponader et al, Blood, 2012). In addition, serum levels of a number of inflammatory cytokines including IL6, IL8, IFNg, and TNFα were decreased by > 50% by day 28 of ibrutinib treatment and remained so by 6 months. This is of specific interest as “pseudoexhausted” T cells from CLL patients were recently shown to secrete high amounts of IFNg, and TNFα (Riches et al, Blood 2013). Thus, the decreased levels of inflammatory cytokines may reflect a reversal of T cell “pseduoexhaustion”. Furthermore, the immunosuppressive cytokine IL10, a Th1-type cytokine that is secreted by CLL cells and activated T cells, was also rapidly and significantly reduced. These in vivo data are consistent with previous in vitro data showing decreased secretion of IL6 and IL10 from T cells upon exposure to ibrutinib (Herman et al, Blood, 2011). Thus, ibrutinib appears to reduce cytokine and chemokine secretion from both CLL and T cells resulting in an overall decrease in inflammatory cytokines. While absolute T-cell numbers showed little change on treatment, we found that ibrutinib reduced the frequency of activated CD4+ T cells (Table). Furthermore, for 3 out of 4 patients, the percentage of Ki67 positive T cells in the peripheral blood decreased on ibrutinib therapy (mean decrease 63%). The frequency of the Th17 T-cell subset was also diminished. Consistently, a decrease in serum levels of IL17 was seen in the two patients having detectable IL17 levels pre-treatment. While changes in the cytokine pattern (decrease in IFNg and IL10) might suggest inhibition of a Th1-type response, there was no change in the ratio of Th1 to Th2 T-cell subsets by immunophenotyping.TableSubset of CD4+ T-cells (measured in 10 patients)Pre-ibrutinib (mean %)At 6 months on ibrutinib (mean %)P (paired t test)Activated cells (HLA-DR+, CD39+)3.91.80.003Th17 (CXCr3neg, CCR6+)1.70.90.04Th1 (CXCR3+, CCR6neg)66570.51Th2 (CXCR3neg, CCR6neg)28390.42 ConclusionsWe here demonstrate a decrease in the levels of inflammatory cytokines and in T-cell activation in CLL patients treated with ibrutinib. Whether this is a direct consequence of BTK inhibition in B-cells or, at least in part, results from inhibition of T-cell signaling remains to be determined. Nevertheless, our data indicate that ibrutinib significantly alters the composition of the tumor microenvironment in CLL, affecting soluble as well as cellular elements. These effects may be important for clinical response and the development of combination therapies and therefore deserve further study.Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia. Chang:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics: Employment, Equity Ownership.

Use of tumor genomic profiling to reveal mechanisms of resistance to the BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL).

7014 Background: Ibrutinib interacts covalently with cysteine 481 of Bruton tyrosine kinase (BTK), resulting in targeted inhibition of B cell receptor signaling. Early trials of ibrutinib mono- or combination therapy enrolled 246 CLL patients receiving a median of 14 months of ibrutinib. 20 patients (8%) experienced progressive disease (PD), including 8 patients with Richter's transformation. Here we examine changes to the CLL genome in 3 patients that acquired resistance to ibrutinib. Methods: Ibrutinib resistance was defined as patients achieving partial response (PR) or better lasting ≥ 6 months, then developing PD without Richter’s transformation. RNAseq and whole exome sequencing (WES) followed by comparative genome analysis was performed at baseline and after PD and confirmed by Sanger sequencing. RNAseq and WES data were aligned using TopHat and BWA software. Single nucleotide variations (SNVs) were identified using SAMtools mpileup. Results: Compared to patients who relapsed from conventional chemotherapy, minimal genomic changes were acquired in ibrutinib resistant patients, reflecting relative genomic stability. SNVs were discovered in 3 patients specific to the relapse sample (Table). 2 out of 3 patients had distinct SNVs that each encode a cysteine-to-serine substitution at position 481of BTK (C481S). Homologous cysteine residues in BMX, ITK, TEC and BLK were wild-type (WT). Ibrutinib inhibited recombinant C481S 25 fold less potently than WT, and could not covalently bind C481S expressed in cells. The third patient had WT BTK, but acquired a potential gain-of-function mutation encoding a R665W substitution in PLCg2, a substrate of BTK, consistent with constitutive PLCg2 activation. Conclusions: Although rare, the acquisition of C481S BTK and R665W PLCg2 mutations in the setting of resistance confirms BTK as an important pharmacologic target of ibrutinib, and suggests mechanisms of ibrutinib resistance. [Table: see text]