Ibrutinib In Chronic Lymphocytic Leukemia Research Articles

Abstract 3150: PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib

Abstract Background: The Bruton agammaglobulinemia tyrosine kinase (BTK) activates B-cell receptor signaling through activation of phospholipase C gamma 2 (PLCG2). Clinical resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) is highly associated with emergence of the BTK C481 mutations that prevent ibrutinib covalent binding. PLCG2 mutations also occur in these ibrutinib-resistant samples but the spectrum of mutations and their occurrence with BTK changes have not been fully delineated. Materials and Methods: All peripheral blood samples with adequate depth of sequencing coverage were included from CLL patients receiving ibrutinib (with or without other therapies) that were submitted from Ohio State University (OSU) to the OSU James Polaris Molecular Laboratory. Genomic DNA was extracted from negatively selected B cells and deep sequencing of the entire coding regions of BTK and PLCG2 performed using a custom Ion Torrent Ampliseq panel. A mean depth of greater than 1000X was obtained with hotspot mutations validated down to 1% variant allele fraction (VAF) in the B cell preparations using orthogonal mutation-specific detection methods. Results: Among 1063 CLL samples from 380 patients who received ibrutinib, BTK C481 resistance mutations were identified in 79 (20.8%) patients including 20 patients that also had co-occurring PLCG2 mutations. 11 patients (2.9%) had PLCG2 mutations without accompanying BTK C481 alterations for a cumulative incidence of PLCG2 mutations in 8.2% of ibrutinib-treated patients. These included previously described mutations in the SH2 and SH3 domain of PLCG2 (R665W, S707F, A708P and L845F) but also previously uncharacterized mutations in the PLCG2 C2 domain that were seen in 12 patients (3.2%). C2 domain mutations, always seen in association with another PLCG2 and/or BTK resistance mutation, affected codons 1140-1144 that include the highly conserved aspartic acid residues that bind calcium and mediate membrane localization in other C2-domain containing proteins. In sequential samples, PLCG2 C2-domain mutations tracked at similar levels to the co-occurring BTK and PLCG2 resistance mutations indicating their presence in the same population of CLL cells. Conclusions: Mutations in three different PLCG2 structural domains commonly co-occur with BTK C481 mutations. The identification of PLCG2 mutations in the calcium-regulated C2 domain expands the possible mechanisms that can produce PLCG2 activation following ibrutinib treatment. The diversity of recurrent mutations observed supports the need for complete PLCG2 sequencing for full characterization of ibrutinib-treated CLL samples. Citation Format: Dan Jones, Jennifer A. Woyach, Weiqiang Zhao, Sean Caruthers, Huolin Tu, Joshua Coleman, John C. Byrd, Amy J. Johnson, Gerard Lozanski. PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2017-3150

Major Bleeding Complications Among Patients Treated with Ibrutinib and Concomitant Antiplatelet, Anticoagulant, or Supplemental Therapy

Background: Ibrutinib, an orally bioavailable small molecular inhibitor of Bruton’s tyrosine kinase (BTK), is an approved therapy for chronic lymphocytic leukemia (CLL), relapsed mantle cell lymphoma (MCL) and Waldenström’s macroglobulinemia (WM). Beyond B lymphocytes, BTK signaling is important for collagen-mediated platelet activation, and BTK inhibition has been associated with primary hemostatic bleeding events (Levade et al Blood 2014). Although serious bleeding events have been uncommon (1-5%) in clinical trial populations, there is limited data describing the potential for increased serious bleeding incidence when ibrutinib is co-administered with other agents affecting the clotting cascade or platelet function.Methods: We conducted a retrospective cohort study to evaluate the incidence of major bleeding in patients receiving ibrutinib concomitantly with antiplatelet agents (non-steroidal anti-inflammatory agents, ADP inhibitors), anticoagulants (heparins, warfarin, novel oral anticoagulants), or supplements with potential anticoagulant activity (vitamin E and fish oil). Major bleeding events were identified using criteria developed by the International Society on Thrombosis and Haemostasis (Schulman et al J Thromb Haemost 2005). Patients 18-89 years of age and treated with ibrutinib for CLL, MCL, or WM between March 1, 2010 and March 1, 2015 were included. The primary endpoint of this study was the incidence of major bleeding events, but we also sought to identify risk factors associated with the development of major bleeding, focusing on potential drug interactions. Based on the historic prevalence of major bleeding in ibrutinib clinical studies, we calculated that at least 20 major bleeding events would need to be identified in order to perform blinded multinomial regression on the collected data of an estimated 400 patients.Results: 437 eligible patients were included in the analysis. Patients were overwhelmingly male (71.4%) and white (94.8%), with a mean age of 67.1 years (range: 29-89). 53.1% received ibrutinib as participants of a clinical trial, and the remainder received standard-of-care ibrutinib treatment. The table (upper panel) summarizes use of concomitant antihemostatic agents by presence or absence of major bleeding events. Characteristics of the major bleeding events are further detailed in the lower panel. The most commonly observed concomitant antihemostatic medication was aspirin, with 147 patients (33.6%) being exposed to aspirin within the study period.Fourteen instances of major bleeding were observed, corresponding to an overall incidence of 3.2%. These major bleeding events all occurred in CLL patients receiving ibrutinib at the standard dose of 420 mg daily. Two patients had platelet counts less than 50 k/µL at time of the bleeding event. One-half of the major bleeding events were observed in the absence of an antihemostatic medication, and 2 of the observed major bleeding events resulted in death (1 received concomitant warfarin). Fourteen patients (3.3%) in the group without major bleeding were on anticoagulation, 4 being warfarin. The most common sites of major bleeding were gastrointestinal (50%), intracranial (14.3%) and thoracic (14.3%). While most patients developing major bleeding permanently discontinued ibrutinib (57.1%), approximately one third of the patients who developed major bleeding subsequently resumed ibrutinib following resolution of the bleeding event. Subsequently, these patients did not experience a recurrent major bleeding event. The rate of major bleeding did not meet power to detect statistical differences in bleeding events when comparing concomitant therapy,Conclusions: Our observed incidence of major bleeding is consistent with previous controlled clinical trials, suggesting similar safety profile when ibrutinib is used outside of a controlled setting. Major bleeding events were uncommon despite the frequent co-administration of antiplatelet agents. However, because we modified practice early to avoid therapeutic anticoagulation during ibrutinib therapy whenever possible, the number of patients receiving such drugs in combination was small and precludes inferences regarding safety. [Display omitted] DisclosuresBlum:Pharmacyclics: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Woyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding. Christian:Pharmacyclics: Research Funding; Janssen: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Impact of Atrial Fibrillation on Subsequent Survival of Patients Receiving Ibrutinib As Treatment of Chronic Lymphocytic Leukemia (CLL): An International Study

IntroductionAtrial fibrillation (AF) occurs in 5-9% of patients treated with ibrutinib for CLL. We previously reported on the clinical characteristics and management outcomes of 56 patients with ibrutinib-associated AF (n=52) or atrial flutter (n=4) retrospectively identified from centers of the FILO group (French Innovative Leukemia Organization, France/Belgium, n=29), MD Anderson Cancer Center (USA, n=21), and Peter MacCallum Cancer Centre (Australia, n=6) (Thompson et al BJH 2016). We found that (1) the median time from the initiation of ibrutinib to AF was 3.8 months (range: 6 - 1410 days); (2) AF recurred or became persistent in 63% despite medical management; (3) AF and its management was associated with serious sequelae including severe cardiac failure (n=3, 1 fatal), ischemic stroke (n=3) and severe bleeding events (n=8); (4) immediate cessation of ibrutinib therapy was required in 22/56 (39%). Given the reported poor outcomes for patients who discontinue ibrutinib for toxicity, we update the results of this cohort with respect to CLL outcomes and survival.AimTo describe long term overall and disease-specific outcomes in patients who develop ibrutinib-associated AF.MethodsThis is an update of an international, retrospective cohort of patients who developed ibrutinib-associated AF. Patients are managed according to local best standard practice. The median follow-up from onset of AF is 21 months.ResultsThe median overall survival after development of AF is 43 months. Twenty-two (39%) patients stopped ibrutinib at the time of AF. Among these patients, 7 (32%) eventually died from CLL progression (n=3), infection (1), cardiac failure (1), pulmonary hemorrhage (1) and colon cancer (1). Of the 34 patients who initially continued ibrutinib, 5 (15%) died; causes of death were: Richter transformation (RT) (n=2), septic shock (1), CLL progression (1), and stroke in the context of AF (1).In order to gain insight into disease control in relationship to AF and its management, we examined PFS from the time of AF onset. The following features had no impact on PFS: single AF episode vs paroxysmal/permanent AF or time from ibrutinib initiation to onset of AF. However, patients who had ibrutinib interrupted at the time of AF onset (n=22) had a significantly inferior PFS (median 19 months) compared with those who had dose reduction without interruption (n=13) or those who continued full dose ibrutinib (n=21, median 27 months, p=0.023, Figure 1). There was a trend toward inferior overall survival in those who interrupted ibrutinib (62% at 3 years) compared with those who continued without interruption (74% at 3 years, p=0.10), but this did not reach statistical significance at the time of analysis.Only 3 of 22 (14%) patients who had initial interruption of ibrutinib successfully restarted ibrutinib following control of AF; all 3 remain in continuous partial remission. Four other patients restarted ibrutinib, but subsequently discontinued due to pulmonary hemorrhage (n=1), stroke (1), CLL progression (1) and RT (1).Among patients who discontinued Ibrutinib permanently, only 9 did so solely because of uncomplicated AF. The others discontinued because of: (a) complications of AF or its management such as cardiac failure (n=1), recurrent AF (n=5) and bleeding events (n=4; one each of hemopericardium, pulmonary hemorrhage, subdural hematoma and gastrointestinal hemorrhage); (b) complications related to CLL such as CLL progression (n=1) and RT (n=2); or (c) other causes such as lung cancer (n=1) or unknown reasons (n=2). Altogether, 21 patients were still on Ibrutinib among 44 patients alive at last follow-up.ConclusionOccurrence of AF at any time during ibrutinib treatment was associated with an initial discontinuation rate of 39%, and only 37.5% of patients remain alive and on drug after a median of 21 months follow-up. Patients who interrupted ibrutinib at the time of AF onset have an inferior PFS compared to those who continued ibrutinib or were managed with dose reductions. This inferior outcome in CLL control is likely related to the low rate of patients (14%) who subsequently successfully restarted ibrutinib. Development of AF was associated with serious complications, including hemorrhage, ischemic stroke and cardiac failure. Management of ibrutinib-associated AF is complex and requires international consensus guidelines and a multidisciplinary approach. [Display omitted] DisclosuresThompson:Pharmacyclics: Consultancy, Honoraria. Levy:Abbive: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Tam:janssen: Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Quinquenel:janssen: Honoraria, Research Funding. Dupuis:ABBVIE: Membership on an entity's Board of Directors or advisory committees; janssen: Honoraria. Cymbalista:Roche: Honoraria; Abbvie: Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Research Funding.

Response to ibrutinib in chronic lymphocytic leukemia improves in quality with time.

Key Clinical MessageUnlike chemoimmunotherapy regimens, which are given for a defined period, ibrutinib, a first‐in‐class Bruton's kinase inhibitor, allows most patients with CLL to remain on treatment for an extended period. Our experience, supported by sequential CT scan images, suggests that long‐term ibrutinib promotes a high response rate that improves in quality with time.

Pharmacokinetic and pharmacodynamic evaluation of ibrutinib for the treatment of chronic lymphocytic leukemia: rationale for lower doses

ABSTRACTIntroduction: Ibrutinib, a first-in-class covalent inhibitor of Bruton’s tyrosine kinase (BTK), is approved in many countries for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) and for previously untreated disease with a 17p deletion and, most recently, as a frontline therapy for CLL. In controlled trials in CLL, ibrutinib produced high response rates and improved survival in both the frontline and relapsed settings. While ibrutinib controls CLL with impressive efficacy, it only infrequently induces complete remissions, particularly of relapsed CLL, and does not eradicate minimal residual disease. Finally, ibrutinib is extremely expensive, has off-target toxicities, and requires indefinite therapy.Areas covered: In this article, we provide an overview of the CLL therapeutic landscape and discuss the pharmacokinetic and pharmacodynamic aspects of ibrutinib. Major clinical trials of ibrutinib in CLL are summarized, and its safety profile explored.Expert opinion: Ibrutinib represents a transformative advance in CLL management and has validated BTK as a therapeutic target in this disease, but has some limitations, leading to the emergence of other BTK inhibitors and mechanism-based combination strategies. Given complete BTK occupancy at lower doses of ibrutinib and declining levels of BTK on ibrutinib therapy, lower doses of ibrutinib in CLL are being explored.

Unusual, spontaneous aneurysm formation in a patient being treated with ibrutinib for chronic lymphocytic leukemia

Unusual, spontaneous aneurysm formation in a patient being treated with ibrutinib for chronic lymphocytic leukemia

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

AbstractAnti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

A Novel Case of Penile Gangrene in a Patient Treated with Ibrutinib for Chronic Lymphocytic Leukemia.

Introduction. Ibrutinib is commonly used for the treatment of patients with CLL in either first-line or relapsed/refractory settings. Case Presentation. We present the case of a 74-year-old Caucasian man with CLL who presented with penile gangrene upon initiation of ibrutinib treatment. Our case is the first showing the complication of penile gangrene associated with ibrutinib use. The gangrene was self-limited upon discontinuing ibrutinib. Conclusion. Our finding describes a very rare yet important adverse event associated with ibrutinib use.

Ibrutinib in chronic lymphocytic leukaemia: alone or in combination?

Ibrutinib in chronic lymphocytic leukaemia: alone or in combination?

Ligands That Mimic the Tissue Microenvironment of Replicating Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL) Protect Ex Vivo Patient Cell Samples from the Cytotoxicity of Combined Treatment with Ibrutinib and Venetoclax (ABT-199)

▪Ibrutinib (IBR) is a small molecule inhibitor of Bruton's Tyrosine Kinase, a key component of the BCR pathway. In phase II studies of single-agent IBR in CLL and MCL, the overall response rate was 89% and 68% respectively. However, only a minority of responses were complete and few are durable, suggesting that drug combinations will be required to achieve broader and more durable responses. In a previous report from our laboratory, Venetoclax (VEN) (GDC-0199; ABT-199), an inhibitor of the anti-apoptotic protein BCL2, showed synergistic cytotoxicity with IBR in MCL cell lines (Axelrod M et al, Leukemia 2014). A clinical trial testing this combination for MCL patients is now open (NCT02419560). Here, we sought to investigate the cytotoxic effects of the IBR and VEN combination in CLL and MCL patient samples and the mechanisms of resistance to drug treatment that might be expected to occur in the tissue microenvironment of these malignancies in patients.Peripheral blood mononuclear cells (PBMCs) from CLL/MCL patients containing circulating neoplastic B cells were incubated with IBR (0.1 μM), VEN (25 nM), and the combination of IBR and VEN for different time intervals. Cytotoxicity following drug treatment was determined by FACS analysis of cleaved PARP in CD5/CD19 double positive cells (Portell CA et al ASH 2014) and loss of CD5/CD19 double positive cells (i.e., neoplastic B cells). Cytotoxicity induced by IBR was variable, but was invariably enhanced by the addition of VEN. In a cohort of CLL and MCL patient samples that were treated (n=14), eleven patients showed synergistic cytotoxicity following treatment with IBR and VEN as evaluated by the Bliss model of independence.Recognizing that CLL and MCL cells populate replication centers (pseudofollicles) of the tissue microenvironment such as lymph node and bone marrow, we attempted to emulate a pseudofollicle in vitro by co-culturing or pre-incubating PBMC with agonistic components of that microenvironment. We screened several cytokines, other agonistic proteins, and supporting cells, including stromal cells (HS-5 cell line), follicular dendritic cells (HK cell line), soluble CD40L, CxCL13, IL-10, IL-2, CpG oligodeoxynucleotides (CpG ODN), BAFF, or IgM. A 12 h pre-incubation with soluble CD40L, IL-10, or CpG ODN generated significant levels of resistance to IBR and VEN. Moreover, the combination of soluble CD40L, IL-10, and CpG ODN resulted in nearly complete resistance to the IBR-VEN combination in cells from three CLL patient samples. The resistance was not overcome by increasing concentrations of IBR up to 10 μM, suggesting that resistance is not due to upregulation of BTK.To gain insight into the mechanism(s) of resistance and ways to overcome it, CLL patient PBMC pre-treated with soluble CD40L, IL-10, and CpG ODN were treated in three-way combination with IBR, VEN and inhibitors of potential resistance pathways known to be downstream from the tested ligands. These included small molecule inhibitors of IKKα and IKKβ, JAK1, 2, and 3, MAP Kinase, or PI3Kδ as well as the anti-apoptotic proteins MCL1, BCL-xL, or Survivin. Cytotoxicity was evaluated with an alamarBlue® assay. The NF-kB, JAK-STAT, PI3Kδ, and anti-apoptotic protein inhibitors all exhibited synergistic interactions with the IBR-VEN combination, as well as substantial cytotoxicity as single agents at higher doses. Inhibitors of the MAP Kinase pathway were ineffective. Taken together our data are consistent with the hypothesis that resistance to IBR, VEN and the combination in patients with CLL or MCL could arise by NF-kB, JAK-STAT, or PI3K signaling from CD40, IL10R and Toll-like receptors.In conclusion, IBR and VEN induced synergistic cytotoxicity in a majority of CLL and MCL patient samples. An in vitro model of the pseudofollicle microenvironment provided protection against cytotoxicity of IBR and VEN, suggesting that the tissue microenvironment provides a niche that supports drug resistance. This resistance was overcome by inhibition of NF-kB, JAK-STAT, or PI3Kδ, or the anti-apoptotic proteins MCL1, BCL-xL, or Survivin. DisclosuresOff Label Use: Ibrutinib and Venitoclax drug combination. This drug combination was used in this study to evaluate cytotoxicity in CLL and MCL ex vivo patient samples. . Portell:AbbVie: Research Funding. Williams:Genentech: Other: Research funding to my institution; Celgene: Consultancy, Other: Research funding to my institution; Takeda: Consultancy, Other: Research Funding to my institution. Petricoin:Theranostics Health: Consultancy, Equity Ownership, Patents & Royalties.

Ibrutinib Significantly Prolonged Survival in a Human Burkitt Lymphoma (BL) Xenograft NSG Mouse Model: Ibrutinib May be a Potential Adjuvant Agent in the Treatment of BL

BACKGROUND: Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH 2012). Children with relapsed or progressive BL develop chemoimmunotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young/Staudt et al, Nat Rev Drug Dicov 2013). Preclinical studies of ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) suggest that its inhibitory effect on cell proliferation is in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk Res 2013), which is higher than the typical plasma concentration range in patients at the recommended dose of 560mg (<0.6uM) (Advani et al, JCO 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was initially approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 and CLL in February 2014, who have received at least one prior therapy. BL, however, is associated with tonic vs chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. These findings suggest that the antitumor activity of ibrutinib in BL, which is associated with tonic BCR signaling, may be less pronounced than in Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), CLL or MCL.OBJECTIVES: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NSG mice.METHODS: Raji Burkitt lymphoma (BL) cells (ATCC) were exposed to ibrutinib (0-10uM, generously provided by Janssen R&D LLC) alone and in combination with dexamethasone (1uM) for 5 days and then MTS cell proliferation assay (Promega) was performed. The IC50 values were determined with CompuSynTM software (Chou and Martin, ComboSyn, 2005) based on data derived from MTS assay. Raji BL cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, 6-8 weeks old) from Jackson laboratory were irradiated (2.5 Gy) and then mice were subcutaneously injected with one million ffluc-zeo tumor cells. Tumor progression and tumor burden were monitored by Bioluminescent Imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with either vehicle or ibrutinib (12.5mg/kg) for 10 days (once a day) at post-tumor cells injection. Analysis of survival rates were determined by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: Ibrutinib compared to control induced a significant dose-dependent decrease in cell proliferation in Raji BL cells (5uM, 0.561 ± 0.170, p<0.03, IC50=5.20uM) following 120 hours (5 days) of ibrutinib treatment. Interestingly, we observed a significant decrease in cell proliferation in Raji BL cells (5uM, 0.213 ± 0.078, p<0.002, IC=0.50 ± 0.374uM) following ibrutinib and 1uM dexamethasone combination treatment following 120 hours (5 days) of treatment. We observed a significant decrease of tumor progression by tumor luminescence signal intensity following ibrutinib treated Raji BL xenografted NSG mice at day 20 (12.5mg/kg, p<0.001 and 25mg/kg, p<0.05) and day 25 (12.5mg/kg, p<0.05 and 25mg/kg, p<0.05) compared to control (Figure 1A).Ibrutinib (12.5 mg/kg) treated mice (n=39) significantly prolonged survival compared to vehicle controls (n=28) (p<0.0001) (Figure 1B).CONCLUSIONS: Ibrutinib (12.5mg/kg) significantly decreased tumor progression and significantly increased survival in Raji BL xenografted NSG mice, suggesting that ibrutinib may be a potential adjuvant agent for therapy in the treatment of BL. Future studies will investigate the efficacy of combination therapy with ibrutinib on survival in BL xenografted NOD/SCID mice. [Display omitted] DisclosuresGalardy:Mission Therapeutics: Research Funding.

Incidence and description of autoimmune cytopenias during treatment with ibrutinib for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is frequently complicated by secondary autoimmune cytopenias (AICs). Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase approved for the treatment of relapsed CLL and CLL with del(17p). The effect of ibrutinib treatment on the incidence of AIC is currently unknown. We reviewed medical records of 301 patients treated with ibrutinib, as participants in therapeutic clinical trials at The Ohio State University Comprehensive Cancer Center between July 2010 and July 2014. Subjects were reviewed with respect to past history of AIC, and treatment-emergent AIC cases were identified. Before starting ibrutinib treatment, 26% of patients had experienced AIC. Information was available for a total of 468 patient-years of ibrutinib exposure, during which there were six cases of treatment-emergent AIC. This corresponds to an estimated incidence rate of 13 episodes for every 1000 patient-years of ibrutinib treatment. We further identified 22 patients receiving therapy for AIC at the time ibrutinib was started. Of these 22 patients, 19 were able to discontinue AIC therapy. We found that ibrutinib treatment is associated with a low rate of treatment-emergent AIC. Patients with an existing AIC have been successfully treated with ibrutinib and subsequently discontinued AIC therapy.

Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia

Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL.

Abstract 2584: Specific antitumor activity of the splicing modulator sudemycin and cooperation with ibrutinib in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in western countries. A core component of the spliceosome, SF3B1, is among the most frequently mutated genes in CLL affecting up to 5-18% of patients. Clinically, SF3B1 mutations correlate with a more aggressive behavior of the disease, poorer clinical outcome and shorter time to treatment, as well as treatment refractoriness. Consistent with the crucial role of the spliceosome in CLL, mutations in other genes of the mRNA splicing, processing and transport machinery are also found to be frequently mutated in this tumor. Thus, our aim was to evaluate the antitumor effect of the spliceosome modulator sudemycin in SF3B1-mutated and -unmutated CLL cells in the in vitro and in vivo settings. For this purpose, cytotoxicity was measured by flow cytometry, in vivo activity of the compound was evaluated in an adoptive transfer mouse model of CLL, splicing products were analyzed by RT-PCR with specific primers and NF-κB activity was determined with the TransAM NF-κB p65 kit. Our results demonstrate that the spliceosome modulator sudemycin showed selective cytotoxicity in primary CLL cells when compared with healthy lymphocytes and tumor cells from other malignancies, with a slight bias for CLL cases with mutations for spliceosome-RNA maturation genes. Consistently, sudemycin exhibited considerable antitumor activity in a xenograft CLL model. At the molecular level, the antileukemic effect of sudemycin involved the splicing modulation of several target genes important for tumor survival, both in SF3B1-mutated and -unmutated cases. Thus, the apoptosis induced by this compound was related to the alternative splicing switch of MCL1 toward its proapoptotic isoform. Sudemycin also functionally disturbed NF-κB pathway through the induction of a spliced RELA variant lacking its DNA binding domain. Importantly, we show that the modulation of the alternative splicing of the inhibitor of Btk(IBTK) by sudemycin leaded to an enhanced antitumor effect in combination with ibrutinib. In conclusion, we provide first evidence that the spliceosome is a relevant therapeutic target in CLL, supporting the use of splicing modulators in combination with ibrutinib as a promising approach for the treatment of CLL patients. Citation Format: Sílvia Xargay-Torrent, Mónica López-Guerra, Laia Rosich, Arnau Montraveta, Jocabed Roldán, Vanina Rodríguez, Neus Villamor, Marta Aymerich, Chandraiah Lagisetti, Thomas R. Webb, Elias Campo, Dolors Colomer. Specific antitumor activity of the splicing modulator sudemycin and cooperation with ibrutinib in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2584. doi:10.1158/1538-7445.AM2015-2584

The splicing modulator sudemycin induces a specific antitumor response and cooperates with ibrutinib in chronic lymphocytic leukemia

Mutations or deregulated expression of the components of the spliceosome can influence the splicing pattern of several genes and contribute to the development of tumors. In this context, we report that the spliceosome modulator sudemycin induces selective cytotoxicity in primary chronic lymphocytic leukemia (CLL) cells when compared with healthy lymphocytes and tumor cells from other B-lymphoid malignancies, with a slight bias for CLL cases with mutations in spliceosome-RNA processing machinery. Consistently, sudemycin exhibits considerable antitumor activity in NOD/SCID/IL2Rγ-/- (NSG) mice engrafted with primary cells from CLL patients. The antileukemic effect of sudemycin involves the splicing modulation of several target genes important for tumor survival, both in SF3B1-mutated and -unmutated cases. Thus, the apoptosis induced by this compound is related to the alternative splicing switch of MCL1 toward its proapoptotic isoform. Sudemycin also functionally disturbs NF-κB pathway in parallel with the induction of a spliced RELA variant that loses its DNA binding domain. Importantly, we show an enhanced antitumor effect of sudemycin in combination with ibrutinib that might be related to the modulation of the alternative splicing of the inhibitor of Btk (IBTK). In conclusion, we provide first evidence that the spliceosome is a relevant therapeutic target in CLL, supporting the use of splicing modulators alone or in combination with ibrutinib as a promising approach for the treatment of CLL patients.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia

AbstractDespite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

Ibrutinib for previously untreated and relapsed or refractory chronic lymphocytic leukaemia with TP53 aberrations: a phase 2, single-arm trial

Patients with chronic lymphocytic leukaemia (CLL) with TP53 aberrations respond poorly to first-line chemoimmunotherapy, resulting in early relapse and short survival. We investigated the safety and activity of ibrutinib in previously untreated and relapsed or refractory CLL with TP53 aberrations. In this investigator-initiated, single-arm phase 2 study, we enrolled eligible adult patients with active CLL with TP53 aberrations at the National Institutes of Health Clinical Center (Bethesda, MD, USA). Patients received 28-day cycles of ibrutinib 420 mg orally once daily until disease progression or the occurrence of limiting toxicities. The primary endpoint was overall response to treatment at 24 weeks in all evaluable patients. This study is registered with ClinicalTrials.gov, number NCT01500733, and is fully enrolled. Between Dec 22, 2011, and Jan 2, 2014, we enrolled 51 patients; 47 had CLL with deletion 17p13.1 and four carried a TP53 mutation in the absence of deletion 17p13.1. All patients had active disease requiring therapy. 35 enrolled patients had previously untreated CLL and 16 had relapsed or refractory disease. Median follow-up was 24 months (IQR 12.9-27.0). 33 previously untreated patients and 15 patients with relapsed or refractory CLL were evaluable for response at 24 weeks. 32 (97%; 95% CI 86-100) of 33 previously untreated patients achieved an objective response, including partial response in 18 patients (55%) and partial response with lymphocytosis in 14 (42%). One patient had progressive disease at 0.4 months. 12 (80%; 95% CI 52-96) of the 15 patients with relapsed or refractory CLL had an objective response: six (40%) achieved a partial response and six (40%) a partial response with lymphocytosis; the remaining three (20%) patients had stable disease. Grade 3 or worse treatment-related adverse events were neutropenia in 12 (24%) patients (grade 4 in one [2%] patient), anaemia in seven (14%) patients, and thrombocytopenia in five (10%) patients (grade 4 in one [2%] patient). Grade 3 pneumonia occurred in three (6%) patients, and grade 3 rash in one (2%) patient. The activity and safety profile of single-agent ibrutinib in CLL with TP53 aberrations is encouraging and supports its consideration as a novel treatment option for patients with this high-risk disease in both first-line and second-line settings. Intramural Research Program of the National Heart, Lung, and Blood Institute and the National Cancer Institute, Danish Cancer Society, Novo Nordisk Foundation, National Institutes of Health Medical Research Scholars Program, and Pharmacyclics Inc.

In Vivo Ibrutinib Abrogates BCR-Dependent Adhesion in Tumor Cells of Patients with Chronic Lymphocytic Leukemia

Ibrutinib, now FDA approved for use in both previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), induces objective clinical responses and is well tolerated (Advani, JCO 2013; Byrd, NEJM 2013). As with other BCR-directed kinase inhibitors, (such as fostamatinib and idelalisib) ibrutinib leads to a treatment-induced transient lymphocytosis that resolves over the first 2-3 months on therapy in patients with CLL. The initial increase in absolute lymphocyte count (ALC) has been shown to be due to the release of tumor cells from the tissue compartments; specifically the lymph nodes, into the peripheral blood (Herman, Leukemia 2014). In vitro ibrutinib reduces the ability of CLL cells to migrate towards chemokines and prevents adhesion of BCR stimulated CLL cells (Ponader, Blood 2012; de Rooij, Blood 2012). We hypothesized that the initial rise in ALC is due to inhibition of CLL cell adhesion. We tested this ex vivo in patients enrolled on a single agent phase II trial of ibrutinib in CLL (NCT01500733) and investigated the mechanism.In order to evaluate the effect of ibrutinib on CLL cell adhesion in vivo we compared peripheral blood tumor cells collected from patients pre-treatment and on day 28 of treatment. Fluorescently labeled cells were plated on fibronectin-coated plates and allowed to adhere for 1 hour. Adhesion was then evaluated using florescence microscopy and quantified using the ImageJ software. After 28 days on ibrutinib, CLL cells displayed a severely reduced adhesion ability (99% reduction, P<0.001, n=13). Next, we tested the effect of ibrutinib on CLL cell adhesion to a confluent stromal cell layer. Although less dramatic, we found a similar reduction in the ability of cells to adhere to the stromal cell layer in all three patients evaluated. As the initial rise in ALC occurs very rapidly in some patients, we evaluated the ability of cells collected on day 2 of ibrutinib therapy and again found a significant inhibition of cell adhesion to fibronectin (81% reduction, P=0.005, n=10). This suggests that even one dose of ibrutinib is sufficient to disrupt CLL cell adhesion.We next sought to determine if ibrutinib could directly interfere with adhesion by causing cells to detach from fibronectin. We found that after one hour of 1µM ibrutinib treatment in vitro there was a significant reduction in the number of CLL cells that remained attached (33% reduction, P=0.03, n=10). This suggests that ibrutinib can not only prevent CLL cell adhesion but can release adhered cells from the microenvironment, consistent with influx of cells from the lymph node into the peripheral blood within hours of starting treatment.To determine whether the observed defect in cell adhesion is due to changes in the expression of CD29/CD49d (VLA-4 or α4β1 integrin), or CD18/CD11a (LFA-1), we measured the expression of these known regulators of CLL cell adhesion in samples obtained pre-treatment and during treatment. While there was inter-patient variability, overall there was no significant change in expression of CD29 after 28 days on treatment. In contrast albeit minor, we found reductions in both CD49d and CD18 (P<.05). When analyzing cells obtained on day 2, we found no significant changes suggesting that alteration of adhesion molecule expression does not account for the rapid increase in ALC. Integrin α4β1 is activated by PKC downstream of BTK. We thus sought to determine whether stimulation with PMA, an activator of PKC, could restore the ability of CLL cells to adhere to fibronectin. Indeed, PMA stimulation of CLL cells obtained from patients after 28 days on ibrutinib partially restored their ability to adhere to fibronectin, suggesting that reduced α4β1 integrin activity contributes to inhibition of adhesion by ibrutinib.In conclusion, ibrutinib abolished CLL cell adhesion at least in part due to direct inhibition of BTK- and BCR-mediated α4β1 integrin activity. Additionally, ibrutinib not only prevented adhesion but also caused the detachment of adhered cells from the extracellular matrix. Together, this suggests that disruption of cellular adhesion is a major contributor to the observed rise in ALC early on treatment.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. DisclosuresWiestner:Pharmacyclics Inc.: Research Funding.

PCN40 - Simulation Model of Ibrutinib for Chronic Lymphocytic Leukemia (CLL) With Prior Treatment

PCN40 - Simulation Model of Ibrutinib for Chronic Lymphocytic Leukemia (CLL) With Prior Treatment

Abstract 4769: Ex-vivo and in-vitro combination strategies with ibrutinib in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) depends on the B-cell receptor (BCR) pathway. Bruton's tyrosine kinase (BTK) is an essential enzyme in BCR and is inhibited by ibrutinib. Though ibrutinib treatment resulted in impressive response rates, most patients only achieve partial remissions. In addition, treatment with ibrutinib elicits lymphocytosis (redistribution of tissue-resident CLL cells from lymph nodes into peripheral blood). We hypothesize that drugs that induce apoptosis in the mobilized CLL cells will provide combination strategy for future clinical trials. We obtained cells from blood of lymphocytosed CLL patients on ibrutinib trial and incubated ex-vivo for 24 h with different drugs currently in clinical trials including BH3 mimetics (ABT-199, ABT-737), PI3K inhibitors (GS-1101, IPI-145), alkylating agent (bendamustine), and BTK inhibitor (ibrutinib). Among these drugs, ABT-199 at 5 and 10 nM, elicited the highest cytotoxicity (35-80% range, median 53%; n = 12). ABT-737 at 5 and 10 nM resulted in a cytotoxic range of 10-67% with a median of 28% in the same 12 samples. ABT-199 and ABT-737-mediated ex-vivo cytotoxicity was compared in 5 patient samples pre- and post- (4 and 12 weeks after starting therapy) ibrutinib. Endogenous cell death prior to ibrutinib was a median 35% (range 5-57%), however after ibrutinib regimen endogenous apoptosis of lymphocytosed cells at week 4 was a median 13% (range 10-48%) and at week 12 was a median 12% (range 4-48%). This suggests that post-ibrutinib lymphocytosed CLL cells exhibit resistance compared to cells prior to ibrutinib therapy. Again, ABT-737 and specially ABT-199 showed maximum cell death; ABT-737 treatment induced a median 32% (range 13-57%) cell death in pre-ibrutinib sample and a median 29% (range 22-68%) in post-ibrutinib samples. ABT-199 treatment on the other hand resulted in 53% cell death in pre and post-ibrutinib samples with a range of 29-63% and 30-71%, respectively. This suggests that pre- and post-ibrutinib treated lymphocytes are similarly sensitive to ABT-mediated cytotoxicity. In-vitro drug combination studies in CLL samples (n = 11) suggested additive or synergistic combination of ibrutinib and ABT-199 or ABT-737 (0.5 and 1 nM) in CLL cells with or without BCR pathway stimulation by IgM. Similar to ex-vivo studies, ABT-199 was more effective than ABT-737. Immunoblots analyzing targets in the BTK signaling pathway and Bcl-2 protein family were performed in the pre- and post-ibrutinib samples after 24 h incubation with the 6 drugs. Preliminary immunoblots analysis suggests a decrease of antiapoptotic Mcl-1 and phospho-Akt Ser473 by weeks 4 and 12 after ibrutinib treatment compared to pre-ibruitinib samples. In contrast, Bcl-2, Bcl-XL, Bim, and total ERK1/2 levels remain stable in pre- and post-samples. Collectively, these data suggest that ABT-199 may be combined with ibrutinib to remove lymphocytosis and for better responses. Citation Format: Fabiola Cervantes-Gomez, Kumudha Balakrishnan, William G. Wierda, Michael J. Keating, Varsha Gandhi. Ex-vivo and in-vitro combination strategies with ibrutinib in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4769. doi:10.1158/1538-7445.AM2014-4769