Inhibition Of ITK Research Articles

Combinatorial inhibition of Tec kinases BTK and ITK is beneficial in ameliorating murine sclerodermatous chronic graft versus host disease.

Graft-versus-host disease (GVHD) is the major factor limiting the widespread use of potentially curative allogeneic hematopoietic stem cell transplant (allo-HSCT). Chronic GVHD is characterized by the activation of alloreactive donor immune cells, especially B- and T-cells, leading to tissue damage and pathogenic fibrosis. In this study, we used highly specific next-generation inhibitors of ITK (PCYC-274), BTK (PCYC-804), and ibrutinib-like BTK/ITK inhibitors (PCYC-914 and PCYC-401) in the B10.D2 → BALB/C model of murine sclerodermatous cGVHD. From the third week onward, allogeneic recipients in each group of respective Tec kinase inhibitors were treated three times weekly with inhibitors at doses of 10 and 30 mg/kg or with saline control via oral gavage. Overall, we found that selective BTK inhibition was less effective than combined ITK/BTK or ITK inhibition in lengthening survival and reducing symptoms of cGVHD. ITK inhibition was most efficacious, with PCYC-274 and PCYC-401 demonstrating a nearly 50 percent reduction in GVHD scoring even at the 10 mg/kg dose, while 30 mg/kg of these compounds almost completely ameliorated GVHD symptomology. BTK/ITK and ITK-treated mice showed significant reductions in overall pathology. Significant reductions in dermal thickness and fibrosis were shown for all treatment groups. There was evidence of mixed Th1 and Th2 cytokine profiles in the skin of mice with dermal cGVHD, as both IFN-gamma and IL-4 were upregulated in the allogeneic control group, while kinase inhibition significantly reduced levels of these cytokines. Using an in vitro model of T-cell polarization, Th1 cell production of TNF-alpha and IFN-gamma were partially blocked by ITK. Th2 cell production of IL-4 was almost completely blocked synergistically by ITK and BTK inhibition. BTK-specific inhibition was unable to block either Th1 or Th2 cytokine production. Taken together, these results confirm previous reports that ITK-focused inhibition inhibits Th1 and Th2 cells. Additionally, the compound's effects on T-cell proliferation were tested by CFSE assay. Pure ITK inhibition was most effective at blocking T-cell proliferation, with no proliferation in PCYC-274-treated cells even at 0.1uM. PCYC-401 and PCYC-914 showed some inhibition at lower doses, with complete inhibition evident at 10uM. PCYC-804 was only partially able to block proliferation even at 10uM. In conclusion, we observed substantial benefit for differential inhibition of Tec kinases in GVHD, with ITK being most efficacious and Th1 cells being more resistant to inhibition, matching the previously reported findings of a Th2 to Th1 selective pressure in cells treated with ibrutinib. Our data warrants the further development of ITK and ITK/BTK inhibitors with specific inhibitory ratios to improve the treatment of GVHD and other T-cell mediated diseases.

Inhibition of ITK Signaling Causes Amelioration in Sepsis-Associated Neuroinflammation and Depression-like State in Mice.

Sepsis affects millions of people worldwide and is associated with multiorgan dysfunction that is a major cause of increased morbidity and mortality. Sepsis is associated with several morbidities, such as lung, liver, and central nervous system (CNS) dysfunction. Sepsis-associated CNS dysfunction usually leads to several mental problems including depression. IL-17A is one of the crucial cytokines that is expressed and secreted by Th17 cells. Th17 cells are reported to be involved in the pathogenesis of depression and anxiety in humans and animals. One of the protein tyrosine kinases that plays a key role in controlling the development/differentiation of Th17 cells is ITK. However, the role of ITK in sepsis-associated neuroinflammation and depression-like symptoms in mice has not been investigated earlier. Therefore, this study investigated the efficacy of the ITK inhibitor, BMS 509744, in sepsis-linked neuroinflammation (ITK, IL-17A, NFkB, iNOS, MPO, lipid peroxides, IL-6, MCP-1, IL-17A) and a battery of depression-like behavioral tests, such as sucrose preference, tail suspension, and the marble burying test. Further, the effect of the ITK inhibitor on anti-inflammatory signaling (Foxp3, IL-10, Nrf2, HO-1, SOD-2) was assessed in the CNS. Our data show that sepsis causes increased ITK protein expression, IL-17A signaling, and neuroinflammatory mediators in the CNS that are associated with a depression-like state in mice. ITK inhibitor-treated mice with sepsis show attenuated IL-17A signaling, which is associated with the upregulation of IL-10/Nrf2 signaling and the amelioration of depression-like symptoms in mice. Our data show, for the first time, that the ITK inhibition strategy may counteract sepsis-mediated depression through a reduction in IL-17A signaling in the CNS.

Tolerability of Acalabrutinib after Prior Ibrutinib Treatment in Patients with CLL: Experience of a Tertiary Cancer Care Center

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by the introduction of Bruton Tyrosine Kinase inhibitors (BTKi). Ibrutinib (IBR) and acalabrutinib (ACA) are two BTKi approved for the treatment of CLL patients (pts) with both frontline and relapsed disease. Based on its kinome inhibitory properties, IBR has more off-target kinase interactions compared to the second-generation BTKi ACA, including inhibition of ITK, HER2, HER4, and TEC, likely leading to increased off-target toxicity (Estupiñán, 2021 and McMullen, 2014). Results from the first randomized phase III trial comparing ACA to IBR in pts with previously treated CLL found a lower incidence of atrial fibrillation (Afib) with ACA (Byrd, 2021). However, there is lack of information on tolerability of ACA in pts treated outside clinical trials who had received prior IBR treatment. Our study aimed, therefore, to evaluate the real-world tolerability of ACA after IBR therapy in pts with CLL with specific attention to cardiovascular effects. Methods: All pts with CLL treated at our institution between 11/2019 and 5/2021 with ACA after prior IBR therapy were included in this study. Pts with less than 6 months of follow up were excluded unless they died prior to that period. Pts with confirmed Richter's Transformation were also excluded. All data were retrospectively obtained through institutional electronic health records, including demographics, clinical and genetic prognostic factors, toxicities, survival outcomes, duration of ACA therapy and reasons for IBR and ACA discontinuation. All grading of adverse events was completed utilizing the Common Terminology Criteria for Adverse Events (CTCAE), International Society on Thrombosis and Hemostasis (ISTH), or European Organization for Research and Treatment of Cancer (EORTC) definitions, as appropriate. All adverse events considered possibly related to BTKi are reported. A waiver of informed consent was granted for this retrospective study. Results: 52 pts treated with ACA and history of prior IBR treatment were identified (Table 1). The median age was 68 years (range, 40-92 years) and 25% were > 75 years old. ACA was administered for a median of 12.4 months (range, 0.4-43.3 months) with 19 pts (37%) requiring a dose reduction at some point during ACA treatment. The most commonly reported adverse effects during IBR treatment included arthralgia/myalgia (31%), Afib (17%), rash (15%), GI upset (12%), and hypertension (HTN, 10%). The reason for IBR treatment discontinuation was adverse effects in 47 pts (90%), progression in 4 pts (8%) and pts preference in 1 (2%). The most commonly reported adverse effects during ACA treatment included GI upset/GERD (25%), headache (17%), arthralgia/myalgia (12%), Afib (10%) and cytopenias (10%). At the time of data collection, 17 (33%) patients had discontinued ACA treatment. The reason for ACA treatment discontinuation was adverse effects in 14 pts (27%), progression in 2 pts (4%) and pt preference in 1 (2%). Specific attention was given to cardiovascular effects since a significant proportion of pts had a past medical history of cardiac comorbidities prior to ACA treatment, including history of HTN in 33 pts (63%) and Afib in 12 pts (23%). New onset Afib developed in 1 pt (2%) and recurrent Afib was observed in 4 pts (8%) during ACA therapy. The 4 pts with recurrent Afib had history of Afib while on IBR therapy. Median time to Afib events with ACA was 4.4 months (range, 0.5-13.3 months). New or worsening HTN developed in only 1 pt while on ACA therapy. Time to HTN episode was 0.2 months. There were few instances of major bleeding with either IBR (2%) or ACA (6%). Two pts (4%) developed an invasive infection while on IBR, no evidence of invasive infection has been reported in the pts receiving ACA. At the time of data collection, 3 patients have died, causes of death included sepsis, respiratory failure and multiorgan failure that were not deemed treatment-related. Conclusion: This real-world experience shows overall good tolerability of ACA after prior IBR therapy in pts with CLL. We observed a lower incidence of Afib (17% vs. 10%) and HTN (10% vs. 2%) during ACA treatment compared to IBR, even if cardiovascular co-morbidities were common among our pts. With a median follow up of 12.4 months (range, 0.4-43.3 months) since the start of ACA therapy, only 27% of pts discontinued treatment because of toxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery of Pteridine-7(8H)-one Derivatives as Potent and Selective Inhibitors of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK) is an attractive therapeutic target in the treatment of cancer, inflammation, and autoimmune diseases. Covalent and noncovalent BTK inhibitors have been developed, among which covalent BTK inhibitors have shown great clinical efficacy. However, some of them could produce adverse effects, such as diarrhea, rash, and platelet dysfunction, which are associated with the off-target inhibition of ITK and EGFR. In this study, we disclosed a series of pteridine-7(8H)-one derivatives as potent and selective covalent BTK inhibitors, which were optimized from 3z, an EGFR inhibitor previously reported by our group. Among them, compound 24a exhibited great BTK inhibition activity (IC50 = 4.0 nM) and high selectivity in both enzymatic (ITK >250-fold, EGFR >2500-fold) and cellular levels (ITK >227-fold, EGFR 27-fold). In U-937 xenograft models, 24a significantly inhibited tumor growth (TGI = 57.85%) at a 50 mg/kg dosage. Accordingly, 24a is a new BTK inhibitor worthy of further development.

Interleukin-2-inducible T-cell kinase (Itk) signaling regulates potent noncanonical regulatory T cells.

Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2‐inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of “noncanonical” CD4+CD25−FOXP3+ Tregs (ncTregs), as well as of “canonical” CD4+CD25+FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft‐versus‐host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft‐versus‐leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk –/− ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Role of ITK signaling in acute kidney injury in mice: Amelioration of acute kidney injury associated clinical parameters and attenuation of inflammatory transcription factor signaling in CD4+ T cells by ITK inhibition

Acute kidney injury (AKI) is a world-wide health problem and linked with increased risk of morbidity/mortality in hospitalized patients and its incidence has been on the rise in the last few decades. AKI is characterized by renal tubular injury which results from interactions between bacterial products and host immune responses which manifests as a rapid deterioration in renal function. Immune system dysfunction induced by sepsis plays a crucial role in AKI through activation of multiple immune cells of both innate and adaptive origin. These cells release pro-inflammatory cytokines such as IL-6, IL-17A, IFN-γ, and reactive oxygen metabolites. Adaptive immune cells, especially T cells also participate in the amplification of renal inflammation through release of pro-inflammatory cytokines such as IL-17A, IFN-γ, TNF-α, and IL-10. Non-receptor protein tyrosine kinases such as ITK play crucial role in T cell through modulation of key downstream molecules such as PLCγ, STAT3, NFkB, NFATc1, and p-38MAPK. However, it has not been explored in CD4+ T cells during AKI. Therefore, this study investigated the effect of ITK inhibitor on AKI linked clinical parameters (serum BUN, creatinine and renal histopathology), downstream signaling molecules in CD4+ T cells (PLCγ, STAT3, NFkB, and NFATc1), Th1/Th2/Treg cell markers (IL-17A, TNF-α, and IL-10), and neutrophil-mediated oxidative inflammation (MPO/carbonyl/nitrotyrosine formation) in mice. Our data exhibit elevated p-ITK levels in CD4+ T cells which is associated with renal dysfunction and elevated Th1/Th17/neutrophilic responses. Blockade of ITK signaling resulted in ameliorated of AKI associated biochemical; parameters through downregulation in transcription signaling in CD4+ T cells and Th1/Th17 immune responses. Therefore, this report suggests that ITK inhibition could be an effective strategy to halt renal dysfunction associated with AKI.

The role of acalabrutinib in adults with chronic lymphocytic leukemia

The treatment landscape of chronic lymphocytic leukemia (CLL) has significantly changed in the past decade. This paradigm shift is due to the introduction of novel agents to the field. The two major classes of drugs that have contributed to this dramatic evolution include the Bruton tyrosine kinase (BTK) inhibitors and BCL2 inhibitors. Ibrutinib was the first-in-class drug which was initially approved by the US Food and Drug Administration (FDA) for the treatment of patients with relapsed/refractory and later for patients with treatment-naïve CLL. Despite encouraging efficacy outcomes, its use has been associated with cardiovascular and gastrointestinal toxicities likely due to off-target inhibition of ITK, TEC and EGFR family kinases. The next generation of BTK inhibitors was developed to be more selective with less off-target inhibition with the prospect to improve tolerability without compromising efficacy. Acalabrutinib, a selective covalent BTK inhibitor, is a second generation BTK inhibitor. The focus of this review is on two major phase III trials that resulted in the FDA approval of acalabrutinib in 2019. The ELEVATE TN trial investigated acalabrutinib with or without obintuzumab versus chlorambucil–obinutuzumab in older and frail patients with previously untreated CLL. The ASCEND trial explored acalabrutinib versus chemoimmunotherapy in patients with relapsed/refractory CLL. Both trials demonstrated superiority of the acalabrutinib-containing arms in terms of both efficacy and tolerability. Unfortunately, the availability of new generation BTK inhibitors has not resulted in mitigating the financial toxicities associated with these potentially life-long treatments.

Abstract 2205: TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma (NHL) that remains incurable with standard therapy. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has proven to be an effective agent for patients with relapsed/refractory MCL, although development of a cysteine to serine mutation at the BTK catalytic site (BTKC481S) or over-activation of the NF-kB pathway can impair MCL response to most BTK inhibitors (BTKis). TG-1701 is a novel irreversible inhibitor highly specific to BTK, with improved selectivity when compared to ibrutinib, currently being evaluated in a phase 1 clinical trial in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) patients. Here we show that, when compared to irreversible and reversible BTKis including ibrutinib, acalabrutinib, and ARQ-531, TG-1701 showed similar cytotoxic activity either in ibrutinib-sensitive or in ibrutinib-resistant MCL cells. We then evaluated the activity of TG-1701 and ibrutinib on anti-CD20 antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), as ibrutinib has previously been show to block rituximab-mediated ADCC due to off-target inhibition of ITK (Kohrt et al., Blood 2014). In sharp contrast with ibrutinib, which partially antagonized rituximab activity, we found that TG-1701 did not impair the anti-CD20-mediated ADCC and ADCP in a multicellular co-culture system that associated MCL to effector cells. In another set of cell-based assays, we showed that the treatment with TG-1701 led to a strong decrease in IL-10 expression, together with a significant increase in IL-2, IL-6 and IL1RN gene transcription, thus associating TG-1701 exposure with the promotion of an immune response within the tumor microenvironment (TME). Accordingly, TG-1701 cooperated with the novel anti-CD20 antibody ublituximab and the novel PI3Kδ-CK1ϵ dual inhibitor umbralisib in reducing the tumor growth in both ibrutinib-sensitive (REC1) and ibrutinib-resistant (UPN-1res) mouse models of MCL. In accordance with in vitro data, the antitumor activity of the drug combination was associated with increased production of IL-2, IL-6 and TNFα, and with an increased infiltration of mouse macrophages and NK cells. Altogether, these data warrant further clinical investigation of TG-1701-mediated engagement of TME-related anti-tumor effect, and clinical evaluation of the triple combination of TG-1701, ublituximab, and umbralisib, is ongoing. Citation Format: Marcelo L. Ribeiro, Diana Reyes-Garau, Meritxell Vinyoles, Marc Armengol, Hari Miskin, Francesc Bosch, Pablo Menendez, Emmanuel Normant, Gael Roue. TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2205.

Ibrutinib, but not zanubrutinib, induces platelet receptor shedding of GPIb-IX-V complex and integrin αIIbβ3 in mice and humans

AbstractThe Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has proven to be efficacious in the treatment of B-cell chronic lymphocytic leukemia (B-CLL) and related diseases. However, a major adverse side effect of ibrutinib is bleeding, including major hemorrhages. The bleeding associated with ibrutinib use is thought to be due to a combination of on-target irreversible Btk inhibition, as well as off-target inhibition of other kinases, including EGFR, ITK, JAK3, and Tec kinase. In this study, we investigated the effects of ibrutinib vs zanubrutinib (a more selective Btk inhibitor) on platelet activation, glycoprotein expression, and thrombus formation. Ibrutinib, but not zanubrutinib, induced a time- and dose-dependent shedding of GPIb-IX complex and integrin αIIbβ3, but not of GPVI and GPV, from the platelet surface. The shedding of GPIbα and GPIX was blocked by GM6001 and TAPI-2, an ADAM17 inhibitor but not ADAM10 inhibitor. Ibrutinib but not zanubrutinib treatment of human platelets increased ADAM17 activation. Pretreatment of C57BL/6 mice with ibrutinib (10 mg/kg), but not zanubrutinib (10 mg/kg), inhibited ex vivo and in vivo thrombus growth over time. Platelets from ibrutinib-treated patients with CLL showed reduced GPIb-IX complex and integrin αIIbβ3 surface expression and reduced ex vivo thrombus formation under arterial flow, which was not observed in zanubrutinib-treated patients. In mice, ibrutinib, but not zanubrutinib, led to increased soluble GPIbα and soluble αIIb levels in plasma. These data demonstrate that ibrutinib induces shedding of GPIbα and GPIX by an ADAM17-dependent mechanism and integrin αIIbβ3 by an unknown sheddase, and this process occurs in vivo to regulate thrombus formation.

Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Dissection of the Effects of JAK and BTK Inhibitors on the Functionality of Healthy and Malignant Lymphocytes.

Despite the emergence of small molecule inhibitors, current treatment strategies for chronic lymphocytic leukemia (CLL) are not curative, and the search for new therapeutic modalities continues. Prosurvival signaling derived from the microenvironment is often mediated via JAK signaling. However, whether JAK inhibitors are useful in CLL therapy has not been studied extensively. JAK inhibitors are valuable therapeutic agents in myelofibrosis and show promising results in graft-versus-host-disease. However, JAK inhibition is associated with an increased infection risk, presumably because of the effect on other immune cells, a feature shared with other kinase inhibitors used for CLL treatment, such as the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib. We compared functional effects of the JAK1/2 inhibitors momelotinib and ruxolitinib, the BTK inhibitors ibrutinib and tirabrutinib, and PI3Kδ inhibitor idelalisib on malignant CLL cells but also on healthy human T, B, and NK lymphocytes. We found several interesting differences among the inhibitors, apart from expected and well-known effects. Momelotinib but not ruxolitinib blocked cytokine-induced proliferation of CLL cells. Momelotinib also reduced BCR signaling, in contrast to ruxolitinib, indicating that these JAK inhibitors in fact have a distinct target spectrum. In contrast to tirabrutinib, ibrutinib had inhibitory effects on T cell activation, probably because of ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-γ production in a mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at identical targets may have differential effects on lymphocyte function. Their unique profile could be strategically employed to balance desired versus unwanted lymphocyte inhibition.

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z9-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees.

ITK signaling differentiates GVT and GVHD after allogeneic bone marrow transplantation by regulating IRF-4, JAK/STAT and Eomesodermin expression

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for patients with hematologic malignancies. Donor T cells contained within the graft prevent tumor recurrence by exhibiting graft-versus-tumor (GVT) effects but also cause graft-versus-host disease (GVHD); therefore, novel treatment strategies are needed to maintain GVT while suppressing GVHD. We investigated the role of TCR-mediated ITK activation in mediating GVT vs. GVHD effects after allo-HSCT. We discovered that T cells from ITK−/−mice display a CD62Lhi CD122+ CD44hi Innate Lymphoid Cell-Like (ILCL) functional phenotype, and higher Eomes but not T bet expression compared to WT. We showed that T cells from ITK−/−mice reduced IRF-4, JAK1 and JAK2 and STAT3. T cells from ITK−/− mice displayed reduced cytokine production but showed preserved cytotoxicity after allo-HSCT. These cells also showed defective upregulation of the chemokine receptors CX3CR3, CXCR1, and CXCR6, which correlated with their reduced migration into GVHD target organs. The defective migration of ITK deficient T cells into GVHD target organs contributed to separation of GVHD and GVT effects, since ITK deficeint T cells cleared intravenously injected but not subcutaneously injected tumor cells in allo-HSCT mice. Moreover, pharmacological ITK inhibition attenuated GVHD and preserved GVT function by wild type CD8+ T cells. Together, our data suggest that ITK inhibition could be used as therapy after allo-HSCT to reduce GVHD while preserving the beneficial GVT effects by donor T cells

ITK inhibition induced in vitro and in vivo anti-tumor activity through downregulating TCR signaling pathway in malignant T cell lymphoma

BackgroundAngioimmunoblastic T cell lymphoma (AITL) is a distinct subtype of peripheral T cell lymphoma and associated with poor outcomes. The activation status of T cell receptor (TCR) signaling has recently become a focus of attention in terms of the therapeutic targets. However, the molecular pathogenesis mechanisms and novel therapeutic targets are largely unknown.MethodsAntibodies specific to phosphorylated ZAP70, ITK and PLCγ1 were used to identify the activation status of intracellular proteins involved in TCR signaling in AITL patients. Malignant T cell lymphoma cells were transduced with a lentiviral construct containing ITK shRNA for cellular and functional assays. The antitumor effects of the selective ITK inhibitor BMS-509744 were determined in vitro and in vivo.ResultsImmunohistochemistry staining showed that more than half of the AITL patients (n = 38) exhibited continuously activated intracellular TCR signaling pathway. Patients positive for phosphorylated ITK showed a lower rate of complete response (20% vs. 75%, P = 0.004) and a shorter progression-free survival (5.17 months vs. 25.1 months, P = 0.022) than patients negative for phosphorylated ITK. Genetic and pharmacological cellular ITK inhibition significantly compromised the proliferation, invasion and migration of malignant T cells. The selective ITK inhibitor BMS-509744 also induced the pro-apoptotic effects and G2/M phase cell cycle arrest in vitro and in vivo. Finally, inhibition of ITK synergistically enhanced the antitumor effect of vincristine and doxorubicin on malignant T cell lymphoma cell lines.ConclusionsOur findings suggest that ITK may be a novel candidate therapeutic target for the treatment of patients with ITK-expressing malignant T-cell lymphomas.

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity.Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point.On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB).Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells.Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells.Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were <10 nM for ibrutinib, acalabrutinib and zanubrutinib. Spebrutinib had an EC50 of 140 nM in hWB, showing the greatest loss of potency versus biochemical assays and hPBMCs, going to the physiologically most relevant cellular experimental conditions (Table 2).Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates.Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested.Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively.No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values <1 µM in Jurkat T cells. Similar results were observed in primary human peripheral T cells.Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. [Display omitted] DisclosuresKaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Modulation of T-Cell Function in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Btki Appears Independent of ITK

Background: Chronic lymphocytic leukemia (CLL) development is associated with global immunodeficiency including T-cell exhaustion. We hypothesise that repairing T cell functions would improve outcome and decrease infectious complications which cause significant morbidity in CLL patients. Chronic B-cell receptor (BCR) activation as well as close interactions with the tumour microenvironment promote survival of malignant CLL B-cells, supporting their ability to induce immune suppression. To date, the most clinically successful approach to BCR-signalling inhibition is by the use of BTK inhibitors (BTKi). It has been suggested that the BTKi Ibrutinib has the ability to modulate T-helper cell polarity from Th2 to Th1 and thus would be a step towards repairing CLL associated T-cell defects (1). We were therefore interested to determine whether the second generation BTKi Acalabrutinib which has no reported inhibitory capacity towards ITK would have similar effects than Ibrutinib in modulating T cell responses.Materials and Methods: To address this question in vivo C57/Bl6 animals 2.5 months of age were injected with 40x10e6 purified CLL B-cells pooled from Eµ-TCL1 mice with CLL. When peripheral blood CLL load reached >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBD) or Acalabrutinib treatment (0.15 mg/l in 2% HPBC) for 21 days. 17 animals were group were analysed. Splenic cells were isolated, the cellular component characterized by CyTOF and T cell function assessed by multi-parameter flow cytometry and T-cell synapse formation assay.Results: Treatment with Acalabrutinib resulted in increased expression of IL2 (p<0.0001) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0016) but not CD8+ T-cells. There was a reduction in Interferon gamma production in both CD4 T-cells (p=0.0463) and CD8+ T-cells (p=0.0064) with Acalabrutinib treatment. In addition, treatment resulted in an increase in CD107a+/CD107a- ratio among both CD44+ and CD44- CD8+ cytotoxic T-cells. This effect was pronounced in the antigen experienced CD44+ cytotoxic T-cells (p<0.0001) but only moderate (p=0.0056) in the CD44- cytotoxic T-cells. Lastly, we find a statistically significant increase in T-cell synapse area (p<0.0001) with Acalabrutinib treatment (Figure 1).Conclusion: We find that treatment with both Ibrutinib and Acalabrutinib result in a similar shift of T cell function with cytokine secretion with increased IL2 and decreased IL4. T-cells in CLL have increased Interferon gamma production (2) and the observed decrease seen with Acalabrutinib is in keeping with a normalization of T cell function. Moreover, overall CD8+ T-cell function is increased with Acalabrutinib treatment as evidenced by an increase in cytotoxic T-cell function and immune synapse formation. We speculate that inhibition of ITK is not the leading cause for this phenomenon as Acalabrutinib does not have inhibitory capacity toward this kinase. These changes suggest that BTKi modulate T cell mediated immune responses indirectly via either their effects in the CLL B-cell or myeloid cells in the tumour microenvironment.ReferencesDubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49.Riches JC, Davies JK, McClanahan F, Fatah R, Iqbal S, Agrawal S, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. 2013;121(9):1612-21. [Display omitted] DisclosuresFrigault:Acerta Pharma: Employment. Gribben:Abbvie: Honoraria; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Unum: Equity Ownership; Roche: Honoraria; TG Therapeutics: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Cancer Research UK: Research Funding; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria.

Interleukin-2-Inducible T-Cell Kinase (ITK) Inhibitors for the Treatment of T-Cell Lymphoproliferative Disorders

Introduction: We previously demonstrated that engagement of the T-cell receptor (TCR) in malignant T-cells leads to ITK-dependent activation of the transcription factors NF-κB and GATA3, thus promoting chemotherapy resistance. TCR-mediated chemotherapy resistance was reversed upon knockdown or pharmacologic inhibition of ITK (Wang, 2017). ITK and resting lymphocyte kinase (RLK) are partially redundant TEC family kinase members involved in TCR signaling (Dubovsky, 2013). Here, we report results from preclinical studies evaluating the use of the ITK-specific inhibitor CPI-818 and the dual ITK/RLK inhibitor CPI-893 in the treatment of T-cell lymphoproliferative disorders.Methods/Results: Kinome screening was performed for CPI-818 and CPI-893. CPI-818 had high specificity for ITK over RLK (IC50 2.3 nM and 260 nM, respectively). In contrast CPI-893 had a high affinity for both ITK and RLK (IC50 0.36 nM and 0.4 nM, respectively). Normal and malignant T-cells from primary patient samples were evaluated for both ITK and RLK by western blot. As anticipated, normal CD3+ T-cells isolated from healthy donors expressed both ITK and RLK (n=3/3). In contrast, malignant T-cells preferentially expressed ITK in 3 of 3 cases and low levels of RLK in a single case. Tissue microarray analysis of cutaneous T-cell lymphoma and peripheral T-cell lymphoma cases further demonstrated ITK expression across multiple subtypes.To confirm on target effects of CPI-818 and CPI-893, Jurkat cells (which express ITK but not RLK) were stimulated with anti-CD3/CD28 beads for 5 minutes in the presence or absence of either agent. Auto-phosphorylation of ITK Y180 and PLC-γ Y783, an ITK substrate, were significantly reduced in the presence of either agent, confirming on target effects. We next evaluated the effect of both drugs on GATA3 expression. Cells were stimulated for 24 hours with anti-CD3/CD28 beads in the presence or absence of these agents (or vehicle control). ITK inhibition with either agent significantly reduced GATA3 expression in these experiments.We next evaluated the effect of CPI-818 and CPI-893 on cell viability in normal T-cells. TCR stimulation for 24 hours increased viability by 359% in the presence of vehicle control when normalized to unstimulated cells (n=3). Treatment with 1 µM CPI-893 reduced the cell viability to only 133% when compared to unstimulated controls (n=3, p = 0.0045). In contrast, treatment with the ITK-specific inhibitor CPI-818 at 1 µM had a numerically smaller, but still statistically significant effect (n=3, average viability 278%, p = 0.0056). We next quantified IL-10 expression by ELISA in cell culture supernatants. Consistent with the observed effect on cell proliferation and viability, only CPI-893 significantly reduced IL-10 production upon TCR stimulation (886 pg/mL vs 11.7 pg/mL, p <0.0001).In order to assess TCR-mediated resistance to chemotherapy in malignant T-cells, T8ML-1 cells (PTCL, NOS cell line) were incubated in the presence or absence of CPI-818 or CPI-893 (both 1 µM) with varying concentrations of vincristine. When compared to TCR-stimulated cells alone, both agents displayed single agent activity, but also sensitized the cells to the effects of vincristine. The IC50 for vincristine in unstimulated cells, TCR-stimulated cells, CP-818 treated stimulated cells, and CPI-893 treated stimulated cells was 0.049, 0.443, 0.066, and 0.091 nM, respectively (n=4 per group). Similar results were observed in primary patient samples treated with romidepsin. Consistent with the observed effect on proliferation and viability, IL-10 production was decreased when compared to TCR stimulated controls (15.97 pg/mL, n=3 for each group) with both CPI-818 (3.47 pg/mL, p <0.0001) and CPI-893 (2.3 pg/mL, p <0.0001).Conclusions: Our results demonstrate that TCR signaling inhibition with the selective ITK inhibitor CPI-818 significantly impaired malignant T-cell growth and proliferation, increased chemotherapy sensitivity, and decreased GATA3 expression. In contrast, this agent had minimal effect on normal T cells; this is likely explained by the expression of both ITK and RLK in most conventional T-cells. Collectively, our data support further preclinical and clinical studies with CP-818 in T-cell lymphoproliferative disorders. DisclosuresBuggy:Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Janc:Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Wilcox:Incyte, Corp: Research Funding.

More Than a BTK Inhibitor; Ibrutinib Profoundly Impacts the Function of Cell Mediated Immunity

Background: Chronic lymphocytic leukaemia (CLL) is associated with profound immune dysfunction, which is often exacerbated by CLL therapies. This study aimed to understand the impact of multiple BTK inhibitors on the function of cytotoxic T cells. BTK, along with ITK, TXK, BMX and TEC is a member of the Tec family of nonreceptor tyrosine kinases which are integral to the downstream signalling of many immune receptors. Critically, BTK and ITK are downstream of the B cell and T cell receptors. Whilst identified as a selective BTK inhibitor, ibrutinib inhibits all five members of the Tec family at clinically meaningful doses (Long et al, JCI, 2017). More selective BTK inhibitors have been developed, including acalabrutinib and zanubrutinib, which have less affinity for ITK but variable inhibition for other Tec family members. Functional impairment of NK cells by ibrutinib is well established (Kohrt et al, Blood 2014), however the effects on T cell function remains unclear. It has been suggested that inhibiting ITK results in skewing of CD4 T cells toward a T helper 1 phenotype whilst CD8 T cell function is preserved due to redundant kinase signalling (Dubovsky et al, Blood, 2013). This is yet to be demonstrated in patients and little is known about the impact of BTK inhibitors on cytotoxic T cell populations.Methods: Peripheral blood mononuclear cells were isolated from CLL patients receiving either ibrutinib or zanubrutinib and from treatment naive CLL patients or age matched healthy donors. T cell proliferation was measured using CellTrace Violet, cultures of whole PBMC were stimulated with CD3/CD28 beads and 20 IU/mL IL-2 and treated with 1uM ibrutinib or zanubrutinib or acalabrutinib or vehicle control for 7 days, n=7. CD8 T cell and NKT cell degranulation in response to CD3/CD28 stimulation was assessed using CD107a mobilisation and IFNγ production over 4 hours, n=6 and n=7. Whole PBMC from patients treated with either ibrutinib or zanubrutinib were stimulated for 24 hours using CD3/CD28 beads, supernatants were analysed using a BD™ Cytometric Bead Array.Results: In vitro treatment with ibrutinib significantly impairs the function of cytotoxic T cells. Both CD4 and CD8 T cells cultured in the presence of ibrutinib had significantly decreased proliferation, whilst zanubrutinib and acalabrutinib did not significantly impact T cell proliferation (Figure 1, p=0.0002 and p<0.0001). Furthermore, CD8 T cells from CLL patients and healthy donors had significant abrogation of degranulation and cytokine production when treated with Ibrutinib but not the more selective BTK inhibitors. Similarly, ibrutinib treated healthy donor NKT cells showed significantly diminished degranulation and IFNγ production (Figure 2, p =0.0004 and p=0.0003). Finally, PBMC isolated from patients after treatment with ibrutinib had muted cytokine production including IL-2 (p= 0.003), IL-17A (p=0.023), TNF (p=0.031) and IL-10 (p=0.016) as compared to PBMC isolated before ibrutinib treatment. However there was no significant change in Th1 or Th2 cytokines.Discussion: Together these results highlight the impact of Ibrutinib on cell mediated cytotoxicity. In both in vitro and ex vivo functional assays ibrutinib, but not more selective BTK inhibitors perturbed proper T cell function. Whilst CD4 T cell proliferation was suppressed Th1 skewing was not observed in ibrutinib treated patients. Furthermore. CD8 T cells had profoundly impaired responses to TCR stimulation. Understanding how BTK inhibitors alter the function of cytotoxic cells is essential for the combination of these therapies with immunotherapies and may inform the use of these therapies in the context of adoptive cellular therapies and transplantation. [Display omitted] DisclosuresTam:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

1268P - NCI 9922: Phase II study of ibrutinib in treatment-refractory distant metastatic cutaneous melanoma (DMCM)

Background: The IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and molecular targeting and/or pharmacologic inhibition of ITK in preclinical melanoma models suppresses cell proliferation without inducing cell death (Carson CCR 2015). Ibrutinib suppresses proliferation of melanoma cell lines in low nM concentrations (Moschos ASCO 2017, TPS9592). We hypothesize that targeting DMCM with ibrutinib will induce antitumor responses, especially in high ITK-expressing melanomas. Methods: This is an open-label, single-arm, Simon’s 2-stage design, multicenter, phase II study for patients (pts) with DMCM refractory to or ineligible for PD-1 and MAPK inhibitors, if BRAFV600-mutant. Given that the IC50 of ibrutinib for ITK is > 10 times than Bruton’s tyrosine kinase’s, we administered ibrutinib at 840mg qd. We hypothesized that an ineffective drug will have a = <5% response rate and = <18% 6-month PFS rate. We present the results of the first stage. Results: 18 pts (13 males; median age 63.5, range 37-82; 14 with M1c disease; 4 with BRAFV600 mutation; 12 with performance status 1 or 2; 4 with resistance to 4 treatments; 5 with resistance to = >5 treatments) were enrolled. Median exposure to ibrutinib was 27.5 days (range 4-155). The most frequent all-grade side effects were fatigue (55%), anorexia (50%), gastrointestinal upset (44%), and anemia (39%). 4 grade IV (hyponatremia, sepsis, cytokine release syndrome, and constipation occurred 6% each) and 9 grade III events [hyponatremia (17%); pneumonia, hypertension, anemia, hypoalbuminemia, dehydration, lymphopenia occurred 6% each] were seen. No antitumor responses were seen. At a median follow-up of 5 months, all pts had progressed (median PFS was 1.3 months, range 0.2-5.5). 15 pts were discontinued from study due to progression and 14 pts had died from melanoma. Median OS was 5 months (range 0.3-10.4 months) in pts who died. Conclusions: In this treatment-refractory DMCM, high-dose ibrutinib did not induce any meaningful clinical benefit; therefore the study will not proceed to stage 2. Correlation between PFS and expression of ITK by melanoma cells and density of tumor-infiltrating T- and B-cells in pretreatment tumor specimens will be reported at the time of the meeting. Clinical trial identification: NCT03427398. Legal entity responsible for the study: NCI-CTEP. Funding: NCI-CTEP, Pharmacyclics. Disclosure: All authors have declared no conflicts of interest.