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The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib’s ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK’s adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB–dependent transcription, activation, and migration in treatment naive CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.

Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu5) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu5 modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu5 served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain (TM) regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine, a prototypical NAM. Three conserved amino acids (Y658, T780, and S808) and two nonconserved residues (P654 and A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TMs 6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Background: Aberrant activation of B-cell receptor (BCR) signaling is considered to be a major oncogenic mechanism that leads to the development and progression of multiple B-cell malignancies. ARQ 531 is a reversible ATP competitive inhibitor of BTK that inhibits ibrutinib-resistant BTK-C481S mutant CLL cells and has demonstrated antitumor activity in CLL, Richter's transformation, and DLBCL mouse models. Objectives: The primary objectives of the clinical study are to assess the safety and tolerability, and to determine the recommended Phase 2 dose and schedule of ARQ 531. The secondary objectives are to assess the pharmacokinetic (PK) profile, pharmacodynamic (PD) activity, and preliminary evidence of anti-tumor activity of ARQ 531. Methods: This is a first in human, Phase 1 dose escalation study in patients with relapsed or refractory CLL/SLL, Waldenstrom's macroglobulinemia, or B-cell NHL who received at least 2 prior lines of systemic therapy. Prior therapy must have included a BTK inhibitor, if FDA approved for their disease. Dose escalation was performed according to a 3+3 study design. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v.4.03. Tumor responses were evaluated per disease specific guidelines. Results: A total of 16 patients enrolled (median age 65.5 years, male 93.8%, 2 DLBCL, 2 follicular lymphoma, 12 CLL/SLL, 5 median prior systemic regimens) and were treated at dose levels of 5, 10, 15, 20 and 30mg QD. No dose limiting toxicities have been reported with ARQ 531. Drug related TEAEs included diarrhea, nausea, vomiting, fatigue, pneumonia, amylase increased, lipase increased, neutrophil count decreased, platelet count decreased, decreased appetite, hypernatraemia, arthralgia, groin pain, dizziness, facial paralysis, headache, tremor and restlessness in one patient (6.3%) each. Drug related grade 3 or worse TEAEs included lipase increased and platelet count decreased in one patient (6.3%) each. No drug related serious TEAEs were reported. Among the 12 patients who have received at least 1 dose of study drug and have at least 1 post-treatment tumor measurement data, 5 achieved stable disease (SD) (1 follicular lymphoma, 1 DLBCL, 3 CLL) and 7 had progressive disease (6 CLL, 1 follicular lymphoma). Three of 5 SD patients (1 follicular lymphoma, 1 DLBCL, 1 CLL) had 35%, 34% and 29% tumor reduction and 2 of them are ongoing on study treatment at 53 and 18 weeks. The CLL patient with 29% tumor reduction had BTK C481S mutation. Preliminary PK data showed ARQ 531 exposure was close to dose proportional and the estimated plasma half-life generally ranged from 20-24 hours. Consistent with increases in exposure, pBTK knockdown was observed. In Cohort 4 (20mg QD), all three patients showed 100% pBTK knockdown at a mean plasma Cmax exposure of 300nM (~4h post dose). Levels of CCL3 protein, a plasma biomarker for BCR pathway activation, was significantly suppressed in CLL patients. Conclusions: ARQ 531 has demonstrated a manageable safety profile to date. Diminished pBTK signaling and CCL3 protein levels have been observed in CLL patients. Additionally, preliminary anti-tumor activity has been observed at doses of ARQ 531 that were not predicted to completely inhibit BTK. Updated safety, PK, biomarker and anti-tumor activity data will be presented at the meeting. Disclosures Flinn: Gilead: Research Funding; Infinity: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Kite: Research Funding; Verastem: Consultancy, Research Funding; Pharmacyclics: Research Funding; Novartis: Research Funding; Curis: Research Funding; Seattle Genetics: Research Funding; Merck: Research Funding; Janssen: Research Funding; ArQule: Research Funding; Calithera: Research Funding; Genentech: Research Funding; Forma: Research Funding; Agios: Research Funding; Constellation: Research Funding; Trillium: Research Funding; Verastem: Research Funding; Portola: Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding; Forty Seven: Research Funding; BeiGene: Research Funding. Savage:ArQule, Inc.: Employment. Eathiraj:ArQule, Inc.: Employment. Tith:ArQule, Inc.: Employment.

Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy. Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines. Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = < 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (>80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier. Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL. Citation Format: Sudharshan Eathiraj, Yi Yu, Ron Savage, Jennifer A. Woyach, Sean D. Reiff, Amy J. Johnson, Brian Schwartz. ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1963.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo. Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. Cancer Res; 76(8); 2125–36. ©2016 AACR.

Introduction: In order to address the issue of acquired resistance to ibrutinib, we sought to characterize the Bruton agammaglobulinemia tyrosine kinase (BTK) inhibitor SNS-062 in preclinical models of chronic lymphocytic leukemia (CLL). Methods: Primary CLL B cells were isolated from the whole blood of consented patients by ficoll density centrifugation and Rosette-Sep negative selection. Annexin V and propidium iodide flow cytometry was used to measure patient CLL cell viability and 7-AAD was used to measure viability in stromal co-culture. CD40 and CD86 expression was evaluated via flow cytometry subsequent to sustained 3.2uM CpG stimulation. BCR signaling in primary CLL cells was investigated by immunoblot following 1 hour treatment and following 1 hour or 24 hours of incubation with SNS-062 in XLA cell lines. ITK inhibition was investigated via immunoblot after stimulation with anti-CD3 and anti-CD28 and incubation with SNS-062 for 1 hour. SNS-062 was used at a concentration of 1uM in preclinical studies unless otherwise noted. Measurement of kinase activity in human recombinant WT BTK or C481S BTK was performed in a FRET kinase assay. Results: Immunoblots of BTK and ERK phosphorylation of XLA cells transfected with WT or C481S BTK demonstrated that SNS-062 inhibition is comparable to that of ibrutinib in WT BTK and greater than that of ibrutinib in C481S BTK. Using a recombinant kinase assay, we found the IC50 of SNS-062 against WT BTK to be 4.6nM and C481S BTK to be 1.1nM, suggesting that SNS-062 retains activity against the mutated BTK variant. Additionally, SNS-062 was found to be six times more potent than ibrutinib and greater than 640 times more potent than acalabrutinib against C481S BTK. SNS-062 demonstrates dose-dependent inhibition of BTK in primary patient CLL cells comparable to ibrutinib via immunoblot for BTK phosphorylation. The viability of primary patient cells treated with 0.1uM, 1.0uM, and 10.0uM SNS-062 for 48 hours was measured to be 96.7%, 96.1%, and 88.1%, respectively, that of the untreated condition. At 48 hours, SNS-062 decreased viability of primary CLL cells in the presence of HS5 stromal protection by 5.5%. SNS-062 was found to decrease CpG induced CD40 and CD86 expression by 8.7% and 15.7%, respectively. Using an in vitro kinase assay, SNS-062 inhibited ITK with an IC50 value of 24nM. An immunoblot of anti-CD3/CD28 stimulated Jurkat cells revealed that SNS-062 decreased the phosphorylation of ERK, implying inhibition of ITK. Conclusion: Unlike ibrutinib, SNS-062 inhibits BTK signaling in the presence of the C481S mutation and may address acquired resistance to covalent BTK inhibitors. SNS-062 decreases B cell activation markers, viability, and stromal cell protection in primary patient CLL cells and was shown to inhibit ITK, suggesting support of T cell mediated antitumor activities. These data support further investigation of this molecule and advancement into clinical trials. Citation Format: Catherine A. Fabian, Sean D. Reiff, Daphne Guinn, Linda Neuman, Judith A. Fox, Wendy Wilson, John C. Byrd, Jennifer A. Woyach, Amy J. Johnson. SNS-062 demonstrates efficacy in chronic lymphocytic leukemia in vitro and inhibits C481S mutated Bruton tyrosine kinase [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 1207. doi:10.1158/1538-7445.AM2017-1207

Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu5) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu5 modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu5 served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain (TM) regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine, a prototypical NAM. Three conserved amino acids (Y658, T780, and S808) and two nonconserved residues (P654 and A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TMs 6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs.

Introduction: The Bruton's tyrosine kinase inhibitor ibrutinib improves survival in chronic lymphocytic leukemia (CLL) compared to standard chemotherapy or immune therapy. In a subset of patients, somatic mutation (C481S) of its binding site results in acquired resistance to ibrutinib therapy with poor clinical outcome. ARQ 531 is an ATP competitive, orally bioavailable, potent inhibitor of BTK and other relevant kinases including the majority of Src and Tec family kinases. Herein we present preclinical data with ARQ 531 in CLL including C481S mutated BTK and its efficacy versus ibrutinib in the TCL1 mouse model of CLL. Methods: Potency of ARQ 531 and its binding kinetics were measured in enzymatic and Surface Plasmon Resonance (SPR) binding assays. Primary CLL B cells were negatively selected and treated with 1μM ARQ 531. BCR signaling was investigated by immunoblot following a 1 hour drug incubation. CLL cells migrating towards CXCL12 and CXCL13 after 4 hours across a 5.0 micron transwell insert were counted by flow cytometry. Annexin V and propidium iodide flow cytometry was used to measure CLL viability over a range of drug concentrations and time. CpG mediated CLL cell activation was measured by CD40 and CD86 expression by flow cytometry. In vivo investigation utilized B6 mice engrafted with 1E7 CD5+/CD19+ TCL1 lymphocytes via tail vein injection. Mice were randomized to treatment with vehicle, ARQ 531, or ibrutinib following the establishment of a CD5+/CD19+ population >10% in peripheral blood. Results: At 72 hours the viability of CLL cells treated with 0.1µM, 1.0µM, or 10.0µM ARQ 531 was found to be 94%, 67%, and 50% that of untreated samples, respectively (p=0.76, p Unlike ibrutinib, ARQ 531 inhibits both wild type and C481S BTK with an IC50 less than 1nM in a biochemical assay, and inhibits phosphorylation of C481S mutated BTK in LV125 transfected HEK293T cells. Cytotoxicity was also observed in CLL cells isolated from patients with C481S BTK mutations, where the viability of cells treated with 1µM ARQ 531 was 72% that of untreated cells at 72 hours (p=0.038). SPR binding assay showed a residence time of 51 minutes in wild type BTK and 56 minutes in C481S mutated BTK. The TCL1 mouse model displays active BCR signaling and responds to treatment with the BTK inhibitors ibrutinib and acalabrutinib (Ponader S, 2012; Herman SEM, 2015). Following establishment of leukemia in the TCL1 transfer model, we randomized mice to treatment with vehicle (n=14), 25 mg/kg ibrutinib (n=6), 50 mg/kg ARQ 531 (n=14) or 75 mg/kg ARQ 531 (n=14) given by daily gavage. ARQ 531 significantly improved survival over both ibrutinib and vehicle (median survival: 36 days vehicle, 53 days ibrutinib, not reached by 74 days 50mg/kg and 75 mg/kg ARQ 531, p Conclusion: The BTK inhibitor ARQ 531 is a potent inhibitor of BTK with promising activity both in vitro and in vivo. Multi-targeted inhibition of cytokine, chemokine, and BCR pathways by ARQ 531 decreases activation, migration, and viability of CLL cells. Unlike ibrutinib, ARQ 531 inhibits activation of C481S mutated BTK variants and maintains cytotoxicity in ibrutinib resistant clones. Additionally, ARQ 531 has demonstrated remarkable efficacy in an in vivo TCL1 adoptive transfer model, improving survival to a greater extent than ibrutinib and potentially restoring granulocyte production. These data justify continued preclinical work and development to facilitate transition to clinical trials. Disclosures Eathiraj: ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

Background: Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression. Methods: Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines. Results: To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1. Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. Disclosures Woyach: Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

7530Background: BTK is an attractive target in CLL. Ibrutinib (IB), a first in class irreversible BTK inhibitor (BTKi), abrogates important survival signals in CLL by proximally blocking multiple p...