Mutations In Bruton's Tyrosine Kinase Research Articles

Genomic Analysis of Ibrutinib Resistance in Waldenstrom Macroglobulinemia

Introduction: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is the first approved therapy for Waldenström's macroglobulinemia (WM), and is highly active in both treatment-naive and relapsing or refractory patients. Although ibrutinib is highly active in WM patients, disease progression can occur. Acquired mutations in BTK at the binding site of ibrutinib (Cys481), or in the protein immediately downstream of BTK, the phospholipase PLCG2, have been identified in half of progressing WM patients on ibrutinib (Xu et al, Blood 2017). However, not all ibrutinib resistant patients harbor these alterations, suggesting that there are other causes of disease progression on ibrutinib. The aim of this study was to identify alternative molecular mechanisms that can drive ibrutinib resistance.Patients and Methods: Five WM patients who progressed while on ibrutinib were studied. Tumor DNA samples at diagnosis, relapse, and germline DNA were available in three patients. For the remaining two, relapse and germline samples were sequenced. DNA was extracted from CD19-selected bone marrow mononuclear cells from patients. Non-CD19 cells from peripheral blood were used as germline controls. Samples were sequenced using whole exome sequencing. Data were analyzed following the Broad Institute's GATK Best Practice Guidelines. Small variants were analyzed using Strelka and MuTect2. Somatic structural variants were detected using Manta, and copy number alterations were called using Control-FREEC.Results: Copy number analysis identified deletions in chromosome 6q in all patients, becoming homozygous in two of them at relapse. In another patient, the homozygous deletion was already present at baseline in a third of the tumor population, and increased at relapse. No other recurrent copy number alterations were detected, though in two patients multiple deletions or gains involving large chromosomal regions were observed. Regarding small variants, relapse samples showed a high proportion of acquired mutations detected at relapse only (median 85%, range 79%-88%) compared to persistent mutations detected at both baseline and relapse (median 15%, range 12%-21%). Three out of the five patients harbored mutations in BTK (two with p.Cys481Ser in the kinase domain and the other a p.Thr62Asn in the PH domain). In patients with a BTK mutation, other alterations were observed in genes related to the B-cell receptor pathway including PLCG2 p.Y495H; CD79B p.D33Y; LYN p.A2Stop and LYN p.A139T.In patients without BTK mutations, we identified several mutations with a putative role in ibrutinib resistance that emerged at relapse including AIP4, an E3 protein-ubiquitin ligase whose substrates are CXCR4, LYN or SYK, in which a recurring p.A646S mutation was observed in two patients; RNF19B, an E3 protein-ubiquitin ligase involved in the cytolytic activity of natural killer cells and cytotoxic T-cells, in which a p.R30G mutation was observed in two patients; FCRL3, an Fc receptor-like 3 that differentially modulates innate immune signaling in B cells, in which a p.E694Q was observed in one patient; Mutations in the negative regulators of Toll-like receptors signaling were observed that included DOK2 p.Y345Stop, and TOLLIP p.M242R and p.R228H which were each observed once in separate patients. Mutant allele frequency clustering analysis by k-means reflected a linear pattern of evolution from baseline to relapse, in which most persistent alterations maintained the same allele frequency (median 18%), and acquired mutations were present in a small proportion of tumor cells (median 9.3%, p<0.01).Conclusions: Our findings depict uniform deletion of 6q, including homozygous loss of 6q, which encompass key regulators of BTK, BCL2, and NFKB; as well as emergence of novel gene mutations, including recurring mutations in E3 ubiquitin ligases, innate immune signaling, and Toll receptor/MYD88 pathway regulators as significant genomic alterations that accompany disease progression on ibrutinib in WM patients. DisclosuresCastillo:Genentech: Consultancy; Beigene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; BMS: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

Outcomes, Causes of Discontinuation and Mutation Profile of Patients with Mantle Cell Lymphoma Who Progressed on Acalabrutinib

Introduction: Acalabrutinib is a Bruton tyrosine kinase (BTK) inhibitor approved for treatment of relapsed patients (pts) with mantle cell lymphoma. We have reported previously that ibrutinib refractory MCL pts have poor survival. However, outcomes, causes of discontinuation, management and the genomic landscape of MCL in pts who discontinued acalabrutinib are rarely reported. Method: We reviewed charts from all MCL pts treated with single agent acalabrutinib (n=28) in the relapsed setting and identified 15 pts who discontinued acalabrutinib and who are described in this analysis. Outcome after discontinuing acalabrutinib is reported. Whole-exome sequencing (WES) with SureSelect Human All Exon V6 was performed on 10 tumor specimens and 5 matched germline samples collected from 9 pts whose MCL progressed on acalabrutinib; among these pts 4 tumors were collected at baseline and 6 were collected after disease progression. One patient had sufficient DNAs available for both time points (baseline and progression). Results: The median duration on treatment with acalabrutinib was 6.5 months (1 to 29 months) and the median number of cycles of acalabrutinib treatment was 6 (range, 1-30). Seven pts had complete remission (CR) as their best response on acalabrutinib, 5 were primary refractory and 3 achieved partial remission. In 12 pts (80%) acalabrutinib was discontinued due to disease progression (2 pts transformed from classic to blastoid and pleomorphic type at progression) and 3 pts were discontinued due to intolerance (one for fatigue and idiopathic encephalopathy, one due to unrelated severe aortic stenosis and another for cytopenias secondary to therapy related myelodysplasia; all three pts were in CR). Nine pts had classic and 3 pts each had blastoid or pleomorphic features before starting acalabrutinib. Overall, median Ki-67 expression was 50% (range, 5-100) and all pts had high a MIPI score. The median number of prior treatments was 1 (range, 1-3); all chemo-immunotherapy (10 pts were previously treated with rituximab-hyper-CVAD) and none with ibrutinib. Two pts who transformed on acalabrutinib received acalabrutinib for a median duration of 12 months (range, 8-16.5). Median follow up after discontinuation was 27 months and the median survival was 25 months (26 months for progression and 1.5 months for intolerance; p Recurrently mutated genes in these tumors included ATM (6/10; 60%), TP53 (4/10; 40%), KMT2C (3/10), MYCN (2/10), NOTCH1 (2/10), NOTCH3 (2/10), and MEF2B (2/10) (Fig. 1B). We did not detect any mutation or copy number alterations in BTK, PLCG2, TRAF2/3 and MYD88 that have been reported previously to be associated with ibrutinib resistance. Compared to tumors at baseline, ATM was mutated at a higher frequency in samples at progression (67% vs. 50%; p=NS). To investigate the mutation evolution on acalabrutinib treatment, mutation profiles, particularly the mutation variant allelic fractions (VAFs), were compared between the baseline and progression samples from pt-1 (Fig. 1C). Mutation of MYCN, MEF2B, ATM, and NOTCH1 were identified in both tumors at similar VAFs, whereas mutation of CARD11 (two mutations), NLRC5 and B2M were detected only at progression. In pt-1, both the NLRC5 and β2M mutations acquired at disease progression were truncating, suggesting loss-of-function alterations. Copy number analysis reveals frequent whole-genome doubling and intensive copy number alterations in all tumors, including recurrent losses of chromosome 9p, 17p, and chromosome 13, indicating chromosomal instability as a driver of disease progression. Conclusion: Patients who progress on acalabrutinib have a poor outcome, and newer therapies are required for their treatment. In this small cohort, we observed non-BTK mutations associated with acalabrutinib resistance and disease progression. Disclosures Nastoupil:Genentech: Honoraria, Research Funding; TG Therappeutics: Research Funding; Spectrum: Honoraria; Gilead: Honoraria; Merck: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Honoraria, Research Funding; Karus: Research Funding; Novartis: Honoraria; Juno: Honoraria. Neelapu:Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Unum Therapeutics: Membership on an entity9s Board of Directors or advisory committees; Kite/Gilead: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity9s Board of Directors or advisory committees. Fowler:Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Wang:Acerta Pharma: Honoraria, Research Funding; MoreHealth: Consultancy; AstraZeneca: Consultancy, Research Funding; Kite Pharma: Research Funding; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Vecabrutinib Is Efficacious In Vivo in a Preclinical CLL Adoptive Transfer Model

Vecabrutinib Is Efficacious In Vivo in a Preclinical CLL Adoptive Transfer Model

Noncovalent inhibition of C481S Bruton tyrosine kinase by GDC-0853: a new treatment strategy for ibrutinib-resistant CLL

AbstractThe 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 naïve 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.

Abstract 1964: Preclinical efficacy of the HSP90 inhibitor SNX-5422 in targeting C481S mutant BTK and ibrutinib resistant CLL

Abstract While chronic lymphocytic leukemia (CLL) is effectively treated by the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, patients that do relapse on ibrutinib (approximately 19%) have an extremely poor clinical outcome without further intervention. Before clinical diagnosis of relapse, signs of molecular relapse can be identified through deep sequencing for mutations in BTK (C481S) and the immediate downstream BTK target, PLCγ2. The BTK C481S mutation (which reduces ibrutinib binding) can be identified at frequencies as low as 5%, and expansion of this mutant close inevitably leads to clinical relapse. Therefore, identifying methods to target this mutant clone before clinical relapse represents a novel and potentially effective therapeutic strategy. HSP90 is a chaperone protein responsible for maintaining the stability of a large number of client proteins involved in cancer development. Cancer cells, particularly those reliant on a mutated oncogene, rely heavily on HSP90 for survival, making this an attractive clinical target. Here we have explored the preclinical efficacy of SNX-5422, a chemically unique, orally bioavailable HSP90 inhibitor, in ibrutinib resistant CLL. We determined that SNX-5422 can degrade C481S mutated BTK by utilizing BTK-null B-cell lines over-expressing wildtype or C481S BTK. Whereas ibrutinib reduces BTK activity in only the wildtype BTK transfected cell line, SNX-5422 degrades both wildtype BTK and C481S mutant BTK and impairs downstream signaling. In fact, C481S mutant BTK shows increased degradation compared to wildtype BTK suggesting that the C481S mutant BTK may rely on HSP90 more for stabilization. We next examined the in vivo efficacy of SNX-5422 in a mouse model of ibrutinib-resistant CLL. Leukemic splenocytes isolated at removal criteria from TCL1 mice continuously treated with ibrutinib were adoptively transferred into syngeneic WT B6 mice. Upon reaching 10% leukemia burden in the peripheral blood, the mice were randomized into 1 of 4 treatment groups: vehicle, SNX-5422 (50 mg/kg MWF), ibrutinib (continuous in drinking water), or SNX-5422 plus ibrutinib (concurrent). Mice treated with the combination of SNX-5422 and ibrutinib exhibit prolonged survival and decreased disease burden in the peripheral blood compared to the vehicle or single agent groups. In summary, we show that SNX-5422 degrades C481S BTK in vitro and prolongs survival when combined with ibrutinib in vivo. We therefore propose that upon molecular relapse, the use of SNX-5422 may prevent clinical relapse and deepen the response to ibrutinib. A phase 1 study is currently open at Ohio State to investigate whether the addition of SNX-5422 to an established dose of ibrutinib can eliminate the C481S mutant clone and prevent or delay disease progression in subjects with CLL (NCT02914327). EH and JAW contributed equally as senior authors. Citation Format: Timothy L. Chen, Bonnie Harrington, Jean Truxall, Ronni Wasmuth, Amy Lehman, Eric Orlemans, John C. Byrd, Jennifer A. Woyach, Erin Hertlein. Preclinical efficacy of the HSP90 inhibitor SNX-5422 in targeting C481S mutant BTK and ibrutinib resistant CLL [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 1964.

Abstract B152: CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant

Abstract Introduction: Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinase. BTK plays a crucial role in the BCR signaling, essential for B-cell development, and BTK has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved BTK inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of BTK to inhibit BTK enzymatic activity, but recent studies suggested that the C481S mutation in BTK disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent BTK inhibitor is highly demanded to overcome ibrutinib resistance. We identified a highly selective, orally available non-covalent inhibitor of BTK, CB1763. Here, we report the characterization of CB1763, capable of potently inhibiting BTK [C481S] mutant, both in vitro and in vivo. Material and Methods: We produced ibrutinib-resistant BTK [C481S] mutant recombinant enzyme to assess efficacy of compounds against the drug-resistant enzyme. To evaluate cellular potency, phosphorylations of BTK and PLC- γ were analyzed by Western blotting in C481S BTK mutant transfected HEK293 cells. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate antitumor efficacy of BTK inhibitors. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: In addition to optimization of CB988, previously reported by our group, we identified CB1763 as a highly selective non-covalent BTK inhibitor. CB1763 exhibited strong inhibitory activity for both wild type and C481S mutant BTKs with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.85 and 0.99 nM for wild type and C481S, respectively). In cellular assays, CB1763 significantly reduced Tyr223 autophosphorylation of BTK protein at nano-molar concentration in C481S BTK mutant transfected HEK293 cells. Interestingly, persistent inhibition of autophosphorylation was observed after washout of CB1763, while no prolonged inhibition was observed by ibrutinib treatment. CB1763 demonstrated excellent antitumor activity in the BTK driven OCI-Ly10 xenograft mouse model. Conclusions: BTK [C481S] mutation has been reported in ibrutinib-relapsed CLL and MCL patients. Orally available novel non-covalent BTK inhibitor, CB1763, strongly inhibited C481S mutant BTK in vitro and in vivo, suggesting that CB1763 has the potential to treat patients who are relapsed/refractory to ibrutinib. Citation Format: Tokiko Asami, Wataru Kawahata, Shigeki Kashimoto, Masaaki Sawa. CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B152.

MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia.

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2, a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR.

Functional Analyses of BTK Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, novel mutations in the Bruton's Tyrosine Kinase (BTK) have been identified in FL (Krysiak et al, Blood 2017). Additional mutations in other components of BCR signaling pathways in FL have been reported as well but functional consequences have not been fully explored.Methods: Using a combination of WES on N=82 FLand BTK resequencing in an extended FL panel we uncovered novel BTK mutations in FL. These BTK mutations are all distinct from the previously identified mutations suggesting that a full complement of BTK mutations has not yet been identified. Using crispr-Cas9-mediated BTK gene targeting in multiple lymphoma cell lines, we generated novel BTK-/- null lines. We introduced all BTK mutations by site-directed mutagenesis into a reference BTK cDNA. Subsequently, we re-introduced HA-tagged wild type and mutated BTK constructs using lentiviral transduction into the BTK-/- cell lines and also into BTK-/- DT40 cells. We measured signal transduction pathway components following BCR cross linking. The location of BTK mutations was modeled onto a 3D-model assembled from various published BTK substructures.Results: Mutations in BTK are distributed along the entire length of the gene and are targeting the PH-, SH2- and catalytic domain, with the catalytic domain carrying the majority of BTK mutations. Interestingly, occasional frame shift mutations as reported and a splice acceptor site mutation by genetic criteria suggest gene inactivation. The 3D modeling data show that all BTK mutations are on the surface of the BTK protein within the various functional domains. The quantitation of BTK protein after reconstitution into various engineered BTK-/- cell lines uncovered lower steady state levels of various mutants especially for mutations targeting the catalytic domain. PLCγ2 remains the best studied BTK substrate and various sites have been reported to be phosphorylated by BTK in vivo (PLCγ2 amino acids 753, 759 and 1217). Following BCR crosslinking in engineered BTK-/- lymphoma cell lines we found only a modest reduction in the phosphorylation of PLCγ2 residue 759 or 1217 demonstrating that these sites are under the control of various tyrosine kinases and not exclusively targeted by BTK. After BCR crosslinking in the BTK-/- cells lines we detected a substantial increase in PLCy2 residues 759 or 1217 phosphorylation over baseline and this increase was abolished by ibrutinib pre-treatment. Therefore, PLCγ2 residue 759 or 1217 phosphorylation is due to multiple BCR stimulated and ibrutinib sensitive tyrosine kinases. In engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK we did not detect major effects by the BTK mutants on the phosphorylation state of PLCγ2 residue 759 or 1217. In addition, phosphorylation of BTK at residue 223 (a major autophosphorylation site) was reduced or absent in multiple BTK mutants. Summarizing these findings, FL-associated BTK mutations do not activate the BTK kinase; instead, multiple BTK mutants are inactivating.Focusing on BCR-regulated signaling pathways, we measured ERK and AKT phosphorylation in the engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK. Following BCR crosslinking, we detected increased p-AKT-473 phosphorylation in most BTK mutants in a total of 4 separate cell lines. In contrast, p-ERK was largely unaffected. Recent reports implicated AKT activation as a major factor in tonic BCR signaling in germinal center type DLBCL. We did not detect elevated AKT phosphorylation prior to BCR stimulation in our BTK reconstituted cell systems but an exaggerated response following BCR engagement. The effects of BTK mutations on protein-protein interactions within the BTK signalosome as well as effects on sensitivity to ibrutinib will be updated at the meeting.Conclusion : Our initial efforts at functional characterization of FL-associated BTK mutations uncovered that despite reduced stability and impaired catalytic activity most BTK mutations resulted in enhanced AKT activation following BCR crosslinking. Given the established pro-growth and pro-survival function of AKT, the data provide a clear reason why FL would select for such mutations. DisclosuresPhillips:Incyte: Other: Travel/Expenses; Pharmacyclics: Consultancy; KITE: Consultancy; Seattle Genetics: Consultancy. Malek:Abbvie: Equity Ownership; Janssen R&D: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding.

Acquired mutations associated with ibrutinib resistance in Waldenström macroglobulinemia

AbstractIbrutinib produces high response rates and durable remissions in Waldenström macroglobulinemia (WM) that are impacted by MYD88 and CXCR4WHIM mutations. Disease progression can develop on ibrutinib, although the molecular basis remains to be clarified. We sequenced sorted CD19+ lymphoplasmacytic cells from 6 WM patients who progressed after achieving major responses on ibrutinib using Sanger, TA cloning and sequencing, and highly sensitive and allele-specific polymerase chain reaction (AS-PCR) assays that we developed for Bruton tyrosine kinase (BTK) mutations. AS-PCR assays were used to screen patients with and without progressive disease on ibrutinib, and ibrutinib-naïve disease. Targeted next-generation sequencing was used to validate AS-PCR findings, assess for other BTK mutations, and other targets in B-cell receptor and MYD88 signaling. Among the 6 progressing patients, 3 had BTKCys481 variants that included BTKCys481Ser(c.1635G>C and c.1634T>A) and BTKCys481Arg(c.1634T>C). Two of these patients had multiple BTK mutations. Screening of 38 additional patients on ibrutinib without clinical progression identified BTKCys481 mutations in 2 (5.1%) individuals, both of whom subsequently progressed. BTKCys481 mutations were not detected in baseline samples or in 100 ibrutinib-naive WM patients. Using mutated MYD88 as a tumor marker, BTKCys481 mutations were subclonal, with a highly variable clonal distribution. Targeted deep-sequencing confirmed AS-PCR findings, and identified an additional BTKCys481Tyr(c.1634G>A) mutation in the 2 patients with multiple other BTKCys481 mutations, as well as CARD11Leu878Phe(c.2632C>T) and PLCγ2Tyr495His(c.1483T>C) mutations. Four of the 5 patients with BTKC481 variants were CXCR4 mutated. BTKCys481 mutations are common in WM patients with clinical progression on ibrutinib, and are associated with mutated CXCR4.

Human NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase

The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control invivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

The Development and Expansion of Resistant Subclones Precedes Relapse during Ibrutinib Therapy in Patients with CLL

▪The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse.All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK.With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter’s or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p<0.01) and MYC abnormalities on FISH (p=0.051). Age<65, del(17p) by FISH, and complex karyotype all associated with discontinuation due to CLL progression (p<0.05). Relapse generally has a poor prognosis, with a median survival from ibrutinib discontinuation for patients with transformation and progressive CLL of 3.9 (95% CI 2.0 to 10.1) and 22.7 (95% CI 13.5 to not reached) months, respectively.46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6).Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan.In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology.These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. DisclosuresWoyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: 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. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

Noninvasive Detection of Ibrutinib Resistance in Non-Hodgkin Lymphoma Using Cell-Free DNA

Background: Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), plays an emerging role in targeted therapy of several lymphoid malignancies. Due to its activity in B-cell receptor (BCR) dependent human B-cell cancers, Ibrutinib is currently approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, Ibrutinib responses can be seen in patients with follicular lymphoma (FL) and in a subset of patients with ABC- (activated B-cell-) like diffuse large B-cell lymphoma (DLBCL) (Wilson et al., Nat Med 2015; Fowler et al., ASH Annual Meeting, 2015). However, resistance mutations in BTK and other genes (e.g., PLCγ2)are a major cause of treatment failure and early identification of those aberrations remains challenging. Circulating tumor DNA (ctDNA) is an emerging biomarker with potential for disease monitoring as well as biopsy-free detection of evolving somatic aberrations. In this case study, we show that noninvasive genotyping of blood plasma allows early detection of resistance mutations duringIbrutinib therapy in a patient with relapsed/refractory follicular lymphoma associated with histological transformation.Methods: We profiled two tumor lymph node (LN) biopsy specimens and 8 plasma samples before, during, and after Ibrutinib therapy of a 67-year-old man with relapsed/refractory FL with evidence of transformation. Preceding Ibrutinib, this patient received 6 lines of lymphoma treatment over a ~3 year disease course, including Rituximab monotherapy, Benadmustine/Rituximab, and R-CHOP. Despite achieving stable disease after 12 weeks, the patient experienced significant disease progression after additional 4 months on Ibrutinib (Figure 1). We profiled these 10 serial samples using CAPP-Seq, a capture-based targeted high-throughput sequencing (HTS) method (Newman et al., Nat Med, 2014), which allows the sensitive identification of emerging somatic aberrations in circulating tumor DNA.Results: We detected the emergence of two independent BTK C481S resistance mutations in the blood plasma during Ibrutinib therapy, which were undetectable immediately before treatment. The major resistance clone harboring the BTK C481S A>T mutation (Figure 1, orange) was identified 2 months after the start of Ibrutinib, with allele frequencies as low as 0.03%. The second resistant subclone emerged 63 days later, carrying a BTK C481S somatic mutation with a substitution of C to G (Figure 1, green). Importantly, despite encoding an identical amino acid mutation, these two adjacent variants were never observed within the samectDNA molecule, demonstrating convergent evolution of independent resistantsubclones. We confirmed the existence of the two resistance mutations by tumor genotyping of a LN biopsy after clinical diagnosis of disease progression. Notably, while the minorsubclone completely disappeared under subsequentimmunochemotherapy with Rituximab/Bendamustine/Lenalidomide (RBL), the major resistance clone reappeared at the time of disease progression and the patient ultimately succumbed to his disease.Conclusions: Resistance mutations in BTK C481S have been described in tumor cells of Ibrutinib-refractory patients with CLL and MCL, with in vitro evidence of responsiveness to BH3-mimetics and SYK inhibitors. This study, to our knowledge, is the first report of its noninvasive detection in the plasma of a patient with Non-Hodgkin lymphoma, with the subsequent partial eradication of a resistant subclone. Therefore, we envision that biopsy-free genotyping will allow early detection of lymphoma patients developing Ibrutinib resistance and will be incorporated into future clinical trials involving targeted therapies. [Display omitted] DisclosuresNewman:Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Varian Medical Systems: Research Funding; Novartis: Consultancy; Roche: Consultancy; Quanticel Pharmaceuticals: Consultancy.

Targeting MALT1 with the Small Molecule Inhibitor MI2 Induces a Caspase-Dependent Apoptosis and Inhibits the NF-κb Pathway in Chronic Lymphocytic Leukemia Primary Cells

Targeting the B-cell receptor (BCR) pathway in chronic lymphocytic leukemia (CLL) with the Bruton Tyrosine Kinase (BTK) inhibitor Ibrutinib has transformed the treatment paradigm of this disease. Ibrutinib is currently indicated for treatment of CLL regardless of the line of therapy. However, ibrutinib is not curative, and relapses secondary to C481S mutation in BTK and gain of function mutations in PLCγ2 (R665W and L845F) are being reported.The protease mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) is the active component of the CARD11-BCL10-MALT1 (CBM) signaling complex. CBM mediates NF-κB activation downstream of BTK and PLCγ2 within the BCR pathway, which makes of MALT1 an ideal therapeutic target in ibrutinib-resistant clones. Notably, the chemical compound MI2 (C19H17Cl3N4O3) binds to- and suppresses the protease activity of MALT1, and was found to be active against activated B-cell-like diffuse large B-cell lymphoma. However, the role of MALT1 inhibition in CLL has not been investigated.We studied the efficacy of MI2 against a cohort of CLL samples (N=21), and explored its mechanism of action. PBMCs from patients with CLL were tested against serial dilutions of MI2 (0.125-8μM) for 48 h in 96-well plates, and MTS assay was performed to quantify cell viability. We observed a dose-dependent cytotoxicity in all samples, with an inhibitory concentration at 25% (IC25) < 2μM observed in the majority of the samples. MI2 cytotoxicity was independent of the IGHV mutational status, CD38 expression, cytogenetics, previous therapy status, and MALT1 protein level (immunoblot on purified CLL cells). To study the drug apoptosis and drug selectivity, we examined apoptotic induction in both CLL cells and T-cells of the same patients. PBMCs from five patients with CLL were treated with MI2 for 48 h and compared to untreated control. At the end of the incubation, cells were stained with CD5, CD19, and Annexin-V, then analyzed using flow cytometry. We found that MI2 induced a dose-dependent apoptosis in CLL cells (defined as CD5+/CD19+) in all samples, with only mild toxicity to their corresponding T-cells (T-cells represent a fraction of each patient’s PBMCs defined as CD5+/CD19-). This minimal toxicity to T-cells was not statistically significant when compared to untreated control T-cells, and may reflect a T-cell receptor signaling-independence of circulating T-cells, which are mostly composed of terminally differentiated effectors. To determine whether the induced apoptosis is caspase-dependent, we treated PBMCs of CLL patients (N=5) with MI2 (0.5-4 μM) in the presence or absence of 100μM of the pan-caspase inhibitor z-VAD-fmk for 48 h. We found that z-VAD-fmk completely prevented the induction of apoptosis in CD19+/CD5+ CLL cells as determined by Annexin-V staining (flow cytometry), and decreased MI2-induced PARP cleavage (Immunoblot).To determine the effect of the microenvironment on MI2 toxicity, we cultured primary CLL cells with and without MI2 (0.5-4µM) for 48 h, in the presence or absence of Nurse-Like Cells (NLC) (N=5) or anti-IgM (N=4). NLCs and anti-IgM cross-linking of BCR are in-vitromodels for the protective effect of the microenvironment. Co-culture with NLCs or with anti-IgM improved the viability of the untreated primary CLL samples. Similar to ibrutinib, MI2 continues to be effective against CLL cells in the presence of NLCs or anti-IgM, but higher concentrations are required to achieve the desired cytotoxicity against CLL cells. There was no cytotoxic effect on NLCs.To investigate the effect of MI2 on tumor biology, 1 μg of total RNA from purified CLL cells (treated for 8 h with MI2, N=3; and untreated control, N=3) was subjected to RNA sequencing. Out of 56,650 tested genes, there were 438 genes whose expression changed ≥2-fold at P<0.05 (312 down- and 126 up-regulated), several genes are known NF-κB targets. The observer-independent method, Gene Set Enrichment Analysis (GSEA), clearly identified the canonical NF-κB among of the top affected pathways.Further studies aim to verify the RNA sequencing data at the protein level, to determine MI2 efficacy against ibrutinib-resistant clones in vitro, and against CLL growth in mice in vivo.Supported by Hope Foundation SWOG Early Exploration and Development (SEED) Award. We thank the Louisiana Cancer Research Center (LCRC) biospecimen core and our patients for the donation of samples to make this research possible. DisclosuresNo relevant conflicts of interest to declare.

Characteristics, Outcome and Pattern of Mutations in Patients with Follicular Lymphoma Who Progressed on BTK Inhibitors (Ibrutinib and acalabrutinib)

Introduction: Acalabrutinib and ibrutinib are Bruton's tyrosine kinase (BTK) inhibitors, which have revolutionized the treatment of B-cell lymphoma/leukemias such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in frontline or relapsed refractory setting. Outcomes of patients with MCL and/or CLL who progressed on ibrutinib therapy are poor. Few reports have shown that ibrutinib resistant patients develop specific BTK mutations which confer resistance. We present the clinical and molecular data on patients with follicular lymphoma (FL) who have progressed on either ibrutinib based regimen or on acalabrutinib.Methods: We selected FL patients enrolled in clinical studies at our institution between 2013 and 2015 who discontinued acalabrutinib or ibrutinib for various reasons. Survival post ibrutinib discontinuation was calculated. We performed whole-exome sequencing on pre and post treatment (paired) samples of 3 FL patients (progressed on ibrutinib) using DNA extracted from formalin fixed paraffin embedded tissue samples. Library preparation and capture were performed using Agilent's SureSelect XT Human All Exon V4. Following capture the enriched libraries were pooled and sequenced in two Illumina HiSeq4000 lanes using a 75nt paired-end configuration.Results: Twenty of 55 patients discontinued treatment with BTK inhibitors; 5 came off from single agent acalabrutinib in relapsed refractory FL (RR-FL) while 15 came off from ibrutinib based regimen (4 as a single agent in RR-FL while 11 were treated with ibrutinib and rituximab in the frontline setting). These patients discontinued BTK inhibitor treatment, after a median of 2.8 months (range 0.9-22 months). Patients who received frontline ibrutinib (n=11), discontinued at a median of 2.8 months (0.9-22 months); previously treated patients discontinued BTK inhibitors at a median of 2.8 months (1.4-7.6 months). The reasons for discontinuation were as follows - 14 disease progression (3 on acalabrutinib and 11 on ibrutinib; 1 of them developed classical Hodgkin lymphoma), 2 non-responders (1 acalabrutinib and 1 ibrutinib) and 4 for other reasons (1 due to fatigue on acalabrutinib, 3 due to ibrutinib - 1 elevated liver enzymes, 1 asymptomatic atrial fibrillation another lost to follow up). Pre-treatment characteristics of the 16 patients who either progressed or were nonresponders to BTK inhibitors were - median age 59 years (range 41-75), 69% had stage 4 disease, 31% had FLIPI score of 3 or 4 and median LDH levels were 457 IU/L (range 290-754). Median follow up after discontinuation for all patients was 14.4 months (0-32 months). Eighteen patients (90%) were alive at the time of last follow up and 2 (10%) have died. Median survival (figure 1) of patients according to the causes of discontinuation was: not reached for both group of patients, those with other causes and those in progressive disease and/or non-responders.All 16 patients who progressed or not responded were treated with additional therapies. At the time of last follow up, 10/16 (62%) were responding to subsequent therapy - 5/10 (62%) achieved CR on lenalidomide-based therapies, 4/10 (40%), responded to chemoimmunotherapy and 1 patient responded to venetoclax-bendamustine and rituximab with CR. None were treated with PI3K inhibitors. WES studies showed EZH2 Y646 mutations in 2 of 3 FL patients in both pre and post treatment samples. Comparison of pre and post treatment samples on 3 patients showed multiple non-recurrent variants (2-12 per patient) in several genes in the post-treatment samples. These included DNMT3A R882H, NOTCH2 A3D and MLL E765G, all of which were noted in the post treatment sample of the patient that progressed to CHL. None of the other detected variants have been implicated in cancer. Of note, none of the samples showed BTK mutations despite adequate coverage.Conclusions: In contrast to other B-cell malignancies such as CLL and MCL, we observed that almost all FL patients who progressed on BTK inhibitors were salvageable by alternate therapies. Absence of BTK gene mutations in paired samples of 3 patients who progressed on ibrutinib suggests alternate mechanisms of drug resistance. [Display omitted] DisclosuresWang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; BeiGene: Research Funding. Fayad:Seattle Genetics: Consultancy, Research Funding. Oki:Novartis: Research Funding. Westin:Spectrum: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees. Fowler:Gilead: Research Funding; Jannsen: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; TG Therapeutics: Consultancy.

The Bruton's Tyrosine Kinase (BTK) Inhibitor ARQ 531 Effectively Inhibits Wild Type and C481S Mutant BTK and Is Superior to Ibrutinib in a Mouse Model of Chronic Lymphocytic Leukemia

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<0.001, p=0.016) in comparison to the 71% relative viability seen with 1µM ibrutinib (p<0.001). ARQ 531 demonstrates dose dependent inhibition of BTK, AKT, and ERK phosphorylation. Stimulation through TLR9 by CpG ligand increases expression of the activation markers CD40 and CD86; ARQ 531 decreased CpG induced expression of CD40 by 25% and CD86 by 40% (p=0.022, p=0.041). ARQ 531 in the presence of CXCL12 or CXCL13 decreased migration by 51% and 66%, respectively (p=0.015, p=0.025), below baseline migration of untreated CLL.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<0.0001). Mice receiving ARQ 531 have consistently lower lymphocyte counts on microscopic examination and reduced CD5+/CD19+ populations via flow cytometry compared to vehicle or ibrutinib. Additionally, we have observed increases in absolute neutrophil count over time in mice receiving ARQ 531. Pharmacokinetic studies are pending; however, in a single oral dose study of 10mg/kg in monkeys, the bioavailability was 72.4% with a CMax of 9µM and a half-life greater than 24 hours.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. DisclosuresEathiraj:ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

PIK3IP1 Inhibition of PI3K in G1 Arrest Induced By CDK4 Inhibition Reprograms MCL for Ibrutinib Therapy

Inhibition of CDK4/6 has emerged as a promising therapy for diverse human cancers. Mantle cell lymphoma (MCL), in which aberrant cyclin D1 expression and CDK4 activity underlie unrestrained proliferation of tumor cells, remains incurable. Targeting Bruton tyrosine kinase (BTK) with ibrutinibin recurrent MCLhas unprecedented single agent activity, with a completion remission (CR) rate of 21%. However, progression on ibrutinib is virtually universal in MCL and is associated with aggressive proliferation of tumor cells and a dismal clinical outcome.By longitudinal integrated RNA- and exome-seq analysis, we previously discovered a mutation in the ibrutinib binding site (BTKC481S) in MCL that was specific to relapse from a durable response, but absent in both primary resistance and resistance following a transient response. Irrespective of mechanism, ibrutinib resistance was associated with enhanced PI3K activation as determined by Western blotting of MCL cells isolated from serial biopsies. Induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4 with palbociclib sensitized to PI3K inhibition independent of this BTK mutation and to ibrutinib when the wild type BTK was retained. In both cases, synergistic killing appears to occur via cooperative inactivation of PI3K. Collectively, our data suggest that dual inhibition of CDK4 and BTK or PI3K restrain the expansion of resistant clones or even eliminate them to deepen or prolong the clinical response to targeting BTK.To test this hypothesis, we initiated a phase I clinical trial of palbociclib plus ibrutinib in recurrent MCL patients in August of 2014. All 5 dose levels included ibrutinib daily and palbociclib administered on days 1-21 of each 28-day cycle (Figure 1). Based on an intent-to-treat analysis of 18 patients, 12 (67%) patients have responded to treatment including 8 (44%) complete responses (CR). The median time to CR was 3 cycles and no responding patients have progressed on study. The therapy was well tolerated (see ASH abstract by Martin et al for clinical information). RNA- and exome-seq were performed to assess the genomic alternations and gene expression on purified MCL cells from samples before therapy, on progression and on treatment from PR patients. Initial analysis did not detect non-synonymous mutation in CDK4, CCND1, or genes in the BCR or PI3K signaling pathway. Hemizygous deletion of Rb, p53, ATM and the INK4A/B locus and amplification of PIK3CA and PIK3CB occurred in 30-50 % of patients before therapy. These copy number variations were predominantly associated with primary resistance (4 subjects) but did not preclude CR or PR. By contrast, expression of PIK3IP1, a putative PI3K inhibitor, was markedly reduced on progression in all primary resistant patients, but not in the PR patient. Function studies demonstrated that PIK3IP1 was required for pG1 sensitization to BTK and PI3K inhibition in MCL cells in vitro. Moreover, PIK3IP1 was induced in pG1 together PI3K inhibition in MCL cells in patients responding to palbociclib alone in a separate clinical trial (Martin, DiLiberto et al, unpublished), suggesting that PIK3IP1 mediates pG1 reprogramming.In summary, the high CR rate and durability support our hypothesis that induction of prolonged early G1 arrest by CDK4 inhibition reprograms MCL cells for deepened and durable targeting of BTK in MCL. Our data further suggest inhibition of PI3K by PIK3IP1 as a potential mechanism to overcome ibrutinib resistance. A phase II multi-center clinical trial is planned to further elucidate the genes and mechanisms that discriminate sensitivity from resistance to dual targeting of CDK4 and BTK. [Display omitted] DisclosuresBlum:Pharmacyclics: Research Funding. Ruan:Pharmacyclics: Research Funding, Speakers Bureau. Martin:Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Other: travel, accommodations, expenses; Novartis: Consultancy; Acerta: Consultancy; Teva: Research Funding.

Updated Results from a Phase II Study of the Fc Engineered CD19 Antibody MOR208 in Combination with Lenalidomide for Patients with Chronic Lymphocytic Leukemia (CLL) and Richter's Transformation or Ibrutinib for Patients with Ibrutinib-Resistant Clones

MOR208 is an Fc engineered CD19 monoclonal antibody with preliminary efficacy in CLL as a single agent (Woyach et al, Blood 2014). Compared to non-engineered antibodies, MOR208 has significantly enhanced antibody dependent cellular cytotoxicity (ADCC), which can be further augmented in vitro with the addition of lenalidomide. Given the potential synergy of these agents, acceptable individual safety profiles, and efficacy of each as a single agent, we chose to combine MOR208 and lenalidomide in patients (pts) with previously treated and previously untreated CLL and in pts who have undergone Richter's Transformation (RT). Because recent data show poor outcomes in pts who relapse after the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib and the presence of mutations in BTK prior to relapse, we included a cohort of ibrutinib-treated pts with identified resistance mutations but no clinical relapse where MOR208 was added to ibrutinib.This study is a single institution phase II trial of MOR208 in combination with lenalidomide or MOR208 in combination with ibrutinib with an initial safety run-in as part of each cohort. In combination with lenalidomide, MOR208 was given at a dose of 1 mg/kg on cycle 1 day 1, 9 mg/kg on days 2, 8, 15, and 22 of cycle 1, and day 1 of cycles 2-12. Lenalidomide was started at a dose of 2.5 mg daily on cycle 1 day 8 and given continuously. The dose of lenalidomide could be escalated up to 10 mg daily in pts without toxicity. After 12 cycles, lenalidomide could be continued indefinitely in responding pts. In combination with ibrutinib, ibrutinib was continued at a dose of 420 mg daily, and MOR208 was given at a dose of 1 mg/kg on cycle 1 day 1, 12 mg/kg on days 2, 8, 15, and 22 of cycle 1, then weekly during cycles 2 and 3, and every other week through cycle 12. Toxicity was assessed using the National Cancer Institute's Common Criteria for Adverse Events (AE) v4.0 for non-hematologic toxicity, and IWCLL 2008 guidelines for hematologic toxicity. For pts on MOR208 plus ibrutinib, variant allele frequency of mutant BTK was measured every 3 cycles.In the previously untreated cohort, 11 pts have been enrolled, with a median age of 62 (range 44-75). 1 pt had both del(17p) and del(11q) on FISH. Grade 3/4 adverse events regardless of attribution have been uncommon and have included hypertension (3 pts), infusion reaction (2 pts), and fatigue, neutropenia, colitis, hyperglycemia, dyspnea, and sinusitis (1 pt each). After a protocol amendment augmenting steroid premedication, no further grade 3 infusion reactions were observed.In the previously treated cohort, 11 pts have been enrolled, with a median age of 70 (range 62-75). 2 pts each had del(17p) and del(11q) on FISH, and 8 have Zap-70 methylated disease. Grade 3/4 toxicities regardless of attribution have included neutropenia (6), hyperglycemia (2), hypertension (2), and thrombocytopenia, fatigue, anemia, upper respiratory infection, catheter related infection, hypercalcemia, hypophosphatemia, infection, respiratory failure, and sepsis in 1 pt each.In the RT cohort, 5 pts have been enrolled, with a median age of 60 (range 55-70). Four had previously treated CLL, and one was previously untreated. Three had del(17p) and 2 had del(11q) on FISH. Grade 3/4 toxicities included hyperglycemia (3) and hyponatremia, thrombocytopenia, and neutropenia in 1 pt each.In the cohort of pts with molecular progression on ibrutinib, 7 pts have been enrolled, with a median age of 62 (range 45-77). Five have del(17p), 1 of whom also has del(11q). Grade 3/4 toxicities have included hypertension (2) and hyperglycemia and hyperuricemia in 1 pt each. Four pts have been on study for at least 3 cycles. One pt had an increase in BTK C481S variant allele frequency (VAF) from 66.3% to 78.8% while the others all have decreased (15.1% to 3.7%, 46.4% to 34.9%, and 67.2% to 18.1%). No pt has developed progressive disease.In conclusion, this Phase II trial in progress demonstrates preliminary safety and activity of the combination of MOR208 and lenalidomide in pts with CLL. This combination also has the potential to positively modulate the immune system, and detailed correlative studies will evaluate T- and NK-cell function. MOR208 appears safe in combination with ibrutinib, and preliminary evidence of activity against CLL cells with BTK C481S has been observed. Trial accrual is ongoing and updated results will be presented at the meeting. DisclosuresWoyach:Morphosys: Research Funding; Karyopharm: Research Funding; Acerta: Research Funding. Awan:Innate Pharma: Research Funding; Novartis Oncology: Consultancy; Pharmacyclics: Consultancy. 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. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Lozanski:Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.

Clinical characteristics and genetic profiles of 174 patients with X-linked agammaglobulinemia: Report from Shanghai, China (2000-2015).

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency. XLA patients typically present with very low numbers of peripheral B cells and a profound deficiency of all immunoglobulin isotypes. Most XLA patients carry mutations in Bruton tyrosine kinase (BTK) gene.The genetic background and clinical features of 174 Chinese patients with XLA were investigated. The relationship between specific BTK gene mutations and severity of clinical manifestations was also examined. Mutations were graded from mild to severe based on structural and functional prediction through bioinformatics analysis.One hundred twenty-seven mutations were identified in 142 patients from 124 families, including 45 novel mutations and 82 recurrent mutations that were distributed over the entire BTK gene sequence. Variation in phenotypes was observed, and there was a tendency of association between genotype and age of disease onset.This report constitutes the largest group of patients with BTK mutations in China. A genotype–phenotype correlation was observed in this study. Early diagnosis of congenital agammaglobulinemia should be based on clinical symptoms, family history, and molecular analysis of the BTK gene.