Mediator Of B-cell Receptor Signaling Research Articles

Orelabrutinib for the treatment of relapsed or refractory MCL: a phase 1/2, open-label, multicenter, single-arm study

AbstractRelapsed or refractory (r/r) mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a poor prognosis. Bruton tyrosine kinase (BTK) is a mediator of B-cell receptor signaling and is associated with the development of B-cell lymphomas. Patients with r/r MCL were enrolled in this phase 1/2 study and treated with orelabrutinib, a novel, highly selective BTK inhibitor. The median number of prior regimens was 2 (range, 1-4). The median age was 62 years (range, 37-73 years). Eligible patients received oral orelabrutinib 150 mg once daily (n = 86) or 100 mg twice daily (n = 20) until disease progression or unacceptable toxicity. A dose of 150 mg once daily was chosen as the preferred recommended phase 2 dose. After a median follow-up duration of 23.8 months, the overall response rate was 81.1%, with 27.4% achieving a complete response and 53.8% achieving a partial response. The median duration of response and progression-free survival were 22.9 and 22.0 months, respectively. The median overall survival (OS) was not reached, and the rate of OS at 24 months was 74.3%. Adverse events (AEs) occurring in >20% of patients were thrombocytopenia (34.0%), upper respiratory tract infection (27.4%), and neutropenia (24.5%). Grade ≥3 AEs were infrequent and most commonly included thrombocytopenia (13.2%), neutropenia (8.5%), and anemia (7.5%). Three patients discontinued treatment because of treatment-related adverse events (TRAEs), but no fatal TRAEs were reported. Orelabrutinib showed substantial efficacy and was well tolerated in patients with r/r MCL. This trial was registered at www.clinicaltrials.gov as #NCT03494179.

Role of Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) as a prognostic marker in Egyptian patients with B-cell Chronic Lymphocytic Leukemia

Abstract Background B-chronic lymphocytic leukemia (B-CLL) is the most common lymphoproliferative in adults and most patients are diagnosed by flow cytometry. High-risk B-CLL has been shown to lack the expression of (LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling. Aim This study aimed to evaluate the expression of CD305 cell surface in patients with B-cell and its correlation with the clinical and prognostic relevance of the disease. Patients and methods This study was performed on 100 patients newly diagnosed with B-CLL (72 males and 28 females) with a median age of (59 y), who were admitted to the Oncology Center of Mansoura University, in addition to 40 healthy individuals who served as a control group. The detection of CD305 and other CDs was performed using specific monoclonal antibodies on a flow cytometer (BD Bioscience FacsCanto II Flow Cytometer, USA) and was further analyzed using flow software (BD Biosciences). Results CD305 was positively expressed in 58% of B-CLL patients compared with 35% of the control group. It had a significant positive correlation with both Hb level and platelet count (P=0.005 and 0.036 respectively), but showed a significant negative correlation with the WBC count (P=0.005). CD38 expression was significantly higher in the CD305-negative group compared with the CD305-positive group (P=0.04). The expression of CD305 was significantly elevated in low B-CLL stages than in higher stages (P<0.05). Conclusion There was a significant negative correlation between CD305 with WBCS. CD38 was a significantly increased expression in the CD305-negative group compared with the CD305-positive group. The number of cases receiving treatment in the CD305-negative group was higher compared with the CD305-positive group. CD305 expression could be used as simple, reliable, inexpensive independent prognostic factors in B-CLL patients with a valuable predictive role for patients who will initiate chemotherapy early.

Discovery of Novel PI3Kδ Inhibitors Based on the p110δ Crystal Structure.

PI3Kδ is a key mediator of B-cell receptor signaling and plays an important role in the pathogenesis of certain hematological malignancies, such as chronic lymphocytic leukemia. Idelalisib, which targets PI3Kδ specifically, is the first approved PI3K inhibitor for cancer therapy. Recently, we carried out virtual screening, cell-based assays, adapta kinase assays, and molecular dynamic analysis to discover novel PI3Kδ inhibitors and identified NSC348884 as a lead PI3Kδ inhibitor. NSC348884 had an excellent docking score, potent PI3Kδ-inhibitory activity, antitumor effects on various cancer cell lines, and a favorable binding mode with the active site of PI3Kδ. Moreover, through the structural modification of NSC348884, we further discovered comp#1, which forms H-bonds with both Val828 and Lys779 in the ATP binding pocket of PI3Kδ, with a more favorable conformation binding to PI3Kδ. In addition, we found that N1, N1, N2-trimethyl-N2-((6-methyl-1H-benzo[d]imidazol-2-yl) methyl) ethane-1,2-diamine might be a potential scaffold structure. Thus, the result of this study provides a far more efficient approach for discovering novel inhibitors targeting PI3Kδ.

CG '806, a First-in-Class Pan-FLT3/BTK Inhibitor, Exhibits Potent Growth Inhibition As a Single Agent and in Combination with a BET Bromodomain Inhibitor or a Bcl2 Inhibitor on Primary AML and CLL Patient Samples

CG '806, a First-in-Class Pan-FLT3/BTK Inhibitor, Exhibits Potent Growth Inhibition As a Single Agent and in Combination with a BET Bromodomain Inhibitor or a Bcl2 Inhibitor on Primary AML and CLL Patient Samples

Development of PROTACs to address clinical limitations associated with BTK-targeted kinase inhibitors.

Chronic lymphocytic leukemia is a common form of leukemia and is dependent on growth-promoting signaling via the B-cell receptor. The Bruton tyrosine kinase (BTK) is an important mediator of B-cell receptor signaling and the irreversible BTK inhibitor ibrutinib can trigger dramatic clinical responses in treated patients. However, emergence of resistance and toxicity are major limitations which lead to treatment discontinuation. There remains, therefore, a clear need for new therapeutic options. In this review, we discuss recent progress in the development of BTK-targeted proteolysis targeting chimeras (PROTACs) describing how such agents may provide advantages over ibrutinib and highlighting features of PROTACs that are important for the development of effective BTK degrading agents. Overall, PROTACs appear to be an exciting new approach to target BTK. However, development is at a very early stage and considerable progress is required to refine these agents and optimize their drug-like properties before progression to clinical testing.

Aberrant ERG expression associates with downregulation of miR-4638-5p and selected genomic alterations in a subset of diffuse large B-cell lymphoma.

ERG (avian v-ets erythroblastosis virus E26 oncogene homolog), an oncoprotein in prostate carcinoma and Ewing's sarcoma is associated with poor prognosis in patients with acute myeloid leukemia and T lymphoblastic leukemia. However little is known about ERG in lymphoma. Here we studied ERG in diffuse large B-cell lymphoma (DLBCL) by immunohistochemistry, fluorescence in situ hybridization (FISH), genome-wide microRNA (miRNA) expression profiling, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and whole exome sequencing (WES). Approximately 30% of de novo DLBCLs (37 of 118) expressed ERG (ERG+). ERG expression showed no significant correlation with DLBCL cell-of-origin classification, patient's age, sex, nodal, or extranodal disease status, tumor expression of p53 or p63. There was no ERG rearrangement in 10 randomly selected ERG+ DLBCLs by FISH. Forty-three miRNAs showed significant differential expression between ERG+ and ERG- DLBCLs. Downregulation of miR-4638-5p was confirmed by real-time RT-PCR. WES not only confirmed known gene mutations in DLBCLs but also revealed multiple novel gene mutations in POLA1, E2F1, PSMD8, AXIN1, GAB2, and GNB2L1, which occur more frequently in ERG+ DLBCLs. In conclusion, our studies demonstrated aberrant ERG expression in a subset of DLBCL, which is associated with downregulation of miR-4638-5p. In comparison with ERG-negative DLBCL, ERG+ DLBCL more likely harbors mutations in genes important in cell cycle control, B-cell receptor-mediated signaling and degradation of β-catenin. Further clinicopathological correlation and functional studies of ERG-related miRNAs and pathways may provide new insight into the pathogenesis of DLBCL and reveal novel targets for better management of patients with DLBCL.

Ibrutinib for the treatment of chronic lymphocytic leukemia

ABSTRACTIntroduction: Chemoimmunotherapy has improved outcomes in chronic lymphocytic leukemia, yet it is not curative, with very high relapse rates, and is associated with a significant risk of toxicities. Moreover, patients with higher-risk genetic abnormalities continue to experience poorer outcomes and lower survival. Recently, novel targeted therapies have been developed to increase efficacy and reduce toxicity.Areas covered: Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase, a mediator of B-cell receptor signaling, which plays a vital role in various B-cell neoplasms. The drug has been approved for the treatment of several hematological malignancies, including chronic lymphocytic leukemia/small lymphocytic lymphoma, where large trials have shown outcomes never seen before even in high-risk patients. The safety profile appeared furthermore favorable, even in elderly and unfit patients.Expert opinion: Therapy with ibrutinib rarely provides MRD-negative complete remission; an indefinite maintenance is therefore needed, with the risk of developing adverse events (AE) or resistance resulting in treatment interruption or discontinuation. Novel, extremely promising, combination strategies, based on the association of ibrutinib with chemoimmunotherapy, anti-CD20 monoclonal antibody or other targeted agents, are currently being investigated, with the goal of achieving greater depth of remission, especially MRD-negativity, and removing the need for indefinite treatment.

Interleukin 4-Induced Gene 1 as an Emerging Regulator of B-Cell Biology and its Role in Cutaneous Melanoma.

Interleukin 4 (IL4)-induced gene 1 (IL4I1) is an oxidase that degrades l-phenylalanine into phenylpyruvate, hydrogen peroxide, and ammonia. In contrast to other amino acid catabolic enzymes (i.e., indoleamine 2,3-dioxygenase and inducible nitric oxide synthase), IL4I1 is expressed not only in an intracellular form but also an active secreted form. Although about 20 yr ago IL4I1 was identified in murine B cells in response to IL4, we only recently established its key role in controlling B-cell receptor-mediated signaling during murine B-cell ontogeny and responses in physiological settings. Genetic IL4I1 invalidation increases the number of tumor-associated B cells and delays development of spontaneous metastatic melanoma in mice that are transgenic for the RET oncogene, without impairing tumor-specific antibody response. Although no consensus exists on phenotype and functions of melanoma-associated B cells, our results in RET mice argue for a protective role, with IL4I1 dampening this benefit. However, regulation of IL4I1 expression in innate-like and conventional B-cell subsets and its impact on B-cell properties are incompletely known, in particular, in cancer settings. This review aims to summarize our present knowledge of B cells in human and murine melanoma and address emerging questions about the impact of IL4I1 on B-cell functions in physiological and cancer settings. We note that during melanoma progression, IL4I1 may selectively be expressed by regulatory B cells and/or indirectly promote B-cell-mediated immunosuppression.

Evaluating the Efficacy, Safety, and Dose Dependent Activities of Entospletinib in Patients with Chronic Lymphocytic Leukemia

Evaluating the Efficacy, Safety, and Dose Dependent Activities of Entospletinib in Patients with Chronic Lymphocytic Leukemia

Results of a Phase 2 Trial Evaluating Efficacy and Safety of Entospletinib (GS-9973) in Patients with Mantle Cell Lymphoma

Introduction: Entospletinib (GS-9973) is an orally bioavailable, selective inhibitor of spleen tyrosine kinase (Syk). Syk is a mediator of B-cell receptor signaling in normal and transformed B-cells. Targeting the B-cell receptor (BCR)-signaling pathway has been the focus in many types of B-cell related hematological malignancies including mantle cell lymphoma (MCL).Methods: This reports the MCL cohort in a phase 2 trial that more broadly evaluated the efficacy and safety of entospletinib (800 mg BID) in patients with relapsed and refractory hematological malignancies (NCT01799889). Tumor response was assessed per Cheson 2007 criteria with imaging planned at weeks 8, 16, 24, and then every 12 weeks. The primary endpoint was PFS at week 16. All efficacy data were assessed by an Independent Review Committee.Results: A cohort of 39 patients with MCL was enrolled. The median age was 72 years (range: 49-92), 64% were male, and a median number of prior regimens were 2 (range: 1-6). Prior therapy included anti-CD20 antibodies (95%), alkylating agents (95%; bendamustine 44%), and anthracyclines (77%). Three patients (8%) have received ibrutinib either as investigational drug (n = 1) or as an approved drug (n = 2), and 1 patient (3%) received idelalisib. Median duration of treatment was 21 weeks (range: 1-87), with 1 patient continuing on treatment. Thirty-five (90%) patients were evaluable for tumor response. Four patients (10%) discontinued prior to initial tumor assessment: death (n = 1), disease progression (n = 2) and investigator’s discretion (n = 1). The most common TEAEs (any grade/≥gr 3, independent of causality) and common lab abnormalities are summarized in Table 1. There were 4 TEAEs that led to study drug discontinuation (all n = 1): cardiac arrest, pruritus, pyrexia, and maculopapular rash. Six deaths were reported, none of which were related to study drug. The ORR was 15% (90% CI: 6.9%, 28.1%), with 6 (15%) patients achieving a PR and 23 (59%) patients maintaining a stable disease. The PFS rate at week 16 was 66% (95% CI: 46%, 79%). Median PFS was 5.6 months (95% CI: 3.6 months, 8.9 months). These results are based on data analysis of June 28, 2016.Conclusion: Entospletinib was well tolerated and demonstrated modest activity in patients with relapsed or refractory MCL. Further development of entospletinib in MCL will focus on the development of combination therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresSharman:Gilead Sciences, Inc.: Honoraria, Research Funding. Kolibaba:Amgen: Research Funding; Celgene: Research Funding; Cell Therapeutics: Research Funding; Genentech: Research Funding; GSK: Research Funding; Janssen: Research Funding; Acerta: Research Funding; Gilead: Consultancy, Research Funding; Novartis: Research Funding; Pharmcyclics: Research Funding; Seattle Genetics: Research Funding; TG Therapeutics: Honoraria, Research Funding. Shustov:Seattle Genetics: Research Funding; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; SPECTRUM: Consultancy, Research Funding; Novartis: Research Funding. Nay:Gilead Sciences, Inc.: Honoraria; Janssen: Honoraria, Other: Advisory board; Celgene: Honoraria, Other: Advisory board; Amgen: Other: Advisory board; Lyndbeck: Honoraria; BMS: Honoraria. Zhang:Gilead Sciences: Employment, Equity Ownership. Shi:Gilead Sciences, Inc.: Employment, Equity Ownership. Forero:University of Alabama at Birmingham: Research Funding. Assouline:BMS: Speakers Bureau; Lundbeck: Consultancy; Janssen: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau.

Abstract B131: Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia

Abstract Spleen tyrosine kinases (Syk) are initiators of B cell receptor-mediated (BCR) signaling events, enabling cells to drive chronic lymphocytic leukemia (CLL) progression. However, the mechanistic principles of biochemical signaling pathways that contribute to CLL pathogenesis are poorly understood. This study investigates Syk-mediated molecular pathways of the BCR signaling network using computational modeling. An expression of ζ chain-associated protein kinase of 70 kD (ZAP-70, Syk-family protein kinase) in the BCR signaling pathway is an adverse prognostic marker in CLL. Therefore, we need a better understanding of molecular mechanisms of Syk family protein kinases and effects of their inhibitors in the BCR signaling pathway. In this work, we dissected several computational models of ZAP-70 and SYK regulation to design an integrated mechanistic description of the BCR signaling pathway, and to explain the observed differences in the clinical behaviors of ZAP-70+ / ZAP-70- phenotypes of CLL patients. Specifically, we characterized the effects of different ZAP-70 and SYK expression and phosphorylation levels on the BCR activation threshold, using stochastic simulations of the BCR signaling network. Preliminary findings suggest that ZAP-70 serves as an adaptor protein that facilitates the recruitment of SYK to the BCR complex. We find that the expression of ZAP-70 enhances the BCR signaling, and therefore correlates with a more aggressive disease course. Our results will help in the development of novel therapies targeting Syk functions to regulate B-CLL responses. Citation Format: Maria P. Frushicheva. Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B131.

Phase 2 Trial of Entospletinib (GS-9973), a Selective Syk Inhibitor, in Indolent Non-Hodgkin's Lymphoma (iNHL)

Background: Spleen tyrosine kinase (Syk) is a mediator of B-cell receptor signaling in normal and transformed B-cells. Entospletinib is an orally bioavailable, selective inhibitor of Syk.Methods: This Phase 2 trial is evaluating entospletinib 800 mg BID in a study of 204 patients with previously treated lymphoid malignancies. Tumor imaging was planned at weeks 8, 16, 24 and then every 12. Tumor response was assessed per Cheson 2007 criteria.Results: A cohort of 69 patients with iNHL (41 follicular lymphoma [FL], 11 lymphoplasmacytoid lymphoma [LPL], 17 marginal zone lymphoma [MZL]) are included in this analysis. Median age was 66 years (range 41 - 89). 58% were male. The median number of prior treatments (Rxs) regimens was 3 (range 1- 14). Prior Rxs included anti-CD20 antibodies (rituximab 99%, ofatumumab 4%), alkylating agents (90%; bendamustine 51%) and anthracyclines (35%). Baseline risk factors: Ann Arbor Stg III-IV (70%), Gr 3a FL (29%), FLIPI ≥3 (34%). Median duration of Rx was 16 weeks with 10 patients continuing on Rx. Entospletinib was generally well tolerated. The most common TEAEs (any Grade/≥Gr 3, independent of causality) were fatigue (54%/13%), nausea (49%/4%), diarrhea(36%/0%), vomiting (26%/0%), headache (23%/1%), pyrexia (23%/3%), decreased appetite (22%/0%), constipation (22%/1%) and common laboratory abnormalities were increased AST (33%/15%), increased ALT (41%/19%), increased total bilirubin (32%/16%), anemia (36%/13%) and neutropenia (38%/13%). 4 patients died while on study from progressive disease. At the time of this analysis, 66 of 69 patients have been treated through first response assessment (1 patient ongoing not reaching first response assessment, 1 patient discontinued due to AE and 1 patient withdrew consent prior to it). 38 out of 61 (62%) patients evaluable for SPD experienced reduced tumor burden, with median duration of Rx 28 weeks (range 4-92). 9/61 (15%) achieved a decrease of ≥ 50% in SPD. The ORR was 13.0% (95% CI: 6.1%, 23.3%), with 7 patients achieving a PR, one LPL patient achieving MR and one patient achieving a CR. Forty-one patients (59.4%) had stable disease. The primary end point of 24 weeks PFS was 48.9% (95% CI: 34.6%, 61.7%). Median PFS was 5.5 months (95% CI: 4.4 months, 8.2 months). There were 39 patients (56.5%) with events of disease progression.Conclusions: Entospletinib monotherapy given with this dose and schedule was well tolerated and demonstrated activity in patients with advanced relapsed iNHL, including those with poor prognostic features. Further development of entospletinib in iNHL will focus on the development of combination approaches with chemotherapy and targeted agents. [Display omitted] [Display omitted] DisclosuresSharman:Calistoga: Honoraria; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; TG Therapeutics, Inc.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Roche: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding. Off Label Use: Management of CLL/SLL and follicular lymphoma. Kolibaba:Takeda Pharmaceuticals International Co.: Research Funding; Genentech: Research Funding; Seattle Genetics, Inc.: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Janssen: Research Funding; GSK: Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding. Abella:Gilead: Employment. Eng:Gilead: Employment. He:Gilead Sciences: Employment. Hu:gilead: Employment. Yasenchak:Seattle Genetics, Inc.: Research Funding.

Phase 2 Trial of Entospletinib (GS-9973), a Selective Syk Inhibitor, in Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL)

Abstract Background: Spleen tyrosine kinase (Syk) is a mediator of B-cell receptor signaling in normal and transformed B-cells. GS-9973 is an orally bioavailable, selective inhibitor of Syk. Methods: This Phase 2 trial enrolled 41 patients with CLL and 15 patients with SLL treated with GS-9973 800 mg BID. Tumor imaging occurred at weeks 8, 16, 24 and then every 12. Response was independently evaluated according to Hallek 2008 as modified by Cheson 2012 for patients with CLL and Cheson 2007 for patients with SLL. Primary endpoint for the study was PFS at 24 weeks. Results: The median ages of CLL and SLL patients were 73 (range 51-89) and 70 (range 57-84), respectively. 68% of CLL subjects and 60% of SLL subjects were male. Ten patients had 17p deletions/TP53 mutations and 17 had SF3B1 or NOTCH1 mutation, or 11q22.3 deletion. The median number of prior regimens for CLL was 2 (range 1-8) and for SLL was 2 (range 1-10). Prior therapies included anti-CD20 antibodies (98%), alkylating agents (86%, [bendamustine 63%]) and fludarabine (66%). 12 CLL and 6 SLL patients are still on treatment; the median duration of treatment for all CLL and SLL patients was 36 weeks The most common treatment emergent AEs (any Grade/≥Gr 3, independent of causality) were fatigue (70%/7%), nausea (54%/2%), diarrhea(48%/0%), cough(34%/0%), dizziness (32%/2%), headache (29%/0%), pyrexia (29%/0%), decreased appetite (27%/2%), upper respiratory tract infection(27%/0%), constipation (23%/0%). Common laboratory abnormalities were increased AST (30%/5%), increased ALT (43%/4%), increased total bilirubin (41%/16%), anemia (50%/7%) and neutropenia (54%/29%). Forty-nine patients were treated for at least 8 weeks and 54 patients had ≥ 1 efficacy assessment, two patients discontinued prior to the first response assessment, one due to AE and one withdrew consent. Per investigator assessment, 51 out of 52 (98%) patients evaluable for SPD experienced reduced tumor bulk; 38 (73%) achieved a decrease of ≥ 50%. The ORR was 62.5% (95% CI: 48.6%, 75.1%), with 35 patients achieving a PR and no subject achieving a CR. Thirteen patients (23.2%) had stable disease. The primary end point of 24 weeks PFS was 72.3% (95% CI: 57.1%, 83.0%). Median PFS was 20.5 months (95% CI: 7.7 months, not reached). There were 24 patients (42.9%) with events, 22 (39%) with disease progression and two deaths (4%) attributed to septic pneumonia and pseudomonal infection which was unrelated to entospletinib by investigator assessment. Among the 35 responding patients, median DOR was 21.3 months (95% CI: 13.2 months, not reached). Results of an independent response assessment are pending and will be presented. Entospletinib was well tolerated and demonstrated substantial activity in patients with CLL, and SLL including those with poor prognostic features. Entospletinib activity seems comparable to that reported by other approved BCR pathway inhibitors with the median PFS reported for Idela of 15.8 months (Blood. 2014;123(22):3390-3397) and Ibrutinib reported 42.6% overall response rate and a PFS of 70-80% @ 12 months (ibrutinib PI) Current studies plans include studying Entospletinib in combination therapy. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Sharman: Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Research Funding; Calistoga: Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Research Funding; TG Therapeutics, Inc.: Research Funding; Janssen: Research Funding. Kolibaba:Janssen: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; TG Therapeutics: Research Funding; GSK: Research Funding; Celgene: Research Funding; Acerta: Research Funding; Seattle Genetics, Inc.: Research Funding; Takeda Pharmaceuticals International Co.: Research Funding; Gilead: Consultancy, Research Funding. Abella:Gilead: Employment. Di Paolo:Gilead Sciences: Employment, Equity Ownership. Eng:Gilead: Employment. Hu:gilead: Employment. He:Gilead Sciences: Employment. Reddy:gilead: Employment. Yasenchak:Seattle Genetics, Inc.: Research Funding.

A Phase 1b, Open-Label, Dose Escalation and Expansion Study Evaluating the Safety and Efficacy of Entospletinib (GS-9973) with Vincristine and Dexamethasone in Adult Subjects with Relapsed or Refractory Acute Lymphoid Leukemia (ALL)

Background: Despite progress in understanding the biology and improvements in the treatment of ALL resulting in the majority of adults entering a complete remission (CR), ultimately most patients relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) after initial cytoreduction in a second or subsequent CR offers a chance of cure in a small, highly selected group of subjects. Therefore, re-induction of a CR is the first step in the treatment of relapsed disease. Current treatments for relapsed ALL are generally unsuccessful and most die of their disease, underscoring the need for novel therapeutic approaches.Spleen tyrosine kinase (SYK) is a mediator of B-cell receptor signaling in normal and transformed B-cells. Increased activity of SYK in ALL cells may play a role in leukemia cell survival and proliferation. Deregulated SYK activity allows growth factor-independent proliferation and transformation of bone marrow-derived pre-B cells in mice. B-ALL results from the abnormal accumulation of transformed progenitor or precursor B lymphocytes (Wossning T, et al. JEM 2006), and inhibition of SYK has been shown to attenuate the growth of B-ALL in vitro and in vivo in mouse models. SYK activation occurs in human B-ALL and is sensitive to small molecule SYK inhibitors; in vitro treatment of primary patient-derived B-ALL cells with a SYK-inhibitor inhibits the proliferation of B-ALL cells (Perova T, et al. Sci Transl Med 2014).Entospletinib is an orally bioavailable, selective inhibitor of SYK which has shown tolerable safety and clinical activity in mature B-cell malignancies (Sharman J, et al. Blood. 2015). In pre-clinical models, the anti-leukemia effect of entospletinib was synergistic with vincristine (M. Axelrod, et al. ASH abstract 2015). We are conducting a Phase 1b trial (NCT02404220) evaluating entospletinib in combination with standard chemotherapy (vincristine and dexamethasone) for the treatment of relapsed or refractory adult Pre B-ALL (figure 1).Methods: This dose escalation trial will follow a 3+3 design. A subsequent dose expansion will enroll 15 additional subjects at the recommended dose level from the dose escalation.Key inclusion criteria for the trial include: previously treated B-ALL (Ph+ or Ph-), ≥18 years of age, ECOG PS≤2 with adequate organ function and resolution of all toxic effects of any prior therapy to ≤Grade 1.Key exclusion criteria include: diagnosis of mature B-ALL (Burkitts leukemia), active or symptomatic CNS disease, history of myelodysplastic syndrome or solid organ transplantation, allogeneic bone marrow progenitor cell transplant within 100 days or on active immunosuppression for GVHD, ongoing/active pneumonitis, and current therapy with proton pump inhibitors.Results: As of 7/21/2015, 3 patients have been enrolled into dose level 1, and 1 patient (64 y.o. female in 1st relapse) has completed the protocol-specified 2 induction cycles of treatment without dose-limiting toxicity. At the end of the 2nd induction cycle, that patient became transfusion independent and bone marrow evaluation showed morphologic CR (MRD positive) with recovery of normal hematopoiesis.Conclusions: The safety of entospletinib administered in combination with vincristine and dexamethasone is being investigated for the treatment of relapsed/refractory adult ALL. [Display omitted] DisclosuresStock:Gilead: Membership on an entity's Board of Directors or advisory committees. Crosswell:KIYATEC (employment); Gilead (common stock ownership and research funding): Employment, Equity Ownership, Research Funding. Chun:Gilead Sciences: Employment. Abella:Gilead: Employment. He:Gilead Sciences: Employment. Eng:Gilead: Employment. Douer:Gilead: Consultancy.

Clinical Activity of Entospletinib (GS-9973), a Selective Syk Inhibitor, in Patients with CLL Previously Treated with an Inhibitor of B-Cell Receptor Pathway Signaling

BackgroundEntospletinib (GS-9973) is an orally bioavailable, selective inhibitor of spleen tyrosine kinase (Syk). Syk is a mediator of B-cell receptor signaling in normal and transformed B-cells. Targeting the B-cell receptor (BCR) signaling pathway has been a successful therapeutic strategy for chronic lymphocytic leukemia (CLL), with both ibrutinib, an inhibitor of BTK (BTKi) and idelalisib, an inhibitor of PI3Kdelta (PI3Ki), approved for this indication. Entospletinib activity in CLL was recently reported, and preclinical data suggested that entospletinib may be effective even in the context of resistance to BTK therapy, including that conferred by activation of PLCγ2. (Liu, Blood -2015-02-626846)MethodsGS-US-339-0102 is an ongoing phase2 trial of entospletinib in CLL and NHL (NCT01799889). The study protocol was amended to add 40 patients in each of 2 CLL cohorts who have been previously treated with BCR signaling pathway (BTK/PI3K) inhibitors. These patients were treated with entospletinib monotherapy (400mg BID) and evaluated using modified Hallek/IWG-CLL criteria every 2-3 months as previously described in Sharman, Blood 2015:125(5).ResultsAs of July 20, 2015, 8 patients with preceding BCR pathway signaling inhibitor treatment have been enrolled, 5 with preceding BTKi therapy (4 with ibrutinib, 1 with AVL-292) and 3 with preceding PI3Ki therapy (idelalisib). The median duration of preceding BTKi treatment was 51 weeks (range 2-85 weeks) and the median duration of preceding PI3Ki treatment was 106 weeks (range 74-168 weeks). Two patients had progressed on prior BTKi and 2 were intolerant (cause missing for 1 patient), while 2 patients progressed on PI3Ki and 1 was intolerant. All 5 patients with preceding BTKi and 2 out of 3 patients with prior PI3Ki remain on entospletinib treatment. Of the 5 patients who were previously treated with BTKi, the ongoing duration of treatment with entospletinib is 8, 8, 13, 25, and 39 weeks. For the 3 patients with preceding PI3Ki, two patients have ongoing treatment of 18 and 26 weeks; one patient stopped treatment and died after 23 weeks due to a cardiac arrest that is not believed to be related to the study drug. The most common treatment-emergent AEs (N=number; any Grade/≥Gr 3, independent of causality) were decreased appetite (3/0), contusion (2/0), dyspepsia (2/0), fatigue (2/0), dehydration (1/1), cardiac arrest (1/1); common laboratory abnormalities were anemia (5/1), neutropenia (4/1), thrombocytopenia (3/2), increased lipase (1/1). Early responses were seen with entospletinib treatment (3 partial response (PR), 1 stable disease, & 3 patients were too early to evaluate) and 1 PD. PR occurred in 1 BTKi and 2 PI3Ki previously treated patients. One patient with preceding PI3Ki developed progressive disease after 8 weeks.ConclusionsEarly experience from this trial with ongoing enrollment demonstrates that entospletinib has clinical activity following therapy with either BTKi or PI3Ki. No additional safety signals were seen from earlier studies. Additional investigation of treatment with entospletinib following progression with B-cell receptor signaling pathway inhibitors is warranted. DisclosuresSharman:Celgene Corporation: Consultancy, Research Funding; TG Therapeutics, Inc.: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Calistoga: Honoraria; Janssen: Research Funding. Shustov:Celegene, BMS: Consultancy, Honoraria, Research Funding. Smith:celegene, spectrum, genentech: Honoraria. Boyd:US Oncology: Research Funding; Celgene: Speakers Bureau; Genentech, Inc.: Research Funding. Kolibaba:Gilead: Consultancy, Research Funding; Seattle Genetics, Inc.: Research Funding; Acerta: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Takeda Pharmaceuticals International Co.: Research Funding; GSK: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapeutics: Research Funding. Abella:Gilead: Employment. He:Gilead Sciences: Employment. Eng:Gilead: Employment. Hu:gilead: Employment. Reddy:gilead: Employment. Mitra:Gilead: Employment. Yasenchak:Seattle Genetics, Inc.: Research Funding.

Encephalomyocarditis Virus 3C Protease Relieves TRAF Family Member-associated NF-κB Activator (TANK) Inhibitory Effect on TRAF6-mediated NF-κB Signaling through Cleavage of TANK

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.

Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo.

Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell receptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, resulting in the phosphorylation of Syk and Erk1/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.

Spleen-specific isoforms of Pax5 and Ataxin-7 as potential proteomic markers of lymphoma-affected spleen.

The splenomegaly, enlargement of spleen, has been observed in several diseases. It has been intended to evaluate histochemical alterations, spleen-specific enzymatic and proteomic markers during splenomegaly, and lympho-proliferative disorders from spleen of mice bearing Dalton's lymphoma. The higher expression of c-fos, c-jun, and MAPK testifies proliferation of lymphocytes. The lower expression of Pax5, higher expression of CD3, and the presence of additional form of Zap-70 suggest hypertrophy of follicles and splenomegaly influenced by weak B-cell receptor-mediated signaling, but activated T-cell receptor-mediated signaling. Simultaneously, lower levels of SOD, NDR2, and MIB2 and higher expression levels of Ataxin-7 and LDH also suggest impact of stress either as a cause or effect of cell proliferation. Spleen-specific isoform of Pax5, NDR2, MIB2, and Ataxin-7 can be considered as spleen-specific unique molecular markers for the evaluation of splenomegaly and lympho-proliferative disorders.

Phase 2 Trial of Entospletinib (GS-9973), a Selective SYK Inhibitor, in Follicular Lymphoma (FL)

Background: Spleen tyrosine kinase (Syk) is a mediator of B-cell receptor signaling in normal and transformed B-cells. Entospletinib is an orally bioavailable, selective inhibitor of Syk (Kd 7.6 nM, no other kinase < 100 nM).Methods: This Phase 2 trial is evaluating Entospletinib 800 mg BID in a 41 subject cohort with previously treated FL in a study of 165 subjects with lymphoid malignancies. Tumor imaging was planned at weeks 8, 16, 24 and then every 12. Tumor response was assessed per Cheson 2007 criteria.Results: For the subjects with FL included in this analysis, median age was 67 years (range 41 – 89), 49% were male. The median number of prior Rx regimens was 2 (range 1-8). Prior treatments (Rxs) included anti-CD20 antibodies (rituximab 100%, ofatumumab 5%), alkylating agents (95%; bendamustine 51%) and anthracyclines (51%). Baseline risk factors: Ann Arbor Stg III-IV (66%), Gr 3a FL (27%), FLIPI ≥3 (34%). At the time of this analysis, 41 subjects with follicular lymphoma were enrolled and 38 subjects have been treated through first response assessment (1 subject ongoing prior to first response assessment, 1subject discontinued due to AE and 1 subject withdrew consent). Entospletinib was generally well tolerated. The most common TEAEs (any Grade/≥Gr 3, independent of causality) were fatigue (46%/12%), nausea (42%/2%), diarrhea(27%/0%), decreased appetite (22%/0%), vomiting (22%/0%) and common laboratory abnormalities were increased AST (37%/17%), increased ALT (34%/20%), increased total bilirubin (27%/5%), anemia (34%/10%) and neutropenia (22%/10%). Reversible Grade 3 or 4 ALT/AST elevations occurred in 8 (19.5%) FL subjects. 3 subjects died while on study: 2 from progressive disease and 1 from acute renal failure investigator reported as unrelated to study drug. Investigator response assessments are available for 29 subjects, 16/29 (55%) subjects experienced reduced tumor bulk measured by SPD; 3 (10%) achieved a decrease of ≥ 50%. CR has not been observed at this early evaluation. 14/41 subjects continue on Rx. Median duration of Rx for all patients was 15 weeks. Among all patients who experienced reduction in tumor volume, median duration of Rx was 25 weeks (range 4-51).Conclusions: Entospletinib monotherapy given with this dose and schedule was generally well tolerated and demonstrated moderate activity in subjects with advanced relapsed FL, including those with poor prognostic features. Updated data with longer follow-up duration will be presented at the meeting. DisclosuresSharman:Gilead Sciences: Research Funding. Klein:Gilead Sciences: Research Funding. Boxer:Gilead Sciences: Research Funding. Kolibaba:Gilead Sciences: Research Funding. Hawkins:Gilead Sciences: Research Funding. Abella:Gilead Sciences: Employment, Equity Ownership. Wu:Gilead Sciences: Employment, Equity Ownership. Yasenchak:Gilead Sciences: Research Funding.

MEK1/2 Inhibition By MEK162 Is Effective Against Chronic Lymphocytic Leukaemia Cells Under Conditions That Mimic Stimulation of B-Cell Receptor-Mediated Signaling

BACKGROUND Chronic Lymphocytic Leukemia (CLL) is characterised by the clonal expansion of apoptosis resistant B-lymphocytes. However, in vitro and in the absence of pro-survival factors primary CLL cells undergo spontaneous apoptosis.B-cell receptor (BCR) signalling plays a major role in the survival and proliferation of CLL cells, which is highlighted by the clinical efficacy of the Btk and PI3-kinase inhibitors, ibrutinib and idelalisib. Mitogen activated protein kinase (MAPK) is an important mediator of signals downstream of both Btk and PI3-kinase but few studies have shown that inhibitors of MEK1/2, a critical component in the MAPK pathway, have any potential benefit for therapy of CLL.METHODS We sought to investigate the potential of the MEK1/2 inhibitor MEK162 against CLL cells in vitro. To mimic the tonic BCR stimulation experienced in vivo, primary CLL cells were stimulated using an immobilised antibody to IgM or were treated with PMA, a less specific B-cell activator which promotes protein kinase C-dependent MAPK-ERK1/2 signaling. Sensitivity to MEK162 and effects on MAPK-ERK1/2 pathway activity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analyses respectively.RESULTS MEK162 treatment of CLL cells cultured in media alone resulted in a modest but significant (P < 0.01) reduction in cell viability; 20µM MEK162 reduced the proportion of viable cells remaining after 48 h to 77.40 +/- 7.81 % relative to vehicle-treated controls. In contrast, BCR stimulation through IgM ligation promoted cell survival 3.2 +/- 1.01 fold and sensitised CLL cells to MEK162; 20µM MEK162 reduced the proportion of viable cells by 56.28 +/- 2.37 % (P < 0.001 relative to control). A similar effect was observed in response to PMA stimulation; cell viability increased 1.78 +/- 0.15 fold and was reduced by 59.55 +/- 10.33 % (P < 0.001 relative to control) following treatment with 20µM MEK162 (Figure 1). At concentrations > 0.05 µM MEK162 was significantly (P < 0.05) more effective against CLL cells stimulated with either anti-IgM or PMA than against cells cultured in media alone.By western blotting we observed low levels of MAPK-ERK1/2 activity in cells cultured in media alone, which we suggest may contribute to the spontaneous apoptosis of these cells and the low degree of sensitivity to MEK162 under these conditions. We confirmed that stimulation with either IgM or PMA results in activation of MAPK-ERK1/2 and show that this response can be effectively blocked using MEK162. The effects of anti-IgM and PMA on cell survival and response to MEK162 were independent of ZAP-70 expression or ATM/TP53 functional status.CONCLUSIONS Our data illustrate the important role of MAPK-ERK1/2 activity in BCR-mediated CLL cell survival and suggest that MEK162 may have potential for CLL therapy. These data highlight the importance of employing appropriate culture conditions in order to make accurate assessments concerning the efficacy of novel agents for the treatment of CLL. [Display omitted] DisclosuresMulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.