B-cell Receptor Signaling Components Research Articles

Mechanistic Elucidation of the Tumor-Promoting Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 in B-Cell Receptor Signaling in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a highly aggressive and incurable type of B-cell non-Hodgkin's lymphoma. B cell receptor (BCR) signaling plays an important role in the pathogenesis of MCL; however, the underlying mechanisms are unknown. Our genome-wide screens identified that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1/CC1) is a central component of BCR signaling and is essential for MCL survival in vitro and in vivo. Among the twelve alternatively spliced human CC1 isoforms, we found that CC1-4L, the longest CC1 isoform is overexpressed in MCL. Structurally, it has the N-terminal domain and three type 2 constant ectodomains, a transmembrane sequence and a signaling cytoplasmic domain with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs or Y493/520). These ITIMs of CC1 can inhibit signals in T cells, NK cells, neutrophils and granulocytes. However, its role in B cells and BCR signaling is not clear. In this study, we have elucidated a novel mechanistic function of CC1 in promoting oncogenic BCR signaling in MCL. First, we studied the role of CC1 in lipid raft formation, which is one of the early events during BCR signaling activation. To study this, we used three panels of MCL lines, Jeko-1-NTC (non-template control), Jeko-1-gCC1 (CC1-knockout) and Jeko-1-gCC1-4L (CC1-knockout restored with CC1-4L isoform). Using high-resolution confocal microscopy, we observed that CC1-expressing Jeko-1 MCL cell line showed significantly increased lipid raft formation when compared to CC1-knockout cells. However, CC1-deficient cells restored with the CC1-4L isoform exhibited increased lipid raft assembly. Further, we also observed that CC1-expressing MCL lines showed increased cytoskeletal assembly, which was observed via increased F-actin and filamin A staining in CC1-expressing MCL lines when compared to CC1-knockout cells. Second, we analyzed the phosphorylation levels of proteins involved in the BCR signaling cascade in a panel of genetically engineered MCL lines, i.e., Jeko-1-NTC, Jeko-1-gCC1, Jeko-1-gCC1-4L, Jeko-1-gCC1-4S (CC1-knockout restored with CC1-4S or short cytoplasmic tail isoform) and Jeko-1-gCC1-mutants (CC1-knockout cells restored with N-terminal domain deletion mutant and ITIM-mutant or YY/FF 493/520). We observed that Jeko-1-NTC and Jeko-1-gCC1-4L cell lines showed significantly increased phosphorylation levels of BCR signaling components such as phospho-SRC (Y416), phospho-SYK (Y319), phospho-AKT (S473) and phospho-BTK (Y223) when compared to Jeko-1-gCC1, Jeko-1-gCC1-4S and Jeko-1-gCC1 mutant MCL lines. This shows that CC1 positively regulates or activates oncogenic BCR signaling in MCL and the N-terminal domain and cytoplasmic tail are required for this function. Next, we analyzed whether CC1 could interact with the BCR signaling components such as CD79B, LYN and SYK using immunoprecipitation (IP) and proximity ligation assay (PLA) in MCL lines. Further, we analyzed the kinetics of BCR activation in order to infer the role of SHP-1 binding to the ITIMs of CC1. Our IP and PLA experiments demonstrated that in Jeko-1 and Mino MCL lines, at the peak (i.e., at 5 and 15 min) of BCR activation, there was significantly increased interaction between CC1 and the BCR-pathway components (CD79B, LYN and SYK), and this interaction subsided by 30 min of IgM activation. However, in the case of CC1 and SHP-1, the interaction was stable throughout the time points and increased by 30 min of IgM activation. These interactions were disrupted in the CC1-N-terminal domain deletion and ITIM mutant MCL lines when compared to the CC-4L expressing MCL line. Finally, we analyzed the interaction between CD79B and SYK in CC1-4L, CC1-ITIM mutant and N-domain deletion mutant expressing MCL lines. Using PLA, we observed increased interaction between CD79B and SYK in CC1-4L expressing cell line compared to other cell lines indicating that functional CC1 is required for the recruitment of SYK to CD79B. In summary, CC1 stabilizes lipid rafts following BCR engagement by anchoring to F-actin and Filamin A. CC1 recruits and activates LYN and SYK at the BCR complex at the peak of BCR activation and subsequently recruits SHP-1 for signal downregulation. These activities of CC1 require intact ITIMs and the N-terminal ligand-binding domain. Hence, CC1 drives oncogenic BCR signaling in MCL and forms an important candidate for MCL immunotherapy.

Tunable B cell response through allosteric modulation of Bruton’s tyrosine kinase

Abstract The B cell receptor (BCR) is essential for both the initial burst of B cell proliferation in response to antigens, and for the differentiation of plasma cells and memory B cells during germinal center reactions. However, the BCR’s precise role in germinal center selection has been difficult to assess: mechanistic investigations into its modulation during the decision of B cell fate suffer from experimental limitations due to the fundamental role of the BCR in B cell biology. Bruton’s tyrosine kinase (BTK), a member of the TEC family, is a key component of BCR signaling. Using a computational screening method based on small molecule docking outside of the BTK active site, we have identified putative allosteric modulators of BTK. In vitro primary B cell Nur77 upregulation, calcium flux, proliferation and plasma cell differentiation confirmed that these molecular probes alter B cell activation strength, kinetics, and fate only when the BCR is engaged. In vivo immunization using an antigen model reveled that BTK agonism strongly favors B cell differentiation into switched plasma cells. BTK agonism, either at the time of the immunization or at the peak of germinal center formation, enhanced humoral immunity, proving for the first time that transient, augmented BCR signaling during germinal center reaction has a direct impact on B cell differentiation ability. In addition to being powerful tools for basic B cell biology investigation, allosteric agonists of BTK could be developed into antigen-licensed B cell adjuvants for vaccines in at risk populations, as well as into cancer immunotherapy drugs which hyperactivate B cells in tumor microenvironments during checkpoint inhibitor blockade therapies.

Modeling the B-cell receptor signaling on single cell level reveals a stable network circuit topology between nonmalignant B cells and chronic lymphocytic leukemia cells and between untreated cells and cells treated with kinase inhibitors.

B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.

Evolution of TP53 abnormalities during CLL disease course is associated with telomere length changes

BackgroundTelomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved.MethodsWe investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity.ResultsAt baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively).ConclusionsOur study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.

MYD88 L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation.In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 L265P or MYD88 WT, alone or in association with CD79B Y196F. In all three cell lines, the co-expression of MYD88 L265P and CD79B Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88L265P in CD79BY196F-associated proximal BCR signaling in DLBCL.To elucidate the potential role of the DOCK8 adapter in MYD88 L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 WT or MYD88 L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 L265P/DOCK8 signals in comparison to MYD88 WT/DOCK8 signals (p<.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 L265P rather than MYD88 WT (p<.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88L265P and CD79B genetic alterations.We next analyzed the consequences of MYD88 L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88L265P/CD79BY196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 WT/CD79B WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88L265P/CD79BY196F DLBCL cell lines but not in lines with MYD88 WT/CD79B WT, highlighting the specific role of DOCK8 in MYD88 L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88L265P/CD79BY196F, but not MYD88WT/CD79BWT, DLBCLs (p<.004, HBL1 and p<.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88L265P/CD79BY196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88L265P/CD79BY196F mutations. DisclosuresShipp: AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Bayer: Other: Institution: Research Grant/Funding; Abbvie: Other: Institution: Research Grant/Funding.

A CASE REPORT OF IBRUTINIB-INDUCED PNEUMONITIS

TOPIC: Lung Pathology TYPE: Medical Student/Resident Case Reports INTRODUCTION: Ibrutinib irreversibly inhibits Bruton's tyrosine kinase, an essential component of B-cell receptor signaling, leading to diminished proliferation and survival of malignant B-cells. Ibrutinib is used to treat leukemias and lymphomas, including chronic lymphocytic leukemia (CLL). Several complications are associated with ibrutinib, below describes a case of ibrutinib induced pneumonitis. CASE PRESENTATION: 64-year-old female recently diagnosed with CLL and started on ibrutinib therapy. Two months later, patient was admitted for progressive dyspnea and nonproductive cough. Initial work-up showed hypoxia, lymphocytic leukocytosis, negative COVID and respiratory viral pathogen panel (RVP). Computed tomography (CT) of chest showed new bilateral ground-glass opacities compared to CT scan three months earlier. She was initially treated with ceftriaxone and azithromycin. Patient had worsening respiratory status and required intubation on day four. Patient did not improve with several antibiotics and antifungals. But given history and borderline elevated beta-D-glucan, she underwent bronchoscopy due to concern for fungal infection. All studies including tuberculosis, histoplasma, aspergillus, coccidioides, blastomyces, pneumocystis jiroveci, fungal and bacterial cultures were negative. All antimicrobials were stopped. Patient had spontaneous pneumothoraxes on hospital day 13 and 15 requiring left needle decompression and bilateral chest tube placement. A sputum culture was repeated which grew few multi-drug resistant pseudomonas. She was treated with 7-day course of meropenem without improvement. Repeat workup showed bilateral ground-glass opacities, negative COVID, RVP, and bronchoscopy with negative fungal antigens, bacterial and fungal cultures. Given concern for ibrutinib induced pneumonitis, steroids were started with subsequent weaning from ventilator support to room air. DISCUSSION: Pneumonitis and pneumothorax are rare complications of ibrutinib therapy. The new ground-glass opacities, not present on imaging prior to starting ibrutinib, dyspnea and hypoxia, no prior lung disease, and no other new medications, is concerning for ibrutinib associated pneumonitis. Infection did not cause the opacities, both extensive infectious workups were unremarkable. The pseudomonas sputum culture was likely colonization from prolonged intubation, as she was afebrile, had stable leukocytosis, and treatment did not change respiratory status. Patient also had spontaneous pneumothoraces on lung protective ventilation. She has no history of pneumothorax, nor underlying condition that would lead to pneumothorax. In addition, oxygenation improved after starting steroids. She was weaned from ventilator support to room air, further supporting ibrutinib associated pneumonitis. CONCLUSIONS: Pneumonitis is a rare, notable complication of ibrutinib therapy. REFERENCE #1: Rai, K. R., MD, & Stilgenbauer, S., MD. (2021, January 07). Selection of Initial Therapy For Symptomatic or Advanced Chronic Lymphocytic Leukemia. Retrieved from https://www.uptodate.com/contents/selection-of-initial-therapy-for-symptomatic-or-advanced-chronic-lymphocytic-leukemia?search=ibrutinib pneumonitissIbrutinib-induced pneumonitis in patients with chronic lymphocytic leukemia. Blood 2016;127 (8): 1064–1067. doi: https://doi.org/10.1182/blood-2015-12-686873 DISCLOSURES: No relevant relationships by Tyler Cooper, source=Web Response No relevant relationships by Lauren Sesemann, source=Web Response

Mutated MYD88 Activates the BCR Component SYK and Provides a Rationale Therapeutic Target in Waldenstrom's Macroglobulinemia

Background: Mutations in the Toll receptor (TLR) pathway are highly prevalent in Waldenstrom's Macroglobulinemia (WM) and ABC DLBCL, wherein mutated MYD88 triggers NF-kB pro-survival signaling through BTK/IRAK. Activation of B-cell receptor (BCR) signaling can also be triggered by activating mutations in CD79A/B in ABC DLBCL, though are rare in WM (5-8%). Despite these findings, there is evidence for BCR activation in WM (Argyropoulos et al, Leukemia 2016). We hypothesized that crosstalk between TLR and BCR might account for aberrant BCR signaling in WM. We therefore performed PhosFLow studies on MYD88 and BCR signaling components and observed the hyperactivation of SYK (pY525-pY526) in MYD88 mutated WM (BCWM.1, MWCL-1) cells compared to wild-type MYD88 expressing control cells. We next treated MYD88 mutated WM cells with a peptide inhibitor of MYD88 homodimerization, and observed that the phosphorylation of SYK at tyrosine 525 and 526 (pY525-pY526) together with phosphorylation of BTK at tyrosine 223 (pY223) was markedly reduced. In contrast, the MYD88 inhibitor had no impact on either SYK or BTK activation in MYD88 wild-type Ramos cells. Knockdown of MYD88 by lentiviral transduction in MYD88 mutated BCWM.1 cells confirmed the reduction of SYK (pY525-pY526) by two shRNAs. Cell viability analysis by CellTiter-Glo® assay showed that the SYK inhibitor R406 was cytotoxic to MYD88 mutated WM cells at pharmacologically attainable drug levels (EC50 0.247 uM for BCWM.1; 0.315 uM for MWCL-1), versus >1 uM for MYD88 wild-type control cells. Furthermore, the combination of R406 with ibrutinib produced synergistic cytotoxicity in MYD88 mutated WM cells. Conclusion: Our findings demonstrate that in addition to TLR path activation, mutated MYD88 activates the BCR component SYK tyrosine kinase. These findings provide the rationale for combined therapeutic targeting of BCR and TLR pathways in MYD88 mutated WM. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Tirabrutinib hydrochloride for B-cell lymphomas.

Bruton tyrosine kinase (BTK) plays an important role in the B-cell receptor (BCR) signaling pathway by mediating proliferation, migration and adhesion in B-cell malignancies. Therefore, the components of BCR signaling, especially BTK, are considered to be attractive therapeutic targets. Ibrutinib, a first-in-class BTK inhibitor, has been approved for the treatment of several types of B-cell malignancies worldwide. However, ibrutinib has off-target activities on non-BTK kinase that are related to adverse effects or might translate into clinical limitations. To overcome these limitations, more specific BTK inhibitors are needed. Tirabrutinib hydrochloride (tirabrutinib) is a potent, highly selective, irreversible oral inhibitor of BTK. Tirabrutinib irreversibly and covalently binds to BTK in B cells and has demonstrated effective in vitro cytotoxicity in many types of B-cell malignancies and in vivo antitumor activity in mouse models. Here, we provide a comprehensive review of the preclinical and clinical activity of tirabrutinib, a drug approved in Japan for relapsed or refractory primary central nervous system lymphoma and all lines of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

Modelling Single Cell B-Cell Receptor Signaling Reveals Enhanced Activity in Primary CLL Cells Compared to Non-Malignant Cells While Fundamental Network Circuit Topology Remains Stable Even with Novel Therapeutic Inhibitors

B-cell receptor (BCR) signalling is central for the pathomechanism of chronic lymphocytic leukemia (CLL). Novel inhibitors of BCR signalling have recently substantially improved treatment of CLL, and a better characterization of the molecular circuitry of leukemic BCR signalling will allow a more refined targeting of this Achilles heel. In order to model malignant and non-malignant BCR signalling, we quantified after stimulation 5 components of BCR signaling (ZAP70/SYK, BTK, PLCy2, AKT, ERK1/2) in single cells from primary human leukemic and non-malignant tissue via phospho-specific flow cytometry over 6 time points. We stimulated cells from 11 patients and non-malignant CD19 negative enriched B-cells from 5 healthy donors by crosslinking the BCR with anti-IgM and/or anti-CD19 and synchronous inhibition of phosphatases with H2O2. As expected, we found more phosphorylation of all BCR signalling components after stimulation in malignant vs non-malignant cells and in IGHV non-mutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2O2 led to higher phosphorylation of BCR components in CLL cells with mutated IGHV genes compared to CLL cells with non-mutated IGHV genes, suggesting a stronger dampening of signalling activity in mutated IGHV CLL by phosphatases. In order to characterize the signalling circuitry, we modelled the connectivity of the cascade components by correlating signal intensities across single cells of the cell populations of single samples (Figure 1). Surprisingly, upon stimulation no substantial differences in network topology were observed between malignant and non-malignant cells. To additionally test for changes in network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib). Ibrutinib and idelalisib acted complementary, but not synergistic, and were similarly effective in IGHV mutated and non-mutated CLL. Effects of idelalisib were the same on malignant and non-malignant cells, whereas ibrutinib was mostly active on CLL cells, not on non-malignant B-cells. Upon stimulation with combinations of IgM and CD19 crosslinking augmented with H2O2, phosphorylation of PLCy2 could not be significantly inhibited by idelalisib or ibrutinib on a timescale of 28mins. We therefore aimed to identify central activating nodes of the BCR signalling cascade using targeted inhibitors. In fact, we found that inhibition of LYN with dasatinib and inhibition of SYK with entospletinib could substantially reduce phosphorylation of PLCy2, BTK and ERK but not AKT after all combinations of BCR stimulation. This suggests additional signalling cascades modulating AKT and a strong impact of SYK/LYN activity on the regulation of PLCy2. In summary, our findings underline the importance of single cell analysis of the dynamic circuitry of B-cell receptor signalling to understand development of resistance mechanisms and potential vulnerabilities. Figure 1: Workflow scheme of the Bayesian network learning and averaging approach. After discretizing the continuous single cell data, an optimal network is derived from each of R bootstrap samples. The Bayesian network learning strategy uses the BDe scoring function and a greedy hill-climbing algorithm to find the network model that represents the resampled data best. An average arc strength for each connection between nodes is derived from the number of occurrences of the respective connection in the set of R best scoring networks. Further averaging among networks derived from different data sets was applied for identifying conditional, temporal, and group-specific differences. Figure 1 Disclosures Döhner: Novartis: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; Bristol Myers Swuibb: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Arog: Research Funding; Seattle Genetics: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria. Stilgenbauer:GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Development of BTK inhibitors for the treatment of B-cell malignancies.

BTK is a key component of B-cell receptor signaling and functions as an important regulator of cell proliferation and survival in B-cell malignancies. The first-in-class BTK inhibitor ibrutinib is a small molecule drug that binds covalently to BTK and has been proved to be an effective treatment for various B-cell malignancies. However, it has off-target activities on non-BTK kinases that are related to side effects or might be translated into clinical limitations, with resistance to ibrutinib also reported. Much progress has been made in the development of more selective and second-generation BTK inhibitors. A recent shift in the mechanisms of action of BTK inhibitors is noteworthy, and novel inhibitors acting through noncovalent BTK inhibition are now being developed. This review describes key characteristics of ibrutinib, including current issues of its clinical use, and summarizes preclinical properties and clinical developments of second-generation BTK inhibitors for the treatment of B-cell malignancies. A review of novel noncovalent BTK inhibitors are also included.

Acalabrutinib in Patients with Relapsed/Refractory (R/R) and High-Risk, Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)

Background: Bruton tyrosine kinase (BTK) is a critical component of B-cell receptor signaling and a validated target for CLL. Acalabrutinib is a highly selective, potent, covalent BTK inhibitor, which has shown promising efficacy and safety in patients with CLL, including high-risk patients. We present preliminary efficacy, safety, and pharmacodynamic results from an ongoing single-center, open-label, phase 2 study of acalabrutinib monotherapy in patients with R/R and high-risk, TN CLL.Methods: Patients with R/R or high-risk (chromosome 17p deletion [del17p] or mutation in TP53 or NOTCH1) TN CLL/small lymphocytic lymphoma (SLL) who met International Workshop on Chronic Lymphocytic Leukemia (IWCLL) 2008 criteria for treatment and had an Eastern Cooperative Oncology Group performance status ≤2 were eligible. Patients who had prior BTK inhibitor therapy were excluded. Patients were randomized to receive oral acalabrutinib 100 mg twice daily (BID) or 200 mg daily (QD) until progressive disease or unacceptable toxicity. The primary endpoint was investigator-assessed overall response rate (ORR) by IWCLL 2008 criteria with modification for lymphocytosis. Secondary endpoints included safety and BTK occupancy. BTK occupancy was measured with a biotin-tagged analogue probe in peripheral blood cells at drug trough time points after 3 days of dosing and after 1, 6, and 12 mo of treatment. BTK occupancy in lymph node samples was measured at drug trough time points after 3 days of dosing.Results: Forty-six patients were enrolled and treated (100 mg BID, n=22; 200 mg QD, n=24). The median age was 64 years (range, 45-83), and 35% (16/46) were TN. Approximately 39% of patients (25% of TN) had bulky lymph nodes ≥5 cm, 37% (50% of TN) had Rai stage III-IV disease at baseline, 76% (88% of TN) had unmutatedIGHV, 21% (40% of TN) had del(17p), 21% (23% of TN) had TP53 mutation, and 47% (54% of TN) had NOTCH1 mutation. As of April 13, 2018, the median time on study for all treated patients was 20 mo (range 1-39), with 89% (41/46) remaining on acalabrutinib. Two patients (9%) in the BID group and 3 patients (13%) in the QD group discontinued treatment due to an adverse event (AE; n=1), progressive disease (n=1), and other reasons (n=3). The patient who discontinued due to progressive disease (BID group) achieved partial response at 2 mo and developed Richter transformation at 6 mo.The ORR was 90% (95% CI: 76, 97) for efficacy evaluable patients (N=39), defined per protocol as patients who had ≥ 6 mo of acalabrutinib (Table). ORR was 95% (75, 100) and 84% (60, 97) for the BID and QD group, respectively. For the intent-to-treat population (N=46), ORR was 80% (66, 91). Most AEs were grade 1/2 and did not require dose delays or modifications. The most common AEs (all grades; >25%) were headache (63%), contusion (50%), diarrhea (43%), upper respiratory tract infection (43%), arthralgia (33%), influenza-like illness (28%), maculo-papular rash (28%), myalgia (26%), and nausea (26%). Grade 3/4 AEs occurred in 33% (15/46) of patients (BID, 27% [6/22]; QD, 38% [9/24]), most commonly (>10%) infections (13%; urinary tract infection, lung infection, hepatitis B reactivation, which led to treatment discontinuation and fatal hepatic failure after 10 mo of treatment, and an invasive pulmonary aspergillosis at 2 mo in the setting of prolonged neutropenia and recent systemic corticosteroid use that led to treatment discontinuation) and neutropenia (11%). Approximately 33% (15/46) of patients (BID, 23% [5/22]; QD, 42% [10/24]) reported serious AEs (all grades), most commonly (>5%) lung infection (7%). No atrial fibrillation was reported, and one grade 1 atrial flutter occurred (BID).On day 4 of cycle 1, median trough BTK occupancy was significantly higher for the BID group versus the QD group in the peripheral blood (95% vs 87%; P<0.001) and in the lymph node (98% vs 90%, P<0.001). Median trough BTK occupancy in the peripheral blood was also higher for the BID group at 1, 6, and 12 mo (range, 98%-99% for BID vs 95%-97% for QD; P<0.05 at all time points).Conclusion: Acalabrutinib monotherapy produced high ORR in R/R and high-risk TN CLL, with an acceptable safety profile. The study was not designed to detect a statistically significant difference in clinical outcomes between the dosing groups. Near complete target coverage (>95%) was more rapidly achieved with 100 mg BID than 200 mg QD dosing in the lymph node and peripheral blood. [Display omitted] DisclosuresNierman:National Institutes of Health: Employment. Covey:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: various patents for ACP-196. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

MYD88 Triggered SYK Activation Promotes BCR Cross-Talk, and Identifies SYK As a Novel Therapeutic Target of Mutated MYD88 Signaling

Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

Targeting Bruton's tyrosine kinase for the treatment of B cell associated malignancies and autoimmune diseases: Preclinical and clinical developments of small molecule inhibitors.

B cell receptor (BCR) signaling plays a key role in B cell development and function. Aberrant BCR signaling has been confirmed as a central driver for the pathogenesis of various B cell malignancies. Bruton's tyrosine kinase (BTK) is a vital component of BCR signaling and exhibits overexpression in various B cell leukemias and lymphomas. Inhibiting BTK has been proved as an efficient way for B cell malignancy intervention. Remarkable achievements have been made in the pursuit of selective BTK inhibitors, represented by the success of the irreversible BTK inhibitors, ibrutinib and acalabrutinib. Constantly emerging agents exhibiting superior efficacy and safety in preclinical and clinical studies provide promising therapeutics for the treatment of B cell malignancies.

Molecular genetics of aggressive B-cell lymphoma.

Molecular genetics of aggressive B-cell lymphoma.

Single-Agent Ibrutinib Demonstrates Efficacy and Safety in Patients with Relapsed/Refractory Marginal Zone Lymphoma: A Multicenter, Open-Label, Phase 2 Study

Single-Agent Ibrutinib Demonstrates Efficacy and Safety in Patients with Relapsed/Refractory Marginal Zone Lymphoma: A Multicenter, Open-Label, Phase 2 Study

In vitro and in vivo study of the role of the mitochondria - shaping protein opa1 in cancer

In vitro and in vivo study of the role of the mitochondria - shaping protein opa1 in cancer

FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas

Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas.Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients.Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p<0.0001). Since in previous studies expression of the proapoptotic member of BCL2 family, HRK, was required for SYK-inhibitor induced cell death in DLBCL cells, we determined the role of FOXO1 activation in HRK expression. HRK expression was dramatically increased in SYK-inhibitor treated control cells, but not in FOXO1-deficient cells. Consistent with this, SYK inhibitor triggered cleavage of HRK transcriptional repressor DREAM only in control cells, but not in FOXO1-depleted cells. HRK induction was blocked by caspase inhibitors. Since AKT is major kinase regulating both FOXO1 activity and HRK induction, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic toxicity (combination index [CI] 0.5-0.8). Combination of inhibitors in FOXO1-depleted cells did not trigger cell death, highlighting the critical effector role of FOXO1. FOXO1 expression was present in 80% of primary DLBCL samples and correlated with SYK activity (p=0.009). High levels of FOXO1 protein expression were associated with longer OS (log rank, p=0.04).Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK. DisclosuresNo relevant conflicts of interest to declare.

Abstract IA09: Therapy of lymphoma inspired by functional and structural genomics

Abstract Diffuse large B cell lymphoma (DLBCL) is a heterogeneous diagnostic category that is comprised of two prominent molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB). These DLBCL subtypes are now viewed as molecularly distinct diseases since they arise from distinct stages of normal B cell development, require distinct recurrent genetic abnormalities to become malignant, have distinct cure rates with current chemotherapy regimens, and respond differentially to targeting agents. We defined a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as the kinase BTK or components of the BCR itself. Over one fifth of ABC DLBCLs have mutations affecting the CD79B or CD79A subunits of the BCR that augment BCR signaling. To attack chronic active BCR signaling therapeutically, we initiated clinical trials in relapsed/refractory DLBCL of ibrutinib, an irreversible and highly selective inhibitor of BTK. Ibrutinib monotherapy induced a high rate of complete and partial responses in ABC DLBCL, while GCB DLBCL tumors rarely responded. Genetic analysis of responding tumors demonstrated an enrichment for those with CD79B mutations, but most responses were observed in tumors with wild type BCR subunits. This observation allowed us to define a non-genetic mechanism of BCR activation that depends on reactivity of the lymphoma BCR to self antigens. In addition to chronic active BCR signaling, the signaling adapter MyD88 is activated by oncogenic mutations in 40% of ABC DLBCL cases. Since these mutant MyD88 isoforms act in parallel to BCR signaling to activate the NF-kB pathway in ABC DLBCL, they might have been predicted to mitigate the clinical effects of ibrutinib. On the contrary, ABC DLBCL tumors with both CD79B and MYD88 mutations were highly responsive to ibrutinib, leading us to demonstrate that the BCR and MyD88 pathways positively regulate one another. To extend the efficacy of ibrutinib, we have identified additional therapeutic targets in the oncogenic signaling pathways in ABC DLBCL including: 1) ubiquitin ligases, such as LUBAC, that are required to assemble active signaling complexes downstream of the BCR; 2) the kinase IRAK4, which mediates oncogenic MyD88 signaling; 3) the PI3 kinase pathway, which is activated by chronic active BCR signaling; 4) BCL2, which is expressed highly in most ABC DLBCL tumors and is the target of recurrent amplifications. Several synergistic, mechanism-based drug combinations that exploit these redundant survival pathways will be presented. Citation Format: Louis M. Staudt. Therapy of lymphoma inspired by functional and structural genomics. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr IA09.

Abstract IA38: Identification of targetable vulnerabilities in lymphoma.

Abstract Diffuse large B- cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma in adults and a clinically and genetically heterogeneous disorder. With current therapy, over 60% of patients with DLBCL can be cured; however, the remaining patients succumb to their disease. DLBCL largely originates in germinal center (GC) B cells, which have high growth rates and increased genomic instability. As a consequence, DLBCLs exhibit multiple low frequency genetic alterations including chromosomal translocations, somatic mutations and copy number alterations (CNAs). We have used several complementary comprehensive strategies to characterize the molecular heterogeneity of DLBCL and identify associated rational therapeutic targets. Using a combination of high-density – SNP arrays, whole exome sequencing, gene expression profiling and pathway analyses, we have comprehensively defined CNAs, candidate driver genes and perturbed signaling pathways in large series of newly diagnosed DLBCLs. The approach revealed a complementary set of CNAs and somatic mutations that decreased p53 activity and perturbed cell cycle regulation. The genetic signature of p53 deficiency and cell cycle deregulation was highly predictive for unfavorable outcome and potentially amenable to targeted therapy. We previously applied consensus clustering methods to the transcriptional profiles of primary DLBCLs and identified 3 highly reproducible tumor groups: BCR (B-cell receptor), OxPhos (oxidative phosphorylation) and HR (host response). “BCR” DLBCLs have increased expression of multiple components of the BCR signaling pathway and increased reliance on BCR-mediated survival signals. We recently characterized distinct BCR/SYK/PI3K-dependent survival pathways in DLBCLs with high or low baseline NF-κB activity and defined specific survival mechanisms in BCR-dependent NF-κB low DLBCLs. In these studies, SYK/PI3K/AKT-dependent cholesterol biosynthesis was found to be a feed-forward mechanism of preserving the integrity of BCRs in lipid rafts in DLBCLs with low- or high-baseline NF-κB. Consistent with the roles of SYK and PI3K/AKT as important mediators of BCR survival signals in primary DLBCLs, SYK amplification and PTEN deletion were identified as selective and largely mutually exclusive genetic alterations in “BCR”-type tumors. These studies suggest potential strategies for specifically targeting BCR-dependent survival signals in defined subtypes of DLBCL. In additional studies, we found that predominant fuel utilization pathways in BCR and OxPhos DLBCLs differ. Whereas BCR DLBCLs primarily utilize aerobic glycolysis, OxPhos DLBCLs largely depend upon fatty acid oxidation and exhibit selective sensitivity to targeted inhibition of this fuel utilization pathway. These findings further define molecular subsets of DLBCLs with distinct metabolic features that are linked to the functional state of upstream BCR signaling components. Citation Format: Margaret Shipp. Identification of targetable vulnerabilities in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA38.

B-cell receptor signaling in the pathogenesis of lymphoid malignancies.

B-cell receptor (BCR) signaling pathway plays a central role in B-lymphocyte development and initiation of humoral immunity. Recently, BCR signaling pathway has been shown as a major driver in the pathogenesis of B-cell malignancies. As a result, a vast array of BCR-associated kinases has emerged as rational therapeutic targets changing treatment paradigms in B cell malignancies. Based on high efficacy in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR signaling pathway. Here, we describe the essential components of BCR signaling, their function in normal and pathogenic signaling and molecular effects of their inhibition in vitro and in vivo.