Bruton's Agammaglobulinemia Tyrosine Kinase Research Articles

B-cells absence in patients diagnosed as inborn errors of immunity: a registry-based study.

Hypogammaglobulinemia without B-cells is a subgroup of inborn errors of immunity (IEI) which is characterized by a significant decline in all serum immunoglobulin isotypes, coupled with a pronounced reduction or absence of B-cells. Approximately 80 to 90% of individuals exhibit genetic variations in Bruton's agammaglobulinemia tyrosine kinase (BTK), whereas a minority of cases, around 5-10%, are autosomal recessive agammaglobulinemia (ARA). Very few cases are grouped into distinct subcategories. We evaluated phenotypically and genetically 27 patients from 13 distinct families with hypogammaglobinemia and no B-cells. Genetic analysis was performed via whole-exome and Sanger sequencing. The most prevalent genetic cause was mutations in BTK. Three novel mutations in the BTK gene include c.115T > C (p. Tyr39His), c.685-686insTTAC (p.Asn229llefs5), and c.163delT (p.Ser55GlnfsTer2). Our three ARA patients include a novel homozygous stop-gain mutation in the immunoglobulin heavy constant Mu chain (IGHM) gene, a novel frameshift mutation of the B-cell antigen receptor complex-associated protein (CD79A) gene, a novel bi-allelic stop-gain mutation in the transcription factor 3 (TCF3) gene. Three patients with agammaglobulinemia have an autosomal dominant inheritance pattern,which includesa missense variant in PIK3CD, a novel missense variant in PIK3R1 and a homozygous silent mutation in the phosphoinositide-3-kinase regulatory subunit (RASGRP1) gene. This study broadens the genetic spectrum of hypogammaglobulinemia without B-cells and presented a few novel variants within the Iranian community, which may also have implications in other Middle Eastern populations. Notably, disease control was better in the second affected family member in families with multiple cases.

Inhibition of Serum- and Glucocorticoid-Regulated Protein Kinase-1 Aggravates Imiquimod-Induced Psoriatic Dermatitis and Enhances Proinflammatory Cytokine Expression through the NF-kB Pathway

Although the anti-inflammatory effect of serum- and glucocorticoid-regulated protein kinase 1 (SGK1) has been established in other diseases, the possible regulatory role of SGK1 in psoriasis and the underlying molecular mechanisms remain largely unknown. In this study, we found that SGK1 expression was decreased in macrophages from patients with psoriasis. Moreover, a specific pharmacological SGK1 inhibitor, EMD638683, significantly enhanced imiquimod-mediated toll-like receptor 7/8 activity and proinflammatory cytokine production in RAW264.7 cells, and this result was confirmed by Sgk1 small interfering RNA. Further mechanistic data showed that SGK1 inhibition increased the phosphorylation of Bruton's agammaglobulinemia tyrosine kinase; moreover, Bruton's agammaglobulinemia tyrosine kinase inhibition abrogated the proinflammatory effects of the SGK1 inhibitor on toll-like receptor 7/8 activation, thereby validating that SGK1 inhibition enhances the toll-like receptor 7/8 pathway by increasing Bruton's agammaglobulinemia tyrosine kinase phosphorylation. In addition, our invivo results showed that SGK1 inhibition significantly increased the secretion of proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, and the infiltration of T helper 17 cells in an imiquimod-induced psoriasis mouse model. Altogether, these results show that SGK1 plays a critical role in the pathogenesis of psoriasis by modulating inflammatory responses in skin lesions, indicating that SGK1‒Bruton's agammaglobulinemia tyrosine kinase signaling could be a novel therapeutic target for the control of psoriasis.

Bruton's agammaglobulinemia tyrosine kinase (Btk) regulates TPA‑induced breast cancer cell invasion via PLCγ2/PKCβ/NF‑κB/AP‑1‑dependent matrix metalloproteinase‑9 activation

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B-lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen-activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor-κB (NF-κB) activation, suggesting an anti-metastatic effect of BTK inhibitors on MCF-7 cells that leads to the downregulation of matrix metalloproteinase (MMP)-9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti-metastatic activity of BTK inhibitors was examined in MCF-7 cells focusing on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MCF-7 cells. The expression and activity of MMP-9 in MCF-7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX-774 (10 µM)] significantly attenuated TPA-induced cell invasion and migration in MCF-7 cells and inhibited the activation of the phospholipase Cγ2/PKCβ signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP-9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA-induced MMP-9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF-κB/activator protein-1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP-9 expression in MCF-7 cells.

Abstract 1207: SNS-062 demonstrates efficacy in chronic lymphocytic leukemia in vitro and inhibits C481S mutated Bruton tyrosine kinase

Abstract Introduction: In order to address the issue of acquired resistance to ibrutinib, we sought to characterize the Bruton agammaglobulinemia tyrosine kinase (BTK) inhibitor SNS-062 in preclinical models of chronic lymphocytic leukemia (CLL). Methods: Primary CLL B cells were isolated from the whole blood of consented patients by ficoll density centrifugation and Rosette-Sep negative selection. Annexin V and propidium iodide flow cytometry was used to measure patient CLL cell viability and 7-AAD was used to measure viability in stromal co-culture. CD40 and CD86 expression was evaluated via flow cytometry subsequent to sustained 3.2uM CpG stimulation. BCR signaling in primary CLL cells was investigated by immunoblot following 1 hour treatment and following 1 hour or 24 hours of incubation with SNS-062 in XLA cell lines. ITK inhibition was investigated via immunoblot after stimulation with anti-CD3 and anti-CD28 and incubation with SNS-062 for 1 hour. SNS-062 was used at a concentration of 1uM in preclinical studies unless otherwise noted. Measurement of kinase activity in human recombinant WT BTK or C481S BTK was performed in a FRET kinase assay. Results: Immunoblots of BTK and ERK phosphorylation of XLA cells transfected with WT or C481S BTK demonstrated that SNS-062 inhibition is comparable to that of ibrutinib in WT BTK and greater than that of ibrutinib in C481S BTK. Using a recombinant kinase assay, we found the IC50 of SNS-062 against WT BTK to be 4.6nM and C481S BTK to be 1.1nM, suggesting that SNS-062 retains activity against the mutated BTK variant. Additionally, SNS-062 was found to be six times more potent than ibrutinib and greater than 640 times more potent than acalabrutinib against C481S BTK. SNS-062 demonstrates dose-dependent inhibition of BTK in primary patient CLL cells comparable to ibrutinib via immunoblot for BTK phosphorylation. The viability of primary patient cells treated with 0.1uM, 1.0uM, and 10.0uM SNS-062 for 48 hours was measured to be 96.7%, 96.1%, and 88.1%, respectively, that of the untreated condition. At 48 hours, SNS-062 decreased viability of primary CLL cells in the presence of HS5 stromal protection by 5.5%. SNS-062 was found to decrease CpG induced CD40 and CD86 expression by 8.7% and 15.7%, respectively. Using an in vitro kinase assay, SNS-062 inhibited ITK with an IC50 value of 24nM. An immunoblot of anti-CD3/CD28 stimulated Jurkat cells revealed that SNS-062 decreased the phosphorylation of ERK, implying inhibition of ITK. Conclusion: Unlike ibrutinib, SNS-062 inhibits BTK signaling in the presence of the C481S mutation and may address acquired resistance to covalent BTK inhibitors. SNS-062 decreases B cell activation markers, viability, and stromal cell protection in primary patient CLL cells and was shown to inhibit ITK, suggesting support of T cell mediated antitumor activities. These data support further investigation of this molecule and advancement into clinical trials. Citation Format: Catherine A. Fabian, Sean D. Reiff, Daphne Guinn, Linda Neuman, Judith A. Fox, Wendy Wilson, John C. Byrd, Jennifer A. Woyach, Amy J. Johnson. SNS-062 demonstrates efficacy in chronic lymphocytic leukemia in vitro and inhibits C481S mutated Bruton tyrosine kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2017-1207

Abstract 3150: PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib

Abstract Background: The Bruton agammaglobulinemia tyrosine kinase (BTK) activates B-cell receptor signaling through activation of phospholipase C gamma 2 (PLCG2). Clinical resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) is highly associated with emergence of the BTK C481 mutations that prevent ibrutinib covalent binding. PLCG2 mutations also occur in these ibrutinib-resistant samples but the spectrum of mutations and their occurrence with BTK changes have not been fully delineated. Materials and Methods: All peripheral blood samples with adequate depth of sequencing coverage were included from CLL patients receiving ibrutinib (with or without other therapies) that were submitted from Ohio State University (OSU) to the OSU James Polaris Molecular Laboratory. Genomic DNA was extracted from negatively selected B cells and deep sequencing of the entire coding regions of BTK and PLCG2 performed using a custom Ion Torrent Ampliseq panel. A mean depth of greater than 1000X was obtained with hotspot mutations validated down to 1% variant allele fraction (VAF) in the B cell preparations using orthogonal mutation-specific detection methods. Results: Among 1063 CLL samples from 380 patients who received ibrutinib, BTK C481 resistance mutations were identified in 79 (20.8%) patients including 20 patients that also had co-occurring PLCG2 mutations. 11 patients (2.9%) had PLCG2 mutations without accompanying BTK C481 alterations for a cumulative incidence of PLCG2 mutations in 8.2% of ibrutinib-treated patients. These included previously described mutations in the SH2 and SH3 domain of PLCG2 (R665W, S707F, A708P and L845F) but also previously uncharacterized mutations in the PLCG2 C2 domain that were seen in 12 patients (3.2%). C2 domain mutations, always seen in association with another PLCG2 and/or BTK resistance mutation, affected codons 1140-1144 that include the highly conserved aspartic acid residues that bind calcium and mediate membrane localization in other C2-domain containing proteins. In sequential samples, PLCG2 C2-domain mutations tracked at similar levels to the co-occurring BTK and PLCG2 resistance mutations indicating their presence in the same population of CLL cells. Conclusions: Mutations in three different PLCG2 structural domains commonly co-occur with BTK C481 mutations. The identification of PLCG2 mutations in the calcium-regulated C2 domain expands the possible mechanisms that can produce PLCG2 activation following ibrutinib treatment. The diversity of recurrent mutations observed supports the need for complete PLCG2 sequencing for full characterization of ibrutinib-treated CLL samples. Citation Format: Dan Jones, Jennifer A. Woyach, Weiqiang Zhao, Sean Caruthers, Huolin Tu, Joshua Coleman, John C. Byrd, Amy J. Johnson, Gerard Lozanski. PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2017-3150

Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib in Canine Models of B-Cell Non-Hodgkin Lymphoma

Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In this study, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy similar to human diffuse large B-cell lymphoma (DLBCL). First, we demonstrated that acalabrutinib potently inhibited BTK activity and downstream effectors in CLBL1, a canine B-cell lymphoma cell line, and primary canine lymphoma cells. Acalabrutinib also inhibited proliferation in CLBL1 cells. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).

WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.

The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca(2+) oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption.

Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

AbstractIbrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib's capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance.

BTK mutations selectively regulate BTK expression and upregulate monocyte XBP1 mRNA in XLA patients.

Mutations in the Bruton agammaglobulinemia tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). Unfolded or misfolded proteins can trigger stress pathways in the endoplasmic reticulum (ER), known as unfolded protein response (UPR). The aim was to clarify the involvement of UPR in XLA pathophysiology. By reverse transcription-quantitative PCR, we evaluated the expression of BTK and 12 UPR-related genes in eight patients. Moreover, we assessed the BTK protein expression and pattern in the patients' monocytes by flow cytometry and fluorescence immunocytochemistry. We found a reduced BTK expression in patients with stop codon mutations (P < 0.02). However, missense mutations did not affect BTK expression. Flow cytometry showed a reduction of BTK in patients which was corroborated by an absent or nonfunctional protein synthesis revealed by immunocytochemistry. In contrast with the other UPR-related genes, X-box binding protein 1 (XBP1) was markedly upregulated in the patients (P < 0.01), suggesting Toll-like receptor (TLR) activation since BTK directly interacts with TLRs as a negative regulator and XBP1 can be activated in direct response to TLR ligation. Different BTK mutations can be identified by the BTK expression. Inasmuch as UPR-related genes were downregulated or unaltered in patients, we speculate the involvement of the TLRs-XBP1 axis in the XLA pathophysiology. Such data could be the basis for further studies of this novel pathomechanism concerning XLA.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia

AbstractDespite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity

Bruton agammaglobulinemia tyrosine kinase (BTK), a cytoplasmic protein tyrosine kinase, is a component of the B-cell receptor signaling pathway. Ibrutinib, a BTK inhibitor, has demonstrated a significant clinical activity against chronic lymphocytic leukemia (CLL) in early clinical trials. Understanding the molecular mechanisms of action would shed light on CLL pathophysiology and provide additional opportunities for the development of new therapies. In this study, we have chosen an in vivo approach by employing an ongoing phase 1b trial of ibrutinib. We prospectively collected and analyzed serial samples from the CLL patients before and after the initiation of ibrutinib. We found that the blockage of cell proliferation was one of the primary effects of ibrutinib against leukemic CLL cells in vivo. Using a co-culture system that induces CLL proliferation in vitro, analysis of several parameters, including Ki-67 expression and bromodeoxyuridine (BrdU) incorporation, revealed that the proliferation of CLL cells was directly inhibited by ibrutinib. Furthermore, activities of BTK and phospholipase Cγ2 as well as downstream signaling molecules, AKT and ERK, were all coordinately downregulated over time in ibrutinib-treated patients. Our findings suggest that the cell proliferation is one of the essential properties of CLL. Blocking cell proliferation via inhibition of BTK-mediated signaling may contribute to clinical responses in ibrutinib-treated patients.

The Potent PI3K-δ,γ Inhibitor IPI-145 Exhibits Differential Activity In Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). Despite aggressive chemoimmunotherapy, ∼40% of patients die of their disease. Molecular differences in the tumor genome are likely to be major contributors to the heterogeneity of clinical response. Several investigators have identified molecular subtypes, including a “cell-of-origin” (COO) signature with activated B-cell-like (ABC) and germinal center B-cell-like (GCB) sub-types (Alizadeh, et al. Nature 2000), and a “consensus clustering” (CC) signature with oxidative phosphorylation, B-cell receptor (BCR)/proliferation, and host response sub-types (Monti, et al. 2005).Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node, relaying signals from cell surface receptors to downstream mediators such as AKT. PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they are critical for cell differentiation, migration, and proliferation. IPI-145 is a potent, oral PI3K-δ,γ inhibitor that has shown clinical activity in the Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies (ClinicalTrials.gov NCT01476657). Activation of the PI3K pathway is an important component of normal BCR signaling and has been implicated in the pathogenesis of DLBCL. To further explore the role of PI3K signaling in DLBCL, a panel of more than 10 DLBCL cell lines of varying molecular profile was treated with IPI-145.PI3K-δ and PI3K-γ were expressed at varying levels across the DLBCL cell lines, without evidence of a correlation with molecular subtype. In a cellular growth inhibition assay, 5 cell lines including 4 GCB (SU-DHL-4, SU-DHL-6, OCI-LY-8, and WSU-DLCL-2) and 1 ABC (Ri-1) subtype were sensitive to IPI-145 treatment in the micromolar (μM) range or below, and 5 cell lines (OCI-LY-3, OCI-LY-7, Pfeiffer, Toledo and U2932) were insensitive to IPI-145 (IC50 >25μM). There was no evidence of a correlation between IPI-145 sensitivity and COO or CC molecular profile in this panel. IPI-145 sensitivity did correlate with evidence of PI3K pathway inhibition as measured by reduction in phospho (p)-AKT. To characterize the kinetics of pathway modulation, phosphorylation of AKT, PRAS40, and S6 was examined following a time-course of IPI-145 treatment in selected cell lines. P-AKT and p-PRAS40 were modulated by 30 minutes, whereas modulation of p-S6 was not detected until after 8 hours. Upon BCR stimulation via antibody-induced crosslinking, some cell lines exhibited enhanced AKT phosphorylation, which was inhibited with IPI-145. The GCB cell line OCI-LY-8 was moderately sensitive to IPI-145 without BCR crosslinking (low μM range) and exhibited enhanced sensitivity to IPI-145 with BCR crosslinking (sub-μM range). These results suggest that intact BCR pathway signaling contributes to IPI-145 sensitivity in DLBCL cell lines. Current efforts are focusing on identification of biomarkers that define IPI-145 responsive DLBCL subsets.IPI-145 activity was also explored in combination with ibrutinib, an irreversible inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK). Interestingly, in the setting of BCR crosslinking, OCI-LY-8 cells exhibited a robust increase in p-AKT which was completely inhibited by IPI-145 but only partially inhibited by ibrutinib. In other cell lines, such as SU-DHL-4, robust inhibition of p-BTK was observed with ibrutinib treatment but not with IPI-145. These findings suggest a potential mechanistic rationale for combination of PI3K-δ,γ and BTK inhibition. Supporting this hypothesis, a combination effect was observed in a cellular growth inhibition assay with IPI-145 plus ibrutinib in the SU-DHL-4 cell line and in the OCI-LY-8 cell line with BCR crosslinking. Studies are ongoing to explore this combination in additional DLBCL cell lines as well as in in vivo DLBCL models.Taken together, these findings suggest that a subset of DLBCL patients might benefit fromPI3K-δ,γ inhibition. Additional work is necessary to determine how to prospectively identify IPI-145-sensitive DLBCL. In addition, preliminary evidence suggests that there may be opportunities to improve targeted therapy options for DLBCL patients with the combination of IPI-145 and ibrutinib. Disclosures:Campbell:Infinity Pharmaceuticals, Inc.: Employment. Thompson:Infinity Pharmaceuticals, Inc.: Employment. Villegas:Infinity Pharmaceuticals, Inc.: Employment. Proctor:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. Stern:Infinity Pharmaceuticals, Inc.: Employment.

II. Therapy of DLBCL based on genomics

II. Therapy of DLBCL based on genomics

Abstract MP069: A Genetic Association Study of D-dimer Levels with 50K SNPs from a Candidate Gene Chip in Four Ethnic Groups: the Candidate Gene Association Resource (CARe) consortium

Introduction: D-dimer, a marker of blood coagulation and fibrinolysis, is a widely-used biomarker of venous thromboembolism. While the coagulation factors V, III, II and fibrinogen alpha chain genes contain known genetic determinants of D-dimer, other determinants are not well characterized, especially in populations other than European Americans (EAs); no data have been reported for Asian American populations. Hypothesis: Large-scale candidate gene association analysis will identify D-dimer related gene variants in EA, African-American (AA), Chinese, and Hispanic populations. Methods: Four cohorts, comprised of 6,848 EA, 2,192 AAs, 670 Chinese, and 1,286 Hispanics from Cleveland Family Study, Cardiovascular Health Study, Framingham Heart Study, and Multi-Ethnic Study of Atherosclerosis in the Candidate Gene Association Resource (CARe) consortium, were included. The inverse normal transformed residual of D-dimer was used in genetic analysis. About 50,000 SNPs located in 2,100 inflammation and atherosclerosis-related genes were genotyped using the custom ITMAT-Broad-CARe (IBC) chip and analyzed by linear regression adjusted for age, sex, study site, and principal components to account for potential population stratification in each cohort and race group. Significance was set at p&lt;2x10-6 after accounting for multiple testing. Results: Twelve SNPs in the coagulation factor V gene and 3 SNPs in the fibrinogen alpha chain genes exceeded the significant p-value threshold in EA. The top SNPs in these two regions were rs6025 (p-value=7.41x10-16) and rs13109457 (p-value=7.52x10-7), respectively. The signals for the remaining significant SNPs in these two regions were no longer statistically significant after adjusting for the known functional SNPs in each region (rs6025 and rs6050, respectively). The top signal at the coagulation factor V gene was replicated in Hispanics but not other race groups. Neither AA nor the other race groups replicated the signals for fibrinogen alpha chain gene. No additional variants in AA, Chinese, or Hispanics showed significant associations with D-dimer, but several SNPs in neuregulin 1 and Bruton agammaglobulinemia tyrosine kinase for AA and vanin 1 for Chinese were suggestively associated with D-dimer levels (p-value&lt;5x10-5). Conclusion: Our study replicated previously reported associations of D-dimer with coagulation factor V and fibrinogen alpha chain variants in EAs and identified coagulation factor V variation in Hispanics. Additional studies are required to confirm the suggestive findings in AAs, Chinese, and Hispanic populations.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

BTK (Bruton agammaglobulinemia tyrosine kinase)

Review on BTK (Bruton agammaglobulinemia tyrosine kinase), with data on DNA, on the protein encoded, and where the gene is implicated.

Dominant-Negative Impact of PAX5/TEL on Downstream Targets of PAX5 and Essential Pre-B Cell Receptor Genes

AbstractAbstract 3231B cell lineage acute lymphoblastic leukemia (ALL) is a common malignancy in childhood, and the pre-B cell receptor (pre-BCR) signalling pathway was previously demonstrated to function as a tumor suppressor. The transcription factor PAX5, a key regulator of B cell development, is frequently involved in chromosomal rearrangements of leukemic blasts. Using high resolution single nucleotide polymorphism (SNP) genomic microarray by us and other groups, several candidate partner genes fused to PAX5 have been detected in samples of pediatric ALL, such as TEL, FOXP1, AUTS2, and C20orf112. Recently, we studied the fusion gene PAX5/TEL and its role in leukemic evolution by Affymetrix HG-U133 plus 2.0 Array of the ALL cell line Nalm6 transfected with a PAX5/TEL construct. PAX5/TEL reduced the expression of PAX5 and its downstream target genes (e.g. CD79A, BACH2, CD19). Moreover, we demonstrated a dominant negative impact of the PAX5/TEL-fusion protein on the binding affinity of wild-type PAX5 to the promoter of CD79A (Iwanski et al., 2009 ASH Abstract No. 3455).To expand our findings, we analyzed the gene expression profile of pediatric ALL samples carrying PAX5/TEL (PAX5/TEL+, n=2) compared to samples with normal PAX5 (n=7) from a genomic ALL study. Samples with normal PAX5 were selected from among 95 B-ALL patients with normal PAX5 status, based on characteristics that most closely matched the two PAX5-TEL+ patients including cytogenetics and current risk stratification. Gene expression data were compiled using the Affymetrix HG-U133A Array, and a heatmap based on the Top 200 probes with the highest expression levels from both sample sets was generated (TIBCO Software Inc.). Notably, the downregulated genes included Bruton agammaglobulinemia tyrosine kinase (BTK; -2.8 fold, FDR < 0.2), an important regulator of pre-BCR signaling, Spleen tyrosine kinase (SYK, -2.3 fold, FDR < 0.2), and IGHM (-5,9 fold; FDR < 0.1), but also significantly up-regulated expression of genes involved in myeloid differentiation, namely Myeloperoxidase (MPO, +24.2 fold, FDR < 0.2), and CCAAT/enhancer binding protein alpha (CEBPA, +3.2 fold, FDR < 0.3), as well as the erythroid genes Aminolevulinate delta-dehydratase (ALAD, +12.2 fold, FDR < 0.2) and the Erythropoietin receptor (EPOR, +7.0, FDR < 0.3).Additionally, we performed a meta-analysis comparing deregulated genes detected in the Nalm6-microarray (PAX5/TEL-MIGR vs. empty vector) to the data from the human B-ALL samples (PAX5/TEL+ vs. normal PAX5). Overall, we identified a set of 35 overlapping genes (FDR < 0.3) that were deregulated in both data sets (21 downregulated, 14 up-regulated). Notably, 10 out of the 21 (47%) downregulated genes are known to be involved in B cell development and BCR signaling, some of them well-recognized as direct PAX5 target genes (e.g. CD79A, CD19, BACH2).Moreover, reporter gene assay with a luciferase reporter construct containing cDNA of the CD19 promoter with PAX5 binding sites (luc-CD19) was performed in Nalm6 cells. Since these cells already express a high level of endogenous PAX5, transcriptional activity of the luc-CD19 reporter plasmid was relatively high in the Nalm6 cells transfected with empty vector, as compared to 293T cells. In contrast, PAX5/TEL-transduced Nalm6 cells displayed a significantly reduced transcriptional activation of the reporter construct (P < 0.01).We also explored if mutation and/or deletion of PAX5 (mut/del PAX5) may have an impact on genes involved in B cell development and the pre-BCR/BCR pathway. Hence, expression files from human ALL samples with mut/del PAX5 (n=50) and B-ALL samples with normal PAX5 (n=95) were analyzed. Notably, only two genes that are known to be involved in B cell development and the pre-BCR pathway were significantly downregulated in samples with mut/del PAX5 compared to normal PAX5, namely CD72, a B cell specific repressor gene activated by PAX5 (-1.51 mean fold, FDR = 0.05), and immunoglobulin heavy constant delta (IGHD), a gene involved in pre-BCR signalling (-1.6 mean fold, FDR = 0.18). These findings suggest no strong influence of mut/del PAX5 on the expression of downstream genes involved in pre-BCR signaling.In conclusion, our results provide further insights into the dominant-negative role of PAX5/TEL and link this fusion gene with the pre-BCR pathway. Disclosures:No relevant conflicts of interest to declare.

Abstract 1613: BTK as a drug target for cancers that harbor inactivating mutations in the DPC4 gene

Abstract The Deleted in Pancreatic Cancer locus 4 (DPC4) is a tumor suppressor gene that is often inactivated in pancreatic and colorectal cancers. In pancreatic adenocarcinomas, the frequency of inactivation of DPC4 is approximately 55%, while in colorectal carcinomas has been reported to be 10% to 35%. The loss of DPC4 has been shown to be associated with the progression and malignancy of pancreatic cancer along with decreased patient survival. We employed a small interfering (siRNA) library based screening strategy to identify potential synthetic lethal partners of the DPC4 gene. We used a kinase focused siRNA library that consisted of two siRNA oligonucleotides for each of the 624 protein kinase genes. A DPC4 isogenic pair, BxPC3-Vector and BxPC3-DPC4 (Wang et al. 2006) were treated by the siRNA oligonucleotides in parallel and the effects of the siRNA oligonucleotides on the growth of the cell lines were then compared. siRNA oligonucleotides that selectively inhibited the cell growth of the BxPC3-Vector cell line were selected as potential positive hits. These genes, once validated, represented potential drug targets that are very specific to cancer cells harboring mutations in the DPC4 gene. One of the top-ranked hits we identified from this screening is the Bruton agammaglobulinemia tyrosine kinase (BTK). With BTK siRNA treatment, BxPC3-Vector cells, which are DPC4 null, showed ∼30% more cell growth inhibition than the BxPC3-DPC4 cells, which are DPC4 wildtype. This selectivity of BTK siRNA against DPC4 null cells was further confirmed in a confirmation screen using two pancreatic cancer cell lines, BxPC3 and PANC-1, which are DPC4 null and DPC4 wildtype, respectively. BTK siRNA also showed significant selectivity against DPC4 deficiency in another pair of DPC4 isogenic colon cancer cell lines, HCT-116 and HCT-116-DPC4-knockout. To validate the BTK siRNA selectivity findings, we evaluated the BTK inhibitor PCI-32765 in our cancer cell line models and observed the same selectively against DPC4 null pancreatic and colon cancer cell lines. These results indicate that BTK is a potential molecular target for pancreas, colon, and other cancers harboring inactivating mutations in the DPC4 gene. (The work was supported by a NCI grant P01CA109552.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1613.

MODELING and Targeting ERG-INDUCED Acute MYELOID LEUKEMIA (AML).

Abstract 475ERG is an oncogene located on the long arm of human chromosome 21 that encodes an ETS transcription factor that has been reported to be involved in normal and aberrant megakaryopoiesis (Salek-Ardakani et al Cancer Res. 2009;69:4665; Stankiewicz et al Blood 2009;113:3337). High ERG expression has also been reported associated with poor prognosis of cytogenetically normal AML (Marcucci et al J Clin Oncol. 2007;25:3337). Thus, deciphering the molecular targets and oncogenic pathways activated by overexpressed ERG protein is likely to lead to more effective therapy for ERG-related myeloid leukemias. Towards these goals we have combined in-vitro and in-vivo approaches. We have created transgenic mice expressing the ERG3 hematopoietic isoform under the VAV promoter. All these mice die from invasive acute megakaryocytic or undifferentiated myeloid leukemia by the age of 5 months. The leukemias are transplantable, and primary growth factor dependent leukemic cell lines, suitable for pharmacological and molecular studies, have been established. To identify ERG target genes and proteins, we have analyzed gene expression in two “mirror-image” cellular systems – shRNA mediated knockdown of ERG in Meg01 megakaryocytic leukemia cells and overexpression of ERG in K562 erythroleukemia cells. Gene Set Enrichment Analysis (GSEA) demonstrated that the “ERG gene-associated expression signature” in these cell lines is enriched with genes that were also identified in primary human AML characterized by ERG overexpression. By chromatin immunoprecipitation, we then identified specific, direct ERG targets and confirmed their expression in samples from ERG transgenic leukemias and primary human “ERG-overexpressing” AMLs. Surprisingly, we observed that the lymphoid kinase Bruton agammaglobulinemia tyrosine kinase (BTK) is an ERG direct target that is upregulated in both ERG overexpressing mouse and human leukemias. Preliminary experiments have demonstrated activity of a BTK inhibitor on ERG transgenic and ERG overexpressing human AML cells. ERG overexpression also induced marked activation of RAS signaling as supported by elevated levels of RAS-GTP and its downstream targets and significant growth inhibitory activity caused by a novel RAS inhibitor, farnesylthiosalicylic acid (Salirasib), that disrupts the spatiotemporal localization of active Ras (Rotblat et al Methods Enzymol. 2008;439:467) was observed. Dramatic growth inhibitory activity has also been induced by two novel compounds that induce megakaryocytic polyploidization, suggesting a potential role for “differentiation” therapy of ERG-related leukemias. Thus we have created a mouse model for ERG-related AML and characterized several genes and biochemical pathways that are susceptible to pharmacological inhibition. Our studies are not only likely to decipher the mechanisms by which ERG overexpression contributes to leukemogenesis, but also provide a platform for preclinical studies of novel therapeutics of these poor prognosis leukemias. Disclosures:No relevant conflicts of interest to declare.

IL-5- and eosinophil-mediated inflammation: from discovery to therapy

IL-5 was originally defined as a T-cell-derived cytokine that triggers activated B cells for terminal differentiation into antibody-secreting plasma cells, at least in mice. Concurrently, IL-5 was recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Over-expression of IL-5 significantly increases eosinophil numbers and antibody levels in vivo. Conversely, mice lacking a functional gene for IL-5 or the IL-5 receptor alpha chain (IL-5Ralpha) display a number of developmental and functional impairments in B-cell and eosinophil lineages. In addition to the Janus kinase-signal transducer and activator of transcription pathway, the tyrosine kinases Lyn and Btk (Bruton agammaglobulinemia tyrosine kinase) are involved, and Ras GTPase-extracellular signal-regulated kinase (Ras-ERK) signals are important for IL-5-dependent cell proliferation and survival. IL-5 critically regulates expression of genes involved in proliferation, cell survival and maturation and effector functions of B cells and eosinophils. Thus, IL-5 plays a pivotal role in innate and acquired immune responses and eosinophilia. In humans, the biologic effects of IL-5 are best characterized for eosinophils. The recent expansion in our understanding of the mechanisms of eosinophil development and activation in the context of IL-5 has led to advances in therapeutic options. A new therapy currently in clinical trials uses humanized mAbs against IL-5 or the IL-5R.