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

Abstract 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. Disclosures Shipp: 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.

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Inhibition of B-Cell Receptor Signaling Disrupts Cell Adhesion in Mantle Cell Lymphoma Via RAC2

Blood ◽

10.1182/blood-2018-99-118598 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1369-1369

Author(s):

Weige Wang ◽

Franzen Carrie ◽

Hui Guo ◽

Jimmy Lee ◽

Yan Li ◽

...

Keyword(s):

Cell Adhesion ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Receptor ◽

Resistant Cell ◽

Advisory Committees ◽

Tumor Tissues ◽

Mantle Cell ◽

Bcr Signaling

Abstract Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib. Methods and materials: In an effort to elucidate the link between BCR signaling and cell adhesion phenotype, we first characterized ibrutinib sensitive and resistant mantle cell lymphoma (MCL) cell lines. We measured cell proliferation and cell growth, and correlated ibrutinib sensitivity with cell adhesion disruption. We then used RNA-sequencing to identify differential pathways between sensitive or resistant cell lines in response to ibrutinib treatment. We validated RNA-Seq findings using cell lines, as well as animal models and human primary MCL tumor tissues and cells. Results: We found that intrinsic sensitivities of MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion gene signatures were simultaneously down-regulated by ibrutinib in ibrutinib-sensitive but not ibrutinib-resistant cell lines. Among the differentially expressed genes in the BCR gene signature, we identified and validated that RAC2, a regulator of cell adhesion, was down-regulated at both RNA and protein levels by ibrutinib only in ibrutinib-sensitive cells. Physical association of RAC2 with BLNK, an early BCR pathway adaptor, was disrupted by ibrutinib uniquely in sensitive cells. RAC2 knockdown with siRNA impaired cell adhesion while RAC2 over-expression rescued ibrutinib-induced reduction in cell adhesion. In a xenograft mouse model, mice treated with ibrutinib demonstrated tumor growth retardation along with down-regulation in RAC2 protein expression. Using immunohistochemical staining, we demonstrated that RAC2 was expressed in ~65% primary MCL tumor tissues with majority of RAC2-positive tumors characterized as being the more aggressive subtypes. Finally, primary MCL cells treated with ibrutinib demonstrated reduced RAC2 that is accompanied by cell adhesion impairment. Conclusions: Our findings uncover a novel cross-talk between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC2. Our study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and new drug development. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Honoraria, Research Funding.

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SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma

Blood ◽

10.1182/blood-2007-07-100115 ◽

2008 ◽

Vol 111 (4) ◽

pp. 2230-2237 ◽

Cited By ~ 224

Author(s):

Linfeng Chen ◽

Stefano Monti ◽

Przemyslaw Juszczynski ◽

John Daley ◽

Wen Chen ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Primary Tumors ◽

Large B Cell Lymphoma ◽

Bcr Signaling ◽

Survival Signals

The role of B-cell receptor (BCR)–mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igα/β immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as “BCR-type” tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

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Pharmacologic Inhibition of B Cell-Receptor-Associated Kinases with CG-806 Induces Apoptosis and Metabolic Reprogramming in Aggressive Non-Hodgkin Lymphoma (NHL) Models

Blood ◽

10.1182/blood-2020-134957 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-29

Author(s):

Elana Thieme ◽

Vi Lam ◽

Nur Bruss ◽

Fei Xu ◽

Stephen E Kurtz ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Cell Receptor ◽

B Cell Receptor ◽

Mitochondrial Mass ◽

Bcr Signaling ◽

Pdx Models

Introduction: Activated B cell receptor (BCR) signaling is a hallmark of NHL. BCR-associated kinases LYN, SYK, BTK and PI3K activate pro-survival signaling pathways including MEK/ERK, AKT/mTOR, and NFκB. While targeting BTK (ibrutinib, acalabrutinib) and PI3K (idelalisib, duvelisib) has shown efficacy in CLL, clinical responses fall short in aggressive NHL, necessitating the development of novel approaches to suppress BCR signaling. CG-806 is a BTK/cluster-selective kinase inhibitor currently under investigation in phase 1 clinical trials for patients with hematological malignancies. CG-806 targets both WT BTK (IC50 ~ 8 nM) and the BTKC481S (IC50 ~ 2.5 nM; www.aptose.com). Here we investigate the anti-tumor effects of CG-806 in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). Methods: CG-806 was provided by Aptose Biosciences, Inc. (San Diego, CA). DLBCL and MCL cell lines were assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), mitochondrial mass and mitophagy. Ibrutinib (ibr) resistance was induced by exposure over 6 months. Primary peripheral blood mononuclear cells were incubated for 24 h in media conditioned by stromal cells engineered to express CD40L or BAFF prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected into the tail vein of NSG mice and tracked weekly by flow cytometry (CD5+ CD19+ CD45+). Upon MCL detection in the peripheral blood, mice began daily treatment with 30.8 or 308 mg/kg CG-806 or vehicle control via oral gavage until moribund. Splenocytes were harvested 1 h after the final drug treatment. Results: CG-806 potently inhibited proliferation of both parental and ibr-resistant MCL cell lines (Mino, JeKo-1) with IC50<0.01 μM at 72 h. DLBCL cell lines (U2932, OCI-LY3 OCI-LY19) demonstrated moderate sensitivity to CG-806 (IC50 0.3-1 μM), while SU-DHL10 was highly sensitive (IC50<0.01 µM). Treatment with CG-806, but not ibrutinib, induced apoptosis of primary MCL cells in CD40L- or BAFF-expressing stromal co-cultures. Following anti-IgM crosslinking of primary cells, treatment with CG-806 decreased phosphorylation of SYK, BTK, AKT and ERK, indicating disrupted BCR signaling. Treatment with CG-806 increased respiratory reserve capacity but did not impact the basal oxygen consumption rate in both parental and ibr-resistant MCL cell lines. Basal extracellular acidification rate (ECAR) was increased following CG-806 treatment, indicating heightened glycolytic activity. Furthermore, CG-806-treated cells demonstrated potent induction of mitophagy accompanied by a reduction in mitochondrial mass. CG-806 slowed expansion of circulating MCL cells and reduced proliferation of spleen-resident MCL cells in both chemo- and ibr-resistant MCL PDX models. CG-806 and ibrutinib extended survival of chemoresistant PDX mice without evidence of toxic events. Treatment with CG-806 led to decreased phosphorylation of SYK, BTK, and AKT but also upregulated expression of BCL2 and BCLX. RNA-seq analysis of spleen-resident cells revealed downregulation of NFκB targets and JAK/STAT signaling in ibr-resistant PDX mice treated with CG-806. This was accompanied by enrichment of metabolic pathways (oxidative phosphorylation, fatty acid metabolism) and MYC targets. Next, we evaluated CG-806 for synthetic lethality in a functional in vitro screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). Consistent with the above observations, synergy was observed between CG-806 and inhibitors of metabolic enzymes (teleglenastat, perhexiline maleate) and BH3-mimetics targeting BCL2/X proteins (venetoclax, AZD4320). Conclusions: Our data demonstrate preliminary efficacy of CG-806 in MCL and DLBCL in vitro and in MCL DPX models. CG-806 treatment led to metabolic reprograming towards glycolysis and induced mitophagy. BCL2 family proteins may be implicated in resistance to CG-806. These results provide rationale for further investigation of CG-806 in aggressive NHL. Disclosures Tyner: Array: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Agios: Research Funding; Aptose: Research Funding. Danilov:Pharmacyclics: Consultancy; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; Celgene: Consultancy; Aptose Biosciences: Research Funding; Bristol-Myers Squibb: Research Funding; Rigel Pharmaceuticals: Consultancy; Karyopharm: Consultancy; BeiGene: Consultancy; Abbvie: Consultancy.

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The Paracaspase MALT1 Acts Independently of Pre-B-Cell Receptor Signaling As a Key Factor in Leukemic Cell Survival in Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-131376 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1288-1288

Author(s):

Nakhle S. Saba ◽

Jade Meyers ◽

Lorena Fontan ◽

Ari Melnick ◽

Adrian Wiestner ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

B Cell Receptor ◽

Gene Sets ◽

Cell Acute Lymphoblastic Leukemia

Despite a central role for B-cell receptor precursor (pre-BCR) pathway in precursor B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data on therapies that aim to disrupt this pathway. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for BCR-mediated NF-κB activation. We recently showed that targeting MALT1 with the small molecule inhibitor MI2 is effective in CLL, including drug-resistant clones (Saba Can Res 2017). We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of inhibiting its activity. First, we tested MALT1 expression by immunoblot in B-ALL using 17 cell lines representing the disease spectrum (7 pro-B: REH, SEMK2, TOM1, RS4;11, NALM21, Z119, BV173; 8 pre-B: HB11;19, NALM6, RCH-ACV, SMS-SB, 697, KASUMI2, KOPN8, HPB-NULL; and 2 mature/Burkitt: 2F7, RAJI), and found that MALT1 was expressed in all cell lines at different levels. To determine sensitivity to MALT1 inhibition we used two molecules: Z-VRPR-fmk, a highly selective MALT1 blocking peptide, and MI2, a small molecule MALT1 inhibitor. Z-VRPR-fmk resulted in a dramatic cell growth inhibition in most of our B-ALL cell lines, with appropriate positive (TMD8) and negative (K562) controls, independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome. We did not observe a clear correlation between MALT1 level and degree of sensitivity to Z-VRPR-fmk. Interestingly, the two ibrutinib-resistant cell lines RS4;11 and 697, were amongst the top sensitive cell lines to MALT1 inhibition. A similar pattern of cell sensitivity was observed when these cell lines were treated with MI2, resulting in an IC50 at 48h of 0.2 µM in RS4;11 and < 0.5 µM in other sensitive cell lines, which is consistent with published data in sensitive DLBCL cell lines (IC50, 0.2-0.5 µM), and our data on the CLL cell line MEC1 (IC50, 0.2 µM). We then tested freshly collected PBMCs from patients with various blood cancers presenting with a leukemic phase against serial dilutions of MI2 for 48h (7 B-ALL, 24 CLL, and 4 CML). In addition, we included normal B-cells collected from five volunteers. Interestingly, B-ALL samples showed the highest sensitivity to MI2, followed by CLL, while the rest were resistant. The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its targets such as A20, CYLD, BCL10, Roquin, Regnase and RelB. Surprisingly, with the exception of the Burkitt cell line 2F7, we did not detect cleavage of these targets at baseline, nor after proteasomal inhibition with MG-132 or following crosslinking of pre-BCR with anti-IgM in pre-B ALL, the latter successfully increased AKT phosphorylation. The constitutive activation of MALT1 in 2F7 was effectively inhibited by Z-VRPR-fmk as determined by a marked reduction in targets cleavage concomitant with an increase in full length proteins. We are expanding the mature B-ALL cell line cohort to include TANOUE, BALL-1, DAUDI, GA-10, and NC-37 cell lines to further explore to role of MALT1 in this disease subset. Collectively, these data highly suggest distinct roles for MALT1 in B-ALL: pro and pre-B-ALL vs. mature B-ALL. To explore the possibility of distinct role for MALT1 in B-ALL, arguably independent of BTK and of signaling through BCR, we used RNA sequencing to determine the changes in gene expression profiling following a 24h treatment with Z-VRPR-fmk in 3 highly sensitive B-ALL cell lines (RS4;11, HPB-NULL, and 697). Out of 39,514 tested genes, there were 160 genes whose expression changed ≥ 2-fold at P < 0.05 (84 down- and 76 up-regulated). Gene Set Enrichment Analysis (GSEA) identified 34 Hallmark and Oncogenic Signatures gene sets relevant to B-ALL that were all downregulated by Z-VRPR-fmk (FDR < 10%, and normalized enrichment score (NES) ≥ 1.50). Among those gene sets, mTOR-S6K and TANK-binding kinase 1 (TBK1)-dependent gene signatures stood out as the most affected. MALT1 was shown to be involved in the ribosomal protein S6 phosphorylation through activation of mTOR/AKT signaling. Indeed, treatment with MI2 and Z-VRPR resulted in a significant decrease in S6 phosphorylation in RS4;11 and SEMK2. In conclusion, MALT1 plays a critical role in B-ALL survival likely through a novel mechanism that involves mTOR-S6K pathway, independently from pre-BCR/BCR signaling. Supported by a grant from the Ladies Leukemia League, Inc., of the Gulf South Region. Disclosures Saba: Kyowa Kirin: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Melnick:Epizyme: Consultancy; Janssen: Research Funding; Constellation: Consultancy. Wiestner:Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding. Burger:AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Pharmacyclics, an AbbVie company: Research Funding; BeiGene: Research Funding. Safah:Celgene: Speakers Bureau; Incyte: Speakers Bureau; Verastem: Honoraria, Speakers Bureau; Jazz: Speakers Bureau; Amgen: Honoraria, Speakers Bureau.

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High ZAP-70 and Differential Expression of B-Cell Receptor Related Genes in Chronic Lymphocytic Leukemia with V3-21 Gene Usage.

Blood ◽

10.1182/blood.v104.11.773.773 ◽

2004 ◽

Vol 104 (11) ◽

pp. 773-773

Author(s):

Dirk Kienle ◽

Alexander Kröber ◽

Dirk Winkler ◽

Daniel Mertens ◽

Annett Habermann ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Mutation Status ◽

Bcr Signaling ◽

Gene Usage ◽

Lower Expression

Abstract V3-21 gene usage defines a distinct genetic subgroup of chronic lymphocytic leukemia (CLL) characterized by a poor clinical outcome regardless of the VH mutation status. V3-21 cases exhibit a highly characteristic B-cell receptor (BCR) structure as demonstrated by homologous CDR3 sequences and a restricted use of VL genes implicating a common antigen involved in tumor pathogenesis of this specific CLL subgroup. To investigate the role of antigenic stimulation in the pathogenesis of V3-21 using CLL, we analyzed the quantitative expression of genes involved in BCR signaling (ZAP-70, SYK, BLNK, LYN, PI3K, PLCG2, FOS), B-cell activation (TRAF3, STAT6, NFKB), and cell cycle or apoptosis control (ATM, BCL-2, BAX, CDK4, CCND1, CCND2, CCND3, p27, E2F1, MYC) in V3-21 cases in comparison to VH mutated (VH MUT) and VH unmutated (VH UM) cases not using the V3-21 gene. To obtain native expression signatures we studied a non-CD19-purified (nPU) cohort (V3-21: 18 cases, equally divided into VH mutated and VH unmutated cases; VH MUT: 17; VH UM: 19) and, for verification, a CD19-purified (PU) cohort (V3-21: 10 cases, equally divided into VH mutated and unmutated; VH MUT: 12; VH UM: 16) to exclude a contamination of the results by non-tumor cells. All cases were analyzed by FISH for +3q, 6q-, +8q, 11q-, +12q, 13q-, 17p-, and t(11;14) to avoid major imbalances of genomic alterations between the subgroups under study. As expected, ZAP-70 expression was higher in VH UM as compared to VH MUT cases in the nPU (p=0.007) as well as the PU cohort (p=0.009). V3-21 cases showed a higher ZAP-70 expression as compared to VH MUT (nPU: p=0.033; PU: p=0.038). This applied also when restricting this comparison to V3-21 mutated cases (nPU: p=0.018). Median ZAP-70 expression in the PU cohort was 1.15 in VH MUT vs. 7.69 in VH UM cases, as compared to 7.05 in V3-21 cases (V3-21 mutated cases: 10.69; V3-21 unmutated: 6.7). Other genes differentially expressed between the V3-21 and VH MUT subgroups in nPU cases were PI3K (p=0.048), PLCG2 (p=0.007), CCND2 (p=0.003), p27 (p=0.003), BCL-2 (p=0.025), and ATM (p=0.006). In addition, a set of genes was detected with a differential expression between V3-21 and VH UM (nPU) including PLCG2 (p=0.014), NFKB (p=0.023), CCND2 (p=0.001), p27 (0.002), and BAX (p=0.028). Notably, except for ZAP-70, all of the differentially expressed genes showed a lower expression in V3-21 as compared to the other subgroups. When comparing the V3-21 mutated and V3-21 unmutated subgroups (nPU), there were no significant gene expression differences except for CDK4, which showed a lower expression in V3-21 unmutated cases. Therefore, cases with V3-21 usage appear to show a rather homogeneous gene expression pattern independently of the VH mutation status, which can be distinguished from VH MUT and VH UM cases not using V3-21. The expression differences observed suggest a role of differential BCR signaling in the pathogenesis of this distinct CLL subgroup. Deregulation of cell cycle, apoptosis, and candidate genes such as ATM indicate the involvement of additional pathways in the pathogenesis of CLL cases using V3-21.

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Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells

Blood ◽

10.1182/blood.v116.21.375.375 ◽

2010 ◽

Vol 116 (21) ◽

pp. 375-375 ◽

Cited By ~ 1

Author(s):

Fatima Talab ◽

Victoria Thompson ◽

John C Allen ◽

Ke Lin ◽

Joseph R Slupsky

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Cell Cultures ◽

Lymphocytic Leukaemia ◽

Cell Receptor ◽

B Cell Receptor ◽

Nfkb Pathway

Abstract Abstract 375 B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn. Disclosures: No relevant conflicts of interest to declare.

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Synergistic Blockade Of Activated B Cell-Like DLBCL Proliferation With a Selective Inhibitor Of IRAK4 In Combination With Inhibition Of The B-Cell Receptor Signaling Network

Blood ◽

10.1182/blood.v122.21.3833.3833 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3833-3833 ◽

Cited By ~ 7

Author(s):

Divya Chaudhary ◽

Nancy Wood ◽

Donna L. Romero ◽

Shaughnessy D. Robinson ◽

Jeremy R Greenwood ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Survival ◽

Cell Lines ◽

Immune Cells ◽

Cell Receptor ◽

B Cell Receptor ◽

Inflammatory Signaling ◽

Bcr Signaling ◽

Equity Ownership

Abstract Toll-Like Receptor (TLR) and IL-1 signaling is mediated by the adaptor protein MyD88 through IRAK4 activation. TLR and IL-1 family ligands activate NFkB through this pathway and stimulate proliferation and cell survival, as well as induce cytokine and chemokine production that can amplify tumor cell survival. The gain-of-function L265P mutation in MyD88 occurs in ∼30% of patients with activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) and ∼90% of Waldenström’s macroglobulinemia. Therefore, inhibition of IRAK4 may be therapeutically relevant in hematologic malignancies containing MyD88 mutations. Recent clinical results with kinase inhibitors strongly support a role for signaling through the B-cell receptor (BCR) pathway in the progression of hematological malignancies including ABC-DLBCL. We were interested to understand the potential utility of selective IRAK4 inhibitors in combination with inhibition of the BCR signaling networks. We have reported previously the identification and characterization of potent and selective IRAK4 inhibitors that are effective in blocking inflammatory signaling in immune cells and demonstrate efficacy in vivo in models of autoimmune disease. ND-2158, a potent (Ki of 1.2 nM) and highly selective IRAK4 inhibitor has been shown to be effective in reducing the proliferation of ABC-DLBCL cell lines. ND-2158 does not decrease cell viability for other cell lines that lack the MyD88 mutation including a germinal center-like DLBCL cell line, BJAB, suggesting that the anti-proliferative effects in ABC-DLBCL cells relate in part to the activating MyD88 mutation. Complete cross-over dose-response proliferation studies of the ABC-DLBCL cell line, OCI-LY10, were conducted using ND-2158 in combination with blockade of key BCR signaling network nodes, using inhibitors of either Btk (ibrutinib), PI3Kdelta (GS-1101), or Syk (P505-15). Isobologram analysis using the Chou-Talalay method revealed that ND-2158 was able to synergistically block cell proliferation in combination with ibrutinib, P505-15, or GS-1101. Interestingly, we find that blockade of SYK, PI3Kdelta, or BTK signaling enhances the potency of ND-2158 in ABC-DLBCL cells. The IC50 values observed in this context are comparable to the potency of ND-2158 when used as a single agent to inhibit inflammatory signaling in immune cells that are not dependent on BCR signaling. The cell proliferation blockade IC50for ND-2158 shifted from an average value of ∼7 μM to 0.19, 0.05, or 0.15 μM, when combined with the IC50 concentrations of the inhibitors of BTK, PI3Kdelta or SYK kinases, respectively. These results suggest that inhibition of both BCR signaling pathways that are amplified in ABC-DLBCL, and IRAK4 signaling activated through MyD88 mutations, are required for a more complete blockade of ABC-DLBCL proliferation. Moreover, we explored ND-2158 combination with lenalidomide, known to be synergistic with BCR and NFkB pathway inhibitors. In contrast to combinations with BCR signaling inhibition, studies with lenalidomide failed to demonstrate an additive or synergistic activity when combined with IRAK4 inhibition in ABC-DLBCL cell lines. Therefore, we conclude that IRAK4 activation, as well as aberrant BCR signaling, are likely to contribute to the proliferative capacity of ABC-DLBCL. We propose that combinatorial therapeutic approaches, including inhibition of IRAK4, may provide benefit for patients with ABC-DLBCL. Disclosures: Chaudhary: Nimbus Discovery Inc.: Employment. Off Label Use: Exploratory inhibitor of IRAK4 for research purposes. Wood:Nimbus Discovery Inc.: Employment. Romero:Nimbus Discovery Inc.: Consultancy, Equity Ownership. Robinson:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Greenwood:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Shelley:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Morin:Nimbus Discovery Inc.: Consultancy. Kapeller:Nimbus Discovery Inc.: Employment. Westlin:Nimbus Discovery Inc.: Employment, Equity Ownership.

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Pre-BCR Signaling Activity Predicts Sensitivity To Syk Kinase Inhibition In B Cell Acute Lymphoblastic Leukemia (B-ALL)

Blood ◽

10.1182/blood.v122.21.614.614 ◽

2013 ◽

Vol 122 (21) ◽

pp. 614-614

Author(s):

Stefan Koehrer ◽

Richard E. Davis ◽

Greg Coffey ◽

Ekaterina Kim ◽

Nathalie Y. Rosin ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

B Lymphocyte ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Lymphocyte Development ◽

Bcr Signaling ◽

Associated Proteins ◽

Cell Acute Lymphoblastic Leukemia ◽

B Lymphocyte Development

Abstract B lymphocyte development proceeds in a stepwise fashion and is tightly linked to the generation of a functional B cell receptor (BCR). At the preB cell stage B lymphocyte progenitors express the precursor B cell receptor (pre-BCR), an immature form of the BCR consisting of two µ heavy chains (µHC) and two surrogate light chains (SLC). Pre-BCR expression marks the proB to preB transition and induces a burst in preB lymphocyte proliferation. In 20% of the cases B cell acute lymphoblastic leukemia (B-ALL) arises from lymphocytes arrested at the pre-BCR positive stage of lymphocyte development (preB-ALL). Due to the essential role of the pre-BCR for preB cell proliferation we hypothesized that pre-BCR signaling also is involved in the maintenance of preB-ALL. Consequently, pharmacological inhibition of Spleen tyrosine kinase (Syk), the main transducer of pre-BCR signaling, may serve as effective treatment for this subtype of B-ALL. We analyzed a panel of six ALL cell lines (SMS-SB, RCH-ACV, Nalm-6, Kasumi-2, 697, KOPN-8) arrested at the pre-BCR+ stage of B lymphocyte development (cytoIgµ+, sIgM-). Assessment of the baseline phosphorylation levels of the pre-BCR associated kinases Lyn, Syk and Btk by immunoblotting and subsequent densitometric analysis allowed us to assign B-ALL cells into groups with either high levels of Lyn, Syk and Btk phosphorylation or with low or absent phosphorylation of these kinases, respectively. Moreover cell lines with highly phosphorylated Lyn, Syk and Btk also exhibited lower surface pre-BCR expression than cell lines with low phosphorylation levels. As pre-BCR activation is followed by its rapid internalization the concomitant presence of low pre-BCR expression and high phosphorylation of pre-BCR associated proteins suggests increased pre-BCR pathway activity. When we investigated the impact of pharmacological inhibition of the pre-BCR associated kinase Syk through the highly specific inhibitor PRT060318, preB-ALL cell lines with highly phosphorylated pre-BCR associated molecules turned out to be more sensitive to Syk inhibition (IC50 < 1.6µM) than preB-ALL cell lines with less phosphorylation (IC50 > 3.9µM). In proliferation assays PRT060318 inhibited preB-ALL proliferation in a dose dependent manner, whereas PRT060318 did not induce apoptosis in concentrations as high as 5µM. This supports the notion that pre-BCR signaling activity may be more relevant for preB-ALL proliferation than for preB-ALL viability. In line with these results the pre-BCR- proB-ALL cell lines REH and RS4;11 were highly resistant to Syk inhibition in all functional assays (IC50 > 10µM), suggesting that pre-BCR expression is a prerequisite for sensitivity to Syk inhibition. To examine the molecular changes following pre-BCR inhibition, ALL cells were treated with increasing concentrations of PRT060318 (100nM-5µM) for two hours and then subjected to immunoblotting. Syk inhibition led to a dose dependent decrease in AKT phosphorylation in all preB-ALL cell lines and subsequently reduced phosphorylation of FOXO transcription factors. In the resistant proB-ALL cell line REH, AKT and FOXO phosphorylation were not affected. Gene expression analysis of the preB-ALL cell lines RCH-ACV and Nalm-6 further suggested that PRT060318 interferes with pre-BCR signaling. Treatment with 1µM PRT060318 for 72h reduced the expression of genes associated with pre-BCR signaling (e.g. BCL6, CD22, PTPN6) and Ingenuity Pathway Analysis identified pre-BCR signaling as the main target of PRT060318 in both cell lines (p<0.05). We are currently validating the GEP analysis by quantitative PCR and immunoblotting. In conclusion, we provide evidence for the efficacy of Syk inhibition in pre-BCR+ ALL. Moreover we were able to correlate the baseline phosphorylation status of pre-BCR associated proteins and pre-BCR expression levels with the sensitivity of preB-ALL to the Syk inhibitor PRT060318. These findings provide a first rationale for the clinical testing of Syk inhibitors in preB-ALL, and suggest that activation status of pre-BCR associated molecules can help in selecting preB-ALL cases that are particularly sensitive to Syk inhibition. Disclosures: Coffey: Portola Pharmaceuticals: Employment.

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Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.284.284 ◽

2014 ◽

Vol 124 (21) ◽

pp. 284-284

Author(s):

Huimin Geng ◽

Christian Hurtz ◽

Dirk Baumjohann ◽

Zhengshan Chen ◽

Wei-Yi Chen ◽

...

Keyword(s):

B Cell ◽

Tyrosine Kinases ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Bcr Signaling ◽

Btk Inhibitor ◽

Bcl6 Expression ◽

Targeting Strategy

Abstract Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL. Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling. Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation. Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells. Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. Disclosures No relevant conflicts of interest to declare.

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Biomarker Predictive Ibrutinib Response Using Profiled ABC-DLBCL Patient Derived Xenografts

Blood ◽

10.1182/blood.v126.23.2759.2759 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2759-2759

Author(s):

Ran Wu ◽

Sheng Guo ◽

Jie Cai ◽

Xuesong Huang ◽

Jie Yang ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Molecular Pathogenesis ◽

Wild Type ◽

Dlbcl Patient ◽

Bruton's Tyrosine Kinase ◽

Single Mutant ◽

Myd88 L265p

Abstract Background. Activated B cell-like (ABC) is a subtype of DLBCL difficult to treat. Activation of B-cell receptor (BCR) signaling pathway, including CD79 transmembrane proteins (A/B subunits) and Bruton's tyrosine kinase (BTK), are required for the disease maintenance. The MYD88 (an adapter for Toll-like receptors in a separate pathway from BCR pathway) activating mutation, L265P, has been suggested to activate BCR pathway (29% of ABC-DLBCL) and thus drives the disease. These DLBCLs seem responsive to ibrutinib (a BTK inhibitor) in the clinics1,2. Majority of MYD88-L265P mutant DLBCL patients also have CD79B mutation (e.g. Y197N), i.e. double mutants (21% of total ABC-DLBCL), hinting collaborative oncogenesis, while some are MYD88-L265P single mutant with wild type CD79B. It remains unclear whether the single and double mutant ABC-DLBCL differ in their molecular pathogenesis as well as response to ibrutinib. Method. We created DLBCL patient derived xenografts (PDX) as experimental system mimicking patients in order to investigate the molecular pathogenesis/response to ibrutinib. We first transcriptome-sequenced the established PDXs. We categorized them into ABC/GCB subtypes along with mutation status of CD79B/MYD88 per transcriptome profiles. We then assessed these PDXs for their response to ibrutinib and trying to correlate antitumor activity to molecular signature. Results. We have created 20 NHL-PDX, with 5 of them transcriptome-sequenced. Clustering analysis per expression profile of 14-genes have classified all these 5 into subclass ABC-DLBCL. Among the 5, one is MYD88-L265P/CD79B-Y197N double mutants (LY2298), one MYD88-L265P single mutant (LY0257), and three wild types (LY2345, LY2214, LY2266). Among the 3 models tested so far, LY2214 (wildtype) and LY0257 (single mutation) did not respond to ibrutinib, but LY2298 (double mutations) did. These results, although small sample sizes, seem to suggest that double mutations are required for the response to ibrutinib, while single mutant and wild type does not. These observation seem to be consistent with the implication from recent clinical study1. We are currently profile and test the remaining models to further confirm these observations. We will also further elucidate the downstream signaling along with the drug response. Conclusion. Our preliminary results confirmed that ibrutinib sensitivity correlates with the concomitant double mutation of CD79B and MYD88. References 1. Wilson, W.H., et al. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma. Nature medicine (2015). 2. Aalipour, A. & Advani, R.H. Bruton's tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib. Therapeutic advances in hematology5, 121-133 (2014). Disclosures No relevant conflicts of interest to declare.

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