Combination of Ibrutinib and BCL-2 or SYK Inhibitors in Ibrutinib Resistant ABC-Subtype of Diffuse Large B-Cell Lymphoma

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the US. DLBCL can progress quickly, and in advanced cases is inconsistently cured with current therapies. Ibrutinib, a first-in-class Bruton's tyrosine kinase (BTK) inhibitor, is approved as a treatment for patients (pts) with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) who have had one prior treatment. The ABC subtype of DLBCL is considered especially high risk and characterized by chronic active B-cell receptor (BCR) signaling, which is blocked by ibrutinib. Recent phase II clinical trial results of ibrutinib as a single agent in DLBCL pts show an overall response rate of 41% in the ABC subtype (Wilson et al. ASH 2012). Responses of various cancers to single kinase targeted therapies are often limited by the cell's ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway. It has been shown that a small number of CLL pts acquire resistance to ibrutinib through mutations in BTK and its substrate phospholipase C gamma 2 (PLCG2) in the B lymphoma cells following prolonged treatments (Woyach et al. NEJM 2014). Such mechanisms may be overcome by combinations of targeted agents. Through screening of wild-type and acquired ibrutinib-resistant ABC-DLBCL cell lines (e.g. expressing BTK C481S), we identify and report herein B-cell lymphoma-2 (BCL-2) and spleen tyrosine kinase (SYK) inhibitors that synergize with ibrutinib in vitro and in vivo. Methods: Gene expression was analyzed by RT-qPCR using TaqMan Gene Expression Master Mix. Human DLBCL cell lines were treated with drugs for 3 days and the effect on cell growth was determined by CellTiter-Glo luminescent cell viability assay. SCID mice were treated when the TMD8 tumors reached 100-150 mm3. Annexin-V-positive and PI-negative population was detected as apoptotic cells in tumor cells at sacrifice. Cell adhesion and migration assays were performed as previously described (Chang et al. Blood 2013). Analysis of clinical samples used for BCL-2 gene expression profiling was performed using Affymetrix microarrays on FFPE specimens from the phase 2 PCYC-1106 trial (NCT01325701) and a rank based statistic (RankProd) was used to determine the significance of gene expression changes. Results: DLBCL cell lines with higher BCL-2 expression were more sensitive to single agent ABT-199 than those with lower expression. Treatment of DLBCL cells with ibrutinib alone increased BCL-2 expression as well as their sensitivity to BCL-2 inhibitors. Combination treatment with BCL-2 inhibitors and ibrutinib completely inhibited tumor growth in murine models of ABC-DLBCL (Figure). Increased apoptotic cell populations were detected in the combination treated tumors compared to either treatment alone. Clinically, pretreatment tissue samples (n=28) from ABC-DLBCL pts who experienced objective responses to ibrutinib (CR+PR) had lower BCL-2 gene expression. A high BCL-2 mutation rate was observed in pts with poor response to ibrutinib (SD+PD). However, none of these mutations occurred in the BH3 or BH1 domains, both of which appear to interact with ABT-199 based on a 3-dimensional co-crystal structure of the inhibitor with BCL-2 (PDB code 4MAN) and further molecular simulation results. These findings suggest the potential benefit from combination therapy. SYK is another downstream mediator of BCR signaling. Pretreatment of DLBCL cells with SYK inhibitors (e.g. R406) increased their sensitivity to ibrutinib. Ibrutinib resistant B-lymphoma cells with either C481S BTK or R665W PLCG2 mutations were re-sensitized to ibrutinib in combination with BCL-2 or SYK inhibitors, inhibiting cell growth, IgM-induced calcium flux, cell adhesion or migration in mutant containing cells. Conclusions: Consistent with previous results from high-throughput combinatorial screenings of drugs interact favorably with ibrutinib (Mathews Griner, et al. PNAS 2013), we found BCL-2 and SYK may function in alternative survival pathways in DLBCL cells upon BTK inhibition. Human B lymphomas harboring ibrutinib-resistant C481S BTK or R665W PLCG2 may be re-sensitized by BCL-2 or SYK inhibitors, both of which provide a rationale for the design of combination therapies. Figure 1 Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth. Figure 1. Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth. Disclosures Kuo: Pharmacyclics: Employment. Crowley:Pharmacyclics: Employment. Xue:Pharmacyclics: Employment. Schweighofer:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment. Hsieh:Pharmacyclics: Employment. Eckert:Pharmacyclics: Employment. Versele:Janssen Pharmaceutica: Employment. Chang:Pharmacyclics, Inc: Employment, Equity Ownership.

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PIM as a Rational Target for B-Cell Lymphomas.

Blood ◽

10.1182/blood.v114.22.3946.3946 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3946-3946

Author(s):

Cristina Gomez-Abad ◽

Helena Pisonero ◽

Juan F Leal ◽

Giovanna Roncador ◽

Jose A. Martinez-Climent ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Specific Inhibitor ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Micromolar Range ◽

Large B Cell ◽

Stat Pathway

Abstract Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

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SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Blood ◽

10.1182/blood.v124.21.926.926 ◽

2014 ◽

Vol 124 (21) ◽

pp. 926-926

Author(s):

Y. Lynn Wang ◽

Jiao Ma ◽

Wei Xing ◽

Pin Lu ◽

Karen Dresser ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Staining Pattern ◽

B Cell Lymphoma ◽

Down Regulation ◽

Dual Inhibitor ◽

Large B Cell Lymphoma ◽

Bcr Signaling ◽

Large B Cell

Abstract Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways. To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%). SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures Pandey: Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

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The Role of PIM1 in the Ibrutinib-Resistant ABC Subtype of Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.699.699 ◽

2015 ◽

Vol 126 (23) ◽

pp. 699-699 ◽

Cited By ~ 1

Author(s):

Hsu-Ping Kuo ◽

Sidney Hsieh ◽

Karl J. Schweighofer ◽

Leo WK Cheung ◽

Shiquan Wu ◽

...

Keyword(s):

Gene Expression ◽

Tumor Growth ◽

B Cell ◽

Repeated Measures ◽

Cell Lymphoma ◽

Xenograft Model ◽

B Cell Lymphoma ◽

Bcr Signaling

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the United States (US). DLBCL is a heterogeneous lymphoma, including the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which have different gene expression profiles, oncogenic aberrations, and clinical outcomes (Alizadeh, Nature 2000; Staudt, Adv Immunol 2005). ABC-DLBCL is characterized by chronic active B-cell receptor (BCR) signaling (Davis, Nature 2010), which is required for cell survival. Thus, the BCR signaling pathway is an attractive therapeutic target in this type of B-cell malignancy. Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling, is covalently bound with high affinity by ibrutinib, a first-in-class BTK inhibitor approved in the US for mantle cell lymphoma and chronic lymphocytic leukemia (CLL) patients (pts) who have received at least one prior treatment, CLL with del17p, and WaldenstršmÕs macroglobulinemia. A recent phase 2 clinical trial of single-agent ibrutinib in DLBCL pts revealed an overall response rate of 40% for ABC-DLBCL (Wilson, Nat. Med 2015); however, responses to single kinase-targeted cancer therapies are often limited by the cellÕs ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway (Nardi, Curr Opin Hematol 2004; Gazdar, Oncogene 2009). The serine/threonine-protein kinase PIM1 is one of several genes exhibiting differential expression in ibrutinib-resistant ABC-DLBCL cells compared with wild-type (WT) cells. We identified and report herein the role of PIM1 in ABC-DLBCL ibrutinib-resistant cells. Methods: PIM1 gene expression was analyzed by RT-qPCR. In vitro, cell viability was assessed in the human ABC-DLBCL cell line HBL-1 after treatment with ibrutinib and/or a pan-PIM inhibitor for 3 days, and the effect on colony formation was determined 7 days post-treatment. PIM1 mutational analysis was performed with clinical tumor biopsy samples from 2 studies, PCYC-04753 (NCT00849654) and PCYC-1106-CA (NCT01325701). PIM1 protein stability was analyzed by treating cells with cycloheximide and examining protein levels at different time points up to 8 hours. Results: Gene expression profiling of ibrutinib-resistant ABC-DLBCL cells revealed an upregulation of PIM1 (15-fold increase compared with WT cells) as well as PIM2 and PIM3. We also found that, compared with single-drug treatment, in vitro cell growth could be synergistically suppressed with a combination of ibrutinib and a pan-PIM inhibitor. This effect was observed in both WT (combination index (C.I.) = 0.25; synergy score = 3.18) and ibrutinib-resistant HBL-1 cells (C.I. = 0.18; synergy score = 4.98). In HBL-1 cells, this drug combination reduced colony formation and suppressed tumor growth in a xenograft model (Figure 1). In 48 DLBCL patient samples with available genomic profiling, PIM1 mutations appeared more frequently in pts diagnosed with ABC-DLBCL compared with GCB-DLBCL (5 out of 6 DLBCL pts with PIM1 mutations were ABC-subtype). 4 of these 5 pts exhibited a poor clinical response to ibrutinib, ie, 80% of ABC-DLBCL pts with PIM1 mutations had progressive disease, compared with only 13 of 26 (ie, 50%) ABC-DLBCL pts without PIM1 mutations. Subsequent characterization of the mutant PIM1 proteins (L2V, P81S, and S97N) confirmed that they were more stable than WT PIM1, suggesting increased protein levels by 2 potential mechanisms (WT PIM1 gene up-regulation or increased mutant PIM1 protein half-life). The impact of these mutations on PIM1 function and ibrutinib sensitivity is under investigation. Conclusions: Ibrutinib-resistant ABC-DLBCL cells have increased PIM1 expression, and synergistic growth suppression was observed when ibrutinib was combined with a pan-PIM inhibitor. PIM1 mutations identified in ABC-DLBCL pts with poor responses to ibrutinib contributed to increased PIM1 protein stability. A better understanding of the role of PIM1 in ibrutinib-resistant ABC-DLBCL tumors could provide a rationale for the design of combination therapies. Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Disclosures Kuo: Pharmacyclics LLC, an AbbVie Company: Employment. Hsieh:pharmacyclics LLC, an AbbVie Company: Employment. Schweighofer:Pharmacyclics LLC, an AbbVie Company: Employment. Cheung:Pharmacyclics LLC, an AbbVie Company: Employment. Wu:Pharmacyclics LLC, an AbbVie Company: Employment. Apatira:Pharmacyclics LLC, an AbbVie Company: Employment. Sirisawad:Pharmacyclics LLC, an AbbVie Company: Employment. Eckert:Pharmacyclics LLC, an AbbVie Company: Employment. Liang:Pharmacyclics LLC, an AbbVie Company: Employment. Hsu:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

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MicroRNA Expression Profiling of Diffuse Large B-Cell Lymphoma Reveals Differences between Germinal Center-Like and Activated B Cell-Like Subtypes.

Blood ◽

10.1182/blood.v106.11.3002.3002 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3002-3002

Author(s):

Charles H. Lawrie ◽

Shamit Soneji ◽

Christopher D. Cooper ◽

Chris Hatton

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Expression Profile ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell ◽

Clinical Cases

Abstract MicroRNAs (miRNA) are a recently discovered class of short non-coding RNA molecules that negatively regulate gene expression. They have been shown to play a critical role in many biological functions. In humans about 320 miRNAs have been identified, some of which are expressed in a cell-specific and developmental stage-specific manner. Recently it has been shown that the expression profile of miRNAs can be used to subtype clinical cases (and cell lines) according to diagnosis with a greater degree of accuracy than traditional gene expression analysis. The identity of miRNAs associated with different lymphoma types however remains poorly defined. Previous expression studies have revealed the presence of at least two subtypes of diffuse large B-cell lymphoma (DLBCL) representing the postulated cell of origin; those that are germinal center B cell derived (GCB-type) and those that are activated B-cell derived (ABC-type). The latter subtype has been linked with poor prognostic outcome. It is not known whether these subtypes are also defined at the miRNA level. Therefore we examined the miRNA expression profile of DLBCL cell lines of defined subtypes as well as sub-populations of B-lymphocytes by microarray analysis. Consistent with recent publications, we found that mir-19a, 19b and 17-5p (part of mir-17-92 cluster) were up-regulated in cell lines but not in normal lymphocyte populations. Furthermore, cluster analysis showed that GCB-type cell lines (SUD-HL4, SUD-HL6 & SUD-HL10) have a distinct miRNA profile from ABC-type cell lines (OCI-Ly3 & OCI-Ly10). Most notably, high levels of expression of mir-155, mir-181b and mir-325 were found in ABC-type cell lines whilst high levels of mir-181a were found in GCB-type cell lines. We looked at expression of mir-155, 181a, 143, 145, 378 and 16 in these cell lines as well as clinical cases of DLBCL by RNase-protection assay. Consistent with the microarray data, we found that mir-155 was expressed in ABC-type cell lines but not GCB-type cell lines whilst the converse was true for mir-181a. Clinical cases showed similar patterns of expression but have still to be sub-typed according to immunohistochemical markers. Although still preliminary, our data suggests that miRNA profiling may be a useful tool in predicting the subtype of DLBCL cases and hence clinical outcome.

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Discovery of DNA G-Quadruplexes As a New Target for B-Cell Receptor Signaling Inhibition in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-116793 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3502-3502

Author(s):

Ying-Zhi Xu ◽

Thomas Raney ◽

Samantha L. Kendrick

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Cd Spectroscopy ◽

B Cell Receptor ◽

Large B Cell Lymphoma ◽

Bcr Signaling ◽

Large B Cell

Abstract Extensive gene expression profiling and RNA interference studies revealed the frequently chemo-resistant activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on constitutive B-cell receptor (BCR) signaling. As such, the clinical importance of BCR signaling inhibition is well appreciated and thus far led to the development of kinase and protease inhibitors. However, this therapeutic approach fails to achieve complete, sustained responses in DLBCL patients because of inherent resistance due to additional genetic lesions in other components of the BCR pathway or acquired kinase mutations. The emerging field of DNA secondary structures support that guanine (G)-rich stretches of DNA capable of adopting G-quadruplex (G4) motifs act as transcription regulatory units, or switches, that can turn gene expression on or off. Targeting G4s is likely to overcome activating kinase mutations by limiting the amount of gene available for translation into protein. Here, we explore a drug discovery effort based on targeting G4 within BCR genes critical for ABC DLBCL cell survival, CD79A, CD79B, CARD11, and MYD88. We first interrogated the BCR-related genes within the hg19 human reference genome for G-rich DNA using a G4 algorithm and discovered each of the four genes contain G4 forming sequences near promoter regions. These G4 elements formed stable G4 structures as determined by circular dichroism (CD) spectroscopy, the standard for visualizing macromolecule secondary structure formation. Melting curves are also generated from CD spectroscopy to determine the thermal stability of a given structure. The CD79A, CD79B, CARD11, and MYD88 G-rich sequences displayed classic, stable G4 structure spectra consisting of negative minima absorption peaks at 240-265 nm and a positive maximum at 260-295 nm with melting temperatures ranging from 62 to 95 °C. We then developed a high-throughput screening assay based on fluorescence resonance energy transfer (FRET) to identify G4 interactive compounds from the NCI Diversity Set IV library (1584 compounds) that uniquely interact with each of the BCR G4 sequences. This screen used the BCR G4 sequences as molecular bait where the 5´-end and 3´-end of the oligomers were labeled with a FAM- and a TAMRA-fluorophore, respectively, such that G4 formation leads to an increase in fluorescence emission (Figure 1). The initial FRET screen tested compounds at a 1:5 molecular ratio of probe to compound and measured the change in fluorescence relative to probe alone. Overall, the screen resulted in a ~1% "hit" rate for each BCR target, except for CD79B, which yielded a lower percent of interactive compounds (0.3%). Seven compounds, which included ellipticine, quinoline, and daunomycin derivatives, were identified to selectively target the CARD11 (n=3), MYD88 (n=3), or CD79A (n=1) G4s relative to other G4, single-stranded, and double-stranded DNA. Of note, all five compounds found to interact with the CD79B G4 also altered FRET of the other BCR G4 sequences. Subsequent FRET validation and CD analyses where each of the BCR sequences was incubated with increasing concentrations of candidate compounds demonstrated dose-dependent effects on G4 structure formation, particularly stabilization of the CARD11 G4 with compound NCI 9037 that resulted in a 300% FRET increase and an 8 °C shift in melting temperature at a 1:10 ratio. This study identifies DNA G4 as a new class of molecular targets for inhibiting an important oncogenic pathway. Discovery of selective compounds in addition to those with "pan" interaction, suggests the CARD11, MYD88, and CD79A G4 have unique folding patterns whereas the CD79B G4 may exhibit more common structural features. These compounds will be used as molecular tools to provide further insight into the structures and mechanisms in which G4 regulate gene transcription. In establishing a high-throughput screen, we discovered compounds for which preclinical development is ongoing and includes evaluation of the effects on BCR target gene and protein expression, inhibition of downstream BCR signaling, and consequent ABC DLBCL tumor growth and survival. This treatment strategy has high potential for leading to a breakthrough in effectively targeting the constitutively active molecules and greatly impacting the clinical management of patients with BCR-dependent DLBCL. Disclosures No relevant conflicts of interest to declare.

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The Novel PI3K/mTOR Dual Inhibitor PQR309 in Pre-Clinical Lymphoma Models: Demonstration of Anti-Tumor Activity As Single Agent and in Combination and Identification of Gene Expression Signatures Associated with Response

Blood ◽

10.1182/blood.v124.21.1782.1782 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1782-1782

Author(s):

Chiara Tarantelli ◽

Eugenio Gaudio ◽

Ivo Kwee ◽

Andrea Rinaldi ◽

Matteo Stifanelli ◽

...

Keyword(s):

Gene Expression ◽

Phase I ◽

B Cell ◽

Cell Lines ◽

Proliferative Activity ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Dual Inhibitor

Abstract Dual PI3K/mTOR inhibitors represent a promising class of anti/cancer compounds, of potential interest in lymphoid neoplasms which present activation of both targeted pathways. PQR309 is a novel, oral, member of this class of compounds and, as single agent, is currently being evaluated in a phase I for patients with solid tumors (NCT01940133). Here, we present the activity of the compound in pre-clinical models of mature lymphoid tumors, also integrating response data with genomic features. Methods. 48 cell lines [27 derived from diffuse large B-cell lymphoma (DLBCL), 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of PQR309 and MTT assays were performed after 72 hrs exposure. A second dual PI3K/mTOR inhibitor, GDC0980, and the PI3Kdelta inhibitor Idelalisib were also used on all the cell lines. IC50, GI50, LC50, and TGI values were used to estimate the cytotoxic and cytostatic effects. PQR309-induced cytotoxic activity was tested by AnnexinV assay. Synergy was assessed by the Chou-Talalay combination index (CI) on 2 DLBCL cell lines (TMD8, U2932) exposed to increasing doses of PQR309 alone or in combination with increasing doses of other drugs for 72 hrs. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. PQR309 showed potent anti-proliferative activity in most of the cell lines tested. The median IC50 was 242 nM (18nM-3.6 mcM), GI50 141 nM (25 nM-1.7 mcM), LC50 2.7 mcM (306 nM->10 mcM), TGI 711 nM (69 nM - >10 mcM). DLBCL (median IC50=166 nM), MCL (234 nM) and SMZL (214 nM) were all more sensitive than ALCL (664 nM) (P=0.005). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. Across the 48 cell lines, PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R=0.95). Idelalisib appeared significantly less active and its pattern of sensitive cell lines was less correlated with PQR309 (R=0.67) or GDC0980 (R=0.71). In DLBCL cell lines, PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 2/2 cells and the baseline p-AKT(Ser 473) levels in 1/1. PQR309 (500 nM, 72 hrs) caused apoptosis in 1/7 cell lines. Synergism or additive effected were observed in 2/2 cells combining PQR309 with the BCL2 inhibitor ABT199 (CI = 0.1 and 0.5), the immunomodulatory drug lenalidomide (0.5 and 0.4), the BTK-inhibitor ibrutinib (0.6 and 0.57) or the proteasome inhibitor bortezomib (0.9 and 0.9), and in 1/2 with the anti-CD20 monoclonal antibody rituximab (0.6), the BET inhibitor JQ1 (0.7) and the chemotherapy agent bendamustine (0.7). We then looked for baseline GEP features associated with sensitivity to PQR309, by comparing very sensitive (IC50 < 200 nM) versus less sensitive DLBCL cell lines (IC50 > 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in B-cell receptor pathway/signaling, kinases regulation, immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. Genes coding for individual proteins involved in PI3K signaling cascade were differentially expressed between the two groups of cells. Conclusions. PQR309 showed promising activity as single agent and in combination providing the basis for phase I/II studied dedicated for lymphoma patients. Baseline features associated with response were identified and are worth of being validated in the context of the next clinical trials. (CT and EG contributed equally to this work) Disclosures Hillmann: Piqur Therapeutics AG: Employment. Fabbro:Piqur Therapeutics AG: Employment. Hebeisen:Piqur Therapeutics AG: Employment. Betts:Piqur Therapeutics AG: Consultancy. Wicki:Piqur Therapeutics AG: Research Funding; University Hospital Basel: Employment. Cmiljanovic:Piqur Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:Piqur Therapeutics AG: Research Funding.

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Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo

Blood ◽

10.1182/blood.v128.22.1845.1845 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1845-1845 ◽

Cited By ~ 1

Author(s):

Sarah Meadows ◽

Anella Yahiaoui ◽

Rick Sorensen ◽

Zhi-Hua Cui ◽

Robert Brockett ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Tumor Regression ◽

Single Agent ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Bet Inhibitor ◽

Large B Cell Lymphoma

Abstract Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL. Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829. Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. Figure 1A Figure 1A. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Meadows: Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.

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The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.2732.2732 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2732-2732

Author(s):

Enrico Derenzini ◽

Ilaria Iacobucci ◽

Elisa Brighenti ◽

Federica Cattina ◽

Richard Eric Davis ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

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HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Inhibits Tumor Growth in Xenograft Models of Human Diffuse Large B-Cell Lymphoma, a Subset of Non-Hodgkins Lymphoma.

Blood ◽

10.1182/blood.v108.11.2519.2519 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2519-2519 ◽

Cited By ~ 2

Author(s):

Ssucheng J. Hsu ◽

Lin A. Esposito ◽

Sharon L. Aukerman ◽

Seema Kantak ◽

Amer M. Mirza

Keyword(s):

Tumor Growth ◽

B Cell ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Non Hodgkins Lymphoma ◽

Xenograft Models

Abstract CD40, a member of the tumor necrosis factor receptor family, is expressed in all human B-cell malignancies and engagement by the CD40 ligand (CD40L) is important for both cell proliferation and cell survival. CD40L has been shown to be co-expressed with CD40 in neoplastic B-cells from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkins Lymphoma (NHL), suggesting the importance of an autocrine CD40/CD40L loop in these malignancies. HCD122 (formerly known as CHIR-12.12) is a fully human, highly potent, IgG1 antagonist anti-CD40 monoclonal antibody (mAb) that blocks CD40/CD40L interactions in vitro and also mediates ADCC. Previous studies showed that HCD122 can mediate ADCC in vitro and has anti-proliferative and anti-tumor activities as a single agent in CLL, MM, and Burkitts Lymphoma in vitro and in vivo. In this study, the activity of HCD122 on a subtype of NHL, Diffuse Large B-Cell Lymphoma (DLBCL) was examined. The DLBCL derived cell lines, RL and SU-DHL-4, were selected for this study based upon in vivo characterization as well as their sensitivity to Rituximab as reported in the literature. These cell lines were subsequently confirmed for the expression of CD40 and CD20 by flow cytometry. The in vivo anti-tumor effects of HCD122 as single agent was demonstrated in these two xenograft models and was compared to Rituximab, an anti-CD20 antibody therapeutic currently approved for the treatment of relapsed or refractory, low-grade or follicular, NHL. HCD122 when administered intraperitoneally weekly at 1 mg/kg significantly reduced tumor growth with a tumor growth inhibition (TGI) of 85.5% (P<0.01) in the RL model. At the same dose and schedule in the RL model, TGI achieved with Rituximab was 31.7% (P>0.05). In the SU-DHL-4 model, an 85% TGI (P<0.01) was observed at the 1 mg/kg dose of HCD122. In comparison, Rituximab at this dose elicited a 57.6% TGI (P<0.05). Additionally, the downstream CD40/CD40L signal transduction pathways were also examined in order to elucidate the molecular mechanism underlying the HCD122-mediated effects in DLBCL. Taken together, these results support the clinical development of HCD122 for the treatment of DLBCL. Currently HCD122 is in Phase I trials for treatment of CLL and MM.

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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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