Therapeutic Targeting of B-Cell Receptor Signaling Pathways

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-27-SCI-27 ◽  
Author(s):  
John C. Byrd
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Low Grade ◽  
B Cell Malignancies ◽  
Bcr Signaling ◽  
Phosphoinositide 3 Kinase ◽  
Continuous Dosing

Abstract Abstract SCI-27 Targeted therapy in hematologic malignancies has achieved significant therapeutic success when relatively selective inhibition is attainable to a target dispensable to the majority of normal cells. The best appreciated example of this is imatinib in chronic myeloid leukemia (CML), in which a single translocation forms a fusion protein involving the tyrosine kinase ABL that molecularly defines the disease. Kinase inhibition of ABL in this setting by imatinib (or other second- and third-generation kinase inhibitors) promotes durable, long-term remission in CML patients. The great majority of B-cell malignancies lack a characteristic translocation, activating mutation, or other aberration that facilitates such targeted therapy as employed in CML. Nonetheless, a common global signaling pathway involving the B-cell receptor (BCR) pathway has emerged as one that might be important to the control of these diseases. Furthermore, mouse models of select kinase targets (phosphoinositide-3 kinase p110 isoform-delta and Bruton's tyrosine kinase [BTK]) have shown that these are dispensable relative to long-term survival. Indeed, knockout or mutation of BTK and p110 delta each have a modest phenotype outside of diminished B-cell development and function. Based upon these preclinical observations, several molecules targeting BCR signaling have come forward to the clinic with exciting results across a wide range of B-cell malignancies. GS-1101 is a selective phosphoinositide-3 kinase p100 delta inhibitor with a very favorable toxicity profile that has shown promising clinical activity in low-grade lymphoma and chronic lymphocytic leukemia (CLL). The toxicity of this orally administered agent is quite modest in the majority of patients and allows sustained continuous dosing. Similarly, ibrutinib is an irreversible inhibitor of BTK and has shown promising clinical potential in an even broader range of B-cell malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, low-grade lymphoma, and CLL. Toxicity with ibrutinib has also been modest, allowing long-term continuous dosing. Notably, each of these agents also produces an atypical mobilization of malignant lymphocytes into the blood soon after treatment. This treatment lymphocytosis is BCR-target-related due to diminished CXCR4/SDF-1 interface between tumor cells and stromal cells in the bone marrow, with egress of these cells to the blood. GS-1101 and ibrutinib are now entering phase III studies for regulatory approval and offer great potential to change the treatment paradigm of both CLL and B-cell non-Hodgkin lymphoma (NHL). Questions moving forward with these agents will include molecular predictors of response, feasibility and efficacy of combining with other effective therapies, and mechanisms of resistance. The scientific session presentation will provide an overview of the most promising BCR signaling agents in CLL and NHL clinical trials. Disclosures: No relevant conflicts of interest to declare.

Targeting B-cell receptor and PI3K signaling in diffuse large B-cell lymphoma

Blood ◽  
2021 ◽  
Author(s):  
Wendan Xu ◽  
Philipp Berning ◽  
Georg Lenz
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Genetic Alterations ◽  
B Cell Receptor ◽  
Special Focus ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category comprising distinct molecular subtypes characterized by diverse genetic aberrations that dictate patient outcome. As roughly one-third of DLBCL patients are not cured by current standard chemo-immunotherapy a better understanding of the molecular pathogenesis is warranted to improve outcome. B-cell receptor (BCR) signaling is crucial for the development, growth and survival of both normal and a substantial fraction of malignant B-cells. Various analyses revealed genetic alterations of central components of the BCR or its downstream signaling effectors in some subtypes of DLBCL. Thus, BCR signaling and the downstream NF-κB and PI3K cascades have been proposed as potential targets for the treatment of DLBCL patients. As one of the main effectors of BCR activation, PI3K mediated signals play a crucial role in the pathogenesis and survival of DLBCL. In this review, we summarize our current understanding of BCR signaling with a special focus on the PI3K pathway in DLBCL and how to utilize this knowledge therapeutically.

scholarly journals Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2

2021 ◽  
Vol 5 (1) ◽  
pp. 185-197
Author(s):  
Wenjun Wu ◽  
Weige Wang ◽  
Carrie A. Franzen ◽  
Hui Guo ◽  
Jimmy Lee ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Mantle Cell ◽  
Bcr Signaling

Abstract Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

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

Blood ◽  
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.

The BCR-Associated Tyrosine Kinase SYK Is Linked to the Activation of AKT in Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1586-1586
Author(s):  
Martina Rudelius ◽  
Denise Sebasigari ◽  
Yoko Tabe ◽  
Theresa Davies-Hill ◽  
Falko Fend ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Negative Regulator ◽  
Akt Pathway ◽  
Akt Activation ◽  
Mantle Cell ◽  
Bcr Signaling

Abstract Mantle cell lymphoma (MCL) is comprised of two major clinical-pathologic subtypes, the more common typical MCL and the blastoid variant (bv-MCL). We recently demonstrated that the PI3K/AKT pathway is preferentially activated in bv-MCL. In all bv-MCL cases, activated phosphorylated AKT (p-AKT) expression was accompanied by the phosphorylation of downstream targets, and pharmacological inhibition of the pathway abrogated or reduced the phosphorylation of AKT and its targets and resulted in cell cycle arrest and apoptosis. Activation of the pathway was neither the result of mutations in the p110 PI3K catalytic subunit, nor the loss of the negative regulator PTEN, in the majority of cases. Since the B-cell receptor (BCR) signaling pathway has been shown to be a major activator of the AKT pathway via SYK/PI3K interactions, the goal of the current study was to gain insight into its possible role in MCL. Initial studies revealed activated SYK in 4 MCL cell lines studied, and immunoprecipitation studies showed a linkage between SYK, the adaptor protein BCAP, and the p85 regulatory subunit of PI3K. To analyze a possible causal connection between SYK and AKT activation, we used both a classical inhibitor approach [piceatannol and SYK-II (Calbiochem)] and a targeted gene knockdown approach employing a lentiviral mirRNA delivery system in two cell lines (Z138C and Granta 519). Functional knock-down and downstream effects were analyzed by western blotting, assessment of PIP3 levels, MTT-test, and flow-cytometry. Using the two approaches, we could block the activation of PI3K/AKT pathway and phosphorylation of AKT downstream targets in the two MCL cell lines, indicating the importance of the B-cell receptor associated SYK kinase in activation of the PI3K/AKT pathway. Activation of BCR signaling components, including SYK, PLCγ2, and LYN, were then assessed by western blotting in 28 primary MCL cases (12 typical and 16 blastoid). Phosphorylated (active) SYK and PLCγ2 (a direct SYK target) were detected in all 28 primary cases (12 blastoid and 16 typical). Surprisingly, there were no significant differences between the two groups, despite the difference in their AKT status. However, there was a distinct difference in the activation state of LYN, as assessed by its phosphorylation at Tyr 396, which was correlated with AKT activation. While Tyr 396 was phosphorylated in the majority of typical MCL, the converse was true for the bv-MCL and cell lines, suggesting that LYN was inactive in the latter. Although LYN has been shown to have both positive and negative roles in BCR activation, the net effect of loss of LYN activity is hyperactivation of B-cell signaling. Given the consistent effect of SYK inhibition on the activity of the PI3K/AKT pathway in the cell lines, we hypothesize that loss of a negative regulator downstream of SYK, possibly LYN, is additionally required to allow activation of the PI3K/AKT pathway in MCL.

The PI3K Delta Inhibitor Idelalisib Interferes With Pre-B Cell Receptor Signaling In Acute Lymphoblastic Leukemia (ALL): A New Therapeutic Concept

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2632-2632
Author(s):  
Nathalie Y. Rosin ◽  
Ekaterina Kim ◽  
Stefan Koehrer ◽  
Zhiqiang Wang ◽  
Susan O'Brien ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Differentially Expressed ◽  
B Cell Malignancies ◽  
Bcr Signaling ◽  
Dose Dependent

Abstract Phosphoinositide-3-kinases (PI3K) play an important role in transmitting signals from surface receptors such as the B-cell receptor (BCR), cytokine receptors and receptor tyrosine kinases, that result in survival and growth of normal and malignant B cells. In mature B-cell malignancies such as CLL and indolent B-NHL, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a p110δ-isoform-specific PI3K inhibitor that is highly active in patients with CLL and indolent NHL. In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. RNA expression of the PI3K isoforms α, β, γ, and δ was detected in all B-ALL cell lines. Protein expression was analyzed by immunoblotting. We noted that in vitro responsiveness to idelalisib treatment was associated with protein expression of the δ isoform and presence of the Pre-BCR. We found that treatment of B-ALL cell lines with idelalisib at concentrations between 10 nM and 5 µM inhibited metabolism and growth of B-ALL cells expressing a pre-BCR, whereas only minor effects were observed in Pro-B. To monitor the expression and phosphorylation of proteins (CD72, Akt, Plcγ2, S6, Vav) involved in BCR and PI3K signaling, we prepared protein lysates of B-ALL cell lines. Cells were treated with idelalisib with/without stimulation of the µ-heavy chain of the (Pre-) BCR. We were able to show an increase of pAktSer473 and pS6Ser235/236 after stimulation, as well as a decrease in a dose dependent manner with idelalisib. The decreased amount of Akt phosphorylation was linear with the sensitivity to idelalisib treatment of the cell lines. To investigate intracellular calcium mobilization which occurs in B cells through pre-BCR stimulation, we treated cells with idelalisib and stimulated them with anti-Igµ. A dose dependent decrease in calcium flux was observed in 5 of 6 pre-B cell lines. To examine the effects of idelalisib treatment on the gene expression of pre-B ALL cells, gene expression profiling (GEP) was performed. This revealed down regulation of several genes involved in MAP-Kinase signaling, (Pre-) BCR signaling and Natural Killer cell (NK-cell) mediated cytotoxicity. For the Pre-BCR signaling pathway several genes were differentially expressed, including genes encoding the following proteins, which were found to be down regulated after 3 days of 1 µM idelalisib treatment: BCL-6, CD72, CD79a, Vav, and Plcγ2. To verify the data of the GEP, qPCR analysis was performed. To further investigate the effects of idelalisib on proteins involved in BCR signaling, six Pre-B-ALL cell lines, as well as one mature and one Pro-B cell line were treated with 1 µM idelalisib and the protein expression was quantified after immunoblotting. Most of the proteins that were differentially expressed on genomic levels have also been differentially expressed on proteomic levels and therefore confirmed the effect on Pre-BCR signaling. In summary, these experiments demonstrate inhibition of Pre-BCR signaling on both gene and protein expression levels via idelalisib treatment of Pre-B-ALL cell lines and support the importance of clinical development of the δ isoform specific PI3K inhibitor idelalisib. Disclosures: Burger: Gilead Sciences Inc: Research Funding.

scholarly journals Distinct Roles of Phosphoinositide-3 Kinase and Phospholipase Cγ2 in B-Cell Receptor-Mediated Signal Transduction

2006 ◽  
Vol 26 (1) ◽  
pp. 88-99 ◽  
Author(s):  
Xuezhi Dai ◽  
Yuhong Chen ◽  
James Schuman ◽  
Zichun Hua ◽  
John W. Adamson ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Phosphoinositide 3 Kinase ◽  
Cell Transition

ABSTRACT During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cγ2 (PLCγ2). PLCγ2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCγ2 using PI3K−/−, PLCγ2−/−, and PI3K−/− PLCγ2−/− B cells. Interestingly, PLCγ2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCγ2. Moreover, whereas PI3K−/− PLCγ2−/− double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K−/− PLCγ2−/− fetal liver cells transplanted into B-cell null JAK3−/− mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCγ2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCγ2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCγ2, activation of PLCγ2 can occur independently of PI3K and that PI3K and PLCγ2 also have distinct functions in BCR signal transduction.

scholarly journals Mechanisms of B Cell Receptor Activation and Responses to B Cell Receptor Inhibitors in B Cell Malignancies

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1396 ◽  
Author(s):  
Dimitar G. Efremov ◽  
Sven Turkalj ◽  
Luca Laurenti
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Receptor Activation ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Downstream Signaling ◽  
Pathway Activation

The B cell receptor (BCR) pathway has been identified as a potential therapeutic target in a number of common B cell malignancies, including chronic lymphocytic leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, and Waldenstrom’s macroglobulinemia. This finding has resulted in the development of numerous drugs that target this pathway, including various inhibitors of the kinases BTK, PI3K, and SYK. Several of these drugs have been approved in recent years for clinical use, resulting in a profound change in the way these diseases are currently being treated. However, the response rates and durability of responses vary largely across the different disease entities, suggesting a different proportion of patients with an activated BCR pathway and different mechanisms of BCR pathway activation. Indeed, several antigen-dependent and antigen-independent mechanisms have recently been described and shown to result in the activation of distinct downstream signaling pathways. The purpose of this review is to provide an overview of the mechanisms responsible for the activation of the BCR pathway in different B cell malignancies and to correlate these mechanisms with clinical responses to treatment with BCR inhibitors.

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

Blood ◽  
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.

scholarly journals Protein Kinase CK1α Sustains B-Cell Receptor Signaling in Mantle Cell Lymphoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Sabrina Manni ◽  
Anna Fregnani ◽  
Laura Quotti Tubi ◽  
Zaira Spinello ◽  
Marco Carraro ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Kinase ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Signaling Cascades ◽  
Mantle Cell ◽  
Bcr Signaling

Mantle Cell Lymphoma (MCL) is still an incurable B-cell malignancy characterized by poor prognosis and frequent relapses. B Cell Receptor (BCR) signaling inhibitors, in particular of the kinases BTK and PI3Kγ/δ, have demonstrated clinically meaningful anti-proliferative effects in B cell tumors. However, refractoriness to these drugs may develop, portending a dismal prognosis. Protein kinase CK1α is an emerging pro-growth enzyme in B cell malignancies. In multiple myeloma, this kinase sustains β-catenin and AKT-dependent survival and is involved in the activation of NF-κB in B cells. In this study, we analyzed the role of CK1α on MCL cell survival and proliferation, on the regulation of BCR-related BTK, NF-κB, PI3K/AKT signaling cascades and the effects of CK1α chemical inhibition or gene silencing in association with the BTK inhibitor Ibrutinib or the PI3Kγ/δ inhibitor Duvelisib. CK1α was found highly expressed in MCL cells as compared to normal B cells. The inactivation/loss of CK1α caused MCL cell apoptosis and proliferation arrest. CK1α sustained BCR signaling, in particular the NF-κB, AKT and BTK pathways by modulating the phosphorylation of Ser 652 on CARD11, Ser 536 p65 on NF-κB, Ser 473 on AKT, Tyr 223 on BTK, as well as the protein levels. We also provided evidence that CK1α-mediated regulation of CARD11 and BTK likely implicates a physical interaction. The combination of CK1α inhibition with Ibrutinib or Duvelisib synergistically increased cytotoxicity, leading to a further decrease of the activation of BCR signaling pathways. Therefore, CK1α sustains MCL growth through the regulation of BCR-linked survival signaling cascades and protects from Ibrutinib/Duvelisib-induced apoptosis. Thus, CK1α could be considered as a rational molecular target for the treatment of MCL, in association with novel agents.

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