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THERAPEUTIC DISRUPTION OF THE BAFF- B-CELL RECEPTOR (BCR) CROSS-TALK IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS
Author(s):
Cody Paiva
,
Cody Paiva
Affiliations:
Oregon Health and Science University,Portland,United States
Taylor Rowland
,
Taylor Rowland
Affiliations:
Oregon Health and Science University,Portland,United States
Bhargava Sreekantham
,
Bhargava Sreekantham
Affiliations:
Oregon Health and Science University,Portland,United States
Olga Danilova
,
Olga Danilova
Affiliations:
Oregon Health and Science University,Portland,United States
Alexey Danilov
Alexey Danilov
Affiliations:
Oregon Health and Science University,Portland,United States
(Abstract release date: 05/18/17) EHA Library. Danilov A. 06/23/17; 181405; S118
Alexey Danilov
Alexey Danilov
Contributions
PDF Abstract
Abstract

Abstract: S118

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45

Location: Hall D

Background
 

Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses, potentially accounting for resistance to BCRi. The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied.

Aims
We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling.

Methods
We established a novel BAFF-expressing stromal co-culture model and employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib). We quantified CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry.

Results
CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X.

BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYK and LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi’s fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. By contrast, pevonedistat, an inhibitor of Nedd8-activating enzyme which we have previously shown to abrogate TNFR-mediated NFκB activation, blocked both canonical and non-canonical NFκB activity in BAFF-simulated CLL cells. SYK inhibitor entospletinib, but not other BCRi’s, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma and abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript.
BAFF receptor signals via the TRAF complex to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that TRAF complex could be directly responsible for SYK activation by BAFF. Indeed, IP experiments demonstrated that SYK directly complexed with TRAF2/3 in BAFF-stimulated neoplastic B-cells.

Conclusion
 

Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL.

Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology

Keyword(s): Chronic Lymphocytic Leukemia, BAFF

Abstract: S118

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45

Location: Hall D

Background
 

Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses, potentially accounting for resistance to BCRi. The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied.

Aims
We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling.

Methods
We established a novel BAFF-expressing stromal co-culture model and employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib). We quantified CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry.

Results
CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X.

BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYK and LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi’s fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. By contrast, pevonedistat, an inhibitor of Nedd8-activating enzyme which we have previously shown to abrogate TNFR-mediated NFκB activation, blocked both canonical and non-canonical NFκB activity in BAFF-simulated CLL cells. SYK inhibitor entospletinib, but not other BCRi’s, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma and abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript.
BAFF receptor signals via the TRAF complex to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that TRAF complex could be directly responsible for SYK activation by BAFF. Indeed, IP experiments demonstrated that SYK directly complexed with TRAF2/3 in BAFF-stimulated neoplastic B-cells.

Conclusion
 

Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL.

Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology

Keyword(s): Chronic Lymphocytic Leukemia, BAFF

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