Ongoing Activation of the BCR, NFκB, and Proliferation Pathways in Mantle Cell Lymphoma: Direct in Vivo Evidence for the Role of the Lymph Node Microenvironnment
Authors: Saba Nakhle , Liu Delong , Herman Sarah E. M. , Grant Cliona , Dunleavy Kieron , Pittaluga Stefania , Wilson Wyndham , Wiestner Adrian 
Published: November 2019

Abstract
Mantle cell lymphoma (MCL) is a mature CD5+ B-cell malignancy that represents ~6% of non-Hodgkin’s lymphoma, and continues to have a high fatality rate approaching 65%. The high rate of clinical responses to the BTK inhibitor ibrutinib in this disease (ORR 68%, CR 23%) was unexpected, given the putative derivation of this malignancy from naïve B cells. Despite considerable advances in MCL research, evidence of BCR engagement in-vivo continues to be very limited. The location of such BCR engagement, and in-vivo consequences on tumor biology remain largely unknown.
Using gene expression profiling (Affymetrix HU133 plus), we previously reported that MCL cells in the lymph node (LN) up-regulate BCR and NFκB target genes, and display higher expression of proliferation genes compared to the peripheral blood (PB). Here we present an extension of our initial report. Using flow cytometry, we measured levels of activated SYK, PLCγ, ERK, AKT, and P65 (RELA) in (CD5+ and CD19+ gated) MCL cells of 2 LN samples and 2 PB samples concomitantly collected from the same patients, and in a cohort of 16 PB samples. Consistent with the observed difference in BCR genes signature score (BCR score), we show that LN-resident MCL cells retain stronger kinases activity (SYK, PLCγ, ERK, AKT, and P65) as evidenced by their remarkably higher degree of phosphorylation when compared to their matched PB. A gene signature consists of a group of co-regulated genes whose combined expression pattern defines a distinct cell type or a cellular process. A gene signature score represents the average of the expression level of its genes. Moreover, levels of pSYK and pAKT strongly correlate with the BCR score (pSYKY348: r =.64, P <.02; pAKTT308: r =.53, P <.05), and levels of pP65 strongly correlate with the NFκB gene signature score (NFκB score) (r =.64, P<.02). pP65 levels also correlated with the BCR score (r =.54, P <.05) suggesting that the BCR pathway is responsible, at least in part, of NFκB activation. Furthermore, the BCR score in MCL whole LN biopsies (N = 30) strongly correlates with the chemokine CCL3 serum levels in the corresponding patients (r =.46; P =.008). CCL3 is one of the most strikingly upregulated genes upon BCR activation and while its role in MCL pathogenesis is unclear, it may serve as a biomarker of BCR activation. In concordance with an increased proliferation in the LN at the gene expression level, we detected a higher level of Ki67-positive MCL cells in the LN compared to PB. (P <.001). To test whether the proliferation signature is a surrogate for tumor proliferation, we studied the correlation between Ki67 positivity and proliferation score in both PB (by flow cytometry) and LN (IHC). We found that Ki67 and proliferation score strongly correlate with a correlation coefficient of 0.88 and 0.79 for PB and LN respectively (P <.0001 for both). The correlation between the BCR score and Ki67 was also high and significant (PB, r =.7, P =.006; LN, r =.39; P =.03)which suggests a role for BCR in driving tumor proliferation. To test ongoing pathway activation in MCL cell lines, mRNA from 10 MCL was profiled on Affymetrix HU133 arrays. BCR, NFκB, and proliferation scores were calculated. The BCR score was distinctly high in SP53 cell line, while the NFκB score was high in SP53 and JVM2, in the later this may be due to EBV infection. Higher baseline levels of activated BCR pathway kinases were observed in SP53 cells when compared to 3 other cell lines (Jeko, UPN1, and JVM2) that represent the spectrum of different levels of BCR and NFκB scores. The proliferation score was comparable in all cell lines. These 4 cell lines were tested against increasing concentration of ibrutinib for 48 and 72 h. Ibrutinib induced a greater growth inhibition in SP53 and JVM2, and resulted in apoptotic induction and cell cycle arrest. Ibrutinib however failed to induce apoptosis in Jeko and UPN1, and resulted only in cell cycle inhibition. Ibrutinib decreased ERK, PLCγ, and P65 phosphorylation in SP53 after 1h incubation.
In conclusion, we demonstrated activation of the BCR, and NFκB pathways in-vivo that is preferentially localized in the LN microenvironment. A high degree of BCR activation correlates with increased tumor proliferation. We also identified SP53 as an MCL cell line with active BCR and NFκB signaling that is ibrutinib sensitive.
Supported by the Intramural Research Program of NHLBI and NCI, NIH. We thank our patients for donating samples to make this research possible.

Disclosures
Wiestner: Pharmacyclics Inc.: Research Funding.