Cody Paiva

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Targeting neddylation effectively antagonizes nuclear factor-κB in chronic lymphocytic leukemia B-cells

Chronic lymphocytic leukemia (CLL) B-cells demonstrate both constitutive and stroma-mediated activation of nuclear factor-κB (NF-κB). NEDD8, a ubiquitin-like protein, regulates activity of Cullin-RING ubiquitin ligases (CRLs) and thus indirectly controls NF-κB activity. Inhibition of CRLs with MLN4924, an investigational agent that targets the NEDD8-activating enzyme, induces accumulation of CRL substrates, including inhibitor of NF-κB (IκB), a negative pathway modulator. We demonstrate that both continuous and pulse treatments with MLN4924 abrogate NF-κB activity in CLL B-cells ex vivo in a CD40L-expressing stromal co-culture system and identify pathways potentially responsible for resistance to MLN4924. To achieve long-lasting therapeutic effects in CLL, combination strategies are likely necessary.

SYK inhibition thwarts the BAFF - B-cell receptor crosstalk and thereby antagonizes Mcl-1 in chronic lymphocytic leukemia

Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

MIR21 is differentially expressed in the lymphoid tissue and modulated by stromal signalling in chronic lymphocytic leukaemia

MicroRNAs contribute to the initiation, progression, and dissemination of malignant clone and serve as important prognostic indicators in chronic lymphocytic leukaemia (CLL) (Calin & Croce, 2009). MIR21 is an oncogenic microRNA; its overexpression in peripheral blood (PB) CLL cells was linked to fludarabine resistance and unfavourable prognosis (Ferracin et al, 2010; Rossi et al, 2010; Ferrajoli et al, 2013). MIR21 promotes survival by blocking expression of apoptosis-related genes, for example via targeting phosphatase and tensin homolog (PTEN) and activation of phosphoinositide-3 kinase (Calin & Croce, 2009). We and others have shown that B-cell receptor (BCR) engagement may upregulate MIR21 in CLL (Humphries et al, 2013; Pede et al, 2013). While recent literature addresses the impact of MIR21 in the malignant B cells circulating in the periphery, few studies have focused on its expression in CLL lymph nodes, where the BCR is most active (Herishanu et al, 2011). The lymphoid tissue constitutes a vital niche where CLL cells receive pro-survival stromal signals and express additional anti-apoptotic BCL2 family members absent in the peripheral cells. In those tissues, some microRNAs demonstrate distinct expression patterns. For example, high MIR155HG (BIC) expression (the primary transcript of MIR155) was found in CLL proliferation centres using in situ hybridization (ISH). However, this technique failed to detect microRNAs expressed at lower levels, including MIR21 (Wang et al, 2008). Here we investigated MIR21 expression in the lymphoid tissue of patients with CLL. MIR21 expression was characterized at single cell resolution in 30 formalin-fixed paraffin-embedded (FFPE) lymph nodes from patients with CLL and four control (tonsil) tissues. We employed a modified combined fluorescence-based ISH (FISH)/immunohistochemistry (IHC) assay as previously reported (Sempere et al, 2010). This assay enabled us to co-detect expression of MIR21 and RNU6-1 RNAs as well as CD20 protein (also termed MS4A1) in FFPE tissues. Briefly, 50 and 30-terminally tagged locked nucleic acid-modified DNA probes against MIR21 (2x-fluorescein tag) and RNU6-1 (2x-biotin tag), both at 50 nmol/l, were hybridized to tissue slides for 75 min at 45°C. Sequential rounds of horseradish peroxidase (HRP)-mediated deposition for 20 min with fluoresceinand rodhamine-conjugated substrates were performed to detect MIR21 and RNU6-1 probes, respectively. A final round of HRP-mediated deposition for 20 min with Dylight594-conjugated substrate was performed to detect CD20. MIR21 was differentially expressed in the lymphoid tissues of patients with CLL (Fig 1A). Of the 30 lymph nodes, MIR21 was detected in 13 (43%); of these, three lymph nodes demonstrated strong staining and 10 showed weak staining. The remaining 17 tissue specimens (56%) stained negative for MIR21 despite RNU6-1 staining. Where present, strong MIR21 expression followed a focal staining pattern. Co-staining for CD20 confirmed MIR21 detection in the neoplastic CLL cells (Fig 1B). In this small cohort, MIR21 positivity did not predict time to first treatment or correlate with CD38 or ZAP70 expression. Interestingly, two of the four control tissues demonstrated strong staining for MIR21, indicating that its expression in the lymphoid organs is not restricted to clonal neoplastic B-cells. Stromal signalling may modulate microRNA expression in CLL. Lymph nodes and bone marrow are the sites of CLL cell proliferation, with expression of gene signatures indicating activation of the BCR and nuclear factor-jB (NF-jB) pathways (Herishanu et al, 2011). We have previously shown that BCR signalling induces MIR21 expression in a subset of CLL, with 5/10 tested samples demonstrating an increase in this microRNA (Humphries et al, 2013). Given the variability of MIR21 expression in primary lymphoid tissues, we then studied if MIR21 was further modulated by stromal signalling in vitro. First, MIR21 expression levels were evaluated in PB. PB CLL cells (n = 33; 90 9% of patients previously untreated) and normal B-cells (from healthy volunteers, n = 7) were processed as described earlier (Humphries et al, 2013). While it was previously reported that RNU6-1, a commonly used normalizer, is differentially expressed between the normal and neoplastic B-cells (Ferrajoli et al, 2013), we did not detect a significant difference in the cycle thresholds between these two groups. Consistent with previous reports, we found increased levels of MIR21 in PB CLL cells compared with normal B-cells, although both groups demonstrated extensive variability in expression of this microRNA (Fig 2A). Similar results were obtained using GAPDH as a normalizer. Subsequently, we modelled lymph node microenvironment in vitro using CD40LG-expressing L cells or control L cells. Stromal co-cultures protect CLL cells from spontaneous apoptosis, while CD40LG-expressing stroma further Correspondence

Pevonedistat, a Nedd8-activating enzyme inhibitor, sensitizes neoplastic B-cells to death receptor-mediated apoptosis

While death receptor ligands (Fas and TRAIL) kill chemoresistant tumor cell lines, related therapies have limited clinical efficacy as single agents. Death receptor signaling is modulated by nuclear factor-κB (NFκB), a family of transcription factors which are constitutively active in B-cell malignancies. We and others have shown that pevonedistat, an investigational inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in B-cell neoplasia. Here we demonstrate that diffuse large B-cell lymphoma, particularly activated B-cell type, and primary chronic lymphocytic leukemia cells are re-sensitized to extrinsic apoptosis by pevonedistat. Pevonedistat enhanced caspase-8 processing following death receptor ligation, and downmodulated cFLIP, a NFκB-regulated protease-deficient caspase homolog. However, treatment with pevonedistat did not modulate death-inducing signaling complex in neoplastic B-cells, suggesting that they were sensitized to death ligands through the mitochondrial pathway. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies.