ALBERT

Description: Human pentraxins are a family of proteins with a unique pentameric structure. Unlike C-reactive protein (CRP), serum amyloid P (SAP) and pentraxin-3 (PTX3) play an opposite role in tissue remodeling. PTX3 induces whereas SAP inhibits the differentiation of CD14+ monocytes into fiborcytes. While in patients with CLL CRP levels are high and were found to be associated with poor overall survival (OS) (Herishanu et al. Ann Med 2017), little is known about the plasma levels or clinical significance of other pentraxins in CLL. Therefore, we obtained plasma sample from 36 randomly chosen treatment-naïve CLL patients and 12 age-matched healthy individuals and, using an enzyme linked immuno-sorbent assay, found that PTX3, CRP and SAP plasma levels were significantly higher in CLL patients than in healthy individuals (P

Description: High dose chemotherapy of Ph+ ALL is rarely curative and clinical responses to protein kinase inhibitors have been transient. Although new regimens combining chemotherapy with Bcr-Abl kinase inhibitors improve survival, the long-term prognosis of patients with Ph+ ALL remains guarded. Thus, novel therapeutic strategies are needed. Hsp90 is a ubiquitous molecular chaperone protein required for the folding, activation and assembly of mediators of signal transduction, cell cycle control, and transcription regulation. The Hsp90 inhibitor EC141 (Biogen Idec, Inc.) blocks the chaperone activity of Hsp90 and induces proteasomal degradation of it’s client proteins. Because Hsp90 is a chaperone of Bcr-Abl we investigated the activity of EC141 against the Ph+ ALL B-cell lines Z-119, Z-181 and Z-33 (Estrov et al. J Cell Physiol166: 618, 1996; Leukemia10:1534, 1996). First we studied the effect of EC141 on Hsp levels in Ph+ ALL cells. EC141 (50 nM) down-regulated the protein levels of Hsp90 and upregulated those of Hsp70. Then, the effect of EC141 on the proliferation of Ph+ ALL cells was evaluated using the MTT assay. EC141 inhibited the growth and metabolic activity of Z-119, Z-181 and Z-33 Ph+ ALL cells in a dose-dependent manner at concentrations ranging from 1 to 100 nM. Similar results were obtained with primary bone marrow cells from patients with Ph+ ALL. Using the ALL blast colony culture assay we found that EC141 inhibited the proliferation of marrow-derived ALL colony-forming cells in a dose-dependent fashion. To explore the mechanism of action Z-181 were incubated cells with increasing concentrations of EC141; immunoprecipitation and Western immunoblotting were used to detect changes in cellular protein levels. EC141 degraded the Bcr-Abl p190 protein and inhibited the phosphorylation of CrkL in a dose-dependent manner. Furthermore, exposure of Z-181 cells to EC141 resulted in a time- and dose-dependent activation of procaspase 3, cleavage of poly (adenosine diphosphate-ribose) polymerase and apoptotic cell death as assessed by Annexin V. Taken together, our data suggest that EC141 degrades the Bcr-Abl p190 protein, inhibits proliferation, and induces apoptosis of Ph+ ALL cells. Additional studies aimed at investigating the in vivo activity of EC141 in Ph+ ALL are warranted.

Description: Janus kinases (JAK) are tyrosine kinases associated with both cytokine receptors and downstream signal transducer and activator of transcription (Stat) proteins. Upon activation of JAK by a variety of cytokines and growth factors, Stats translocate to the nucleus and promote transcription of target genes. Constitutive activation of Stat proteins in AML has been associated with poor prognosis and AG490, an inhibitor of this pathway, was shown to suppress AML cell proliferation in vitro. WP-1066 represents a further development of AG490 with biological activity at significantly lower concentrations. Therefore, we studied the effects of WP-1066 on the AML cell lines OCIM2 and K562 and on fresh bone marrow aspirates obtained from five newly diagnosed AML patients. We found that WP-1066 inhibited the proliferation of OCIM2 and K562 cells and of fresh marrow AML blast colony-forming cells in a dose-dependent fashion at concentrations ranging from 0.5 to 3 μM. WP-1066 completely abrogated the growth of leukemia cells at a concentration of 3 μM. Furthermore, WP-1066 induced a cell cycle arrest of OCIM2 and K562 cells. Incubation of AML cells with 2 μM of WP-1066 resulted in a time-dependent accumulation of OCIM2 and K562 cells in the sub-G0 phase of the cell cycle. Those leukemia cells underwent apoptotic cell death as assessed by annexin V-FITC. Incubation of OCIM2 cells with 0.5 to 3 μM WP-1066 for 2 hours induced a dose-dependent apoptosis in 52% of the cells. A 4 hour exposure of either OCIM2 or K562 cells to 2 μM of WP-1066 induced caspase 3 activation and PARP cleavage. As expected, WP-1066 inhibited Stat3 and Stat5 phosphorylation in K562 and OCIM2 cells both in a time- and dose-dependent manner, confirming that inhibition of the JAK-Stat pathway is its mechanism of action. Overall, our data showing that WP-1066 inhibits the JAK-Stat pathway, suppresses proliferation, induces cell cycle arrest and apoptosis of AML cells, suggest that the activity of this compound warrants further exploitation aimed at developing WP-1066 for future therapy of AML.

Description: Introduction: While in CLL cells phosphorylation of STAT3 on serine 727 residues is constitutive, phosphorylation of STAT3 on tyrosine 705 residues is inducible. Cytokines, such as IL-6, or IgM antibodies that activate CLL cells' BCR, induce tyrosine phosphorylated (p) STAT3. However, whereas IL-6 induces tyrosine pSTAT3 phosphorylation within 15 minutes, IgM induces pSTAT3 within ≥ 2-4 hours. The reason for the delayed IgM-induced phosphorylation is unknown. Like STAT3, the transcription factor NF-κB is constitutively activated in CLL cells and stimulation of the BCR activates NF-κB. Whether BCR stimulation upsurges NF-κB's transcriptional activity has not been elucidated. Because IL-6 is an NF-κB-target gene and, like IL-6, IgM antibodies induce tyrosine pSTAT3, we wondered whether prolonged stimulation with IgM antibodies induces tyrosine pSTAT3 via NF-κB-mediated induction of IL-6 in CLL cells. Methods: We incubated peripheral blood CLL cells in the presence or absence of IgM antibodies or IL-6, and harvested the cells at different time points. Total RNA was extracted using TRIzol (Life technology), cDNA was synthesized with Super Script First synthesis System for RT-PCR (Invitrogen), and NF-κB-target gene expression was quantified using RT-PCR (Invitrogen Life Sciences). To measure the levels of tyrosine pSTAT3 we used flow cytometry and to assess binding of NF-κB (p65) to DNA we utilized an electromobility shift assay (EMSA) using an NF-κB-binding site labelled DNA probe. Results: The transcriptional activity of NF-κB was studied using a PCR array that profiles the expression of 83 NF-κB-target genes. To reduce the 'noise' from stochastic variability in gene expression we first identified a core of genes that are expressed in cells from all patients' samples. To that aim we ranked the Ct values in each array and considered all genes that were amplified earlier than the cycle in the 75th percentile. Using this approach we identified 35 genes (42% of genes represented in the array) that were amplified in all 6 patients' samples. Annotation analysis revealed that the key pathways common to these 35 genes included 'Positive regulation of the NF-κB cascade', 'Inflammation' and 'Negative regulation of apoptosis'. Applying stringent criteria we identified 5 genes common to all cases that were amplified prior to the cycle representing the 25th percentile. Most amplified genes detected in all samples prior to stimulation (28/35, 80%) were also detected after 4 h of IgM stimulation, confirming that NF-κB is constitutively activated in CLL cells. However, 19 addition genes (19/83, 23%of the genes in the array) were detected in all IgM-stimulated but not in unstimulated cells. Remarkably, IL-6 was detected in all cases only after IgM stimulation. Furthermore, the delta-delta Ct method identified an IgM-induced time-dependent increment in IL-6 and IL-8, suggesting that IL-6 expression is dependent on stimulation of the BCR. Indeed IL-6 neutralizing antibodies significantly reduced the levels of tyrosine pSTAT3 in CLL cells incubated for 18 h with IgM antibodies. In addition, EMSA studies using CLL cells from 4 different patients showed that stimulation of the BCR with IgM antibodies increased the binding of NF-κB to DNA in a time-dependent manner. Moreover, the JAK2 inhibitor Ruxolitinib attenuated the NF-κB-DNA binding, suggesting that long exposure to IgM antibodies induces activation of NF-κB, a process mediated in part by IL-6 that activates the JAK2/STAT3 pathway. Conclusions: The BCR of CLL cells is stimulated in the bone marrow and lymph nodes. However, whereas the immediate effects of BCR stimulation have been excessively studied, the successive effect BCR stimulation is poorly understood. We found that stimulation of the BCR induces tyrosine phosphorylation of STAT3 via NF-κB-mediated induction of IL-6, a process that requires protracted BCR stimulation. Although NF-κB is constitutively activated in CLL cells, continuous activation of the BCR further activates NF-κB. Continuous stimulation of the BCR increases the levels of IL-6 that, upon binding to its receptor, activates STAT3 that in turn activates NF-κB. Taken together, our data suggest that agents, such as Ruxolitinib, that inhibit the successive effects of BCR activation, would become effective therapeutic agents in CLL. Disclosures Rozovski: Novartis: Other: Advisory board. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy.