Background: How IB kinase K171E and K171T mutations mediate lymphomagenesis is not known. Results: We performed biochemical, molecular modeling and TALEN-based knockin studies on wild-type and mutant IKK. Conclusion: Mutant IKK molecules are constitutively active in an activation-loop phosphorylation-independent manner. Significance: These results have broad implications for the function of positively charged residues in all activation loop-dependent kinases.
Survival, proliferation, and resistance to chemotherapy in CLL cells have been shown to be consistently associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by the BCR. Key molecules in this pathway are LYN and SYK (Spleen tyrosine kinase), as well as PI3K, BTK (Bruton’s tyrosine kinase), and others. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for suppression of LYN kinase. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle repeated every 28 days, while effective up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, all had responses according to IWCLL criteria as follows: The median time to progression was 21 months. In the first week multiple blood samples were taken for analysis of target inhibition and subsequent apoptosis. The schedule of administered agents was altered in the first week to determine which components were associated with which downstream effects. Hence, dasatinib was given on Day 1, no treatment was administered on Day 2, F and R without dasatinib on Day 3, dasatinib with FR on Day 4, and dasatinib with F on Day 5. Initial in vitro studies revealed inhibition of phosphorylation of Lyn at 6 hours after patients were given dasatinib on Days 1 and 4, with recovery by 24 hours. Day 3 treatment with FR but without dasatinib showed no such inhibition at 6 hours. Assessment of global tyrosine phosphorylation in CLL cells showed this same pattern, including that of Syk phosphorylation, specifically. Flow cytometry for annexin-V demonstrated that apoptosis was greatest on Day 4 after 6 hours of exposure to all 3 drugs. We conclude the following: 1) the combination of dasatinib with FR, as seen in the first 10 patients of this study, was associated with excellent responses in blood and lymph nodes as assessed by physical exam, 2) the combination was well tolerated with mainly hematologic toxicity, 3) the inhibition of phosphorylation of Lyn and Syk was associated with apoptosis and clinical response. This combination may have therapeutic promise in advanced CLL and is worthy of further investigation. Disclosures: Off Label Use: Dasatinib use in CLL is off-label. This trial shows that dasatinib may be beneficial in the treatment of CLL. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding. Fathi:Seattle Genetics, Inc.: Advisory/Scientific board membership Other, Research Funding; Millennium: Research Funding; Agios: Membership on an entity’s Board of Directors or advisory committees; Teva: Membership on an entity’s Board of Directors or advisory committees.
Survival, proliferation, and resistance to chemotherapy in CLL cells have been consistently shown to be associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by it. Key molecules in this pathway are Lyn and Syk (Spleen tyrosine kinase), as well as PI3K, Btk (Bruton’s tyrosine kinase), and ERK. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for the direct suppression of Lyn kinase and Btk. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle, repeated every 28 days, for up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, the schedule of treatment was altered to determine signal transduction effects and resultant apoptosis of the CLL cells due to the different components of the regimen. Hence, only dasatinib was given on Day 1, only fludarabine and rituximab on Day 3, and all 3 drugs on Day 4. Blood samples were obtained from the patients before dosing and at 6 hours after treatment to measure effects on the CLL cells, which were isolated by standard Ficoll separation and frozen until the assays could be performed. For these 10 patients the median time to progression (TTP) was 21 months, and the initial clinical response was as follows: Site CR CRi PR SD PD ORR 95% CI Blood 4 2 4 0 0 100% 74%, 100% Nodes 6 1 1 1 1 80% 49%, 96% CT 2 0 2 6 0 40% 15%, 70% IWCLL 2 0 2 5 1 40% 15%, 70% Key: CR=complete response in blood = < 4,000/ul lymphocytes, CR in nodes = no nodes palpable by PE, CRi = complete response with incomplete blood recovery, PR=partial response, SD=stable disease, PD=progressive disease, ORR=overall response rate, CI=confidence interval. IWCLL = International Workshop on CLL criteria. The patterns of signal transduction in response to the various drugs at 6 hours are shown in aggregate for the patients below. The numbers represent the ratio of phosphorylated protein to total protein at the times indicated with respect to their baseline levels set as 100%. The phosphorylation sites tested were p-Lyn (Y416), p-Syk (Y352), p-ERK1/2 (T202/Y204). Phosphorylation at 6 h after indicated treatment – % of baseline ± SE (N=7): Day 1 (D) Day 3 (F+R, no D) Day 4 (D+F+R) p-Lyn/Lyn: 42% ± 3% 207% ± 63% 58% ± 13% p-Syk/Syk: 34% ± 15% 122% ± 67% 36% ± 15% p-ERK/ERK : 64% ± 22% 168% ± 40% 56% ± 22% The patterns of signal transduction for the 3 patients with the most favorable outcome (TTP and OS) were compared to that for the 4 patients with the poorest clinical outcome. The baseline ratios of phospho-ERK1/2 to ERK1/2 (pre-treatment) correlated most strongly with outcome. Those with a good outcome exhibited low basal p-ERK/ERK (mean 1.0, range 0.1 to 1.8 percent), while patients with a poor outcome exhibited high basal p-ERK/ERK (mean 22.1, range 2.2 to 65.6 percent). Conclusions: For most patients, dasatinib inhibits (directly or indirectly) phosphorylation of Lyn kinase, Syk kinase, and ERK1/2 in the first 6 hours. The degree of apoptosis (to be presented, but not shown here) resulting from this is variable and is probably affected by activation of the PI3K/Akt pathway and other pathways. The long-term clinical outcome of our patients correlated strongly with the baseline phosphorylation of ERK1/2, suggesting that future treatments of patients with CLL might benefit from targeting ERK directly, or by targeting other molecules in the MAPK pathway. Disclosures Off Label Use: Dasatinib for treatment of CLL is off-label use. We present a rationale for its use in CLL patients.. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Attar:Agios: Employment. Fathi:Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.
1258 Poster Board I-280 Background An inhibitory signaling pathway involving sialic acid 9-O-acetyl esterase (SIAE), sialic acid binding lectins (Siglecs) particularly Siglec-2/CD22, the Lyn tyrosine kinase, and the SH2 domain containing tyrosine phosphatase, SHP-1, attenuates B cell receptor signaling and sets a threshold for B cell activation. A key step in the process is the requirement that SIAE access N-glycans on Siglec ligands and remove 9-O-acetyl groups from terminal αa2,6 linked sialic acid moieties. Siglec-ligand interaction is followed by phosphorylation of ITIM tyrosines on CD22 by Lyn, and the recruitment of SHP-1 by CD22 resulting in signal attenuation (Cariappa et al., J.Exp.Med.2009, 206, 125). While Lyn has both positive and negative signaling functions, knockout mice studies suggest that inhibitory functions are dominant. Previous studies have shown that CLL cells overexpress active Lyn at the protein level and that Lyn is localized to sites beyond the plasma membrane. Although cell surface expression of CD22 is reduced in CLL, it is not known if CD22 can be accessed in CLL cells by promiscuously active Lyn. We sought to ask if cancer progression in CLL involves the evolution of mechanisms to evade inhibitory signaling, thus tipping the balance towards positive, pro-proliferative signaling by Lyn. Methods CLL B cells from patient and control subjects were isolated. Immunoprecipitation and Western blot approaches were used to quantitate the total cellular levels of Lyn and CD22αa and β proteins at the protein level, the ratio of CD22 phosphorylated on an inhibitory tyrosine to total CD22, recruitment of SHP-1 by CD22, the activation of Syk, the expression of c-Cbl and the recruitment of PI3K by c-Cbl in CLL and control B cells. Results A modest decrease in total CD22αa and β proteins was observed in CLL but a dramatic reduction in the proportion of ITIM-phosphorylated CD22, and a reduction in the recruitment of SHP-1 by CD22 in CLL B cells. Decreased inhibitory signaling in CLL correlates with an increase in active Syk. An increase in c-Cbl protein levels was observed and an increased recruitment of p85PI3K was observed specifically in Zap-70 positive CLL. Conclusions Defective inhibitory signaling may contribute to disease progression in CLL. This defect probably results from the inability of CD22 to access 9-O-deacetylated ligands even in the presence of active Lyn. Enhanced constitutive BCR signaling prevails in all CLL patients but in Zap70+ CLL patients p85PI3K is more readily recruited by c-Cbl. Disclosures Hochberg: Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau; Enzon: Speakers Bureau.
Conclusions Our study demonstrates that B cell-intrinsic IFN-g receptor signals promote lupus pathogenesis via formation of spontaneous, autoimmune GCs. In addition, we have uncovered a novel cell-intrinsic program whereby IFN-g, together with BCR-, TLR-and/or CD40 signals, orchestrates B cell expression of the GC master transcription regulator BCL-6. Our combined findings suggest that this IFN-g signalling program may be a potential therapeutic target in SLE.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.