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Cell signaling initiated by the B cell receptor is critical to normal development of B lymphocytes, most notably at the transitional B cell stage. Inhibition of this signaling pathway with the syk inhibitor, fostamatinib, has produced significant efficacy in lymphoid malignancies and autoimmune conditions. Here, we demonstrate that short-term use of fostamatinib impairs B lymphocyte development at the transitional stage without affecting mature B cell populations. Additionally, IL-10 producing B cells remained relatively constant throughout the treatment period. These findings provide insight into the mechanism of action of B cell receptor inhibition in autoimmune disease. As the development of agents targeting B cell receptor signaling proceeds, monitoring for long-term consequences as well as functional evaluation of B cell subsets may further improve our understanding of this rapidly growing class of novel agents.

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Certain normal and malignant B-cells rely upon “tonic” B-cell receptor (BCR)-mediated survival signals. BCR signaling induces the phosphorylation of the associated Ig alpha and beta proteins and the recruitment and activation of spleen tyrosine kinase (SYK); thereafter, SYK initiates downstream events and amplifies the original BCR signal. A subset of DLBCL exhibits coordinate overexpression of BCR pathway components, including SYK, and relies upon tonic BCR signaling. FosD is an orally available inhibitor of SYK under development for rheumatoid arthritis, and has demonstrated significant in vitro activity against BCR-dependent NHLs. We have performed the first clinical trial of the oral SYK inhibitor, FosD, in patients (pts) with a variety of relapsed or refractory B-cell non-Hodgkin’s lymphomas. We first enrolled 13 pts in a Phase I component, exploring two dose levels (200 and 250 mg BID) of FosD. Pts (Follicular [FL], 5; CLL/SLL, 2; mantle [MCL], 3; DLBCL, 3) were heavily pretreated. On the first day of FosD dosing at 200 mg and 250 mg, the maximum plasma concentrations were 668 ± 258 ng/mL and 1020 ± 781 ng/mL, and the AUC0–4 estimates were 1800 ± 602 ng*h/mL and 2590 ± 1900 ng*h/mL, respectively. Plasma concentrations increased approximately 2-fold with continued administration, but beyond Day 29 there was no apparent change in FosD concentrations measured over time. Dose limiting toxicity in Phase I was neutropenia; and 200 mg BID was chosen for Phase II evaluation. We then enrolled 68 pts with relapsed/refractory NHL in 3 separate disease cohorts: DLBCL (23); FL (21) and other B-cell NHL (24) including SLL/CLL (11), MCL (9) marginal zone/MALT (3) and lymphoplasmacytic NHL (1). The median age of pts in phase II was 61 (range 41–87); pts received a median of 5 prior therapies, including ASCT (16; 12 with DLBCL) and radioimmunotherapy (8). FosD was overall very well tolerated; there were 4 cases of febrile neutropenia reported. Eight pts required dose modification for neutropenia (2), hypertension (2), liver function test abnormalities (2), fever (1) and mucositis (1). Six pts withdrew before initial response evaluation (AE, 5; noncompliance, 1). Best responses by disease were: DLBCL 21% (4PR; 1CR); SLL/CLL 54% (6PR); FL 10% (2PR); MCL 11% (1PR). Stable disease was observed in an additional 23 patients, including 12 with FL, 4 with DLBCL, 4 with MCL, 2 with CLL/SLL and 1 with MALT. As of 7/15/08, 16 pts were treated for more than 200 days, and 14 pts remain on study. Median PFS is 4.5 months, and only 5 pts have died (4 with DLBCL of disease progression and 1 with CLL of infection following therapy). Several pts with CLL/SLL experienced a transient increase in circulating lymphocytes in the setting of responding nodal disease. Of the 6 responding pts with CLL, Zap-70 status was positive (3), negative (1) and unknown (2); cytogenetics were trisomy 12 (1), del13 (1), normal (1), complex (1) and unknown (2). We conclude that disrupting BCR-induced signaling by inhibiting SYK kinase represents a safe and well-tolerated novel therapeutic approach to NHL. In addition to significant responses in DLBCL and CLL/SLL, prolonged stable disease was observed in pts with FL. FosD should be developed further, as a single agent and in rational combinations, for BCR-dependent B-cell NHLs.

Backround: Anti CD-20 radioimmunotherapy (RIT) is effective therapy for indolent B-cell NHL, and under investigation in more aggressive histologies. Most data on safety and efficacy of RIT is from the pre-rituximab era, and the effect of rituximab exposure on RIT in pts with NHL is unknown. Gopal et al recently demonstrated that exposure to rituximab correlated with inferior tumor response and alteration in tumor: organ dosimetry ratio both in vitro and in mouse models following therapy with iodine-131 tositumomab (Blood 112:830). Two ongoing SWOG trials evaluating RIT consolidation therapy provide a unique opportunity to evaluate the impact of prior rituximab on pharmacodynamics of iodine-131 tositumomab in humans. S0016 enrolls previously untreated pts with follicular NHL, and iodine-131 tositumomab consolidation is administered after 6 cycles of CHOP. S0433 enrolls previously untreated pts with DLBCL, and iodine-131 tositumomab is administered after 6 cycles of CHOP with rituximab, and 2 additional cycles of CHOP alone. As rituximab leads to B-cell depletion for 6 months or more, we hypothesized the residence time of iodine-131 tositumomab would differ in pts exposed recently to rituximab compared to no prior rituximab. Methods: Prospective pts at the University of Rochester enrolled in S0016 and S0433 were analyzed. Residence times of iodine-131 tositumomab were calculated using serial imaging on a Picker XP 2000 gamma camera. Rituximab levels were performed within one week prior to dosimetric iodine-131 tositumomab administration using ELISA. Medians were used to summarize the data, and the 2-tailed Mann-Whitney-Wilcoxon test was used for hypothesis testing. Results: 16 pts (6 female) on S0016 and 12 pts (6 female) on S0433, were identified, with median ages of 54.5 and 69.5 respectively. All pts had advanced stage disease, and median BMI and creatinine were similar for both groups. Pts on S0433 had a median time from rituximab to RIT of 78.6 days (range 58–98 days). Despite this, rituximab levels were present at time of iodine-131 tositumomab in all pts measured (N=9; median rituximab level 37.2 ug/ml, range 15.6–61.69). Median absolute lymphocyte count appeared lower in the S0433 group compared to the S0016 group (600 vs 1050 /ul), but this difference was not significant (p=0.12). Pts on S0433 (all had received rituximab prior to iodine-131 tositumomab consolidation) had significantly longer RIT residence times when compared to those on S0016, (not treated with prior rituximab): 115 hours vs. 107 hours; p=0.02. Therapeutic doses of iodine-131 tositumomab were not significantly different between the two studies (S0433: 72 mCi vs. S00016: 78 mCi p=0.59). Conclusions: Our results indicate that prior therapy with rituximab results in a longer residence time of iodine-131 tositumomab when used as consolidation after chemotherapy. Measurable rituximab levels at time of RIT suggest that rituximab-induced B-cell depletion decreases clearance of RIT, possibly allowing for longer exposure times. The significance of this longer residence time is unknown but it could be associated with greater toxicity to normal organs, and could be indicative of decreased tumor binding. If confirmed in larger studies, these findings could have profound implications on RIT administration in the context of rituximab. Rituximab-induced B-cell depletion could obligate the need for unlabeled antibody dosing prior to RIT, and may affect dosimetry of RIT. Prospective studies of RIT in the rituximab era should evaluate the impact of prior rituximab and RIT residence time on toxicities and outcomes in pts treated with RIT.

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes involved in neutrophil maturation, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε ). It also represses expression of neutrophil secondary granule proteins (SGPs), including LF, which are downstream targets of C/EBPε . We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. Additionally, we have demonstrated that overexpression of CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Conversely, knocking down CDP expression by RNA interference in 32D cells upregulates levels of LF mRNA in uninduced cells. NB4 cells are an acute promyelocytic cell line that contains the t(15;17) PML-RARα translocation and can be induced toward neutrophil maturation with all-trans-retinoic Acid (ATRA). We have previously demonstrated that NB4 cells uniformly fail to express LF and all the SGP genes upon ATRA induction. In further studies, we showed that this failure of expression occurs despite appropriate binding of C/EBP factors known to upregulate SGP expression. We further demonstrated persistent binding of CDP to the LF promoter in NB4 cells upon ATRA induction, suggesting that failure of LF expression was due to persistent binding of CDP rather than a failure of C/EBP transactivation. In order to test this hypothesis, we knocked down CDP expression in NB4 cells using short hairpin RNA (shRNA) constructs and examined the effect of reduced CDP levels on SGP expression. ShRNA CDP constructs were introduced into NB4 cells by nucleofection using an Amaxa® nucleofector. Half the transfected cells were induced with ATRA, while the other half remained uninduced. After 24-hours of induction, total RNA was extracted from the cells and RTqPCR performed to assess the levels of SGP mRNA. We demonstrate that decreased CDP expression in ATRA- induced NB4 cells is sufficient to restore LF, collagenase, and gelatinase expression in the leukemic cell line. SGP expression remains absent in uninduced CDP-shRNA NB4 cells, as well as in both uninduced and induced cells transfected with an empty vector. Our observations reaffirm the critical role played by CDP in regulating the expression of LF and other SGP proteins during neutrophil development. How the PML-RARα gene product functions to alter CDP activity in NB4 cells at the molecular level, thereby restricting SGP expression, is currently being addressed.