In chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated on serine 727 and plays a role in the pathobiology of CLL. However, what induces constitutive phosphorylation of STAT3 is currently unknown. Mass spectrometry was used to identify casein kinase 2 (CK2), a serine/threonine kinase that co-immunoprecipitated with serine phosphorylated STAT3 (pSTAT3). Furthermore, activated CK2 incubated with recombinant STAT3 induced phosphorylation of STAT3 on serine 727. Although STAT3 and CK2 are present in normal B- and T-cells, STAT3 is not constitutively phosphorylated in these cells. Further study found that CD5 and BLNK co-expressed in CLL, but not in normal B- or T-cells, are required for STAT3 phosphorylation. To elucidate the relationship of CD5 and BLNK to CK2 and STAT3, STAT3 was immunoprecipitated from CLL cells and CK2, CD5, and BLNK were detected in the immunoprecipitate. Conversely, STAT3, CD5, and BLNK were in the immunoprecipitate of CLL cells immunoprecipitated with CK2 antibodies. Furthermore, siRNA knockdown of CD5 or BLNK, or treatment with CD5-neutralizing antibodies significantly reduced the levels of serine pSTAT3 in CLL cells. Finally, confocal microscopy determined that CD5 is cell membrane bound and fractionation studies revealed that the CK2/CD5/BLNK/STAT3 complex remains in the cytoplasm, whereas serine pSTAT3 is shuttled to the nucleus. Implications These data show that the cellular proteins CK2, CD5, and BLNK are required for constitutive phosphorylation of STAT3 in CLL. Whether this protein complex phosphorylates other proteins or inhibiting its activity would have clinical benefit in patients has yet to be determined.

In chronic lymphocytic leukemia (CLL) cells, both interleukin-6 (IL-6) and the B-cell receptor (BCR) activate Janus kinase 2 (JAK2) and induce the phosphorylation of signal transduction and activator of transcription 3 (STAT3) on tyrosine 705 residues. However, whereas IL-6 phosphorylates STAT3 within 15 minutes, stimulation of the BCR with anti–immunoglobulin M (IgM) antibodies phosphorylates STAT3 in 2–4 hours. Here we show that this process takes longer because it requires transcriptional activity of NF-κB. Using an electromobility shift assay, we found that incubation with IgM antibodies for 4 or 18 hours, but not 15 minutes, increased NF-κB DNA-binding of CLL cells and increased binding was translated to increased transcriptional activity. Hence, 42% of the 83 NF-κB target genes were constitutively expressed in all CLL cells prior to any inducible stimuli. However, activation of the BCR increased the number of NF-κB target genes with detectable expression by 23%. Remarkably, prolonged incubation with anti-IgM antibodies induced a time-dependent transcription, production, and secretion of IL-6 protein. The IgM-induced production of IL-6 prompted the phosphorylation of STAT3 on tyrosine residues. This effect was inhibited by the JAK1/2 inhibitor of the JAK/STAT3 pathway ruxolitinib. Taken together, these results suggest that in CLL cells, constitutive tonic activation of NF-κB can be further enhanced by the BCR and that the BCR-induced activation of the JAK/STAT3 pathway depends on the NF-κB–induced production of IL-6.

Introduction: PRM-151, a recombinant human pentraxin-2 molecule, has been shown to prevent and reverse fibrosis in animal models of myelofibrosis (MF) by postulated targeting differentiation of fibrocytes (essential cells in fibrotic process) from monocytes. In the first stage of a two-stage trial, 27 patients (pts) with primary myelofibrosis (PMF), post-essential thrombocythemia/polycythemia vera (post-ET/PV) MF, and Grade 2 or 3 bone marrow fibrosis (BMF) received PRM-151 10 mg/kg IV ± ruxolitinib (RUX) for 6 cycles (24 weeks). A reduction in BMF, decrease in symptoms (MPN-SAF Total Symptom Score [TSS]), and a reduction of palpable splenomegaly were observed, along with a favorable safety profile. Pts experiencing clinical benefit were allowed to continue beyond 24 weeks in an open label extension (OLE) study. Here, we report interim efficacy and safety data for 18 pts enrolled in the OLE, who had been treated for up to 35 cycles (140 weeks). Methods: All pts in the OLE received a monthly infusion of PRM-151 10 mg/kg IV. Pts receiving PRM-151 alone or in combination with RUX in the main study continued with their respective drugs. Safety and hematology parameters were assessed monthly, and MPN-SAF TSS Q3 months. BM biopsies were obtained at baseline, end of main study, and at physician discretion in the OLE. These were evaluated centrally by two independent, blinded hematopathologists for the degree of reticulin and collagen fibrosis. Immunostaining for fibrocytes was performed on available biopsies. The primary objective of the OLE was to assess safety and efficacy of long-term administration of PRM-151. Results: Of the 18 pts enrolled in the OLE, 9 received PRM-151 as single agent, and 9 in conjunction with RUX. Baseline characteristics included median age of 66 years (51-78); 8 pts were DIPSS Int-1, 9 Int-2, and 1 high-risk; 6 (33%) had Hgb < 100 g/L, 12 (67%) had PLT < 100 x 109/L; 7 (39%) had PLT <50 x 109/L; 8 pts had PMF, 10 had post-ET/PV MF; 13 pts (72%) had a palpable spleen, and 9 (50%) had Grade 3 BMF. Median time on study for these 18 pts was 30.9 months (15.9-47.2 months). All pts experienced at least 1 adverse event (AE), with 8 pts (44%) reporting a possible study drug-related AE. No unexpected AEs were observed, and no deaths were reported during the study period. The mean best percent change (by palpation) in spleen size from baseline was −37%, with a median percent reduction of −26.1% (Figure). The mean best percent improvement in MPN-SAF TSS was −54%, with a median percent reduction of TSS of −64%. Pts in the PRM-151 monotherapy group showed similar improvements in MPN-SAF TSS and splenomegaly as those receiving PRM-151 + RUX (Figure). Complete data on hematology parameters will be available at the time of presentation. Improvement in BM reticulin grade was observed in 5 of 7 pts (71%) with Grade 2 BMF at baseline, and in 4 of 9 pts (44%) with Grade 3 BMF at baseline. Of the pts with Grade 3 BMF at baseline, 2 of 9 pts (22%) had a 2-grade reduction (Table). In addition, 5 of 6 pts (83%) with Grade 2 collagen fibrosis at baseline had a 1-grade reduction, with 2 of 6 pts (33%) improving to Grade 0. Also, 2 of 7 pts (28%) with Grade 3 collagen fibrosis had a reduction of 1-grade with 1 of 7 (14%) improving to Grade 0 (Table). Eight of 18 pts had BM fibrocyte immunostaining done: mean fibrocyte count in the hematopoietic BM decreased from 378.0 ± 255.3 per mm2 (11.2 ± 7.7% of total cells) at study baseline to 81.3 ± 86.5 per mm2 (2.0 ± 2.1% of total cells; 7 of 8 pts evaluable) at cycle 25, with continuous reduction in fibrocyte numbers observed at interim. Over the same period, improvement in BM reticulin grade was detected in 4 of 7 pts (57%), and collagen fibrosis grade was decreased in 3 of 7 pts (43%). Conclusions: This long-term follow-up study of 18 pts with MF who received PRM-151 for a median of 31 months showed the drug to be well tolerated. An overall improvement in BM reticulin and collagen fibrosis grade, as well as reductions in MPN-SAF TSS and splenomegaly were observed. In a subset of pts for whom bone marrow fibrocyte immunostaining data were available, a mean reduction in fibrocyte counts was observed, as well as improved bone marrow reticulin and collagen fibrosis grade. These data warrant confirmation in a larger controlled study. Figure. Figure. Disclosures Verstovsek: Incyte: Consultancy; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees. Pozdnyakova:Promedior, Inc.: Consultancy. Mesa:Promedior: Research Funding; Incyte: Research Funding; Ariad: Consultancy; Novartis: Consultancy; Celgene: Research Funding; Galena: Consultancy; Gilead: Research Funding; CTI: Research Funding. Foltz:Incyte: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Roche: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Promedior: Research Funding. Ritchie:Astellas Pharma: Research Funding; NS Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; ARIAD Pharmaceuticals: Speakers Bureau; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Palmer:Novartis: Research Funding. Kremyanskaya:Incyte: Research Funding. van den Blink:Promedior, Inc.: Employment. Gupta:Promedior, Inc.: Employment. Gotlib:Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Promedior: Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Assessing albumin, ALC and PNI might improve prognostication in patients with myelofibrosis and could assist in recognition of patients under increased risk of death.

Summary Background Disease-related symptoms impair the quality of life of countless patients with chronic lymphocytic leukemia (CLL) who do not require systemic therapy. Currently available therapies are not specifically aimed at symptom control. Because stimulation of the B-cell receptor activates Janus kinase (JAK)-2 in CLL cells and the JAK2 inhibitor ruxolitinib improves symptoms of patients with myelofibrosis, we hypothesized that ruxolitinib would improve disease-related symptoms in CLL patients. Methods Ruxolitinib (10 mg twice daily) was administered to symptomatic CLL patients who did not require systemic therapy for CLL. Scores on the brief fatigue inventory (BFI), CLL module of the MD Anderson symptom inventory (MDASI) and symptom-associated interference in daily activities (interference score; IS), were assessed prior to treatment and after 3 months of treatment. Plasma cytokine/chemokine levels were measured at baseline and at 3 months. Findings Forty-one CLL patients (25 untreated and 16 previously treated) were enrolled. Thirty-two (78%) of the participants experienced ≥20% reduction in the average BFI score or in the average MDASI score. 59% of the participants had ≥2 units reduction in worst fatigue score in 24 hours as assessed by the BFI. The mean percentage reductions in BFI, MDASI, and IS scores were >42% (p<0.0001). Improvements in the three symptom scores correlated with reductions in levels of IL-6, C-reactive protein, CXCL10, osteopontin, TNF-α, ICAM-1/CD54, VCAM-1/CD106, and beta-2 microglobulin. Furthermore, treatment with ruxolitinib increased and then decreased lymphocyte counts to baseline levels or lower. Grade 3/4 cytopenias were recorded in three patients. Interpretation In CLL patients, ruxolitinib significantly improved disease-related symptoms, reduced cytokine and chemokine levels, and increased and then decreased lymphocyte counts, likely through mobilization followed by apoptosis of CLL cells. Further studies aimed at testing the therapeutic efficacy of ruxolitinib in CLL are warranted. Funding Supported by the Incyte Corp., MD Anderson Cancer Center Support Grant CA016672 and Award Number P01 CA049639 from the National Cancer Institute.