Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL
Authors: Rozovski Uri , Harris David M. , Li Ping , Liu Zhiming , Manshouri Taghi , Veletic Ivo , Jain Preetesh , Ferrajoli Alessandra , Burger Jan A. , Bose Prithviraj , Thompson Philip A. , Jain Nitin , Verstovsek Srdan , Wierda William G. , Keating Michael J , Estrov Zeev E. 
Published: November 2019

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<.001, P.002 and P<.001, respectively). Because PTX3 induces differentiation of CD14+ monocytes into fiborcytes known to induce bone marrow (BM) fibrosis in myelofibrosis (MF) (Verstovsek et al. J Exp Med, 2016), and because a retrospective analysis of 176 CLL patients (Tadmor et al. Cancer, 2013) detected reticulin fibrosis in CLL patients' BM and showed that the degree of BM fibrosis was associated with thrombocytopenia, anemia, elevated β2M, 11q- (P<.0015), and OS (P<.0001), we analyzed BM biopsy specimens of 8 randomly selected CLL patients and 3 healthy donors. Using fluorescent immuno-histochemistry we detected grade 1 to 2 reticulin fibrosis and an increased number of fibrocytes co-expressing CD45, CD68 and pro-collagen-I in 8 of 8 CLL patients' but not in normal donors' BM sections.
Whereas CRP and SAP are produced by the liver, PTX3 was found to be released from macrophages and endothelial cells. Because in patients with CLL is characterized by an increased number of circulating CLL cells, we wondered whether CLL cells produce PTX3. Using flow cytometry we found that approximately 50% of CD5+/CD19+ CLL cells co-expressed intracellular PTX3, and using Western immunoblotting we detected PTX3 protein in 7 out of 7 CLL patients' cell extracts but not in normal B cells. Therefore we sought to determine why CLL cells produce PTX3. In MF, characterized by constitutive activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, high PTX3 levels constitute an independent indicator of disease burden, clonal expansion and OS (Veletic, et al. Br J Haemat 2019). In CLL STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3. Therefore we postulated that phosphoserine STAT3 induces the production of PTX3 in CLL cells.
To test our hypothesis we performed western immunoblotting and found PTX3 protein in 6 of 6 CLL patients' cell extracts, and by using flow cytometry we found that intracellular phosphoserine STAT3 and PTX3 are co-expressed in 77% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of PTX3 and with DNA of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Sequence analysis of the PTX3 gene promoter identified four γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites spanning within 630 base pairs upstream the PTX3 start codon. To determine which putative binding site binds STAT3 we designed three different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated all 3 STAT3-putative binding sites at high affinity. Similar results, obtained with IL-6-stimulated MM1 cells, further confirmed these findings. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-PTX3 DNA complexes with DNA probes, designed to detect the PTX3 gene promoter binding sites, and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the PTX3 gene promoter. To further confirm these data, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and PTX3 mRNA levels.
In conclusion, we found that constitutively activated STAT3 activates the PTX3 gene and induces the production of high levels of PTX3 in patients with CLL. PTX3, known to induce differentiation of CD14+ monocytes into fibrocytes, enhances the production of BM fibrocytes that induce BM reticulin fibrosis in patients with CLL.

Disclosures
Burger: Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; BeiGene: Research Funding; AstraZeneca: Honoraria. Bose:Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding. Wierda:Cyclcel: Research Funding; AbbVie: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Acerta Pharma Inc: Research Funding; Loxo Oncology Inc.: Research Funding; Sunesis: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding.