CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB
Authors: Rozovski Uri , Harris David M. , Li Ping , Liu Zhiming , Jain Preetesh , Ferrajoli Alessandra , Burger Jan A. , Bose Prithviraj , Thompson Philip A. , Jain Nitin , Wierda William G. , Keating Michael J. , Estrov Zeev E. 
Published: October 2019

Abstract
The two most common cytogenetic abnormalities in patients with chronic lymphocytic leukemia (CLL) are deletion (del) 13q (found in more than 50% of patients) and trisomy (tri)-12 (detected in approximately 15% of patients). These cytogenetic abnormalities are associated dissimilar clinical presentations, disease course and treatment outcome. Unlike CLL patients with del13q, patients with tri12 present with a high disease burden, experience a high rate of Richter's transformation and a poorer treatment outcome.
The signal transducer and activator (STAT)-3 and its target B cluster lymphoma (BCL)-2 gene play similar roles in the pathobiology of CLL. Through distinct pathways, STAT3, BCL2 and BCL2-related proteins provide CLL cells with survival advantage. While a unique protein complex consisting of CD5-CK2-BLNK-STAT3 constitutively phosphorylates and activates STAT3, several mechanisms such as hypomethylation of the BCL2 gene promoter or deletion of regulatory microRNAs (miRs) such as miR15 and miR16, increase BCL2 levels in CLL cells. Whereas both STAT3 and BCL2 protect CLL cells from apoptosis, STAT3 also induces cellular proliferation and contributes to CLL cell transformation. Because both pathways are commonly operative in CLL cells, we wondered whether an aberrantly activated or dissimilar pathway differently affects CLL cells with del13q and tri12.
To test this hypothesis, we isolated circulating CLL cells from 16 treatment naïve patients, 8 with a sole del13q and 8 with a sole tri12 cytogenetic abnormality. Using Western immunoblotting and densitometry with normalization to the β-actin protein level, we found significantly higher levels of BCL2, BIM and BAD in CLL cells with del13q than in CLL cells with tri12. Conversely, STAT3 and phosphoserine STAT3 levels were significantly higher in CLL cells with tri12 than in CLL cells with del13q. To test whether dissimilar transcription activity levels were responsible for these differences we assessed BCL2, BIM, BAD, MCL1, and STAT3 mRNA levels in the same patient samples using qPCR. Consistent with the protein level results, higher mRNA levels of BCL2 and BCL2 family members were found in CLL cells with del13q as compared with their levels in CLL cells with tri12. Conversely, STAT3 mRNA levels were similar in del13q and tri12 CLL cells. Because mutations in genes such as NOTCH-1 and BIRC3, found in approximately 30% of patients with tri12, are thought to induce activation of the NF-κB pathway and because we have previously found that unphosphosphorylated STAT3, whose levels were found to be higher in cells with del13q, activates NF-κB, we performed an electomobility shift assay (EMSA) and found higher levels of DNA-NF-κB complexes in 8 samples of CLL cells with tri12 than in 8 samples with del13q. Because STAT3 protein levels were higher in del13q whereas STAT3 mRNA levels were similar in del13q and tri12 CLL cells, we reasoned that post translational modification might be responsible for these differences. To test this hypothesis we immunoprecipitated STAT3 from 4 del13q and 4 tri12 CLL cell samples and, using Western immunoblotting with anti-ubiquitin antibodies, we detected significantly higher levels of ubiquitinated STAT3 in del13q than in tri12 CLL cells, suggesting that STAT3 ubiquitination is higher in CLL cells with del13q.
Taken together, our data revealed a strong association between del13q and tri12 cytogenetic abnormalities and different molecular pro-survival -proliferation pathways. CLL cells with del13q express high levels of anti-apoptotic BCL2 family member proteins, whereas CLL cells with tri12 express high levels of phosphorylated STAT3 and activated NF-κB. Whether different additional survival and proliferation pathways contribute the disparate clinical features of CLL patients with del13q and tri12 warrants further investigation.

Disclosures
Bose: Pfizer, Inc.: Research Funding; Celgene Corporation: Honoraria, Research Funding; Astellas Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding; Constellation Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Research Funding. Thompson:AbbVie: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Research Funding. Jain:Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Astra Zeneca: Research Funding; Servier: Research Funding; Genentech: Research Funding; Incyte: Research Funding; BMS: Research Funding; Incyte: Research Funding; Abbvie: Research Funding; Pharmacyclics: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Infinity: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Abbvie: Research Funding; Infinity: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Cellectis: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wierda:Genentech: Research Funding; AbbVie, Inc: Research Funding.