Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes by regulating the activity of their enhancers. BTKi treatment reduces enrichment of enhancer marks (H3K4me1 and H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. We also find that enhancer-regulated APOBEC3 expression contributes to replication stress in malignant B cells. In total we demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in an NFATc1-dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.

Patients with large granular lymphocytic leukemia (LGLL) frequently present with neutropenia. When present, anemia is usually accompanied by neutropenia and/or thrombocytopenia and isolated anemia is uncommon. We evaluated a cohort of 244 LGLL patients spanning 15 years and herein report the clinicopathologic features of 34 (14%) with isolated anemia. The patients with isolated anemia showed a significantly male predominance (p = 0.001), a lower level of hemoglobulin (p < 0.0001) and higher MCV (p = 0.017) and were less likely to have rheumatoid arthritis (p = 0.023) compared to the remaining 210 patients. Of the 34 LGLL patients with isolated anemia, 13 (38%) presented with pure red cell aplasia (PRCA), markedly decreased reticulocyte count and erythroid precursors, and more transfusion-dependence when compared to non-PRCA patients. There was no other significant clinicopathologic difference between PRCA and non-PRCA patients. 32 patients were followed for a median duration of 51 months (6-199). 24 patients were treated (11/11 PRCA and 13/21 non-PRCA patients, p < 0.02). The overall response rate to first-line therapy was 83% [8/11 (72.7%) for PRCA, 12/13 (92.3%) for non-PRCA], including 14 showing complete response and 6 showing partial response with a median response duration of 48 months (12-129). Half of non-PRCA patients who were observed experienced progressive anemia. During follow-up, no patients developed neutropenia; however, 5/27 (18.5%) patients developed thrombocytopenia. No significant difference in overall survival was noted between PRCA and non-PRCA patients. In summary, this study demonstrates the unique features of LGLL with isolated anemia and underscores the importance of recognizing LGLL as a potential cause of isolated anemia, which may benefit from disease-specific treatment. LGLL patients with PRCA were more likely to require treatment but demonstrated similar clinicopathologic features, therapeutic responses, and overall survival compared to isolated anemia without PRCA, suggesting PRCA and non-PRCA of T-LGLL belong to a common disease spectrum.

Richter syndrome (RS) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. RS is known to be associated with a number of genetic alterations such as TP53 and NOTCH1 mutations. However, it is unclear what immune microenvironment changes are associated with RS. In this study, we analyzed expression of immune checkpoint molecules and infiltration of immune cells in nodal samples, and peripheral blood T-cell diversity in 33 CLL and 37 RS patients. Compared to CLL, RS nodal tissue had higher PD-L1 expression in histiocytes and dendritic cells (median 16.6% vs. 2.8%, P < 0.01) and PD1 expression in neoplastic B cells (median 26.0% vs. 6.2%, P < 0.01), and higher infiltration of FOXP3-positive T cells (median 1.7% vs. 0.4%, P < 0.01) and CD163-positive macrophages (median 23.4% vs. 9.1%, P < 0.01). In addition, peripheral blood T-cell receptor clonality was significantly lower in RS vs. CLL patients (median [25th-75th], 0.107 [0.070-0.209] vs. 0.233 [0.111-0.406], P = 0.046), suggesting that T cells in RS patients were significantly more diverse than in CLL patients. Collectively these data suggest that CLL and RS have distinct immune signatures. Better understanding of the immune microenvironment is essential to improve immunotherapy efficacy in CLL and RS.