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Inhibitors of Bruton’s Tyrosine Kinase (BTKi) and phosphoinositide 3-kinase (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Here we demonstrate in vitro and in vivo the essential roles of PI3K-δ for CLL B-cell survival and migration and of PI3K-γ in T-cell migration and macrophage polarization; and more efficacious inhibition in CLL-cell burden by dual inhibition of PI3K-δ,γ. We also report an ibrutinib-resistant CLL case, whose clone exhibited BTK and PLCγ2 mutations, responded immediately to single agent duvelisib with a redistribution lymphocytosis followed by a partial clinical remission associated with subsequent modulation of T and myeloid cells. CLL samples from patients progressed on ibrutinib were also responsive to duvelisib in patient-derived xenografts irrespective of BTK mutations. Our data support dual inhibition of PI3K-δ,γ as a valuable approach for therapeutic interventions, including patients refractory to BTKi.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) is linked to mutagenesis and occurs within lymphoid tissues. CpG DNA (ODN-2006) and IL-15, a cytokine found within stromal cells of B-CLL-infiltrated tissues, manifest striking synergy in promoting in vitro growth of B-CLL cells (J Immunol 195:901–923, 2015). Synergy is in part explained by an ODN-induced, NF-kB (p65)-dependent elevation of IL-15Rα and IL-2/15Rβ (CD122) mRNA and protein (J Immunol 201:1570–1585, 2018). Most recently, we examined the downstream consequences of IL-15 signaling through experiments involving intracellular immunofluorescence staining, pharmacologic inhibitors, RT-PCR, and western blotting. Levels of activated pAKTSer473 and pSTAT5Tyr694 were significantly elevated soon after IL-15 exposure to ODN-primed B-CLL cells; furthermore, STAT5 remained activated throughout cycling. Pharmacologic inhibitors of PI-3K or STAT5 blocked IL-15-driven growth, indicating the functional relevance of these pathways. RT-PCR experiments +/− these inhibitors show that IL-15 signaling in B-CLL is characterized by PI-3K & STAT5-dependent rise in cyclin-D2 and repression of two important DNA damage response (DDR) mediators, ATM and TP53BP1, known to dampen growth and facilitate DNA repair. Repression of the latter two proteins was confirmed by western blotting and immunofluorescence studies. We conclude that IL-15 signaling in TLR9-primed B-CLL can provoke a rapid burst in growth via mechanisms resembling those recently described in CD8 T cells. Nonetheless, because dividing B-CLL cells additionally upregulate the mutagenic enzyme, AICDA (Blood 120: 4802–4811, 2012), these simultaneous events may augment B-CLL mutagenesis.

B-cell chronic lymphocytic leukemia (B-CLL) clones express BCR specific for microbes and/or apoptotic cells and elevated TLR-9. In patients, B-CLL clonal expansion occurs within lymphoid tissues. We recently reported that CpG DNA (ODN-2006) and IL-15, found within stromal cells of CLL-infiltrated lymphoid tissues, manifest synergy in promoting in vitro B-CLL growth (J. Immunol 195:901–923, 2015). This study explores the mechanism responsible by using purified B-CLL from peripheral blood to assess the temporal requirements for IL-15; effect of ODN on expression of IL-15 receptors (IL-15Rα and CD122 (IL-2/15Rβ)); and IL-15-induced activation of AKT and STAT5. Experiments with CFSE-labeled B-CLL show that IL-15 is critical during a 20–36 h window after ODN priming. Neutralizing mAbs to IL-15 or CD122 fully abrogate IL-15-driven growth when added during this interval. RT-PCR/immunofluorescence studies show that ODN triggers rapid increases in mRNA/protein for IL-15Rα and CD122 that are blocked by IkBα inhibitor. Furthermore, ODN-primed B-CLL exposed to IL-15 manifest elevated pAKT(Ser473) and pSTAT5(Tyr694) levels. Both PI-3K/AKT and STAT5 pathways are functionally relevant, as indicated by growth abrogation by pharmacologic inhibitors, LY294002, pimozide or STAT5 Inh II. Importantly, activated STAT5 levels are high in cycling CFSE-labeled B-CLL; furthermore, extended divisions were blocked by delayed (day 4) administration of STAT5 inhibitors or neutralizing mAbs to IL-15/CD122. Together, findings show that ODN + IL-15-driven growth represents coordinated early functions of TLR-9 and CD122 as well as later CD122 signaling during clonal expansion. This synergy may help drive in vivo growth of B-CLL clones in patients.

Cladribine is a purine analog with impressive activity in patients with low-grade lymphoproliferative disorders. We studied the combination of cladribine with other antineoplastic drugs against two human-derived B-cell lymphoma cell lines in vitro. Cladribine was combined with cisplatin, daunorubicin, chlorambucil, paclitaxel or etoposide. Under the experimental conditions studied, only the combination of cladribine and cisplatin showed significantly increased cytotoxicity compared to the effect of either drug alone.

Over the past decade, major breakthroughs have been made in both the molecular understanding and the treatment of chronic lymphocytic leukemia (CLL). In this article, old and new concepts of CLL biology are explored and insights into the relevance of the newer prognostic factors are discussed. The therapeutic landscape has changed dramatically with the advent of purine analogs, monoclonal antibodies, and combination therapy. As opposed to older agents, these new therapies commonly yield complete remissions. This improvement has spurred a debate as to new goals in treating CLL patients.

Existence and degree of somatic mutation of expressed Ig V genes stratify CLL cases into unmutated (U-CLL) and mutated (M-CLL), clinically distinct subgroups. CLL cells likely receive survival cues via interactions through their BCR, TLR, and cytokine/chemokine receptors. We reported ex vivo expression of CCR4 and its induction on CLL cells stimulated in vitro. In assays of chemotactic behavior in response to CCL17, migrated cells showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 (a known marker of CLL prognosis) in the CCR4-mediated downstream pathway. Dendritic cell-derived CCL17 may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells in peripheral organs. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, and in 3/15 (20%) M-CLL cases, and promoted TLR-9-mediated cell proliferation in 13/15(87%) M-CLL cases and 6/16 U-CLL cases. In addition, CCL17 promoted cell survival and down-regulated pro-apoptotic molecules Bid and PUMA in 6/16 cases studied. These pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating BCR- and TLR-9-mediated signaling and migration in CLL. Inhibition of CCR4:CCL17 interaction in vivo could represent a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival.

Natural antibodies (Abs) and Abs from patients with chronic lymphocytic leukemia (CLL) bind a subset of apoptotic cells that expose intracellular myosin on their surface. To test the mechanisms that produce myosin exposed apoptotic cells (MEACs), Jurkat T cells were induced to undergo intrinsic (spontaneous or camptothecin-induced) or extrinsic (Fas ligand- or anti-Fas-induced) apoptosis. After prolonged incubation by all methods, a high percentage of apoptotic cells became MEACs. Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator is involved. Consistent with this, a caspase-3 inhibitor reduced both apoptosis and MEAC formation, whereas a caspase-1 inhibitor had no effect. To test if caspase-3 cleavage resulted in membrane exposure of myosin, cytoplasmic and membrane protein extracts were examined for caspase-3 cleaved myosin fragments. MEAC extracts predominantly exhibited the expected 149 and 94 kDa myosin fragments in the membrane fraction. In contrast, live cell extracts exhibited predominantly the full-length myosin protein in the cytoplasmic fraction. These results indicate that MEAC formation occurs during intrinsic and extrinsic apoptosis when caspase-3 is activated. Caspase-3 cleavage of myosin produces fragments that reside in the cell membrane, where they become available to react with CLL and natural Abs.

CLL is a lymphoproliferative disorder characterized by an accumulation of CD5+ B cells. Somatic mutation of Ig V genes and cell surface CD38 expression by CLL cells stratifies cases into clinically distinct subgroups. CLL cells are likely sustained by the stimulation via BCR, TLR, and cytokine/chemokine:ligand interactions. Here we report on CCR4 expression and function in CLL. Cases with high numbers of CD38+ cells (≥30%) showed significantly higher percentages of CCR4-expressing cells (52.8 ± 5.2; p< 0.01) than counterparts with low numbers of CD38+ cells (

Chronic lymphocytic leukemia (CLL) mAbs containing an IGHV1-69, IGHD3-16 and IGHJ3 rearrangement with a nearly identical or stereotyped sequence (subset 6) recognize non-muscle myosin heavy chain IIA (MYHIIA). Immunohistochemistry showed that MYHIIA becomes exposed on the surface of apoptotic cells. Flow cytometry with 7-amino-actinomycin D and Annexin V-Phycoerytherin showed that MYHIIA exposure (and concomitant subset 6 CLL mAb binding) occurs in a subgroup of both early and late apoptotic cells. Because other CLL mAbs bind apoptotic cells, the binding of MYHIIA exposed apoptotic cells (MEACs) to a panel of CLL mAbs, only two from subset 6, was measured by flow cytometry. Most CLL mAbs (16/26) bound MEACs well. The MEAC binding level inversely correlated with the degree of IGHV mutation. CLL patients with unmutated IGHV (≤ 2%) tend to have shorter survival times. Similarly, CLL mAbs with high binding to MEACs correlated with shorter patient survival. Natural antibodies are characterized by unmutated IGHV and binding to apoptotic cells. Thus, binding of normal serum antibodies to MEACs was tested. Serum samples had reactivities comparable to CLL mAbs with high binding to MEACs, whereas an antigen-selected human antibody specific for CD20 had little MEAC reactivity. Thus, many CLL leukemic clones may derive from natural antibody-expressing B cells, such as B-1 cells, and stimulation by MEACs could lead to development and/or expansion of CLL leading to poor patient outcome.