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Purpose: Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. Experimental Design: Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. Results: We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. Conclusions: Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

Effects of inhibition of PI3K isoforms on CLL-cell proliferation, survival, and migration.

Purpose:Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL.Experimental Design:Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib.Results:We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells.Conclusions:Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

IC50 values of PI3K isoform inhibitors used. Duvelisib5, IPI-3063 (PI3K-d inhibitor)5, IPI-549 (PI3K-g inhibitor used in vitro)50, IPI-5243 (PI3K-g inhibitor used in vivo).

Purpose Intolerance is the most common reason for kinase inhibitor (KI) discontinuation in CLL. Umbralisib a novel, highly selective PI3Kδ/CK1e inhibitor, is active and well tolerated in CLL patients. This phase 2 trial evaluated umbralisib in CLL patients who are intolerant to prior BTK or PI3K inhibitor therapy. Patients and methods In this phase 2 trial (NCT02742090), umbralisib was initiated at 800 mg oral daily in CLL patients requiring therapy per investigator discretion who were intolerant to prior BTK or PI3K inhibitor therapy, until progression or toxicity. Primary endpoint was progression-free survival (PFS). Secondary endpoints included time to treatment failure and umbralisib safety profile. DNA isolated from buccal swabs was genotyped for polymorphisms in CYP3A4, CYP3A5 and CYP2D6. Results Fifty-one patients were enrolled (44 BTKi and 7 PI3Kδi intolerant). Median age was 70 years (range 48-96), median of 2 prior lines of therapy (1-7), 24% had del17p and/or TP53 mutation, and 65% were IGHV unmutated. Most common AEs leading to prior KI discontinuation were rash (27%), arthralgia (18%), and atrial fibrillation (16%). Median progression free survival (PFS) was 23.5 months (95% CI 13.1-not estimable). 58% of patients were on umbralisib for a longer duration than prior KI. Most common (≥5%) grade ≥3 AEs on umbralisib (all causality) were neutropenia (18%), leukocytosis (14%), thrombocytopenia (12%), pneumonia (12%), and diarrhea (8%). Six patients (12%) discontinued umbralisib due to an AE. Eight patients (16%) had dose reductions and were successfully re-challenged. Conclusions Umbralisib is safe and effective in this BTK and alternate PI3K inhibitor intolerant CLL population. These are the first prospective data to confirm that switching from a BTK or alternate PI3K inhibitor to umbralisib can result in durable, well tolerated responses.

Hairy cell leukemia (HCL) is a rare chronic B cell lymphoproliferative disorder characterized by high prevalence of BRAF V600E mutation.1,2 Purine nucleoside analogues can achieve an overall response rate (ORR) of 90 to 100% and complete responses (CR) of 80 to 95% and remain the mainstay of first-line treatment in HCL.3,4 However, approximately 30-40% patients will experience recurrent disease and relapse-free survival rates decrease with repeated courses of purine analogue-based treatments with cumulative myelotoxicity and immune suppression.5,6 The genetic basis of HCL was first uncovered a decade ago when Tiacci and colleagues reported that BRAF V600E is a key mutation in HCL,7 a finding that was further validated by subsequent studies.8,9 Based on these findings, we conducted a multicenter phase 2 clinical trial in the U.S. evaluating the efficacy and safety of vemurafenib, an oral BRAF inhibitor, in patients with relapsed/ refractory (R/R) HCL. We previously published the initial outcome of 26 U.S. patients together with the Italian study conducted by Tiacci et al.7 Vemurafenib monotherapy induced 96-100% ORR and 35-42% CR rates in both studies. With a median follow-up duration of 11.7 months, we observed relapses in 29% of the patients but the long-term clinical outcome and response or acquired resistance to vemurafenib retreatment have not been previously reported. Herein, we report the long-term follow-up data of the entire patient cohort in the completed U.S. clinical trial (NCT01711632) including the ORR, relapse free survival, clinical factors associated with improved survival as well as outcomes after retreatment with vemurafenib or alternative agents.

Duvelisib (IPI-145), a first-in-class, oral, dual PI3K-δ,γ inhibitor, has been approved by the FDA for the treatment of patients with chronic lymphocytic leukemia (CLL), small lymphocytic and follicular lymphoma. Duvelisib potently inhibits PI3K-δ and PI3K-γ, thereby offering a novel therapeutic approach. Here we aimed to understand how duvelisib targets CLL cells and the tumor microenvironment (TME) and hence how it affects CLL, even with BTK-resistant disease. We first assessed the distinct contributions of PI3K-δ and PI3K-γ on CLL cell survival, proliferation, and migration in vitro by using PI3K-δ (PI3K-δi) or PI3K-γ (PI3K-γi) specific inhibitors in addition to duvelisib. We found a significant reduction in viable CLL cells after exposure to PI3K-δi and duvelisib. By measuring Ki67 and p-AKT levels, we found duvelisib and PI3K-δi more potently block CLL cell proliferation than PI3K-γi. PI3K-δi and duvelisib also significantly reduced the migration of CLL B cells into the spleen relative to control. In contrast, PI3K-γi inhibited T cell not B cell homing in vitro and in vivo. We then investigated the impact of single versus dual inhibitory agents on myeloid cells. Tumor-associated macrophages (TAMs) of the “M2 phenotype” contribute to a pro-tumor TME by preventing the induction of T cell mediated anti-tumor immunity. We expanded murine bone marrow derived monocytes and then polarized them to M2 macrophages, in the absence or presence of PI3Ki. At 100nM, PI3K-γi, but not PI3K-δi, significantly inhibited M2 polarization. Duvelisib significantly reduced Arg1 expression at doses equal to or above 10nM. While co-culturing leukemic B cells with M2-polarized murine macrophages increased CLL-cell survival, addition of duvelisib to such co-cultures significantly reduced CLL cell survival. Altogether, duvelisib sensitizes primary CLL cells to apoptosis and abrogates M2 cell-mediated CLL-cell survival. Finally, in a patient-derived xenograft mouse model (PDX), we demonstrated the essential roles of PI3K-δ and PI3K-γ in the CLL TME. Specifically, we found a more efficacious inhibition in CLL cell burden by dual inhibition of PI3K-δ,γ. Also, samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in PDX, irrespective of BTK mutations. In support of this, we identified an ibrutinib resistant CLL patient, with a clone exhibiting BTK and PLCγ2 mutations who responded immediately to single agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with subsequent modulation of T and myeloid cells. In conclusion, our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B cell numbers and T and myeloid cell pro-leukemia functions, supporting the use of duvelisib as a potentially valuable therapeutic intervention, including for patients refractory to BTKi. Citation Format: Shih-Shih Chen, Jacqueline C. Barrientos, Gerardo Ferrer, Priyadarshini Ravichandran, Michael Ibrahim, Yasmine Kieso, Jeffery L. Kutok, Marisa Peluso, Sujata Sharma, David T. Weaver, Jonathan A. Pachter, Kanti R. Rai, Nicholas Chiorazzi. Duvelisib eliminates CLL B Cells, impairs CLL-supporting cells, and overcomes ibrutinib resistance in a patient-derived xenograft model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4933.

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy., publishedVersion

Alterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

How the genomic features of a patient’s cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo. Analysis of growth dynamics in a dataset from 107 patients with chronic lymphocytic leukaemia (CLL) reveals both exponential and logistic patterns of growth, which are associated with differences in genetic attributes and clinical outcomes.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell–associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15–driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15–driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15–induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

International audience; Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Em-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma.Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eμ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation.

The previous edition of the consensus guidelines of the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), published in 2008, has found broad acceptance by physicians and investigators caring for patients with CLL. Recent advances including the discovery of the genomic landscape of the disease, the development of genetic tests with prognostic relevance, and the detection of minimal residual disease (MRD), coupled with the increased availability of novel targeted agents with impressive efficacy, prompted an international panel to provide updated evidence- and expert opinion–based recommendations. These recommendations include a revised version of the iwCLL response criteria, an update on the use of MRD status for clinical evaluation, and recommendations regarding the assessment and prophylaxis of viral diseases during management of CLL.

Chylothorax, which is defined as the presence of chyle in the pleural space, is often caused by malignancy. However, chylothorax as a result of underlying CLL is exceedingly rare in the literature. Chyle contains fat soluble vitamins and lymphocytes, meaning that its collection into the pleural space may further exacerbate the immunosupressed state of an individual with CLL. Here, we report three cases of patients with CLL who developed chylothorax, and their management. Chylothorax, although rare with CLL, should be considered in the differential diagnosis when patients with CLL present with pleural effusions, especially if recurrent. Discovery of a chylothorax may indicate the need for further treatment of CLL.

Background. CLL-like monoclonal B-ceII lymphocytosis (MBL) is considered a requisite precursor of CLL, with 1-2% of subjects annually progressing to CLL requiring therapy. The role of immune alterations leading to and operating in MBL and controlling progression to CLL is not well characterized. Since an increased frequency of immune-related conditions associated with immune dysfunction exists prior to CLL diagnosis (Landgren et al, Br J Haematol 2007 and Blood 2007; Andersen et al, Leukemia 2020), immune alterations likely exist in MBL. We have examined the serum protein profiles of MBL and CLL in search of clues linking immune dysfunction with malignant transformation. Objectives. 1. Characterize the serum proteomic profiles of MBL individuals, IGHV-mutated CLL (M-CLL) and IGHV-unmutated CLL (U-CLL) patients, and age-matched healthy controls (HC). 2. Compare the serum proteomic profiles between the groups. 3. Assess the effect of IGHV mutation status on serum proteomic profiles in CLL. Methods. A total of 12 MBL, 12 M-CLL, 12 U-CLL and 12 age-matched HC were studied. Patients were cared for at Northwell Health, New York (n=25) and Hospital del Mar, Barcelona (n=11). All patients were treatment naive except for one U-CLL patient who was treated one year beforehand. Serum samples were collected, and their protein levels measured employing SOMAmers (modified single-stranded DNA aptamers; SomaLogic) to quantify relative levels of 1,310 proteins. P-values Results. Overall, the levels of 862 proteins differed significantly between groups: MBL vs. HC: 10 downregulated (d) and 24 upregulated (u); M-CLL vs. HC: 206d and 6u; U-CLL vs. HC: 54d and 40u; M-CLL vs. MBL: 384d and 5u; U-CLL vs. MBL: 74d and 24u; and M-CLL vs. U-CLL: 35d and 0u. IPA highlighted a role for the pro-inflammatory HMGB1 pathway in several comparisons. First, an activated HMGB1 pathway was predicted in MBL compared to HC, together with activation of phagocytes, and an inhibition of the systemic immune suppressor TGF-β. Second and consistent with the former, U-CLL patients displayed an activated HMGB1 pathway compared to HC, along with other signs of immune stimulation (activated NFkB and Th1 pathways, maturation of dendritic cells, and inhibition of TGF-β) and leukemic progression (activated progression of tumor, and leukocyte extravasation). Third and contrary to the above, the HMGB1 pathway was inhibited when comparing M-CLL to HC, in line with a global immune suppression signature (inhibited PRR, GM-CSF, FcεRI, IL1, IL8, TNF, Th1, STAT3 and NFkB pathways, in addition to inhibited cell movement, viability and activation). Notably, inhibition of immune pathways was predicted for both M-CLL and U-CLL compared to MBL (diminished TNF and IL6 signaling, and reduced cell movement), although the greatest differences were seen for M-CLL vs. MBL comparison, including blockade of the HMGB1 pathway in M-CLL. Finally, the M-CLL vs. U-CLL comparison suggested inhibited INFγ, IL2, IL3, IL4, NFkB, and decreased T lymphocyte stimulation and movement of tumor cells in M-CLL patients. Conclusions. An increased pro-inflammatory signature with involvement of the HMGB1 pathway was identified in MBL and U-CLL compared with HC, whereas the opposite was seen for M-CLL. Since MBL most often exhibit mutated IGHV (91% of cases in our cohort), these findings suggest immune stimulation as a characteristic feature in the pre-leukemic and U-CLL leukemic stage that surprisingly is not operative in M-CLL. Consistent with this, M-CLL displayed a global immune suppression (HMGB1 pathway inhibition), whereas U-CLL exhibited signs of immune stimulation (HMGB1 pathway activation) compared to HC which may relate to distinct capabilities of the two subtypes to interact with the microenvironment. Lastly, the increased inflammatory signature identified in MBL, which are mainly IGHV-mutated, was lessened in CLL, mainly in M-CLL patients. This is consistent with a decreased influence of immune imbalance and the HMGB1 pathway associated with IGHV-mutated clonal expansions. Acknowledgments. GB is supported by a grant from Fundación Alfonso Martín Escudero. Disclosures Allen: Alexon: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees. Rhodes: Conquer Cancer Foundation Young Investigator Award: Other: Grant/Research Support; AbbVie, Genentech, Pharmacyclics, TG Therapeutics: Other: Consultant.

Background: The development of Richter Transformation (RT) in patients (pts) with chronic lymphocytic leukemia (CLL) while on targeted agents has been reported to have a median survival of 2.3-3.5 months (mo). Treatment is usually anthracycline-based combination regimens, though responses are short-lived. Phase 2 trial of combined venetoclax-REPOCH achieved a median progression free survival (PFS) of 16 mo. We studied the use of ibrutinib in combination with obinutuzumab, cyclophosphamide, doxorubicin, vincristine, and prednisone (I-OCHOP) JUN2017-SEP2018 (ClinicalTrials.gov: NCT03145480). During the conduct of the study, ibrutinib plus RCHOP in untreated diffuse large B cell lymphoma (NCT01855750) reported more treatment-emergent serious adverse events when compared to RCHOP, leading to higher rates of treatment discontinuation with toxicity more prominent in elderly pts. Since the majority of pts with CLL are elderly, enrollment was stopped. We report the outcomes of 3 participants. Methods: Informed consent was provided through IRB-approved protocol. Subjects were enrolled in an open-label phase II trial combining ibrutinib with O-CHOP. Ibrutinib 560mg was taken daily starting cycle 1 day 1 (C1D1) continuously until progression or start of new anticancer treatment. Obinutuzumab was given per prescription insert starting on C1D1 with CHOP for up to 6 cycles. Pts were allowed to discontinue study drugs to pursue allogeneic stem cell transplant (allo-SCT). Pts aged 18-80 years old with adequate organ function, Eastern Cooperative Oncology Group performance status ≤ 2, and histologically confirmed evidence of RT from CLL were eligible to participate. Prior targeted agent use was allowed until 24 hours prior to dosing. Chemo- or immuno-therapy was not allowed within 21 and 10 days of C1D1 respectively. The primary endpoint was investigator-assessed overall response rate (ORR) per revised response criteria for malignant lymphoma. Secondary outcomes included hematological improvement, PFS, overall survival (OS), and quality of life. Pts were allowed to pursue allo-SCT after achieving a response and were followed for PFS/OS. Descriptive analyses were utilized, and PFS/OS was calculated from initial treatment to death or last follow-up. Results: Three pts consented and started therapy: two women and one man with a median [range] age of 53 [36-73] years, all had high-risk disease (Figure 1). Median Cumulative Illness Rating Scale at baseline 2 [0-4]. No large cell lymphoma involved the bone marrow. All pts developed RT while on a novel agent (ibrutinib, acalabrutinib, or venetoclax monotherapy respectively). All enrolled immediately after RT diagnosis and received I-OCHOP. Participants had overall good adherence to ibrutinib therapy (mean missed doses per 28-day treatment cycle was 0.88 doses) while on trial. Two pts developed neutropenia (n=1, grade 3 and n=1, grade 4; neither febrile) that resolved by time of withdrawal from study. 2 pts had anemia (grade 1 and 2), and one participant had thrombocytopenia (grade 2), neither requiring transfusion support. All 3 pts reported fatigue (all grade 2 or less) that on average improved during treatment as assessed by FACIT questionnaire (mean Δ from baseline to end of treatment: -5). Figure 1 lists other adverse events. Of 3 enrolled pts, 2 pts achieved remission (1 complete, 1 partial) and undetectable minimal residual disease (detection sensitivity 0.01%) in the bone marrow post 4 cycles I-OCHOP and underwent allo-SCT and were followed for progression. Figures 2 and 3 show the PET responses after 2 cycles of I-OCHOP in 2 participants with bulky lymphadenopathy. Time to allo-SCT for the 2 pts was 3.7 and 3.9 mo from C1D1. After transplant, one pt relapsed with RT in the central nervous system after 20 mo and died from pneumonia 5 mo later; the other pt is alive 49.5 mo later with no evidence of disease. The third pt had rapid progression and came off study. Median PFS for the 3 pts was 23.5 mo [1.9-NR] and OS 29.1 mo [6.7-NR] as of datacut July 30, 2021. Conclusions: Despite historically poor outcomes reported after RT while on a targeted agent, ibrutinib addition to anthracycline-based regimen seemed well tolerated. Two patients achieved a rapid and deep responses irrespective of prior BTK inhibitor use and were able to pursue allo-SCT which correlated with durable PFS/OS. Figure 1 Figure 1. Disclosures Rhodes: Conquer Cancer Foundation Young Investigator Award: Other: Grant/Research Support; AbbVie, Genentech, Pharmacyclics, TG Therapeutics: Other: Consultant.

Richter's transformation (RT) is a complication of chronic lymphocytic leukemia (CLL) characterized by the development of an aggressive lymphoma, typically diffuse large B cell lymphoma (DLBCL). RT is associated with high expression of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation of non-immunoglobulin genes. Current frontline therapy of CLL, ibrutinib, eliminates AID + CLL cells and alters tissue microenvironment to Th1-biased niches; however approximately 10-20% of CLL patients develop RT upon ibrutinib treatment. In addition, ibrutinib leads to a clinical response in only ~40% of RT patients. Thus, RT remains an important unmet challenge for CLL patients and the mechanism of RT development during ibrutinib treatment is poorly understood. To determine the contributions of AID and T cells in RT development, and to understand the efficacy of ibrutinib in the treatment of RT, we first created patient-derived xenografts (PDXs) with samples from three RT patients who were refractory to (immuno)chemotherapy but had not previously received ibrutinib. In each case, the B-lymphocytes were larger in size and expressed lower levels of CD5 but higher levels of activation markers compared with the CLL cell counterpart. In PDXs, ibrutinib treatment led to significantly less RT cells in spleen with percentage of inhibition ranging from 28-60% for all three cases. Notably, AID levels were similar in RT cells residing in spleen of ibrutinib-treated and control mice. The numbers of T cells were also similar, although Th1:Th2 ratios were significantly increased by ibrutinib (control vs ibrutinib group: 33% vs 42%, P=0.01). Interestingly, the growth and survival of RT cells located in bone marrow (BM) were not affected by ibrutinib. We then investigated the efficacy of ibrutinib in the prevention and treatment of RT in a novel strain of TCL-1 mice, TCL1/Igκ-AID mice, that have AID overexpressed solely in B lymphocytes. These mice develop accelerated CLL disease, have shorter overall survival (OS), and bear increased numbers of AID signature mutations in genes that are drivers in CLL and DLBCL. First we injected primary splenocytes from a TCL1/Igκ-AID mouse (A-31) into wild-type C57BL/6 mice. A week later, mice were treated with vehicle or ibrutinib continuously for 3 weeks (N=5 per group, duplicated experiments). At the end of treatment, RT cells were found in 25% of recipients in the control group and only in 10% of ibrutinib-treated recipients. Ibrutinib-treated animals had overall smaller spleens and lymph nodes (LNs) filled with significantly less CLL or RT cells. Importantly, the balance of Th1/Th2 cytokines was also significantly changed by ibrutinib (Th1/Th2 in control vs ibrutinib: 25% vs 47%, P=0.01). However, again RT cells in BMs of treated and control animals were similar. We then repeated the experiment to determine overall survival by giving the mice vehicle or ibrutinib continuously. Compared to the TCL1/Igκ-AID (A-31) donors, recipients treated with control vehicle developed more aggressive CLL with shorter OS. However, Ibrutinib did not change the OS; both control and ibrutinib treated recipients had similar OS with all the mice developed and died from RT eventually. Finally, to determine the efficacy of ibrutinib in the treatment of RT in TCL1/Igκ-AID mice, we sorted A-31-derived RT cells collected from LNs, and injected these into C57BL/6 mice. Mice then received treatment for 3 weeks. Notably, ibrutinib did not change the numbers or sizes of LNs or spleens. The numbers of RT cells at all sites, including BM, remained the same. In both groups, the number of T cells and the Th1:Th2 were not altered by ibrutinib. In summary, our data suggest that ibrutinib is somewhat effective against primary RT cells in the spleen in PDX models but not at all in the aggressive TCL1/IgK-AID mouse model. This might be because ibrutinib does not reduce AID levels in tissue-resident RT cells. Notably, although RT cells in spleens and LNs respond to ibrutinib, those residing in BM are resistant in both PDX and TCL1/IgK-AID models. This suggests different clones or different ibrutinib-responsiveness of RT cells in primary versus secondary lymphoid tissues. Overall, these results suggest that ibrutinib does not prevent or cure RT. Additionally, TCL1/Igκ-AID mice might provide a model to study the development, prevention, and treatment of RT without the influence of prior chemo- or other therapies. Disclosures Wirtz: Abbvie: Current Employment. Szafer-Glusman: AbbVie: Current Employment, Other: Stock or other ownership.

Phosphatidylinositol 3-kinase-delta (PI3Kδ) signaling is critical for proliferation, survival, homing, and tissue retention of malignant B cells. Idelalisib, a selective oral inhibitor of PI3Kδ, has shown considerable single-agent activity in patients with heavily pretreated chronic lymphocytic leukemia (CLL). This study evaluated the safety and clinical activity of idelalisib in combination with bendamustine (IB) or rituximab (IR) or both (IBR) in patients with relapsed or refractory (R/R) CLL. Idelalisib was given continuously at 100 or 150 mg twice daily in combination with rituximab (375 mg/m2 weekly × 8 doses), bendamustine (70 or 90 mg/m2, days 1 and 2 every 4 weeks × 6 cycles) or BR (rituximab, 375 mg/m2 every 4 weeks and bendamustine, 70 mg/m2, days 1 and 2 every 4 weeks × 6 cycles). The primary endpoint was safety; secondary endpoints included overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Fifty-two patients (median age 64 years) with a median of 3 prior therapies were enrolled. ORR was 84.6% (89.5% IR group, 77.8% IB group, and 86.7% IBR group). The overall median PFS was 25.6 months, and median DOR was 26.6 months. The most common grade ≥3 adverse events (≥10% of patients) were pneumonia (19.2%), diarrhea (13.5%), and febrile neutropenia (17.3%). Idelalisib-based combination therapy with bendamustine and/or rituximab was highly active, resulting in durable tumor control in patients with heavily pretreated R/R CLL. However, its tolerability profile suggests that these regimens should be used cautiously in this patient population. ClinicalTrials.gov ID: NCT01088048.

Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

The clinical course of patients with recently diagnosed early stage chronic lymphocytic leukemia (CLL) is highly variable. We examined the relationship between CLL-cell birth rate and treatment-free survival (TFS) in 97 patients with recently diagnosed, Rai stage 0-II CLL in a blinded, prospective study, using in vivo 2H2O labeling. Birth rates ranged from 0.07 to 1.31% new cells per day. With median follow-up of 4.0 years, 33 subjects (34%) required treatment by NCI criteria. High-birth rate was observed in 44% of subjects and was significantly associated with shorter TFS, unmutated IGHV status and expression of ZAP70 and of CD38. In multivariable modeling considering age, gender, Rai stage, expression of ZAP70 or CD38, IGHV mutation status and FISH cytogenetics, only CLL-cell birth rate and IGHV mutation status met criteria for inclusion. Hazard ratios were 3.51 (P=0.002) for high-birth rate and 4.93 (P

ROR1 is an oncoembryonic orphan receptor found on chronic lymphocytic leukemia (CLL) B cells, but not on normal postpartum tissues. ROR1 is a receptor for Wnt5a that may complex with TCL1, a coactivator of AKT that is able to promote development of CLL. We found the CLL cells of a few patients expressed negligible ROR1 (ROR1Neg), but expressed TCL1A at levels comparable to those of samples that expressed ROR1 (ROR1Pos). Transcriptome analyses revealed that ROR1Neg cases generally could be distinguished from those that were ROR1Pos in unsupervised gene-expression clustering analysis. Gene-set enrichment analyses demonstrated that ROR1Neg CLL had lower expression and activation of AKT signaling pathways relative to ROR1Pos CLL, similar to what was noted for leukemia that respectively developed in TCL1 vs ROR1xTCL1 transgenic mice. In contrast to its effect on ROR1Pos CLL, Wnt5a did not enhance the proliferation, chemotaxis, or survival of ROR1Neg CLL. We examined the CLL cells from 1568 patients, which we randomly assigned to a training or validation set of 797 or 771 cases, respectively. Using recursive partitioning, we defined a threshold for ROR1 surface expression that could segregate samples of the training set into ROR1-Hi vs ROR1-Lo subgroups that differed significantly in their median treatment-free survival (TFS). Using this threshold, we found that ROR1-Hi cases had a significantly shorter median TFS and overall survival than ROR1-Lo cases in the validation set. These data demonstrate that expression of ROR1 may promote leukemia-cell activation and survival and enhance disease progression in patients with CLL.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia of unknown etiology. Multiple studies suggest that the structure of the variable domains of the surface IGs on these cells, and signaling through them, play key roles in developing the disease. Hence, CLL appears to be driven by antigen–BCR interactions, and identifying the selecting antigens involved in this process is an important goal. We studied the antigen-binding characteristics of 23 CLL-derived, recombinantly-expressed IGs with 5 pathogenic bacteria, determining that CLL IGs differ in bacterial reactivity based on IGHV gene use, mutation status, and association with IGHD and IGHJ genes (“stereotypy”). Although most bacterial-reactive IGs followed the paradigm that IGHV-unmutated IGs were more auto-/poly-reactive, several did not. In addition, some CLL IGs were bacterial mono-reactive, and these displayed IGKV use biases. These findings are consistent with CLL B cells being driven into the leukemogenic process by bacterial as well as auto- antigens.

Within a period of just over a decade, managing chronic lymphocytic leukemia (CLL) has become more effective and yet more challenging than ever before. The important improvement in the treatment of CLL can be ascribed to the availability of many new options, mainly with the development of novel targeted therapies, such as ibrutinib, idelalisib, duvelisib and venetoclax. There are now newer tests that reliably define high-risk patients, and treatment plans can be tailored accordingly. Overall, this indeed is a new era in the treatment of patients with CLL. However, despite this progress, CLL remains an incurable disease and continues to remain challenging. In this brief review, the authors highlight the many great choices available to clinicians who manage patients with CLL and focus on the sequencing of these choices based on the available data.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15–producing cells are prevalent within B-CLL–infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB–driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rβ (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36–48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti–IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

The field of chronic lymphocytic leukemia (CLL) has witnessed considerable change since the time clinical staging was introduced in clinical practice in 1975. Over the years, the prognostication in CLL has expanded with the addition in late 90s of mutational status of variable region of immunoglobulin heavy chain (IGHV), and chromosomal analyses using fluorescent in situ hybridization (FISH). More recently, stereotypy of BCR (B cell receptor) and whole exome sequencing (WES) based discovery of specific mutations such as NOTCH1, TP53, SF3B1, XPO-1, BIRC3, ATM, and RPS15 further refined the current prognostication system in CLL. In therapy, the field of CLL has seen major changes from oral chlorambucil and steroids prior to 1980s, to chemo-immunotherapy (CIT) with fludarabine, cyclophosphamide, rituximab (FCR) to the orally administered targeted therapeutic agents inhibiting kinases in the B cell receptor (BCR) signaling pathway such as Ibrutinib (BTK inhibitor) and Idelalisib (p110 PI3Kδ inhibitor) and novel anti-CD20 mAb's (monoclonal antibodies) such as obinutuzumab. This progress is continuing and other targeted therapeutics such as Bcl2 antagonists (Venetoclax or ABT-199) and finally chimeric antigen receptor against T cells (CART) are in the process of being developed. This review is an attempt to summarize the major benchmarks in the prognostication and in the therapy of CLL. The topic allocated to us by Dr Ayalew Tefferi and Dr Carlo Brugnara is very appropriate to reminisce what our understanding of chronic lymphocytic leukemia (CLL) was in 1976 and how rapidly have the advances occurring in this field affected the patients with CLL.

Bruton's tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.