Bruton Tyrosine Kinase Inhibitor Ibrutinib Research Articles

Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

A single-cell multiomics analysis of IRF4 in mediating treatment resistance in ibrutinib-treated chronic lymphocytic leukemia (CLL).

7051 Background: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib (ibr) has revolutionized the treatment of CLL, showing efficacy in the majority of patients. Nevertheless, resistance occurs in a subset of patients, often with dismal clinical outcomes. Ibr resistance occurs through several mechanisms, including mutations affecting BTK or PLCG2, or upregulation of alternative survival pathways. We investigated at the single-cell level the molecular mechanisms that underlie ibr resistance in CLL cells and their interactions with non-malignant tumor microenvironment (TME) cell populations. Methods: We retrospectively identified 7 CLL patients with ibr resistance (as second line therapy). Samples were obtained before ibr treatment and at progression. Chromium Single Cell Multiome ATAC + Gene Expression (GEX) (10x Genomics) was applied to a mixture of CD19+CD5+ and CD19- cells to jointly analyze DNA accessibility and gene expression in the same single nuclei of CLL B and TME cells. An average of 4,800 cells (range 2,600-15,000) per sample was retained after QC filtering. Principal component analysis (PCA) was performed on GEX counts. ATAC peaks were called with MACS2, and latent semantic indexing was used for dimensionality reduction. We then computed a weighted nearest neighbour graph, identified clusters using the SLM algorithm and performed cell type assignment with established marker genes. Results: We identified 28 distinct cell clusters, representing CLL B-cells or cell types that constitute the TME, including CD4+ and CD8+ T cells, monocytes, dendritic and NK cells. CLL B-cell clusters were patient and timepoint-specific, while TME clusters were occupied by cells from multiple patients. Five patients showed multiple distinct CLL B-cell clusters pre-treatment or at progression, reflecting cellular heterogeneity within the tumor compartment. Interestingly, some clusters that were dominated by post-treatment CLL B-cells also included cells from pre-treatment samples, suggesting pre-existence of the clone giving rise to the relapse. For each patient, we performed pairwise differential expression between CLL B-cell clusters. Gene ontology analysis revealed increased activation of IRF4 and MHC-related pathways in CLL B-cells at progression compared to pre-treatment in 6 and 4 out of 7 patients, respectively. The elevated IRF4 pathway activity was underpinned by increased chromatin accessibility in IRF4 signature genes. Conclusions: Differential blockade of B-cell receptor signaling by ibr has been reported to downregulate activity of IRF4, a key transcription factor in B-cell activation. Our results suggest that CLL B-cells that are resistant to ibrutinib have re-activated IRF4 activity beyond pre-treatment levels, implying a potential role for IRF4 in ibrutinib resistance.

Comparison of zanubrutinib (zanu) and acalabrutinib (acala) in B-cell malignancies: An adverse event (AE)-based analysis.

e19049 Background: Acala and zanu are 2nd generation Bruton tyrosine kinase inhibitors (BTKis) that have demonstrated improved safety profiles vs 1st generation BTKi ibrutinib in clinical trials. While head-to-head comparison of zanu vs acala is lacking, a recent meta-analysis of clinical trials provided a comprehensive, indirect comparison of the AE profiles in B-cell malignancies (Hwang, Hemasphere. 2023;7(S3):1134). This study aims to evaluate costs and impacts on quality of life (QoL) for zanu vs acala using their AE profiles. Methods: A health economic model was developed to determine cost and QoL associated with AE management for zanu and acala. The incidence rates ( IRs) of all grade and grade ≥3 AEs of interest (n=21, including bleeding events, hypertension, atrial fibrillation, cytopenias, infections, headache, arthralgia, diarrhea) were taken from the meta-analysis. Additional model inputs, such as disutility, average duration of AE, and unit cost of each AE were sourced from published literature and publicly available cost databases in consultation with clinical experts. The model was developed from the US perspective, considering Medicare costs, inflated to 2023 USD. Model outcomes were AE management cost (= IR * unit cost) and quality-adjusted life years (QALYs) lost due to AE (= IR * Disutility * Duration/365.25). Results: In the base-case scenario (considering all AEs), treating a hypothetical cohort of 1000 patients (pts) with zanu instead of acala yielded cost savings of $124K and 3.7 QALY gains (i.e., 3.7 years extra in full health). Subgroup analysis for severe (grade ≥3) and non-severe AEs (grade 1-2) demonstrated similar trends. A sensitivity analysis limited to AEs significantly different between zanu and acala (n=13) yielded consistent results. Additionally, a probabilistic sensitivity analysis, conducted with 1000 iterations to account for uncertainty in model parameters, confirmed robustness of the model, indicating stable conclusions across a wide range of parameter uncertainties. Conclusions: This analysis suggested that zanu is cost-saving and associated with added health benefits compared to acala in terms of AE management in pts with B-cell malignancies. Should these results derived from meta-analysis of clinical trial data be applicable to real-world pt populations across different indications, the potential cost savings could be considerable. These results could be further improved if the differences in efficacy of zanu vs acala are also incorporated. [Table: see text]

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

New Means and Challenges in the Targeting of BTK.

Bruton's tyrosine kinase (BTK) is central to the survival of malignant and normal B lymphocytes and has been a crucial therapeutic target of several generations of kinase inhibitors and newly developed degraders. These new means for targeting BTK have added additional agents to the armamentarium for battling cancers dependent on B-cell receptor (BCR) signaling, including chronic lymphocytic leukemia and other non-Hodgkin lymphomas. However, the development of acquired resistance mutations to each of these classes of BTK inhibitors has led to new challenges in targeting BTK as well as novel insights into BCR signaling. The first-generation covalent BTK inhibitor ibrutinib is susceptible to mutations affecting the covalent binding site, cysteine 481 (C481). Newer noncovalent BTK inhibitors, such as pirtobrutinib, overcome C481 mutation-mediated resistance but are susceptible to other kinase domain mutations, particularly at residues Threonine 474 and Leucine 528. In addition, these novel BTK inhibitor resistance mutations have been shown biochemically and in patients to cause cross-resistance to some covalent BTK inhibitors. Importantly, newer generation covalent BTK inhibitors zanubrutinib and acalabrutinib are susceptible to the same mutations that confer resistance to noncovalent inhibitors. The BTK L528W mutation is of particular interest as it disrupts the kinase activity of BTK, rendering it kinase dead. This observation suggests that BTK may act independently of its kinase activity as a scaffold. Thus, the timely development of BTK degrading proteolysis targeting drugs has allowed for degradation, rather than just enzymatic inhibition, of BTK in B-cell lymphomas, and early clinical trials to evaluate BTK degraders are underway.

Abstract 5842: An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5842.

Abstract A038: Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib

Abstract Ovarian clear cell carcinoma (OCCC) is a difficult to treat histological subtype of ovarian cancer, demonstrating resistance to standard chemotherapy. New approaches are required in order to identify compounds with the potential to target this tumor. OCCC are characterized by mutations in ARID1A and/or ARID1B (AT-rich interactive domain-containing proteins) that encode for members of the mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex. We have used an epigenetic library consisting of 160 compounds that targets epigenetic readers, writers, erasers, and transcriptional modulators, to screen seven OCCC cell line models (RMG1, JHOC5, OVTOKO, OVMANA, OVISE, OV207 and TOV21G) and three non-OCCC ovarian cancer cell line models (A2780, COV434 and OVCAR8). Cell lines were seeded in triplicate into 96-well plates and treated with all 160 compounds at both 5 µM and 0.5 µM, as well as with a DMSO vehicle control, for 72 hours. Following treatment, cell viability was determined using a MTS assay and each data point normalized to the DMSO control. Based on the results of viability assays, drugs were sought that preferentially targeted OCCC relative to non-OCCC cell lines. Drugs that increased cell proliferation in two or more of the OCCC lines were removed from the analysis, as were drugs that did not impact upon viability. Drug 152, ibrutinib, inhibited cell viability between 53 to 78% in 6 of the 7 OCCC cell lines at 5 µM, with an average inhibition of 64%. In non-OCCC cell lines, the average cell viability after treatment with 5 µM ibrutinib was 91% (range, 84 – 103%). Ibrutinib is a small molecule inhibitor of Bruton tyrosine kinase (BTK) that has been approved by the FDA for treatment of a number of hematological malignancies including mantle cell lymphoma, small lymphocytic lymphoma and chronic lymphocytic leukemia. BTK functions in B-cell receptor signaling, regulating B cell proliferation and survival. Ibrutinib works by irreversibly blocking the kinase activity of BTK. Interestingly, ibrutinib binding sites are also present in other tyrosine kinases, including EGFR, raising the possibility of off-target effects that can function to decrease tumor cell viability. In addition to hematological malignancies, ibrutinib is being investigated in clinical trials for the potential to impact on solid tumors including non-small cell lung cancer, certain oesophagogastric carcinoma, advanced carcinoid and pancreatic neuroendocrine tumors, squamous cell carcinoma of the head and neck, renal cell carcinoma and glioblastoma, including in some cases in combination with other therapeutic drugs including cisplatin, temozolomide and nivolumab. Following organoid drug testing, off-label use of ibrutinib in a patient with low-grade serous ovarian cancer has recently shown promising results. Research is on-going to determine the effectiveness of ibrutinib in pre-clinical models of OCCC, including analyses of the effectiveness of ibrutinib in ARID1A and ARID1B wild-type and mutant CRISPR-engineered isogenic cell line panels of OCCC. Citation Format: Yue Ma, Kristie Ann Dickson, Natisha Field, Tao Xie, Nham Tran, Deborah Joy Marsh. Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A038.

Waldenström Macroglobulinemia: Targeted Agents Taking Center Stage.

With the worldwide approval of the oral covalent Bruton tyrosine kinase (BTK) inhibitors ibrutinib and zanubrutinib for treating patients with Waldenström macroglobulinemia (WM), targeted agents have certainly taken center stage in the therapeutic landscape of WM. This review discusses the biological and clinical data supporting current and up-and-coming targeted agents in WM. Bruton tyrosine kinase inhibitors induce fast, deep, and durable responses in patients with WM, comparable to chemoimmunotherapy; however, there is a glaring absence of comparative studies between these regimens. The high response and progression-free survival rate and the ease of administration of BTK inhibitors must be balanced against their specific adverse-event profile with unique toxicity (e.g., bleeding and cardiac arrhythmia) and the indefinite duration of the therapy. Novel targeted agents of interest include BCL2 antagonists (e.g., venetoclax and sonrotoclax) and non-covalent BTK inhibitors (e.g., pirtobrutinib and nemtabrutinib), among others. The therapeutic landscape of patients with WM will benefit from the robust participation of patients in clinical trials.

CAR-modified Cellular Therapies in Chronic Lymphocytic Leukemia: Is the Uphill Road Getting Less Steep?

The clinical development of chimeric antigen receptor (CAR) T-cell therapy has been more challenging for chronic lymphocytic leukemia (CLL) compared to other settings. One of the main reasons is the CLL-associated state of immune dysfunction that specifically involves patient-derived T cells. Here, we provide an overview of the clinical results obtained with CAR T-cell therapy in CLL, describing the identified immunologic reasons for the inferior efficacy. Novel CAR T-cell formulations, such as lisocabtagene maraleucel, administered alone or in combination with the Bruton tyrosine kinase inhibitor ibrutinib, are currently under investigation. These approaches are based on the rationale that improving the quality of the T-cell source and of the CAR T-cell product may deliver a more functional therapeutic weapon. Further strategies to boost the efficacy of CAR T cells should rely not only on the production of CAR T cells with an improved cellular composition but also on additional changes. Such alterations could include (1) the coadministration of immunomodulatory agents capable of counteracting CLL-related immunological alterations, (2) the design of improved CAR constructs (such as third- and fourth-generation CARs), (3) the incorporation into the manufacturing process of immunomodulatory compounds overcoming the T-cell defects, and (4) the use of allogeneic CAR T cells or alternative CAR-modified cellular vectors. These strategies may allow to develop more effective CAR-modified cellular therapies capable of counteracting the more aggressive and still incurable forms of CLL.

NLRP3 Inflammasome-Mediated Platelet Activation and Thrombus Formation in Sickle Cell Mice Can be Targeted By the BTK Inhibitor Ibrutinib

Introduction: Platelets are abnormally activated in sickle cell disease (SCD) and are involved in promoting vascular occlusions and thrombosis. The NOD like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mediator that has recently been found to be upregulated in platelets in SCD. Our recent discovery of Bruton Tyrosine Kinase (BTK) as a regulator of the NLRP3 inflammasome has provided an opportunity to suppress NLRP3 inflammasome activation in platelets via the BTK inhibitor ibrutinib. The role of the NLRP3 inflammasome in regulating platelet activation in SCD and its potential targetability through BTK inhibition is poorly understood. Methods: Townes SCD mice, which express human sickle hemoglobin (HbSS), and control mice (HbAA) at age 16 to 28 weeks were used for this study. Where indicated, mice were injected with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control 4 hours prior to blood collection. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) with confocal microscopy. We measured platelet surface expression of P-selectin (CD62P), platelet ATP secretion, platelet spreading, platelet aggregation, and in vitro thrombus formation using flow cytometry, luminescence, scanning electron microscopy, whole blood impedance aggregometry, and a flow chamber with blood perfused at shear rates of 1,700 s -1, respectively. Isolated platelets or blood samples were treated with the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Data are shown as mean ± SD. Results: We found increased platelet P-selectin expression (CD62P%: 74.57 ± 3.03 vs 35.18 ± 0.73, p<0.01), platelet ATP secretion (AUC: 16.24 ± 2.17 vs 3.16 ± 0.94, p<0.01), platelet spreading (spreading area, µm 2: 5.47 ± 1.97 vs 2.35 ± 0.23, p<0.01), platelet aggregation (AUC: 68.18 ± 6.84 vs 36.28 ± 3.29, p<0.01), and thrombus formation (thrombus area %: 64.24 ± 14.64 vs 27.44 ± 5.99, p<0.05) in blood from SCD mice as compared to control mice. NLRP3:ASC speck formation (Pearson coefficient: 0.62 ± 0.03 vs 0.38 ± 0.02, p<0.001) and caspase-1 activation levels (RFUs: 476.6 ± 54.84 vs 237.8 ± 50.09, p<0.001) were elevated in platelets from SCD mice as compared to control mice, indicating platelet NLRP3 inflammasome activation in SCD mice. Next, we investigated the role of the NLRP3 inflammasome in regulating platelet function in SCD mice. Treatment of mice with the NLRP3 inhibitor MCC950 resulted in a reduction of upregulated platelet P-selectin expression (CD62P%: 33.55 ± 17.21 vs 74.23 ± 9.95, p<0.001), platelet ATP secretion (AUC: 5.98 ± 1.2 vs 13 ± 2.91, p<0.001), platelet spreading (spreading area, µm 2: 2.42 ± 0.75 vs 5.47 ± 1.97, p<0.001), platelet aggregation (AUC: 39.85 ± 2.5 vs 64.65 ± 6.5, p<0.001), and thrombus formation (thrombus area %: 41.63 ± 7.78 vs 70.22 ± 5.5, p<0.001) (MCC950 vs vehicle) in SCD mice but had no effect in control mice. We next investigated the effect of the BTK inhibitor ibrutinib. Injection of ibrutinib interfered with elevated platelet NLRP3 inflammasome activation in SCD mice, as shown by quantification of intraplatelet NLRP3:ASC specks (Pearson coefficient: 0.56 ± 0.04 vs 0.72 ± 0.03, p<0.001) and platelet caspase-1 activity (RFUs: 193 ± 55.24 vs 386.8 ± 88.08, p<0.001) (ibrutinib vs vehicle) but had no effect in control mice. Ibrutinib treatment decreased upregulated platelet P-selectin (CD62P%: 37.81 ± 10.65 vs 64.74 ± 17.73, p<0.01), platelet ATP secretion (AUC: 6.78 ± 0.24 vs 13.9 ± 1.12, p<0.001), platelet spreading (spreading area, µm 2: 2.32 ± 0.79 vs 6.45 ± 0.99, p<0.001), platelet aggregation (AUC: 40.08 ± 3.56 vs 62.13 ± 3.99, p<0.001), and thrombus formation (thrombus area, %: 44.42 ± 8.24 vs 66.97 ± 11.83, p<0.001) (ibrutinib vs vehicle) in SCD mice. The NLRP3 activator nigericin reversed these inhibitory effects of ibrutinib in SCD mice, indicating that ibrutinib interferes with platelet activation and thrombus formation in SCD mice through the NLRP3 inflammasome. Conclusion: The NLRP3 inflammasome is a critical mediator of platelet activation and thrombus formation in SCD mice, which is targetable by the BTK inhibitor ibrutinib, making it a potentially useful therapeutic approach for managing the prothrombotic state in SCD.

Aberrant Heterochromatin Silences Immune Response Genes in Chronic Lymphocytic Leukemia

Introduction: Recent studies have shown that clonal B cells derived from patients with chronic lymphocytic leukemia (CLL) have altered histone modification landscapes compared to B cells from healthy controls. We have also shown that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells, implicating the interaction between oncogenic signaling and epigenetic machinery in CLL (Wang et al., Blood Cancer J. 2022; Wang et al., Cancer Res (2023) 83 (7_Supplement): 4725). However, the majority of the chromatin studies in CLL concentrate on the active enhancers associated with oncogene expression; there is a very limited understanding of lost enhancers in CLL: 1) What is the function of lost enhancers; 2) How are these lost enhancers maintained in CLL; 3) Can these lost enhancers be detected at the precursor state of CLL, monoclonal B-cell lymphocytosis (MBL); 4) if this aberrant chromatin signature affected by CLL treatment. Methods: We systematically mapped the genome-wide histone modification profile (histone marks: H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, and H3K36me3) in 22 previously untreated CLL samples, 6 high count MBL samples, and peripheral blood B cells from 6 healthy donors by Cleavage Under Targets and Tagmentation (CUT&Tag). We also analyzed B cells from 8 patients before and after one year of continuous Bruton tyrosine kinase inhibitor (BTKi) ibrutinib treatment. The genomic regions with a decrease in both H3K4me1 and H3K27ac enrichment (analyzed by Deseq2, fold change > 1, p < 0.01) were named lost enhancers. We used CRISPR-mediated epigenetic editing to study the role of gained H3K9me3 on EBF1 gene expression. Results: Compared to normal B cells, the lost enhancers (genomic regions defined by decreased enrichment of H3K4me1 and H3K27ac) in CLL B cells showed increased heterochromatin marks (H3K9me3 and H3K27me3) enrichment. Importantly, these enhancers are associated with genes downregulated in CLL and involved in the immune response, indicating the source of epigenetic impact on impaired immunity in CLL. We further found that this aberrant chromatin structure also exits in clonal B cells from the precursor stage of MBL, implicating the importance of epigenetic silencing in CLL evolution. In particular, we found the lost enhancer signature across the gene locus EBF1, an important transcription factor that defines B cell lineage and regulates normal B cell activation. Using CRISPR-mediated epigenetic editing, we demonstrated that the gain of H3K9me3 is sufficient to silence the EBF1 enhancer and its gene expression. Finally, we found that while BTKi ibrutinib treatment partially inhibits the CLL-gained enhancers in some patients, it failed to restore the lost enhancers in CLL B cells. Conclusion: In summary, we have elucidated an epigenetic silencing mechanism that may contribute to the loss of physiological function of normal B cells, which places the clonal B cells at a selection advantage during CLL evolution and increases the risk of serious infections in CLL and MBL ( Fig 1).

Combined Inhibition of Protein Kinase CSNK2 and BET Proteins As a Novel Therapeutic Strategy for Mantle Cell Lymphoma

Currently available therapeutic approaches for Mantle Cell Lymphoma (MCL), including the Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib, acalabrutinib and zanubrutinib, are not curative. Therefore, the discovery of novel molecules amenable of drug targeting is a research priority in the MCL field. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain (B) and extra terminal (ET) (BET) protein family that regulate chromatin modifications and assembly of transcriptional complexes. Inhibition of BET by available compounds have shown promising anticancer effect. In MCL, BET inhibitors, such as INCB054329 or JQ-1, have been shown to increase apoptosis through downregulation of the AKT-mTOR, ERK, and other B Cell Receptor (BCR)-triggered cascades. We and others have previously demonstrated that the S/T kinase CSNK2 supports chronic BCR activation in MCL by upregulating the AKT/PI3K and NF-κB pathways. Inactivation of CSNK2 results in efficacious induction of MCL cell death. Notably, BRD4 activity is regulated by CSNK2 and double inhibition of BRD4 and CSNK2 has shown significant activity in other tumor models. The most effective and tested CSNK2 chemical inhibitor is CX4945 (silmitasertib), but very recently a novel compound, SGC-CK2, has been developed. No data are available on the effects of SGC-CK2 on MCL cells. In this work, we have evaluated the effects of the combined inhibition of CSNK2 and BET proteins on MCL cell growth and survival signaling pathways. CSNK2 inhibitors employed were CX4945 and SGC-CK2. CSNK2 gene silencing was achieved with the generation of anti- CSNK2 short hairpin-RNA IPTG-inducible MCL cell clones. BET inhibitors used were JQ-1 and INCB054329. Cell survival, apoptosis and proliferation were investigated by AnnexinV/PI staining and FACS analysis, by Western blot (WB) detection of PARP cleavage and Mcl1 expression and by other cell proliferation assays. The synergic action of BET and CK2 inhibitors was assessed by the Chou-Talalay combination index method. CK2 expression and survival-related signaling components were analyzed by WB, qRT-PCR and protein expression assays. CX4945 and the novel inhibitor SGC-CK2 displayed a potent anti-tumor activity on MCL cells as judged by the effective induction of apoptosis and proliferation arrest. A synergistic effect of CSNK2 and BET inhibitors was observed in all the MCL cell lines tested, (Jeko-1, Rec-1, including Granta-519 that are known to be much less sensitive to ibrutinib) and with all the inhibitors tested. The same results were obtained in the CSNK2 gene silencing models. Preliminary data showed that the cooperative cytotoxic effect of CK2 inhibition and BET inhibition on MCL apoptosis was observed also in MCL patient derived B cells, but not in healthy B lymphocytes form buffy coat. CSNK2 inactivation was associated with strong down-regulation of c-Myc and Mcl-1 proteins and with hampered activation of NF-κB and PI3K/AKT-dependent signaling. Surprisingly, BET protein inhibition caused increased Mcl-1 protein expression and augmented Ser 529 p65-NF-kB phosphorylation in Granta-519 and Rec-1 cells. Remarkably, CK2 inactivation led to a robust reduction of the BET inhibitor-induced increase of Mcl1, and NF-kB Ser 529 phosphorylation, thus counteracting BET-inhibitors-evoked compensatory pathways that could favor apoptosis resistance. Therefore, combined CSNK2 and BET proteins inhibition could represent an innovative strategy for chemotherapy and BTKi-resistant MCL.

CXCR4 S338X Promotes Resistance to Second Generation BTK Inhibitor and BCL-2 Inhibitor in WM Cells

Introduction: Waldenström macroglobulinemia (WM) is an IgM-secreting lymphoplasmacytic lymphoma. CXCR4 somatic mutations are common in WM and are associated with clinical resistance to Bruton's tyrosine kinase (BTK) inhibitors and other therapeutic agents. CXCR4 mutations occur nearly exclusively with MYD88 L265Pmutation, with CXCR4 S338X constituting the most common in WM patients. The aim of this study was to further investigate the effect of CXCR4 S338X on BTK inhibitors (ibrutinib and zanubrutinib) and a BCL-2 inhibitor (venetoclax) resistance. Methods: MWCL-1 cells were transfected with scramble, CXCR4 or CXCR4 S338Xplasmids using Lipofectamine 3000 followed by neomycin selection. CXCR4 expression was confirmed using qPCR and flow cytometry. Transfected cells were cultivated in presence of ibrutinib (5μΜ), zanubrutinib (5μΜ) and venetoclax (1μΜ) for 48 hours. Proliferation was estimated using CCK-8 colorimetric assay while cell cycle was evaluated by propidium iodide staining and flow cytometry. Apoptosis was estimated by Annexin V/7-AAD staining and flow cytometry while the expression of apoptotic genes was determined by qPCR. The statistical analysis was performed using Student's t-test and Graphpad Prism. Results: Drug-induced cytotoxicity was observed only in the case of venetoclax in CXCR4 S338X transfectedcells (p= 0.0007) while increased cytotoxicity was noted in the presence of ibrutinib, zanubrutinib and venetoclax in the case of scramble-transfected cells (p=0.038, p=0.006 and p=0.0006, respectively). Moreover, no significant difference in proliferation rate was noted between CXCR4 S338Xtransfectedcells treated with the therapeutic agents and CXCR4 S338X untreated cells, underlining the S338X role to promote cell proliferation. Interestingly, CXCR4 S338X transfectedcells exhibited apoptosis resistance not only compared to scramble-transfected cells but also in comparison with CXCR4-transfected cells, associated with resistance to all three drug agents (p=0.004, p=0.003 and p=0.034, respectively). In the case of scramble-transfected cells, an increase in apoptosis was demonstrated in parallel with an increase in caspases 8, 9 and 3 in presence of ibrutinib (p=0.006, p=0.0001 and p=0.025, respectively) and zanubrutinib (p=0.027, p=0.007 and p=0.052, respectively), whereas no statistically significant increase in caspase gene expression was observed in the case of CXCR4 S338Xtransfectedcells, highlighting S338X role in the inhibition of intrinsic and extrinsic apoptosis pathways. Lastly, the presence of S338X mutation acted as a mitotic promoting factor when cells treated with the aforementioned drugs showed a high proportion of cells in G2/M phases of cell cycle compared to scramble and CXCR4 controls. Conclusions: The above data demonstrate the role of CXCR4 S338X mutation in drug resistance and suggest the necessity to develop therapeutic strategies to address this issue and overcome this therapeutic barrier in WM. Data on more cell lines and downstream signaling will be presented at the meeting.

Toxicity, Progression-Free Survival, and Quality of Life of Patients Treated with Zanubrutinib Versus Ibrutinib: A Q-TWiST Analysis from the ALPINE Study in Relapsed or Refractory Chronic Lymphocytic Leukemia

Introduction: Zanubrutinib is a second-generation Bruton's tyrosine kinase inhibitor (BTKi) with enhanced specificity over the first-generation BTKi ibrutinib. Patients treated with zanubrutinib had a significantly longer progression-free survival (PFS) versus those treated with ibrutinib in the global, phase 3, randomized ALPINE trial in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) (NCT03734016). Quality-adjusted time without symptoms of disease and toxicity (Q-TWiST) is a clinical tool to assess overall benefits and risks of cancer therapies by integrating progression, survival, treatment toxicity, and patient quality of life (QoL) into a single metric. It provides valuable insights into the quality and duration of improved health states. To gain a comprehensive understanding of the benefits and risks associated with zanubrutinib versus ibrutinib in terms of quality-adjusted survival, a Q-TWiST analysis was performed within the ALPINE trial. Methods: Patients in the ALPINE trial were followed for a median duration of 29.6 months. For the Q-TWiST analysis, overall survival (OS) of each patient was partitioned into 3 mutually exclusive health states: TOX (time before disease progression with toxicity after randomization); TWiST (time from randomization to disease progression without toxicity); and REL (time after disease progression until death or censoring). Survival curves corresponding to TOX, PFS, and OS were estimated by Kaplan-Meier method with a monthly cycle. Restricted mean survival time for each health state was derived from the area under the Kaplan-Meier curve (TWiST = PFS - TOX; REL = OS - PFS). Q-TWiST for each treatment was estimated as the mean time spent in each health state weighted by its respective QoL, denoted by health states' utility value (U; 0 [indicates death] to 1.0 [indicates “perfect” health]). Relative Q-TWiST gain was estimated by dividing the absolute Q-TWiST gain by mean OS with ibrutinib. To ensure the comparability of populations and treatments across published Q-TWiST studies, the base-case analysis focused on a high-risk population (i.e., patients with chromosome 17p deletion, TP53 mutation, or both). In the base case, the TOX health state included adverse events (AEs) of grade 2 or higher. Standard utilities were applied, with a value of 0.5 assigned to both TOX and REL and a value of 1.0 assigned to TWiST. A sensitivity analysis was performed in an intent-to-treat population with same-grade AEs considered for TOX and standard utilities. Results:In the base case, the mean durations of TOX, TWiST, and REL health states for zanubrutinib (N = 73) were 11.54 months, 14.45 months, and 1.70 months, respectively. The corresponding durations for ibrutinib (N = 71) were 11.38 months, 11.09 months, and 3.78 months, respectively. The mean difference (95% CI) for zanubrutinib versus ibrutinib was 0.16 months (−0.18, 0.51) for the TOX state, 3.36 months (2.6, 4.2) for the TWiST state, and −2.08 months (−2.6, −1.6) for the REL state. The mean duration of Q-TWiST was 21.07 months for zanubrutinib versus 18.67 months for ibrutinib. The estimated difference in means (95% CI) for Q-TWiST gain was significantly higher for zanubrutinib versus ibrutinib (2.40 months; 1.9, 2.9; P<0.001), and the relative Q-TWiST gain was 9.14% (Figure). In the sensitivity analysis, using the intent-to-treat population, the mean difference (95% CI) for Q-TWiST gain was 1.30 months (1.0, 1.6; P=0.05) for zanubrutinib versus ibrutinib, and the relative Q-TWiST gain was 4.63%. Conclusions: This Q-TWiST analysis demonstrated a statistically significant gain in quality-adjusted survival with zanubrutinib compared with ibrutinib in patients with high-risk R/R CLL. The slightly longer TOX duration in patients receiving zanubrutinib versus those receiving ibrutinib could be explained in part by better treatment adherence. The results of this Q-TWiST analysis, which integrates both the length and quality of survival in addition to efficacy and toxicity, provide valuable insights that may help to inform clinical decision-making in the treatment of patients with R/R CLL.

Fixed Duration Mosunetuzumab Plus Polatuzumab Vedotin Has Promising Efficacy and a Manageable Safety Profile in Patients with BTKi Relapsed/Refractory Mantle Cell Lymphoma: Initial Results from a Phase Ib/II Study

Background: New treatment options are needed for patients (pts) with relapsed/refractory (R/R) mantle cell lymphoma (MCL), especially for pts with progressive disease (PD) after Bruton's tyrosine kinase inhibitor (BTKi) therapy. Outcomes for R/R MCL pts with PD after the BTKi ibrutinib are very poor, demonstrated by a median overall survival of 2.9 months (Martin et al. Blood 2016). While chimeric antigen receptor (CAR) T-cell therapies are approved for pts with R/R MCL; treatment is associated with high toxicity and difficulty with accessibility. Mosunetuzumab (Mosun) is a CD20xCD3 T-cell engaging bispecific antibody that engages and redirects T cells to eliminate malignant B cells. Polatuzumab vedotin (Pola) is a CD79b-targeted antibody-drug conjugate that inhibits cell division and induces apoptosis in B cells. This ongoing Phase Ib/II study (NCT03671018) is investigating Mosun in combination with Pola (M-Pola) in pts with R/R B cell non-Hodgkin lymphoma. Here, we report initial safety and efficacy data from an ongoing Phase II expansion cohort of pts with R/R MCL who had received prior BTKi therapy. Methods: Eligiblepts had histologically confirmed R/R MCL and had received ≥2 prior regimens (including an anti-CD20 agent, BTKi, and anthracycline- or bendamustine-based therapy). Mosun was administered subcutaneously (SC) by step-up dosing on Days (D) 1 (5mg), 8 (45mg), and 15 (45mg) of Cycle (C) 1, then 45mg on D1 of every cycle from C2D1, every 3 weeks for a total of 17 cycles. Pola (1.8mg/kg intravenous [IV] infusion) was administered on D1 of C1-6. All pts received corticosteroid premedication prior to C1 of treatment. From C2, premedication was optional for pts who did not experience cytokine release syndrome (CRS) in the previous cycle. Response was assessed by investigators using Lugano 2014 criteria (Cheson et al. J Clin Oncol 2014). Results: As of October 27, 2022, 20 pts had received M-Pola (n=10 active on treatment, n=1 completed treatment, n=9 discontinued Mosun treatment). Median age of all pts was 68.0 (range: 44-82) years, 75% were male, 95% had Ann Arbor stage III/IV disease, and 40% had a MIPI score ≥6 at study entry. Median number of prior lines of therapy (LOT) was 3 (range: 2-9). All pts had received prior BTKi therapy; 7 pts (35%) had received prior CAR T-cell therapy; and 85% (17/20) of pts were refractory to their last LOT. Median time since last LOT was 1.6 (range: 0-39) months. The proportions of pts with high-risk MCL factors at baseline were: 65% of pts with Ki-67 proliferation index ≥50%; 50% with blastoid/pleomorphic variants; and 20% with TP53 mutation. The overall median time on study was 7.2 (range: 0.4-17.5) months. The most common (≥25%) Mosun and/or Pola-related adverse events (AEs) in all pts (n=20) were CRS (50%), injection site reaction (50%), fatigue (45%), dyspnea (35%), paresthesia (30%), diarrhea (30%), myalgia (30%), infusion-related reaction (25%), and nausea (25%). Two (10%) Grade (Gr) 5 AEs occurred, both of which were due to COVID-19 pneumonia; neither were deemed treatment-related by the investigator. Ten pts (50%) experienced CRS events; 9 pts experienced Gr 1 events, and 1 pt experienced a Gr 2 event, which was managed with tocilizumab and low-flow oxygen. One pt was managed with corticosteroids and no pts required pressors or ICU admission. All CRS events resolved by the data cut-off. Treatment-related neurologic AEs, potentially consistent with ICANs, occurred in 3 pts (15%; Gr 1 confusional state and Gr 2 amnesia, 1 pt; Gr 1 agitation, 1 pt; and Gr 2 memory impairment, 1 pt). Neuropathy occurred in 3 pts (15%); all events were Gr 1. Four pts (20%) had AEs leading to treatment discontinuation: 1 pt with Gr 3 uveitis (related to M-Pola); 1 pt with Gr 3 pneumonitis (related to M-Pola); and 2 pts with Gr 5 COVID-19 pneumonia (both unrelated to M-Pola). Overall response rate (ORR) and complete response (CR) rates were 75% and 70%, respectively (Figure). Best ORR and CR rates were generally consistent in high-risk MCL subgroups (Figure). Median duration of CR was not evaluable (NE; 95% confidence interval: 3.8-NE). Conclusions: Fixed-duration Mosun SC in combination with Pola IV induced high CR rates in pts with R/R MCL who had previously received BTKi therapy, including those in high-risk subgroups and those who had received prior CAR T-cell therapy. CRs were observed early and persisted during the follow up period. M-Pola had a manageable safety profile, and all CRS events were low grade.

Overcoming the therapeutic limitations of EZH2 inhibitors in Burkitt's lymphoma: a comprehensive study on the combined effects of MS1943 and Ibrutinib.

Enhancer of zeste homolog 2 (EZH2) and Bruton's tyrosine kinase (BTK) are both key factors involved in the development and progression of hematological malignancies. Clinical studies have demonstrated the potential of various EZH2 inhibitors, which target the methyltransferase activity of EZH2, for the treatment of lymphomas. However, despite their ability to effectively reduce the H3K27me3 levels, these inhibitors have shown limited efficacy in blocking the proliferation of lymphoma cells. To overcome this challenge, we employed a hydrophobic tagging approach utilizing MS1943, a selective EZH2 degrader. In this study, we investigated the inhibitory effects of two drugs, the FDA-approved EZH2 inhibitor Tazemetostat, currently undergoing clinical trials, and the novel drug MS1943, on Burkitt's lymphoma. Furthermore, we assessed the potential synergistic effect of combining these drugs with the BTK inhibitor Ibrutinib. In this study, we evaluated the effects of combination therapy with MS1943 and Ibrutinib on the proliferation of three Burkitt's lymphoma cell lines, namely RPMI1788, Ramos, and Daudi cells. Our results demonstrated that the combination of MS1943 and Ibrutinib significantly suppressed cell proliferation to a greater extent compared to the combination of Tazemetostat and Ibrutinib. Additionally, we investigated the underlying mechanisms of action and found that the combination therapy of MS1943 and Ibrutinib led to the upregulation of miR29B-mediated p53-upregulated modulator of apoptosis PUMA, BAX, cleaved PARP, and cleaved caspase-3 in Burkitt's lymphoma cells. These findings highlight the potential of this innovative therapeutic strategy as an alternative to traditional EZH2 inhibitors, offering promising prospects for improving treatment outcomes in Burkitt's lymphoma.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Drug-drug interactions and dose management of BTK inhibitors when initiating nirmatrelvir/ritonavir (paxlovid) based on physiologically-based pharmacokinetic models

ObjectiveCo-administration of Bruton's tyrosine kinase (BTK) inhibitors with nirmatrelvir/ritonavir is challenging because of potential drug-drug interactions (DDIs). However, clinical trials specifically evaluating such DDIs are absent. To evaluate and quantify the DDIs between them and provide rational dose management strategies of BTK inhibitors, we conducted this study using physiologically-based pharmacokinetic (PBPK) models. MethodsPhysicochemical properties and pharmacokinetic parameters were acquired from the published literature and databases. The PBPK models were developed using Simcyp® software. These models were validated by comparing with published literature values. The successfully validated PBPK models were used to simulate the plasma concentration-time profiles and DDIs in a virtual healthy population receiving BTK inhibitors alone or with ritonavir. ResultsSimulated plasma concentration-time profiles and pharmacokinetic parameters of each drug were in agreement with clinically observed values from literatures. Ritonavir increased ibrutinib maximum plasma concentration (Cmax) and the area under plasma concentration-time curve (AUC) 33- and 53.88-fold, respectively, increased zanubrutinib Cmax and AUC 2.57- and 3.18-fold, respectively, and increased acalabrutinib Cmax and AUC 3.85- and 6.54-fold, respectively. Based on our simulations, dose-adjustment strategies may consist of ibrutinib at 25 mg q48h, zanubrutinib at 80 mg twice-daily and acalabrutinib at 25 mg twice-daily with nirmatrelvir/ritonavir. ConclusionsThe PBPK models predicted the in vivo pharmacokinetics and the DDIs of BTK inhibitors and ritonavir. The prospective simulations not only provided scientific evidence regarding rational dosing management strategies when initiating nirmatrelvir/ritonavir therapy but also provided a reference for the design of clinical DDIs study that may save resources and time. SummaryPaxlovid could increase Cmax and AUC0-τ of BTK inhibitors (ibrutinib, zanubrutinib and acalabrutinib), and dose adjustment strategy of ibrutinib (25 mg q48h), zanubrutinib (80 mg q12h) and acalabrutinib (25 mg q12h) should be considered when combination with nirmatrelvir/ritonavir.

Treatment with idelalisib in patients with chronic lymphocytic leukemia – real world data from the registry of the German CLL Study Group

Idelalisib in combination with rituximab is an efficacious treatment for patients suffering from chronic lymphocytic leukemia (CLL) with known limitations due to toxicities. However, the benefit after prior Bruton tyrosine kinase inhibitor (BTKi) therapy remains unclear. For this analysis, 81 patients included in a non-interventional registry study of the German CLL study group (registered at www.clinicaltrials.gov as # NCT02863692) meeting the predefined criteria of a confirmed diagnosis of CLL and being treated with idelalisib containing regimens outside clinical trials were considered. 11 patients were treatment naïve (13.6%) and 70 patients (86.4%) pretreated. Patients had median of one prior therapy line (range 0–11). Median treatment duration with idelalisib was 5.1 months (range 0–55.0 months). Of 58 patients with documented treatment outcome, 39 responded to idelalisib containing therapy (67.2%). Patients treated with the BTKi ibrutinib as last prior treatment prior to idelalisib responded in 71.4% compared to a response rate of 61.9% in patients without prior ibrutinib. Median event free survival (EFS) was 15.9 months with a 16 versus 14 months EFS in patients with ibrutinib as last prior treatment or not, respectively. Median overall survival was 46.6 months. In conclusion, treatment with idelalisib appears to have a valuable impact in patients being refractory to prior ibrutinib therapy even though there are limitations in our analysis due to the low number of patients included.

Waldenström's macroglobulinemia - clinical symptoms and review of therapy yesterday, today and tomorrow.

Waldenström macroglobulinemia (WM) is a lymphoplasmocytic lymphoma with immunoglobulin M monoclonal protein. The incidence of this disease is very low (0.4/100,000), so that this disease can be regarded as an orphan's disease. It means that new drugs are often tested and registered for more frequent diseases. In this review we will focus on the efficacy of the new drugs for WM. The current treatment options for symptomatic WM patients include alkylating agent cyclophosphamide and anti-CD20 monoclonal antibodies. Therapy with rituximab and bendamustin resulted in longer therapeutic response then therapy with rituximab, cyclophosphamide and dexamethasone. Many drugs, used in multiple myeloma (MM), shoved promising results in WM patients. Bortezomib is effective in WM, but its neurotoxicity is higher in WM than in MM patients. Therefore, new proteasome inhibitors, carfilzomib and ixazomib, are better tolerated as documented in several studies. New types of antiCD20 antibody (obinutuzumab) can be used in patients with rituximab intolerance. in five of our patients with WM, obinutuzumab and bendamustin reached deeper responses than therapies administered in previous lines of therapy. Oral Bruton tyrosine kinase (BTK) inhibitor ibrutinib alone and in combination with rituximab have extended the treatment options for WM patients. New BTK inhibitors (e. g. acalabrutinib, zanubrutinib, and vecabrutinib) were tested and their lower toxicity (atrial fibrillation) was documented. Moreover, the BCL2 inhibitor venetoclax is newly tested. New antiCD20 antibody (obinutuzumab) is of advantage in patients with WM with rituximab intolerance as well as bendamustin and new proteasome inhibitors (ixazomib and carfilzomib) or new BTK inhibitors with lower cardiotoxicity. Many of the abovementioned drugs do not have official registration for WM and can be administrated with the consent of the health care provider only. Thus, this work brings evidence of their efficacy.