Bruton's Tyrosine Kinase Inhibitor Research Articles

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [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 534.

Abstract 6409: A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients

Abstract Purpose Functional dysregulation of B-cells leads to a variety of autoimmune disorders and blood cancers. Multiple B-cell depleting therapies (e.g., BTK inhibitors, antibodies or CAR-Ts) are currently in active clinical trial investigation. Following therapeutic elimination of pathogenic B-cells, it is critical to monitor the productive regeneration of fully functional B-cells as they mediate innate and adaptive immune responses. To this end, we developed a multiparametric flow cytometry assay for exquisite monitoring of early stages of B cell development, maturation, and antibody production to correlate it with treatment outcomes and determine if continuous depletion of B-cells is necessary to maintain clinical benefits for patients. Study Design We designed a high throughput 12-parameter flow cytometry panel incorporating primary differentiation markers namely, CD45, CD3, CD38, CD10, CD27, CD19, CD20, IgD, Lambda & Kappa Light Chains required for identification of different developmental stages of B-cells in peripheral blood using an in vitro diagnostic grade flow cytometry system (BD FACSLyric). The optimized method underwent robust fit-for-purpose analytical validation per CLSI H62 guidelines with emphasis on establishing assay sensitivity, specificity, and reproducibility across experimental days, operators, and instruments. Results We were able to reliably identify different stages of B-cell development, namely, Naïve (CD19+CD20+IgD+CD27-), Unswitched/Marginal (CD19+CD20+IgD+CD27+), Memory (CD19+CD20+IgD-CD27+), Transitional (CD19+CD20+IgD+CD27-CD10+), Antibody Secreting Cells (CD19+CD20-IgD-CD27+CD38hi) as well as clonality (CD19+CD20+Lambda+ or Kappa+). The lower limit of quantitation was determined to be around 60 events, based upon analysis of 90 data points with good reproducibility (CV range: 0-11.7%) across experimental days, operators and instruments demonstrating sensitive detection and robust performance. Conclusion In summary, we have developed and validated a 12-parameter flow cytometry assay focusing on monitoring the regeneration of different subsets of B-cells in various B-cell malignancies (Non-Hodgkin’s Lymphoma, B CLL) and autoimmune disorders (Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus). The high reproducibility/robustness of the assay demonstrates its suitability for use in clinical trials as a valuable tool for safety monitoring and disease evaluation enabling development of novel therapeutic agents. Citation Format: Amit Kumar Mehta, Ruby Tandon, Anil Pahuja, Jane Gao, Kevin Nguyen, Naveen Dakappagari, Zeni Alfonso. A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients [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 6409.

Abstract 7583: Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer

Abstract The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), as the core component of CBM complex (CARD9/10/11/14-BCL10-MALT1), plays an essential role in activating the NF-κB pathway and regulating the cell proliferation, differentiation and immunity. Inhibiting of MALT1 is considered a promising therapeutic approach for the treatment of several non-Hodgkin B-cell lymphomas, chronic lymphocytic leukemia (CLL), solid tumors and autoimmune diseases. Herein, we report the discovery of a novel potent MALT1 allosteric inhibitor, BPI-530616, which strongly inhibited MALT1 protease activity with subnanomolar IC50, and low off-target activity on other proteases. BPI-530616 can inhibit the proliferation of B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell line, OCI-ly3, bearing CD79b and CARD11 mutations. The cell line is intrinsically resistant to covalent and non-covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Meanwhile, BPI-530616 suppressed the cellular cytokine secretion (e.g.IL-10, IL-2) or MALT1 direct substrate cleavage (e.g. CYLD). In vivo, BPI-530616 dose-dependently suppressed tumor growth and inhibited the cleavage of CYLD in OCI-ly3 xenograft mouse model. To address the role of MALT1 inhibitor in the contest of immune-oncology, a CT26 syngeneic mouse model was set up. Oral administration of BPI-530616 exhibited a good antitumor effect comparable to that of the anti-PD1 antibody, with no observable adverse effects. Additionally, BPI-530616 displayed desirable drug-like properties, including low clearance and good oral bioavailability across multiple pre-clinical species. Taken together, our research has led to the discovery of a novel, potent, selective, and orally bioavailable small molecule MALT1 inhibitor, BPI-530616. It demonstrated anti-tumor efficacy in the DLBCL animal model and showed promising effects in an immune-oncology animal model. Citation Format: Yuzhen Zhou, Bang Fu, Jie Chen, Weidong Cai, Xiao Sun, Yinlong Li, Wei Ren, Qichao Shen, Guolong Du, Xiaojun Zhou, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Cheng Yang, Xiangyong Liu, Jing Guo, Hao Wu, Lieming Ding, Jiabing Wang, Hong Lan. Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer [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 7583.

Abstract 7140: A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents

Abstract Background: Plant-derived compounds, including flavonoids, can be potent anti-cancer agents, but low bioavailability, lack of specificity, and complexity in their synthesis can hamper their clinical development. Here, we present seven novel flavonoid derivatives, and test them for their anti-tumor activity in various models derived from hematological cancers and solid tumors. Methods: MTT assessed anti-proliferative activity after 72h of exposure to increasing concentrations of drugs. Tubulin Polymerization Assay was performed with 3-fold dilutions (15-0.02 μM), compared to paclitaxel (1-3 μM) or vinblastine (3 μM) as references. The OncolinesProfiler analysis was used to compare IC50 fingerprints determined on the Oncolines cell line panel to the IC50 fingerprints of 213 pre-profiled reference drugs. Results: SDN207, SND210, SND216, SND462, SND504, SND524, and SND562 were designed and synthesized using a flavonoid polyphenol scaffold selectively modified to markedly increase cytotoxicity towards cancer cells. All seven compounds showed dose-dependent anti-proliferative activity across 15 lymphoma cell lines derived from germinal center B-cell-like (GCB) diffuse large B cell lymphoma (DLBCL) (n=6), activated B cell-like (ABC) DLBCL (n=2), mantle cell lymphoma (n=4), and marginal zone lymphoma (MZL) (n=3). Activity in the nM range was seen with five compounds: SDN207 (median IC50 1.6 nM; 95%CI, 1.3-3.2), SND210 (1.4 nM; 95%CI, 0.9-3.3), SND216 (19.5 nM; 95%CI, 10-38 nM), SND562 (28.9 nM; 95%CI, 15.4-57.7), and SND524 (46.7 nM; 95%CI, 32-133.3). Both other compounds had a median IC50 of 2 μΜ (SND462, 95%CI, 1.1-4.5 μM; SND504, 95%CI, 0.5-3.9 μM). There were no differences based on lymphoma histology. The anti-tumor activity was maintained in MZL cell lines with acquired resistance to BTK and PI3K inhibitors (n=3). Six compounds were also assessed for their anti-tumor activity in models other than lymphomas (SDN207, n=31; SND210, n=20; SND462, n=4; SND504, n=4; SND524, n=9; SND562, n=35), confirming the higher activity for SDN207, SND210, SND562, and SND524. Regarding the mechanism of action, up to now, a tubulin polymerization assay was performed for three compounds. Microtubule depolymerization (SND207), and microtubule polymerization (SND524, SND562) were observed at concentrations as low as 20 nM. The OncolinesProfiler identified the tubulin-targeting agents vinblastine, vincristine, paclitaxel, and docetaxel among the top reference compounds most correlated with SND207 and SND210 (i.e., Pearson correlation > 0.5). Conclusions: Novel compounds developed using plant-derived skeletons have shown in vitro anti-tumor activity in lymphomas, leukemias, and solid tumors, including models of resistance to targeted agents. Microtubule targeting appeared as a possible mechanism of action for at least four of them. Citation Format: Alberto J. Arribas, Eleonora Cannas, Eugenio Gaudio, Paulina Biniecka, Dan Stoicescu, Francesco Bertoni. A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents [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 7140.

Evaluating population-level outcomes in Chronic Lymphocytic leukemia in the era of novel therapies using the SEER registry

In the last decade, novel agents such as BTK and BCL-2 inhibitors have revolutionized treatment of CLL/SLL, with clinical trials showing improved overall survival compared to chemotherapeutic agents. However, studies examining whether they have improved overall survival at the population level are lacking. We evaluated this by conducting a retrospective analysis of CLL/SLL patients registered in the National Cancer Institute’s surveillance epidemiology and end results (SEER) database, analyzing overall survival (OS) in periods pre- and post-availability of novel agents, along with demographic information. Our results showed that median OS significantly improved over time [7.8 years (2000–2005), 9.1 years (2006–2013), and not reached (2014–2018) (p < 0.001)]. Compared to diagnosis in 2014–2018, diagnosis in earlier periods was associated with higher mortality risk (2000–2005-HR 1.32, 95 % CI 1.28–1.37, p < 0.001: 2006–2013-HR 1.09, 95 % CI 1.06–1.13, p < 0.001). Lower mortality risk was seen in patients age < 85 years whereas median household income of <$75000 was associated with higher mortality. Our study provides real-world data suggesting a possible multifactorial contribution to improvement in survival, including availability of novel agents, better monitoring, and supportive care. They also show discrepancies in overall survival for CLL/SLL patients due to socioeconomic status and demographic factors.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P&amp;lt;0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p&amp;lt;0.001) and hyperproliferative Ki67+ GCB cells (p&amp;lt;0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value &amp;lt;0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p&amp;lt;0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p&amp;lt;0.01), and vs. these cell lines cultured in vitro (all p&amp;lt;0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p&amp;lt;0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p&amp;lt;0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p&amp;lt;0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [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 7586.

Abstract 676: The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma

Abstract Successive generations of BTK inhibitors and CAR T-cell therapy have transformed the treatment of mantle cell lymphoma (MCL). However, treatment resistance has inevitably emerged, creating significant medical need. Recently a novel, clinical stage small molecule, BTM-3566, has been described that targets a mitochondrial process essential for Diffuse Large B-cell lymphoma (DLBCL) survival. BTM-3566 activates the mitochondrial protease OMA1, triggering the ATF4 integrated stress response (ISR). BTM-3566 has robust in vitro efficacy in DLBCL cell lines irrespective of genomic background and elicits complete tumor regression in multiple CDX and PDX mouse models of DLBCL. Based on the activity observed in DLBCL, we further explored the effectiveness of BTM-3566 in MCL models in vitro and in xenograft mouse models in vivo. BTM-3566 inhibited cell proliferation across a panel of MCL cell lines, irrespective of resistance status to ibrutinib and venetoclax (IC50s of 142.5-687.6 nM). Following a 24-hour treatment with BTM-3566, a dose-dependent increase in apoptosis was observed in four tested MCL cell lines—JeKo-1, JeKo-IBN-R, Mino, and Mino-VEN-R, as determined by increased caspase-3 activation and PARP cleavage. Similarly, western blot analysis revealed dose-dependent reductions in MCL-1 and c-MYC, accompanied by an increase in the transcription factor ATF4, indicative of activation of the stress response. We evaluated the in vivo efficacy of BTM-3566 in the JeKo-CDX mouse model. Daily oral administration of BTM-3566 at 10, 20 and 30 mg/kg (n = 5 for each group) in a 5-day on and 2-day off schedule resulted in tumor regression at all three doses (Treatment vs. Vehicle, p &amp;lt; 0.01). We expanded our investigation to PDX mouse models, including those derived from patients resistant to multiple therapies. Four PDX models encompassing naive, rituximab-resistant, BTKi-CAR T dual-resistant and BTKi-VEN-CAR T triple-resistant models were evaluated. BTM-3566 treatment results in nearly complete tumor growth inhibition associated with robust reductions in tumor burden in treated mice across all four PDX models (reduction in tumor weight at the study endpoint, p &amp;lt; 0.001). Notably, no toxicity was observed during the treatment period. In summary, BTM-3566 is effective in treating MCL in vitro and in vivo in preclinical studies. Citation Format: Yang Liu, Heng-Huan Lee, Joseph McIntosh, Yijing Li, Tianci Zhang, Wei Wang, Matthew J. Kostura, Michael Wang. The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma [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 676.

In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function

AbstractBruton’s tyrosine kinase (BTK) is an enzyme needed for B-cell survival, and its inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has, for more than 2 decades, generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using nonlymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas–mediated knockin mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and the morphology of lymphoid organs, as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control and mounted similar humoral immune responses as Y223F and wild-type animals. Collectively, our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase Cγ2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this posttranslational modification is dispensable for the function of BTK.

Cardiovascular events reported in patients with B-cell malignancies treated with zanubrutinib

AbstractFirst-generation Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, has been associated with an increased risk of cardiovascular toxicities. Zanubrutinib is a more selective, next-generation BTK inhibitor. In this analysis, incidence rates of atrial fibrillation, symptomatic (grade ≥2) ventricular arrhythmia, and hypertension were evaluated in a pooled analysis of 10 clinical studies with zanubrutinib monotherapy in patients (N = 1550) with B-cell malignancies and a pooled analysis of head-to-head studies comparing zanubrutinib with ibrutinib (ASPEN cohort 1; ALPINE). Among the 10 studies, most patients (median age, 67 years) were male (66.3%) and had CLL/SLL (60.5%). Overall incidence and exposure-adjusted incidence rates (EAIR) for atrial fibrillation, symptomatic ventricular arrhythmia, and hypertension were lower with zanubrutinib than ibrutinib. Despite a similar prevalence of preexisting cardiovascular events in ASPEN and ALPINE, atrial fibrillation/flutter incidence rates (6.1% vs 15.6%) and EAIR (0.2 vs 0.64 persons per 100 person-months; P < .0001) were lower with zanubrutinib than with ibrutinib. Symptomatic ventricular arrhythmia incidence was low for both zanubrutinib (0.7%) and ibrutinib (1.7%) with numerically lower EAIR (0.02 vs 0.06 persons per 100 person-months, respectively) for zanubrutinib. The hypertension EAIR was lower with zanubrutinib than ibrutinib in ASPEN but similar between treatment arms in ALPINE. The higher hypertension EAIR in ALPINE was inconsistent with other zanubrutinib studies. However, fewer discontinuations (1 vs 14) and deaths (0 vs 6) due to cardiac disorders occurred with zanubrutinib versus ibrutinib in ALPINE. These data support zanubrutinib as a treatment option with improved cardiovascular tolerability compared with ibrutinib for patients with B-cell malignancies in need of BTK inhibitors. These trials were registered at www.ClinicalTrials.gov as # NCT03053440, NCT03336333, NCT03734016, NCT04170283, NCT03206918, NCT03206970, NCT03332173, NCT03846427, NCT02343120, and NCT03189524.

Simultaneous Determination of Ibrutinib, Dihydroxydiol Ibrutinib, and Zanubrutinib in Human Plasma by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry.

Ibrutinib and zanubrutinib are Bruton tyrosine kinase inhibitors used to treat mantle cell lymphoma, chronic lymphocytic leukemia, and small lymphocytic lymphoma. Dihydroxydiol ibrutinib (DHI) is an active metabolite of the drug. A liquid chromatography-tandem mass spectrometry method was developed to detect ibrutinib, DHI, and zanubrutinib in human plasma. The method involved a protein precipitation step, followed by chromatographic separation using a gradient of 10 mM ammonium acetate (containing 0.1% formic acid)-acetonitrile. Ibrutinib-d5 was used as an internal standard. Analytes were separated within 6.5 minutes. The optimized multiple reaction monitoring transitions of m/z 441.1 → 304.2, 475.2 → 304.2, 472.2 → 455.2, and 446.2 → 309.2 were selected to inspect ibrutinib, DHI, zanubrutinib, and the internal standards in positive ion mode. The validated curve ranges included 0.200-800, 0.500-500, and 1.00-1000 ng/mL for ibrutinib, DHI, and zanubrutinib, respectively. The precisions of the lower limit of quantification of samples were below 15.5%, the precisions of the other level samples were below 11.4%, and the accuracies were between -8.6% and 8.4%. The matrix effect and extraction recovery of all compounds ranged between 97.6%-109.0% and 93.9%-105.2%, respectively. The selectivity, accuracy, precision, matrix effect, and extraction recovery results were acceptable according to international method validation guidelines. A simple and rapid method was developed and validated in this study. This method was used to analyze plasma concentrations of ibrutinib and zanubrutinib in patients with mantle cell lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, or diffuse large B-cell lymphoma. The selected patients were aged between 44 and 74 years.

Scaffold-Oriented Asymmetric Catalysis: Conformational Modulation of Transition State Multivalency during a Catalyst-Controlled Assembly of a Pharmaceutically Relevant Atropisomer.

A new class of superbasic, bifunctional peptidyl guanidine catalysts is presented, which enables the organocatalytic, atroposelective synthesis of axially chiral quinazolinediones. Computational modeling unveiled the conformational modulation of the catalyst by a novel phenyl urea N-cap, that preorganizes the structure into the active, folded state. A previously unanticipated noncovalent interaction involving a difluoroacetamide acting as a hybrid mono- or bidentate hydrogen bond donor emerged as a decisive control element inducing atroposelectivity. These discoveries spurred from a scaffold-oriented project inspired from a fascinating investigational BTK inhibitor featuring two stable chiral axes and relies on a mechanistic framework that was foreign to the extant lexicon of asymmetric catalysis.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Safety and efficacy of zandelisib plus zanubrutinib in previously treated follicular and mantle cell lymphomas.

The combination of the phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor zandelisib with the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib was hypothesized to be synergistic and prevent resistance to single-agent therapy. This phase 1 study (NCT02914938) included a dose-finding stage in patients with relapsed/refractory (R/R) B-cell malignancies (n = 20) and disease-specific expansion cohorts in follicular lymphoma (FL; n = 31) or mantle cell lymphoma (MCL; n = 19). The recommended phase 2 dose was zandelisib 60 mg on Days 1-7 plus zanubrutinib 80 mg twice daily continuously in 28-day cycle. In the total population, the most common adverse events (AEs; all grades/grade 3-4) were neutropenia (35%/24%), diarrhoea (33%/2%), thrombocytopenia (32%/8%), anaemia (27%/8%), increased creatinine (25%/0%), contusion (21%/0%), fatigue (21%/2%), nausea (21%/2%) and increased aspartate aminotransferase (24%/6%). Three patients discontinued due to AEs. The overall response rate was 87% (complete response [CR] = 33%) for FL and 74% (CR = 47%) for MCL. The median duration of response and progression-free survival (PFS) were not reached in either group. The estimated 1-year PFS was 72.3% (95% confidence interval [CI], 51.9-85.1) for FL and 56.3% (95% CI, 28.9-76.7) for MCL (median follow-up: 16.5 and 10.9 months respectively). Zandelisib plus zanubrutinib was associated with high response rates and no increased toxicity compared to either agent alone.

Molecular Insights into the Impact of Mutations on the Binding Affinity of Targeted Covalent Inhibitors of BTK.

Targeted covalent inhibitors (TCIs) have witnessed a significant resurgence in recent years, particularly in the kinase drug discovery field for treating diverse clinical indications. The inhibition of Bruton's tyrosine kinase (BTK) for treating B-cell cancers is a classic example where TCIs such as ibrutinib have had breakthroughs in targeted therapy. However, selectivity remains challenging, and the emergence of resistance mutations is a critical concern for clinical efficacy. Computational methods that can accurately predict the impact of mutations on inhibitor binding affinity could prove helpful in informing targeted approaches─providing insights into drug resistance mechanisms. In addition, such systems could help guide the systematic evaluation and impact of mutations in disease models for optimal experimental design. Here, we have employed in silico physics-based methods to understand the effects of mutations on the binding affinity and conformational dynamics of select TCIs of BTK. The TCIs studied include ibrutinib, acalabrutinib, and zanubrutinib─all of which are FDA-approved drugs for treating multiple forms of leukemia and lymphoma. Our results offer useful molecular insights into the structural determinants, thermodynamics, and conformational energies that impact ligand binding for this biological target of clinical relevance.

A real-world pharmacovigilance study of FDA Adverse Event Reporting System (FAERS) events for Bruton’s tyrosine kinase inhibitors (BTKis) single and its combination therapy

ABSTRACT Background Bruton’s tyrosine kinase inhibitors (BTKis) are targeted treatments for B-cell tumors but have significant side effects. This study assesses and contrasts the side effects of BTKis alone and its four combination therapies. Research design and methods The reporting odds ratio (ROR) was used to analyze the data on three BTKis monotherapies and combinations of ibrutinib with rituximab, obinutuzumab, venetoclax, and lenalidomide in the FDA Adverse Event Reporting System (FAERS) database up to December 2022. Results We analyzed the top 20 PTs for each treatment regimen. In monotherapies, atrial fibrillation (ROR (95% CI): 9.88 (9.47–10.32)) in zanubrutinib and rash (6.97 (5.42–8.98)) in acalabrutinib had higher associations. In combinations, infection (6.86 (6.11–7.70)), atrial fibrillation (27.96 (22.61–34.58)) and myelosuppression (10.09 (8.89–11.46)) were vital signals when ibrutinib was combined with obinutuzumab, and pyrexia (4.22 (2.57–6.93)) had a high signal value when combined with lenalidomide. Hemorrhage had a lower signal value when combined with venetoclax compared to ibrutinib alone (2.50 (2.18–2.87) vs 3.60 (3.52–3.68)). Conclusions The ibrutinib-obinutuzumab combo has the highest risk of infection, atrial fibrillation, and myelosuppression, and the ibrutinib-lenalidomide combo has the highest risk of pyrexia. However, the ibrutinib-venetoclax combo has a lower risk of hemorrhage than monotherapy.

Disrupting pro-survival and inflammatory pathways with dimethyl fumarate sensitizes chronic lymphocytic leukemia to cell death

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.

Matching-Adjusted Indirect Comparison of Brexucabtagene Autoleucel (ZUMA-2) and Pirtobrutinib (BRUIN) in Patients with Relapsed/Refractory Mantle Cell Lymphoma Previously Treated with a Covalent Bruton Tyrosine KinaseInhibitor.

Patients with relapsed/refractory (R/R) mantle cell lymphoma (MCL) often require multiple lines of treatment and have a poor prognosis, particularly after failing covalent Bruton tyrosine kinase inhibitor (cBTKi) therapy. Newer treatments such as brexucabtagene autoleucel (brexu-cel, chimeric antigen receptor Tcell therapy) and pirtobrutinib (non-covalent BTKi) show promise in improving outcomes. Without direct comparative evidence, an unanchored matching-adjusted indirect comparison was conducted to estimate the relative treatment effects of brexu-cel and pirtobrutinib for post-cBTKi R/R MCL. Using logistic propensity score models, individual patient-level data from ZUMA-2 brexu-cel-infused population (N = 68) were weighted to match pre-specified clinically relevant prognostic factors based on study-level data from the BRUIN cBTKi pre-treated cohort (N = 90). The base-case model incorporated the five most pertinent factors reported in ≥ 50% of both trial populations: morphology, MCL International Prognostic Index, number of prior lines of therapy, disease stage, and prior autologous stem cell transplant. A sensitivity analysis additionally incorporated TP53 mutation and Ki-67 proliferation. Relative treatment effects were expressed as odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (CIs). In the base-case model, brexu-cel was associated with higher rates of objective response (OR 10.39 [95%CI 2.81-38.46]) and complete response (OR 10.11 [95%CI 4.26-24.00]), and improved progression-free survival (HR0.44 [95%CI 0.25-0.75]), compared to pirtobrutinib. Overall survival and duration of response favored brexu-cel over pirtobrutinib but the differences crossed the bounds for statistical significance. Findings were consistent across the adjusted and unadjusted analyses. Findings suggest that brexu-cel may offer clinically and statistically significant benefits regarding objective response, complete response, and progression-free survival compared to pirtobrutinib among patients with R/R MCL after prior cBTKi therapy. Given the short follow-up and high degree of censoring in BRUIN, an analysis incorporating updated BRUIN data may provide more definitive overall survival results.

NFKBIE mutations are selected by the tumor microenvironment and contribute to immune escape in chronic lymphocytic leukemia.

Loss-of-function mutations in NFKBIE, which encodes for the NF-κB inhibitor IκBε, are frequent in chronic lymphocytic leukemia (CLL) and certain other B-cell malignancies and have been associated with accelerated disease progression and inferior responses to chemotherapy. Using in vitro and in vivo murine models and primary patient samples, we now show that NFKBIE-mutated CLL cells are selected by microenvironmental signals that activate the NF-κB pathway and induce alterations within the tumor microenvironment that can allow for immune escape, including expansion of CD8+ T-cells with an exhausted phenotype and increased PD-L1 expression on the malignant B-cells. Consistent with the latter observations, we find increased expression of exhaustion markers on T-cells from patients with NFKBIE-mutated CLL. In addition, we show that NFKBIE-mutated murine CLL cells display selective resistance to ibrutinib and report inferior outcomes to ibrutinib treatment in NFKBIE-mutated CLL patients. These findings suggest that NFKBIE mutations can contribute to CLL progression through multiple mechanisms, including a bidirectional crosstalk with the microenvironment and reduced sensitivity to BTK inhibitor treatment.

Targeting BTK in B Cell Malignancies: From Mode of Action to Resistance Mechanisms.

The B cell receptor (BCR) signaling pathway plays a crucial role in B cell development and contributes to the pathogenesis of B cell neoplasms. In B cell malignancies, the BCR is constitutively active through both ligand-dependent and ligand-independent mechanisms, resulting in continuous Bruton tyrosine kinase (BTK) signaling activation, which provides a survival and proliferation advantage to the neoplastic clone. Among B cell malignancies, those in which the most significant results were obtained by treatment with BTK inhibitors (BTKi) include chronic lymphocytic leukemia, mantle cell lymphoma, lymphoplasmacytic lymphoma, and diffuse large B cell lymphoma. Covalent BTKi (namely ibrutinib, acalabrutinib, and zanubrutinib) functions by irreversibly blocking BTK through covalent binding to the cysteine residue 481 (Cys-481) in the ATP-binding domain. Despite the high efficacy and safety of BTKi treatment, a significant fraction of patients affected by B cell malignancies who are treated with these drugs experience disease relapse. Several mechanisms of resistance to covalent BTKi, including Cys-481 mutations of BTK, have been investigated in B cell malignancies. Non-covalent BTKi, such as pirtobrutinib, have been developed and proven effective in patients carrying both Cys-481-mutated and unmutated BTK. Moreover, targeting BTK with proteolysis-targeting chimeras (PROTACs) represents a promising strategy to overcome resistance to BTKi in B cell neoplasms.

Iridium-Catalyzed Asymmetric Hydrogenation of Heteroaromatics with Multiple N Atoms via Substrate Activation: An Entry to 4,5,6,7-Tetrahydropyrazolo[1,5-a]pyrimidine-3-carbonitrile Core of a Potent BTK Inhibitor.

The chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine is the key core skeleton of potent Bruton's tyrosine kinase (BTK) inhibitor Zanubrutinib, and the catalyst-controlled asymmetric hydrogenation of planar multinuclear pyrimidine heteroarenes with multiple N atoms could provide an efficient route toward its synthesis. Owing to the strong aromaticity and poisoning effect toward chiral transition metal catalyst, asymmetric hydrogenation of pyrazolo[1,5-a]pyrimidines with multiple nitrogen atoms is still a challenge for synthesizing the chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]-pyrimidine. Herein, an efficient iridium-catalyzed asymmetric hydrogenation of pyrazolo[1,5-a]pyrimidines has been developed using substrate activation strategy, with up to 99% ee. The decagram scale synthesis further demonstrated the potential and promise of this procedure in the synthesis of Zanubrutinib. In addition, a mechanistic study indicated that the hydrogenation starts with 1,2-hydrogenation.