Covalent Bruton Tyrosine Kinase Inhibitor Research Articles

Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

Optimizing BTK Inhibition in Waldenström Macroglobulinemia.

Bruton tyrosine kinase (BTK) inhibitors have become a standard of care in the treatment of patients with Waldenström macroglobulinemia (WM) and are the only medications approved by the FDA to treat these patients. As more patients with WM are treated with BTK inhibitors in the United States and worldwide, it is essential to optimize this therapy by selecting the patients who are more likely to benefit from it, and by managing the unique adverse effects associated with these agents. Herein, we propose a genomic-driven approach to selecting patients with WM who are more likely to experience fast, deep, and durable responses to BTK inhibitors, and provide practical strategies for managing adverse effects, including BTK inhibitor dose reductions, switching to other BTK inhibitors, and abandoning BTK inhibitor therapy. Ongoing clinical trials are evaluating covalent and noncovalent BTK inhibitors alone and in combination, as well as BTK degraders, with exciting results, making the horizon for BTK-targeting therapies in WM bright and hopeful.

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.

Taking the Next Step in Double Refractory Disease: Current and Future Treatment Strategies for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell lymphoproliferative disease with a high annual incidence in Western countries. As B-cell receptor (BCR) signaling and intrinsic apoptotic resistance play critical roles in the development and survival of CLL cells, therapeutic approaches targeting these pathways have been extensively investigated to tackle this incurable disease. Over the last decade, several Phase 3 trials have confirmed the superior efficacy of covalent Bruton tyrosine kinase inhibitors (cBTKis) and venetoclax, a selective B-cell lymphoma 2 (BCL2) inhibitor, over chemoimmunotherapy. This has been demonstrated in both the treatment-naïve and relapsed/refractory (RR) settings and includes patients with high-risk molecular features. However, these drugs are not curative, with patients continuing to relapse after treatment with both cBTKis and BCL2is, and the optimal treatment strategy for these patients has not been defined. Several novel agents with distinct mechanisms have recently been developed for CLL which have demonstrated efficacy in patients who have previously received cBTKis and BCL2i. In particular, novel BCR-signaling targeting agents have shown promising efficacy in early-phase clinical trials for RR-CLL. Furthermore, cancer immunotherapies such as bispecific antibodies and chimeric antigen receptor T-cells have also shown anti-tumor activity in patients with heavily pretreated RR-CLL. Personalised approaches with these novel agents and combination strategies based on the understanding of resistance mechanisms have the potential to overcome the clinical challenge of what to do next for a patient who has already had a cBTKi and venetoclax.

An Updated Comparison of Overall Survival with Brexucabtagene Autoleucel (Brexu-cel) CAR T-Cell Therapy (ZUMA-2) Versus Standard of Care (SCHOLAR-2) in Patients with Relapsed/Refractory Mantle Cell Lymphoma (R/R MCL) Previously Treated with a Covalent Bruton Tyrosine Kinase Inhibitor (BTKi)

An Updated Comparison of Overall Survival with Brexucabtagene Autoleucel (Brexu-cel) CAR T-Cell Therapy (ZUMA-2) Versus Standard of Care (SCHOLAR-2) in Patients with Relapsed/Refractory Mantle Cell Lymphoma (R/R MCL) Previously Treated with a Covalent Bruton Tyrosine Kinase Inhibitor (BTKi)

Pirtobrutinib: A New and Distinctive Treatment Option for B-Cell Malignancies.

The objective was to evaluate the efficacy/safety of pirtobrutinib in the treatment of B-cell malignancies and distinguish it from other available Bruton's tyrosine kinase (BTK) inhibitors. A literature search of PubMed (January 2021 through November 2023) and Clinicaltrials.gov was conducted using terms pirtobrutinib, Jaypirca, and LOXO 305. Licensing trials of available BTK inhibitors were also reviewed. Relevant English-language clinical trials were evaluated. Pirtobrutinib was approved by the US Food and Drug Administration for the treatment of relapsed/refractory mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) based largely on a phase 1/2 study in B-cell malignancies. Pirtobrutinib demonstrated a 73% overall response rate (ORR) in the CLL population and 58% in MCL. Pirtobrutinib has activity in patients resistant to earlier-generation, covalent BTK inhibitors. In fact, the ORRs were similar in BTK-pretreated and naïve patients. Adverse effects include fatigue, diarrhea, bleeding, and infection. Atrial fibrillation, a class effect of BTK inhibitors, may be less common with pirtobrutinib. Compared with earlier-generation BTK inhibitors, pirtobrutinib is more selective for BTK and binds noncovalently to the receptor. Ongoing studies are evaluating pirtobrutinib's use in multiple B-cell malignancies and comparing it with other BTK inhibitors. The characteristics of pirtobrutinib render it useful in the treatment of B-cell malignancies no longer responding to a previous BTK inhibitor, and results from ongoing clinical trials may support future expanded use.

Treatment patterns in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma post covalent Bruton tyrosine kinase inhibitor treatment: a Japanese claims database study.

Standard treatment has not been established for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after discontinuation of covalent Bruton tyrosine kinase inhibitor (cBTKi) therapy. This retrospective, administrative database (Medical Data Vision) study described the patient characteristics, treatment patterns, and factors associated with receiving post-first-cBTKi treatment in Japanese patients with CLL/SLL. Patients aged ≥18 years with confirmed CLL/SLL diagnosis and treated with anti-neoplastic drugs indicated for CLL/SLL between March 2013 and February 2022 were included. Patient characteristics at baseline (first line), first cBTKi exposure (first-cBTKi), post-first-cBTKi treatment received, and the treatment sequence of CLL drugs received first line through third line, were described. Time-to-event analyses used the Kaplan-Meier method. Multivariable logistic regression analysis was used to explore factors associated with receiving post-first-cBTKi treatment among patients who discontinued first-cBTKi treatment. Among 2,424 eligible patients (median age: 72.0 years, 61.9% male), 450 (18.6%) received cBTKi in any treatment line. Among patients treated with cBTKi, 273 (60.7%) discontinued treatment; 56.0% of them (n = 153/273) received subsequent treatment. Median duration of post-first-cBTKi treatment was 2.2 months (95% confidence interval [CI]: 1.8, 3.5). The most common regimens post-first-cBTKi were cBTKi therapy (47.7%), bendamustine-based therapy (17.0%), and venetoclax-based therapy (13.1%). Patients aged <75 years (odds ratio [OR] [95% CI]: 2.0 [1.2, 3.4]) and those who did not receive blood transfusion during cBTKi treatment (OR [95% CI]: 2.3 [1.3, 4.1]) were more likely to receive post-first-cBTKi treatment. In conclusion, Japanese patients with CLL/SLL received various treatments for short duration after first-cBTKi discontinuation.

Abbv-101, a Highly Potent and Selective Clinical Stage Bruton Tyrosine Kinase Degrader for the Treatment of B-Cell Malignancies

Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion and homing to key lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. In preclinical studies, the BTK degrader ABBV-101 has shown potent in vitro and in vivo efficacy in BCR pathway-dependent leukemia/lymphoma models, including covalent BTK inhibitor resistant BTK-C481S systemic mouse CLL and human DLBCL models. ABBV-101 degrades BTK-WT and BTK-C481S with similar sub-nanomolar potency in the human DLBCL cell line TMD8 which translates to potent cellular growth inhibition. In addition to the BTK-C481S mutation, ABBV-101 demonstrates similar potent activity against novel BTK mutations associated with resistance to reversible BTK inhibitors in the clinic, an emerging unmet need. ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Further, ABBV-101 induces complete tumor regressions in multiple non-GCB DLBCL PDX models, demonstrating deeper and more durable response than covalent and reversible inhibitors. Combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Kinome and global proteomics profiling showed ABBV-101 to be highly selective, a designed feature that may enable favorable tolerability in patients. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501

Correlation between Real-World Progression-Free Survival (rwPFS) and Overall Survival (OS) in Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Who Relapsed or Are Refractory, Intolerant, Resistant to Treatment with a Bruton's Tyrosine Kinase Inhibitor (BTKi)

Introduction: Given the long maturation time of OS, PFS is often used in oncology and hematology as a surrogate for OS. However, the association between the two endpoints varies across cancer types. In CLL clinical trial populations, PFS and OS were shown to be strongly correlated in patients receiving at least two lines of therapy (LOT). The present study used real-world individual patient data to evaluate the strength of correlation between real-world PFS (rwPFS) and OS in CLL/SLL patients who relapsed or were resistant/refractory/intolerant to treatment with a covalent BTKi. Methods: A retrospective chart review was conducted of adult CLL/SLL patients at Dana-Farber Cancer Institute who were treated with at least one covalent BTKi and relapsed or were resistant/refractory/intolerant to the treatment and have discontinued BTKi. Patients were included if they had records available from 6 months before to 12 months after initiation of the first observed (index) BTKi, and were excluded if they were treated with a non-hormonal antineoplastic agent for other primary malignancies in the period between CLL/SLL diagnosis and receipt of their first BTKi. Demographic and clinical variables, oncologic and medical histories, treatments, and clinical outcomes were abstracted from patients' medical charts. Time to event endpoints were described using Kaplan-Meier curves. Time to progression (TTP) was defined as time from index to progression. OS was defined as time from index to death; patients were censored at the last visit if they did not die. rwPFS was defined as the time to progression or death; patients who did not progress or die were censored at index BTKi treatment discontinuation. Fleischer's method was used to estimate the individual-level Pearson correlation coefficient between rwPFS and OS among patients treated with BTKi in any line of treatment. Confidence intervals (CIs) were calculated by bootstrapping (1000 replicates). Results: A total of 104 patients who initiated treatment with the index BTKi from 07/2011 to 09/2019 and relapsed or were resistant/refractory/intolerant to the treatment were included in the analysis. Median (interquartile range [IQR]) age at BTKi initiation was 66.7 (60.3-73.0) years, median (IQR) time from index BTKi initiation to last clinical visit or death was 5.7 (3.3-7.0) years. Among patients with a known index BTKi LOT (N=102 [98%]), 28% received the index BTKi in first line, 32% in second line, 23% in third line, 12% in fourth line, and 6% in or after the fifth line. An assessment date for response to the index BTKi and for whether a patient had progressed was available for 95 patients (91%) ( Table 1). Among these patients, progression after index BTKi treatment was observed for 48 (51%) patients. Median (95% CI) time from BTKi initiation to progression was 3.7 (2.6, 4.1) years. More than one-third of patients (35%) died and median (95% CI) OS was 8.9 (6.7, not reached) years. 7 patients died without progressing and cause of death was available for 5 of these patients: 3 died because of an infection, 1 because of a secondary malignancy, and 1 because of other unspecified causes. 58% patients progressed or died and median (95% CI) rwPFS was 3.2 (2.6, 3.9) years. The correlation (95% CI) between rwPFS and OS was 0.79 (0.63, 0.94). Conclusions: This analysis found a strong, positive, and significant correlation between rwPFS and OS in patients with CLL/SLL who received treatment with a covalent BTKi and relapsed on or were resistant/refractory/intolerant to the treatment. These findings reinforce the hypothesis that rwPFS may be a useful early endpoint for evaluating the effectiveness of BTKi treatment outside of clinical trials among the patient population of CLL/SLL patients who relapsed or were refractory/resistant/intolerant to BTKi, at time points when OS is not mature. However, further analysis of the change in rwPFS that predicts an improvement in OS (surrogate threshold effect) is required to establish rwPFS as a valid surrogate for OS.

Preclinical Efficacy and Mechanism for a Novel BTK-Protacs Against Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL) that exhibits diverse clinical courses and biological contexts. Given the significance of Bruton tyrosine kinase (BTK) as a therapeutic target in MCL, this study aimed to design a range of BTK-PROTACs. The objective was to develop efficient BTK-PROTACs capable of degrading BTK proteins, inhibiting the proliferation of MCL cells, and overcoming resistance to both covalent and non-covalent BTK inhibitors in both in vivo and in vitro settings. Methods:A series of BTK-PROTACs were gradually screened by Western blot and MTS assays, and the best active drug was finally determined. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. RNA sequencing identified differentially expressed genes and important signaling pathways that influenced by BTK-PROTACs. BTKi-resistant cell lines were constructed to evaluate the ability of BTK-PROTACs to degrade BTK protein and suppress the proliferation of MCL cells. R esults: BTK-PROTACs C23 demonstrated effective degradation of BTK protein and inhibition of tumor cell growth across various MCL cell lines. Additionally, C23 exhibited the ability to induce cell apoptosis, activate the ubiquitin pathway, and inhibit the B-cell receptor (BCR), NF-κB, and PI3K-AKT-mTOR signaling pathways. Importantly, C23 effectively degraded BTK in both Z138-BTK C481S and Z138-BTK L528W cells, suggesting its potential to overcome resistance to both covalent and non-covalent BTK inhibitors. In our in vivo experiments using MCL xenograft NSG mouse models, BTK-PROTACs C23 significantly impeded tumor proliferation in Z138-BTK WT, Z138-BTK C481S, and Z138-BTK L528W MCL models. Additionally, C23 induced the degradation of BTK in tumor tissues. Notably, HE staining revealed no significant adverse effects of BTK-PROTACs C23 on normal organs and tissues. Moreover, routine blood tests and liver function tests showed no impairment of hematopoietic or hepatic function caused by BTK-PROTACs C23. C onclusion: In this study, we successfully designed and synthesized BTK-PROTACs C23, which demonstrated a remarkable ability to degrade BTK protein and effectively suppress the proliferation of MCL cells both in vitro and in vivo. Importantly, BTK-PROTACs C23 showed promising potential to overcome resistance to both covalent and non-covalent BTK inhibitors.

Trial in Progress: A Phase 1b Study of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Patients with Previously Treated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma or Non-Hodgkin Lymphoma

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited by toxicity related to off-target kinase inhibition and acquired resistance due to BTK C481 mutation. AS-1763 is a potent, highly selective, orally available, and non-covalent BTK inhibitor, equipotent against both wild-type and C481S-mutated BTK with sub-nanomolar IC50 values (Kawahata et al. J Med Chem 2021; 64,14129-14141). In a Phase 1 single-ascending dose study in healthy volunteers, AS-1763 was well tolerated and safe at doses of 5-600 mg and achieved maximum inhibition of B cell activation at doses of 100 mg and above at 1-2 h post-dose and the duration of inhibitory effect increased with dose [Arimura et al. AACR 2022; Cancer Res 2022;82(12_Suppl)]. In preclinical studies, AS-1763 demonstrated a broad spectrum of inhibition against BTK mutations that confer resistance to other non-covalent BTK inhibitors in addition to the C481 mutation. Study Design and Methods: This study is an open-label, multi-center, Phase 1b study of oral AS-1763 in patients with advanced chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) and other B-cell non-Hodgkin lymphomas (NHLs) who have failed or are intolerant to at least two prior lines of systemic therapy. Prior therapy with a covalent BTK inhibitor is permitted; prior use of a noncovalent BTK inhibitor is excluded. This study consists of two parts, i.e., dose escalation and expansion parts. The dose escalation part will follow a 3+3 design with a starting dose of 100 mg BID. Each cycle will be 28 days. Three dose levels will be selected for dose expansion part based on a comprehensive review of data from dose escalation. The dose expansion part consists of 2 cohorts, i.e., Cohort 1 for CLL/SLL and Cohort 2 for B-cell NHL. In each cohort, the first 30 patients will be allocated to the 3 dose levels (10 patients for each), then the same provisional recommended Phase 2 Dose (RP2D) will be selected for Cohort 1 and Cohort 2 after 30 patients have enrolled in either cohort. After the provisional RP2D is selected, up to 28 patients in Cohort 1 and up to 15 patients in Cohort 2 may be treated at the provisional RP2D. In this study, up to 110 patients will be enrolled. Treatment will continue for 24 cycles or until disease progression, occurrence of unacceptable toxicity or discontinuation because of other reasons. Eligible patients include CLL/SLL, Waldenstrӧm macroglobulinemia (WM), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and follicular lymphoma (FL). Patients with transformed disease (e.g., Richter's transformation) prior to or during screening are not eligible. Key exclusion criteria include CNS involvement by systemic lymphoma, stem cell transplant or CAR-T therapy &amp;lt;30 days, and clinically significant cardiovascular disease. The primary objective of the dose escalation part is to determine the maximum tolerated dose/dose-limiting toxicity. Key secondary endpoints include the evaluation of safety profile and tolerability, pharmacokinetics properties, and preliminary anti-tumor activity based on overall response rate (ORR) by investigator according to iwCLL 2018 for CLL, IWW6 for WM, and Lugano Treatment Response Criteria for MCL, MZL, SLL and FL. The primary objective of the dose expansion part is to assess the preliminary anti-tumor activity based on ORR by the Safety Monitoring Committee (SMC). Key secondary objectives are to investigate safety, tolerability, and pharmacokinetic profiles and to assess the preliminary antitumor activity based on ORR by investigator and best overall response, duration of response, progression-free survival and overall survival by investigator and the SMC. This trial will evaluate several key exploratory objectives including evaluation of minimal residual disease negativity, characterization of patient subsets defined by mutation status of BTK and phospholipase C gamma 2 (PLCG2), and characterize BTK and PLGC2 gene mutation before and after disease progression. Changes in the level of biomarkers such as CCL3 and CCL4 chemokines and B-cell receptor pathway signaling will be evaluated prior to, during therapy, and at time of disease progression. The RP2D will be determined based on all the data generated in the study. The study has been registered on ClinicalTrials.gov (NCT05602363).

Pirtobrutinib, a Highly Selective, Non-Covalent (Reversible) BTK Inhibitor in Relapsed/Refractory Follicular Lymphoma: Results from the Phase 1/2 BRUIN Study

Background: Follicular lymphoma (FL) is a chronic and incurable disease requiring multiple lines of therapy for patients (pts) with relapsed/refractory (R/R) disease. Covalent Bruton tyrosine kinase inhibitors (cBTKi) have transformed the management of select B-cell malignancies, in particular chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, despite the transformative impact in CLL and MCL, the efficacy of single-agent cBTKi has been limited in pts with R/R FL, with an overall response rate (ORR) of 20.9% to 37.5% (Gopal et al, J Clin Oncol, 2018; Bartlett et al, Blood, 2018). Pirtobrutinib, a highly selective, non-covalent (reversible) BTKi, inhibits both wildtype and C481-mutant BTK with equal low nM potency and has a favorable oral pharmacology that enables continuous BTK inhibition throughout the once-daily dosing interval. Pirtobrutinib has demonstrated promising efficacy and tolerability in pts with poor-prognosis B-cell malignancies following prior therapy, including cBTKi. Here, we report the safety and efficacy of pirtobrutinib in a cohort of pts with R/R FL from the BRUIN study (NCT03740529). Methods: Pts with previously treated B-cell malignancies were eligible for treatment with pirtobrutinib monotherapy in either the dose escalation or expansion portion of the multicenter phase 1/2 BRUIN study. FL diagnosis required pathologic review of an adequate biopsy. Key endpoints included investigator-assessed ORR per Lugano 2014 criteria, duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. A data cutoff date of 05 May 2023 was utilized. Results: Among the 48 pts with FL, 45 (94%) pts received the recommended phase 2 dose of pirtobrutinib (200 mg once daily) as their starting dose. Pts had a median age of 64.5 years (range, 37.0-85.0), were mostly male (60%, n=29), and had a median of 3 (range, 1-12) prior lines of therapy. Most pts (81%, n=39) had Ann Arbor stage III/IV disease. The Follicular Lymphoma International Prognostic Index (FLIPI) risk was low (0-1) in 9 (19%) pts, intermediate (2) in 13 (27%) pts, high (3-5) in 23 (48%) pts, and missing in 3 (6%) pts. Overall, 43 (90%) pts had received chemotherapy plus an anti-CD20 antibody, 17 (35%) pts had received a PI3K inhibitor, 14 (29%) pts had received lenalidomide, 6 (13%) pts had received an autologous stem cell transplant, and 4 (8%) pts had received CAR T-cell therapy. Of the 4 (8%) pts who had received a prior cBTKi, 3 discontinued due to disease progression and 1 discontinued for intolerance. The ORR was 50.0% (95% confidence interval [CI], 35.2-64.8), including 7 (14.6%) complete responses and 17 (35.4%) partial responses (Figure). An additional 12 (25.0%) pts had stable disease. Among 4 pts who had received prior cBTKi, 3 achieved partial response and 1 had stable disease. With a median follow-up of 18.4 months (interquartile range, 10.1-21.0) among 24 responding pts, median DoR was 5.5 months (95% CI, 3.7-not estimable [NE]), and the 18-month estimated DoR rate was 41.0% (95% CI, 20.1-60.9). Median PFS was 5.8 months (95% CI, 3.8-8.1), and the 18-month estimated PFS rate was 32.3% (95% CI, 19.1-46.2). Median OS was NE, and the 18-month estimated OS rate was 78.3% (95% CI, 62.1-88.1). The efficacy outcomes by FLIPI risk category are presented in the Table. The median time on treatment was 7.6 months (range, 0.6-42.2), with 14 (29.2%) pts still receiving pirtobrutinib. The most frequent treatment-emergent adverse events (TEAEs), regardless of attribution, were diarrhea (29.2%, n=14), fatigue (25.0%, n=12), and nausea (22.9%, n=11). TEAEs of hemorrhage/hematoma (6.3%, n=3), hypertension (4.2%, n=2), and atrial fibrillation/flutter (2.1%, n=1) were infrequent. The most frequent grade ≥3 TEAEs were infection (18.8%, n=9) and neutropenia/neutrophil count decreased (14.6%, n=7). Only 1 (2.1%) pt had a treatment-related adverse event (rash) that led to pirtobrutinib discontinuation. Conclusions: In this cohort of heavily pre-treated pts with R/R FL, pirtobrutinib showed potential efficacy and was well tolerated, including in pts with high risk FLIPI.

Analysis of Ventricular Arrhythmias and Sudden Death with Acalabrutinib from 5 Prospective Clinical Trials

Background: The approval of ibrutinib, the first covalent Bruton tyrosine kinase inhibitor (BTKi), provided an effective non-chemotherapy option for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). While generally well tolerated, ibrutinib demonstrated significant toxicities in clinical studies, particularly cardiovascular adverse events such as hypertension, atrial fibrillation, and ventricular arrhythmias (VAs). Post hoc analysis of the phase 3 FLAIR trial in patients with previously untreated CLL reported an unexplained sudden or cardiac death rate of 0.5 patients per 100 patient-years with ibrutinib plus rituximab (Hillmen et al. Lancet Oncol. 2023). Acalabrutinib is a next-generation, more selective, irreversible BTKi approved to treat CLL/SLL and relapsed/refractory mantle cell lymphoma. Herein, the rates of nonfatal and fatal VAs and sudden deaths (SDs) from 5 acalabrutinib clinical trials of patients with CLL were analyzed. Methods: Incidence and relative risk of nonfatal and fatal VAs and SDs were analyzed using pooled data from 5 prospective acalabrutinib clinical trials (nonrandomized trials: CL-001 [acalabrutinib monotherapy], CL-003 [acalabrutinib + obinutuzumab]; randomized trials: CL-006 [acalabrutinib vs ibrutinib], CL-007 [acalabrutinib ± obinutuzumab vs chlorambucil + obinutuzumab], CL-309 [acalabrutinib vs idelalisib + rituximab OR bendamustine + rituximab]). Acalabrutinib-containing arms were pooled as a single group; comparators, which were standard-of-care therapies, were pooled as a separate group. Events were identified by comprehensive data extraction of terms capturing nonfatal and fatal VAs and SDs (terms are defined in Table 1 footnote). Nonfatal events were analyzed with and without the inclusion of premature ventricular contractions (PVCs). A subset of fatal events also was analyzed after adjudication through manual clinical review by the sponsor's medical, clinical, and safety leadership. Results: In total, 1299 patients received acalabrutinib (cumulative exposure, 4568.4 patient-years) and 585 received standard-of-care therapies (comparator group) (941.3 patient-years). Considering only fatal VAs and SDs, 5 (0.4%) and 3 (0.5%) patients had an event in the acalabrutinib and comparator groups, respectively, via comprehensive data extraction ( Table 1). Based on exposure-adjusted event rates per 100 patient-years, fatal VAs and SDs were numerically less frequent for acalabrutinib versus comparators; however, relative risks did not statistically favor either group. This trend persisted when events were adjudicated through manual clinical review. After adjudication, 2 of 5 patients in the acalabrutinib group and 2 of 3 patients in the comparator group were determined to have fatal VA or cardiac-related SD. Nonfatal VAs were reported in 11 (0.8%) patients in the acalabrutinib group and 3 (0.5%) patients in the comparator group. When PVCs were excluded, 2 patients in each group (acalabrutinib, 0.2%; comparator, 0.3%) had nonfatal VAs. These nonfatal VAs consisted of ventricular fibrillation (acalabrutinib, n=2 [grades 2 and 4]; comparator, n=1 [grade 4]) and unspecified VA (comparator, n=1 [grade 1]). Although the acalabrutinib group had a lower exposure-adjusted event rate per 100 patient-years than the comparator group, relative risk did not favor either group. With both fatal and nonfatal VAs (with or without PVCs), time to event was notably longer with acalabrutinib than with comparators ( Table 1). A sensitivity analysis excluding the ibrutinib-treated patients from the comparator cohort yielded overall similar results (data not shown). Conclusions: This pooled analysis of prospective acalabrutinib clinical trials demonstrates that the risk of VA and SD with acalabrutinib is low and similar to that of standard-of-care therapies when adjusted for exposure. The current analysis with more than 1200 patients treated with acalabrutinib points to favorable safety outcomes and no specific trend with SDs and VAs. Additional analyses with larger cohorts from the clinical development program and all postmarketing sources will continue to further characterize the safety profile of acalabrutinib.

Matching-Adjusted Indirect Comparison (MAIC) of Brexucabtagene Autoleucel (Brexu-cel) and Pirtobrutinib in Patients with Relapsed/Refractory (R/R) Mantle Cell Lymphoma (MCL) Previously Treated with a Covalent Bruton Tyrosine Kinase Inhibitor (cBTKi)

Introduction: Patients with MCL often require multiple lines of therapy and exhibit a generally unfavorable prognosis, particularly following failure of cBTKi therapy. Therapies such as chimeric antigen receptor (CAR) T-cell therapies and non-covalent BTKi therapies have shown promise in improving outcomes. Brexu-cel (formerly known as KTE-X19) is the only CAR T-cell therapy approved in the US for R/R MCL based on results from a phase 2 multicenter, single-arm trial (ZUMA-2; NCT02601313) in patients with R/R MCL who had 1-5 prior therapies, including a BTKi. Pirtobrutinib, a highly selective, non-covalent BTKi, was recently approved in the US for treatment of R/R MCL after at least two lines of systemic therapy including a BTKi, based on ongoing results from a multicenter phase 1/2 study (BRUIN; NCT03740529). In the absence of a direct head-to-head trial comparing brexu-cel and pirtobrutinib, we performed an unanchored MAIC to estimate the relative treatment effects of these therapies in the post-BTKi setting for R/R MCL. Methods: Individual patient-level data were available for ZUMA-2 (N=68; 35.8 months median follow-up as of July 24, 2021 data cut-off) whereas study-level data were available for BRUIN (N=90 in the BTKi pre-treated cohort; 23.5 months median follow-up as of July 29, 2022 data cut-off). Longer-term overall survival (OS) data for ZUMA-2 from the July 23, 2022 data cut-off (46.1 months median follow-up) were available and used. Logistic propensity score models were used to weight the brexu-cel infused population from ZUMA-2 to match the aggregate baseline characteristics of the BTKi pre-treated cohort in BRUIN. These baseline characteristics represented clinically relevant prognostic factors which were pre-specified based on input from clinical experts and data availability. The base-case model incorporated the top five most pertinent factors, which were reported in at least 50% of patients in both trials: blastoid morphology, MCL International Prognostic Index, number of prior lines of therapy, disease stage, and prior autologous stem cell transplantation. A sensitivity analysis additionally incorporated TP53 mutation (missing in &amp;gt;50% of patients in ZUMA-2 and BRUIN) and Ki-67 proliferation index (missing in &amp;gt;50% of patients in BRUIN). Several prognostic factors (response to prior BTKi therapy, response to last therapy, and duration on prior BTKi therapy) could not be considered due to not being reported for BRUIN. The effective sample size was calculated as a measure of the degree of precision after weighting, reflecting the extent of overlap in the distribution of covariates between the trial populations. Relative treatment effects were expressed as odds ratios or hazard ratios with 95% confidence intervals. Outcomes included objective response (ORR), complete response (CR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: In the base-case MAIC, a significant difference in the odds of ORR and CR in favor of brexu-cel versus pirtobrutinib was observed (Table 1). Similarly, brexu-cel demonstrated a significant improvement in PFS (Table 2). OS and DOR point estimates were in favor of brexu-cel when compared to pirtobrutinib, although not statistically significant. These findings were consistent with unadjusted naïve analyses as well as the sensitivity analyses including adjustment for TP53 mutation and Ki-67 proliferation index. Conclusions: While acknowledging the inherent limitations of an unanchored indirect comparison, our findings suggest that brexu-cel offers clinically and statistically significant efficacy benefits in terms of ORR, CR, and PFS compared to pirtobrutinib in patients with R/R MCL after prior cBTKi therapy. Both treatments were not statistically different in terms of OS and DOR although the estimated hazard ratios indicated a favorable trend for brexu-cel; however, given the relatively shorter follow-up and the high degree of censoring in BRUIN, an updated analysis incorporating longer follow-up data with more events from BRUIN could provide more reliable results. Although efforts were made to ensure a robust approach to the selection of prognostic factors for inclusion in the model, the possibility of some residual confounding variables cannot be completely ruled out. Nevertheless, these results provide valuable insights that may help inform treatment decisions for this population.

Acquired Mutations in Patients (Pts) with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) That Progressed in the ALPINE Study

Introduction:Pts who receive covalent Bruton tyrosine kinase inhibitors (cBTKis) for CLL can develop drug resistance, leading to disease progression. Often, cBTKi resistance results from the emergence of subclones with mutations at the cBTKi binding site (C481). Less frequent, non-C481 mutations, including gatekeeper residue T474 and kinase-impaired L528 mutations, have been reported in pts with progression on cBTKis. Most characterizations of cBTKi resistance mutations are derived from small or retrospective pt populations. To gain further insight into the genetic mechanisms of cBTKi resistance in a randomized population of pts with CLL, we performed next-generation sequencing (NGS) on samples from pts who had progression on zanubrutinib (zanu) or ibrutinib (ibr) in the phase 3 ALPINE study (NCT03734016; Brown et al. NEJM. 2023). Methods:Progressive disease (PD) in ALPINE pts was assessed by independent review committee (IRC; n=139; zanu, n=54; ibr, n=85) and/or by investigator (INV; n=132; zanu, n=54; ibr, n=78) using Hallek et al ( Blood. 2008) criteria. Peripheral blood samples were collected at or after PD from 57 pts (zanu, n=26; ibr, n=31); 94.7% (54/57) had paired baseline samples. In 57 pts with PD, the median study follow-up was 25.7 mo; the median number of prior lines of therapy was 1 (range: zanu, 1-3; ibr, 1-7). Four pts (zanu, n=2; ibr, n=2) had Richter transformation (RT) at PD. A high-sensitivity NGS panel (PredicineHEME) with full exon coverage of 106 genes, including BTK, PLCG2 and 27 putative CLL driver genes (Knisbacher et al. Nat Gen. 2022), was used for mutation analysis. All acquired mutations in BTK and PLCG2were reported at a variant allele frequency (VAF) of ≥0.25%. For all other genes, pathogenic mutations (assessed using VarSome) with a VAF of ≥1% are reported. Del(17p), del(11q), trisomy 12, del(13q), IGHV mutation status, and complex karyotype (CKT) status were assessed in baseline samples. Results: No BTK mutations were detected at baseline, but 2 pts had PLCG2 mutations (zanu, R589C; ibr, E914STOP). To determine rates of BTK/PLCG2 or CLL driver gene mutations, only pts with paired baseline and PD samples and without RT at PD (n=52; zanu, n=24; ibr, n=28) were included (Table 1). Nine pts acquired BTK/ PLCG2 mutations: 8 (zanu, n=5 [20.8%]; ibr, n=3 [10.7%]) had BTK mutations, and 2 (both in ibr; 7.1%) had PLCG2 mutations; 1 pt had both BTK and PLCG2 mutations. Of 18 single nucleotide variants (SNV) in BTK, 77.8% (n=14; zanu, n=11; ibr, n=3) were at C481. Non-C481 mutations were detected in 12.5% of pts (3/24) with progression on zanu (Table 2). All 9 pts with acquired BTK/ PLCG2 mutations had PD assessed by IRC and INV during treatment, except 1 ibr pt who had PD assessed by INV after treatment. Median treatment duration was shorter in pts with PD without BTK mutations (zanu: n=19; 16.8 mo; range, 4.4-33.3 mo; ibr: n=25; 15.9 mo; range, 5.9-29.4 mo) than in pts with PD with acquired BTK mutations (zanu: n=5; 29.7 mo; range, 18.4-34.6 mo; ibr: n=3; 30.8 mo; range, 11.8-34.5 mo). At baseline, SNV or indels in 18/27 driver genes were observed in 48/52 pts. Most frequent: NOTCH1 (n=21), TP53 (n=19), and ATM (n=8). Copy number aberrations (CNA) in 9/27 driver genes were observed in 23 pts. Most frequent: CCND2 (n=10, amplification [amp]), ATM (n=8, deletion [del]), TP53 (n=6, del), and KMT2D (n=6, amp). At PD, acquired SNV were observed in 3 pts ( SETD2, ASXL1, and SF3B1 n=1 each; all ibr). Acquired CNA in driver genes were observed in 10 pts ( KRAS amp: zanu, n=3; NRAS amp: ibr, n=2; CDKN1B amp: zanu, n=2, ibr, n=1; BIRC3 del: ibr, n=2). Acquisition of driver gene mutations was not associated with del(17p), IGHV mutation, or CKT status. Conclusion: Of the 52 pts, most (82.6%) did not have acquired BTK or PLCG2 mutations. Among the zanu pts, 3/24 (12.5%) developed non-C481 BTK mutations. This rate was lower than that reported by Woyach et al (ICML 2023); shorter follow-up and fewer prior therapies in the ALPINE study may explain this discrepancy. Additionally, these data suggest that BTK and/or PLCG2 mutations are not the sole factors driving PD in this pt population; the presence or acquisition of mutations in driver genes with known negative prognostic value in CLL may also contribute to PD in pts without BTK or PLCG2 mutations. Given the low incidence of non-C481 mutations in ALPINE pts with PD, pts with CLL who were treated with cBTKis are likely to remain sensitive to other BTK-targeting therapies.

Extended Follow-up and Resistance Mutations in CLL Patients Treated with Acalabrutinib

Background: Covalent Bruton tyrosine kinase inhibitor (cBTKi) is a standard treatment for chronic lymphocytic leukemia (CLL). Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib. Mutations in BTK C481 and to a lesser extent PLCG2 are frequently detected in ibrutinib-treated pts at the time of progressive disease (PD). In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib. A recent study identified 3 pts with BTK C481 mutations and no pts with PLCG2 mutations among 8 acalabrutinib-treated pts with progressive disease (PD) (Black, et al. Blood 2022). A second study found a kinase-dead BTK L528W mutation in 7/13 zanubrutinib-treated pts (Blombery, et al. Blood Adv 2022). Although cBTKi share the same mechanism of action by binding to BTK C481, the latter report discloses differences in clonal selection and underscores the need for further investigation in pts treated with next generation cBTKi. In a phase 2 study of acalabrutinib in pts with treatment naïve (TN) CLL or small lymphocytic lymphoma (SLL) with 17p deletion, or TP53 or NOTCH1 mutation, or relapsed/refractory (R/R) CLL/SLL regardless of cytogenetics or somatic mutations (Sun, et al. Blood 2020), we sought to understand clonal evolution during treatment. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 48 pts at baseline, after 6 months, then annually on treatment until PD. Current analysis includes 14 PD pts [excluding 3 Richter transformation (RT) pts] comprising 10 R/R and 4 TN CLL pts. Additionally, paired baseline and PD bone marrow (BM) from 6 pts and lymph node (LN) samples from 2 pts were included. Targeted next-generation sequencing of ~700 recurrently mutated genes in hematological malignancies (Collins, et al. Leuk Lymphoma. 2021) was performed using DNA isolated from CD19 + enriched cryopreserved PBMCs and BM aspirates, as well as fresh-frozen core LN biopsies. Filtered variants in putative CLL driver genes (Knisbacher, et al. Nat Genet. 2022) with variant allele frequencies (VAF) ≥1% for BTK and PLCG2, ≥5% for variants with known oncological significance, and ≥7.5% for all other variants were included in downstream analysis. Results: After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years. Best response was complete remission in 2/20 (10%) pts and partial remission in 18/20 (90%) pts with CLL PD. IGHV was unmutated in all but one PD patient. At baseline, 34 CLL driver genes were mutated in 41/45 pts (91%; excluding 3 RT pts) with most mutations in NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE. During acalabrutinib treatment but before PD, 14/45 pts (31%; 8 R/R and 6 TN) acquired mutations in 11 driver genes, most commonly in BRAF (n=4), CREBBP (n=4), and KMT2D (n=2). At the time of PD, 11/14 pts (79%) had newly acquired mutations in 4 genes, predominantly BTK (n=6) and PLCG2 (n=4). BTK C481 mutations (VAF 2-42%) were detected in 6/14 (43%; 4 R/R and 2 TN) pts and T474I mutation (VAF 4-46%) in 3/14 (21%; 1 R/R and 2 TN) pts. Paired BM samples in 2 pts and LN samples in 2 pts showed the same BTK mutations as PBMCs with similar VAFs. PLCG2 mutations (VAF 1-8%) were found in 4/14 (29%; 2 R/R and 2 TN) pts. BTK C481 co-occurred with BTK T474I in 3 pts, but on different sequencing reads suggesting independent subclones, and with a PLCG2 mutation in 1 pt. There have been no pts with BTK L528 mutations. In conclusion, mutations in BTK and PLCG2 are the predominant resistance mechanism to acalabrutinib. Among BTK mutations, C481 was the most common. All T474I mutations co-occurred with C481.

Treatment Effectiveness with Venetoclax-Based Therapy after Bruton Tyrosine Kinase Inhibitors in Chronic Lymphocytic Leukemia: An International Real-World Study

Introduction: Venetoclax has demonstrated consistent efficacy and manageable toxicity in patients with previously untreated or relapsed/refractory (R/R) chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL). However, evidence on venetoclax use in patients previously treated with Bruton tyrosine kinase inhibitors (BTKi) is limited. This real-world evidence (RWE) study assessed clinical outcomes of patients who received venetoclax after covalent BTKi (cBTKi) therapy, i.e., response rates, time to next treatment or death (TTNT-D), and progression-free survival (PFS), overall and stratified by line of therapy. Methods: The CLL Collaborative Study of Real-World Evidence (CORE), a retrospective, international, observational study (23 centers) provided data for this analysis. Adult patients diagnosed with CLL/SLL were included if they received a venetoclax-based regimen after discontinuation of a cBTKi-based regimen. Baseline characteristics at venetoclax initiation were summarized using descriptive statistics. Overall response rate (ORR) was calculated as the proportion of patients with complete response (CR) or partial response (PR) among patients with documented responses as recorded in the patients' medical charts based on physician assessment. TTNT-D was measured from date of venetoclax initiation to change of treatment/death (event) or end of follow-up, while PFS was measured from date of venetoclax initiation to disease progression/death (event) or end of follow-up. Outcomes were reported for the overall population and stratified by line of therapy (1L→2L and 2L→3L) and also by with and without CT/CIT exposure prior to cBTKi for 2L→3L. Results: Of the 2,020 patients included in CORE, 1,287 (63.7%) received a cBTKi-based regimen in ≥1 line of therapy; 184 patients (14.3%) discontinued cBTKi (intolerance: 83 [45.1%], progression: 78 [42.4%]) and initiated venetoclax-based therapy (115 venetoclax monotherapy; 69 venetoclax combined with rituximab or obinutuzumab). Mean age at venetoclax initiation was 68.6 (median: 68.2) years old, 69.0% were male, and 31.0% had Medicare. Among tested patients, 41/61 (67.2%) had unmutated IGHV, and 28/109 (25.7%) had 17p deletion or TP53 mutation at venetoclax initiation. Average follow-up time from initiation of venetoclax therapy was 19.6 months (median: 16.6). Of the 127 patients (69.0%) with a documented response, the ORR was 78.0% (CR: 43.3%, PR: 34.6%). The median TTNT-D for these patients was 39.5 months (95% CI: 30.4, not reached [NR]) with 12- and 18-month rates of 82.3% and 72.4%, respectively. The median PFS was 43.2 months (95% CI: 31.9, NR), with 12- and 18- month PFS rates of 82.8% and 75.1%, respectively. Among patients who started venetoclax-based therapy post-cBTKi as 1L→2L (n=65), the ORR was 84.1% (CR: 54.5%, PR: 29.5%, out of 44 documented). The median TTNT-D for these patients was NR (95% CI: 31.9, NR) but the 12- and 18-month rates were 85.0% and 73.9%, respectively. The median PFS was 43.2 months (95% CI: 39.5, NR) with 12- and 18- month rates of 86.4% and 81.8%, respectively. Among patients who started venetoclax-based therapy post-cBTKi as 2L→3L (n=67), the ORR was 78.3% (CR: 41.3%, PR: 37.0%, out of 46 documented). The median TTNT-D for these patients was 44.2 months (95% CI: 37.0, NR) with 12- and 18-month rates were 83.1% and 76.5%, respectively. The median PFS for these patients was 44.1 months (95% CI: 31.8, NR) with 12- and 18- months rates were 85.2% and 80.4%, respectively. The results for ORR, TTNT-D, and PFS were also similar for those with and without CT/CIT exposure prior to cBTKi. Conclusions: In this multicenter real-world study, results demonstrate that venetoclax is effective overall and also when used either in 2L or 3L following cBTKi therapy. Additionally, even with prior CT/CIT exposure, results support that venetoclax-based therapy remains effective as a therapeutic option. Thus, this study demonstrates that venetoclax-based therapy post-cBTKi is associated with durable remission. These results are especially timely as the CLL treatment paradigm continues to evolve with multiple treatment options available, providing clinicians with valuable evidence to inform modern clinical practice. Further analysis of this study with larger cohorts and longer follow-up time will be undertaken as part of future work to grow this body of evidence.

Real-World Treatment Patterns, Overall Survival, Healthcare Resource Utilization, and Costs Among U.S. Elderly Patients with Mantle Cell Lymphoma (MCL) after Failure of Covalent Btki Treatment

Background: Over the past decade, Bruton's tyrosine kinase inhibitors (BTKis) have emerged as standard of care treatment for relapsed/refractory mantle cell lymphoma (R/R MCL). Despite improvements in progression-free survival with currently available covalent BTKis (cBTKis), response duration can be limited as patients eventually experience disease progression or treatment intolerance. Limited real-world evidence on post-cBTKi clinical and economic outcomes exists for these patients. The goal of this study was to examine treatment patterns, overall survival (OS), healthcare resource use (HRU), and costs in elderly U.S. patients with R/R MCL in the third-line (3L) setting who received first-generation or second-generation cBTKi treatment in prior lines of therapy (i.e. 1L or 2L). Methods: This retrospective study used 2010-2019 U.S. Medicare claims (100%), the most recently available data at the time of the analysis, to identify elderly (≥66 years) patients with a new MCL diagnosis between 01/01/2010 and 12/31/2018 (index date = first MCL diagnosis date). Patients were required to have continuous fee-for-service Medicare coverage in the 12-months pre- and a minimum of 12-months post-index. An algorithm to identify lines of therapy (LOT) was developed using prior published studies. Patients were included in our final sample if they had evidence of use of a cBTKi (i.e. ibrutinib or acalabrutinib, the two available BTKIs during our study period) in the 1L and/or 2L setting and had progressed to 3L treatment. Outcomes included patient clinical and sociodemographic characteristics, treatment patterns, all-cause and MCL-related HRU and costs, and median OS after 3L treatment initiation. Results: The final sample contained 230 elderly patients with R/R MCL receiving 3L treatment who had cBTKi use in a prior LOT. The vast majority (&amp;gt;95%) of these patients had received their cBTKi in the 2L setting; the most common cBTKi in the 1L (100%) and 2L (&amp;gt;93%) setting was ibrutinib. Patients had a mean age of 75.0 years (SD=5.9) and 21.7% were age &amp;gt;80 years. Over two-thirds (67.4%) were male and majority (93.9%) were white. About one-quarter (25.7%) of the patients received a cBTKi (17.8% ibrutinib and 7.8% acalabrutinib) in the 3L setting. Other common 3L treatments included standard chemotherapy (26.1%), lenalidomide (18.7%), and bortezomib (18.3%). Over a median follow-up of 1.6 years, 38.7% of the patients progressed to fourth-line (4L) treatment and the mean time to next treatment (i.e. end of 3L to start of 4L) was 2.8 months. Median OS from 3L treatment initiation was 9.4 months and the 1-year and 3-year survival rates were 43.7% and 17.9%, respectively. All-cause hospitalization was common (73.6%) and 44.7% used hospice services in the 12 months after 3L treatment initiation. All-cause total costs were substantial ($145,726) in the 12 months following 3L treatment initiation and 87% of these costs were MCL-related ($126,526). Inpatient and outpatient costs in the 12 months following 3L treatment initiation were $32,520 and $19,370, respectively (these costs were higher in the subgroup of patients who progressed to 4L therapy). MCL-related prescription costs ($70,127) represented over half (55%) of MCL-related total spending; most of those costs were for Part D oral agents ($43,009) rather than Part B IV-administered agents ($27,119). Conclusions: In this real-world study of elderly U.S. patients with R/R MCL, we found that 3L patients treated with currently available cBTKis in prior lines had poor OS. Limited options for 3L treatment existed, with over half of patients using another cBTKi or standard chemotherapy. Further, rates of HRU were high and healthcare costs were substantial in the 3L setting. Our study suggests there is a large unmet need in elderly patients with R/R MCL who previously failed a cBTKi, highlighting the importance of ongoing development with novel therapeutics aiming to improve outcomes in R/R MCL.

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

TL-895, a Highly Selective, Covalent Inhibitor of Bruton's Tyrosine Kinase (BTK), Sensitizes Myeloproliferative Neoplasm (MPN)-Blast Phase Stem Cells to Navtemadlin By Targeting Intrinsic Dysregulated MDM2/p53 and NF-Κb Pathways and Disrupting the Protective Tumor Microenvironment (TME)

We recently reported that navtemadlin (nvtm), a potent, orally available, selective inhibitor of MDM2 (MDM2i), can deplete/eliminate TP53WT MPN-blast phase (BP) stem cells (SC) in an MPN-BP patient (pt)-derived xenograft (PDX) model (Wang EHA 2023). Single agent nvtm has demonstrated significant clinical activity in pts with advanced myelofibrosis (MF) (Vachhani EHA 2023, Verstovsek Future Oncology 2022). We hypothesized that effects of nvtm on MPN-BP, which is characterized by multiple SC subclones, could be enhanced by combination with agents that target additional MPN-BP SC vulnerabilities, such as hyperactivation of p65 subunit of NF-κB (Fisher Leukemia 2017; our unpublished data) and dependence on TME support. BTK and bone marrow (BM) kinase X-linked (BMX) are key Tec family members expressed in cells of myeloid origin. Here, we show BTK is constitutively activated in MPN-BP SC subpopulations relative to normal donor (ND) hematopoietic SCs (HSC) and progenitors [phospho (p)-BTK Y233 mean fluorescence intensity fold change (FC)]: CD34 +CD38 - cells: 1.34±0.18, HSC/multipotent progenitor (MPP): 1.50±0.23, multi-lymphoid progenitor (MLP): 1.59±0.59, megakaryocyte-erythroid progenitor (MEP): 1.56±0.49; p &amp;lt;0.05]. TL-895 is a highly selective, potent, orally available, second-generation covalent inhibitor of BTK and BMX, currently under clinical evaluation for treatment of pts with CLL (NCT02825836) and MF (NCT04655118). We used multi-modal single-cell analysis to study mechanistic effects of combination TL-895 + nvtm on dysregulated intracellular signaling pathways in primary MPN-BP SC, and investigated the combination in a PDX model of MPN-BP. In vitro treatment with TL-895 alone (100 nM-5 µM) did not affect growth of ND CD34 + cells [FC (TL-895 vs. vehicle, veh): 2.5 µM: 1.27±0.11, 5 µM: 0.97±0.14, n=3)] but modestly decreased growth of primary TP53WT MPN-BP CD34 + cells, in a dose dependent fashion [FC (TL-895 vs. veh): 2.5 µM: 0.88±0.11, 5 µM: 0.62±0.12, n=4. p&amp;lt;0.05). Addition of TL-895 (5 µM) to nvtm (400 nM) resulted in much greater reductions in MPN-BP CD34 + and CD34 +CD38 - cell numbers, along with increased apoptosis (n=6, p&amp;lt;0.05, TL-895 + nvtm vs. veh, TL-895, or nvtm alone). By contrast, at these doses, combination TL-895 + nvtm did not affect ND HSC subpopulations (n=3). We previously demonstrated that, multiple MPN-BP SC subclones contribute to the development of MPN-BP irrespective of clone size, and minor subclones may be responsible for clonal evolution and disease progression in most cases (Wang JCI insight 2022). Strikingly, single cell mutational profiling and epitope analysis of primary MPN-BP CD34 + cells treated with TL-895 + nvtm showed depletion of all 8 distinct MPN-BP SC subclones with differing variant allele frequencies, each containing 2-3 oncogenic mutations in WT1, ETV6 and/or KRAS. Combination TL-895 + nvtm treatment depleted 7 of 8 subclones to a greater degree than either agent alone (Fig. 1). Bulk RNA-seq and gene set enrichment analysis showed that combination TL-895 + nvtm synergistically upregulated p53 pathway gene transcription ( CDKN1 HDM2, DDB2, BTG2, GADD45A, FAS, FUCA1, S100A10, ATF3) in primary MPN-BP CD34 + cells. Mass cytometry analysis and intracellular flow cytometry of MPN-BP CD34 + cells treated with combination TL-895 + nvtm further revealed significant (p&amp;lt;0.05) upregulation of p53 and BAX proteins and decreases in the anti-apoptotic factors BCL-xL and activated NF-κB (p-p65), as well as significant reductions in DNA synthesis and protein markers associated with glycolysis (p-AMPK, MCT1). Downregulation of NF-κB activation might result from decreases in the upstream modulators p-S6 and p-4EBP1 (p&amp;lt;0.05, TL-895 + nvtm vs. veh). Treatment in vivo withTL-895 promoted dose-dependent egress of hCD45 +CD34 + cells from BM into the spleen and peripheral blood (PB) of MPN-BP PDX mice (Fig. 2). Furthermore, a limited course of combination TL-895 + nvtm treatment for 3 cycles led to clearance of hCD45 +CD34 + cells from the BM, spleen and PB of MPN-BP PDX mice (Fig. 2); neither treatment had this effect when administered alone. Our results indicate that BTKi therapy might increase susceptibility of MPN-BP SC to MDM2i therapy, by upregulating p53 activity and dampening NF-κB signaling, and also by disrupting protective TME interactions that sustain MPN-BP SC. This novel combination merits further clinical investigation in advanced phase MPN.