Citations

Abstract Early-phase clinical trials of Bruton’s tyrosine kinase (BTK) degraders have demonstrated efficacy in patients with BTK inhibitor-resistant chronic lymphocytic leukemia (CLL). How clinical resistance to BTK degraders arises is unknown. Here we sequenced serial CLL samples from patients enrolled in the phase I trials of zelebrudomide and bexobrutideg and observed recurrent expansion of preexisting BTK A428D mutations at relapse. Unlike previously studied BTK inhibitor resistance mutations, BTK A428D conferred pan-resistance to BTK inhibitors and degraders. In the absence of BTK-directed therapies, however, cells bearing BTK A428D exhibited a competitive disadvantage. A crystal structure of BTK A428D revealed that the mutant aspartate clashes with the adenine ring of ATP and the adenine-mimetic moiety of BTK inhibitors and degraders. Combining BTK degraders with venetoclax mitigated the expansion of BTK A428D. These results provide the molecular basis for clinical resistance to BTK degraders and will inform the development of next-generation BTK degrader therapies.

Abstract Early-phase clinical trials of BTK degraders in relapsed/refractory CLL have shown response rates of over 80%, including in patients with BTK mutations that confer resistance to conventional BTK inhibitors (BTKi). The molecular basis of resistance to BTK degraders is unknown and, given the promise of this class of drugs, would hold implications for therapy sequencing and the design of next-generation BTK degraders. To this end we identified five patients at Memorial Sloan Kettering who developed progressive disease while being treated on the phase 1 trials of zelebrudomide (NCT04830137, 4 patients) or bexobrutideg (NCT05131022, 1 patient). In two patients, one treated with zelebrudomide and the other with bexobrutideg, a pre-existing BTK A428D mutation had expanded at progression, rising from <1.5% at the start of treatment to >25%. In both cases, the increase in frequency of BTK A428D coincided with the loss of BTK degradation in peripheral blood B cells. No mutation in cereblon, the ligase utilized by these BTK degraders, was detected. To validate these clinical findings we generated wild-type and BTK A428D knock-in single-cell clones in TMD8 and HBL1 cells. BTK A428D conferred resistance to BTKi, including ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib, as well as the clinical-stage BTK degraders zelebrudomide, bexobrutideg, BGB-16673, and AbbVie-1 (Compound 1, WO 2023183811). BTK A428D’s pan-resistance is unique compared to previously studied BTKi resistance mutations which remain susceptible to at least one BTKi and all clinical-stage BTK degraders tested. To understand how BTK A428D confers pan-resistance to BTKi and BTK degraders we conducted surface plasmon resonance studies which demonstrated that BTKi and all four clinical-stage BTK degraders tested are unable to bind BTK A428D. We next solved the crystal structure of apo BTK A428D to 1.8Å, which revealed that the mutant aspartate, which is larger and negatively charged compared to the wild-type alanine, sterically blocks BTKi and degraders from accessing residues in the hinge region of the kinase that are required for drug binding. We next asked why BTK A428D, despite conferring pan-resistance, has only been detected in 1.1% of patients treated with BTK inhibitors. Intriguingly, we noted that in one of our index patients, the frequency of BTK A428D declined and became undetectable after the withdrawal of zelebrudomide, suggesting a fitness defect. To assess this hypothesis, we compared the proliferation rates of five TMD8 wild-type and five BTK A428D single-cell clones; the BTK A428D clones grew more slowly and were consistently outcompeted in mixing studies. In summary, low-frequency BTK A428D mutations can expand under the selection pressure of BTK degraders and confer clinical resistance by preventing drug binding. The mutation, however, is outcompeted in the absence of BTK-targeted therapies. These findings provide rationale for the investigation of combination therapies and the development of next-generation BTK degraders capable of circumventing BTK A428D. Citation Format: Quinlan Sievers, Hao Lu, Allison Cool, Stefan Grajewski, Tim Kong, Mark Noviski, Xiaoli Mi, Emel Ficici, Ratul Mukerji, James Iuliano, Jordan Ye, Mateo Sanchez Garcia de los Rios, Hugo Bousquet, Sarah Whelan, May Tan, Christopher Phelps, Jahan Raman, Ryan Notti, Lizbeth Mendoza Navarrete, Samantha Sekeres, Eria Lamkin, Eduardo Bravo, Mark Ewalt, Prioty Islam, Anthony Mato, Lindsey Roeker, Justin Taylor, Meghan Thompson, Gwenn Hansen, Omar Abdel-Wahab. BTK A428D is a recurrent pan-resistance mutation to BTK degraders in clinical trials [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr PR004.

Abstract BTK degraders promote proteasomal degradation of BTK and can overcome resistance to covalent and noncovalent BTK inhibitors in patients with relapsed CLL. Currently the molecular basis for BTK degrader resistance is unknown. We therefore set out to understand BTK degrader resistance based on observations from the phase 1 trial of zelebrudomide (NX-2127) (NCT04830137), the first BTK degrader to enter clinical studies. Targeted 500-gene sequencing was performed on blood and/or tissue from four CLL patients treated with zelebrudomide. In one patient previously treated with chemoimmunotherapy, ibrutinib, venetoclax, and pirtobrutinib, BTK A428D was detected in the baseline blood sample with variant allele frequency (VAF) of <2%, along with BTK V416L (13%) and mutations in TP53 (60%) and SF3B1 (37%). Following daily treatment with 100mg zelebrudomide and subsequent dose escalation to 200mg, at 6 months the VAF of all four mutations increased, with BTK A428D VAF increasing to 6% in the blood and 29% in a lymph node. BTK A428D was detected at baseline but not at progression in a second patient. To evaluate the impact of BTK A428D on drug response, we transduced BTK-dependent TMD8 cells with BTK WT, C481S, L528W, and A428D, and found that BTK A428D uniquely conferred resistance to all FDA-approved BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, pirtobrutinib) as well as three BTK degraders under clinical evaluation (zelebrudomide, bexobrutideg, BGB-16673). To model the subclonal nature of BTK A428D we conducted a competition assay in which TMD8 cells expressing GFP + BTK A428D (5%) were mixed with TMD8 cells expressing mCherry + BTK WT (95%); over 9 days the GFP + BTK A428D expanded from 5% to 88-97% with all four BTKi and all three BTK degraders. In vitro kinase assays revealed that the BTK A428D mutant protein lacks kinase activity yet facilitates calcium flux and ERK/AKT signaling upon B-cell receptor activation, suggesting a scaffolding effect similar to other kinase deficient BTK mutations. To determine whether BTK A428D affects BTK degradation, we developed a reporter that fuses the BTK kinase domain to GFP. BTK A428D showed minimal degradation following 20 hours of treatment with a titration of all three BTK degraders. Similarly, we knocked BTK A428D into the endogenous BTK locus of HBL1 cells and confirmed a lack of degradation at doses up to 1 µM of all 3 degraders. Consistent with these findings, surface plasmon resonance revealed that the FDA-approved BTK inhibitors as well as zelebrudomide, bexobrutideg, BGB-16673, and AbbVie-1 (Compound 1 from WO 2023183811) are unable to bind to BTK A428D. Additionally, an in-cell NanoBRET assay demonstrated that zelebrudomide was unable to mediate the interaction between cereblon and BTK A428D. Strikingly, NanoBRET also demonstrated that a panel of promiscuous kinase-binding compounds was unable to bind to BTK A428D. The lack of binding of both selective and promiscuous kinase binders to BTK A428D led us to speculate that the mutation disrupted the ATP binding pocket to an unusual degree. We therefore solved the crystal structure of apo BTK A428D and found that, in addition to its predicted steric clash with ligand binding in the hinge region, the mutation led the protein to crystallize with its activation loop occupying the catalytic cleft, rendering it inaccessible. This is the first apo BTK structure observed in this autoinhibited conformation, and explains the observed binding profile. We hypothesized that the addition of a second targeted agent such as venetoclax might mitigate expansion of the A428D subclone. To test this, we introduced the BTK A428D mutation into the endogenous BTK gene in 3% of TMD8 cells and then exposed them to 0.1µM of zelebrudomide, bexobrutideg, or BGB-16673 +/- venetoclax 0.1µM. Combining the BTK degraders with venetoclax slowed the emergence of the BTK A428D resistant population and, in the case of zelebrudomide, eradicated both the wild-type and A428D clones. In summary, we identified and validated BTK A428D as a clinically relevant acquired pan-resistance mutation that confers resistance by blocking access to the ATP binding pocket. This mechanism differs from other kinase dead BTK mutations in which the kinase's enzymatic pocket remained accessible.These data also provide rationale for combining BTK degraders with BCL2 inhibitors, particularly in patients with BTK A428D mutations, or high-risk mutations such as in TP53.

AbstractFunctionally gradient materials emulate nature's ability to seamlessly blend properties through variations in material composition, unlocking advanced engineering applications such as biomedical devices and high‐performance composites. Additive manufacturing, particularly stereolithography, enables sophisticated 3D geometries with diverse materials. However, current stereolithography‐based multi‐material 3D printing is constrained by time‐intensive material switching and compromised interfacial properties. To overcome these challenges, we present dynamic fluid‐assisted micro continuous liquid interface production (DF‐µCLIP), a high‐speed multi‐material 3D printing platform that integrates varying compositions in a fully continuous fashion. By utilizing the polymerization‐free “dead zone”, vliquid resins are seamlessly replenished within a resin bath equipped with dynamic fluidic channels and a synchronized material supply system. DF‐µCLIP achieves ultra‐fast printing speeds of 90 mm/hour with 7.4 µ m pixel‐1 resolution while enabling on‐the‐fly material transitions. This strategy enhances mechanical strength at multi‐material interface through entangled polymer networks and promotes seamless material transitions between distinct materials ilike fragile hydrogels and rigid polymers, addressing interfacial failure caused by mismatch of swelling behavior. Additionally, dynamic material replenishment with real‐time composition control enables continuous gradient printing instead of the conventional step‐wise controlled gradient. Demonstrations include polymers with gradient color transitions and gradient carbon nanotube (CNT) composites with seamlessly varying conductivity.

ABSTRACTBackgroundContrast‐enhanced magnetic resonance neurography (ceMRN) can enhance brachial plexus visualization and quality of imaging. However, the interval between contrast injection and scanning that provides the highest‐quality images is not known.MethodsFifteen patients underwent brachial plexus imaging using the 3D T2‐NerveView sequence with a scanning duration of 5 min. A consecutive six‐phase scan was initiated immediately at the start of contrast agent injection. Subsequently, all patients' images were classified into six groups according to the phases: group A (phase 1, delay 0 min), group B (phase 2, delay 5 min), group C (phase 3, delay 10 min), group D (phase 4, delay 15 min), group E (phase 5, delay 20 min), and group F (phase 6, delay 25 min). The image quality in each group was assessed based on nerve signal (signalnerve), muscle signal (signalmuscle), lymph node signal (signallymph node), background noise (BN), signal‐to‐noise ratio (SNR), contrast‐to‐noise ratio (CNR), and subjective score.ResultsSignalnerve, signalmuscle, BN, and SNR did not significantly differ among the six groups (p > 0.05). However, significant differences (p < 0.05) were observed in signallymph node (F = 16.067), CNR (F = 9.495), and subjective score (χ2 = 23.586). As the scanning delay increased, signallymph node intensity gradually increased whereas the CNR gradually decreased. The subjective score was significantly higher in groups B (4.83 ± 0.24), C (4.90 ± 0.21), D (4.87 ± 0.30), E (4.83 ± 0.31), and F (4.83 ± 0.31) than in group A (4.47 ± 0.30).ConclusionWe recommend performing brachial plexus ceMRN 5 min after contrast injection. With this delay, the brachial plexus can be visualized optimally with minimal interference from background signals.

Background: We have found that the vascular large conductance Ca 2+ -activated K + channels (BK) are targeted to caveolae. The goal of this study is to determine the role of caveolae in BK regulation by angiotensin II (AII) in normal and diabetic rats. Results: In coronary arterial smooth muscle cells (CASMC) from control rats, AII (2 μ M) inhibited BK by 40.5% from 227±26 pA/pF to 135±14 pA/pF (HP −60 mV, TP 150 mV, n=7, p<0.05). However, AII had no effects on BK in CASMC transfected with caveolin-1 siRNA (vs 50% inhibition in cells transfected with control siRNA) or in cells from Cav1−/− mice, suggesting the AII effects are caveolae-dependent. Density gradient analysis of rat vessel homogenates showed that caveolin-1, BK, Gq, cSrc, and NOX-1 all reside in the caveolae-rich fraction, where AT 1 R was absent unless the animal was pre-treated with AII, suggesting receptor translocation upon agonist activation. However, CASMC from streptozotocin-induced diabetic rats showed significant reduction in BK (61.5±10.6 pA/pF) with no response to AII (61.2±15.7 pA/pF, n=8). Vascular BK, caveolin-1, AT 1 R, cSrc, and NOX-1 expression in caveolae were all upregulated by 1.6- to 3.1-fold in diabetes. Density gradient analysis of diabetic vessels showed the presence of AT 1 R in the caveolae-rich fraction without AII pretreatment, suggesting that the AT 1 R signaling pathway might be constitutively active in diabetes. This was accompanied by a 2-fold increase in BK tyrosine phosphorylation and BK tyrosine nitration. The AII effects on BK function in diabetic CASMC were abolished by pretreatment (1 hr) with PKC inhibitor or by LavA (cSrc inhibitor) and can be mimicked by incubation (2 hr) of control CASMC with peroxynitrite. Conclusion: AT 1 R-BK-enzyme complex in the caveolae microdomain is critical in vascular BK regulation. Enhanced AII-signaling facilitates BK post-translational modification, resulting in BK dysfunction in diabetes.

Abstract The clinical use of surface imaging has increased dramatically, with demonstrated utility for initial patient positioning, real‐time motion monitoring, and beam gating in a variety of anatomical sites. The Therapy Physics Subcommittee and the Imaging for Treatment Verification Working Group of the American Association of Physicists in Medicine commissioned Task Group 302 to review the current clinical uses of surface imaging and emerging clinical applications. The specific charge of this task group was to provide technical guidelines for clinical indications of use for general positioning, breast deep‐inspiration breath hold treatment, and frameless stereotactic radiosurgery. Additionally, the task group was charged with providing commissioning and on‐going quality assurance (QA) requirements for surface‐guided radiation therapy (SGRT) as part of a comprehensive QA program including risk assessment. Workflow considerations for other anatomic sites and for computed tomography simulation, including motion management, are also discussed. Finally, developing clinical applications, such as stereotactic body radiotherapy (SBRT) or proton radiotherapy, are presented. The recommendations made in this report, which are summarized at the end of the report, are applicable to all video‐based SGRT systems available at the time of writing.

Background: Reduced left atrial (LA) compliance (LAC) contributes to pulmonary venous hypertension in heart failure with preserved ejection fraction (HFpEF); however the underlying mechanisms are poorly defined. We hypothesized that microvascular inflammation with rarefaction and impaired nitric oxide (NO) signaling, LA titin hypophosphorylation and fibrosis may each contribute to reduced LAC in HFpEF. Methods: Invasive LA pressure-volume (PV) analyses (open-chest; pericardiotomy; admittance catheter) were performed in 9 aged dogs with experimental hypertension (bilateral renal wrap + DOCA; HFpEF) compared to 13 sham-operated young adult dogs (control). LAC was measured at end-LA reservoir during acute preload reduction (cava occlusion). LA tissue and LA microvascular function were analyzed. Results: Compared to controls, the curvilinear end-LA reservoir PV relationship ( Fig A ) was shifted upward/leftward in HFpEF (LA diastolic stiffness constant 0.08 vs 0.16 mmHg/mL in controls, p=0.005). LA microvascular density (MVD; CD31 stain) tended to be lower in HFpEF than controls (2154±578 vs 2650±523 vessels/mm 2 , p=0.07) and LA MVD negatively correlated with the LA diastolic stiffness constant (r=-0.66, p=0.006). LA microvessels from HFpEF dogs showed impaired endothelium-dependent shear stress-mediated dilation compared to controls, consistent with impaired NO signaling ( Fig B ). Titin isoform expression was similar between groups, but isoform N2B (stiffer) was (p=0.03) and N2BA (compliant) tended to be (p=0.09) hypophosphorylated in HFpEF LA appendage (LAA) tissue. Fibrosis (Picrosirius Red) was similar between groups in the LA free wall and LAA, but increased in areas adjacent to the pulmonary veins in HFpEF (27±12% vs controls 15±8%, p=0.01). Conclusion: In experimental HFpEF, LAC was reduced in association with evidence of LA microvascular rarefaction and impaired NO signaling, titin hypophosphorylation and regional increases in LA fibrosis.

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.