Kong, Tim

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.

Abstract Myeloproliferative neoplasms (MPNs) are typically ascribed to hyperactive JAK-STAT signaling via mutations in JAK2, CALR, or MPL. Here, we characterize how activating mutations in the calcium sensor STIM1, previously established as the cause of Stormorken Syndrome (SS), a rare congenital disorder, are sufficient to drive development of myelofibrosis (MF). We further uncover distinct interactions between STIM1 activity and JAK2 versus CALR driven MPNs. We initially identified two SS patients with activating mutations in STIM1 (R304W and S88G) and features of MF, including marked reticulin fibrosis with megakaryocytic hyperplasia and atypia. This led us to hypothesize that altered calcium signaling may play a role in the development of MF in both SS and MPNs. Single cell RNA sequencing performed on samples from both patients demonstrated elevated NFκB inflammatory signaling in monocyte populations, suggesting a link between altered STIM1 and store operated calcium entry (SOCE) activity and aberrant inflammation, thereby contributing to MF development. To corroborate these findings from our SS patients, we generated a knock-in mouse model of the heterozygous R304W mutation. Induction of hematopoietic-specific expression of mutant Stim1 with Vav-Cre recapitulated the hematological features of SS, including elevated SOCE activity and thrombocytopenia. We also recapitulated MF features including the development of bone marrow fibrosis and severe osteosclerosis, with the mice developing splenomegaly as young as 9 weeks old. In ex vivo megakaryocyte differentiation assays with our Stim1 R304W knock-in mice, we also observed aberrant cytoskeletal organization and elevated TGFβ mRNA expression. When induction was driven universally through CMV-Cre, we also validated that mice recapitulated multiple SS features, including thrombocytopenia, tubular aggregate myopathy, growth deficiency, and the development of bone marrow fibrosis as young as 10 weeks. We additionally identified a separate cohort of 9 related individuals with SS and STIM1 mutations (L92V) as well as a separate patient with the R304W mutation. In ex vivo megakaryocytic differentiation assays, cells from SS patients demonstrated defective thrombopoiesis with significantly decreased proplatelet formation, and displayed aberrant cytoskeletal formation within the pro-platelet termini, in conjunction with increased expression of TGFβ. Further supporting a role for STIM1 in MPNs, we identified upregulation of STIM1 expression in MPN patient CD34+ hematopoietic stem/progenitors' cells (HSPCs), we well as megakaryocyte progenitors and platelets, including those with CALR or JAK2 mutations. To evaluate the functional role of STIM1 in MPNs, we generated patient-derived xenograft (PDX) models with CALR and JAK2-mutantpatient samples subjected to CRISPR ablation of STIM1. In an initial CALR-mutant PDX experiment, abrogation of STIM1 led to decreased human CD45+ cell engraftment in conjunction with prolonged survival. In a second CALR-mutant PDX experiment, STIM1 targeting did not overtly impact engraftment or survival. In contrast, CRISPR ablation of STIM1 in JAK2-mutant PDX models led to exacerbated disease phenotypes, including increased human CD45+ cell engraftment, increased splenomegaly, and early lethality. Altogether, this study represents the first demonstration of MF development in patients with Stormorken Syndrome and reveals a previously unrecognized role of aberrant SOCE underlying SS and MPNs. We recapitulated these findings in a novel conditional mouse model. These findings also illustrate unique interactions between STIM1 and JAK2 and CALR driven MPNs. These observations have important implications for the development of targeted therapeutic approaches for these disorders.

Activating mutations in the calcium sensor STIM1 underlie Stormorken Syndrome (SS), a rare congenital disorder that causes thrombocytopenia and tubular aggregate myopathy. We recently identified two patients with SS and confirmed mutations in STIM1 (R304W and S88G), who were found to have features consistent with myelofibrosis (MF), as indicated by bone marrow biopsies demonstrating megakaryocyte hyperplasia and atypia in conjunction with marked reticulin fibrosis. The unusual MF findings in these two patients raised the hypothesis that altered calcium signaling represents a shared hallmark of congenital platelet disorders such as Stormorken Syndrome and myeloproliferative neoplasms (MPNs) including MF. Analyzing gene expression data from public datasets, we found increased expression of STIM1 in megakaryocyte progenitors and platelets, as well as CD34+ hematopoietic stem/progenitor cells (HSPCs), from MPN patients (including those with CALR or JAK2 mutations) vs healthy controls. These findings led us to hypothesize that dysregulation of STIM1 and store operated calcium entry (SOCE) signaling may contribute to altered megakaryopoiesis and development of fibrosis in both SS and MPNs. To further delineate these processes, we analyzed samples from our two SS patients, demonstrating constitutive activation of SOCE causing increased calcium flux, as well as decreased aggregation in mature platelets. We also performed transmission electron microscopy (TEM) and identified platelets with abnormal granularity in dense and alpha granules. We also performed single cell RNA-seq on SS patient peripheral blood mononuclear cells, which revealed enhanced NFkB inflammatory signaling, suggesting that altered signaling driven by STIM1 mutations may drive aberrant inflammation to contribute to MF development. To corroborate these initial observations, we identified a separate family cohort of 9 individuals in Italy with SS and confirmed STIM1 mutation (L92V). In ex vivo megakaryocytic differentiation assays, cells derived from these patients demonstrated a defect in thrombopoiesis with significantly decreased pro-platelet formation and adhesion to extracellular matrix. To further characterize the effects of the gain of function STIM1 mutations seen in our patients, we developed a novel, conditional knock-in mouse model of the heterozygous R304W mutation in the coiled coil (CC) domain of Stim1. Induction of hematopoietic-specific expression of mutant Stim1 via Vav-Cre resulted in recapitulation of hematological features of SS, including thrombocytopenia and a mild bleeding tendency, with confirmation of increased SOCE activity and calcium flux. Notably, we confirmed the development of bone marrow fibrosis, as well as findings of severe osteosclerosis, in these mice. TEM imaging confirmed that platelets from Stim1R304W/+Vav-Cre mice exhibit abnormal granularity, similar to what we observed from our SS patients. Noting that we identified increased STIM1 expression in samples from MPN patients harboring either JAK2 or CALR mutations, we further characterized the role of STIM1 in MPN disease development. NSGS mice were engrafted with CALR-mutant CD34+ cells subjected to CRISPR ablation of STIM1. Recipient mice exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to exacerbated disease phenotypes, as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Taken together, these findings suggest a striking, differential relationship between mutant JAK2 and CALR and STIM1 activity and their relationship to MF development. Altogether, this study represents the first demonstration of MF development in patients with Stormorken Syndrome, and reveals a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying SS and MPNs. We further demonstrate recapitulation of these hematologic features in a novel knock-in Stim1R304W/+Vav-Cre mouse model. These findings implicate an important role for STIM1and SOCE activity in MF development, and further uncover important distinctions between JAK2 vs CALR mutations and their interaction with altered STIM1 activity. These studies may contribute to the development of novel therapeutic approaches for these disorders.

MB and VA equal contributors AB and STO co-corresponding authors This study was initiated following evaluation of a 32 year-old woman who presented with a history of thrombocytopenia identified in childhood who subsequently developed features of myelofibrosis (MF). A bone marrow biopsy demonstrated hypercellularity in conjunction with megakaryocyte hyperplasia and marked reticulin fibrosis. Molecular testing for JAK2, CALR, and MPL mutations was negative. Given the unusual association between congenital thrombocytopenia and MF in this patient, exome sequencing was performed, revealing a heterozygous R304W mutation in the coiled coil (CC) domain of STIM1. Activating mutations in the CC and EF hand domains of STIM1 have been associated with Stormorken syndrome, a rare congenital platelet disorder associated with abnormal store operated calcium entry (SOCE). We subsequently identified a second patient with MF associated with a STIM1 activating mutation. This individual was found to have severe thrombocytopenia at birth, and exome sequencing revealed a heterozygous S88G mutation in the EF hand of STIM1. A bone marrow biopsy obtained at 6 months of age revealed atypical megakaryocytes and grade 1-2 MF. A repeat biopsy at age 2 showed persistence of stable MF. The unusual finding of MF in these two patients suggested the possibility of altered calcium signaling as a shared mechanism driving congenital platelet disorders such as Stormorken syndrome and myeloproliferative neoplasms (MPNs) including MF. In support of this notion, we identified elevated STIM1 expression in MF vs normal megakaryocyte progenitors, as well as in platelets from patients with essential thrombocythemia (ET) vs healthy controls. Additionally, we found that STIM1 expression was significantly elevated in CD34+ hematopoietic stem/progenitor cells (HSPCs) from both ET and MF patients vs healthy controls. Notably, STIM1 expression was increased in both JAK2 and CALR-mutant MF patients. Collectively, these findings provide evidence of aberrant STIM1 expression in MPN patient cells. To determine the functional role of STIM1 in MPN disease development, colony assays and patient-derived xenograft (PDX) experiments were performed with MF patient CD34+ cells subjected to CRISPR ablation of STIM1. Strikingly discordant results were observed with JAK2 vs CALR-mutant patient samples. Abrogation of STIM1 in CALR-mutant CD34+ cells led to decreased colony formation, and NSGS mice engrafted in parallel with STIM1-targeted cells exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. These findings suggest an important role for STIM1 in CALR-mutant MPN disease phenotypes. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to increased colony formation and exacerbated disease phenotypes in vivo as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Similar results were obtained in experiments utilizing pharmacologic inhibitors of SOCE activity. Taken together, these findings indicate that the consequences of aberrant STIM1 activity may be context-dependent relating to specific MPN driver mutations. To expand these observations, we identified a separate cohort of 9 family members in Italy with Stormorken syndrome and confirmed STIM1 EF hand mutations. In ex vivo megakaryocytic differentiation assays, cells from affected individuals exhibited a defect in proplatelet formation. These observations were corroborated by initial analyses of a newly generated Stim1 R304W conditional knock-in mouse which recapitulated the characteristic thrombocytopenia found in patients with Stormorken syndrome. In summary, this study represents the first demonstration of MF development in patients with Stormorken syndrome, thereby uncovering a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying Stormorken syndrome and MPNs. Our findings suggest distinct mechanisms relating to the interaction between JAK2 vs CALR mutation and altered STIM1 activity. Further studies of these relationships may have important ramifications for potential therapeutic approaches targeting these pathways.