Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

NRX-0492 degrades wild-type and C481 mutant BTK and demonstrates in vivo activity in CLL patient-derived xenografts.

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting.

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

Self-reactivity on a spectrum: A sliding scale of peripheral B cell tolerance.

Efficient mechanisms of central tolerance, including receptor editing and deletion, prevent highly self-reactive B cell receptors (BCRs) from populating the periphery. Despite this, modest self-reactivity persists in (and may even be actively selected into) the mature B cell repertoire. In this review, we discuss new insights into mechanisms of peripheral B cell tolerance that restrain mature B cells from mounting inappropriate responses to endogenous antigens, and place recent work into historical context. In particular, we discuss new findings that have arisen from application of a novel in vivo reporter of BCR signaling, Nur77-eGFP, expression of which scales with the degree of self-reactivity in both monoclonal and polyclonal B cell repertoires. We discuss new and historical evidence that self-reactivity is not just tolerated, but actively selected into the peripheral repertoire. We review recent progress in understanding how dual expression of the IgM and IgD BCR isotypes on mature naive follicular B cells tunes responsiveness to endogenous antigen recognition, and discuss how this may be integrated with other features of clonal anergy. Finally, we discuss how expression of Nur77 itself couples chronic antigen stimulation with B cell tolerance.

Optimal Development of Mature B Cells Requires Recognition of Endogenous Antigens.

It has long been appreciated that highly autoreactive BCRs are actively removed from the developing B cell repertoire by Ag-dependent receptor editing and deletion. However, there is persistent debate about whether mild autoreactivity is simply tolerated or positively selected into the mature B cell repertoire as well as at what stage, to what extent, under what conditions, and into which compartments this occurs. In this study, we describe two minor, trackable populations of B cells in B1-8i Ig transgenic mice that express the VH186.2 H chain and recognize a common foreign Ag (the hapten 4-hydroxy-3-nitrophenylacetyl) but differ in L chain expression. We use the Nur77-eGFP reporter of BCR signaling to define their reactivity toward endogenous Ags. The less autoreactive of these two populations is strongly counterselected during the development of mature B1a, follicular, and marginal zone B cells. By genetically manipulating the strength of BCR signal transduction via the titration of surface CD45 expression, we demonstrate that this B cell population is not negatively selected but instead displays characteristics of impaired positive selection. We demonstrate that mild self-reactivity improves the developmental fitness of B cell clones in the context of a diverse population of B cells, and positive selection by endogenous Ags shapes the mature B cell repertoire.

Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery.

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.

Control of autoreactive B cells by IgM and IgD B cell receptors: maintaining a fine balance.

A substantial fraction of mature naïve B cells recognize endogenous antigens, and this autoreactivity must be controlled to prevent autoantibody secretion. Selective downregulation of the IgM BCR on autoreactive B cells has long been appreciated, and recent findings illustrate how this might impose tolerance. The BCR isotype maintained on autoreactive B cells, IgD, is less sensitive to endogenous antigens than IgM. This reduced sensitivity may be conferred by structural properties of IgD and/or differential association with activating and inhibitory co-receptors. Once activated, autoreactive B cells are normally excluded from rapid plasma cell responses, but they can enter the germinal center and lose their autoreactivity through a mutation-selection process termed clonal redemption.

IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate.

Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn-/- B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells.

Nur77 Is Upregulated in B-1a Cells by Chronic Self-Antigen Stimulation and Limits Generation of Natural IgM Plasma Cells.

B-1a cells are a unique population of innate-like B cells with a highly restricted and self-reactive BCR repertoire. Preimmune "natural" IgM produced by B-1a-derived plasma cells is essential for homeostatic clearance of cellular debris and forms a primary layer of protection against infection. In this study, we take advantage of a fluorescent reporter of BCR signaling to show that expression of the orphan nuclear hormone receptor Nur77 is upregulated under steady-state conditions in self-reactive B-1a cells in response to chronic Ag stimulation. Nur77-deficient mice exhibit elevated natural serum IgM (but not IgG) and marked expansion of IgM plasma cells of B-1a origin. Moreover, we show that Nur77 restrains the turnover of B-1a cells and the accumulation of immature IgM plasma cells. Thus, we identify a new critical negative-regulatory pathway that serves to maintain B-1a cells in a quiescent state in the face of chronic endogenous Ag stimulation.

An extracatalytic function of CD45 in B cells is mediated by CD22.

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal "ignorance."