1050 - NX-2127 and NX-5948, two clinical stage cereblon-recruiting BTK degraders, facilitate T-cell functionality in CLL

Introduction: Inhibitors targeting Bruton’s tyrosine kinase (BTK) altered the treatment paradigm in chronic lymphocytic leukemia (CLL). In addition to direct anti-neoplastic effects, BTK inhibitors ibrutinib and acalabrutinib were found to favorably modulate T-cell function in pre-clinical and clinical studies of CLL. Specifically, treatment with ibrutinib down modulated expression of T-cell exhaustion markers, shifted T-cell polarization towards a pro-inflammatory TH1 phenotype, and improved overall T-cell number and repertoire diversity.
However, resistance to BTK inhibitors eventually occurs due to the development of mutations in BTK. As a result, Targeted Protein Degradation has emerged as a strategy to circumvent acquired resistance to BTK inhibition. The compounds NX-2127 and NX-5948 were designed to induce BTK degradation by recruiting the E3 ubiquitin ligase, cereblon (CRBN), which was originally recognized for its modulation of the immune system. However, only NX-2127, and not NX-5948, was designed to maintain CRBN immunomodulatory function. The effects of BTK degraders on T-cell functionality are not known. Furthermore, it is unclear to what extent CRBN modulation contributes to pre-clinical (and ultimately clinical) activity of BTK degraders. Here, we began to address these questions using the bifunctional NX-2127 and immunomodulatory neutral NX-5948 compounds.

Methods: Peripheral blood mononuclear cells were isolated from CLL patients. CLL cells were treated with drugs for 24 hours and BTK, Aiolos, and Ikaros levels were assessed by immunoblotting. CD3+ T-cells were purified using Dynabeads. For apoptosis and activation assays, CD3+ T-cells were stimulated with αCD3/CD28 and concurrently treated with drugs. T-cells were then analyzed through flow cytometry for apoptosis (Annexin V) and activation (CD69, CD25, HLA-DR, PD-1, and CD38) after 24, 48, and 72 hours. For polarization assays, naïve CD4+ T-cells were stimulated with αCD3/CD28 and treated with drugs for 7 or 14 days under TH1/Treg/TH2-differentiation conditions. Flow cytometry was used to detect IFN-γ and IL-2 (TH1); CD25+FoxP3+ T-cells (Tregs); and IL-4 (TH2). For immunological synapse assays, αCD3/CD28-stimulated CD3+ T-cells and CLL cells were treated with drugs for 24 hours. T-cells and CLL cells were then combined at a ratio of 1:1, stained with CellTracker, F-actin, and granzyme B, and imaged using immunofluorescent confocal microscopy. NX-2127 and NX-5948 were provided by Nurix Therapeutics, Inc. BTK inhibitor ibrutinib and CRBN modulator lenalidomide were used as controls at 1 µM concentrations.

Results: Treatment of CLL cells with 0.1-1 µM NX-2127 and NX-5948 resulted in degradation of BTK. Furthermore, NX-2127, but not NX-5948, induced dose-dependent degradation of Aiolos and Ikaros (known CRBN substrates), thereby confirming that only NX-2127 possessed CRBN immunomodulatory activity.
Exposure of CD3+ T-cells to 0.1-1 µM NX-2127 or NX-5948 was not accompanied by either apoptosis or reduced cell proliferation for up to 72 hours. Neither early (CD69) nor late (CD25, HLA-DR, and PD-1) activation of CD4+ and CD8+ T-cells was disrupted in the presence of either compound. Interestingly, we observed an increase in the activation marker CD38 within CD4+ and CD8+ T-cells treated with NX-2127, but not with NX-5948. A similar effect was observed with both ibrutinib and lenalidomide.
In vitro polarization assays indicated that treatment with 1 µM NX-2127, but not NX-5948, resulted in a dramatic and significant upregulation of both IFN-γ and IL-2 under TH1-polarizing conditions, to a degree which significantly exceeded the effect of either ibrutinib or lenalidomide. At the same time, BTK degraders did not alter T-cell differentiation under TH2-polarizing conditions as indicated by unchanged IL-4 expression. Additionally, NX-2127 and lenalidomide significantly reduced differentiation of Tregs when used in higher concentrations (1 µM). This effect was not observed with either immunomodulatory-neutral NX-5948 or ibrutinib.
Immunofluorescent confocal microscopy revealed that NX-2127 enhanced immunological synapse formation to levels comparable to those of lenalidomide. Synapses were not induced by ibrutinib.

Conclusions: CRBN-recruiting BTK degraders NX-2127 andNX-5948 induce degradation of BTK in primary CLL cells and do not interfere with T-cell activation and survival in vitro. More importantly, CRBN-immunomodulatory activity was required to upregulate CD38 (an IFN response gene), promote T-cell differentiation towards a TH1 phenotype, downregulate Treg differentiation, and facilitate immunological synapse formation, suggesting NX-2127 has the potential to reverse the immunosuppressive environment of CLL. Overall, our findings provide a strong rationale for continued investigation of these BTK degraders in CLL and lymphoid malignancies.