B-cell receptor (BCR) signaling plays an essential role in normal B-cell development and is a key driver of proliferation and survival of chronic lymphocytic leukemia (CLL) B cells. BCR signals through BTK to NF-ĸB. Targeted inhibition of BTK with ibrutinib, a first-in-class covalent BTK inhibitor, is highly effective in treating CLL and related B-cell malignancies. However, progressive disease on continuous therapy with ibrutinib has been associated with mutations in BTK and PLCG2. Most patients with acquired resistance and progressive CLL on ibrutinib have mutations in BTK affecting the cysteine at position 481 (C481) to which ibrutinib covalently binds. The common C481S mutation increases IC50 with ibrutinib from low nM to μM concentrations. The emergence of specific BTK mutations in ibrutinib resistant CLL further validate BTK as a valuable therapeutic target in CLL. Therefore, agents that can target C481 mutant BTK are of great interest, and non-covalent BTK inhibitors have entered clinical trials. Here we investigated an alternative approach using a chimeric targeting molecule: NRX0492. NRX0492 combines two molecules: a "hook" that binds BTK with a "harness" that recruits ubiquitin ligases that mediate proteasome-dependent degradation of BTK. In cell line models, NRX0492 induce both wild-type and C481S mutant BTK degradation efficiently and specifically.
Here we sought to define the on-target effects of NRX0492 in primary CLL cells in vitro and in vivo and to test NRX0492 against ibrutinib-resistant primary CLL cells. CLL cells were treated at densities of 5 × 106 cells/ml in 24-well plates with NRX0492, ibrutinib, the "hook" moiety devoid of the ubiquitin ligase binding moiety, or DMSO. BTK levels were quantified by Western blotting and flow cytometry. The mean ED50 and ED90 of NRX0492 for BTK degradation at 4 hours was 0.18nM and 0.5nM, respectively. At ED90 concentrations, mean cell viability of cultures treated with NRX0492 was 97.5%, and not different from viability of controls. Incubation at ED50 up to 24 hours also did not induce cell death. A lack of substantial cytotoxic activity with BTK degradation is in line with observations with BTK inhibitors under similar conditions. BTK degradation was achieved rapidly; in time-course experiments of CLL cells treated with 0.2nM NRX0492 the mean half-live of BTK protein was 2.7 hours (range 2.4h-3h), and at 2nM 99% of BTK was degraded within 4 hours.
Next, we tested the rate of recovery of BTK expression after drug washout. After 0.2nm NRX0492 treatment for 4 hours, mean BTK levels were 55% of pre-treatment. After washout and continued culture of cells in complete medium for 96 hours, BTK levels were 41% of pre-treatment. In additional experiments with washout of NRX0492 after 4 hours, we found the lowest levels of BTK 48hours post-treatment, indicating a sustained drug effect. There was no significant difference in the efficacy of BTK degradation between CLL samples subdivided by IGHV mutation status or FISH categories. At 0.2nM NRX0492 for 4 hours, mean remaining BTK was 54% in IGHV mutated and 55% in IGHV unmutated samples (P =0.78), and 60% vs 50% for del(13q) vs del(17p) (P =0.18). Next, we studied samples from two patients progressing on ibrutinib with classic C481S mutations at cancer cell fractions of 50% and 84%, respectively. Both patients remained on ibrutinib at the time of sample collection. After 4 hours of treatment with 0.2nM NRX0492, BTK levels were 50% and 15% of DMSO-treated controls, respectively, consistent with degradation of mutant but not ibrutinib bound BTK. Wild-type BTK bound by ibrutinib is not degraded because ibrutinib prevents NRX0492 binding to BTK.
In conclusion, we find NRX0492 is highly effective in degrading BTK in CLL cells. NRX0492 is effective at sub nM concentrations with ED50 ~0.2nM and ED90 ~0.5nM in primary CLL cells. Onset of BTK degradation was rapid and sustained for days after drug washout. NRX0492 was effective across CLL risk groups and equally effective in degrading wild-type BTK and C481S-mutant BTK. Studies testing NRX0492 in vivo in PDX models are ongoing and will inform dosing regimens for clinical studies.
Disclosures
Kelly: Nurix Therapeutics: Employment. Robbins:Nurix Therapeutics: Employment. Tan:Nurix Therapeutics: Employment. Ingallinera:Nurix Therapeutics: Employment. Sands:Nurix Therapeutics: Employment. Baskar:NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.
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