Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia
Journal of Hematology & Oncology volume 15, Article number: 166 (2022)
Inhibitors of B cell receptor (BCR) signaling such as the Bruton’s tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.
To the editor
Many antagonists of B cell receptor (BCR) signaling, especially inhibitors of Bruton’s tyrosine kinase (BTK), have demonstrated clinical efficacy in the treatment of chronic lymphocytic leukemia (CLL). While therapeutically effective, BTK inhibition alone is not sufficient to eliminate disease, with only a small subset of patients treated with the BTK inhibitor (BTKi) ibrutinib reaching complete responses and rarely achieving undetectable minimal residual disease (uMRD) . Previous studies showed prolonged BTK inhibition increases dependence on B Cell Lymphoma 2 (BCL2), an anti-apoptotic protein highly expressed in CLL . In a phase 1 clinical trial, patients with relapsed/refractory (R/R) CLL treated with the BCL2 inhibitor venetoclax had an overall response rate of 79%; however, only 5% of patients had uMRD in the bone marrow . The combination of ibrutinib and venetoclax is preclinically synergistic and clinically effective [2, 4, 5]. Nemtabrutinib (formerly ARQ 531, MK-1026) is a potent, reversible BTKi that has demonstrated greater inhibition of BTK’s downstream targets compared to ibrutinib, as well as improved efficacy in R/R disease and Richter’s Transformation . Therefore, we aimed to determine if combination nemtabrutinib and venetoclax would be similar or superior to combination ibrutinib and venetoclax in preclinical testing.
We observed significantly decreased viability of primary CLL cells treated with venetoclax compared to vehicle (p < 0.0001); though not statistically significant, the addition of ibrutinib or nemtabrutinib further increased cytotoxicity by 6 and 10%, respectively, compared to venetoclax alone (Fig. 1A). In primary CLL cells with C481S BTK, venetoclax significantly decreased viability compared to vehicle (p = 0.0074). While not statistically significant due to small sample size, nemtabrutinib decreased cell viability and ibrutinib was ineffective (Fig. 1B). Next, we investigated the effects of combining venetoclax and BTKis on BCR signaling (Fig. 1C). Both BTKis inhibited autophosphorylation of BTKY223 (ibrutinib vs. stimulated vehicle p = 0.0021, nemtabrutinib vs. stimulated vehicle p = 0.0024), which was unaffected by addition of venetoclax (Fig. 1D). As previously shown, ERK phosphorylation was inhibited to a greater degree with nemtabrutinib than ibrutinib (nemtabrutinib vs. stimulated vehicle p < 0.0001, nemtabrutinib vs. ibrutinib p = 0.0461), and, as expected, this inhibition was not affected by the addition of venetoclax (Fig. 1E) .
We additionally sought to determine the dependence on BCL2-family proteins in CLL cells from patients treated with nemtabrutinib in a clinical trial at our institution. BH3 profiling of samples from 3 nemtabrutinib treated patients showed cytochrome c release in response to interaction with BIM, BAD, and Y4ek peptides, indicating that these CLL cells are dependent on BCL2 and BCL-xL (Fig. 1F) . This suggests venetoclax sensitivity and supports the potential clinical benefit of combining nemtabrutinib and venetoclax.
Next, we tested the in vivo efficacy of venetoclax in combination with either BTKi using the Eμ-TCL1 adoptive transfer model (Fig. 2A) . Due to the large study size, splenocytes from multiple Eμ-TCL1 donors were required. Peripheral blood disease was monitored weekly (Fig. 2B). As a result of using different donor clones, we observed high variability between recipient groups, as previously reported by Kater et al.  including groups that did not have BCR-dependent disease as evidenced by lack of response to ibrutinib. These groups were excluded from analysis, but data are presented in Additional file 1: Fig. S1. We assessed the survival of 48 mice engrafted with splenocytes from a single donor with BCR-dependent disease (Fig. 2C, primary statistical analysis in legend). Ibrutinib-treated mice reached a median survival of 56.5 days while mice treated with ibrutinib and venetoclax had a median survival of 66 days. Nemtabrutinib-treated mice reached a median survival of 81.5 days while mice treated with nemtabrutinib and venetoclax reached a median survival of 92 days. Interestingly, mice treated with venetoclax alone did not prolong survival compared to vehicle-treated mice. Mice treated with nemtabrutinib alone or in combination had significantly prolonged survival compared to vehicle-treated mice (p = 0.0238, p < 0.0001, respectively). Notably, the combination of nemtabrutinib and venetoclax significantly prolonged survival compared to the combination of ibrutinib and venetoclax (p = 0.0415).
The data presented here further demonstrate the benefit of combining BTKis with venetoclax in CLL and suggest that nemtabrutinib is a rational BTKi to combine with venetoclax. Our in vivo data especially support the combination of nemtabrutinib plus venetoclax as an alternative to ibrutinib plus venetoclax. Further investigation, both preclinically and clinically, is warranted.
B cell receptor
Bruton’s tyrosine kinase
Bruton’s tyrosine kinase inhibitor
Chronic lymphocytic leukemia
Undetectable minimal residual disease
B cell lymphoma 2
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NK Tert cells (Riken, RCB2350) were provided by Dr. Jan Burger (MD Anderson).
5 R35 CA197734 (JCB), Some experiments were performed using nemtabrutinib supplied by ArQule. Both JAW and KAR are Scholars in Clinical Research of the Leukemia & Lymphoma Society.
Ethics approval and consent to participate
Blood samples were obtained from CLL patients at The Ohio State University, who consented to an Institutional Review Board–approved tissue procurement protocol or who were enrolled in Institutional Review Board–approved clinical trial of nemtabrutinib in CLL. All patients gave written informed consent in accordance with the Declaration of Helsinki. All animal protocols were reviewed and approved by The Ohio State University Institutional Animal Care and Use Committee.
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Availability of data and materials
The data that support the findings of this study are available on request from the corresponding author.
JCB reports consulting, stock ownership with Vincerx, consulting with AstraZeneca, Syndax, Janssen, Pharmacyclics, Acerta, and Trillium that are outside of the submitted work. JAW has consulted for Abbvie, AstraZeneca, Beigene, Genentech, Janssen, Loxo, Newave, Pharmacyclics, and Schrodinger and receives research funding from Abbvie, Janssen, Morphosys, and Schrodinger. KAR receives research funding from Genentech, AbbVie, Novartis, and Janssen, consults for AstraZeneca, Beigene, Genentech, AbbVie, Pharmacyclics, and Innate Pharma and received travel funding from AstraZeneca. DS has received honoraria from Newave.
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Kaplan–Meier estimated survival of all recipient mice (n=93) treated with vehicle, venetoclax (100 mg/kg), nemtabrutinib (75 mg/kg), ibrutinib (25 mg/kg), venetoclax (100 mg/kg) and nemtabrutinib (75 mg/kg), or venetoclax (100 mg/kg) and ibrutinib (25 mg/kg).
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Muhowski, E.M., Ravikrishnan, J., Gordon, B. et al. Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia. J Hematol Oncol 15, 166 (2022). https://doi.org/10.1186/s13045-022-01386-1