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Letters to the Editor
essential for the activation of ERK, a downstream mole- cule in BCR signaling.12 Compared to DMSO-treated cells, BTK inhibition substantially decreased phospho- SHP2(Y542) in WT cells, whereas only vecabrutinib specifically inhibited SHP-2 phosphorylation in both mutant cell lines (Figure 2B).
Other proteins that showed a change after drug treat- ment were ribosomal protein pS6Ka1(T573); FAK pro- tein pPTK2(Y397); pHSB1(S82); transcription factor pERK1/2(T202/Y204); transcription factors pSTAT3(S727); pSTAT3(Y705); transcription factors ETS1 and GATA1; cell cycle proteins PLK1, and phospho- p27(T198); B-cell protein PD1; and cleaved caspase 7 (Figure 2B). In general, among WT and mutant variants, changes in these proteins were more pronounced in WT cells than in mutants.
Of the two drugs, vecabrutinib produced deeper or similar responses compared to those to ibrutinib in WT cells for all proteins (Figure 2B). Consistent with a prior report,12 treatment with ibrutinib and vecabrutinib caused a 2-log decrease in phospho-ERK in BTKWT cells after treatment. Furthermore, vecabrutinib treatment resulted in a 1-log decline in cells harboring mutant BTK, whereas ibrutinib had no effect (Figure 2B). A hallmark protein that is affected by ibrutinib is p70S6K or S6 kinase. The target protein of this kinase is S6 ribosomal protein, which initiates protein synthesis upon phospho- rylation. Impressively, compared to ibrutinib, vecabruti- nib profoundly decreased phospho-S6K in all three cell types. Among all proteins, phosphorylated ERK and S6K were consistently and substantially decreased in all three cell types. These two proteins may serve as biomarkers for the effect of vecabrutinib (Figure 2B).
Ibrutinib produced a larger decrease in PD1 levels than vecabrutinib did. Parallel to PD1 protein levels, ser- ine(727) and tyrosine(705) phosphorylation of STAT3 has been shown to be reduced in CLL cells after ibrutinib treatment.13 STAT3 and STAT5 activation through phos- phorylation has been associated with inflammation and carcinogenesis.14 STAT3 is constitutively active in CLL cells, but this activation is mitigated by ibrutinib treat- ment.13,15 Vecabrutinib treatment decreased Y705 and S727 phosphorylation on STAT3 in WT and mutant cell lines. Interestingly, in mutant cells, a decline in S727 phosphorylation occurred only with vecabrutinib (Figure 2B). Although extensive cell death was not seen with either of these drugs, RPPA data revealed that vecabruti- nib treatment increased cleaved caspase 7 in all trans- duced cell lines.
Finally, we tested vecabrutinib in primary CLL cells from five patients with either WT or mutant BTK (Figure 3A). Cell death ranged from 0 up to 21% after 24 hours of vecabrutinib, being higher in BTKWT than in BTKmutant samples (Figure 3B). BTK with C481S and C481R alter- ations had the lowest apoptosis. In concert, immunoblot results showed that vecabrutinib inhibited BCR pathway (phosphorylation of BTK, ERK, and S6) signaling in BTKWT and in BTKT474F (gate-keeper mutation) (Figure 3C, D). Consistent with the RPPA data, cells expressing the BTKC481S and BTKC481R variants had minor changes (patient 3). In patients 4 and 5, phospho-ERK either remained the same or increased; these two patients had C481S (catalytic domain) and T474I (gate-keeper) double mutations (Figure 3E). Super-resistance to irreversible BTK inhibitors or variable sensitivity to reversible non- covalent BTK inhibitors has been reported for cells har- boring T474 variants along with the cysteine 481 substi- tution.16
In summary, our study provides a nonclinical character-
ization of vecabrutinib. This reversible BTK inhibitor is selective in kinome profiling and inhibits phosphoryla- tion of BTK and PLCg2 in preclinical whole-cell assays. Ibrutinib-sensitive and -resistant model systems demon- strated inhibition of the BCR signal transduction cascade in both BTKWT and BTKmutant B-cell lines.6 Data were con- sistent in CLL cells, but the impact on BTKC481S and BTKC481R was modest in primary cells. The results of a clinical trial of vecabrutinib in B-cell malignancies will be reported soon.7
Burcu Aslan,1 Stefan Edward Hubner,2 Judith A. Fox,3 Pietro Taverna,3 William G. Wierda,2 Steven M. Kornblau2 and Varsha Gandhi1,2
1Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; 2Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX and 3Sunesis Pharmaceuticals, Inc., South San Francisco, CA, USA
Correspondence:
VARSHA GANDHI - vgandhi@mdanderson.org. doi:10.3324/haematol.2021.279158
Received: May 6, 2021.
Accepted: September 1, 2021.
Pre-published: September 9, 2021.
Disclosures: related to this work, VG has sponsored research agreements with Sunesis Pharmaceuticals and Loxo Oncology. Outside of this work, VG has received research support from AbbVie, Acerta, AstraZeneca, ClearCreek Bio, Dava Oncology, Gilead,
and Pharmacyclics. JAF and PT were employees of Sunesis Pharmaceuticals. The other authors have no conflicts of interest.
Contributions: BA designed and performed the experiments, analyzed the results, and wrote the first draft of the manuscript;
SEH performed the bioinformatics analyses of the RPPA data;
JF and PT contributed other nonclinical and clinical investigations of vecabrutinib, provided data that are included in Figure 1, and reviewed the manuscript;WGW identified ibrutinib-resistant patients and provided samples; SMK supervised SEH and analyzed RPPA data; VG conceptualized and supervised the research, obtained funding, analyzed the data, and wrote and revised the manuscript.
Acknowledgments: the authors thank Drs. Joseph R. Marszalek, Christopher P. Vellano, Michael Peoples, and Mikhila Mahendra for the creation of MEC-1 cell lines that overexpress WT and mutant BTK. The authors thank LaKesla Iles for technical assistance and Tamara Locke, Scientific Editor, Research Medical Library, for editing this article.
Funding: this work was supported in part by The University of Texas MD Anderson Cancer Center Moon Shot Program, a Sponsored Research Agreement from Sunesis Pharmaceuticals, and by the NIH/NCI through award number P30CA016672.
References
1. Burger JA. Treatment of chronic lymphocytic leukemia. N Engl J Med. 2020;383(5):460-473.
2. Timofeeva N, Gandhi V. Ibrutinib combinations in CLL therapy: scientif- ic rationale and clinical results. Blood Cancer J. 2021;11(4):79.
3. Honigberg LA, Smith AM, Sirisawad M, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A. 2010;107(29):13075-13080.
4. Woyach JA, Ruppert AS, Guinn D, et al. BTKC481S-mediated resistance to ibrutinib in chronic lymphocytic leukemia. J Clin Oncol. 2017; 35(13):1437.
5.Burger JA, Landau DA, Taylor-Weiner A, et al. Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition. Nat Commun. 2016;7(1):1-13.
6. Aslan B, Kismali G, Chen LS, et al. Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant
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