BTK kinase activity is dispensable for the survival of diffuse large B-cell lymphoma

Abstract

Inhibitors targeting Bruton's tyrosine kinase (BTK) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treatment as a result of mutations in BTK. Here, by a combination of structural modeling, in vitro assays, and deep phospho-tyrosine proteomics, we demonstrated that four clinically observed BTK mutations—C481F, C481Y, C481R, and L528W—inactivated BTK kinase activity both in vitro and in diffused large B-cell lymphoma (DLBCL) cells. Paradoxically, we found that DLBCL cells harboring kinase-inactive BTK exhibited intact B cell receptor (BCR) signaling, unperturbed transcription, and optimal cellular growth. Moreover, we determined that DLBCL cells with kinase-inactive BTK remained addicted to BCR signaling and were thus sensitive to targeted BTK degradation by the proteolysis-targeting chimera. By performing parallel genome-wide CRISPR-Cas9 screening in DLBCL cells with WT or kinase-inactive BTK, we discovered that DLBCL cells with kinase-inactive BTK displayed increased dependence on Toll-like receptor 9 (TLR9) for their growth and/or survival. Our study demonstrates that the kinase activity of BTK is not essential for oncogenic BCR signaling and suggests that BTK’s noncatalytic function is sufficient to sustain the survival of DLBCL. Inhibitors targeting Bruton's tyrosine kinase (BTK) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treatment as a result of mutations in BTK. Here, by a combination of structural modeling, in vitro assays, and deep phospho-tyrosine proteomics, we demonstrated that four clinically observed BTK mutations—C481F, C481Y, C481R, and L528W—inactivated BTK kinase activity both in vitro and in diffused large B-cell lymphoma (DLBCL) cells. Paradoxically, we found that DLBCL cells harboring kinase-inactive BTK exhibited intact B cell receptor (BCR) signaling, unperturbed transcription, and optimal cellular growth. Moreover, we determined that DLBCL cells with kinase-inactive BTK remained addicted to BCR signaling and were thus sensitive to targeted BTK degradation by the proteolysis-targeting chimera. By performing parallel genome-wide CRISPR-Cas9 screening in DLBCL cells with WT or kinase-inactive BTK, we discovered that DLBCL cells with kinase-inactive BTK displayed increased dependence on Toll-like receptor 9 (TLR9) for their growth and/or survival. Our study demonstrates that the kinase activity of BTK is not essential for oncogenic BCR signaling and suggests that BTK’s noncatalytic function is sufficient to sustain the survival of DLBCL. During B cell development, antigen-mediated cross-linking of the B cell receptor (BCR) activates the nonreceptor tyrosine kinases Bruton's tyrosine kinase (BTK) (1Burger J.A. Wiestner A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances.Nat. Rev. Cancer. 2018; 18: 148-167Google Scholar). BTK then promotes the activation of phospholipase Cγ2 (PLCγ2), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to elicit an increase of intracellular Ca2+ (2Takata M. Kurosaki T. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2.J. Exp. Med. 1996; 184: 31-40Google Scholar, 3Fluckiger A.-C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. et al.Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.EMBO J. 1998; 17: 1973-1985Google Scholar). The resulting Ca2+ flux activates diverse transcriptional programs to promote B cell proliferation and differentiation (4Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration.Nature. 1997; 386: 855-858Google Scholar, 5Liu Q.-H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. et al.Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli.J. Immunol. 2005; 174: 68Google Scholar). Oncogenic mutations, microbial antigens, or autoantigens can co-opt BCR signaling to support the growth and/or survival of malignant B cells, resulting in B-cell leukemias and lymphomas (6Lenz G. Davis R.E. Ngo V.N. Lam L. George T.C. Wright G.W. et al.Oncogenic CARD11 mutations in human diffuse large B cell lymphoma.Science. 2008; 319: 1676Google Scholar, 7Marcucci F. Mele A. Hepatitis viruses and non-Hodgkin lymphoma: epidemiology, mechanisms of tumorigenesis, and therapeutic opportunities.Blood. 2011; 117: 1792-1798Google Scholar, 8Young R.M. Wu T. Schmitz R. Dawood M. Xiao W. Phelan J.D. et al.Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 13447Google Scholar, 9Davis R.E. Ngo V.N. Lenz G. Tolar P. Young R.M. Romesser P.B. et al.Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.Nature. 2010; 463: 88-92Google Scholar, 10Morin R.D. Mendez-Lago M. Mungall A.J. Goya R. Mungall K.L. Corbett R.D. et al.Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.Nature. 2011; 476: 298-303Google Scholar, 11Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.-H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Google Scholar). Inhibitors that target BTK (BTKi) have emerged as breakthrough therapies for treating a variety of B-cell malignancies, such as chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, and Waldenström’s macroglobulinemia (12Burger J.A. Tedeschi A. Barr P.M. Robak T. Owen C. Ghia P. et al.Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia.N. Engl. J. Med. 2015; 373: 2425-2437Google Scholar, 13Dreyling M. Jurczak W. Jerkeman M. Silva R.S. Rusconi C. Trneny M. et al.Ibrutinib versus temsirolimus in patients with relapsed or refractory mantle-cell lymphoma: an international, randomised, open-label, phase 3 study.Lancet. 2016; 387: 770-778Google Scholar, 14Dimopoulos M.A. Trotman J. Tedeschi A. Matous J.V. Macdonald D. Tam C. et al.Ibrutinib for patients with rituximab-refractory Waldenström's macroglobulinaemia (iNNOVATE): an open-label substudy of an international, multicentre, phase 3 trial.Lancet Oncol. 2017; 18: 241-250Google Scholar). However, durable response to BTKi is hampered by acquired resistance after prolonged treatment (15Furman R.R. Cheng S. Lu P. Setty M. Perez A.R. Guo A. et al.Ibrutinib resistance in chronic lymphocytic leukemia.N. Engl. J. Med. 2014; 370: 2352-2354Google Scholar, 16Woyach J.A. Furman R.R. Liu T.-M. Ozer H.G. Zapatka M. Ruppert A.S. et al.Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.N. Engl. J. Med. 2014; 370: 2286-2294Google Scholar). First- and second-generation BTKi, such as ibrutinib, acalabrutinib, and zanubrutinib, inhibit the kinase activity of BTK by binding to its ATP-binding pocket and then covalently modifying a cysteine residue at position 481 (C481) of BTK (17Pan Z. Scheerens H. Li S.-J. Schultz B.E. Sprengeler P.A. Burrill L.C. et al.Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase.ChemMedChem. 2007; 2: 58-61Google Scholar, 18Guo Y. Liu Y. Hu N. Yu D. Zhou C. Shi G. et al.Discovery of zanubrutinib (BGB-3111), a novel, potent, and selective covalent inhibitor of Bruton’s tyrosine kinase.J. Med. Chem. 2019; 62: 7923-7940Google Scholar). Correspondingly, the most common mechanism of BTKi resistance occurs through mutations changing this reactive cysteine into serine (C481S) and less frequently into phenylalanine, tyrosine, or arginine (C481F, C481Y, and C481R) (19Maddocks K.J. Ruppert A.S. Lozanski G. Heerema N.A. Zhao W. Abruzzo L. et al.Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia.JAMA Oncol. 2015; 1: 80-87Google Scholar, 20Hamasy A. Wang Q. Blomberg K.E.M. Mohammad D.K. Yu L. Vihinen M. et al.Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.Leukemia. 2017; 31: 177-185Google Scholar). In addition, several non-C481 mutations have been observed in patients resistant to irreversible BTKi (19Maddocks K.J. Ruppert A.S. Lozanski G. Heerema N.A. Zhao W. Abruzzo L. et al.Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia.JAMA Oncol. 2015; 1: 80-87Google Scholar, 21Sharma S. Galanina N. Guo A. Lee J. Kadri S. Slambrouck C.V. et al.Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL.Oncotarget. 2016; 7: 68833-68841Google Scholar) and, more recently, in relapsed chronic lymphocytic leukemia patients treated with the noncovalent BTK inhibitor pirtobrutinib (22Wang E. Mi X. Thompson M.C. Montoya S. Notti R.Q. Afaghani J. et al.Mechanisms of resistance to noncovalent Bruton’s tyrosine kinase inhibitors.N. Engl. J. Med. 2022; 386: 735-743Google Scholar). Whereas previous studies have clarified their mechanism of resistance to BTKi, whether these BTK mutations affect the biochemical activity and/or function of BTK in malignant B cells are not well understood. In an attempt to explore the biochemical impact of clinically observed BTK mutations, we first docked ATP into the active site of BTK kinase domain. In the resulting structural model, the adenine ring of ATP formed hydrogen bonds with the side chain of T474 and the backbone of M477 in BTK. In addition, the phosphate group of ATP was locked in the active site of BTK by forming two hydrogen bonds with the side chain of K430. Residues C481 and L528 were located below the binding pocket and showed no direct interaction with ATP (Fig. 1, A and B). Next, we evaluated whether clinically observed BTK C481 and non-C481 mutations might affect ATP binding. Whereas C481S was not expected to affect the mode of ATP binding, substitutions of C481 by bulky side chains of phenylalanine (C481F), tyrosine (C481Y), or arginine (C481R) were all predicted to generate steric clashes to the sugar ring or the phosphate group of ATP (Fig. 1B). Similarly, leucine at position 528 mutated to tryptophan (L528W) caused a steric clash to the adenine ring of ATP (Fig. 1B). To verify these predictions, we purified recombinant BTK kinase domains and performed the thermal shift assay. We observed that ATP stabilized the kinase domains of BTK WT, C481S, and C481R but did not stabilize the kinase domains of BTK C481F, C481Y, and L528W (Fig. 1, C and D). Compared to phenylalanine and tyrosine, arginine may be more flexible at the active site due to the lack of an aromatic ring in its side chain. Thus, ATP may gain access to the active site and stabilize the kinase domain of BTK C481R. These observations prompted us to examine the kinase activity of BTK mutants using an in vitro kinase assay. By incubating the BTK kinase domain with either a peptide substrate derived from PLCγ2 or a protein substrate (the SH3 domain of BTK containing an auto-phosphorylation site), we observed a near complete lack of kinase activity of BTK C481F, C481Y, and L528W in vitro (Fig. 1E). Notably, although C481R did not show a defect in ATP binding in the thermal shift its kinase activity was with that of the WT BTK (Fig. 1E). these that four clinically observed BTK mutations, C481F, C481Y, C481R, and BTK kinase activity in To examine whether and L528W BTK kinase activity in malignant B cells, we and two diffused large B-cell lymphoma (DLBCL) cell of the B-cell R.E. Ngo V.N. Lenz G. Tolar P. Young R.M. Romesser P.B. et al.Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.Nature. 2010; 463: 88-92Google as of their to ibrutinib (Fig. We a to BTK WT, or L528W in and cells at near their BTK (Fig. and then their to Whereas of WT BTK did not the of or to ibrutinib, of BTK and L528W resistance to ibrutinib in both cell (Fig. These cell in which BTK was by ibrutinib, us to examine the biochemical and of BTK BTK is a tyrosine we deep phospho-tyrosine to of in DLBCL cells following BTK by were by an Y. Li L. M. Liu X. T. Cheng et tyrosine by a Chem. 2016; Scholar) by by (Fig. We from and cells of these BTK and showed in both cell following ibrutinib treatment (Fig. Moreover, we to BCR signaling, resulting in the of of these BTK BTK and showed in both cell following ibrutinib treatment (Fig. We deep to the of cells BTK with ibrutinib to their with of cells. the that showed to cells, four were common in BTK BTK and (Fig. BTK is a auto-phosphorylation site of BTK H. Wahl M.I. Tam C. et of function by a site the SH3 1996; Scholar). BTK be in and BTK cells but was in cells BTK or L528W and of BCR by increased BTK in and BTK cells but not in BTK or cells and observations were in cells (Fig. B and To from deep proteomics, we in to a BTK L528W which was less sensitive to ibrutinib the (Fig. A and B). In the proliferation of BTK L528W was from that of cells (Fig. with deep BTK was in BTK L528W cells (Fig. BTK is to PLCγ2 and R. M. J. A. et in phospholipase Cγ2 essential for the function in B-cell Chem. Scholar). However, deep showed that PLCγ2 was not by the of BTK kinase activity Moreover, the lack of PLCγ2 and and BTK kinase activity in both and cells and B and observations were in L528W cells (Fig. the of PLCγ2 are by a kinase in DLBCL cells. We whether BTK kinase activity was for oncogenic BCR signaling by Ca2+ flux following BCR By a of B-cell lymphoma cell we found that of displayed Ca2+ flux following (Fig. A and B). the cell cells displayed Ca2+ flux and by with ibrutinib the of the Ca2+ flux in cells by (Fig. A and B). Ca2+ flux was observed in BTK L528W cells to cells, and the Ca2+ flux in BTK L528W cells no be by ibrutinib (Fig. A and B). These that cells BTK kinase activity oncogenic BCR from in we BCR signaling in the lymphoma cell in which induced Ca2+ flux (Fig. A and B). not on BCR signaling for we thus a BTK of (Fig. The resulting BTK cells showed Ca2+ flux following (Fig. We were that BTK did not Ca2+ were observed in previous studies Kurosaki T. J.A. of signaling by the tyrosine kinases and Chem. Scholar, A. L. C. F. et BTK mutants ibrutinib resistance through activation of the kinase 2022; that mechanisms to Ca2+ flux in BTK cells. We then various BTK mutants and found all of the Ca2+ flux in BTK cells to the as WT BTK (Fig. C and D). These that the kinase activity of BTK is for the of flux to oncogenic BCR BCR signaling activates transcription factors to sustain the growth and survival of malignant B cells (4Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration.Nature. 1997; 386: 855-858Google Scholar, 5Liu Q.-H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. et al.Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli.J. Immunol. 2005; 174: 68Google Scholar). We to examine whether the of BTK kinase activity might affect of BCR signaling By cells treated with or ibrutinib, we a of genes and genes in cells and that ibrutinib treatment genes in signaling and of cell and with previous studies (Fig. A. E. et clinically active BTK inhibitor B-cell and and in chronic lymphocytic Scholar, S. et tyrosine kinase inhibitor chronic lymphocytic leukemia cell survival and in vitro and in Scholar). by ibrutinib did not with (Fig. By these genes as a we found that were at in cells BTK C481S or kinase-inactive BTK and These that of BTK kinase activity not affect of BCR We a cell growth to examine whether DLBCL cells in BTK kinase activity remained on BCR signaling for growth and survival (Fig. To we of and BTK L528W cells and their on the (Fig. We then an BTK and found that cells from both and BTK L528W cells were at (Fig. The of cell due to of BTK be by either BTK and or kinase-inactive BTK and (Fig. We cells with that target several genes of the BCR signaling of the BCR B cell protein kinase C of the and a B We observed that both cells and BTK L528W cells were on these genes for survival (Fig. these that DLBCL harboring BTK mutations not BCR signaling for growth and survival. DLBCL cells with kinase-inactive BTK remained addicted to BTK, we that targeted BTK degradation by the proteolysis-targeting might be an to BTKi resistance Y. Zhao X. N. H. Wu Y. Y. et BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell 2018; Scholar). Thus, we a BTK to the to BTK (Fig. treatment induced the degradation of both WT BTK and BTK L528W in cells, which be by the inhibitor and the (Fig. A and B). and cells harboring kinase-inactive BTK mutations were sensitive to the of (Fig. we the of In cells treated with for BTK and were the two (Fig. and By previous genome-wide screening in J.D. Young R.M. S. Wright G.W. M. et oncogenic signalling in lymphoma.Nature. 2018; we found that was not essential for the survival of (Fig. Thus, we that the activity of is a result of BTK the BTK promotes the degradation of kinase-inactive BTK mutants to resistance to irreversible To explore whether BTK mutations might result in of DLBCL we performed a parallel genome-wide CRISPR-Cas9 screening in and L528W cells. of the we cells for 3 and then performed to the of in cells (Fig. of genes in L528W cells to cells (Fig. and these that Toll-like receptor 9 and 3 formed a interaction (Fig. is a of the Toll-like receptor which to the and microbial to signaling E. A. A. et after from the to in the Immunol. Scholar). and are for and to to 2008; Scholar, E. B. The interaction the protein and and 9 is for 2007; Scholar, T. S. T. Y. N. et protein with receptor is for Exp. Med. 2007; Scholar). In addition, targeting the nonreceptor tyrosine kinase were in L528W cells to cells (Fig. We the cell growth (Fig. to from the CRISPR-Cas9 with targeting BTK L528W cells were with to cells (Fig. To in the cell growth we the of cells BTK and L528W and their BTK with with the cells of BTK were with their (Fig. In with targeting or BTK kinase-inactive cells and were with BTK cells and observations were in cells and we that DLBCL cells with kinase-inactive BTK are more on signaling for their growth and/or survival. Our study a of clinically observed BTK mutations that not resistance to irreversible BTKi but the kinase activity of BTK. These mutations affect two in BTK, and with containing bulky side resulting in steric to ATP binding. By the biochemical and of these BTK mutants in DLBCL cell we the that kinase-inactive BTK mutants were as at BCR signaling as their WT observations of kinase-inactive BTK mutants have been in et M. Wahl M.I. Kurosaki T. et of Bruton's tyrosine kinase is sufficient to signaling in B Immunol. 2: Scholar) observed that a kinase-inactive BTK calcium flux and activation in cells. In addition, BTK and mutants have been to lack auto-phosphorylation activity in and cells A. Wang Q. Blomberg K.E.M. Mohammad D.K. Yu L. Vihinen M. et al.Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.Leukemia. 2017; 31: 177-185Google Scholar, A. L. C. F. et BTK mutants ibrutinib resistance through activation of the kinase 2022; Scholar). with BTK’s noncatalytic activity of kinase activity is for oncogenic BCR signaling to support the growth and survival of malignant B cells. protein kinases are as their noncatalytic such as of the of signaling and of transcription N. for the of protein 2016; Scholar). Our parallel CRISPR-Cas9 screening in DLBCL cells with versus kinase-inactive BTK an increased of BTK kinase-inactive cells on and Thus, of BTK kinase activity in a of cellular these genes were is a of the of tyrosine kinases and is in cells and B lymphocytes J.D. Lewis R.M. kinase in cells of 7: Scholar). of have been in various of such as and leukemia G. M. T. et role for cell kinase of and in proliferation and survival of Chem. Scholar, Y. Liu Y. S. E. M. D. et of kinases and for leukemia but not chronic Scholar). In we demonstrated that DLBCL cells with kinase-inactive BTK displayed increased dependence on to DLBCL cells with BTK. with a study the activation of in malignant B cells with kinase-inactive BTK A. L. C. F. et BTK mutants ibrutinib resistance through activation of the kinase 2022; Scholar). and mechanisms of activation in DLBCL cells with kinase-inactive BTK a of the receptor in cells, is a receptor for from and viruses H. T. T. S. H. et receptor Scholar). the of and to or responses A. A.R. W. A. et of by and responses that cell Scholar, A. W. S. M. activates cells through receptor Scholar, J. A. S. R. A. Toll-like receptor of by Exp. Med. Scholar). studies have that BTK was for signaling in cells and B cells C. by and 9 requires Bruton's tyrosine kinase.J. Chem. 2007; Scholar, S. Lam Bruton's tyrosine kinase activation and in B Chem. 2008; Scholar). In addition, the of BCR with and MYD88 into a in DLBCL cells J.D. Young R.M. S. Wright G.W. M. et oncogenic signalling in lymphoma.Nature. 2018; Scholar). These observations that BTK the BCR and signaling with the of BTK kinase activity in DLBCL cells, although not BCR signaling, may the BCR and signaling, resulting in increased on The biochemical and and kinase-inactive BTK BTKi the treatment for various B cell been that BTKi by the BTK’s Our study the the mechanism of of BTKi such as ibrutinib not BTK kinase activity but oncogenic BCR that BTK kinase activity is for oncogenic BCR signaling, we that BTKi in clinical may target BCR signaling by BTK’s noncatalytic the for BTK’s noncatalytic in various of B cell protein cell and are in of cell and are in human lymphoma cell were in with and and were to be on a using a assay. BTK kinase domain was to with the and ATP was to the of was with of on for were performed using a The was increased from to with an of and of at to The was as the to the of the first of BTK kinase domain and PLCγ2 peptide or recombinant BTK SH3 domain were with the kinase and and with ATP were at for and was using the was by was determined with using to A of cells were treated with ibrutinib for by a with of deep are in were with and in the with in at for cells were and in for an at were performed using a using an with was performed by with a the were as in which was as the of to was as the the calcium flux were determined with cellular was purified from cells treated with or ibrutinib for was performed by were to the human using B. with Scholar) by with B. from with or a 2011; Scholar). Differential were performed with M.I. W. S. of and for with 2014; Scholar) with the following and less of genes was performed with G. Wang Y. an for Scholar). cell were with the at a of of cells by were to 3 using and to or The human N. M. M. et to activity and of 2016; Scholar) was into and BTK L528W cells at a of and a of cells cells were for 3 for was in performed on from cells. were by W. H. Xiao T. L. M.I. F. et of essential genes from 2014; Scholar) to cells were well in were treated with of ibrutinib or with a survival was using the cell was by was determined with using to the and were performed with was to the the two more two was to were performed was were and was of have been in the of the the study are from the W. R. M. Li C. Z. X. et for 2015; Scholar, S. T. J. for 2019; Scholar, D. and peptide in 2017; Scholar, F. and sensitive with Scholar, X. Li L. Q. Wang Q. Z. Y. et and of a novel inhibitor targeting the binding Chem. Scholar, M. S. L. et and of 2017; Scholar, A.J. J.A. Y. M. M. et and for protein Scholar). and Liu are and of and are and of and no We Lu and Lu for with and for cell and and Liu for and H. Y. and Y. C. Liu and T. H. H. Y. Y. J. F. and W. J. Z. H. and T. H. H. Y. Y. J. F. W. Z. H. and T. H. was by from the of and of and and and of to T. H. The no role in study and or the to the for

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