Abstract

Simple Summary

   Glucocorticoid therapy resistance in B-cell malignancies is often
   associated with constitutive activation of tyrosine kinases. Novel
   anticancer drugs targeting hyperactivated tyrosine kinases, such as
   Bruton’s tyrosine kinase (BTK), have, therefore, gained much interest
   over the past few decades and have already been approved for clinical
   use. In this study, we compared the therapeutic efficacy of the
   phytochemical kinase inhibitor withaferin A with the clinically
   approved BTK inhibitor ibrutinib to target hyperactivated tyrosine
   kinase signaling in glucocorticoid-resistant multiple myeloma cells.
   Our results demonstrate that withaferin A-induced cell death of
   glucocorticoid-resistant MM1R cells involves covalent cysteine
   targeting of multiple Hinge-6 domain type tyrosine kinases of the
   kinase cysteinome classification, including BTK.

Abstract

   Multiple myeloma (MM) is a hematological malignancy characterized by
   plasma cells’ uncontrolled growth. The major barrier in treating MM is
   the occurrence of primary and acquired therapy resistance to anticancer
   drugs. Often, this therapy resistance is associated with constitutive
   hyperactivation of tyrosine kinase signaling. Novel covalent kinase
   inhibitors, such as the clinically approved BTK inhibitor ibrutinib
   (IBR) and the preclinical phytochemical withaferin A (WA), have,
   therefore, gained pharmaceutical interest. Remarkably, WA is more
   effective than IBR in killing BTK-overexpressing glucocorticoid
   (GC)-resistant MM1R cells. To further characterize the kinase inhibitor
   profiles of WA and IBR in GC-resistant MM cells, we applied
   phosphopeptidome- and transcriptome-specific tyrosine kinome profiling.
   In contrast to IBR, WA was found to reverse BTK overexpression in
   GC-resistant MM1R cells. Furthermore, WA-induced cell death involves
   covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of
   the kinase cysteinome classification, including inhibition of the
   hyperactivated BTK. Covalent interaction between WA and BTK could
   further be confirmed by biotin-based affinity purification and confocal
   microscopy. Similarly, molecular modeling suggests WA preferably
   targets conserved cysteines in the Hinge-6 region of the kinase
   cysteinome classification, favoring inhibition of multiple B-cell
   receptors (BCR) family kinases. Altogether, we show that WA’s
   promiscuous inhibition of multiple BTK family tyrosine kinases
   represents a highly effective strategy to overcome GC-therapy
   resistance in MM.

   Keywords: withaferin A, BTK, multiple myeloma, therapy resistance,
   glucocorticoids, ibrutinib

1. Introduction

   MM is a hematological malignancy of terminally differentiated plasma
   cells and is currently the second most common adult blood cancer
   [[56]1]. MM often results in development of end-organ diseases, such as
   anemia, hypercalcemia, renal insufficiency, and bone lesions, making it
   an illness of considerable clinical and social impact [[57]2,[58]3]. In
   recent years, important therapeutical advancements in the field of MM
   have been made, increasing the life expectancy of patients by six to
   ten years [[59]4,[60]5]. These novel therapies have mostly been
   developed based on an improved understanding of the biology of myeloma
   cells and their interaction with the bone marrow (BM) environment
   [[61]6]. They include proteasome inhibitors [[62]7], immunomodulatory
   drugs [[63]8], glucocorticoids (GCs) [[64]9], monoclonal antibodies
   [[65]10], and histone deacetylase inhibitors [[66]11]. However, MM
   still remains an incurable disease as the majority of patients
   eventually relapse and become refractory to existing therapies
   [[67]12]. Acquisition of resistance to anticancer drugs, therefore,
   remains the main barrier in treating MM [[68]4,[69]13].

   One of the cellular pathways mediating drug resistance in many B-cell
   malignancies is the BCR signaling pathway [[70]14]. Under physiological
   conditions, the BCR is activated upon ligation of antigen and promotes
   survival, function and development of B-cells [[71]15]. After initial
   antigen binding, the immune receptor tyrosine activation motif domains
   CD79A and CD79B are phosphorylated by Src family kinases Lyn and Syk
   and recruit other adaptor proteins and tyrosine kinases (TK), a key
   example being Bruton’s tyrosine kinase (BTK) [[72]16]. BTK ultimately
   orchestrates activation of downstream effectors of BCR signaling, such
   as nuclear factor-κB (NF-κB) and nuclear factor of activated T cells
   (NFAT), through PLC-γ2 and phosphoinositide 3-kinase (PI3K)
   phosphorylation [[73]14]. In MM, BTK is often constitutively activated,
   thereby modulating survival signals and therapy resistance
   [[74]17,[75]18,[76]19,[77]20]. As a result, BTK kinase inhibitors have
   received growing pharmacological interest and have already shown
   promising therapeutic responses in B-cell malignancies in the clinic
   [[78]21]. More particularly, the covalent-binding BTK inhibitor IBR has
   shown to be a potent anticancer drug in chronic lymphocytic leukemia
   (CLL), mantle cell lymphoma, diffuse large B-cell lymphoma, and MM by
   interfering with B-cell homing, survival and microenvironment-mediated
   drug resistance [[79]17,[80]18,[81]22,[82]23,[83]24,[84]25].
   Suppression of BTK hyperactivation is also key to the therapeutic
   efficacy of GCs in B-cell leukemias, where IBR has been shown to
   improve GC therapy response [[85]26,[86]27,[87]28,[88]29]. In clinical
   trials of MM, combination therapies investigating IBR efficiency
   demonstrated encouraging responses and a manageable safety profile
   [[89]30,[90]31].

   Although preliminary clinical data revealed the beneficial effects and
   acceptable safety profile of IBR in MM, clinical studies in other
   B-cell malignancies have linked IBR use with adverse effects, including
   diarrhea, fatigue, nausea, and rashes [[91]32,[92]33]. More important,
   IBR therapy is associated with a significant increase in the occurrence
   of ventricular arrhythmias and sudden cardiac death [[93]34,[94]35].
   The underlying mechanisms of these severe side effects are not well
   understood but could be partially explained by the off-targets
   interaction of IBR with interleukin-2-inducible T-cell kinase (ITK),
   epidermal growth factor (EGFR), and PI3K [[95]36]. Another pressing
   issue that has arisen since IBR has been applied in the clinic is the
   development of therapy resistance (reviewed in [[96]37]). Nearly
   one-third of patients diagnosed with B-cell malignancies display
   primary resistance against IBR therapy, while many others acquire
   resistance over time. These major issues accompanying the therapeutic
   use of IBR have sparked the development of second and third-generation
   BTK inhibitors (e.g., acalabrutinib), characterized by higher
   selectivity and potency profiles [[97]38]. Unfortunately, acquired
   resistance to these novel BTK inhibitors has already been described in
   some B-cell malignancies as well [[98]39]. As a result, alternative
   treatment strategies to suppress BTK hyperactivation in
   therapy-resistant hematological malignancies are currently being
   investigated in high-throughput combinatorial screenings of clinically
   approved and preclinical investigational compound libraries [[99]40].
   Given that nearly half of the agents used in cancer therapy today are
   either natural products or derivatives thereof, novel BTK-targeting
   lead compounds may be identified from this vast arsenal of chemically
   active structures [[100]41,[101]42,[102]43]. Interestingly, withaferin
   A, a withanolide phytochemical isolated from Withania somnifera, is one
   of the top investigational compounds prioritized for IBR combination
   therapy to target chronic active BCR signaling [[103]40].

   WA reveals broad-spectrum therapeutic activities in several
   (drug-resistant) cancer cell types [[104]44], including B-cell lymphoma
   and MM [[105]45,[106]46,[107]47]. Of particular interest, some of WA’s
   antitumor effects have been attributed to its ability to covalently
   target kinase activity [[108]48,[109]49,[110]50,[111]51,[112]52].
   Accordingly, innovative phosphopeptidome kinome activity profiling, RNA
   sequencing, in silico docking simulations, and chemo-affinity
   approaches were combined in this study to characterize BTK
   hyperactivation and TK inhibitor therapy response of WA and IBR in
   GC-resistant MM cells.

2. Results

2.1. GC Therapy Resistance in Multiple Myeloma Is Associated with
Hyperactivation of Tyrosine Kinases

   GC therapy-sensitive MM1S and -resistant MM1R cell lines derived from a
   single MM patient have previously been described as cell models to
   study the etiology of GC therapy resistance and to evaluate novel
   classes of chemotherapeutic drugs [[113]53,[114]54]. To investigate the
   vulnerability of GC-resistant MM1R cells for specific clinical TK
   inhibitor drugs, we compared the tyrosine kinome activity profiles of
   GC-resistant MM1R and GC-sensitive MM1S cell lysates by means of a
   PTK-specific phosphopeptide array (PamChip), containing 144 conserved
   peptides corresponding to TK specific substrates [[115]55,[116]56].
   Overall, TK activity was consistently higher in MM1R cells compared to
   MM1S cells ([117]Figure 1a and [118]Figure S1). Identification of the
   20 most significant differential hyperphosphorylated peptides (adjusted
   p-value (FDR) < 0.01) in MM1R compared to MM1S predicted
   hyperactivation of multiple (non) receptor TK in MM1R, such as SYK,
   DDR, ABL, ZAP70, FAK2, BRK, BTK, ITK and FGR ([119]Figure 1b).
   Subsequent MetaCore pathway analysis showed that the hyperactivated
   kinases in MM1R cells are involved in cell proliferation, cell cycle
   regulation, cell adhesion, cancer, therapy resistance, immune response,
   T-cell receptor signaling and BCR signaling ([120]Supplementary Figure
   S2).

Figure 1.

   [121]Figure 1
   [122]Open in a new tab

   (a) Heatmap showing phosphorylation intensities of peptides serving as
   substrates for tyrosine kinases. Figure shows hyperphosphorylated (red)
   or hypo-phosphorylated (blue) peptides in MM1R (n = 3) and MM1S (n = 3)
   samples. (b) Ranking of hyperactivated kinases in MM1R versus MM1S
   cells based on the top 20 significant differentially phosphorylated
   peptides. Fill color of the bars is based on the kinase specificity
   score, indicating the specificity of differences in kinase activity
   with respect to the quantity of peptides used for predicting the
   corresponding kinase (c) Heatmap representation of differentially
   expressed genes (logFC >|1|, p < 0.01) in MM1R versus MM1S cells as
   determined by RNA sequencing. n = 3 biologically independent replicates
   per cell line. (d) Ranking of the top overexpressed kinases in MM1R
   versus MM1S cells based on their log2-fold change as determined by RNA
   sequencing. Fill colors of the bars are a measure for kinase activity
   as measured via the PTK-specific phosphopeptide array. (e) Relative
   Bruton’s tyrosine kinase (BTK) mRNA levels in MM1R and MM1S cells. Data
   are plotted as the mean ± s.d., n = 3 biologically independent
   replicates (** p = 0.0035, unpaired t-test). (f) Western immunoblot
   detection and quantification of basal BTK and GAPDH protein levels in
   MM1R and MM1S cells. Data are plotted as the mean ± s.d., n = 3
   biologically independent replicates (* p = 0.0385, unpaired t-test).

   To investigate whether the observed TK hyperactivation is also
   reflected at the transcriptome level, RNA sequencing analysis of basal
   gene expression in MM1S and MM1R cells was conducted. Results were
   analyzed with the R-package DESeq2 [[123]57], using a selection
   criterion of a minimal FDR < 0.01. Upon comparing gene expression
   patterns in both cell lines, 1383 differentially expressed genes (DEG)
   (logFC > 1 or logFC < −1) could be identified ([124]Figure 1c).
   Interestingly, from all hyperactivated TK in MM1R cells listed before,
   BTK was identified as the strongest upregulated TK in GC-resistant MM1R
   cells ([125]Figure 1d, is included in the top 20 most significant DEG
   ([126]Table 1). Upregulation of BTK in MM1R cells was validated by qPCR
   and Western blot analysis ([127]Figure 1e–f) and is in line with
   previous observations [[128]17,[129]58]. Taken together, these findings
   suggest that BCR signaling and BTK in MM1R cells may represent an
   attractive target to kill GC-resistant MM1R cells.

Table 1.

   Overview of top 20 most significantly differentially expressed genes
   (logFC > 1 or logFC < −1) between glucocorticoid (GC)-resistant MM1R
   and GC-sensitive MM1S cells.
   Gene Ontology Term Symbol Gene ID Name Log2FC p-Adj.
   ECM and cell–cell adhesion RELN * 5649 Reelin 2.9 7.4 × 10^−160
   PLXNB2 23654 Plexin B2 1.8 7.1 × 10^−96
   PODXL2 * 50512 Podocalyxin like 2 2.6 6.8 × 10^−83
   ESAM 90952 Endothelial cell adhesion molecule 2.0 1.7 × 10^−80
   PRKX * 5613 Protein kinase X-linked 1.1 8.1 × 10^−79
   ACP5 54 Acid phosphatase 5, tartrate-resistant 4.8 2.0 × 10^−77
   GPCR signaling GNG7 2788 G protein subunit gamma 7 1.5 5.7 × 10^−99
   UTS2R 2837 Urotensin 2 receptor 1.9 5.4 × 10^−77
   BCR signaling BTK * 695 Bruton’s tyrosine kinase 2.8 1.5 × 10^−216
   TNFRSF8 943 TNF receptor superfamily member 8 3.7 6.9 × 10^−93
   CD52 1043 CD52 molecule 3.5 5.6 × 10^−83
   mRNA/protein stability CTAG2 30848 Cancer/testis antigen 2 8.5 4.0 ×
   10^−168
   LINC01518 101929397 Long intergenic non-protein Coding RNA 1518 7.9 1.4
   × 10^−147
   CMTR1 23070 Cap methyltransferase 1 −1.1 9.6 × 10^−115
   TMEM25 84866 Transmembrane protein 25 4.8 6.0 × 10^−79
   Cell cycle regulation CDKN2A 1029 Cyclin-dependent kinase inhibitor 2A
   9.4 1.7 × 10^−210
   Cytoskeleton TUBB4A 10382 Tubulin beta 4A class IVa 3.6 6.1 × 10^−165
   Inflammation NLRP11 204801 NLR family pyrin domain containing 11 4.9
   1.2 × 10^−220
   Transmembrane transport SLC38A5 92745 Solute carrier family 38 member 5
   1.8 1.7 × 10^−92
   ABCG2* 9429 ATP-binding cassette subfamily G member 2 5.6 2.0 × 10^−84
   [130]Open in a new tab

   * Genes associated with therapy resistance. Abbreviations: ECM,
   extracellular matrix; GPCR, G-protein coupled receptor; BCR, B-cell
   receptor.

2.2. The Tyrosine Kinase Inhibitor Profile of Withaferin A and Ibrutinib Show
a High Degree of Similarity

   Taking into account the hyperactivation of BCR-BTK kinase signaling in
   MM1R cells (see 2.1.), we next tested their sensitivity for the
   clinically approved BTK inhibitor IBR as well as WA, a top prioritized
   investigational phytotherapeutic compound identified in a
   high-throughput drug screening against chronic BCR signaling [[131]40].
   MM1R cells were treated for 24 h with different concentrations of WA or
   IBR kinase inhibitors, and the relative % cell survival/cell death was
   evaluated by MTT assay. Both compounds were effective in killing
   GC-resistant MM1R cells in a dose-dependent manner, although WA is the
   more potent cell death inducer (IC50 = 1.7 µM) since its IC50 was > 10
   times lower than the one of IBR (IC50 = 27.9 µM) ([132]Figure 2a).
   Remarkably, both WA and IBR also induce cell death in GC-sensitive MM1S
   cells lacking BTK overexpression, although IC50 values were higher than
   MM1R cells (IC50[WA] = 1.9 µM, IC50[IBR] = 49.3 µM) ([133]Figure 2a).

Figure 2.

   [134]Figure 2
   [135]Open in a new tab

   (a) Relative cell viability of MM1 cells upon 24 h exposure to
   increasing concentrations of BTK inhibitor ibrutinib (IBR) or
   withaferin A (WA). Data are plotted as the mean ± s.d., n = 3
   biologically independent replicates. (** p < 0.01, *** p < 0.001 **** p
   < 0.0001, ANOVA). (b) Heatmap representation of hyperactivated or
   inhibited kinases in MM1R versus MM1S cells, or following 15 min IBR or
   WA treatment, n = 3 biologically independent replicates per treatment
   group. (c) Close-up heatmap representation of [136]Figure 2b showing
   inhibited BCR-related kinases in MM1R versus MM1S or following 15 min
   IBR or WA treatment, n = 3 biologically independent replicates per
   treatment group. (d) Kinase trees displaying the tyrosine kinase
   targets of IBR (left) and WA (right). Kinase trees were generated with
   the CORAL web tool ([137]http://phanstiel-lab.med.unc.edu/CORAL/,
   accessed on 30 November 2020).

   To explore the mode of action of these compounds, we measured
   corresponding cellular changes in TK activities by phosphopeptidome
   based tyrosine kinome profiling of MM1R cells exposed to either WA (1
   µM) or IBR (1 µM). Similarly, and as mentioned above, cell lysates of
   treated cells were analyzed through PTK-specific phospho-peptide
   arrays, after which the top activated or inhibited kinases were
   identified based on the significant differences in phospho-intensities
   of the PTK peptide substrates. We found that WA and IBR both inhibited
   most of the hyperactivated kinases in MM1R cells, with largely
   overlapping, though promiscuous, TK inhibitor profiles ([138]Figure 2b,
   annotation of heatmap rows can be found in [139]Supplementary Figure
   S3). At the level of BCR signaling, clear inhibition of BTK kinase
   activity can be observed in the presence of IBR, as expected, and WA
   ([140]Figure 2c,d). Besides BTK, multiple BCR signaling kinases, such
   as ZAP70, BLK, FLT3, TEC, and SYK, are also targeted by both WA and IBR
   ([141]Figure 2c). In line with previous studies, which already revealed
   that IBR could trigger off-target BCR-BTK independent kinase inhibitor
   (side) effects [[142]59], we also identified additional IBR-responsive
   TK in MM1R cells ([143]Figure 2c, [144]Figure S3). Of special note,
   whereas most binding affinities of IBR have been determined in vitro
   ([145]Supplementary Figure S4), we provide the first cell-based
   integrated tyrosine kinome activity map in MM1R cells in the presence
   of the IBR inhibitor. Although WA inhibits similar BCR family kinases
   as IBR, variations in kinase inhibitor specificity/potency of WA in
   comparison to IBR treatment may explain differences in the therapeutic
   efficacy of both compounds in MM1R cells ([146]Figure 2a,d).

2.3. WA Inhibits BCR-BTK Kinase Activity by Transcriptional Downregulation
and Covalent Cysteine-Dependent Targeting of BTK

   To further characterize how WA decreases BTK kinase activity in MM1R
   cells, we next checked whether WA treatment changes BTK mRNA and
   protein expression levels. Panther pathway enrichment analysis of RNA
   sequencing data from WA-treated MM1R cells already revealed that DEGs
   are significantly enriched in B-cell activation ([147]Figure 3a). More
   particularly, WA significantly decreased BTK mRNA expression (Log2FC =
   −0.525, FDR = 0.023). This was further confirmed by qPCR and western
   immunoblot experiments. As can be observed from [148]Figure 3b,c, WA
   was able to lower BTK expression in a time-dependent manner, both at
   the mRNA and protein level. Similar WA-specific changes could also be
   observed in U266 cells, another GC-resistant multiple myeloma cell line
   sensitive to WA treatment ([149]Figure 3b,c and [150]Figure S5). In
   line with previous studies [[151]60,[152]61,[153]62], we did not
   observe any changes in BTK expression after the IBR treatment of MM1R
   cells indicating that IBR mainly targets BTK (hyper)phosphorylation and
   not total BTK protein levels ([154]Supplementary Figure S6).

Figure 3.

   [155]Figure 3
   [156]Open in a new tab

   (a) Panther pathway enrichment analysis of significant (FDR < 0.05)
   differentially expressed genes of WA-treated MM1R cells as determined
   by RNA sequencing. (b) Relative BTK mRNA levels of MM1R, MM1S and U266
   cells treated with WA for 3 h. Data are plotted as the mean ± s.d., n =
   3 biologically independent replicates (* p = 0.0453, ** p = 0.0015,
   ANOVA) (c) Western blot detection and quantification of BTK and GAPDH
   expression levels after WA treatment in MM1R and U266 cells. Data are
   plotted as the mean ± s.d., n = 3 biologically independent replicates.
   (* p < 0.05, ** p < 0.01, ANOVA).

   Since WA contains several reactive nucleophilic groups, which can
   covalently bind to kinase sulfhydryl groups of cysteines through
   Michael addition [[157]48,[158]63,[159]64], we also evaluated the
   potential covalent binding of WA to BTK by pulldown experiments with
   biotinylated WA (WABI). Pull-down experiments with WABI in MM1R cells
   indeed confirmed cysteine-dependent binding to BTK, which can be
   blocked by excess amounts (1 mM) of the reducing agent dithiothreitol
   (DTT) ([160]Figure 4a). Along the same line, covalent WA-BTK
   interaction could be confirmed in U266 cells ([161]Figure 4b). The
   biological relevance of this covalent interaction could further be
   validated in washout experiments where MM1R cells were exposed to
   increasing concentrations of WA for 15 min, after which it was washed
   away with PBS. Although WA cells were only briefly treated with WA, the
   cell viability of MM1R cells was still affected in a dose-dependent
   manner 24 h post-treatment ([162]Supplementary Figure S7). The growth
   inhibition remains the strongest in the unwashed cells, suggesting the
   contribution of non-covalent interactions of WA as well. H-bond and van
   der Waals interactions between WA and target molecules, such as Hsp90,
   have indeed been reported to contribute to the anticancer mechanism of
   WA [[163]65]. Alternatively, covalent binding by WA may be weaker than
   a normal covalent binding and become reversed by washout experiments
   [[164]66,[165]67,[166]68]. Finally, by confocal microscopy, we could
   also demonstrate colocalization of BTK and WABI in MM1R cells
   ([167]Figure 4c).

Figure 4.

   [168]Figure 4
   [169]Open in a new tab

   (a) Western immunoblot detection of BTK levels before and after
   pulldown with biotinylated WA (WABI), following 2 h WABI treatment in
   MM1R in the presence or absence of excess thiol donor DTT (1 mM). (b)
   Western immunoblot detection of BTK levels before and after pulldown
   with biotinylated WA (WABI), following 2 h WABI treatment in U266 cells
   in the presence or absence of excess thiol donor DTT (1 mM). (c)
   Confocal imaging of colocalization of BTK expression and WABI
   localization in MM1R cells.

2.4. Covalent C481 Targeting of BTK by WA in Hinge-6 Domain of the Protein
Kinase Cysteinome Classification Reduces Survival of Glucocorticoid Resistant
Multiple Myeloma MM1 Cells

   Human protein kinases are composed of two highly conserved domains,
   namely catalytic and regulatory domains. It has been shown that the
   catalytic domain can be further classified based on the positions of
   the gatekeeper amino acids and cysteines hosted in the catalytic pocket
   [[170]69,[171]70,[172]71,[173]72]. Based on the positions of cysteines
   present across the human kinome, Leproult et al. reclassified the human
   kinome into a cysteinome according to the cysteine positions relative
   to the ATP binding pocket ([174]Table 2) [[175]69,[176]73].
   Interestingly, most kinases inhibited by WA treatment, including BTK,
   BLK and EGFR, were found to be highly enriched in Hinge-6 domain type
   kinases ([177]Table 2). Multiple sequence alignment of these Hinge-6
   domain orthologs revealed the presence of a conserved glycine-cysteine
   motif, suggesting that WA covalently binds to this site within its TK
   targets ([178]Figure 5a). Through molecular docking studies, we found
   that the conserved cysteine site within BTK (Cys481) is indeed
   accessible and favorable (−4.93 kcal/mol) for WA binding
   ([179]Supplementary Table S1). The docking results predict that
   covalent bond formation occurs via the C[4]-OH group of WA and the
   Cys481 residue of BTK and that this interaction is further stabilized
   via hydrogen bond formation with surrounding Leu482, Tyr485 and Gly480
   amino acids ([180]Figure 5b,c). Given that IBR also covalently targets
   the Cys481 residue of BTK, these results suggest that WA interacts with
   BTK in a similar manner as IBR [[181]63]. By using the NanoBRET Target
   Engagement Intracellular Kinase assay, where the affinity of WA for BTK
   can be analyzed by competitive displacement of a fluorescent NanoBRET
   Tracer bound to BTK, we could further confirm that WA can displace the
   WT BTK protein, but not the mutated C481S BTK protein in HEK-293 cells
   ([182]Figure 5d). This suggests that the Cys481 residue of BTK is the
   main binding site of WA. Furthermore, we show that silencing of
   endogenous WT BTK reduces cell MM1R viability and can be rescued upon
   overexpression of C481S BTK overexpression. ([183]Figure 5e,
   [184]Figure S8). Of special note, C481S BTK overexpression cannot
   completely rescue WA-induced cell death in MM1R, confirming that WA
   kinase effects on cell viability are not limited to BTK alone and may
   involve additional hinge 6 domain type kinase targets of the cysteinome
   classification in MM1R cells. Along the same line, GC-sensitive MM1S
   cells lacking BTK overexpression are also sensitive to WA treatment
   through promiscuous covalent cysteine targeting of alternative cell
   survival tyrosine kinases expressed in MM1S cells ([185]Figure S9a–c).

Table 2.

   Kinase cysteinome classification. Summarized from [[186]73]. Bold
   highlights kinases representing the main tyrosine kinase targets of
   withaferin A.
   Site Subsite Representative Kinases
   Gatekeeper region GK MOK
   GK + 1 SgK494
   GK − 1 MAP2K4, MKK3, MAP2K6, KHS1, KHS2, GCK
   DFG region DFG + 1 MAP3K8, MOS, MAP3K4, PINK1
   DFG + 2 PKCz, PKCi, AKT1, AKT2, AKT3, PKCg, SGK1F, SGK2
   DFG − 1 PBK, TGFbR2, CDKL3, CDKL2, PRP4, MNK2, MNK1
   Glycine rich loop region Glycineloop WNK4, WNK1, WNK2, WNK3, HER3
   Glycineloop 1 ZAK
   Glycineloop 2 SgK496, MEKK1, PLK2, PLK3, PLK1, RSK1
   Glycineloop 3 SgK493
   Glycineloop5 FGFR1, FGFR2, FGFR3, FGFR4
   Hinge binding region Hinge 1 FGFR4, TTK, MAPKAPK2, MAPKAPK3
   Hinge 2 IKKa, IKKb, LKB1, NEK4, Wee1, SLK, FLT4, KDR
   Hinge 3 Ron, FGR, SgK494, Kit, CSFR, FLT3
   Hinge 4 SgK110, BubR1, LKB1, TBK1
   Hinge 5 PINK1, EphB3
   Hinge 6 MAP2K7, TEC, TXK, ITK, BTK, BMX, BLK, HER2, EGFR, HER4, JAK3
   Hinge 7 JNK1, JNK2, JNK3
   Roof region Roof sheet HER3
   [187]Open in a new tab

Figure 5.

   [188]Figure 5
   [189]Open in a new tab

   (a) Multiple sequence alignment of Hinge 6 domain-type kinases. The
   conserved GC-motif between different orthologs is indicated. Alignment
   was performed with Clustal Omega
   ([190]https://www.ebi.ac.uk/Tools/msa/clustalo/ accessed on 30 November
   2020). (b) Crystal structure of BTK (PBD id: 6TFP) in complex with WA
   (PubChem CID: 26537) covalently bound to Cys481. (c) Interaction
   between WA and BTK structure visualized using Ligplot [[191]74],
   showing the covalent bond formation of the C4-OH group of WA with the
   SH group of Cys481 from BTK. (d) Inhibition of wild-type (WT) BTK and
   mutated (C481S) BTK by WA in HEK-293 cells. Data are plotted as mean ±
   s.d., n = 3 biologically independent replicates. (e) Relative viability
   of MM1R cells treated for 24 h with increasing concentrations of WA,
   upon BTK silencing (siBTK) in presence or absence of C481S BTK
   overexpression. Data are plotted as mean ± s.d., n = 3 biologically
   independent replicates (ns = p > 0.05, * p = 0.0309, *** p < 0.0001,
   ANOVA).

3. Discussion

   A common feature of many B-cell malignancies is their ability to
   develop therapy resistance through increased BCR signaling and BTK
   activity. Along the same line, increased BCR signaling has been shown
   to reduce the therapeutic efficacy of GCs in B-cell leukemias
   [[192]26,[193]27,[194]28,[195]29]. Although MM cells are typically
   lacking active BCR, BTK overexpression and signaling has been
   associated with increased therapy resistance in the disease
   [[196]18,[197]75]. Accordingly, we compared the anticancer effect of
   the clinically approved BTK kinase inhibitor IBR and the preclinical
   phytotherapeutic kinase inhibitor WA to kill GC therapy-resistant MM
   cells via suppression of BTK hyperactivation. Phosphopeptidome based
   tyrosine kinome profiling confirmed hyperactivation of multiple BCR
   family kinases besides other receptor TK families (FGFR, PDGFR, DDR) in
   GC therapy-resistant MM1R cells. Furthermore, hyperactivated BCR
   kinases could completely be suppressed with both kinase inhibitors, WA
   and IBR. Nonetheless, kinase tree representations of both kinase
   inhibitor profiles revealed quantitative and qualitative differences in
   specificity and potency, which may underly the differences in potency
   of WA and IBR to kill GC-resistant MM cells. Biotin
   affinity-purification experiments confirmed a cysteine-dependent
   covalent interaction between WA and BTK, similar to IBR-BTK Cys481
   binding [[198]63]. This is supported by molecular modeling studies,
   which revealed favorable covalent WA binding to the conserved Cys481
   residue in BTK. In addition, NanoBRET Target Engagement Intracellular
   Kinase assays confirm WA-dependent inhibition of WT BTK kinase
   activity, which is lost upon C481S mutation. Interestingly, most
   kinases inhibited by WA treatment, including BTK, BLK and EGFR, belong
   to the Hinge-6 domain-type kinases, according to the kinase cysteinome
   classification. The latter suggests broad redundant kinase inhibitory
   effects of WA through covalent cysteine targeting. Accordingly, BTK
   silencing experiments could only partially mimic the therapeutic cell
   death effects of WA in MM1R, whereas overexpression of the C481S kinase
   mutant could only partially protect against WA. Similarly, cancer
   therapeutic effects of WA could also be observed in GC-sensitive MM1S
   cells, which lack BTK overexpression, through covalent targeting of
   alternative cell survival kinases expressed in MM1S. As such, the
   therapeutic efficacy of WA against different cancer cell types may
   strongly depend on its promiscuous nucleophilic cysteine reactivity
   towards the cellular repertoire of hyperactivated tyrosine cell
   survival kinases. Additionally, the anticancer effects of WA may also
   partially rely on its non-covalent interactions with target proteins.
   H-bond interactions with terminal hydroxyl groups of WA were shown to
   be sufficient to deform protein complexes through naïve hindrance
   [[199]65]. Alternatively, the covalent interaction of WA may be weaker
   than that of the normal covalent bond and be reversible in washout
   experiments [[200]76]. Taunton and coworkers demonstrated that drug
   efficacy could be optimized by finetuning warhead residence time and
   exploiting the intrinsic reversibility of the reaction moiety
   [[201]66,[202]77]. In this respect, partially reversible covalent
   binding characteristics of WA may promote more promiscuous tyrosine
   kinase targeting than IBR and explain the higher efficacy of WA than
   IBR in overcoming MM drug resistance. Finally, WA-mediated inhibition
   of BTK kinase activity was also accompanied by a time-dependent
   decrease in BTK mRNA and protein expression, not observed for IBR,
   suggesting that WA targets BTK hyperactivation at multiple levels
   [[203]62]. For example, WA may indirectly decrease BTK activity by
   reducing kinase protein levels by inhibition of Sp1-dependent
   transcription [[204]78], by post-transcriptional microRNA silencing
   mechanisms [[205]53,[206]79], or by decreasing kinase stability via
   heat shock chaperone proteins [[207]45,[208]47].

   Although WA clearly suppresses hyperactivated BTK family kinases
   signaling, some limitations of our study need to be acknowledged. Most
   notably, mainly GC therapy-resistant MM cell models were studied here.
   Further preclinical in vitro and in vivo studies are required to fully
   appreciate the therapeutic efficacy of WA to overcome multidrug
   resistance by covalent inhibition of hinge 6 cysteine domain BTK family
   kinases. In addition, small qualitative and/or quantitative variations
   in specificity/potency observed for the promiscuous BCR TK inhibitor
   profiles of WA and IBR may not be sufficient to explain the large
   difference in the therapeutic efficacy of WA as compared to IBR against
   GC-resistant MM cell lines. Of special note, whereas nM concentrations
   of IBR are effective against B-cell lymphoma cancer cells, much higher
   μM concentrations seem to be required to kill BTK overexpressing MM
   cells [[209]17,[210]80,[211]81,[212]82,[213]83], presumably because of
   the presence of highly redundant BCR/NFKB survival pathways
   (PI3K/Akt/mTOR/Syk) in MM, which compensates for pharmacological BTK
   inhibition [[214]37]. Yet, despite the presence of these compensatory
   pathways, complete silencing of BTK expression is cytotoxic to MM1R
   cells, emphasizing BTK’s involvement in myeloma cell survival. This is
   in line with previous observations made in BTK KO mouse models, where
   complete loss of BTK expression resulted in a more significant decrease
   in splenic B-cell numbers compared to mice harboring BTK mutations
   [[215]84]. Because BTK also possesses crucial shuttling and scaffold
   activities, complete silencing of BTK is likely more detrimental to
   B-cells due to additional loss of non-kinase functions [[216]85].

   Furthermore, covalent cysteine binding of WA has also been reported to
   Ser/Thr kinases [[217]86] and phosphatases [[218]87], which were not
   included in our phosphopeptidome screening approach. Indeed, in
   addition to BTK, BCR signaling is fine-tuned by Ser/Thr kinases (for
   example, IKK2, PKCβ) [[219]15,[220]88] and phosphatases (i.e., SHIP1,
   PTEN) [[221]89,[222]90]. Finally, chemoproteomic strategies have
   identified additional WA non-kinase target proteins, which may further
   strengthen the potential cancer therapeutic efficacy of WA
   [[223]45,[224]91]. Therefore, functional silencing approaches with
   shRNA libraries will be informative to further distinguish druggable
   key cancer targets from adverse off-targets to defeat therapy
   resistance in MM by WA [[225]87,[226]92]. Further research determining
   the safety and pharmacokinetic profile of WA is needed as well.
   Preliminary (pre)clinical toxicology studies of WA in different cancer
   models revealed that WA administration is generally well tolerated with
   limited to no adverse toxicity reported [[227]93,[228]94,[229]95]. Yet,
   no toxicology data of WA have been collected in B-cell malignancies,
   such as MM.

4. Materials and Methods

4.1. Cell Culture and Cell Viability Assays

   GC-sensitive MM1S (CRL-2974) and GC-resistant MM1R MM cell lines
   (CRL-2975) have been described previously and were purchased from ATCC
   [[230]53]. GC-resistant U266 cells were kindly provided by Dr. Eva
   Lion, Head of Tumor Immunology Group of the Laboratory of Experimental
   Hematology (University of Antwerp). Cells were cultivated in RPMI-1640
   supplemented with 10% fetal bovine serum (E.U approved; South American
   origin) and 1% penicillin/streptomycin solution (Invitrogen, CA, USA).
   The cell lines were additionally supplemented with 1% MEM Non-Essential
   amino acids and 1% sodium pyruvate (Invitrogen, CA, USA). Each cell
   line was maintained at 37 °C in 5% CO[2] and 95% air atmosphere and
   95–98% humidity. Cell viability was assessed by colorimetric assay with
   3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
   (Sigma-Aldrich, St. Louis, MO, US) as previously described [[231]53].

4.2. Cell Lysis and Peptide Array-Based TK Activity Profiling

   WA- or BTK-treated and untreated MM1S and MM1R cells (n = 3
   biologically independent samples per cell line per treatment) were
   lysed in M-PER lysis buffer containing 1:100 Halt’s protease and
   Pierce™ phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA).
   The lysates were collected and centrifuged at >13,000× rpm for 15 min
   at 4 °C. The supernatants were transferred to pre-chilled Eppendorf
   tubes and flash-frozen immediately on dry ice. Protein concentrations
   were determined by the Pierce™ BCA method (Thermo Scientific, Waltham,
   MA, USA) [[232]96]. Cellular kinase activities were measured by
   TK-specific phosphopeptide arrays (PamChip, PamGene International B.V.
   the Netherlands) according to the manufacturer’s protocol as previously
   described [[233]56,[234]97]. The differences in peptide phosphorylation
   signal intensity between different experimental setups were analyzed in
   a linear mixed model by using BioNavigator 6.3 software integrated with
   R (Bioconductor) for statistical analysis (paired two-sided t-test and
   unpaired t-tests). FDR were calculated using Benjamini–Hochberg method
   (adjusted p-values < 0.05) [[235]55]. The upstream kinases were
   correlated with the identified PTK-specific phosphopeptide fingerprints
   according to the human (phospho)protein reference database
   [[236]98,[237]99,[238]100,[239]101]. Peptide lists showing differential
   phosphorylation intensities were further cross-compared with
   phosphorylation specific datasets of the Kinexus kinase predictor
   [[240]102]. The kinases’ ranking was based upon scoring penalties of
   sensitivity and specificity of observed peptide phosphorylation changes
   in the tested experimental conditions. The significance of the upstream
   kinase scoring functions was calculated by Fisher’s exact test. Pathway
   enrichment of differentially phosphorylated protein IDs was analyzed
   via MetaCore (Clarivate Analytics, Philadelphia, PA, USA) and Ingenuity
   pathway analysis (IPA) software (Qiagen, Germany) [[241]103].

4.3. cDNA Conversion Quantitative Real-Time PCR

   Total RNA (1 µg) extracted from each sample was converted into cDNA
   with the Go Script reverse transcription system (Promega, Madison, WI,
   USA) following the manufacturer’s protocol. Next, qPCR analysis was
   carried out using the GoTaq qPCR master mix (Promega, Madison, WI, USA)
   according to the manufacturer’s instructions. In brief, a 25 µL
   reaction volume mix per sample was prepared, containing 12.5 µL GoTaq
   qPCR master mix, 0.4 µM forward and reverse primer and nuclease-free
   water. The following PCR program was applied on the Rotor-Gene Q qPCR
   machine of Qiagen: 95 °C for 2 min, 40 cyclic denaturations (95 °C, 15
   s) and annealing/extension (60 °C, 30 s), and dissociation (60–95 °C).
   Each sample was run in triplicate. The median value of the triplicates
   was taken to calculate the ΔΔCt-values using GAPDH as the normalization
   gene. Primer sequences are listed in [242]Supplementary Table S2.

4.4. RNA Extraction and RNA Sequencing

   The RNeasy mini kit (Qiagen, Germany) was used to extract total RNA
   from untreated, or WA exposed MM1S and MM1R cells (n = 3 biologically
   independent samples per cell line per treatment) according to the
   manufacturer’s protocol. Isolated, pure total RNA was then quantified
   and qualified using an Epoch^TM microplate spectrophotometer (BioTek,
   Winusky, VT, USA). RNA samples were stored at −80 °C and subsequently
   shipped to BGI (BGI Group, Beijing, China), where RNA integrity was
   determined using the 2100 Bioanalyzer system (Agilent Technologies, CA,
   USA). All 12 samples with acceptable quality levels (RNA content >80
   ng/µL, 28 s/18 s ≥ 1.0 and RIN ≥ 7.0) were included for sequencing
   library preparation. Prepared libraries were 2 × 50 bp paired-end
   sequenced using the BGISEQ-500 platform (BGI Group, China). RNAseq data
   were deposited in the NCBI GEO database ([243]GSE162475).

   The quality of the RNA sequencing reads was assessed using FastQC
   (v0.11.5, Babraham Institute, Cambridge, UK) [[244]104]. STAR (v2.7.3a,
   Cold Spring Harbor Laboratory, CA, USA) [[245]105] was subsequently
   used to map reads to the human reference genome build 37 (hg19) and to
   generate a read count table summarizing the counts per gene. Finally,
   differential gene expression analysis was performed using the DESeq2
   package [[246]57], and pathway analysis was performed with Panther
   (Protein Informatics, Celera Genomics, CA, USA) [[247]106].

4.5. Antibodies and Reagents

   WA was purchased from Alta Vista Phytochemicals (Hyderabad, India), and
   biotinylation of WA was performed by Dr. P. Van der Veken (WA-BT;
   Universiteit Antwerpen, Belgium). Both formulations were stored as 20
   mM stocks in DMSO at −20 °C as previously described [[248]48,[249]107].
   IBR (IMBRUVICA^®) stock solutions were obtained from Pharmacyclics
   (Sunnyvale, CA, USA). Antibodies BTK (3533) and GAPDH (2118S) were
   obtained from Cell Signaling Technology (Danvers, MA, USA).

4.6. Cell Viability after WA Washout

   After 15 min treatment of MM1R cells with increasing concentrations of
   WA (with or without DTT), cells were washed extensively with PBS (3 × 5
   min) and left to grow for an additional 24 h in WA-free supplemented
   RPMI-1640 medium. Once incubation was complete, cell viability was
   determined by colorimetric MTT assay as described above. MM1R cells
   continuously treated with WA for 24 h were included to allow for a
   direct comparison of covalent and non-covalent effects on cell
   viability.

4.7. Protein Extraction and Western Immunoblot Analysis

   For western immunoblot analyses, cell pellets were lysed in 0.5 mL RIPA
   buffer (150 mM NaCl, 0.1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl pH 8)
   supplemented with protease inhibitors (Complete Mini^®, Roche, CA,
   USA). Soluble protein extracts were obtained after 15 min incubation on
   ice, followed by brief sonication and centrifugation at 16 g for 20 min
   at 4 °C. Samples were separated by sodium dodecyl
   sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred
   onto nitrocellulose membranes (Hybond C, Amersham) following standard
   protocols. After blocking, membranes were incubated overnight at 4 °C
   with the primary antibodies, followed by dye-conjugated secondary
   antibodies (polyclonal goat anti-rabbit HRP, #P0448, Dako). Bound
   complexes were detected with the Amersham Imager 680 (Cytiva, Austria)
   and quantified by Image J software (Max Planck Institute, Dresden,
   Germany) [[250]108]. Original immunoblot images are provided in
   [251]Supplementary Table S4.

4.8. WA-Biotin-Based Affinity Purification

   WA-BT affinity purification and co-precipitation were performed as
   previously described [[252]48]. In short, MM1R and U266 cells were
   seeded in 10 cm culture dishes and incubated for 24 h at 37 °C. Cells
   were treated with biotinylated WA (1 µM, 2.5 µM, 5 µM or 7.5 μM as
   indicated) or left untreated for 2 h. Cells were then lysed in 1 mL
   lysis buffer (5 mM Tris pH 7.6, 1% Triton-X100 and 5 mM EDTA,
   supplemented with Complete™ protease inhibitor cocktail) and incubated
   with Neutravidin beads overnight. Beads were centrifuged for 5 min at
   500 g after which the supernatant was removed. Beads were subsequently
   washed 5 times with lysis buffer. Total protein lysates, or
   coprecipitates, were separated by SDS–PAGE and electrotransferred onto
   a nitrocellulose membrane. Blots were probed using the appropriate
   antibodies, and the immunoreactive proteins were detected using the
   Amersham Imager 680 (Cytiva, Austria).

4.9. Immunofluorescence Confocal Microscopy

   After treatment with WABI, MM1R cells were fixed in 4% (v/v)
   formaldehyde. Cells were stained with rhodamine–phalloidin (Molecular
   Probes, Thermo Fisher Scientific Inc., Waltham, MA, USA) to visualize
   the cellular morphology, Hoechst 33,258 (Sigma-Aldrich, St. Louis, MO,
   USA) to visualize the nucleus and streptavidin-Alexa Fluor555
   (Molecular Probes, Thermo Scientific, MA, USA) to visualize
   biotinylated WA. To determine WABI colocalization with specific
   proteins (BTK), WABI-treated MM1R cells were immunostained with primary
   (anti-rabbit BTK, Cell Signaling Technology, mAb#8547) and
   corresponding labeled secondary antibodies (anti-rabbit Alexa
   Fluor-488, Molecular Probes, Thermo Scientific, MA, USA). Cells were
   then placed on glass slides and mounted with coverslips using
   Fluoromount^TM (Sigma-Aldrich, St. Louis, MO, US)Confocal imaging was
   carried out by a laser-scanning microscope equipped with a
   Plan-Apochromat 63X/1.40 Oil DIC objective lens and excitation
   wavelengths 405, 488, 561 and 640 nm (Zeiss LSM 800, Carl Zeiss,
   Germany). At least 20 different microscopic fields were analyzed for
   each sample using ZEN imaging software (Carl Zeiss). ZEN lite^TM (Carl
   Zeiss, Germany) was used to perform image reconstruction and
   presentation.

4.10. Covalent Docking of WA with BTK

   In silico molecular docking studies of the BTK protein (PDB id: 6TFP)
   with WA (PubChem CID: 26537) were performed as previously described
   [[253]109]. Briefly, BTK and WA structures were energy minimized with
   Swiss-PdbViewer (v4.1) [[254]110] and UCSF Chimera (Geneva Biomedical
   Institute, Geneva, Switzerland) [[255]111], respectively. Molecular
   docking and calculation of the docking scores were performed with
   Autodock4 (v4.2.6, University of California, Berkeley, CA, USA). The
   obtained docking solutions were evaluated based on their scoring, and
   the generated poses were clustered based on the ligand’s cluster ranks.
   Finally, the clusters were differentiated based on the covalent bond
   lengths and prime energies. Final docking results were visualized with
   LIGPLOT (v.4.5.3, University College London, UK) [[256]74] and PyMOL
   (v.2.4, Schrödinger, NY, USA) [[257]112] software.

4.11. Cell Transfections and BRET Measurements

   BTK- and BTK(C481S)-NanoLuc^® fusion vectors were purchased from
   Promega and used in BRET target engagement experiments. HEK-293 cells
   were transfected with the NanoLuc^® fusion vectors using FuGENE HD
   (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
   Briefly, fusion vectors were diluted into transfection carrier DNA
   (Promega, Madison, Wisconsin, USA) at a mass ratio of 1:10, after which
   FuGENE HD was added at a ratio of 1:3. The FuGENE HD complexes were
   then added to HEK-293 cells (1:20 ratio) at a density of 2 × 10^5 per
   mL. Subsequently, cells were plated onto white, 96-well plates
   (Corning, NY, USA) at a density of 2 × 10^4 cells/well and left to
   incubate for 24 h at 37 °C and 5% CO[2]. After incubation, cells were
   equilibrated for 2 h with the NanoBRET tracer reagent (K11, Promega,
   Madison, WI, USA) and increasing WA concentrations (0.5–10 µM). To
   measure BRET, NanoBRET NanoGlo substrate and extracellular NanoLuc
   inhibitor (Promega, Madison, WI, USA) was added according to the
   manufacturer’s protocol, and filtered luminescence was measured on a
   GloMax Discover luminometer equipped with 450 nm BP filter and 600 nm
   LP filter. MilliBRET units (mBU) were calculated by multiplying raw
   BRET values by 1000 and plotted with GraphPad Prism.

4.12. Nucleofection of siBTK and C481S BTK

   The BTK(C481S)-NanoLuc^® Fusion Vector was purchased from Promega, and
   siBTK was purchased from GE-Healthcare Bio-sciences (Accell Human BTK
   siRNA, EQ-003107–00–0005, 29121299, sequence supplied in [258]Table
   S3). MM1R cells were transfected with siBTK alone or siBTK combined
   with BTK(C481S) using the Nucleofector IIb device (Lonza, Switzerland)
   according to the manufacturer’s instructions. In short, 2.10^6 MM1R
   cells were resuspended in supplemented nucleofector solution, after
   which 300 nM siBTK was added with or without 2 µg BTK(C481S). To each
   nucleofection reaction, an additional 2 µg of pmaxGFP™ Vector was added
   as an internal positive control (transfection efficiency = 51.3 ±
   2.4%). Resuspended cells were subsequently transferred to a cuvette and
   transfected using the O-020 nucleofector program. After nucleofection,
   a pre-equilibrated medium was added, and cells were transferred to a
   96-well plate at a density of 80.000 cells/well. Eight hours
   post-transfection, transfection efficiency and GFP expression were
   assessed with fluorescence microscopy. If a GFP signal was present,
   cells were treated with increasing concentrations of WA and cell
   viability was measured after 24 h using the MTT colorimetric method.
   BTK mRNA expression of nucleofected cells was further confirmed by
   quantitative real-time PCR.

5. Conclusions

   Although cysteine code classification has been developed to explain the
   selectivity of different electrophiles classes, many cysteine-reactive
   natural products have shown promiscuous therapeutic anticancer
   activities with poorly characterized targets
   [[259]87,[260]113,[261]114]. In a high-throughput compound library
   screening, the cysteine reactive phytochemical WA was identified as a
   top prioritized investigational compound to suppress hyperactivated BCR
   cancer signaling, together with IBR [[262]63]. Given that WA possesses
   broad-spectrum therapeutic activities in several (drug-resistant)
   cancer cell types, including B-cell lymphoma and MM [[263]46,[264]51],
   we further characterized its molecular mechanisms of action in
   GC-resistant MM cells by combining innovative transcriptomic and
   phosphor-peptidomic kinase (activity) profiling approaches. Our
   findings demonstrate that the TK inhibitory profile of WA in
   GC-resistant MM cells is similar to that of IBR and that
   hyperphosphorylation of many BCR-related kinases, including BTK, is
   inhibited. Because WA also covalently targets BTK in a
   cysteine-dependent manner, we hypothesize that WA inhibits BTK
   autophosphorylation and activation in an IBR-like fashion. We
   additionally found that WA silences BTK expression in GC-resistant MM
   cells at the mRNA and protein level, suggesting that WA suppresses BTK
   cancer signaling via a dual mechanism. Since WA displays high a
   similarity with IBR mode of action, but with a more potent effect on
   cell death of GC-resistant MM cells, further structure-function
   analysis of WA-analogs may allow (pre)clinical optimization of highly
   effective withanolide BCR inhibitors to expand the arsenal of cancer
   drugs against therapy-resistant B-cell malignancies.

Acknowledgments