Bruton tyrosine kinase (BTK) was originally identified as a non-receptor tyrosine kinase whose deficiency is responsible for X-linked agammaglobulinemia (XLA) in humans [1, 2, 3]. Patients with XLA suffer from severe antibody deficiency and recurrent infections due to a differentiation defect causing the absence of mature B lymphocytes [4, 5, 6]. Conversely, inhibition of BTK has been shown to ameliorate various forms of leukemia and lymphoma [7, 8], with the premier example being chronic lymphocytic leukemia (CLL) [9, 10, 11]. Ibrutinib is the first such inhibitor of BTK to be clinically applied [7, 8, 10, 11]. However, there remain uncertainties as to how it alters signaling mediated by BTK in vivo, ultimately affecting development, physiology, and behavior at the organismal level. In addressing these questions, a genetically tractable in vivo animal model would be most useful.

Drosophila melanogaster (hereinafter Drosophila) is an outstanding model organism due to its amenability to experimental manipulations at the molecular, cellular, and organismal levels. The Btk29A type 2 isoform encoded by the gene Btk29A is the sole ortholog of human BTK in Drosophila: the other isoform, Btk29A type 1, has a unique N-terminus and distinct expression and is thus structurally and functionally separable from Btk29A type 2 [12]. Btk29A type 2 has been shown to play an essential role in dorsal closure in postembryonic development [13], a morphogenetic process to suture the left and right halves of the exoskeleton. Btk29A type 2 is also pivotal for stem cell niche functions, e.g., germ cell proliferation and differentiation, as well as follicle precursor migration in the ovary at the adult stage [13, 14, 15, 16, 17, 18]. In the ovary, the Btk29A type 2 protein was shown to bind to $\beta$-catenin and to phosphorylate specific tyrosine residues of $\beta$-catenin to modulate Wnt4 signaling, thereby regulating germ cell development [19, 20]. Furthermore, Btk29A type 2 was shown to mediate behavioral habituation in adult flies, which was abrogated by ibrutinib [18]. Thus, Drosophila will offer an ideal platform to evaluate the actions of ibrutinib on the BTK ortholog. Here we show that dietary ibrutinib recapitulates the effects of the Btk29A mutant on the morphogenesis of external structures and germ cell development, likely through its interference with $\beta$-catenin–Btk29A interactions.

Visual inspection of the notum of adult flies suggested that feeding ibrutinib throughout the larval stage results in the widening of the distance between left and right homologous bristles along the midline (Fig. 1A–D,G–J), a phenotype also observed in Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutants (Fig. 1E,F,P). Measurements of the distance between the midline and innermost bristles in the control and test groups of flies demonstrated that feeding ibrutinib significantly widens this distance (Fig. 1P, Supplementary Fig. 1), suggesting a defect in dorsal closure in the ibrutinib-fed flies. Failures in dorsal closure were also manifested as an incomplete fusion of bilateral tergites in Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutants (Fig. 1K–M), which was phenocopied by feeding an ibrutinib-containing diet to flies of control genotypes (Fig. 1N,O,Q). It remains an open question as to whether flies with a severe thorax phenotype concordantly have a severe dorsal phenotype. In addition, we noted that a substantial proportion of ibrutinib-fed flies display distorted wing structures, particularly a loss of certain regions near the wing margin (Fig. 2C cf. Fig. 2A,B). Similar wing phenotypes were observed with an equivalent frequency in Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutant flies that were not treated with ibrutinib (Fig. 2C,E).

Fig. 1.

Dorsal nota and tergites of Drosophila melanogaster. (A–O) Dorsal nota (A–J) and tergites (K–O) of male wild-type (A, B, K and I, J, O), Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$/+ (C, D, L and G, H, N), or Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ (E, F, M) flies without (A–F and K–M) or with the administration of 10 µL of 50 µM ibrutinib onto 2.6 mL fly culture media (G–J and N and O), throughout the life cycle. The boxed region in (A, C, E, G, and I) is enlarged in (B, D, F, H, and J), respectively. (P and Q) Quantitative analysis of suture levels in the dorsal notum and tergite. (P) The distances were measured for a bilateral bristle pair that were positioned closest to the midline. Shown are the distances relative to the mean inter-bristle distance of wild-type flies (wt) for indicated fly groups. The numbers of flies examined are indicated in parentheses for each fly group in (P) and (Q). The data for untreated wild-type flies are indicated as wt, whereas the data for flies treated with solvent DMSO only are indicated as wt: ctrl (these designations were also used in the other figures). The statistical significance of differences was evaluated by Student’s t-test: **p 0.01 (P) or by Fisher’s exact test: **p 0.01 (Q).

Fig. 2.

Wing phenotypes in flies deficient in functional Btk29A type 2. (A–F) Wings of wild-type (A, D, and F) flies without (A) or with ibrutinib (D and F) and Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$/+ (B) and Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ (C and E) flies without ibrutinib throughout the life cycle. (G) The proportion of flies (%) with wing defects is indicated in each panel. **Statistically significant at p 0.01 by Fisher’s exact test.

We then examined, in wild-type flies, the possible effects of ibrutinib feeding on oogenesis, during which Btk29A mutations are known to induce overproduction of cystoblasts (see below), which are directly derived from germ stem cells (GSCs); a cystoblast differentiates into cystocytes that transform into an oocyte and nurse cells after four rounds of divisions [21, 22]. This process of germ cell proliferation takes place in the anterior tip of an ovariole called the germarium, which contains one to three GSCs, several cystoblasts, and tens of cystocytes in wild-type flies [21]. Feeding ibrutinib caused underproduction of cystoblasts (Fig. 3E,F,K and L cf. Fig. 3A,G), as identified by the presence of spectrosomes (recognized as round structures rich in actin), often leading to a complete lack of cystoblasts in a substantial proportion of germaria (Fig. 3M). This is in sharp contrast to the germaria of Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutants, where supernumerary cystoblasts were usually observed (Fig. 3B,H,M). However, when Btk29A was knocked down with Btk29A RNAi in escort cells that support the maintenance of GSCs in the germarium, some ovaries of the manipulated flies carried an empty germarium (Fig. 3D,J,M). We infer that GSCs failed to reproduce themselves as the result of a strong bias toward differentiating into cystoblasts, resulting ultimately in the empty germarium. Conversely, when a GSC was maintained even upon accelerated cystoblast differentiation, the number of cystoblasts would increase. Because the Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutation removes the type 2 Btk29A isoform, sparing the type 1 isoform, it might be that the type 1 isoform partially compensated for the lack of type 2 so that the GSC was maintained and produced more cystoblasts. In contrast, the UAS-Btk29A RNAi transgene likely knocked down both type 1 and type 2 isoforms, leading to contrasting outcomes, i.e., loss of the GSCs and cystoblasts.

Fig. 3.

Btk29A deficiency impairs germ cell production in the ovary. (A–F) Enlarged views of the germarium at the anterior tip of the ovary, which contains the niche where germ stem cells (GSCs) divide into cystoblasts while reproducing GSCs themselves. (G–L) Schematic drawings of the germarium are shown below the photographs to highlight germline cells containing spectrosomes. Compared with the wild-type germarium (A and G), the Btk29A${}^{\text{𝑓𝑖𝑐𝑃}}$ mutant germarium (B and H) contains excessive cystoblast-like cells immunopositive to monoclonal antibody 1B1, which labels the spectrosome (green), a round structure characteristic of GSCs and cystoblasts. The anti-Vasa antibody was used to counterstain all germline cells (magenta). The majority of wild-type females fed on diets containing ibrutinib (E and K) carried germaria containing a reduced number of cystoblasts. Some wild-type females treated with ibrutinib (F and L) had germaria devoid of any germline cells, indicative of GSC loss. (M) The results of quantitative analysis of the numbers of cystoblasts contained in the fly germaria of different groups are shown. The proportions of germaria containing different numbers of spectrosome-containing cells are shown; red: 0–1 cells; yellow: 2–3 cells; blue: $>$4 cells (see the Supplementary Tables for details. **Statistically significant at p 0.01 by Fisher’s exact test).

Finally, we examined whether ibrutinib affects molecular interactions between Btk29A and $\beta$-catenin. As an experimental system, we chose the primate cell line Cos7 rather than flies because the former express no endogenous Btk, simplifying the interpretation of the assay result, while the latter has two Btk29A isoforms under complex control by other kinases, which complicates the analysis of Btk29A-$\beta$-catenin interactions. Indeed, our previous experiment demonstrated that fly Btk29A phosphorylates endogenous $\beta$-catenin at its Y142 when transfected into Cos7 cells [19]. Btk29A carrying an HA tag was transfected into Cos7 cells, and the cell lysates were subjected to immunoblotting with an anti-pY142 $\beta$-catenin antibody. The tyrosine residue at position 142 (Y142) is one of the major Btk29A-dependent phosphorylation sites of $\beta$-catenin in vivo [19]. The results showed that incubation of cells with 10 µM ibrutinib reduced the amount of a Y142-phosphorylated population of endogenously expressed $\beta$-catenin (Fig. 4A), and this reduction was statistically significant (Fig. 4B). We suggest that ibrutinib interferes with Btk29A–$\beta$-catenin interactions, thereby manifesting defects in oogenesis and other morphogenetic processes in vivo. It remains to be examined whether the phosphorylation of Armadillo Y150 homologous to $\beta$-catenin Y142 is inhibited by ibrutinib in the fly ovary.

Fig. 4.

Ibrutinib reduces phosphorylation of Y142 $\beta$-catenin in Cos7 cells. (A) Western blotting of Cos7 cell lysates probed with the anti-pY142 $\beta$-catenin antibody (upper panel), the anti-HA antibody that reacts with Btk29A type 2::HA (middle panel), or anti-$\beta$-catenin antibody (lower panel) in the absence (-) or presence (+) of ibrutinib (shown above the panels). Lysates were immunoprecipitated with an anti-$\beta$-catenin antibody and probed with anti- anti-pY142 $\beta$-catenin antibody. Cos7 cells were transfected with an empty vector (indicated as Control) or Btk29A type 2 tagged with HA, while $\beta$-catenin was endogenously expressed. (B) Quantification of western blot results. The signal intensity of bands was evaluated by band-area densitometry measurements and normalized by the amount of endogenous anti-$\beta$-catenin. **p 0.01, ***p 0.001, n.s., statistically non-significant.

In the present work, we showed that ibrutinib faithfully reproduced some of the Btk29A mutant phenotypes in Drosophila when orally administered during the larval and adult stages, an observation consistent with the view that BTK and its ortholog are the major targets for ibrutinib in both humans and flies. It is also reminded that ibrutinib does not distinguish between the two Btk29A isoforms because they share the same kinase domain. Our results also point to the possibility that Drosophila, with its genetic tractability and rich collections of molecular resources, may provide an ideal in vivo experimental system for elucidating the mode of action of therapeutic drugs that target BTK. Drosophila will be particularly effective when used to screen chemicals that potentially act on BTK for the development of novel therapeutic drugs to control CLL because a large number of flies can be easily obtained for rounds of assays, and due to their short life cycle, life-long chronic effects of compounds can be evaluated in only a few weeks. Visible phenotypes such as a collapse in the dorsal closure and wing formation and other external structures [23, 24] will help to promptly reveal the effects of compounds without dissection of tissues or any other experimental handling, making it possible to enrich promising candidate molecules within a short period of time.

While our phenotypic and biochemical data presented in this and other works suggest functional homology between human BTK and fly Btk29A [25, 26], one caveat is that we have no direct evidence that ibrutinib exerts its effects on BTK/Btk29A through the same mechanism in humans and flies. In inhibiting human BTK, irreversible inhibitors, including ibrutinib, bind to the ATP pocket, in which Cystine481 plays an important role in inhibitor–BTK interactions [11, 27]. However, fly Btk29A harbors a cystine-to-serine replacement at the corresponding position. Irreversible inhibitors, including ibrutinib, were found to be less effective for patients with CLL carrying the cystine-to-serine substitution in BTK. It remains to be clarified how ibrutinib modulates the Btk29A functions in flies. It might be envisaged that fly Btk29A has a higher order structure, distinct from that of human BTK, which compensates for the effect of cystine-to-serine replacement that reduces the binding of ibrutinib and other irreversible inhibitors.

Feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants. Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

NHK—Investigation, writing, reviewing, editing; CIES, BFN, YE, RZ—Conceptualization, reviewing; DY—Writing, reviewing, editing, conceptualization, and interpretation of data for the work. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Not applicable.

We thank Yoshimi Takamura for secretarial assistance. The authors are grateful to the Bloomington Drosophila Stock Center (BDSC) and Vienna Drosophila Resource Center (VDRC) for providing fly strains.

This work was supported in part by a MEXT Grant-in-Aid to DY (21H04790), a Grant-in-Aid for JSPS Fellows to NHK, and grants from the Center for Innovative Medicine to CIES, the Swedish Cancer Society (22 2361 Pj 01 H) to CIES, the Stockholm County Council (ALF-project) to CIES, and The Swedish Cancer Society (201044 PjF) to YE.

The authors declare no conflict of interest.