Dysregulation in the phosphoinositide 3-kinase (PI3K) signalling pathway has continually been implicated in multiple types of disease such as cancers, immune metabolic diseases and auto-immune conditions (1). This signalling pathway is critical in modulating immune cell functions such as homeostasis, metabolism and proliferation. In lymphocytes, the activation of class 1 PI3Ks is controlled by antigen receptor, co-stimulatory receptors, cytokines and chemokine receptors (2). Of the four class I PI3K enzymes, PI3Kδ and PI3Kγ are selectively expressed in immune cells (3, 4) PI3Kδ has critical functions in B cells as well as T cells (5), whereas the functions of PI3Kγ in T cells, macrophages and neutrophils are most well established (6). We found that chronic lymphocytic leukemia cells upregulate PI3Kγ and this isoform has non-redundant functions in chemokine responses (7).

Autoimmune diseases result from chronic lymphocyte activation targeting self-antigens resulting from disruption of normal mechanisms maintaining immune tolerance (8, 9). There are over 100 auto-immune diseases affecting a wide range of the population (3-5%), and although there is paucity of information on the exact etiological events leading up to the disease, certain factors such as genetics, environment, sex, diet have been attributed as risk factors (10–13). Systemic lupus erythematosus (SLE) is an autoantibody-mediated disease characterized manifested by a broad spectrum of antibodies targeting nuclear proteins and nucleic acids and immune-complex deposition in the kidney (8). The traditional treatment options for autoantibody-mediated conditions such as SLE include immune suppressive drugs (cyclophosphamide) and non-specific anti-inflammatory (corticosteroids) (9). While more specific and effective treatment options are clearly needed, only two new treatments have been approved in the last 60 years: antibodies targeting the B cell growth factor BAFF (Belimumab) (14) or the type 1 interferon receptor (Anifrolumab) (15).

Several lines of evidence implicate the PI3K pathway in autoimmunity. The inhibitory phosphatase PTEN is primarily known as a tumour suppressor, but has also been described as one of the “gatekeepers” of immunological tolerance (16). Reduced expression of PTEN in SLE patient B cells was suggested as one mechanism of PI3K pathway dysregulation and breaking tolerance (17). Another PI phosphatase SHIP is also known to restrain autoreactive B cells, with acute deletion of SHIP leading to development of autoimmune disease in mice (18). Thus, uncontrolled PI3K pathway activity is implicated in both driving malignant B cell proliferation and driving activation of autoreactive B cells. While the specific PI3K isoforms important in the context of autoreactive B cells remain unknown, treatment with a PI3Kδ-specific inhibitor was able to restore B cell tolerance in PTEN +/- x SHIP +/- mice (19).

Once activated, class I PI3Ks phosphorylate inositol lipid headgroups in the plasma membrane leading to the generation of two major phosphoinositide (PI) species PI(3,4,5)P3 and PI(3,4)P2. These PI products are key players that function in the binding and activation of other downstream signaling molecules with a PI-binding motif (20, 21). The tandem PH domain containing proteins TAPP1 and TAPP2 are adaptor proteins recruited to the plasma membrane by binding specifically to PI(3,4)P2 via their C-terminal PH domains (22, 23). In B cells, TAPP adaptor proteins have functions in regulating germinal centre responses and humoral immunity that depend on PI(3,4)P2 binding. This has been demonstrated by introducing point mutations to TAPP1 and 2 genes within their PI-binding regions, thereby compromising their ability to bind PI(3,4)P2 (24). Uncoupling TAPPs from PI(3,4)P2 resulted in dysregulated activation of the kinase Akt, another PI3K-dependant signaling molecule, as well as dysregulated B cell metabolic programming (25). These TAPP mutant mice develop chronic B cell activation and germinal centres, generate autoantibodies and ultimately develop a lupus-like autoimmune disease.

PI3K pathway inhibition with kinase inhibitor drugs has been extensively explored in anti-cancer therapy, based on genetic or regulatory abnormalities leading to constitutive pathway activity in various cancers (26). Currently approved PI3Ki such as Idelalisib and Alpelisib potently inhibit specific class I PI3Ks isoforms (delta and alpha respectively). Duvelisib is another new generation class I PI3Ki that has been developed to exert dual PI3Kδ/γ inhibition in B cell malignancies (7, 27–31). Several studies have found that dual PI3Kδ/γ inhibitor can also have anti-inflammatory activity in mouse models (32, 33); for example, in an arthritis disease model, individual inhibition of PI3Kδ or γ played a subtle role in diminishing joint disease, while the dual inhibition of PI3Kδ/γ was superior in reducing the induction and progression of inflammatory cells and neutrophils into the joints (34). The impact of PI3Kδ/γ inhibition in the context of autoantibody-mediated disease remains to be explored.

Here we sought to assess the potential of current clinical-grade PI3K inhibitors as therapy for SLE by treating TAPP KI mice with the dual PI3Kδ/γ inhibitor Duvelisib. We find that treatment with dual PI3Kδ/γ inhibitor diminished chronic GC and plasma cells as well as autoantibody levels within these mice. These findings indicate that PI3K inhibition pathway is a potential therapeutic target in controlling SLE type autoimmune disease.

Novel and innovative strategies for targeting autoantibody-producing B cells in diseases such as SLE are urgently needed. The use of small molecule kinase inhibitors to shut down chronic BCR signaling is a concept that is now well established for treatment of B cell malignancies, with several clinically approved drugs being widely used and more in development. Several lines of evidence suggest that targeting BCR signaling may be of benefit in SLE. A significant fraction of SLE susceptibility genes identified in genome-wide association studies are B cell signaling molecules, indicating that genetic perturbations in B cell signaling pathways can underlie development of SLE (40, 41). Numerous mouse models with disruptions in BCR and co-receptor signaling have been found to lead to development of SLE-like disease pathology (42–44). Finally, as described below a growing number of studies have indicated that treatment of mouse autoimmunity models with pre-clinical kinase inhibitors can reduce disease pathology. Together this evidence suggests that some clinically approved kinase inhibitors initially developed for treatment of B cell malignancies could potentially be repurposed for treatment of autoantibody-mediated disease.

The PI3K pathway is most well-known for its roles in cancer, however multiple lines of evidence have implicated it in autoantibody-mediated disease. Mouse models exhibiting elevated PI3K pathway activity due to deficiency in the activity of PI phosphatases such as SHIP1 or PTEN were found to develop autoantibody-mediated pathology (18, 45). Indeed the key inhibitory receptor on B cells FcγRIIB functions to recruit SHIP (46), and this inhibitory circuit has been implicated in human and mouse SLE studies (47). PTEN has been described as a “gatekeeper” of B cell tolerance, and has been found to be dysregulated in human SLE patients (16, 48). Recent studies found that PI3Kδ gain-of-function mutations led to generation of autoantibodies (49). While most studies indicate that PI3Kδ is the most critical isoform activated by BCR signaling, we chose to examine a dual PI3Kδ/γ inhibitor after noting that plasma cells can downregulate PI3Kδ and increase PI3Kγ expression ( Figure 1 ). Another potential benefit of this approach is that both the PI3Kδ and γ isoforms are restricted to hematopoietic cells, unlike PI3Kα inhibitors being actively developed for treatment of solid tumours (50).

In this study we have assessed a PI3K inhibitor drug developed and approved for treatment of B cell leukemia and lymphoma in the TAPP KI mouse model. We previously found that this model exhibits chronic B cell activation and germinal center responses driven by dysregulated PI3K pathway activity (24, 25, 44). Our findings here indicate that the dual PI3Kδ/γ inhibitor Duvelisib can significantly reduce chronic GC and plasma cells responsible for auto-antibody production. The significant reduction of autoantibodies observed within a four-week treatment period is remarkable given that we are reversing established disease and that the half-life of pre-existing IgG antibodies is in the order of weeks. However, in most cases, the reduction was only partial in that it did not reduce autoantibodies to the baseline level of C57BL6 mice. Interestingly, the treatment selectively altered the profile of autoantibodies produced, with the majority showing a trend of reduced levels, but only ~20% being significantly lower. Consistent with a partial reduction in autoantibodies, we found evidence for reduced IgG deposition in kidney and improvement of glomerulonephritis. Improvements in kidney pathology could potentially reflect the effect of PI3Kδ/γi on other elements of the inflammatory process beyond the autoantibodies themselves.

While PI3Kδ/γ inhibition can clearly impact directly on B cells, it is likely to also impact on other cells of the immune system including T cells and myeloid cells. Indeed, we observed reductions in T_FH cell populations, which could be due either to direct effects of the treatment on T cells or indirect effects since interactions with GC B cells are known to promote T_FH activation and differentiation. Both PI3Kδ and PI3Kγ are expressed and functional in CD4+ T cells and PI3Kδ has been shown to be important for T_FH development and function (51, 52). It should be noted that the aim of our study was to assess the potential of repurposing a clinically approved PI3K inhibitor and was not designed to assess B cell-intrinsic versus extrinsic affects.

Other studies have examined the impact of pre-clinical inhibitor compounds selectively targeting PI3Kδ or PI3Kγ on development of various pathologies in mouse models of SLE (53–55); however their impact on B cell subsets and autoantibody profiles were not determined. An inhibitor targeting Bruton’s tyrosine kinase (Btk), whose activity is dependent on binding to PIP3 produced by PI3K, was also found to inhibit generation anti-DNA antibodies in the MRL/Lpr mouse model (56). One study reported that treatment of NZB mice with IPI-145/Duvelisib for 20 weeks reduced anti-DNA antibodies and kidney pathology, consistent with our findings (32). More recently the Cambier group used an elegant model of inducible PI3K pathway dysregulation to acutely reactivate anergic BCR transgenic B cells and demonstrated that even low doses of PI3Kδ-specific inhibitor Idelalisib could prevent generation of autoantibodies in this context (19). Interestingly, reactivation of anergic B cells via deletion of the protein phosphatase SHP1 was not reversed by Idelalisib (19). Thus, our results extend previous findings, which collectively support the concept that dual PI3Kδγ inhibition can reverse autoimmunity driven by PI3K dysregulation.

As an expanding class of small molecule therapeutics, PI3K inhibitors are clearly highly bioactive with broad potential for applications in diseases beyond the initial applications for treatment of B cell malignancies. Notably, inhibitors of class I PI3Ks have been investigated in a number of other autoimmune and inflammatory disorders, including asthma/COPD, collagen-induced arthritis, experimental autoimmune encephalitis and type 1 diabetes (57). While PI3Ki are effective in B cell malignancies, their use has been limited by unexpected toxicities including T cell mediated inflammation (58, 59). Inflammatory toxicities have been largely observed in chronic lymphocytic leukemia patients treated with PI3Kdelta or delta/gamma inhibitors for prolonged periods of time (60, 61). As our study assessed a short-term treatment regimen, we did not assess the development of inflammatory toxicities, which are likely related to impairments in normal regulatory functions of lymphocytes and macrophages (62–64). Thus, a current challenge in developing and repurposing PI3K inhibitors will be to optimize specificity and dosing regimens to maximize benefits while minimizing unwanted effects depending on the disease context.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by University of Manitoba Animal Care Committee.

FA and SH carried out experimental work. FA and AM analyzed data. FA and AM wrote the paper. All authors contributed to the article and approved the submitted version.