Expanding the scope of PI3K-δ inhibition: Leniolisib treatment in PRKCD deficiency

Protein kinase C δ (PKCδ) deficiency is an inborn error of immunity (IEI) caused by autosomal recessive variants in PRKCD that result in PKCδ loss-of-function (1, 2, 3). PKCδ is a ubiquitously expressed serine-threonine kinase involved in cell proliferation, differentiation, and apoptosis. Once activated, PKCδ is modulated by phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (4).

Although the interaction between PKCδ and PI3K remains incompletely defined, PKCδ may inhibit spleen tyrosine kinase, influencing downstream PI3K-dependent signals in B lymphocytes (4, 5) (Fig. 1 A). Furthermore, previous studies have shown that pharmacological inhibition of PRKC activity leads to AKT hyperphosphorylation, suggesting that the PI3K/AKT signaling pathway is negatively regulated by PKC (6). PKCδ also impacts the function of other immune cells (4). Knockout (Prkcd^−/−) mice have disrupted immune homeostasis, resulting in autoimmunity, lymphoproliferation, and B cell infiltration (3, 7, 8).

Since 2013, PKCδ deficiency has been reported in 21 patients (Table 1, 3, 9, 10, 11, 12, 13, 14, 15, 16, 17), revealing a diverse clinical phenotype paralleling the murine model. Patients most commonly present with early-onset and often severe autoimmunity, particularly lupus-like disease with multiorgan involvement, including immune-mediated cytopenias, nephritis, and systemic inflammation. Lymphoproliferative features such as persistent lymphadenopathy and splenomegaly are frequently observed. Recurrent and sometimes severe infections are also reported, involving both common bacterial pathogens and opportunistic organisms. Most cases present with a combination of autoimmunity, lymphoproliferation, and susceptibility to infections, while some exhibit a singular phenotype. Manifestations overlap with other disorders, including autoimmune lymphoproliferative syndrome, monogenic systemic lupus erythematosus, common variable immunodeficiency, and chronic granulomatous disease (3). Accordingly, therapeutic strategies vary (Table 1) and mainly include immunomodulatory agents such as mTOR inhibitor rapamycin. Hematopoietic stem cell transplantation (HSCT) was performed in two cases (9).

Leniolisib, a selective PI3Kδ inhibitor, is approved only for the treatment of activated PI3Kδ syndrome (APDS) in patients aged ≥12 years (18). APDS is characterized by increased AKT/mTOR signaling due to hyperactive PI3Kδ (19, 20). Inhibition restored immune cell function while attenuating lymphoproliferation (21, 22, 23). Here we present the first successful application of leniolisib for the treatment of PRKCD deficiency.

A 14-year-old male offspring of consanguineous parents (first cousins) was diagnosed with a pathogenic homozygous variant in PRKCD (NM_006254.4, c.1352+1G>A), leading to protein loss (1, 2). The parents were unavailable for segregation testing, but an older brother who experienced primary immunodeficiency with lymphoproliferation, autoimmunity, and predominant renal involvement had the same biallelic variant. The sibling clinical picture was characterized by membranous glomerulonephritis leading to chronic kidney disease in a solitary kidney, organizing pneumonia with splenomegaly and lymphadenopathy, and recurrent infections. Since early childhood, the proband experienced multiple hospitalizations for invasive bacterial and viral infections, including multidrug-resistant Staphylococcus epidermidis sepsis, human herpesvirus six encephalitis, recurrent Mycoplasma pneumoniae pneumonia, perforated Pseudomonas aeruginosa otitis media, and two episodes of respiratory syncytial virus pneumonia. Immunophenotyping revealed reduced memory B cells, increased transitional B cells, elevated immunoglobulin (Ig)M, and hypogammaglobulinemia that were treated with Ig replacement therapy and antibacterial prophylaxis. Moreover, he experienced autoimmunity and lymphoproliferation. In fact, by 12 years of age, he developed alopecia areata, trilineage cytopenia, autoimmune hepatitis, enteritis, splenomegaly, fluctuating lymphadenopathies, and thymic hyperplasia. Table 2 summarizes baseline findings.

To address autoimmunity and lymphoproliferation, rapamycin was selected as a first-line treatment. Hepatic cytolysis and spleen size reduced, while cytopenias improved. Fluctuating lymphadenopathies, leukopenia, and spleen lesions persisted; thymic hyperplasia progressed. Despite the adequate rapamycin dose and plasma levels at the upper-end of therapeutic range, during rapamycin treatment, the patient developed severe, asymptomatic, infiltrative lung disease characterized by a restrictive pattern on lung function tests, reduced diffusing capacity, and evidence of interstitial involvement. Differential considerations, including PRKCD-related disease progression and potential hypersensitivity to rapamycin, precluded establishing the cause of lung involvement (24). Because the pulmonary involvement could not be clearly attributed to PRKCD-related progression versus drug toxicity, and because the patient’s sirolimus trough levels were already at the upper end of the therapeutic range, further dose escalation was not considered safe. Rapamycin treatment was therefore withdrawn due to lack of efficacy and/or potential causative role in lung disease.

Given ongoing multisystem disease activity and the need to prevent further organ damage, we considered several therapeutic alternatives to sirolimus. A steroid-sparing strategy was prioritized in a preadolescent patient experiencing significant growth restriction, delayed pubertal development, and immunodeficiency. B cell depletors were deemed unsuitable due to anecdotal association with disease relapse and lack of effectiveness in the patient’s brother. In addition, it is increasingly clear that prolonged B cell depletion is not without significant consequences beyond secondary hypogammaglobulinemia. This approach entails risks such as sustained B cell aplasia, which cannot be fully mitigated by Ig replacement, particularly in a preadolescent with PRKCD deficiency affecting both B and T cell compartments.

Mycophenolate mofetil and other broad immunosuppressants were considered suboptimal due to limited evidence of durable control in PRKCD deficiency and the risk of cumulative toxicity in a preadolescent with underlying immunodeficiency. HSCT was not pursued due to multiorgan involvement, the uncertain impact of the PRKCD variant on extra-hematopoietic tissues, and limited data on outcomes.

Compassionate use of leniolisib was based on a precision, pathway-guided rationale with multiple clinical, molecular, and laboratory indicators of PI3K/AKT/mTOR pathway hyperactivity (Fig. 1). The index patient’s clinical and immune phenotypes overlapped with APDS. His T cells exhibited ex vivo hyper-phosphorylation of S6—an established downstream surrogate marker of the PI3K/AKT/mTOR axis, analogous to pAKT and used here as a proxy for mTOR activation (25)—at levels comparable to those observed in APDS patients. These findings align with the partial clinical response observed during rapamycin treatment and highlighting the in vivo relevance of pathway hyperactivity. We observed ex vivo normalization of stimulated phospho-S6 (pS6) in patient CD4⁺ and CD8⁺ T cells after experimental addition of rapamycin or idelalisib (a selective PI3Kδ inhibitor). Research suggests that PRKCD variants impair the inhibitory function of PKCδ on the PI3K pathway, strengthening the use of PI3Kδ inhibition to target underlying immune dysregulation (5). We performed the same in vitro pS6 assay, with addition rapamycin and idelalisib, in the older sibling obtaining comparable results—again consistent with mTOR pathway hyperactivity and normalization of S6 phosphorylation to healthy-donor levels upon pharmacologic inhibition (Fig. 1 C). The sibling was not considered a suitable candidate for in vivo administration of leniolisib for the end-stage kidney disease, which may have unanticipated impact on pharmacokinetics and plasma concentrations.

Based on this rationale, leniolisib was administered according to a compassionate-use protocol in this 14-year-old patient (weight 34.8 kg) and titrated to a maintenance dose of 40 mg twice daily; details of dose escalation are provided in the supplementary material. At the time of writing, the patient has received leniolisib for a total of 10 mo. Fig. 2 depicts the timeline of leniolisib treatment. The patient did not experience serious adverse events. Nonserious adverse events commonly reported among patients with APDS receiving leniolisib such as infections, skin rashes, gastrointestinal symptoms, fatigue, neutropenia, and elevated liver enzymes were not observed.

Sustained disease control and multiparameter improvement were evident (Table 3). The patient engaged in academic, social, and athletic activities. Self-perceived quality of life improved. Neither infections requiring hospitalization nor mild recurrent infections were reported. One episode of self-limiting enteritis was reported after the observation period. Trimethoprim/sulfamethoxazole was discontinued 8 mo after leniolisib initiation. Alopecia areata resolved without the need for topical glucocorticoids. No new autoimmune manifestations were observed. Cervical and axillary lymphadenopathy resolved.

Lung volumes and alveolar diffusing capacity improved despite the patient never undergoing prescribed respiratory physiotherapy and current tobacco use. Computed tomography (CT) scans performed before and after treatment revealed favorable structural changes related to interstitial lung disease, reduction of bronchiectasis and air trapping, and resolution of the tree-in-bud pattern (Fig. 3 A). Magnetic resonance imaging (MRI) and ultrasonography showed a progressive reduction in the size of the spleen (52.4%; Fig. 3 B), lymph nodes, and thymus (44.6%; Fig. 3 C) (Table S1). Splenic lesions resolved, and structural homogeneity was maintained.

Cytopenias resolved, with hemoglobin and platelets levels and white blood cell counts remaining within reference ranges (Table 3).

At presentation, absolute circulating B cell counts were at the lower end of the age-adjusted range, in the context of global lymphopenia. Immunophenotyping showed expansion of transitional B cells, reduction of memory B cells, and expansion of CD21^low B cells, consistent with a block in B cell maturation. Serum IgM was elevated (hyper-IgM pattern). During treatment, T and B cell populations improved (Fig. 3, D–F and Table 3). CD3⁺, CD4⁺, and CD4⁺ naive T cells increased. The percentage and absolute counts of total and naive B cells increased. We observed an early decrease in transitional and CD21^low B cells together with a fall in serum IgM, suggesting a partial release of this maturational block along the B cell lineage. Memory B cells showed only minimal early increase, which is expected to require longer time to recover.

During treatment, pS6 levels in CD4⁺ and CD8⁺ T cells were comparable to healthy controls (Fig. 3 G). No differences in pS6 levels were detected in samples obtained 10 h (corresponding to half-life of leniolisib) or 1 h (corresponding to maximum plasma concentration) after leniolisib intake, suggesting stable normalization of mTOR activity between doses.

This was the first compassionate use of leniolisib for the treatment of an IEI other than APDS (26). Leniolisib was well tolerated and improved clinical and laboratory parameters that were only partially controlled with rapamycin. Quality of life also improved: fatigue, infections, lymphoproliferation, or cytopenias were not reported after initiating leniolisib. The patient did not require corticosteroids, B cell depletors, or additional antibiotic treatment while receiving leniolisib.

Lung performance improved, with enhancements in lung volumes and alveolar diffusing capacity. The absence of complete disease reversal after rapamycin withdrawal suggests that pulmonary involvement was due to underlying disease.

Spleen size reduction and improvement in splenic structural changes were observed; the latter was present during rapamycin therapy. Episodic, persistent lymph node enlargements, which continued with rapamycin treatment, resolved during leniolisib treatment. Blood cell counts, which previously fluctuated during rapamycin therapy, normalized. Leniolisib treatment improved immunophenotype abnormalities and was consistent with outcomes reported among patients with APDS (21, 22, 23). mTOR hyperactivity (elevated pS6) in CD4⁺ and CD8⁺ T cells reduced to healthy control levels following treatment.

The molecular mechanisms underlying the impact of leniolisib in PRKCD deficiency and the precise interaction between PI3K and PKCδ require further elucidation. While findings from a single patient cannot be generalized, this experience supports the use of PI3Kδ inhibitors for the treatment of relevant IEIs besides APDS.

Clinical trials in APDS utilize a prescribed weight-based dosing strategy, but it is unknown if this dosing strategy is optimal in disorders such as PRKCD deficiency. Thus far, our patient has maintained a good response to 40 mg twice daily, but adjustments over time to account for growth and observed degree of clinical response will likely be needed.

One limitation of working with ultra-rare diseases is challenges compiling large cohorts of patients. International collaboration to identify cases is warranted to determine efficacious therapeutic strategies. Comprehensive selection of patients based on clinical, genetic, immunophenotypic, and molecular features—particularly those associated with AKT/mTOR pathway hyperactivity—will optimize efficacy and safety. Trials using selected patient populations may help establish leniolisib as an alternative to mTOR inhibitors, particularly in cases of inadequate disease control or adverse effects. Long-term monitoring will be essential to evaluate the overall safety and efficacy of leniolisib.

This case highlights the capability of leniolisib to address immunodeficiency and lymphoproliferation associated with PRKCD deficiency, paving the way for broader clinical applications in other IEIs and multifactorial immune-mediate diseases. In the era of computational drug repurposing, exploring the application of existing drugs for rare diseases is essential. This case supports the notion that healthcare professionals should rationally explore the application of exiting molecules for diseases where treatments are lacking or nonexistent.

Treatment protocol details are presented in Table S2, including dose-escalation strategy; clinical, laboratory, and imaging evaluations related to safety monitoring; and outcome achievements. Compassionate use of leniolisib was approved on April 26, 2024, by the Regional Pediatric Local Ethics Committee at Meyer Children’s Hospital, Florence. Both the legal representative and patient provided written informed consent before treatment initiation. Pharming Group N.V. provided free access to leniolisib for compassionate use.

Safety was assessed throughout the intervention period to monitor adverse events and ensure timely management. Adverse events were classified as serious, of special interest, or nonserious, with predefined thresholds for reporting and intervention. The “expected” adverse events (consistent with safety profile listed in prescribing information of leniolisib) included infections, gastrointestinal symptoms, skin manifestations, cytopenia, and elevation of liver enzymes. If safety thresholds were exceeded, adjustments or discontinuation of treatment were planned.

Primary outcomes were selected to assess therapeutic impact on disease and patient health, based on clinical, imaging, and laboratory evaluations.

Key metrics:

Assessment of overall health status and health-related quality of life (36-Item Short Form Health Survey) (27), incidence of infections, absence of progression in preexisting autoimmune-associated manifestations (e.g., no increase in hepatic cytolysis markers, no extension of alopecia lesions, and no exacerbation of gastrointestinal involvement), assessment of newly emerging autoimmune phenomena, incidence of noninfectious lymphadenopathy episodes, and lung function assessment, including lung volumes and alveolar diffusing capacity.

Longitudinal assessment of lung, spleen, lymph nodes, and thymus size, along with characterization of structural abnormalities, evaluated through ultrasound, MRI (Achieva 3 Tesla, Philips) or high-resolution CT (HR-CT; TC Revolution, GE Medical Systems). Lung HR-CT was acquired using a lung parenchyma window with consistent acquisition and reconstruction parameters. Spleen MRI was acquired via T2-weighted Turbo Spin Echo sequence in the axial plane. Spleen volumetry was calculated using the IntelliSpace Portal (Philips). CT of the thymus was acquired using a mediastinal window inspiration. Thymus volumetry was calculated using the IntelliSpace Portal (Philips). CT of lymph nodes was acquired using a mediastinal window.

Changes in hemoglobin concentration (g/dl), platelet count (×10³/μl), absolute white blood cell count (×10³/μl), and lymphocyte count (cells/μl).

Variations in the percentage and absolute number of naive B and T cells; transitional B cells and senescent T cells (percentage; cells/μl); plasma concentrations of IgM and IgA with IgG replacement therapy (mg/dl); variations in stimulated pS6 in CD4⁺ and CD8⁺ T cells (geometric mean of mean fluorescence intensity). Reference ranges for immune subsets and IgM were age-matched and obtained from literature (28, 29, 30, 31). Ranges were as follows: B cells (CD19⁺), 226–370 cells/μl; naive B cells (CD27⁻IgD⁺), 171–293 cells/μl; transitional B cells (CD24⁺⁺CD38⁺⁺), 10–30 cells/μl −3.9 to 7.8%; CD21^low B cells (CD21^lowCD38^low), 2–10 cells/μl −0.9 to 3.3%; CD3⁺ T cells, 954–2,332 × 10⁶/L; CD4⁺ T cells, 610–1,446 × 10⁶/L; CD8⁺ T cells, 282–749 × 10⁶/L; naive CD4⁺ T cells (CD45RA⁺CCR7⁺CD4⁺), 230–770 × 10⁶/L; naive CD8⁺ T cells (CD45RA⁺CCR7⁺CD8⁺), 240–710 × 10⁶/L; senescent CD8⁺ T cells (CD57⁺CD8⁺), 10–40 cells/μl −1.83 to 25%; and IgM, 42.4–197 mg/dl.

Peripheral blood mononuclear cells (PBMCs, 2 × 10⁶) were plated in 96-well plates at the concentration of 2 × 10⁵ cells per well in 200 μl of RPMI overnight at 37°C. For the stimulation, the plate was precoated with Mouse Anti-CD3 human (10 µg/μl) (86022706; Sigma-Aldrich) in 100 μl phosphate-buffered saline for 2 h. After dispensation of cells, 1 μl of Mouse Anti-Human CD28 (Purified NA/LE Mouse Clone CD28.2 [RUO], 555725, BD Biosciences) was added to each well.

After 24 h, PBMCs from the patient and a healthy control were stained for Mouse Anti-Human CD4 APC (555349; BD Pharmingen), Mouse Anti-Human CD8 PerCP-Cy5.5 (Clone SK1, 341050; BD Biosciences), and Rabbit Anti-Human Phospho-S6 Ribosomal Protein Antibody (Ser235/236) (BK2211LCST, Cell Signaling Technology). For secondary staining, Goat anti-Rabbit Ig Human ads-FITC antibody (Cat. No: 4010-02; Southern Biotech) was utilized. Cal101 and rapamycin were added before stimulation and used as inhibitors for PI3Kδ and mTOR, respectively, as previously described (32). All samples were collected with a FACSCanto flow cytometer and analyzed with FlowJo software. Data were analyzed using a two-sided independent samples Kruskal–Wallis test with Bonferroni correction.

Table S1 presents longitudinal spleen and thymus volume measurements over time, while Table S2 details the treatment regimen and the associated monitoring and follow-up plan.

The data presented in this article are not readily available because of ethical and privacy restrictions. Requests to access the dataset should be directed to the corresponding author. Fig. 1 A was modified using https://BioRender.com.

The authors thank the patient who took part in this case study and their family. Pharming Technologies, B.V. provided leniolisib at no cost and had no input in data interpretation. Pharming Group, Inc. funded manuscript editorial assistance by Precision AQ. Phospho-S6 assays were performed with the support of Fondazione Spedali Civili di Brescia and Fondazione Camillo Golgi. The data are available in the published article and its online supplemental material.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions: Lorenzo Lodi: conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, supervision, validation, visualization, and writing—original draft, review, and editing. Valentina Guarnieri: conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, supervision, validation, visualization, and writing—original draft, review, and editing. Matilde Peri: data curation, formal analysis, and writing—original draft. Manuela Baronio: data curation, investigation, methodology, resources, and writing—review and editing. Silvia Ricci: supervision, validation, visualization, and writing—review and editing. Clementina Canessa: conceptualization. Francesca Lippi: conceptualization and resources. Marta Voarino: conceptualization. Elisa Calistri: investigation, resources, validation, and visualization. Laura Pisano: resources. Anna Perrone: formal analysis and visualization. Grazia Fenu: resources and visualization. Giuseppe Indolfi: conceptualization, data curation, supervision, validation, and writing—original draft, review, and editing. Vassilios Lougaris: data curation, formal analysis, funding acquisition, methodology, resources, validation, and writing—review and editing. Rebecca A. Marsh: supervision and writing—review and editing. Chiara Azzari: conceptualization, methodology, project administration, resources, supervision, validation, and writing—review and editing (18).