Treatment of ADA2 deficiency with autophagy inhibitors

Abstract

The invention relates to the use of autophagy inhibitors in treating or alleviating symptoms caused by ADA2 (adenosine deaminase 2) deficiency.

Description

[0001] TREATMENT OF ADA2 DEFICIENCY WITH AUTOPHAGY INHIBITORS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to treating or alleviating symptoms caused by ADA2 (adenosine deaminase 2) deficiency.

[0004] The invention relates to novel medical uses of autophagy inhibitors such as Chloroquine (CQ) and Hydroxychloroquine (HCQ).

[0005] BACKGROUND OF THE INVENTION

[0006] ADA2 deficiency (DADA2) is a hereditary autoinflammatory disease caused by biallelic mutations in the ADA2 gene. The disease manifests with a diverse phenotype of inflammation and immunodeficiency. Many patients improve upon TNF inhibition but bone marrow failure often persists warranting treatment escalation including hematopoietic stem cell transplantation. The pathophysiology of DADA2 on a cellular level is poorly understood and mechanistic studies have to date largely been performed in model systems requiring overexpression of mutant ADA2.

[0007] Transfected into HEK293T cells, pathogenic ADA2 variants show retention in the endoplasmic reticulum and impaired secretion. In primary DADA2 monocytes, ADA2 protein expression is however minimal to absent despite normal mRNA expression for some variants, suggesting rapid protein degradation of mutant ADA2

[0008] [Chen et al. (2023) J Allergy Clin Immuno! 152, 771-782; Greiner-Tollersrud et al. (2020) bioRxiv.162990],

[0009] SUMMARY OF THE INVENTION

[0010] The present invention shows that inhibition of autophagy increases protein expression of endogenous mutant ADA2 in monocytes from DADA2 patients.

[0011] The invention is further summarised in the following statements:

[0012] 1. A composition comprising an autophagy inhibitor for use in treating or alleviating symptoms caused by ADA2 (adenosine deaminase 2) deficiency.

[0013] 2. The composition comprising an autophagy inhibitor for use according to statement 1, wherein the symptoms are one or more of vasculitis, autoinflammation, immunodeficiency, and hematologic defects.

[0014] 3. The composition comprising an autophagy inhibitor for use according to statement 1 or 2, wherein the autophagy inhibitor is Chloroquine (CQ) or Hydroxychloroquine (HCQ). 4. The composition comprising an autophagy inhibitor for use according to any one of statements 1 to 3, wherein the ADA2 mutation leading to ADA2 (adenosine deaminase 2) deficiency is selected from the group consisting of Tyr453Cys AlalO9Asp, Gly47Ala, Hisll2Gln, Argl69Gln, Gly47Arg, Pro251Leu, Gly47Val, Trp264Ser, Thrll9Ala, Glyl42Ser, Phe212deL

[0015] 5. The composition comprising an autophagy inhibitor for use according to any one of statements 1 to 4, wherein the ADA mutation leading to ADA2 (adenosine deaminase 2) deficiency is selected from the group consisting of: c.973-2A>G / c.dell240-1442, c,140G>T / c.dell240-1442,

[0016] C.140G >T / C.140G >T, c,140G>T / c.506G>A, c.973-2A>G / c.506G>A,

[0017] C.139G >A / C.139G >A, c.973-2A>G / c.973-2A>G, and c.973-2A>G / c.506G>A.

[0018] 6. A method of treating or alleviating symptoms caused by ADA2 (adenosine deaminase 2) deficiency comprising the step of administering an autophagy inhibitor such as Chloroquine (CQ) or Hydroxychloroquine (HCQ).

[0019] DETAILED DESCRIPTION

[0020] Figure legend

[0021] Figure 1: Expression of mutant ADA2 upon inhibition of autophagy.

[0022] (A) ADA2 protein expression in CD14+ monocytes from healthy controls (HC), DADA2 heterozygous carriers (CR) or DADA2 patients (P) incubated for 20-24h with 50 pM chloroquine (CQ), 100 nM bafilomycin Al (Baf) or 2 pg / mL monensin (MN). (

[0023] B) ADA2 protein expression in CD14+ monocytes from healthy controls (HC), DADA2 heterozygous carriers (CR.) or DADA2 patients (P) incubated for 20-24h with 100 nM bafilomycin Al (Baf) or 200 nM delanzomib (DZM).

[0024] (C) ADA2 protein expression in CD14+ monocytes from healthy controls (HC) after 24-hour incubation with 2000 ng / mL GM-CSF and / or 50 pM chloroquine (CQ).

[0025] (D) ADA2 protein expression in GM-CSF differentiated human monocyte-derived macrophages treated with 50 pM chloroquine (CQ) for 24h.

[0026] (E) CD14+ monocytes from healthy control HC4 and DADA2 patient P4 were incubated with or without GM-CSF for the indicated time and subsequently treated with 50 pM chloroquine (CQ), 100 nM bafilomycin Al (Baf) or 2 pg / mL monensin (MN) for 24h. ADA2 protein expression was determined by western blot. (F) Protein levels of LC3-I and LC3-II were determined by western blot in CD14+ monocytes from healthy controls (HC), DADA2 heterozygous carriers (CR) and DADA2 patients (P) at baseline or after 24h-incubation with or without 2000 ng / mL GM-CSF. LC3-II expression was normalized to p-actin.

[0027] (G) ADA2 protein expression in whole cell lysates of CD14+ monocytes extracted immediately after magnetic cell selection. Samples from DADA2 patient P6 were taken prior to (i) and four days after initiation of treatment with hydroxychloroquine (ii). Samples from HC12 were taken at both time points in parallel with the patient's samples. ADA2 was detected using anti-ADA2 antibody clone EPR.25430-131 (#ab288296; abeam) in all depicted blots.

[0028] The ADA gene encodes adenosine deaminase (EC 3.5.4.4), an enzyme that catalyses the irreversible deamination of adenosine and deoxyadenosine in the purine catabolic pathway.

[0029] Adenosine deaminase 2 deficiency (DADA2) is a complex systemic autoinflammatory disorder in which vasculopathy / vasculitis, dysregulated immune function, and / or hematologic abnormalities may predominate. Inflammatory features include intermittent fevers, rash (often livedo racemosa / reticularis), and musculoskeletal involvement (myalgia / arthralgia, arthritis, myositis). Vasculitis, which usually begins before age ten years, may manifest as early-onset ischemic (lacunar) and / or haemorrhagic strokes, or as cutaneous or systemic polyarteritis nodosa. Hypertension and hepatosplenomegaly are often found. More severe involvement may lead to progressive central neurologic deficits (dysarthria, ataxia, cranial nerve palsies, cognitive impairment) or to ischemic injury to the kidney, intestine, and / or digits. Dysregulation of immune function can lead to immunodeficiency or autoimmunity of varying severity; lymphadenopathy may be present and some affected individuals have had lymphoproliferative disease. Hematologic disorders may begin early in life or in late adulthood, and can include lymphopenia, neutropenia, pure red cell aplasia, thrombocytopenia, or pancytopenia. Of note, both interfamilial and intrafamilial phenotypic variability (e.g., in age of onset, frequency and severity of manifestations) can be observed; also, individuals with biallelic ADA2 pathogenic variants may remain asymptomatic until adulthood or may never develop clinical manifestations of DADA2.

[0030] Up to 60 ADA2 disease mutants are described. The most frequent occurring mutations are Tyr453Cys AlalO9Asp, Gly47Ala, Hisll2Gln, Argl69Gln, Gly47Arg, Pro251Leu, Gly47Val, Trp264Ser, Thrll9Ala, Glyl42Ser, Phe212deL A non-limited list of autophagy inhibitors includes:

[0031] Chloroquine (CQ) and Hydroxychloroquine (HCQ) : These are antimalarial drugs that are also used as autophagy inhibitors. They raise the pH of lysosomes, inhibiting their fusion with autophagosomes.

[0032] 3-Methyladenine (3-MA): 3-MA is a widely used autophagy inhibitor that inhibits class III phosphoinositide 3-kinase (PI3K) and interferes with autophagosome formation. Bafilomycin Al : Bafilomycin Al is an antibiotic that inhibits the vacuolar-type H + ATPase (V-ATPase), which leads to an increase in lysosomal pH and inhibits autophagosome-lysosome fusion.

[0033] Spautin-1 : Spautin-1 is an inhibitor that promotes the degradation of the class III PI3K complex components, Beclinl and VPS34, thereby inhibiting autophagy.

[0034] Wortmannin: Wortmannin is a potent inhibitor of phosphoinositide 3-kinases (PI3Ks) and can block autophagosome formation.

[0035] LY294002: LY294002 is another PI3K inhibitor that can block autophagy by interfering with the class III PI3K complex.

[0036] Perifosine: Perifosine is an Akt inhibitor that indirectly inhibits autophagy by blocking the Akt / mTOR pathway, which is a negative regulator of autophagy.

[0037] Dihydroartemisinin (DHA) : DHA is a derivative of the antimalarial drug artemisinin and has been shown to inhibit autophagy by disrupting lysosomal function.

[0038] Lys05: Lys05 is a lysosomotropic agent that disrupts lysosomal function and inhibits autophagy.

[0039] Trehalose: Trehalose is a natural disaccharide that can inhibit autophagy by reducing the cellular levels of mTOR, a key regulator of autophagy.

[0040] Lysosomal localization of ADA2 may be related to the degradation of misfolded ADA2 was mediated by autophagy.

[0041] To test this, CD14+ monocytes from healthy control (HC) and DADA2 patients were treated with different inhibitors of autophagy resulting in a strong increase in ADA2 expression in the patients' cells (Figure 1A). This was observed independently of the patients' genotype (table 1), including the c.dell240-1442 variant resulting in deletion of exon 9 expressed by Pl and P2.

[0042] In P9 - a patient homozygous for the splice site variant c.973-2A>G that exhibits nonsense-mediated mRNA decay - no increase in ADA2 protein levels was detected. Interestingly, PIO showed detectable levels of monocytic ADA2 expression already at baseline although she shared the genotype of P7 who did not differ from the rest of the cohort.

[0043] Incubation with the proteasome inhibitor delanzomib did not alter mutant ADA2 expression (Figure IB). In HC CD14+ monocytes, inhibition of autophagy tended to cause a decrease in intracellular ADA2 protein levels (Figure 1C). In human monocyte-derived macrophages, inhibition of autophagy only caused a slight shift in molecular weight (Figure ID). In fact, mutant ADA2 levels after ten days of macrophage differentiation with GM-CSF were similar to those achieved by inhibition of autophagy in monocytes over 24 hours (Figure IE), highlighting cell type-specific differences in managing mutant ADA2.

[0044] Autophagy may not only mediate the degradation of mutant ADA2 but also determines the fate of DADA2 monocytes. By analyzing autophagic flux, we observed increased levels of lipidated LC3B in CD14+ monocytes of several DADA2 patients (Figure IF).

[0045] Patient P6 of our cohort was started on the drug (dosage) due to insufficient treatment response and persistent cytopenia under ciclosporin. CD14+ monocytes were isolated right before treatment initiation and four days after starting HCQ. We observed a 1.3-fold increase in intracellular ADA2 protein levels (Figure 1G). No difference was seen in HC monocytes that were isolated from donor HC12 in parallel with the patient's cells (Figure 1G). ADA2 enzyme activity in the patient's serum did not change with HCQ treatment, at least not after this short time span. The patient tolerated the therapy well but showed no clinical improvement within the first weeks of treatment. This case report suggests that inhibition of autophagy affects protein expression of mutant ADA2 in vivo.

[0046] U937 and Thpl models with knock-in of relevant pathogenic variants of ADA2 (leading to mRNA decay, to impaired secretion or maintained secretion of an ADA2 mutant without adenosine deaminase activity) are used in identifying small molecules that modulate DADA2 disease, with the secretion of ADA2 and measured ADA2 enzyme activity as a read-out.

[0047] Table 1. Cohort characteristics

Claims

CLAIMS1. A composition comprising an autophagy inhibitor for use in treating or alleviating symptoms caused by ADA2 (adenosine deaminase 2) deficiency.

2. The composition comprising an autophagy inhibitor for use according to claim 1, wherein the symptoms are one or more of vasculitis, autoinflammation, immunodeficiency, and hematologic defects.

3. The composition comprising an autophagy inhibitor for use according to claim 1 or 2, wherein the autophagy inhibitor is chloroquine (CQ) or hydroxychloroquine (HCQ).

4. The composition comprising an autophagy inhibitor for use according to any one of claims 1 to 3, wherein the ADA2 mutation leading to ADA2 (adenosine deaminase 2) deficiency is selected from the group consisting of Tyr453Cys AlalO9Asp, Gly47Ala, Hisll2Gln, Argl69Gln, Gly47Arg, Pro251Leu, Gly47Val, Trp264Ser, Thrll9Ala, Glyl42Ser, Phe212deL5. The composition comprising an autophagy inhibitor for use according to any one of claims 1 to 4, wherein the ADA mutation leading to ADA2 (adenosine deaminase 2) deficiency is selected from the group consisting of: c.973-2A>G / c.dell240-1442, c,140G>T / c.dell240-1442, C.140G >T / C.140G >T, c,140G>T / c.506G>A, c.973-2A>G / c.506G>A, C.139G >A / C.139G >A, c.973-2A>G / c.973-2A>G, and c.973-2A>G / c.506G>A.

Images