Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6-blockade-refractory idiopathic multicentric Castleman disease

Abstract

Background: Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype.

Methods: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus.

Results: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months.

Conclusion: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904).

Funding: Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.

Keywords: Cytokines; Hematology; Immunology; Lymphomas.

Conflict of interest statement

Conflict of interest: DCF is the index case. DCF receives research support from Janssen Pharmaceuticals. TSU is a coinventor on US patent 10,001,483 B2, assigned to the U.S. Government, with a portion of royalties going to employee-inventors under PL 99-502. TSU has research support through Cooperative Research and Development Agreements with the National Cancer Institute, Celgene Corporation, and Merck, and through a clinical trial agreement from Roche and the Fred Hutchinson Cancer Research Center.

Figures

Figure 1. Study schema.

Flow of 3 IL-6 blockade–refractory iMCD patients (iMCD-1, iMCD-2, iMCD-3) with TAFRO syndrome for whom translational studies were performed and sirolimus was administered. Sirolimus trough was maintained at 5–10 ng/mL. iMCD clinical assessment included MCD Overall Symptom Score, Clinical Benefit Response, and Cheson Criteria.

Figure 2. Clinical course and elevation of VEGF-A and sIL-2Rα prior to disease flare for iMCD-1.

(A) Select laboratory values, dates of initiation of disease flares (dotted vertical lines; defined by hypoalbuminemia (< 3.5 g/dL), elevated CRP (> 10 mg/L), anemia (hemoglobin < 13.5 g/dL), renal dysfunction (creatinine > 1.3 mg/dL), constitutional symptoms, and fluid accumulation), and treatment regimens administered throughout iMCD-1’s disease course (n = 1). CRP closely parallels disease status. IVIg, intravenous immunoglobulin; ACE, doxorubicin (adriamycin)-cyclophosphamide-etoposide; VDT, bortezomib (velcade)-dexamethasone-thalidomide; CRP, C-reactive protein. (B) Serum levels of sIL-2Rα (normal < 1022 pg/mL) and VEGF-A (normal < 86 pg/mL) from 1 year before to 1 year after iMCD-1’s fifth disease flare (onset indicated by dotted vertical line; duration by shaded region), with CRP included for reference. Arrows indicate when sIL-2Rα and VEGF-A rose above the ULN.

Figure 3. Serum proteomics and pathway analyses identify VEGF-A, sIL-2Rα, and PI3K/Akt/mTOR signaling as candidate therapeutic targets for iMCD-1.

(A) Heatmap of the analytes whose levels increase (blue) or decrease (orange) by at least 2-fold in the same direction between flare and remission for iMCD-1’s third and fifth flares, as measured by Myriad RBM DiscoveryMAP (n = 1). Analytes are presented in ascending order from left to right based on the log2 (flare/remission) fold-change at the fifth flare, compared with remission. Key provides the color intensity for a given fold change. (B, C) Enrichment analysis, using Enrichr, of Myriad RBM DiscoveryMAP gene sets for metabolic pathways for iMCD-1. Results of the top 5 enriched gene sets (FDR < 0.01, rank ordered by combined score) from the (B) third flare and (C) fifth flare when proteins with log2 (flare/remission) greater than 2 were analyzed for KEGG pathway gene sets. Colored cells represent gene members in specific pathways that were found to be greater than 4-fold up (blue) or 4-fold down (orange) during flare compared with remission.

Figure 4. Increased CD8+ T cell activation, VEGF-A levels, and mTOR signaling in IL-6 blockade–refractory iMCD.

(A–C) Flow cytometry of PBMCs gated for live nonnaive CD8+ T cells. PBMCs were obtained from iMCD-1, iMCD-2, and iMCD-3 at onset of a relapse of flare (n = 3, represented by iMCD-1 flare), and from 3 age-matched healthy controls (Healthy control). Nonnaive CD8+ T cells were gated for expression of CD38 and HLA-DR. The percentage of cells within the gated regions is provided for each (black rectangle: CD38+; red rectangle: CD38+HLA-DR+). Mean with SEM is presented. Unpaired 1-tailed Student’s t test was performed between the 3 iMCD flare samples and 3 age-matched healthy controls. No abnormalities were observed in the CD4+ T cell population (data not shown). (D) Circulating VEGF-A levels were measured for iMCD-1 and iMCD-3 at the time of relapse as part of routine clinical care; VEGF-A for iMCD-2 was measured with a clinical grade assay (ARUP Laboratories) (n = 3). Healthy control range (9–86 pg/mL) is shown. (E–I) Immunohistochemistry was performed on lymph node tissue and representative images are provided of a reactive (Reactive) (E), an autoimmune lymphoproliferative syndrome (ALPS) (F), and an iMCD (iMCD) (G) lymph node immunostained (brown) in parallel with an antibody against phosphorylated ribosomal protein S6 (phospho-S6), a marker of mTOR activation, and counterstaining with hematoxylin (blue) (scale bars: 300 μm). (H) Quantification of germinal center staining intensity and (I) quantification of interfollicular staining intensity, shown as the percentage of pixels stained positive, as well as the breakdown of weak, medium, or strong staining, for reactive (green circle; n = 6), ALPS (red square; n = 5), and iMCD (blue triangle; n = 3). Dot plots along with the means are presented. Statistical significance was tested by comparing the centered log-transformed ratios by a 1-tailed Mann-Whitney U test. *P < 0.05.

Figure 5. Decreased T cell activation and VEGF-A levels following therapeutic inhibition of mTOR in IL-6 blockade–refractory iMCD.

(A–C) Flow cytometry of PBMCs gated for live nonnaive CD8+ T cells. PBMCs from iMCD-1, iMCD-2, and iMCD-3 at onset of a relapse of flare (n = 3, represented by iMCD-2 flare), as per Figure 4A–C, were compared with PBMCs after a remission was achieved with mTOR inhibition for all 3 patients (represented by iMCD-2 remission). Nonnaive CD8+ T cells were gated for expression of CD38 and HLA-DR. The percentage of cells within the gated regions is provided for each (black rectangle: CD38+; red rectangle: CD38+HLA-DR+). Paired 1-tailed Student’s t test was performed between the 3 iMCD flare samples and 3 remission samples, P < 0.05. (D–E) Circulating VEGF-A levels were measured by a clinical grade assay (ARUP Laboratories) for iMCD-2 (D) and as part of routine clinical care for iMCD-1 (data presented in Figure 2B) and iMCD-3 (E) at the time of relapse and at 3 subsequent time points after sirolimus was initiated. Healthy control range for both VEGF-A assays was 9–86 pg/mL. (F) Plot displaying the percentage change in baseline symptom score as determined by the MCD-related overall symptoms score for iMCD-1, iMCD-2, and iMCD-3 (n = 3).