Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6 blockade–refractory idiopathic multicentric Castleman disease
David C. Fajgenbaum, Ruth-Anne Langan, Alberto Sada Japp, Helen L. Partridge, Sheila K. Pierson, Amrit Singh, Daniel J. Arenas, Jason R. Ruth, Christopher S. Nabel, Katie Stone, Mariko Okumura, Anthony Schwarer, Fábio Freire Jose, Nelson Hamerschlak, Gerald B. Wertheim, Michael B. Jordan, Adam D. Cohen, Vera Krymskaya, Arthur Rubenstein, Michael R. Betts, Taku Kambayashi, Frits van Rhee, Thomas S. Uldrick
David C. Fajgenbaum, Ruth-Anne Langan, Alberto Sada Japp, Helen L. Partridge, Sheila K. Pierson, Amrit Singh, Daniel J. Arenas, Jason R. Ruth, Christopher S. Nabel, Katie Stone, Mariko Okumura, Anthony Schwarer, Fábio Freire Jose, Nelson Hamerschlak, Gerald B. Wertheim, Michael B. Jordan, Adam D. Cohen, Vera Krymskaya, Arthur Rubenstein, Michael R. Betts, Taku Kambayashi, Frits van Rhee, Thomas S. Uldrick
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Clinical Research and Public Health Hematology Immunology

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 3 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 3 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 3 IL-6 blockade–refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus substantially attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all 3 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 This study was supported by the Castleman’s Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and the National Heart Lung and Blood Institute.

Authors

David C. Fajgenbaum, Ruth-Anne Langan, Alberto Sada Japp, Helen L. Partridge, Sheila K. Pierson, Amrit Singh, Daniel J. Arenas, Jason R. Ruth, Christopher S. Nabel, Katie Stone, Mariko Okumura, Anthony Schwarer, Fábio Freire Jose, Nelson Hamerschlak, Gerald B. Wertheim, Michael B. Jordan, Adam D. Cohen, Vera Krymskaya, Arthur Rubenstein, Michael R. Betts, Taku Kambayashi, Frits van Rhee, Thomas S. Uldrick

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Figure 4

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

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Increased CD8+ T cell activation, VEGF-A levels, and mTOR signaling in I...
(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.