Integrated stress response and immune cell infiltration in an ibrutinib‐refractory mantle cell lymphoma patient following <scp>ONC</scp>201 treatment (2018)

Mantle cell lymphoma (MCL) is an incurable subtype of non-Hodgkin lymphoma (NHL). The Bruton tyrosine kinase (BTK) inhibitor ibrutinib achieves a 68% overall response rate in relapsed/refractory MCL patients, but 69% will progress within 2 years and their median survival is only 5·8 months (Cheah et al, 2015). ONC201, a first-in-class small molecule that selectively antagonizes the G protein-coupled receptor (GPCR) dopamine receptor D2 (DRD2) (Madhukar et al, 2017a), activates the integrated stress response (ISR) that attenuates protein translation and induces activating transcription factor 4 (ATF4) and C/EBP homology protein (CHOP), which transcriptionally regulate genes involved in apoptosis that include TNFRSF10B (also termed death receptor 5, DR5) and TNFSF10 (also termed tumour necrosis factor-related apoptosis-inducing ligand, TRAIL) (Allen et al, 2016). Preclinical evidence suggests that ONC201 lacks cross-resistance with other endoplasmic reticulum stress-inducing agents, such as bortezomib, and exhibits pro-apoptotic activity in refractory cancer models, including ibrutinib-refractory MCL (Ishizawa et al, 2016). This involves sustained pharmacodynamic effects, such as ISR activation, that outlive the systemic half-life of ONC201 and rationalize its infrequent dose schedule (Ishizawa et al, 2016; Kline et al, 2016). ONC201 prolonged the survival of transgenic Eμ-myc mice that spontaneously develop B-cell lymphoma (Allen et al, 2013). Profiling of >1000 human cancer cell lines in vitro (Allen et al, 2016) identified B cell malignancies as the most ONC201-sensitive tumour type, explained by malignant B cell expression of DRD2 (Meredith et al, 2006) and pronounced sensitivity to the ISR. A 66-year-old man with refractory advanced MCL was enrolled in a Phase I trial of ONC201 at a dose of 125 mg ONC201 orally once every 3 weeks. Prior therapy included R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), ibrutinib plus rituximab, rituximab plus bendamustine and bortezomib, and carfilzomib plus ibrutinib. MCL was present in the rectum (Fig 1A: pre-treatment) and colon. Pre-treatment tumour tissue was positive for DRD2 and negative for DRD5 (a member of the D1-like subfamily of dopamine receptors that oppose DRD2 signaling effects, Fig 1B). Pharmacokinetic analysis after start of treatment revealed a maximum ONC201 serum concentration of 800 ng/ml (~2 μmol/l) (Fig 2A), with a half-life of 11·5 h, and area under the curve of 9·2 h μg/l. Serum prolactin induction was observed as a surrogate marker of target engagement (Fig 2B). Rectal biopsies collected 17 and 16 days after the third and seventh cycles (immediate off study), respectively, revealed induction of CHOP and TNFRSF10B (Fig 2C, D; Figure S1), indicating therapeutic exposure to ONC201 that resulted in target engagement in the tumour. After 7 cycles, a repeat colonoscopy showed new disease in the ileum and sigmoid, with persistent disease in the rectum, and the patient was removed from the study and observed. Six months later, an extensive re-evaluation including biopsies of the stomach, duodenum, terminal ileum, ascending colon, descending colon, sigmoid colon and rectum unexpectedly revealed only a small cyclin D1+SOX11+ lymphoid infiltrate in one rectal biopsy (Fig 1A: 6 months off-study). At the time, rectal biopsies revealed a significant increase in CHOP (Fig 2C), CD45+ lymphocytes and CD8+ T cells (Fig 2E, Figure S2), but not TNFRSF10B (Fig 2D) or CD56+ Natural Killer (NK) cells or granzyme B+ cells (Figures S3 and S4). This is the first patient with MCL to receive ONC201. He exhibited ibrutinib-refractory disease and experienced a transient anti-tumour effect after 3 cycles of ONC201. Since DRD5 mediates acquired and innate resistance to ONC201 through missense gain-of-function mutation and overexpression (Madhukar et al, 2017b), respectively, and our patient had DRD5-negative MCL, this could suggest an association with the initial response. Hallmarks of activation and sustained pharmacodynamics of ISR (i.e. induction of CHOP/TNFRSF10B) were present 16–17 days after cycles 4 and 7 (immediate off study). The most striking induction of these biomarkers was TNFRSF10B, a TNFSF10 death receptor that is induced by CHOP and has been shown to mediate ONC201-induced cell death (Allen et al, 2013; Kline et al, 2016). The transient response was followed by progression of disease and ONC201 was stopped. Six months later, only minimal residual disease was detected. We thus explored the possibility of a delayed enhancement of the immune system by ONC201. Prior studies have found that DRD2 is expressed on the surface of immune cells (e.g. NK cells) and that DRD2 antagonism can result in immune cell activation (Zhao et al, 2013). While no significant induction of NK cells was observed in immediate pre-treatment biopsies obtained from the patient, a significant increase in CD45+ lymphocytes was evident in biopsies taken 6 months off-study. Further immunophenotyping of tumour biopsies indicated infiltration of CD8+ T cells, but not NK cells, at the same time point. CD8+ T cells are known to be cytotoxic to tumour cells and could have contributed to the minimal residual MCL that was observed despite the ~5-month washout period. It is possible that the time points of the biopsies were such that activation of NK cells, and other immune cells that are more rapidly activated, were not observed and thus the relevance of other immune cells cannot be completely discounted. Further clinical evaluation of ONC201 in NHL is warranted and the possibility of delayed immune-mediated anti-tumour effects should be considered, along with the role of DRD2 and DRD5 expression in the tumour. J. Romaguera designed the research study, analysed the data and wrote the paper. H. Lee performed the research, analysed the data and helped write the paper. R. Tarapore and L Schalop analysed the data and wrote the paper. V. Prabhu and J. Allen performed and analysed the data and wrote the paper. A Sloza, E. Sadimin and J. Schenkel performed the research. C. Ok and M. Wang performed the research, analysed the data and wrote the paper. M. Badillo analysed the data. Figure S1. Quantification of CHOP and TNFRSF10B expression in rectum biopsies following ONC201 treatment. Figure S2. CD8 staining in rectum biopsies following ONC201 treatment. Figure S3. CD56 staining in rectum biopsies following ONC201 treatment. Figure S4. Granzyme B staining in rectum biopsies following ONC201 treatment. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.