Purpose: Based on preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab (anti-PD-L1) together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. Patients and Methods:We designed a single arm Phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced urothelial carcinoma who had previously progressed on ICI therapy with PD-1 or PD-L1 targeting agents were eligible. Pre-planned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. Results: Safety run-in enrolled 6 patients and Phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease, exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and stable disease. Furthermore, high IL-6 and IL-8 levels in the patients’ plasma associates with short survival. Conclusions: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

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Supplementary data

Supplementary Tables 1

Supplementary Tables Supplementary Table 1: Patient and tumor sample information Supplementary Table 2: Tumor mutation burden Supplementary Table 3: Unfiltered whole exome sequencing mutation analysis Supplementary Table 4: Filtered whole exome sequencing mutation analysis Supplementary Table 5: Immunohistochemistry scores of tumors Supplementary Table 6: FACS panel for PBMC analysis Supplementary Table 7: Geometric mean fluorescence intensity (GMFI) from PBMC FACS Supplementary Table 8: Representativeness of Study Participants

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Figure S1

Supplementary Figure 1: Patient recruitment and sample collection overview.

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Figure S2

Supplementary Figure 2: Modest treatment-induced DNA methylation changes in UC tumors.

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Supplementary Figure 3: Copy number variation detected by SeSAMe from DNA methylation data.

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Supplementary Figure 4: Weak correlation of TE expression induction with DNA methylation change and viral mimicry activation.

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Supplementary Figure 5: Overall tumor mutation burden and frequency of mutations in cancer-associated genes in UC tumors.

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Supplementary Figure 6: Gene Set Enrichment Analysis reveals immune-related pathways discriminate PD and SD tumors.

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Supplementary Figure 7: Representative IHC staining of additional PD and SD tumors in pre-treatment and post-treatment timepoint from additional patients.

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Supplementary Figure 8: PD-L1 levels in tumor or tumor microenvironment does not associate with tumor progression or control.

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Supplementary Figure 9: CD8 infiltration in tumor is associated with survival.

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Supplementary Figure 10: Peripheral immune cell dynamics induced by combination therapy.

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Supplementary Figure 11: Peripheral immune cells are activated mid-treatment cycle.

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Supplementary Figure 12: HLA-DR and NKG2D abundance on peripheral lymphocytes associate with longer progression free survival.

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