The epigenetic immunomodulator, HBI-8000, enhances the response and reverses resistance to checkpoint inhibitors

Abstract

Background: Treatment with immune checkpoint inhibitors (ICIs) targeting CTLA-4 and the PD-1/PD-L1 axis is effective against many cancer types. However, due in part to unresponsiveness or acquired resistance, not all patients experience a durable response to ICIs. HBI-8000 is a novel, orally bioavailable class I selective histone deacetylase inhibitor that directly modifies antitumor activity by inducing apoptosis, cell cycle arrest, and resensitization to apoptotic stimuli in adult T cell lymphoma patients. We hypothesized that HBI-8000 functions as an epigenetic immunomodulator to reprogram the tumor microenvironment from immunologically cold (nonresponsive) to hot (responsive).

Method: Mice bearing syngeneic tumors (MC38 and CT26 murine colon carcinoma and A20 B-cell lymphoma were treated daily with HBI-8000 (orally), alone or in combination with PD-1, PD-1 L, or CTLA-4 antibodies. MC38 tumors were also analyzed in nanoString gene expression analysis.

Results: HBI-8000 augmented the activity of ICI antibodies targeting either PD-1, PD-L1 or CTLA-4, and significantly increased tumor regression (p < 0.05) in the above models. Gene expression analysis of the treated MC38 tumors revealed significant changes in mRNA expression of immune checkpoints, with enhanced dendritic cell and antigen-presenting cell functions, and modulation of MHC class I and II molecules.

Conclusions: These findings suggest that HBI-8000 mediates epigenetic modifications in the tumor microenvironment, leading to improved efficacy of ICIs, and provide strong rationale for combination therapies with ICIs and HBI-8000 in the clinical setting.

Precis: As an HDACi, HBI-8000 plays an important role in priming the immune system in the tumor microenvironment. The current preclinical data further justifies testing combination of HBI-8000 and ICIs in the clinic.

Keywords: Checkpoint blockade; Dendritic cells; Epigenetics; HDAC; Histone deacetylase; Immune checkpoint; PD-1; PD-L1.

Fig. 1

Tumor growth inhibition (TGI) in mice treated with ICI, HBI-8000, or their combination. Syngeneic MC38 (A-D), A20 (E & F) and CT26 (G & H) tumors were implanted in C57BL/6 or BALB/c mice and were allowed to grow until the mean tumor volume was ~ 100 mm³. Animals were then randomized into groups with equivalent mean tumor volumes and treated with the indicated therapeutic agents. Data shown represent the median tumor volume for each treatment group at the indicated day post-initiation of therapy (A, C, E and G), as well as the individual tumor volumes per animal (B, D, F and H). I Dosing regimens and intervals

Fig. 2

Immune cell-types and pathways modulated by PD-1 Ab, HBI-8000, or their combination. Syngeneic MC38 tumors were implanted in C57BL/6 mice and allowed to grow until the mean tumor volume was ~ 100 mm³. The mice were then randomized into groups of 20 mice with equivalent mean tumor volumes and treated with the indicated therapeutic agents. At days 7, 14, and 17, groups of 20 mice were killed, and the tumors were excised, fixed in formalin, and embedded in paraffin. Tumor sections were then processed for nCounter gene expression analysis as described in the Methods. A. Plots of the immune cell types in the TME modulated by PD-1 Ab, HBI-8000, or their combination at days 7, 14, and 17 for each treatment group. B. Immune checkpoints (PD1, PD-L1, CTLA4, CD86, CD276, and CD244) modulated by PD-1 Ab, HBI-8000, or their combination. The data depict the mRNA expression levels for each gene at days 7, 14, and 17. Statistical significance is as indicated in the graphs. Individual mice were tagged according to the antitumor response. Red circles () represent TGI > 75%, inverted green triangles () TGI from 25% through 75%, and blue squares () were assigned to mice with TGI < 25%

Fig. 3

Expression analyses of TNFα, KLRD1, CCR5, CCL2, CD137, and IRF4. Experiments and data analyses as described in Fig. 2

Fig. 4

TME immune response-relevant markers modulated by PD-1 Ab, HBI-8000, or their combination. Expression of IL-2Rα, CD8α, CCR1, ENTPD1, GZMB, and PRF1 in tumors isolated from mice in the Vehicle, HBI-8000, PD-1 Ab, and the combination of HBI-8000 and PD-1 Ab groups

Fig. 5

Expression of cytokine/chemokine receptors, MHC class I and class II are modulated by PD-1 Ab, HBI-8000, or their combination. nCounter data analyses (as explained in the Methods and in Fig. 2) identified significant differences in the expression of IL-7R, CXCR6, CX3CR1, CXCR3, H2-Aa, H2-Eb1, H2-D1, and H2-K1 in tumors treated with PD-1 Ab, HBI-8000, or their combination compared to the Vehicle-treated group

Fig. 6

ICI (PD-L1 Ab) plus HBI-8000 reverses resistance to PD-1 Ab therapy and rescues mice with MC38 tumors progressing on PD-1 Ab therapy. Mice implanted with MC38 tumors were treated with PD-1 Ab as a first-line therapy for 18–21 days, at which point mice displaying stable or slow tumor growth were randomized into 1 of 6 s-line treatment groups, including Vehicle, HBI-8000, PD-1 Ab, PD-1 Ab plus HBI-8000, PD-L1 Ab, and PD-L1 Ab plus HBI-8000. Data shown represent median tumor growth (Fig. 6A) and survival (Fig. 6B) in each treatment cohort. * denote statistical significance (p < 0.05) when compared HBI-8000 plus anti-PD-L1 vs. anti-PD-1 monotherapy