Chemoresistance is a major obstacle that limits the benefits of cisplatin-based chemotherapy in various cancers, including hepatocellular carcinoma. De-regulation of the poly(ADP-ribose) polymerase 1 (PARP1)/high-mobility group box 1 (HMGB1) signaling pathway has been proposed as an important mechanism involved in cisplatin-resistance. In this study, we investigated therapeutic potential of a natural flavonoid Morin hydrate against cisplatin-induced toxicity using the HepG2^DR multi-drug resistant cell line, which is derived from the HepG2 human hepatocellular carcinoma cell line. HepG2^DR cells were exposed to cisplatin and Morin hydrate alone or together after which autophagy and apoptotic signaling pathways were monitored by fluorometric assay and Western blot analysis. Xenograft mouse models were performed to confirm the in vitro effect of Morin hydrate. PARP1 was hyper activated in cisplatin-resistant HepG2^DR cells. Cisplatin-induced PARP1 activation resulted in chemoresistance via increased autophagy. The cisplatin/Morin hydrate combination was effective in the reversal of the HepG2^DR cell resistance via suppression of PARP1-mediated autophagy by regulating the HMGB1 and microtubule-associated protein 1A/1B light chain 3B (LC3) I/II. Moreover, PARP1 inhibition by 4-amino-1,8-naphthalimide or autophagy inhibition by a knockdown of the autophagy-related 5 (ATG5) gene resulted in sensitizing the HepG2^DR cells to cisplatin (CP) through activation of the c-Jun N-terminal kinase (JNK) pathway. In a mouse xenograft model, the treatment of cisplatin with Morin hydrate reversed the increased expression of PARP and HMGB1 and significantly suppressed tumor growth. These findings indicate dysregulated expression of PARP1 confers cisplatin-resistance via autophagy activation in HepG2^DR cells. Morin hydrate inhibits cisplatin-mediated autophagy induction, resulting in increased susceptibility of HepG2^DR cells to cisplatin cytotoxicity. The combination of Morin hydrate with cisplatin may be a promising therapeutic strategy to enhance the efficacy of conventional chemotherapeutic drugs.

Purpose: Intratumoral injection of Clostridium novyi-NT, lacking alpha toxin, germinates and subsequently replicates in the tumor hypoxic regions, causing cell lysis and inflammation. This phase 1b study investigated the safety and synergistic effects of pembrolizumab and C. novyi-NT in advanced solid tumors. Experimental Design: Patients with injectable solid tumors received one intratumoral (IT) C. novyi-NT injection (Day 8) across four dose cohorts (3x104 to 100x104 spores) with pembrolizumab (200mg intravenous (IV) every 3 weeks, Day 0 to 24 months). Sixteen patients were enrolled with a median age of 62.5 years and the most common tumor types being breast cancer and melanoma. Results: The combination therapy demonstrated a manageable safety profile, with one grade 3 dose-limiting toxicity (DLT) of abscess formation and the remainder of treatment-related adverse events (TRAEs) in ≥ 10% of patients being grade 1 or 2 injection site reaction (25%), pyrexia (19%), pruritus (13%), leukopenia (13%) and anemia (13%). The maximum tolerated dose (MTD) was determined as pembrolizumab IV 200mg every 3 weeks and a single dose of 100x104 C.novyi-NT spores IT on Day8. The confirmed overall objective response rate of 25%(n=4) was observed among patients with undifferentiated nasopharyngeal squamous carcinoma, squamous cell carcinoma of the base of the tongue, vulvar melanoma, and chordoma. Among these patients we obtained 3 partial responses and one complete response. Median duration of response was 10.93 months, and stable disease was observed in 69% patients. Conclusion: This study highlights the potential of the C. novyi-NT and pembrolizumab combination as a promising therapeutic strategy for treatment-refractory solid tumors.

The cross-talk between apoptosis and autophagy influences anticancer drug sensitivity and cellular death in various cancer cell lines. However, the fundamental mechanisms behind this phenomenon are still unidentified. We demonstrated anti-cancerous role of cisplatin (CP) and morin hydrate (Mh) as an individual and/or in combination (CP-Mh) in hepatoma cells and tumor model. Exposure of CP resulted in the production of intracellular reactive oxygen species (ROS)-mediated cellular vacuolization, expansion of mitochondria membrane and activation of endoplasmic reticulum (ER)-stress. Consequently, Cyt c translocation led to the increase of Bax/Bcl-2 ratio, which simultaneously triggered caspase-mediated cellular apoptosis. In addition, CP-induced PARP-1 activation led to ADP-ribosylation of HMGB1, which consequently developed autophagy as evident by the LC3I/II ratio. Chemically-induced inhibition of autophagy marked by increased cell death signified a protective role of autophagy against CP treatment. CP-Mh abrogates the PARP-1 expression and significantly reduced HMGB1-cytoplasmic translocation with subsequent inhibition of the HMGB1-Beclin1 complex formation. In the absence of PARP-1, a reduced HMGB1 mediated autophagy was observed followed by induced caspase-dependent apoptosis. To confirm the role of PARP-1-HMGB1 signaling in autophagy, we used the PARP-1 inhibitor, 4-amino-1,8-naphthalimide (ANI), HMGB1 inhibitor, ethyl pyruvate (EP), autophagy inhibitors, 3-methyl adenine (3-MA) and bafilomycin (baf) and small interfering RNAs (siRNA) to target Atg5 in combination of CP and Mh. Exposure to these inhibitors enhanced the sensitivity of HepG2 cells to CP. Collectively, our findings indicate that CP-Mh in combination served as a prominent regulator of autophagy and significant inducer of apoptosis that maintains a homeostatic balance towards HepG2 cells and the subcutaneous tumor model.

Supplementary Table S3. Patient-Level Data on MSI Status, TMB, Injection Site, Tumor Size, and Prior IO Exposure.

Supplementary File S1. Study protocol.

Supplementary Table S4. Anti-tumor efficacy in injected lesion(s) and best overall response per RECISTv1.1 among enrolled participants.