Background:Sorafenib is a potent inhibitor against Raf kinase and several receptor tyrosine kinases that has been approved for the clinical treatment of advanced renal and liver cancer. Combining sorafenib with other agents has been shown to improve its antitumour efficacy by not only reducing the toxic side effects but also preventing primary and acquired resistance to sorafenib. We have previously observed that tetrandrine exhibits potent antitumour effects in human hepatocellular carcinoma. In this study, we investigated the synergistic antitumour activity of sorafenib in combination with tetrandrine.Methods:This was a two-part investigation that included the in vitro effects of sorafenib in combination with tetrandrine on cancer cells and the in vivo antitumour efficacy of this drug combination on tumour xenografts in nude mice.Results:Combined treatment showed a good synergistic antitumour effect yet spared nontumourigenic cells. The potential molecular mechanism may be mainly that it activated mitochondrial death pathway and induced caspase-dependent apoptosis in the cancer cells. Accumulation of intracellular reactive oxygen species (ROS) and subsequent activation of Akt may also be involved in apoptosis induction.Conclusion:The antitumour activity of sorafenib plus tetrandrine may be attributed to the induction of the intrinsic apoptosis pathway through ROS/Akt signaling. This finding provides a novel approach that may broaden the clinical application of sorafenib.

BACKGROUND AND PURPOSETetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications to treat diverse diseases. Tetrandrine induced apoptosis or, at low concentrations, autophagy of human hepatocellular carcinoma cells. Here we have tested the effects of inhibitors of autophagy such as chloroquine, on the response to low concentrations of tetrandrine in cancer cells. EXPERIMENTAL APPROACHCultures of several cancer cell lines, including Huh7, U251, HCT116 and A549 cells, were exposed to tetrandrine, chloroquine or a combination of these compounds. Cell viability and content of reactive oxygen species (ROS) were measured and synergy assessed by calculation of the combination index. Western blot and RT-PCR assays were also used along with fluorescence microscopy and histochemical techniques. KEY RESULTSCombinations of tetrandrine and chloroquine were more cytotoxic than the same concentrations used separately and these effects showed synergy. Such effects involved increased ROS generation and were dependent on caspase-3 but independent of Akt activity. Blockade of tetrandrine-induced autophagy with 3-methyladenine or bafilomycin-A1 induced apoptosis in cancer cells. Lack of p21 protein (p21 −/− HCT116 cells) increased sensitivity to the apoptotic effects of the combination of tetrandrine and chloroquine. In a tumour xenograft model in mice, combined treatment with tetrandrine and chloroquine induced ROS accumulation and cell apoptosis, and decreased tumour growth. CONCLUSIONS AND IMPLICATIONSThe combinations of tetrandrine and chloroquine exhibited synergistic anti-tumour activity, in vitro and in vivo. Our results suggest a novel therapeutic strategy for tumour treatment.

Cyclin-dependent kinases (CDKs) 4/6 inhibitors are a powerful class of therapeutic drugs for the treatment of advanced metastatic breast cancer. However, the currently approved CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib have dose-limiting toxicities that require treatment holidays or reductions to sub-optimal doses, thus limiting full target inhibition. The residual CDK4/6 activity, together with persistent signaling through CDK2/cyclin E are among key resistant mechanisms that can compromise full clinical benefit. RGT-419B is a 3rd generation CDK inhibitor with an optimized kinase activity spectrum that has been discovered employing a Computer Accelerated Rational Design technology platform. RGT-419B has potent sub-nM CDK4 activity with desired degrees of selectivity against kinases such as CDK6, CDK9 and GSK3β to enable full target engagement with an improved safety profile. Furthermore, single digit nM CDK2 kinase activity has been incorporated into the design of RGT-419B to combat Cyclin E/CDK2-driven resistance. In vitro, RGT-419B showed more robust activity against palbociclib-resistant ER+ breast cancer cells than abemaciclib. In ER+ T47D breast cancer cells with overexpression of Cyclin E1, RGT-419B exhibited better antiproliferation activity than either abemaciclib or palbociclib. RGT-419B also demonstrated more durable in vivo tumor growth inhibition when compared with abemaciclib in an ER+ breast cancer xenograft model. The optimized kinase activity spectrum of RGT-419B provides an opportunity to treat ER+ breast cancer patients refractory to the existing CDK4/6 inhibitors, as either a single agent or in combination with other therapies. Citation Format: Zhi Xie, Jing Han, Zhilong Hu, Hu He, Xianqiang Sun, Fei Zhang, Jing Lin, Lili Yao, Xinjuan Wang, Liufeng Mei, Yangyang Liu, Guoyun Zhu, Xi Chen, Xiaotian Zhu, Wenge Zhong. Targeting resistance to current CDK4/6 therapies by RGT-419B, an inhibitor with optimized kinase activity spectrum [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-22.

Background: HPK1 (MAP4K1) is a serine/threonine Ste20-related protein kinase that belongs to the mitogen-activated protein kinase (MAPK) family. HPK1 is mainly expressed in hematopoietic cells and serves as a negative regulator of anti-tumor immunity through modulating the activation of lymphocytes and dendritic cells. Upon TCR activation, HPK1 phosphorylates the adaptor protein SLP76 at Ser376 to destabilize the SLP76 microclusters, which leads to the attenuation of the TCR signaling. The reported anti-tumor efficacy data from HPK1 knockout and kinase-dead knock-in mouse models support HPK1 as a novel intracellular I/O target. Methods: Regor CARD platform (Computer Accelerated Rational Discovery) was deployed to identify potent and selective inhibitors of HPK1. Biochemical assays and primary human pan T cell-based cellular assays were utilized to support the structure-activity relationship (SAR) analysis and inhibitor optimization. In vitro and in vivo target engagement studies were conducted in Jurkat T cells and mouse splenocytes, respectively. In vivo efficacy study data were generated using CT-26 syngeneic tumor mouse model. Results: Highly potent HPK1 inhibitors with good selectivity against the liable immune kinases were identified. RGT-197, one of the lead inhibitors demonstrated a significant increase of IL-2 secretion in primary human pan T cells upon TCR activation. It inhibited TCR-induced phosphorylation of SLP76 at Ser-376 in vitro and in vivo. Anti-tumor efficacy was observed in CT-26 tumor-bearing mice by oral dosing of RGT-197 as a single agent and in combination with an anti-PD1-monoclonal antibody. Furthermore, RGT-197 increased the level of cytokines important for anti-tumor immunity in vivo. Conclusion: RGT-197, a potent and selective HPK1 inhibitor, provides a potential opportunity as a small molecule I/O agent to boost anti-tumor immunity either as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Hao Liu, Lei Wu, Song Feng, Wei Huang, Huijuan Li, Jing Lin, Yangyang Liu, Liufeng Mei, Baoqi Ren, Julie Xie, Lili Yao, Wenge Zhong. A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P241.

Background: KRAS is the most frequently mutated oncogene with high prevalence in non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and pancreatic cancers (PAC). FDA currently approved sotorasib provides breakthrough therapy for cancer patients with KRASG12C mutation, however there is still high unmet medical need for new agents that target a broader KRAS mutated tumors. A new opportunity emerges to develop a pan KRAS inhibitor by suppressing the upstream guanine nucleotide exchange factor (GEF) protein son of sevenless 1 (SOS1). SOS1 is a key activator of KRAS and facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state. Binding to its catalytic domain, small molecule SOS1 inhibitor prevents KRAS activation and suppresses cancer cell proliferation. Material and Methods: Regor’s unique Computer Accelerated Rational Design (CARD) technology platform was applied to identify potent and selective SOS1 inhibitors. Biochemical assays and cellular assays were utilized to drive the structure-activity relationship (SAR). In vitro and in vivo target engagement were confirmed. In vivo efficacy study data were generated using lung and pancreatic cancer xenograft mouse models with KRAS mutation. Results: In vitro, RGT-018 blocked the interaction of KRAS::SOS1 with high selectivity, and inhibited proliferation of a broad spectrum of mutant KRAS-driven cancer cells as a single agent. Robust anti-proliferation activity was observed when RGT-018 was combined with MEK, KRASG12C, EGFR or CDK2/4/6 inhibitors in vitro. Oral administration of RGT-018 inhibited tumor growth and suppressed KRAS signaling pathway activation in tumor xenografts. Furthermore, combination with MEK or KRASG12C inhibitors led to profound tumor regression. Conclusions: The pharmacological properties of RGT-018 represent an attractive drug candidate with oral bioavailability for combination with targeted agents to treat a broader patient population driven by mutant KRAS. Citation Format: Fei Xiao, Kailiang Wang, Xinjuan Wang, Huijuan Li, Zhilong Hu, Wei Huang, Xiaoming Ren, Teng Feng, Lili Yao, Jing Lin, Chunlai Li, Liufeng Mei, Zhuanzhuan Zhang, Yangyang Liu, Xi Chen, Xiaotian Zhu, Wenge Zhong, Zhi Xie. Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 477.