Chk1 inhibition potently inhibits phosphorylation and activation of the STAT3 transcription factor in human multiple myeloma cells

A recent discovery by Dr. Grant’s team published in the American Association of Cancer Research journal Molecular Cancer Research describes a previously unknown link between the DNA damage response-related protein Chk1 and the STAT3 transcription factor, which plays an important role in the survival and proliferation of numerous malignancies, including multiple myeloma (MM).

Highlights of the study:

• Chk1, conventionally viewed as a serine-threonine kinase, represents an important component of the DNA damage response in cancer cells exposed to single-strand DNA breaks and replication stress. The present study demonstrated for the first time that interruption of Chk1 e.g., by pharmacologic Chk1 inhibitors, prevented the activation of the STAT3 transcription factor in MM cells by blocking tyrosine705 phosphorylation of the STAT3 trans-activation domain.

• These actions were associated with down-regulation of downstream pro-survival targets of STAT3 e.g., MCL-1, BCL-xL, and c-Myc, and induction of MM cell death both in vitro and in vivo.

• Collectively, these findings identify a novel, non-canonical function of Chk1 as a STAT3 tyrosine kinase activator, and raise the possibility that Chk1 inhibitors may represent valuable additions to the therapeutic armamentarium for MM and potentially other STAT3-dependent malignancies.

The STAT3 transcription factor has long been known as an important contributor to tumorigenesis, particularly in the case of MM cells. It promotes the survival of malignant cells, and protects them from the lethal effects of chemotherapeutic agents, in part by triggering microenvironmental forms of drug resistance. However, like most transcription factors, targeting STAT3 pharmacologically has been very challenging. The Chk1 kinase represents an important component of the DNA damage response to various genotoxic agents which induce replication stress. Inhibitors of Chk1 spare CDC25A/C phosphatases from degradation, leading to dephosphorylation/activation of CDK1, resulting in inappropriate transit of cells with DNA damage through the G2M boundary, culminating in mitotic catastrophe and cell death.

The Grant laboratory, using a gene expression profiling strategy, found that MM cells exposed to a Chk1 inhibitor exhibited a dramatic reduction in expression of STAT3-dependent genes, including MCL-1, BCL-xL, and c-Myc. Notably, they found Chk1 inhibitors potently antagonized phosphorylation of the STAT3 transactivation domain on a tyrosine 705 phosphorylation site which plays a critical role in STAT3 dimerization and nuclear transport. It is noteworthy that these agents were active at concentrations 1-2 logs

lower than standard STAT3 antagonists. Significantly, it was demonstrated via an in vitro kinase assay that Chk1 represented a kinase responsible for STAT3 Tyr705 phosphorylation (Figure 1). This led in turn to down-regulation of the previously mentioned proteins, events shown to play a functional role in Chk1-mediated MM cell death. Importantly, these findings were recapitulated in an in vivo mouse model, suggesting that Chk1 inhibitors may represent a viable approach to STAT3 interruption in the clinical setting. The significance of these findings is that Chk1 antagonists, or potentially inhibitors acting upstream of this kinase, may represent a novel and potentially more effective strategy to disrupt STAT3 cytoprotective signaling in patients with MM or other malignancies. It is also possible that this strategy may be particularly effective against STAT3-dependent malignancies. Based on these considerations, discussions with the NCI are ongoing to test this concept through the NCI’s Experimental Therapeutics Clinical Trials Network, of which the Massey Cancer Center and VCU are a part.

This work was supported by R01 CA205607, P30 CA16059, and 5UM1CA186644-06. It was also supported by the Massey Cancer Center Mouse Models and Flow Cytometry shared resources.

Publication:

* Zhou L, Pei X, Zhang Y, Ning Y, Li L, Hu X, Chalasani SL, Sharma K, Nkwocha J, Yu J, Bandyopadhyay D, Sebti SM, Grant S. Chk1 Inhibition Potently Blocks STAT3 Tyrosine705 Phosphorylation, DNA-Binding Activity, and Activation of Downstream Targets in Human Multiple Myeloma Cells. Mol Cancer Res. 2022 Mar 1;20(3):456-467. DOI: 10.1158/1541-7786.MCR-21-0366

About the Investigators: Steven Grant is Professor of Internal Medicine, Human and Molecular Genetics, and Biochemistry, VCU Health Science Center, as well as the Shirley and Sture Gordon Olsson Professor of Oncology. He is also Associate Director for Translational Research and co-Leader of the Developmental Therapeutics program of the Massey Cancer Center. Dr. Said Sebti is Professor of Pharmacology and Associate Director of Basic Science, Massey Cancer Center. Liang Zhou was an Instructor of Internal Medicine, VCU. Lin Li and Xiaoyan Hu are post-doctoral Research Associates in the Department of Internal Medicine, VCU. Kanika Sharma and Jewel Nkwocha are research specialists. Johnathan Yu was a VCU undergraduate. Xinyan Pei, Yu Zhang, and Sri Lakshmi Chalassani were post-doctoral research associates in the Department of Internal Medicine, VCU.