A Novel Inhibitor of HSP70 Induces Mitochondrial Toxicity and Immune Cell Recruitment in Tumors

Abstract

The protein chaperone HSP70 is overexpressed in many cancers including colorectal cancer, where overexpression is associated with poor survival. We report here the creation of a uniquely acting HSP70 inhibitor (HSP70i) that targets multiple compartments in the cancer cell, including mitochondria. This inhibitor was mitochondria toxic and cytotoxic to colorectal cancer cells, but not to normal colon epithelial cells. Inhibition of HSP70 was efficacious as a single agent in primary and metastatic models of colorectal cancer and enabled identification of novel mitochondrial client proteins for HSP70. In a syngeneic colorectal cancer model, the inhibitor increased immune cell recruitment into tumors. Cells treated with the inhibitor secreted danger-associated molecular patterns (DAMP), including ATP and HMGB1, and functioned effectively as a tumor vaccine. Interestingly, the unique properties of this HSP70i in the disruption of mitochondrial function and the inhibition of proteostasis both contributed to DAMP release. This HSP70i constitutes a promising therapeutic opportunity in colorectal cancer and may exhibit antitumor activity against other tumor types. Significance: These findings describe a novel HSP70i that disrupts mitochondrial proteostasis, demonstrating single-agent efficacy that induces immunogenic cell death in treated tumors.

HSP70 Inhibition Blocks Adaptive Resistance and Synergizes with MEK Inhibition for the Treatment of NRAS-Mutant Melanoma

“…Like MEKi, HSP90 inhibitors have not shown promise as monotherapies, but these inhibitors synergize with chemotherapy and targeted therapies in preclinical and early-phase clinical studies, thus supporting the use of this combination (49). Notably, the HSP70 inhibitor we used here-in has been shown to enhance the immune response to melanoma tumors (35), thus supporting the combination of this compound with immune checkpoint inhibitors as well. Along these lines, other members of the HSP70 family like the mitochondrial-localized member GRP75 (HSPA9) are also emerging targets for melanoma (50).…”

“…To do this, we first tested whether treatment with an HSP70 inhibitor would cause ID3 to localize in a detergent-insoluble fraction of the cell (due to misfolding). We performed a soluble-insoluble fractionation of WM4265.2, M93-047, and 1205Lu cell lines treated with our novel HSP70 inhibitor, AP-4-139B; whereas AP-4-139B is tagged with a triphenylphosphonium to increase distribution of the compound to the mitochondria, this inhibitor is broadly distributed, and it affects the solubility of client proteins in the cytosol and nucleus as well (35). Treatment of cells with increasing doses of AP-4-139B resulted in an increase of ID3 in the insoluble fraction in each of the cell lines tested, comparable with a known client, EGFR (Fig.…”

Inhibition of the Heat Shock Protein A (HSPA) Family Potentiates the Anticancer Effects of Manumycin A

“…It seems that pan-HSPA inhibition can target the redundant network of HSPA paralogs to overcome their joint pro-survival activity. In this context it is noteworthy that non-cancerous epithelial cells are relatively insensitive to pan-HSPAs inhibition [26,36].…”

“…Disruption of vital pro-cancerous inter-protein interactions, examples of which are the HSPAs-BAG3 interaction [33] or HSPAs-DNAJ interaction [36], seems the most important. These mechanisms can trigger apoptosis in apoptosis-competent NSCLC cells, exert cytostatic or non-apoptotic death-promoting effects in apoptosis-defective NSCLC cells [26], or lead to perturbations in mitochondrial proteostasis, secretion of danger-associated molecular patterns (DAMP), and increased recruitment of immune cells in primary and metastatic models of colorectal cancer [36]. Given that HSPAs can interact with components of the immune system, a type of immunogenic HSPA inhibitor would potentially be useful to modulate anticancer immune responses.…”

Overcoming Immunotherapy Resistance by Targeting the Tumor-Intrinsic NLRP3-HSP70 Signaling Axis

“…This is also true for the activity of the synthetic HSP70 inhibitor, MAL3-101, previously demonstrated to suppress proliferation and growth of Merkel cell carcinoma tumor cells in vitro and in vivo , respectively [ 85 ]. While there have been few studies linking the inhibition of HSP70 with anti-tumor immunity, a recent study performed in the MC38 and CT26 murine colon cancer cell lines as well as the HT-29 human colon cancer cell line demonstrated the HSP70 inhibitor, AP-4-139B, to induce an immunogenic form of cell death that promotes the recruitment of both CD4 + and CD8 + T cells [ 86 ]. Finally, another HSP70 inhibitor, minnelide, has also been found to have anti-tumor properties and is currently being investigated in a series of clinical trials focused on various cancer types ( Table 1 ) [ 87 , 88 ].…”