Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981’s anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.

Background & Aims The epithelial barrier is the host’s first line of defense against damage to the underlying tissue. Upon injury, the epithelium plays a critical role in inflammation. The IκB kinase β (IKKβ)/nuclear factor-κB pathway was shown to be active in the esophageal epithelium of patients with esophageal disease. However, the complex mechanisms by which IKKβ signaling regulates esophageal disease pathogenesis remain unknown. Our prior work has shown that expression of a constitutively active form of IKKβ specifically in esophageal epithelia of mice ( Ikkβca Esophageal Epithelial Cell-Knockin ( EEC-KI ) ) is sufficient to cause esophagitis. Methods We generated ED-L2/Cre;Rosa26-Ikkβca +/L ;Stat3 L/L ( Ikkβca EEC-KI ;Stat3 Esophageal Epithelial Cell Knockout ( EEC-KO ) ) mice, in which the ED-L2 promoter activates Cre recombinase in the esophageal epithelium, leading to constitutive activation of IKKβ and loss of Stat3. Esophageal epithelial tissues were collected and analyzed by immunostaining, RNA sequencing, quantitative real-time polymerase chain reaction assays, flow cytometry, and Western blot. Ikkβca EEC-KI mice were treated with neutralizing antibodies against interleukin (IL)23p19 and IL12p40. Results Here, we report that Ikkβca EEC-KI mice have increased activation of epithelial Janus kinase 2/STAT3 signaling. Stat3 deletion in Ikkβca EEC-KI mice attenuated the neutrophil infiltration observed in Ikkβca EEC-KI mice and resulted in decreased expression of genes related to immune cell recruitment and activity. Blocking experiments in Ikkβca EEC-KI mice showed that STAT3 activation and subsequent neutrophil recruitment are dependent on IL23 secretion. Conclusions Our study establishes a novel interplay between IKKβ and STAT3 signaling in epithelial cells of the esophagus, where IKKβ/IL23/STAT3 signaling controls neutrophil recruitment during the onset of inflammation. GEO accession number: GSE154129.

Although cancer immunotherapy has revolutionized cancer treatment, patient response to immunotherapy remains varied. Despite progress, the mechanisms limiting cancer immunotherapy are not yet fully understood. A low number of tumor infiltrating T cells (cold tumor) is one of the limiting factors for cancer immunotherapy. Agents that enhance immunotherapy by shifting cold tumors to hot tumors will greatly benefit cancer immunotherapy. In prostate cancer, the majority of tumors are known to be cold and hence, cancer immunotherapy is not the ideal treatment option. Here, we use a prostate cancer model as an example to demonstrate that modulating the tumor microenvironment through altering autophagy will change the tumor cytokine secretion profile, which in turn attracts immune lymphocytes into the tumor microenvironment. In tandem, we have identified a candidate compound known as ESK981 for such a purpose. Method A small molecule library was used for screening autophagy activity and cytokine secretion. Various types of human cancer cell lines (prostate, renal, bladder, breast etc.) and multiple syngeneic mouse lines were examined for autophagy activity as well as an in vitro response to interferon stimulation with or without ESK981. A syngeneic mouse prostate cancer was used for the in vivo examination of autophagy as well as the anti-tumor effect by ESK981 monotherapy and/or in combination with anti-PD-1 therapy. Conclusion We have discovered a robust, novel autophagy-modulating small molecule, named ESK981, for the treatment of various cancer types as a monotherapy. In addition, we have demonstrated that autophagy has an essential role in the anti-tumor effect of immunotherapy, especially for anti-PD-1 in syngeneic prostate cancer model. Therefore, the use of a small molecule such as ESK981 to target autophagy can enhance immunological infiltration induced by cancer immunotherapy, such as immune checkpoint blockade, for non-immunogenic tumors. Conflict of Interest Statement A.M.C. is a co-founder and serves on the Scientific Advisory Board of Esanik Therapeutics, Inc. which owns to the rights to the clinical development of ESK981. Esanik Therapeutics, Inc. did not fund or approve the conduct of this study. Funding This project is supported by Prostate Cancer Foundation. Citation Format: Yuanyuan Qiao, Jae E. Choi, Josh N. Vo, Jean C. Tien, Lisha Wang, Lanbo Xiao, Stephanie A. Simko, Andrew D. Delekta, Nathan B. Hodge, Parth Desai, Kristin Juckette, Alice Xu, Fengyun Su, Rui Wang, Xuhong Cao, Xiaoju Wang, Xiaoming Wang, Javed Siddiqui, Zhen Wang, Amélie Bernard, Ester Fernandez-Salas, Nora M. Navone, Ke Ding, Eeva-Liisa Eskelinen, Elisabeth I. Heath, Daniel J. Klionsky, Weiping Zou, Arul M. Chinnaiyan. Therapeutic targeting autophagy to sensitize cancer immunotherapy in various cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4153.

Achalasia is an esophageal motility disorder characterized by the functional loss of myenteric plexus ganglion cells in the distal esophagus and lower esophageal sphincter. Histological changes have been reported in the esophageal mucosa of achalasia, suggesting its involvement in disease pathogenesis. Despite recent advances in diagnosis, our understanding of achalasia pathogenesis at the molecular level is very limited and gene expression profiling has not been performed. We performed bulk RNA-sequencing on esophageal mucosa from 14 achalasia and 8 healthy subjects. 65 differentially expressed genes (DEGs) were found in the distal esophageal mucosa of achalasia subjects and 120 DEGs were identified in proximal esophagus. Gene expression analysis identified genes common or exclusive to proximal and distal esophagus, highlighting regional differences in the disease. Enrichment of signaling pathways related to cytokine response and viral defense were observed. Increased infiltration of CD45+ intraepithelial leukocytes were seen in the mucosa of 38 achalasia patients compared to 12 controls. Novel insights into the molecular changes occurring in achalasia were generated in this transcriptomic study. Some gene changes observed in the mucosa of achalasia may be associated with esophagitis. Differences in DEGs between distal and proximal esophagus highlight the importance of better understanding regional differences in achalasia.