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20 μM TFP-treated group. The cell were recorded in 2 h after seeding on 6-well plate equipped with x10 objective lens. The video were recorded by Cell Observer (Carl Zeiss, Germany).

Supplementary Figure S1. TFP induces morphological change of U87MG cells, and cell death; Supplementary Figure S2. Body weight is not changed during TFP injection in in vivo glioblastoma skin xenograft model; Supplementary Figure S3. IP3R1, 2 and 3 shRNA knockdown test by western blot and functional knockdown test using GBL 28 primary glioblastoma cells; Supplementary Figure S4. TFP reduces cell viability of several GBL cells;Supplementary Table S1. IP3R 1, 2, 3 and CaM shRNA sequences

0.9% saline-treated control group. The cell were recorded in 2 h after seeding on 6-well plate equipped with x10 objective lens.

Background: Axl, Mer and CSF1 receptor tyrosine kinases play vital roles in promoting the immunosuppressive tumor microenvironment (TME) by affecting myeloid functions (e.g., tumor associated macrophage [TAM], myeloid derived suppression cell [MDSC]) and promoting epithelial-to-mesenchymal transition (EMT). Thus, simultaneous inhibition of Axl, Mer and CSF1R may be an effective strategy for TME modification. Q702 is a novel Axl/Mer/CSF1R kinase inhibitor that affects the immune components (modulating TAM and MDSC populations, inducing CD8+ T cell infiltration and increasing IFN-ɣ in CD8+ T cell) as well as changes in malignant cells such as increasing MHC I on the tumor cells of syngeneic mouse models. These nonclinical results suggest that Q702 monotherapy or Q702 combination with conventional therapies may have considerable potential as a novel treatment strategy for patients with advanced solid tumors. Methods: This is a Phase 1, Multicenter, Open-label, Dose-Escalation, Safety, Pharmacodynamic, Pharmacokinetic Study of Q702 with a Cohort Expansion at the recommended phase 2 dose (RP2D) in Patients with Advanced Solid Tumors (NCT04648254). Q702 was administered orally for seven days every other week. Peripheral blood samples were obtained on days 1,8,15, and 21. Axl, Mer and CSF1R target engagement is assessed by the quantifications of soluble Axl, Mer and M-CSF in plasma by Luminex xMAP® technology or ELISA. The pharmacodynamic biomarker changes are measured by flow cytometry for immune cell population shifts and IFN-ɣ levels in specific immune cells. Results: PK and PD biomarker samples from 22 patients with various tumor types (e.g. colon, pancreas, esophageal) from the dose escalation phase (4 mg to 240 mg) have been analyzed. Pharmacokinetic studies demonstrated dose proportional increase in Cmax and AUClast of Q702 and its two active metabolites which have activity against Axl and/or CSF1R. Axl and CSF1R target engagement by Q702 treatment is observed in a dose dependent manner. From the 60 mg cohort, target engagement for Axl and CSF1R reached a inhibitory level that was observed in nonclinical models. In the pharmacodynamic biomarker analysis, IFN-ɣ in CD8+ T cells and non-T cell populations is increased. Monocytes and M-MDSC population are decreased in peripheral blood. Conclusion: Up to 240 mg, Q702 has demonstrated the intended pharmacologic activity with acceptable safety profile. In biomarker analysis, immune modulation activity is exerted by Axl/Mer/CSF1R inhibition. Further assessment of pharmacokinetics, pharmacodynamics, safety and antitumor activity will be performed at the expansion phase at the RP2D in patients with selected advanced tumors. Citation Format: Bae Jung Choi, Devalingam Devalingam, Angela Alistar, Anthony El-Khoueiry, Alain Mita, Hwankyu Kang, Jinho Choi, Hyunji Ahn, Jeongjun Kim, Seung-Joo Lee, Yeong-In Yang, Jiye Ahn, Borami Jeon, Jaeseung Kim, Kiyean Nam. Patient pharmacodynamic biomarker and pk evaluation results from an ongoing phase I dose-escalation study of q702, an axl, mer and csf1r kinase inhibitor in patients with advanced solid tumors [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 2255.

Calcium (Ca2+) signaling is an important signaling process, implicated in cancer cell proliferation and motility of the deadly glioblastomas that aggressively invade neighboring brain tissue. We have previously demonstrated that caffeine blocks glioblastoma invasion and extends survival by inhibiting Ca2+ release channel inositol 1,4,5-trisphosphate receptor (IP3R) subtype 3. Trifluoperazine (TFP) is an FDA-approved antipsychotic drug for schizophrenia. Interestingly, TFP has been recently reported to show a strong anticancer effect on lung cancer, hepatocellular carcinoma, and T-cell lymphoma. However, the possible anticancer effect of TFP on glioblastoma has not been tested. Here, we report that TFP potently suppresses proliferation, motility, and invasion of glioblastoma cells in vitro, and tumor growth in in vivo xenograft mouse model. Unlike caffeine, TFP triggers massive and irreversible release of Ca2+ from intracellular stores by IP3R subtype 1 and 2 by directly interacting at the TFP-binding site of a Ca2+-binding protein, calmodulin subtype 2 (CaM2). TFP binding to CaM2 causes a dissociation of CaM2 from IP3R and subsequent opening of IP3R. Compared with the control neural stem cells, various glioblastoma cell lines showed enhanced expression of CaM2 and thus enhanced sensitivity to TFP. On the basis of these findings, we propose TFP as a potential therapeutic drug for glioblastoma by aberrantly and irreversibly increasing Ca2+ in glioblastoma cells. Mol Cancer Ther; 16(1); 217–27. ©2016 AACR.

TPS2673 Background: Immune checkpoint inhibitors directly targeting T cell activation have been successfully used in the treatment of various malignancies, nevertheless, the durable ORRs are low for certain indications. The low ORRs have been attributed to the immune suppressive tumor microenvironment (TME), composed of innate immune suppressive components such as tumor associated macrophages (TAM) and myeloid-derived suppression cells (MDSC). The potential contributions of innate immune modulation to anti-tumor immunity, suggest the need for the novel strategies to elicit a more efficient/robust immune response against the targeted malignant cells. Axl, Mer and CSF1R receptor tyrosine kinases play vital roles in promoting an immune suppressive TME by affecting TAM and MDSC populations and by decreasing antigen presentation on tumor cells. Q702 is a novel Axl/Mer/CSF1R inhibitor, able to modulate the TAM and MDSC population leading to CD8+ T cell activation and to increase antigen presentation of the tumor cells in syngeneic animal models. Q702, as a monotherapy, shows significant tumor growth inhibition in multiple syngeneic tumor models, and demonstrates synergistic effects with anti-PD-1 treatment particularly in high myeloid containing tumor models. Interestingly, intermittent administration of Q702 monotherapy demonstrates a more favorable immune cell population changes, possibly through preventing immune exhaustion secondary to negative feedback with continuous activation. These results suggest that Q702 monotherapy or in combination with existing therapies have a good potential to become a novel treatment strategy for patients with advanced solid tumors. Methods: “A Phase 1, Multicenter, Open-label, Dose-Escalation, Safety, Pharmacodynamic, Pharmacokinetic Study of Q702 with a Cohort Expansion at the RP2D in Patients with Advanced Solid Tumors. (NCT04648254)” is open and recruiting patients at 4 US investigative sites. Patients with histologically or cytologically confirmed advanced or metastatic solid tumors, that have progressed following SOC or for which there is no SOC which confers clinical benefit are being enrolled in this study. The study follows a standard dose escalation. The study will enroll up to 78 patients. The primary endpoint is to establish safety, PK profile and define the recommended phase 2 dose. The secondary and exploratory endpoints include establishing pharmacokinetic/pharmacodynamic relationship, potential biomarkers and preliminary anti-tumor activity. Clinical trial information: NCT04648254.

Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival time in mice models of glioblastoma. In attempt to identify an effective trifluoperazine analog, fourteen compounds have been synthesized and biologically in vitro and in vivo assessed. Using MTT assay, compounds 3dc and 3dd elicited 4–5 times more potent inhibitory activity than trifluoperazine with IC 50 = 2.3 and 2.2 μM against U87MG glioblastoma cells, as well as, IC 50 = 2.2 and 2.1 μM against GBL28 human glioblastoma patient derived primary cells, respectively. Furthermore, they have shown a reasonable selectivity for glioblastoma cells over NSC normal neural cell. In vivo evaluation of analog 3dc confirmed its advantageous effect on reduction of tumor size and increasing the survival time in brain xenograft mouse model of glioblastoma. Molecular modeling simulation provided a reasonable explanation for the observed variation in the capability of the synthesized analogs to increase the intracellular Ca 2+ levels. In summary, this study presents compound 3dc as a proposed new tool for the adjuvant chemotherapy of glioblastoma.