Introduction
Two-pore channel 2 (TPC2) represents an exceptional ion channel due to its localization on lysosomes1. TPC2 is overexpressed in several cancer types, e.g. in prostate cancer, and its expression is associated with poor survival probability2. To date, a role of TPC2 in leukemia has not been investigated. Pharmacologic inhibition of TPC2 can be performed with tetrandrine (Tet), a bisbenzylisoquinoline alkaloid3. However, its use is restricted due to unfavorable toxicity in animal studies4. In our study, we aim to elucidate the role of TPC2 function in multidrug resistant leukemia and, concurrently, identify efficacious TPC2 inhibitors by screening a library of synthetic bisbenzylisoquinoline derivatives (BBIQDs).
Results
Firstly, via qPCR analysis, we detected a 2-fold increased TPC2 mRNA expression in a vincristine-resistant (VCR-R) CEM (VCR-R CEM, T-ALL) cell line, compared with the parental CCRF-CEM cell line. Secondly, we knocked out TPC2 in VCR-R CEM cells using the CRISPR-Cas9 system to investigate its involvement in cell growth and treatment response. By studying cell proliferation over a period of six days, we found that the doubling time of TPC2-deficient cells was increased to 30 h, compared with 24 h for the wildtype (wt) cell line. Additionally, treatment of TPC2 knockout (ko) cells with VCR resulted in increased sensitivity versus wt, as indicated by proliferation (IC50 wt: 3.3 µM, IC50 ko: 1.6 µM, 72 h) and apoptosis (EC50 wt: 3.0 µM, EC50 ko: 1.7 µM, 48 h) assays. Next, a library of BBIQDs was synthesized and screened for the ability to inhibit TPC2 function using the whole endolysosomal patch clamp method. Our small molecule hits SG-005 and SG-094 inhibited TPC2 current density by 50% and 70%, respectively, giving novel TPC2 inhibitors with either similar or even increased potency, compared to Tet (50%). Treatment of VCR-R CEM cells with Tet, SG-005 and 94 effectively inhibited proliferation (IC50: 5-15 µM, 48 h). Toxicity was assessed by propidium iodide exclusion assays using Peripheral Blood Mononuclear cells (PBMCs, n=3 healthy donors), indicating reduced toxicity of SG-094 (<5% dead cells vs. 25% dead cells for Tet and SG-005, 20 µM, 48 h treatment). Additionally, SG-094 was well tolerated in vivo when applied to a mouse model (90 nmol/kg/d on three consecutive days). Remarkably, combination of VCR (0.01 and 0.1 µM) with Tet, SG-005 or 94 (1 and 5 µM) synergistically increased treatment response of VCR-R CEM cells (wt) and of B ALL patient-derived xenograft (PDX) cells from a relapse patient (Bliss values: 1.1-2.4).
Methods
CellTiter-Blue® cell proliferation assays, qPCR mRNA expression, propidium iodide exclusion and analysis of apoptosis were performed as described by the manufacturers. Whole endolysosomal patch clamp, analysis of specific apoptosis of PDX cells and generation of the TPC2 ko model using the CRISPR-Cas9 system were performed as described previously3, 5, 6. Analysis of doubling time was performed by counting cells daily using trypan blue staining and a Vi-CELL XR device as indicated by the manufacturer.
Conclusion
Taken together, we detected an upregulated mRNA expression of endolysosomal TPC2 in VCR-R CEM cells, indicating that it represents a potential target for therapeutic intervention for difficult-to-treat leukemia phenotypes. Furthermore, we could show that genetic ablation of the ion channel results in a reduced proliferation rate and an increased sensitivity towards VCR treatment. Finally, we successfully identified pharmacologic TPC2 inhibitors which show antiproliferative effects when applied as monotherapy and synergistically enhance treatment response of VCR-R CEM cells and PDX cells when combined with VCR.
References S. Patel, B. S. Kilpatrick, Biochim Biophys Acta Mol Cell Res1865, 1678-1686 (2018). F. Li, J. P. Ji, Y. Xu, R. L. Liu, Clin Transl Oncol, (2019). O. N. Nguyen et al., Cancer Res77, 1427-1438 (2017). H. Jin et al., Chem Res Toxicol24, 2142-2152 (2011). F. Koczian et al., Haematologica104, 546-555 (2019). F. A. Ran et al., Nat Protoc8, 2281-2308 (2013).
Disclosures
No relevant conflicts of interest to declare.
Biological and Clinical Significance of the CCR5/CCL5 Axis in Hepatocellular Carcinoma
