CLIC1 is an Attractive Candidate for the Initial Binding Target of the Novel Peptide MTI‐101
Authors: Freeman Sarah , Elzamzamy Osama , Geldenhuys Werner , Hazlehurst Lori 
Published: April 2020

Acute Myeloid Leukemia (AML) is a hematologic cancer characterized by the increase in proliferation and block in differentiation of myeloid cells. AML is incurable, and most patients who initially respond to first line therapies will relapse. The majority of AML therapies target apoptosis as a method of tumor cell death, and resistance to apoptosis correlates with patient relapse. New therapies are needed to target alternative cell death pathways to overcome this apoptotic resistance. MTI‐101 is a novel cyclic peptide found to induce necrotic cell death in multiple myeloma, prostate cancer, and AML cell lines. MTI‐101 was initially discovered based on a cell adhesion phenotypic screen and shown to induce robust calcium flux and caspase independent cell death. MTI‐101 was found to have increased potency in relapsed multiple myeloma patient samples over newly diagnosed patients, making it an attractive new line of therapy for hematologic cancers with high relapse rates. The main objective of this project is to identify the binding target of MTI‐101. Based on our recent findings, Chloride Intracellular Channel 1 (CLIC1) has emerged as an attractive putative target of MTI‐101. Using a biotin conjugated analog of MTI‐101, we found CLIC1 to be present in the MTI‐101 drug binding complex. Moreover, our preliminary data indicate that MTI‐101 inhibits chloride efflux from AML U937 cells. Finally, CLIC1 was increased in the binding complex in cell lines depleted of the adhesion receptors CD44 and Integrin Alpha 4 & 6; a finding that correlates with increased MTI‐101 induced cell death. To determine the initial binding target of MTI‐101, we developed an assay to measure intracellular Ca2+ levels and cell death by using the Ca2+ indicator Fluo‐4 AM and DAPI as a marker for cell death. We utilized CRISPR to deplete the adhesion receptors CD44, and Integrins Alpha 4, Alpha 6, and Beta 1, which were previously found in the MTI‐101 drug binding complex by Mass Spectrometry. These knockout cell lines were used to re‐probe for the MTI‐101 binding complex using the biotin‐tagged MTI‐101. Lastly, we analyzed chloride efflux in the presence and absence of MTI‐101. Our data showed that depletion of CD44, Integrins Alpha 4 and Alpha 6, and Integrin Beta 1 contributed to a significant increase in MTI‐101 induced cell death. Additionally, we saw an increase in CLIC1 presentation in the MTI‐101 binding complex in the knockout cell lines compared to the wild type by Mass Spec. CLIC1’s increased levels in the drug binding complex of the knockout cell lines correlates with increased MTI‐101 induced Ca2+ flux and cell death compared to wild type U937 cells. Finally, we observed a robust inhibition of chloride efflux as evidenced by decreased MQAE in cells treated with MTI‐101. Based on our data, we hypothesize that MTI‐101 binds a CLIC1 containing complex and inhibits chloride efflux by directly binding CLIC1. This data suggests CLIC1 is an attractive candidate for the initial binding target of MTI‐101, and future studies will focus on depleting CLIC1 and determining whether CLIC1 is essential for MTI‐101 induced cell death.

Support or Funding Information

NIGMS U54GM104942, R44 CA221554, NCI 1R01CA195727‐01

Depletion of CD44 and Integrins Alpha 4, Alpha 6, and Beta 1 significantly increased MTI‐101 induced cell death at 10uM and 20uM doses. Significance compared within drug dose to Vector Control dose response. (**** p<0.0001, ANOVA, Post Hoc: Dunnett’s multiple comparison test). 4 replicates per day, 3 independent days, total N of 12. Mean and Standard Error shown.

Figure 1

MTI‐101 inhibits chloride efflux as evidenced by decreased MQAE fluorescence compared to the untreated control. (****p<0.0001, Unpaired t‐test with Welch’s correction.) 5 Replicates per day, 3 independent days for N of 15. Mean and Standard Error shown.

Figure 2