In Vitro Characterization of TP-2846: A Novel Tetracycline Antileukemia AgentCorey Fyfe1, Ajay Bhargava2, Juan Ballesteros3, Alicia Robles3, Edward E. MacKee4, Cuixiang Sun1, Xiao-Yi Xiao1, and Jacques Dumas1

1Tetraphase Pharmaceuticals, Inc., Watertown, MA; 2Shakti Bioresearch, LLC, Woodbridge, CT; 3Vivia Biotech, S.L., Madrid, Spain, 4Central Michigan University College of Medicine

Poster # 4802

AACR (American Association for Cancer Research), March 29 – April 3, 2019, Atlanta, Georgia

ConclusionsTP-2846 shows potent anti-proliferative activity against AML cell lines in vitro as well as activity against frozen primary AML samples in an ex vivo study. TP-2846 appears to inhibit mitochondrial translation and induce growth arrest in leukemic cells.

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Contact:Tetraphase Pharmaceuticals Medical Informationtetraphase@druginfo.com(833) 793-7282

IntroductionThe potential for a mitochondrially targeted therapy for the treatment of cancers has been proposed both through the development of targeted novel agents and repurposing currently approved antimicrobial therapies with anti-mitochondrial activity (Skrtic 2011, Cohen and Saneto 2012). Prior research has shown that the altered metabolic function of cancer cells, and especially therapy resistant cells, can push them towards a reliance on oxidative phosphorylation and increased overall mitochondrial biomass (Lamb 2015, Alam 2016, Ozsvari 2017). Early investigations with tigecycline, a tetracycline derivative antibiotic (glycylcycline), showed activity in cancer cell lines through mitochondrial inhibition or the miRNA-199b-5p-HES-1-pAKT pathways, as well as some promising activity in tumor xenograft models. However, tigecycline failed to show activity at the maximum tolerated dose in a phase 1 study (Skrtic 2011, Jitkova 2014, Reed 2016, Yang 2016). TP-2846 was identified through a medicinal chemistry program screening for tetracycline derivatives with anti-proliferative activity (Abstract #3857). TP-2846 showed potent in vitro activity in cell proliferation assays against leukemic and solid tumor cell lines. Cell-free transcription/translation assays support activity against mitochondrial ribosomes. Mechanistic studies on the activity of TP-2846 against various cell lines support a linking of two proposed pathways showing inhibition of mitochondrial activity and cellular respiration as well as promotion of apoptosis in cancer cells. Additionally, ex vivo assays performed on samples from AML patients showed promising inhibitory activity of TP-2846.

MethodsCell proliferation assays

Cell lines were purchased from the ATCC, DSMZ, and JRCB, and grown in RPMI 1640/10% FBS or ATCC modified RPMI 1640/10% FBS in a humidified 37° C/5% CO2 incubator. Cell proliferation assays were performed in 96-well white-walled cell culture treated plates (Corning 3903) using the Promega Cell Titer Glo luminescent assay according to manufacturer instructions. Cells were plated in 50 µL volume at approximately 1x105 cells/mL and incubated overnight. Compound dilutions were added in 50 µL volume (T0) and incubated for 72 hours.

Cell-free transcription/translation assays

Mitochondrial transcription/translation assays were performed in the McKee laboratory as previously described (McKee 2006).

Western blots and qPCR

Western blot assays were performed by Shakti BioResearch. Incubation and media conditions were as noted above with the addition of pen-strep. Cells were plated in 6 well plates and treated for 18 hours with compound. Cell lysates were prepared with lysis buffer from Cell Signaling Technology. Equivalent amounts of protein samples were run on NuPAGE 4-12% Bis-Tris gels with MOPS/SDS running buffer and transferred to a nitrocellulose membrane using iBLOT (Invitrogen). Primary antibodies were purchased from BosterBio (COX-1), Santa Cruz Laboratories (COX-4), and Cell Signaling Technologies (β-Actin). HRP-conjugated secondary antibodies were purchased from Bio-Rad. Detection was performed with Pierce ECL Plus Substrate.

Results (cont’d)Methods (cont’d)Gene expression assays were performed similarly, with cells grown in 1 mL culture in 24-well plates. Cells were treated with compound for 18-24 hours with triplicate wells per growth condition. Total RNA and miRNA samples were prepared using Thermo mirVana isolation kits. Samples for quantitation were prepared using Thermo High Capacity cDNA and TaqMan Advanced miRNA kits. Assays were run on a Thermo StepOne Plus instrument with results analyzed by ΔΔCT method using commercially available primer and probe sets from Thermo Scientific. GAPDH was used as the endogenous control for mRNA samples while miR-361-5p was used as the endogenous control for miRNA samples.

Vivia ex vivo assays

TP-2846 was screened as a single agent against 15 frozen bone marrow samples from AML patients in the Vivia Native Environment cell depletion assay. Cell depletion dose response curves were measured by flow cytometry at five compound concentrations after 48 hours of exposure.

Figure 1. TP-2846 retains activity against a venetoclax-resistant AML cell line

ResultsCell proliferation assays

MV4-11 HL-60 MOLT4 K-562 CCRF-CEM MOLM-14 CESS CEM/C1 ARH-77 KASUMI-1 OCI-AML-2 OCI-AML-3

TP-2846 0.13 0.22 0.06 0.35 0.30 0.24 0.24 0.19 0.51 0.06 0.33 0.31

Tigecycline 2.26 5.7 1.7 2.7 4.65 2.5 3.297 2.49 4.72 4.37 5.2 5.81

Table 1. TP-2846 activity against leukemic cell lines

IC50 values presented in µM as an average of minimum three replicate experiments.

TP-2846 exhibits potent anti-proliferative activity against a number of leukemic cell lines, including p53 mutant cell lines, with an average 20-fold potency advantage over tigecycline.

TP-2846 Doxorubicin Daunorubicin

K562 0.4956 0.3341 0.02155

K562ADM 1.126 5.345 1.393

Shift 2.27 15.99 64.64

Table 2. TP-2846 activity against anthracycline-resistant cells

IC50 values presented in µM

TP-2846 was active against K562ADM cell lines expressing elevated MDR1/ABCB1/P-gp, a common mechanism of resistance to anthracycline compounds.

Cell-free transcription/translation assaysFigure 2. Activity of TP-2846 in mitochondrial transcription/translation assays

TP-2846 showed potent inhibition of mitochondrial protein synthesis in an isolated intact mammalian mitochondrial protein synthesizing system.

Western blots and qPCRFigure 3. The impact of TP-2846 exposure on mitochondrial protein levels in MV4-11

MV4-11 cells treated for 18 hours, equal amount of protein loaded and analyzed on 4-12% gel. COX-1 (mitochondrial cytochrome oxidase-1) rabbit primary antibody (PA1317) diluted 1:1000, COX-4 mouse primary antibody (SC376381) diluted 1:400. Treatment with TP-2846 shows depletion of mitochondrially expressed COX-1 protein but not nuclear encoded COX-4, relative to β-Actin control.

Figure 5. The impact of TP-2846 exposure on mitochondrial and cell proliferation gene expression in U87 glioma cells

Consistent with literature report for tigecycline, elevations in miR199-b-5p were seen with exposure to TP-2846. Overall gene expression changes in U87 cells mirrored those in AML lines, showing a pattern consistent with loss of mitochondrial function and cell cycle inhibition.

Figure 6. The effect of TP-2846 on 15 frozen patient AML samples in Vivia’s native environment cell assay

Vivia ex vivo assays

TP-2846 exhibited potent anti-proliferative activity tested as a single agent against AML tumor cells and tumor stem cells, including cytarabine-resistant lines, when screened in an ex vivo native environment cell depletion assay. Normalized activity for all lines is shown on the left, combined activity against tumor cells and tumor stem cells is shown on the right.

Figure 4. The impact of TP-2846 exposure on mitochondrial and cell proliferation gene expression in MV4-11

Expression of mitochondrially expressed COX-1 and ATP6 as well as regulatory gene HES-1 were elevated in cells exposed to TP-2846 while CDK4 was downregulated in response to TP-2846. Gene expression patterns are consistent with loss of mitochondrial function and cell cycle inhibition.