TR57, an Inhibitor of the Integrated Stress Response, Is Synergistic with Venetoclax Against CLL Cells, Independent of Their TP53 Status
Authors: Fatima Narjis , Shen Yandong , Crassini Kyle R , Iwanowicz Edwin , Christopherson Richard , Best Giles , Mulligan Stephen P. 
Published: November 2019

Introduction
Despite recent advances in the treatment of chronic lymphocytic leukemia (CLL), a significant proportion of patients still relapse with drug-resistant disease. Patients with deletions and/or mutations in TP53 remain a poor prognostic sub-group. There remains a need for on-going research on identifying novel treatment strategies.
We have investigated the therapeutic potential of TR57 (Madera Therapeutics, USA), as a single agent and in combination with the Bcl-2 inhibitor venetoclax in CLL. TR57 is an inhibitor of the integrated stress response (ISR) and has shown efficacy in pre-clinical studies of breast cancer.
Methods
TR57 and venetoclax, as single agents or in combination, were studied against primary CLL cells co-cultured with CD40L-expressing fibroblasts to mimic the tumour microenvironment (TME). The effects of the drugs were also assessed against an OSU-CLL TP53 knock-out cell line generated using CRISPr Cas-9. Cell viability was assessed using the mitochondrial membrane potential dye DiIC1(5), propidium iodide (PI) and flow cytometry. Cytotoxic synergy between TR57 and venetoclax was determined by calculating combination indices (CI) using the CompuSyn software, with CI values of <1 indicative of synergy. Primary CLL cells were stimulated into cycle primary using Dsp30 in combination with IL-2. Cell cycle distribution and proliferation were analysed by flow cytometry using PI and CFSE, respectively. The migratory and adhesive capacities of primary CLL cells was assessed using stroma-derived factor 1a and by assessing expression of CXCR4 and CD49d. The mechanisms of the synergy between TR57 and venetoclax were studied by immunoblotting. Statistical analyses were performed using the students t-test with P-values of < 0.05 considered significant.
Results
TR57 was cytotoxic towards primary CLL cells in a nanomolar range, with IC50 values of 38 ± 1.38 nM and 287 ± 50.45 nM against cells in medium and stromal cell co-culture, respectively. No significant difference was observed in the sensitivity of samples with ATM or TP53 aberrations (n=12).
Synergy between TR57 and venetoclax against CLL cells in stromal cell co-culture was consistent with a significant (P < 0.001) decrease in the IC50 for both drugs (Figure 1A). A CI value of 0.13 was calculated at a fractional effect of 0.5.
TR57 was cytotoxic towards OSU-CLL and OSU-CLLTP53ko cells, with IC50 values of 3.98 ± 1.03 nM and 90.7 ± 3.51 nM, respectively. Synergy between TR57 and venetoclax was evident in both lines with a CI = 0.1 at Fa 0.5. Dose-response analyses in the OSU-CLLTP53ko line are shown in Figure 1B.
Following stimulation of primary CLL cells with Dsp30/IL-2 we observed a significant (P < 0.01) increase in the proportion of cells in S, G2 and M phases, which was consistent with an increase in cell proliferation. TR57 and venetoclax in combination had a greater effect than individual drug treatments, significantly reduced the proportion of cells in these cell cycle phases and the proliferative fraction of cells.
TR57 and venetoclax in combination also had a significantly greater effect on the migratory capacity (P < 0.05) of CLL cells and on the expression of CXCR4 and CD49d (P < 0.001) than either drug as a single agent.
Immunoblotting of primary CLL and OSU-CLL cells showed that treatment with TR57 decreased expression of Grp78, which supports the notion that this drug may function through inhibition of the ISR. We also observed that TR57, alone and in combination with venetoclax, down-regulated the expression of the pro-survival Mcl-1 and Bcl-2 proteins, up-regulated expression of the pro-apoptotic Noxa and Bax proteins and inhibited the phosphorylation of AKT and ERK1/2-MAPK.
Conclusions
The data presented demonstrate that TR57 is cytotoxic towards CLL cells under in vitro conditions that mimic the TME and cells with lesions of the TP53 pathway. The synergy observed between TR57 and venetoclax suggests that the drug combination may be highly effective at limiting the survival and proliferation of CLL cells as well as their ability to migrate to, and be retained within, the TME. The mechanisms of this synergy include a shift towards a pro-apoptotic balance in Bcl-2 family proteins and inhibition of signalling via the AKT and ERK1/2-MAPK pathways. Collectively, these data suggest that TR57 alone and in combination with venetoclax may be a highly effective treatment strategy for high risk CLL.

Disclosures
Iwanowicz: Madera Therapeutics, LLC: Other: President;Ownership.