The Preclinical Activities of PTC596, a Novel Tubulin Binding Agent That Down-Regulates BMI1, Alone and in Combination with Bortezomib in Multiple Myeloma
Authors: Nagai Yurie , Mimura Naoya , Rizq Ola , Isshiki Yusuke , Oshima Motohiko , Rizk Mohamed , Saraya Atsunori , Koide Shuhei , Nakajima-Takagi Yaeko , Miyota Makiko , Oshima-Hasegawa Nagisa , Tsukamoto Shokichi , Mitsukawa Shio , Takeda Yusuke , Ohwada Chikako , Takeuchi Masahiro , Iseki Tohru , Nakaseko Chiaki , Sheedy Josephine , Weetall Marla , Iwama Atsushi , Sakaida Emiko 
Published: November 2019

Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM).
Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed.
Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021).
Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p<0.0001 vs. control, p=0.0003 vs. PTC596, p=0.0104 vs. bortezomib). Severe body weight loss was not observed in the combination-treated mice except one mouse which needed washout period until recovery. White blood cell count and hemoglobin level in peripheral blood of mice were not significantly changed during the treatment with PTC596 or even the combination. Survival of the host mice treated with the combination was significantly prolonged as compared with the control or either single treatment (p<0.0001 vs. control, p=0.0002 vs. PTC596, p=0.0072 vs. bortezomib).
Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients.

Disclosures
Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.