- ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumors and lymphomas (NCT02503423) where preliminary activity has been described in a group of T-cell lymphomas (1)
- Herein, together with its well-characterized pro-apoptotic effect (2), we describe a new role for ASTX660 as an immunomodulatory molecule capable of promoting an anti-tumor immune response in pre-clinical models of T-cell lymphoma. These data add to the description of ASTX660’s mode of action and our ongoing understanding of the preliminary clinical efficacy that has been reported.
1. Samaniego F, et al., Hematological Oncology. 2019;37(S2):527.
2.Ward GA et al., Mol Can Ther. 2018;17(7):1381-91
- The mitogen activated protein kinase (MAPK) pathway is frequently dysregulated in cancer, leading to activation of the downstream kinases ERK1/2 (ERK). Phosphorylation of ERK substrates such as p90RSK (RSK) leads to cancer cell proliferation.
- Clinical efficacy can be limited by toxicity, so it is important to establish an optimal, tolerated dose schedule which maximises efficacy. Preclinical studies investigating the duration of target engagement required for efficacy can inform on dose schedules to be tested in the clinic.
- A number of compounds under clinical development target ERK activity directly: we have recently described the development of a novel, potent and selective small molecule inhibitor of ERK, the lead compound, using fragment-based drug discovery1.
Background: ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumor and lymphomas (NCT02503423). IAP antagonists have been reported to exhibit broad immuno-modulatory effects on both the innate and adaptive immune systems .
Aims: We have investigated the profile of ASTX660 in preclinical T cell lymphoma models and evaluated ASTX660’s ability to enhance immune mediated killing of T cell lymphoma cells, both in vitro and in vivo.
Methods: ASTX660 was tested in a panel of human and mouse tumor cell lines, assessing apoptosis, necroptosis and immunogenic cell death (ICD). ASTX660 was tested in vitro alone or with recombinant death receptor ligands (TNFa, FASL or TRAIL) and with or without caspase-8/RIPK inhibitors to demonstrate mechanism of action. Target engagement along with induction of apoptosis, necroptosis and ICD markers were analysed by Western blotting, and flow cytometry. Murine tumor models in immunocompetent and immunocompromised mice were utilised to evaluate the efficacy of ASTX660 in the presence or absence of an effective immune response. The Nanostring IO360 panel was used to assess immune cell recruitment.
Results: ASTX660 antagonised IAPs in cell lines, as indicated by a decrease in cIAP1 protein levels and disruption of the XIAP:SMAC protein complex. In murine T cell lymphoma cell lines (BW5147, EL4 and L5178Y), ASTX660 treatment was associated with an increase in apoptosis or necroptosis and ICD biomarkers. In immunocompetent mice, administration of ASTX660 delivered a complete regression of BW5147 tumor growth, which was not seen in mice deficient in T and B cells. These mice remained refractory to subsequent rechallenge after initial complete regression. Biomarker evaluation from this model indicated a potent immunogenic/necroptotic response after ASTX660 dosing and upregulation of immune effector cells.
1. Michie J. et al., The Immuno-Modulatory Effects of Inhibitor of Apoptosis Proteins Antagonists, Cells, 2020, 9(1), 207.
2. A. Hollebecque et al., AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2019.
Background: The tumour suppressor p53 is activated in response to various stress signals to induce transcriptional changes leading to cellular responses such as cell cycle arrest and apoptosis. Activity of p53 is tightly regulated by the E3 ubiquitin ligase MDM2, which inhibits p53 function by, for example, targeting it for proteasomal degradation. Targeting the MDM2-p53 interaction to restore p53 function, is therefore, a promising strategy for cancer therapy and a number of these compounds are in clinical development including ASTX295 (NCT03975387). ASTX295 is a novel, orally bioavailable MDM2 antagonist developed through structure-based drug design that has demonstrated potent activity in a range of p53 wild-type pre-clinical models.
Aims: We investigated the therapeutic potential of ASTX295 alone and in combination with decitabine, a DNA- hypomethylating agent, in AML.
Methods: Primary blasts were isolated from AML patient samples using a combination of antibodies against CD34, CD33, CD45 and CD117. A panel of AML cell lines and primary AML blasts were treated with decitabine and ASTX295 at a range of concentrations, alone and in combination. After treatment, viability was assessed by Alamar blue assay or induction of apoptosis by flow cytometry using a fluorescent caspase substrate or Annexin V.
Effects of drug combinations were analysed using the Combenefit software based on different mathematical models (Loewe, bliss & HSA). Target engagement was confirmed by western blotting.
Results: When tested in a panel of p53 wild-type AML cell lines, ASTX295 exerted a strong anti-proliferative effect in which GI50 <30 nM was observed in 9 out of 11 cell lines. Additionally, p53 activation by ASTX295 triggered apoptosis in both AML cell lines, and primary AML blasts isolated from patients.
Activity of ASTX295 was further enhanced by combining with decitabine. Treatment of AML cell lines with ASTX295 and decitabine showed an increase in growth inhibitory effect and apoptosis compared to respective single agent treatments. This combinatory effect, as assessed by Combenefit, was also observed in primary AML blasts in which 7 of 12 samples tested demonstrated increased apoptosis at or above 300 nM ASTX295 and 100 nM decitabine. Target engagement of ASTX295 and decitabine was confirmed by upregulation of p53 transcriptional targets and decreased DNMT-1 expression.
Summary/Conclusion: Our findings demonstrate that the combination of ASTX295 with decitabine exhibits potent activity against p53 wild-type AML cells, and thus merits further investigation.