2020 AACR: ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, promotes an anti-tumor immune response in pre-clinical models of T-cell lymphoma

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ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, promotes an anti-tumor immune response in pre-clinical models of T-cell lymphoma

Summary

  • 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.

References:
1. Samaniego F, et al., Hematological Oncology. 2019;37(S2):527.
2.Ward GA et al., Mol Can Ther. 2018;17(7):1381-91

2020 EHA: Characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, in mouse models of T cell lymphoma

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Characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, in mouse models of T cell lymphoma

Abstract:

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.

References:
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.

 

2020 EHA: ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine

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ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine

Abstract:

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.

2020 EHA: Comparative results of azacitidine and decitabine from a large prospective Phase 3 study in treatment naïve acute myeloid leukemia (TN-AML) not eligible for intensive chemotherapy

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Comparative results of azacitidine and decitabine from a large prospective Phase 3 study in treatment naïve acute myeloid leukemia (TN-AML) not eligible for intensive chemotherapy

 

Abstract:

Background: Older patients with TN-AML who are ineligible for intensive chemotherapy have limited therapeutic options and poor outcomes. Hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DEC) have been the standard of care in this population for more than a decade and were approved in Europe for patients not candidates for intensive chemotherapy or patients not candidates for hematopoietic cell transplant. However, there is no direct efficacy and safety comparative data of AZA and DEC from a prospective large randomzied study. We took advantage of the largest randomized trial for patients with TN-AML who were not eligible for intensive chemotherapy, ASTRAL-1, to compare efficacy and safety of AZA vs DEC in patients randomized to these 2 treatments

Aims: To compare clinical outcomes between AZA and DEC in TN-AML patients not eligible for intensive chemotherapy

Methods: ASTRAL-1 is a global randomized Phase 3 trial which enrolled 815 patients with TN AML who were not eligible for intensive chemotherapy using stringent criteria including age ≥ 75 year or comorbidities including ECOG PS 3. Patients were randomized 1:1 to either Guadecitabine (G), a next generation HMA (60 mg/m2/d SC days 1-5) or a preselected Treatment Choice (TC) of AZA (75 mg/m2/d IV or SC days 1-7), DEC (20 mg/m2/d IV days 1-5), or low dose Ara-C (LDAC) (20 mg SC BID days 1-10). AML diagnosis and responses were assessed by an independent central pathologist blinded to randomization assignment. Responses were recorded using IWG 2003 criteria. Rates of Complete Response (CR) and Overall Survival (OS) were co-primary endpoints.

Results: 815 patients were randomized to G (408) or TC (407). Preselected TCs were DEC (43%), AZA (42%), or LDAC (15%). Of 407 patients randomized to TC, 338 (83%) were treated with either AZA (171 patients) or DEC (167 patients). Baseline variables were well balanced between AZA and DEC patients with no statistically significant differences in baseline characteristics: median age 76 y for both treatments, with poor PS 2-3 in 47.4% vs 53.9%, poor risk cytogenetics 38% vs 33.5%, secondary AML 38% vs 36.5%, BM blasts > 30% in 63.7% vs 71.3%, and TP53 mutations in 12.9% vs 11.3% for AZA vs DEC respectively. Median follow up was 25.5 months and median number of treatment cycles was 6 for AZA (range 1,31), and 5 for DEC (range 1,34). The ITT analyses showed a CR rate of 17.5% vs 19.2% (p= 0.70); and overall CR (CR+CRp+CRi) of 22.2% vs 25.1% (p= 0.53) for AZA vs DEC respectively. Median OS was 8.7 vs 8.2 months for AZA vs DEC respectively with Hazard Ratio of 0.97 (95% CI 0.77, 1.23; log rank p= 0.8). Additional subgroup analyses by baseline characteristics and molecular genetic mutations will be presented at the meeting. There was no statistically significant difference in the incidence of Grade ≥ 3 AEs (88.9% vs 87.4%), serious AEs (81.9% vs 76.0%), or 30-day all-cause mortality (11.7% vs 7.8%) for AZA vs DEC respectively. There was a trend of higher 60-day all-cause mortality on AZA (20.5%) vs DEC (13.2%) (p= 0.07).

Conclusions/Summary: This is the largest comparison of clinical outcomes associated with AZA and DEC for patients with TN AML not eligible for intensive chemotherapy who were treated in the same prospective study. While patients were randomized between G and each of AZA and DEC separately with no direct randomization of AZA vs DEC, the patients’ characteristics were well balanced in patients randomized to the two HMA treatments. There were no significant differences in CR, overall CR, OS, or safety between AZA and DEC.

Ramsey, et al., Oral Azacitidine and Cedazuridine Approximate Parenteral Azacitidine Efficacy in Murine Model

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Ramsey, H.E., Oganesian, A., Gorska, A.E. et al. Oral Azacitidine and Cedazuridine Approximate Parenteral Azacitidine Efficacy in Murine Model. Targ Oncol 15, 231–240 (2020). https://doi.org/10.1007/s11523-020-00709-x

Abstract:

Background: DNA methyltransferase inhibitors (DNMTis) improve survival for patients with myelodysplastic syndromes (MDS) and those with acute myeloid leukemia (AML) unable to receive standard cytotoxic chemotherapy and are, accordingly, the backbone of standard-of-care treatment for these conditions. Standard regimens with DNMTIs, decitabine (DEC) or azacitidine (AZA) include daily subcutaneous (s.c.) or intravenous (i.v.) administration for 5–7 consecutive days. Attempts to provide the therapy orally have been limited given rapid clearance of the agents by the enzyme cytidine deaminase (CDA), which is ubiquitous in the gut and liver as part of first-pass metabolism. Recently, cedazuridine (CDZ), an oral inhibitor of CDA, was successfully combined with DEC to approximate the pharmacokinetics of i.v. DEC in patients.

Objective: To determine if an oral dosing strategy might be feasible in the clinic with AZA, we attempted to increase the bioavailability of oral AZA through the use of CDZ, in a murine model.

Methods: Following pharmacokinetic and pharmacodynamic assessment of oral AZA dosed with CDZ in murine and monkey models, we tested this regimen in vivo with a human cell line-derived xenograft transplantation experiment (CDX). Following this we combined the regimen with venetoclax (VEN) to test the efficacy of an all-oral regimen in a patient-derived xenograft (PDX) model.

Results: Parenteral AZA and oral AZA + CDZ exhibited similar pharmacokinetic profiles, and efficacy against human AML cells. Tumor regression was seen with AZA + CDZ in MOLM-13 CDX and PDX models.

Conclusions: We conclude that oral AZA when combined with CDZ achieves successful tumor regression in both CDX and PDX models. Furthermore, the combination of AZA + CDZ with VEN in a PDX model emulated responses seen with VEN + AZA in the clinic, implying a potential all-oral VEN-based therapy opportunity in myeloid diseases.

Garcia-Manero et al., Oral cedazuridine/decitabine: a phase 2, pharmacokinetic/pharmacodynamic, randomized, crossover study in MDS and CMML

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Garcia-Manero et al, “Oral cedazuridine/decitabine: a phase 2, pharmacokinetic/pharmacodynamic, randomized, crossover study in MDS and CMML”. Blood. 2020 Apr 13. pii: blood.2019004143. doi: 10.1182/blood.2019004143. [Epub ahead of print]

Abstract:

This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with
cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-
1/2- or high-risk myelodysplastic syndromes (MDS), or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral
cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral
cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage
and then as a single fixed-dose combination (FDC) tablet. Primary endpoints: mean decitabine systemic exposure (geometric least-squares
mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), % long interspersed nuclear element 1
(LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and
treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1%, 106.5%) and 97.6% (80.5%, 118.3%)
for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical
responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events
regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35
mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar
efficacy. ClinicalTrials.gov NCT02103478.

Ye, et al. “ASTX660, an antagonist of cIAP1/2 and XIAP, increases antigen processing machinery and can enhance radiation-induced immunogenic cell death in preclinical models of head and neck cancer”

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https://www.tandfonline.com/doi/full/10.1080/2162402X.2019.1710398

Abstract:

Inhibitor of apoptosis protein (IAP) antagonists have shown activity in preclinical models of head and neck squamous cell carcinoma (HNSCC), and work across several cancer types has demonstrated diverse immune stimulatory effects including enhancement of T cell, NK cell, and dendritic cell function. However, tumor-cell-intrinsic mechanisms for this immune upregulation have been largely unexplored. In this study, we show that ASTX660, an antagonist of cIAP1/2 and XIAP, induces expression of immunogenic cell death (ICD) markers in sensitive HNSCC cell lines in vitro. Experiments in syngeneic mouse models of HNSCC showed that ASTX660 can also enhance radiation-induced ICD in vivo. On a functional level, ASTX660 also enhanced killing of multiple murine cell lines by cytotoxic tumor-infiltrating lymphocytes, and when combined with XRT, stimulated clonal expansion of antigen-specific T lymphocytes and expression of MHC class I on the surface of tumor cells. Flow cytometry experiments in several human HNSCC cell lines showed that MHC class I (HLA-A,B,C) was reliably upregulated in response to ASTX660 + TNFα, while increases in other antigen processing machinery (APM) components were variable among different cell lines. These findings suggest that ASTX660 may enhance anti-tumor immunity both by promoting ICD and by enhancing antigen processing and presentation.

 

Mita, et al. “A Phase 1 Study of ASTX660, an Antagonist of Inhibitors of Apoptosis Proteins, in Adults With Advanced Cancers or Lymphoma”

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https://clincancerres.aacrjournals.org/content/early/2020/01/03/1078-0432.CCR-19-1430.long

 

Abstract

Purpose: This first-in-human, phase 1 study evaluated ASTX660, an oral, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins in patients with advanced solid tumors or lymphoma. Experimental Design: ASTX660 was administered orally once daily on a 7-day-on/7-day-off schedule in a 28-day cycle. Dose escalation followed a standard 3+3 design to determine the maximum tolerated dose and recommended phase 2 dose (RP2D). Dose expansion was conducted at the RP2D. Results: Forty-five patients received ASTX660 (range 15-270 mg/d). Dose-limiting toxicity of grade 3 increased lipase with or without increased amylase occurred in 3 patients at 270 mg/d and 1 patient at 210 mg/d. The maximum tolerated dose was determined to be 210 mg/d and the RP2D 180 mg/d. Common treatment-related adverse events included fatigue (33%), vomiting (31%), and nausea (27%). Grade ≥3 treatment-related adverse events occurred in 7 patients, most commonly anemia (13%), increased lipase (11%), and lymphopenia (9%). ASTX660 was rapidly absorbed, with maximum concentration achieved at ~0.5‒1.0 hour. An ~2-fold accumulation in area under the curve exposures was observed on day 7 vs 1. ASTX660 suppressed cellular inhibitor of apoptosis protein-1 in peripheral blood mononuclear cells, which was maintained into the second cycle beyond the off-therapy week at the 180-mg/d RP2D and above. Clinical activity was seen in a patient with cutaneous T-cell lymphoma. Conclusions: ASTX660 demonstrated a manageable safety profile, and exhibited evidence of pharmacodynamic and preliminary clinical activity at the 180-mg/d RP2D. The phase 2 part of the study is ongoing.

Dittmann, et al. “Next-generation hypomethylating agent SGI-110 primes acute myeloid leukemia cells to IAP antagonist by activating extrinsic and intrinsic apoptosis pathways”

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https://www.nature.com/articles/s41418-019-0465-8

Abstract

Therapeutic efficacy of first-generation hypomethylating agents (HMAs) is limited in elderly acute myeloid leukemia (AML) patients. Therefore, combination strategies with targeted therapies are urgently needed. Here, we discover that priming with SGI-110 (guadecitabine), a next-generation HMA, sensitizes AML cells to ASTX660, a novel antagonist of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) and X-linked IAP (XIAP). Importantly, SGI-110 and ASTX660 synergistically induced cell death in a panel of AML cell lines as well as in primary AML samples while largely sparing normal CD34+ human progenitor cells, underlining the translational relevance of this combination. Unbiased transcriptome analysis revealed that SGI-110 alone or in combination with ASTX660 upregulated the expression of key regulators of both extrinsic and intrinsic apoptosis signaling pathways such as TNFRSF10B (DR5), FAS, and BAX. Individual knockdown of the death receptors TNFR1, DR5, and FAS significantly reduced SGI-110/ASTX660-mediated cell death, whereas blocking antibodies for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or FAS ligand (FASLG) failed to provide protection. Also, TNFα-blocking antibody Enbrel had little protective effect on SGI-110/ASTX660-induced cell death. Further, SGI-110 and ASTX660 acted in concert to promote cleavage of caspase-8 and BID, thereby providing a link between extrinsic and intrinsic apoptotic pathways. Consistently, sequential treatment with SGI-110 and ASTX660-triggered loss of mitochondrial membrane potential (MMP) and BAX activation which contributes to cell death, as BAX silencing significantly protected from SGI-110/ASTX660-mediated apoptosis. Together, these events culminated in the activation of caspases-3/-7, nuclear fragmentation, and cell death. In conclusion, SGI-110 and ASTX660 cooperatively induced apoptosis in AML cells by engaging extrinsic and intrinsic apoptosis pathways, highlighting the therapeutic potential of this combination for AML.

2019 ASH: Oral Azacitidine and Cedazuridine Approximate Azacitidine Efficacy in a Murine Model

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Abstract # 1376 – Oral Azacitidine and Cedazuridine Approximate Azacitidine Efficacy in a Murine Model

Authors: Haley E. Ramsey1-2, Maria P. Arrate1, Londa Fuller1, Agnieszka E. Gorska1, Kelli Boyd3-4, Melissa A. Fischer1-2, Aram Oganesian5, Mohammad Azab5, and Michael R. Savona1,2,4

1Department of Medicine, 2Cancer Biology Program, 3Department of Pathology, Microbiology and Immunology, 4Vanderbilt-Ingram Cancer Center; Vanderbilt University School of Medicine, Nashville, TN USA; 5Astex Pharmaceuticals, Inc., Pleasanton, CA

Background: DNA methyltransferase inhibitors (DNMTi) induce remissions and improve survival for patients with MDS and those with AML unable to receive standard cytotoxic chemotherapy. Accordingly, DNMTi therapy is the backbone of SOC treatment for MDS and AML. Given the inconvenience, pain, and general detriments to QOL with SQ or IV therapy daily for 5-7 days every month with azacitidine (AZA) or decitabine (DAC), many have attempted to provide the therapy orally, but encountered difficulties with this method of administration given rapid first-pass clearance via the enzyme cytidine deaminase (CDA) which is ubiquitous in the gut and liver. Recently, DAC was combined with cedazuridine (CDZ), an oral CDA inhibitor, in a fixed-dose (35mg/100mg) combination tablet (ASTX727) to approximate the pharmacokinetics of IV DAC (Savona et al Lancet Haematology 2019). To determine if a similar strategy might be feasible with AZA, we attempted to increase the bioavailability of oral AZA with CDZ in a murine tumor model.

Methods: We measured GI50 in AML cell lines treated with vehicle (DMSO), AZA, and AZA/CDZ combination. Although cancer cell lines produce CDA, total levels are negligible in comparison CDA in the gut and liver. For this reason, we then studied CDZ, AZA, and the AZA/CDZ combination in a systemic model of AML in immunocompromised mice. NSGS mice were sub-lethally irradiated and administered MOLM-13 AML cells via tail vein injection. At day seven post-transplant, engrafted mice were randomized to receive 2.5 mpk i.p. AZA(n=8), 2.5 mpk oral AZA(n=8), 3 mpk CDZ + oral AZA(n=6), or CDZ alone at 30 mpk (n=7). All oral AZA and CDZ was administered via oral gavage daily for 7 consecutive days; i.p. therapy was similarly given for 7 consecutive days. During treatment, the kinetics of MOLM-13 expansion was defined by detection of human AML in the blood as detected by flow cytometry. At approximately three weeks after transplant, CDZ-only treated mice became moribund, and all experimental groups were sacrificed for analysis of chimerism.

Results: After 72 hour of treatment, no differences were noted in viability of cell lines between AZA and AZA+ CDZ in vitro. In the xenograft model, as expected, i.p. AZA-treated mice had significant decreases in leukemic expansion in the bone marrow and spleen, whereas CDZ alone did not (AZA i.p. vs CDZ, p = 0.004 and <0.001). More importantly, whereas oral AZA alone failed to decrease AML expansion in both the bone marrow and spleen of treated mice (oral AZA vs CDZ., p = 0.677 and 0.249, respectively), the addition of CDZ to oral AZA led to significant decreases in AML in both bone marrow and spleen (Oral AZA + CDZ vs CDZ, p = 0.012 and 0.004, respectively). Likewise, addition of CDZ to oral AZA showed no significant differences in activity against AML compared to traditional AZA i.p. dosing in either bone marrow or splenic tissue (p= 0.204 and 0.224, respectively). Furthermore, in a Kaplan-Meier survival analysis, mice treated with CDZ alone die within 21 days of transplant, but both i.p. AZA and oral AZA + CDZ treated mice had a 50% extended survival with no significant difference in survival between i.p. AZA and oral AZA + CDZ treated mice (p= 0.502). H&E staining revealed no significant bone marrow toxicity in treated mice, suggesting that AZA preferentially effected transplanted MOLM-13 AML cells.

Conclusion: Consistent with previous preclinical and clinical studies with ASTX727, the oral AZA approximated AZA anti-tumor activity when co-treated with CDA-inhibitor CDZ. These preliminary data provide rationale for the development of CDZ + oral AZA therapy as a fixed dose combination (ASTX030) in myeloid disease. Clinical trials evaluating ASTX030 are being planned.