Shibuya, et al. “SMAC Mimetics Synergistically Cooperate with HDAC Inhibitors Enhancing TNF-α Autocrine Signaling”; Cancers 2023

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https://www.mdpi.com/2072-6694/15/4/1315

Abstract:

The overexpression of inhibitor of apoptosis (IAP) proteins is strongly related to poor survival of women with ovarian cancer. Recurrent ovarian cancers resist apoptosis due to the dysregulation of IAP proteins. Mechanistically, Second Mitochondrial Activator of Caspases (SMAC) mimetics suppress the functions of IAP proteins to restore apoptotic pathways resulting in tumor death. We previously conducted a phase 2 clinical trial of the single-agent SMAC mimetic birinapant and observed minimal drug response in women with recurrent ovarian cancer despite demonstrating on-target activity. Accordingly, we performed a high-throughput screening matrix to identify synergistic drug combinations with birinapant. SMAC mimetics in combination with an HDAC inhibitor showed remarkable synergy and was, therefore, selected for further evaluation. We show here that this synergy observed both in vitro and in vivo results from multiple convergent pathways to include increased caspase activation, HDAC inhibitor-mediated TNF-α upregulation, and alternative NF-kB signaling. These findings provide a rationale for the integration of SMAC mimetics and HDAC inhibitors in clinical trials for recurrent ovarian cancer where treatment options are still limited.

2022 ACoP: A Population Pharmacokinetic Model of Tolinapant in Subjects with Advanced Solid Tumors and Lymphomas

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View Poster: A Population Pharmacokinetic Model of Tolinapant in Subjects with Advanced Solid Tumors and Lymphomas

Abstract:

 Methods: Data from dose-escalation stage (Phase 1) and dose-expansion stage (Phase 2) from clinical study ASTX660-01 were included. Subjects recruited into Phase 1 received tolinapant in either powder (15, 30, 60, 120 and 180 mg; n=16) or capsule formulation (180, 210 and 270 mg; n=27). Subjects in Phase 2 only received capsule formulation (90, 120, 150 or 180 mg). A population PK model was developed with NONMEM v. 7.3 using first-order conditional estimation with eta-epsilon interaction (FOCE-I). Model selection was based on goodness-of-fit plots, objective function values, prediction and variance corrected visual predictive check (pvcVPC), and model plausibility. Confidence intervals (CIs) around the parameters were computed using the sampling importance resampling (SIR) method.[1]

Results: The data comprised 3427 tolinapant concentration measurements from 163 subjects (Phase 1; n=43, Phase 2; n=120) aged 23 to 84 years. tolinapant PK was best described using a two-compartment nonlinear elimination model with absorption described by a transit compartment model. The rate of absorption was dependent on formulation and was described by separate transit rate constants (KTR) for powder and capsules. Population estimates of Michaelis-Menten constant (Km), maximum elimination rate (Vmax), and apparent central volume of distribution (Vc/F) were 918.3 ng/mL, 72.2 ng/L and 488.6 L, respectively. Between subject variability included on Vmax and Vc/F were 21.3% and 40%, respectively. An additive error model on log transformed data was used to account for the unexplained residual variability. All parameters were estimated with acceptable precision. The predictive performance of the model assessed by the pvcVPC indicated that the data were adequately described by the model.

Conclusion: A population PK model was developed for tolinapant in subjects with advanced solid tumors and lymphomas. This work is the first description of the tolinapant PK, and the next steps are to explore the exposure and efficacy relationships and investigate the anti-tumor activity of tolinapant in one or more selected tumor types.

Reference:

1. Dosne AG, Berstrand M, Harling K, Karlsson MO. Improving the estimation of parameter uncertainty distributions in nonlinear mixed effects models using sampling importance resampling. J Pharmacokinet Pharmacodyn, 43(6):583–596, 2016.

2022 TCLF – Encore Presentation: Preliminary Analysis of the Phase II Study Suing Tolinapant (ASTX6660) Monotherapy in 98 Peripheral T-Cell Lymphoma and 51 Cutaneous T-Cell Lymphoma Subjects with Relapsed Refractory Disease

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Preliminary Analysis of the Phase II Study Suing Tolinapant (ASTX6660) Monotherapy in 98 Peripheral T-Cell Lymphoma and 51 Cutaneous T-Cell Lymphoma Subjects with Relapsed Refractory Disease
 

Abstract:

Background:

There are limited treatment options for patients with PTCL and CTCL, especially when front line therapy has failed. Tolinapant (ASTX660) is a novel oral non-peptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which also induces necroptosis. Tolinapant is being evaluated in a first-in-human ongoing Phase I/II study in subjects with advanced solid tumors and lymphoma (NCT02503423). Phase I and the initial Phase II results were previously reported (Mita et al. Clin Cancer Res, 2020; Mehta et al., EHA 2019).

 

Aim:

Here we report the preliminary efficacy and safety analysis for the Phase II PTCL and CTCL cohorts.

Methods:

This is a single-arm open-label Phase II study. To be eligible, subjects must have documented progressive disease and received at least two prior systemic therapies. Subjects received tolinapant at the recommended Phase II dose of 180 mg/day on Days 1 to 7, and 15 to 22 in a 28-day cycle. The primary endpoint is investigator assessed best overall response rate (ORR) to either the Lugano criteria (PTCL) or Global Assessment (CTCL). Adverse events (AEs) are assessed per CTCAE V4.03. The efficacy data set is based on subjects who had tumor evaluation at baseline and at least 1 post-treatment evaluation, unless they died or stopped treatment due to progression or toxicity. The safety data set is based on all subjects that received at least one dose of tolinapant.

Results:

There were 98 PTCL subjects and CTCL 51 subjects that received drug with 98 and 50 evaluable, respectively. Enrollment is closed with a minimum of 6 months follow-up on all subjects at the time of the data cut (05JAN2022). Subject characteristics: median (range) age PTCL 62.5 (27, 82) and CTCL 62 (24,87), median number of previous therapies PTCL 3 (0-8) and CTCL 6 (1-10). Among all subjects, the most common related AEs of any grade (≥ 15%) were: lipase elevation (35%), amylase elevation (25%), rash (combined listings) (24%), ALT elevation (15%), and AST elevation (15%). Related AEs ≥ Grade 3 (≥ 5%) were: lipase elevation (15%), rash (9%), and amylase elevation (7%). Pancreatitis was identified in 2 subjects (1%) (both Grade 4). There were no related ≥ Grade 3 AEs for diarrhea, nausea or vomiting; for related Grade 2 AEs there was a 5% incidence of diarrhea and 1% incidence of nausea/vomiting.

The ORR for PTCL is 22%, including 9 complete responses (CRs) and 12 partial responses (PRs). The ORR in CTCL is 26% including 2 CRs and 11 PRs. The median durability of response for PTCL is 133 (Q1-Q3; 69 – 280) days and for CTCL is 148 (Q1-Q3; 103 – 294) days.

Conclusions:

In this Phase II study, the novel oral agent tolinapant has shown meaningful single-agent clinical activity against PTCL and CTCL with a manageable safety profile. A new PTCL study combining tolinapant with oral decitabine/cedazuridine in relapsed/refractory PTCL is currently enrolling subjects.

This abstract has been presented at EHA 2022.

2022 TCLF: Trials-In-Progress, A Phase 1-2, Open-Label Study of the Safety, Pharmacokinetics, Pharmacodynamics, and Preliminary Activity of Tolinapant in Combination with Oral Decitabine/Cedazuridine and Oral Decitabine/Cedazuridine Alone in Subjects with Relapsed/Refractory Peripheral T-cell Lymphoma

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Trials-In-Progress, A Phase 1-2, Open-Label Study of the Safety, Pharmacokinetics, Pharmacodynamics, and Preliminary Activity of Tolinapant in Combination with Oral Decitabine/Cedazuridine and Oral Decitabine/Cedazuridine Alone in Subjects with Relapsed/Refractory Peripheral T-cell Lymphoma

Rationale

There are limited treatment options for patients with PTCL after front line therapy has failed. Tolinapant (ASTX660) is a novel oral non-peptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which also induces necroptosis in T-cell lymphoma models (Ferrari et al., Blood Advances, 2021). An ongoing Phase 1/2 study demonstrates an overall response rate (ORR) of >20% in relapsed/refractory PTCL with single agent tolinapant (Michot et al., EHA 2022). While there are limited studies using hypomethylating agents (HMAs) in PTCL, a recent prospective study showed 40% ORR (Wong et al., Leukemia, 2022). Preclinical data demonstrate decitabine treatment leads to re-expression of gene expression critical for necroptosis and synergy between decitabine and tolinapant in T-cell tumor models (Ward et al. ASH 2021; Manavalan et al; EHA abstract 2022). These data suggest that this combination may have synergistic activity in PTCL. There are minimal overlapping toxicities between the study drugs and no expected drug-drug interactions. Oral decitabine and cedazuridine (an inhibitor that enhances the oral bioavailability of decitabine) is an oral fixed dose combination of the two drugs with pharmacokinetic equivalence to IV decitabine. This combination was recently approved in the US, Canada, and Australia for the treatment of intermediate and high-risk MDS and CMML.

Study Design

ASTX660-03 is a Phase 1-2, open-label study investigating the safety and efficacy of combination tolinapant and oral decitabine/cedazuridine treatment in relapsed/refractory PTCL. To be eligible, subjects with ECOG PS ≤2 must have received at least two prior systemic therapies with evidence of documented progressive disease with at least one measurable lesion by CT. Subjects with CD30-positive disease must have received, be ineligible for, or intolerant to brentuximab vedotin. Key exclusion criteria include ejection fraction <50%, QTc >470 msec, and the use of concomitant medications that are either strong or moderate CYP3A4 inhibitors/inducers.

There is a lead-in phase to confirm tolerability of the MDS-approved regimen of oral decitabine/cedazuridine is tolerated in a PTCL population. Phase 1 is randomized to oral decitabine/cedazuridine alone or in combination with tolinapant. The combination arm will have escalation of tolinapant in dose ranges that have shown efficacy in PTCL. The oral decitabine/cedazuridine only arm will enroll 20-24 subjects. Once the combination arm reaches recommended Phase 2 dose/maximum tolerated dose there will be a dose expansion of 20 subjects in the combination arm prior to the initiation of the combination dosing in Phase 2, with an enrollment goal of 102 subjects. There will be no formal analysis in Phase 1. In Phase 2, there will be efficacy analysis for every 34 subjects, without a pause in enrollment. Anticipated study opening is May 2022.

 

2022 EHA: Preliminary Analysis of the Phase II Study Suing Tolinapant (ASTX6660) Monotherapy in 98 Peripheral T-Cell Lymphoma and 51 Cutaneous T-Cell Lymphoma Subjects with Relapsed Refractory Disease

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Preliminary Analysis of the Phase II Study Suing Tolinapant (ASTX6660) Monotherapy in 98 Peripheral T-Cell Lymphoma and 51 Cutaneous T-Cell Lymphoma Subjects with Relapsed Refractory Disease

Abstract:

Background: There are limited treatment options for patients with Peripheral T-cell lymphoma (PTCL) and Cutaneous T-cell lymphoma (CTCL), especially when front line therapy has failed. Tolinapant (ASTX660) is a novel oral nonpeptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which also induces necroptosis in T-cell lymphoma models (Ferrari et al., Blood Advances, 2021). Tolinapant is being evaluated in a first-in-human ongoing Phase I/II study in subjects with advanced solid tumors and lymphoma (ClinicalTrials.gov NCT02503423). Phase I results were previously reported (Mita et al. Clin Cancer Res, 2020) and the recommended phase 2 dosing (RP2D) was established. Initial results for Phase II were previously reported at EHA 2019 (Mehta et al., EHA 2019, # PS1073).

Aims: Here we report the preliminary efficacy and safety analysis for the Phase 2 PTCL and CTCL cohorts.

Methods: Methods: This is a single-arm open-label Phase II study. To be eligible, subjects must have evidence of documented progressive disease and received at least two prior systemic therapies. Subjects received treatment with tolinapant at the RP2D 180 mg/day on Days 1 to 7, and 15 to 22 in a 28-day cycle. The primary endpoint is best overall response rate (ORR) as assessed by the investigator according to either the Lugano criteria (PTCL) or Global Assessment (CTCL). Adverse events (AEs) are assessed per CTCAE v4.03. The efficacy data set is based on subjects who had tumor evaluation at baseline and at least 1 post-treatment tumor evaluation visit, unless they died or stopped treatment earlier due to clinical progression or toxicity. The safety data set is based on all subjects that received at least one dose of tolinapant.

Results: Results: As of the data cut of January 5, 2022, there were 98 subjects with PTCL and 51 subjects with CTCL that received drug and 98 and 50 subjects that were evaluable respectively. The study is currently closed to enrollment with a minimum of 6 months follow-up on all subjects at the time of the data cut. Subject characteristics: median (range) age PTCL 62.5 (27, 82) and CTCL 62 (24,87), median number of previous therapies PTCL 3 (0-8) and CTCL 6 (1-10). Among all subjects, the most common related AEs of any grade (≥ 15%) were: lipase elevation (35%), amylase elevation (25%), rash (combined listings) (24%), ALT elevation (15%), and AST elevation (15%). Related AEs ≥ Grade 3 (≥ 5%) were: lipase elevation (15%), rash (combined listings) (9%), and amylase elevation (7%). Pancreatitis was identified in 2 subjects (1%) (both Grade 4). There were no related ≥ Grade 3 AEs for diarrhea, nausea or vomiting; for related Grade 2 AEs there was a 5% incidence of diarrhea and 1% incidence of nausea/vomiting.

The ORR for PTCL is 22%, including 9 complete responses (CRs) and 12 partial responses (PRs). The ORR in CTCL is 26% including 2 CRs and 11 PRs. The median durability of response for PTCL is 133 (Q1-Q3; 69 – 280) days and for CTCL is 148 (Q1-Q3; 103 – 294) days. Pharmacodynamic and correlative analysis is ongoing with preliminary analysis suggesting an immunomodulatory antitumoral effect of tolinapant (Ferrari et al., Blood Advances, 2021).

Summary/Conclusion: In this Phase II study, the novel oral agent tolinapant has shown meaningful clinical activity against PTCL and CTCL with a manageable safety profile. These results support the continued development of tolinapant for the treatment of R/R PTCL and CTCL. A drug combination study using tolinapant in R/R PTCL is being developed.

2022 EHA: COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA

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COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA

Abstract:
Background: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In a Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in preclinical studies.

Aims: Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death, such as necroptosis, by rational combination of our clinical compounds in
preclinical models of TCL.

Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1,respectively, by Western blotting in mouse and human cell lines. Levels of key necroptosis biomarkers (RIPK3, MLKL) were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. Karpas-299 cells genetically-manipulated to express RIPK3 were used to demonstrate involvement of necroptosis in drug-induced cell death (Cytotox NIR) in vitro. Cell death was monitored by viability (CellTiterGlo)or real-time microscopy (IncuCyte) assays. Levels of key immunomodulatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma. Levels of methylation in RIPK3 promoter regions were measured by pyrosequencing after bisulfite conversion. Comparative changes in gene expression were measured by RT-qPCR.

Results:
TCL cell lines treated with tolinapant, decitabine or both led to depletion of cIAP1 and DNMT1 in TCL cell lines,
demonstrating target engagement of both agents. The combination of tolinapant and decitabine synergistically
reduced viability of some human T-cell lymphoma cell lines.
Some cell lines, including Karpas-299, were resistant to tolinapant treatment and showed low expression of RIPK3,
which was found to be due to promoter methylation. Increased expression of RIPK3 in Karpas-299 by genetic
manipulation or by decitabine treatment resulted in enhanced lytic cell death upon tolinapant treatment.
Decitabine and tolinapant treatments resulted in expression of cytokines, chemokines and DAMPs, suggesting
potential for immune activation and the effects were enhanced when combined. Furthermore, normally silenced
cancer/testis antigen expression was increased by decitabine, potentially increasing the immunogenicity of the cells.
Evaluation of the combination of agents in mouse models suggested that increased necroptosis signal and immune-potentiating biomarker modulation can be achieved in vivo.

Summary/Conclusion: These data demonstrate that hypomethylating agents enhance immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. In addition, modulation of cytokine response and cancer/testis antigen expression could enhance anti-tumour response. These findings provide a strong rationale to explore this combination clinically.

2021 ASH: Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

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Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Abstract:

Introduction:  Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP.  In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).

Methods:  On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).

Results:  Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.

Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.

Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.

By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3-/- and MLKL-/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.

Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.

Conclusion:  These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway.  This finding provides strong rationale to explore this combination clinically.

Ferrari, et al. “Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma”, 2021

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Blood Advances. 2021 Oct 26;5(20):4003-4016.

DOI: 10.1182/bloodadvances.2020003955

2020 EORTC-NCI-AACR: The non-peptidomimetic cIAP1/2 and XIAP antagonist tolinapant promotes an anti-tumour immune response in T-cell lymphoma

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The non-peptidomimetic cIAP1/2 and XIAP antagonist tolinapant promotes an anti-tumour immune response in T-cell lymphoma

Summary

Tolinapant (ASTX660) is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP) [1], which is currently being tested in a first in human phase I-II clinical trial in patients with advanced solid tumours and lymphomas (NCT02503423) [2]. IAP antagonists have been reported to exhibit broad immuno-modulatory effects on both the innate and adaptive immune systems. We have investigated the profile of tolinapant in preclinical T cell lymphoma models and evaluated tolinapant’s ability to enhance immune mediated killing of T cell lymphoma cells, both in vitro and in vivo.

References:

  1. G Ward et al., 2018, Mol Cancer Therapeutics Jul;17(7):1381-1391
  2. A Hollebecque et al., 2019, AACR-NCI-EORTC International Conference on Molecular
    Targets and Cancer Therapeutics

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