2021 ASH: ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics

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ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics

Abstract:
Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075).
Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance.

Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27.

We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38–leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009).

Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining “stemness” of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced “stemness” of AML cells (Figure 1c).

Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress.

References:
1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992.
2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4.

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

Identification of potent small molecule allosteric inhibitors of SHP2

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Identification of potent small molecule allosteric inhibitors of SHP2

Summary
SHP2 is a ubiquitously expressed protein tyrosine phosphatase required for growth factor signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes.

Genetic knockdown and pharmacological inhibition of SHP2 inhibits proliferation of RTK-driven cancer cell lines and suppresses RAS/MAPK signalling.

SHP2 inhibitors are a promising therapeutic approach as RTK deregulation often leads to a wide range of cancers and several compounds are being tested in the clinic.

Using our fragment-based screening approach, PyramidTM, we identified fragment hits binding to the tunnel region<sup>1</sup> between the phosphatase domain and the C-SH2 domain of SHP2 which were improved using structure-guided design.

Here we describe the optimisation of mM fragment hits into potent SHP2 antagonists with in vitro and in vivo anti-tumour activity.

Combined inhibition of SHP2 and ERK enhances anti-tumor effects in preclinical models

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Combined inhibition of SHP2 and ERK enhances anti-tumor effects in preclinical models

Summary

MAPK signalling is frequently dysregulated in cancer. The pathway can Panel composition and the number of responding cell lines Examples of dose-response curves Anti-tumor activity of SHP2i, ASTX029 and combination in MIA PaCa-2 xenograft be targeted by inhibition of different nodes and is tightly regulated by feedback mechanisms. Resistance to single-agent therapies frequently occurs through several different mechanisms including upregulation of receptor tyrosine kinases (RTKs), therefore, combination therapies are of interest.

The Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is a key regulator of MAPK pathway downstream of RTKs and upstream of RAS, whilst ERK acts at the bottom of the pathway phosphorylating multiple substrates.

We investigated the potential of targeting the MAPK pathway through a combination of SHP2 and ERK inhibition in preclinical models. Using a SHP2 inhibitor (SHP2i) discovered by our structure-based drug discovery programme and ASTX029, an ERK inhibitor in a Phase I-II clinical trial (NCT03520075), we tested panels of cell lines representing various indications and genetic backgrounds in vitro and confirmed enhanced tumor growth inhibition by the combination in a xenograft model.

The clinical candidate, ASTX029, is a novel, dual mechanism ERK1/2 Inhibitor and has potent activity in MAPK-activated cancer cell lines and in vivo tumor models

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The clinical candidate, ASTX029, is a novel, dual mechanism ERK1/2 Inhibitor and has potent activity in MAPK-activated cancer cell lines and in vivo tumor models

Summary

  • The MAPK signaling pathway is commonly upregulated in human cancers due to oncogenic mutations of upstream
    components such as BRAF or KRAS.
  • MAPK pathway inhibition has been clinically validated by BRAF and MEK inhibitors.
  • As the final node in the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers, including those resistant to upstream inhibition.
  • Previously we described the fragment-based discovery of a chemical series targeting ERK[1]. Here we disclose for the first time the structure of the clinical candidate, ASTX029.

References:

  1. Heightman et al., (2018). J Med Chem 61; 4978

2020 AACR: Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2

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2020 AACR: Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2

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 AACR: Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity

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Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity

Summary

  • 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.

2019 AD/PD: Discovering small-molecule modulators of autophagy for neurodegeneration

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Discovering Small-Molecule Modulators of Autophagy for Neurodegeneration

Summary

  • Autophagy is a major protein degradation pathway with proven roles in protecting neurons against accumulation of aggregation-prone proteins and obsolete mitochondria. Genetics of human-disease and mouse-knockout studies highlight the connection between autophagy dysfunction and neurodegeneration, and therefore finding a way to augment autophagic-flux seems a promising therapeutic strategy (Rubinsztein DC, Bento CF & Deretic V. Journal of Experimental Medicine 2015; Bento CF et al. Annual Review of Biochemistry 2016).
  • Fragment-based drug discovery (FBDD) has the potential to deliver potent, selective and CNS-penetrant small-molecules capable of inducing autophagy in the brain. For that purpose, the use of cell-based assays that accurately monitor the ability of small-molecules to modulate autophagy in a high-throughput- and unbiased-manner is instrumental.

2018 EORTC: A novel ERK1/2 inhibitor has potent activity in NRAS-mutant melanoma cancer models

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A novel ERK1/2 inhibitor has potent activity in NRAS-mutant melanoma cancer models

Summary

  • NRAS mutations occuring 15- 20% of melanoma cancer patients. Currently there is no approved molecularly targeted therapy for NRAS mutant melanoma, an indication which is associated with aggressive clinical outcome and a poor prognosis.
  • The NRAS mutation leads to constitutive activation of the MAPK pathway. AsERK1/2(ERK) is the primary downstream effect or of the MAPK pathway, its direct inhibition may provide an attractive therapeutic approach for the treatment of NRAS-mutant melanoma.
  • As previously described, using fragment-based drug discovery we have identified a novel and selective inhibitor of ERK which inhibits in vitro ERK catalytic activity as well as ERK phosphorylation 1.
  • Here, we demonstrate the in vitro and in vivo activity of a novel, highly potent, selective ERK inhibitor in models of NRAS-mutant melanoma.