Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I-II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381-91. ©2018 AACR.

Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors.

The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen against DDR1 and identified fragments that bound either at the hinge or in the back pocket associated with the DFG-out conformation of the kinase. Modeling based on crystal structures of potent kinase inhibitors facilitated the "back-to-front" design of potent DDR1/2 inhibitors that incorporated one of the DFG-out fragments. Further optimization led to low nanomolar, orally bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell lines.

DNA-PK-A candidate driver of hepatocarcinogenesis and tissue biomarker that predicts response to treatment and survival.

PURPOSE: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate. EXPERIMENTAL DESIGN: PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy. RESULTS: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo. CONCLUSIONS: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance.

Inhibition of HSP90 by AT13387 delays the emergence of resistance to BRAF inhibitors and overcomes resistance to dual BRAF and MEK inhibition in melanoma models.

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions in which vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together, these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R and SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced, and growth in 3D culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma.

Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.

Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway.

Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.

Further characterisation of the cellular activity of the DNA-PK inhibitor, NU7441, reveals potential cross-talk with homologous recombination.

PURPOSE: Inhibition of DNA repair is emerging as a new therapeutic strategy for cancer treatment. One promising target is DNA-PK, a pivotal kinase in double-strand break repair. The purpose of this study was to further characterise the activity of the DNA-PK inhibitor NU7441, giving some new insights into the biology of DNA-PK. METHODS: We used NU7441, a potent DNA-PK inhibitor, to evaluate potential pharmacodynamic markers of DNA-PK inhibition, inhibition of DNA repair and chemo- and radio-potentiation in isogenic human cancer cells proficient (M059-Fus1) and deficient (M059 J) in DNA-PK. RESULTS: NU7441 strongly inhibited DNA-PK in cell lines (IC(50) = 0.3 µM) but only weakly inhibited PI3 K (IC(50) = 7 µM). The only available anti-phospho-DNA-PK antibody also recognised some phosphoprotein targets of ATM. NU7441 caused doxorubicin- and IR-induced DNA DSBs (measured by γ-H2AX foci) to persist and also slightly decreased homologous recombination activity, as assessed by Rad51 foci. Chemo- and radio-potentiation were induced by NU7441 in M059-Fus-1, but not in DNA-PK-deficient M059 J cells. DNA-PK was highly expressed in a chronic lymphocytic leukaemia sample but undetectable in resting normal human lymphocytes, although it could be induced by PHA-P treatment. In K652 cells, DNA-PK expression was not related to cell cycle phase. CONCLUSION: These data confirm NU7441 not only as a potent chemo- and radio-sensitiser clinical candidate but also as a powerful tool to study the biology of DNA-PK.

A dual function for Pex3p in peroxisome formation and inheritance.

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.