Preclinical characterization and clinical trial of CFI-400945, a polo-like kinase 4 inhibitor, in patients with relapsed/refractory acute myeloid leukemia and higher-risk myelodysplastic neoplasms.

CFI-400945 is a selective oral polo-like kinase 4 (PLK4) inhibitor that regulates centriole duplication. PLK4 is aberrantly expressed in patients with acute myeloid leukemia (AML). Preclinical studies indicate that CFI-400945 has potent in vivo efficacy in hematological malignancies and xenograft models, with activity in cells harboring TP53 mutations. In this phase 1 study in very high-risk patients with relapsed/refractory AML and myelodysplastic syndrome (MDS) (NCT03187288), 13 patients were treated with CFI-400945 continuously in dose escalation from 64 mg/day to 128 mg/day. Three of the 9 efficacy evaluable AML patients achieved complete remission (CR). Two of 4 AML patients (50%) with TP53 mutations and complex monosomal karyotype achieved a CR with 1 patient proceeding to allogenic stem cell transplant. A third patient with TP53 mutated AML had a significant reduction in marrow blasts by > 50% with an improvement in neutrophil and platelet counts. Responses were observed after 1 cycle of therapy. Dose-limiting toxicity was enteritis/colitis. A monotherapy and combination therapy study with a newer crystal form of CFI-400945 in patients with AML, MDS and chronic myelomonocytic leukemia (CMML) is ongoing (NCT04730258).

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer.

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.

Activity of the novel polo-like kinase 4 inhibitor CFI-400945 in pancreatic cancer patient-derived xenografts.

BACKGROUND: Polo-like kinase 4 (PLK4) plays a key role in centriole replication. Hence PLK4 inhibition disrupts mitosis, and offers a novel approach to treating chromosomally unstable cancers, including pancreatic cancer. CFI-400945 is a first in class small molecule PLK4 inhibitor, currently undergoing early phase clinical trials. RESULTS: Treatment with CFI-400945 significantly reduced tumor growth and increased survival in four out of the six models tested. Consistent with PLK4 inhibition, we observed reduced expression of the proliferation marker Ki-67 associated with an increase in nuclear diameter during treatment with CFI-400945. Additionally, treatment with CFI-400945 resulted in a significant reduction of tumor-initiating cells. DISCUSSION: These results support the further investigation of PLK4 as a drug target in pancreatic cancer. METHODS: Sensitivity to CFI-400945 was tested in a series of six patient-derived pancreatic cancer xenografts, selected to represent the range of growth characteristics, genetic features, and hypoxia found in pancreatic cancer patients.

Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones as potent PLK4 inhibitors with oral antitumor efficacy.

Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein 'directly-linked' aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.

Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent.

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine Kinase, TTK.

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).

Targeting Mitosis in Cancer: Emerging Strategies.

The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.

The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents.

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.

The discovery of Polo-like kinase 4 inhibitors: identification of (1R,2S).2-(3-((E).4-(((cis).2,6-dimethylmorpholino)methyl)styryl). 1H.indazol-6-yl)-5'-methoxyspiro[cyclopropane-1,3'-indolin]-2'-one (CFI-400945) as a potent, orally active antitumor agent.

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.

Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression.

Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer initiation. Genetic loss of Gclm prevents a tumor's ability to drive malignant transformation. Intriguingly, these findings can be replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death in vitro and in vivo, demonstrating the importance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.

The discovery of Polo-like kinase 4 inhibitors: design and optimization of spiro[cyclopropane-1,3'[3H]indol]-2'(1'H).ones as orally bioavailable antitumor agents.

Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.

Discovery of inhibitors of the mitotic kinase TTK based on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)-acetamides and carboxamides.

TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50=3.6nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.

Functional characterization of CFI-400945, a Polo-like kinase 4 inhibitor, as a potential anticancer agent.

PLK4 was identified as a promising therapeutic target through a systematic approach that combined RNAi screening with gene expression analysis in human breast cancers and cell lines. A drug discovery program culminated in CFI-400945, a potent and selective PLK4 inhibitor. Cancer cells treated with CFI-400945 exhibit effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication, mitotic defects, and cell death. Oral administration of CFI-400945 to mice bearing human cancer xenografts results in the significant inhibition of tumor growth at doses that are well tolerated. Increased antitumor activity in vivo was observed in PTEN-deficient compared to PTEN wild-type cancer xenografts. Our findings provide a rationale for the clinical evaluation of CFI-400945 in patients with solid tumors, in particular those deficient in PTEN.

Organization of vagal afferents in pylorus: mechanoreceptors arrayed for high sensitivity and fine spatial resolution?

The pylorus is innervated by vagal mechanoreceptors that project to gastrointestinal smooth muscle, but the distributions and specializations of vagal endings in the sphincter have not been fully characterized. To evaluate their organization, the neural tracer dextran biotin was injected into the nodose ganglia of rats. Following tracer transport, animals were perfused, and their pylori and antra were prepared as whole mounts. Specimens were processed to permanently label the tracer, and subsets were counterstained with Cuprolinic blue or immunostained for c-Kit. Intramuscular arrays (IMAs) in the circular muscle comprised the principal vagal afferent innervation of the sphincter. These pyloric ring IMAs were densely distributed and evidenced a variety of structural specializations. Morphometric comparisons between the arbors innervating the pylorus and a corresponding sample of IMAs in the adjacent antral circular muscle highlighted that sphincter IMAs branched profusely, forming more than twice as many branches as did antral IMAs (means of 405 vs. 165, respectively), and condensed their numerous neurites into compact receptive fields (∼48% of the area of antral IMAs) deep in the circular muscle (∼6µm above the submucosa). Separate arbors of IMAs in the sphincter interdigitated and overlapped to form a 360° band of mechanoreceptors encircling the pyloric canal. The annulus of vagal IMA arbors, putative stretch receptors tightly intercalated in the sphincter ring and situated near the lumen of the pyloric canal, creates an architecture with the potential to generate gut reflexes on the basis of pyloric sensory maps of high sensitivity and fine spatial resolution.

Architecture of vagal motor units controlling striated muscle of esophagus: peripheral elements patterning peristalsis?

Little is known about the architecture of the vagal motor units that control esophageal striated muscle, in spite of the fact that these units are necessary, and responsible, for peristalsis. The present experiment was designed to characterize the motor neuron projection fields and terminal arbors forming esophageal motor units. Nucleus ambiguus compact formation neurons of the rat were labeled by bilateral intracranial injections of the anterograde tracer dextran biotin. After tracer transport, thoracic and abdominal esophagi were removed and prepared as whole mounts of muscle wall without mucosa or submucosa. Labeled terminal arbors of individual vagal motor neurons (n=78) in the esophageal wall were inventoried, digitized and analyzed morphometrically. The size of individual vagal motor units innervating striated muscle, throughout thoracic and abdominal esophagus, averaged 52 endplates per motor neuron, a value indicative of fine motor control. A majority (77%) of the motor terminal arbors also issued one or more collateral branches that contacted neurons, including nitric oxide synthase-positive neurons, of local myenteric ganglia. Individual motor neuron terminal arbors co-innervated, or supplied endplates in tandem to, both longitudinal and circular muscle fibers in roughly similar proportions (i.e., two endplates to longitudinal for every three endplates to circular fibers). Both the observation that vagal motor unit collaterals project to myenteric ganglia and the fact that individual motor units co-innervate longitudinal and circular muscle layers are consistent with the hypothesis that elements contributing to peristaltic programming inhere, or are "hardwired," in the peripheral architecture of esophageal motor units.

The discovery of PLK4 inhibitors: (E)-3-((1H-Indazol-6-yl)methylene)indolin-2-ones as novel antiproliferative agents.

The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.

Vagal afferent innervation of the lower esophageal sphincter.

To supply a fuller morphological characterization of the vagal afferents innervating the lower esophageal sphincter (LES), specifically to label vagal terminals in the tissues forming the LES in the gastroesophageal junction, the present experiment employed injections of dextran biotin into the nodose ganglia of rats. Four types of vagal afferents innervated the LES. Clasp and sling muscle fibers were directly and prominently innervated by intramuscular arrays (IMAs). Individual IMA terminals subtended about 16° of arc of the esophageal circumference, and, collectively, the terminal fields were distributed within the muscle ring to establish a 360° annulus of mechanoreceptors in the sphincter wall. 3D morphometry of the terminals established that, compared to sling muscle IMAs, clasp muscle IMAs had more extensive arbors and larger receptive fields. In addition, at the cardia, local myenteric ganglia between smooth muscle sheets and striated muscle bundles were innervated by intraganglionic laminar endings (IGLEs), in a pattern similar to the innervation of the myenteric plexus throughout the stomach and esophagus. Finally, as previously described, the principle bundle of sling muscle fibers that links LES sphincter tissue to the antropyloric region of the lesser curvature was innervated by exceptionally long IMAs as well as by unique web ending specializations at the distal attachment of the bundle. Overall, the specialized varieties of densely distributed vagal afferents innervating the LES underscore the conclusion that these sensory projections are critically involved in generating LES reflexes and may be promising targets for managing esophageal dysfunctions.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

Sprouty proteins inhibit receptor-mediated activation of phosphatidylinositol-specific phospholipase C.

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.

A pathologic link between Wilms tumor suppressor gene, WT1, and IFI16.

The Wilms tumor gene (WT1) is mutated or deleted in patients with heredofamilial syndromes associated with the development of Wilms tumors, but is infrequently mutated in sporadic Wilms tumors. By comparing the microarray profiles of syndromic versus sporadic Wilms tumors and WT1-inducible Saos-2 osteosarcoma cells, we identified interferon-inducible protein 16 (IFI16), a transcriptional modulator, as a differentially expressed gene and a candidate WT1 target gene. WT1 induction in Saos-2 osteosarcoma cells led to strong induction of IFI16 expression and its promoter activity was responsive to the WT1 protein. Immunohistochemical analysis showed that IFI16 and WT1 colocalized in WT1-replete Wilms tumors, but not in normal human midgestation fetal kidneys, suggesting that the ability of WT1 to regulate IFI16 in tumors represented an aberrant pathologic relationship. In addition, endogenous IFI16 and WT1 interacted in vivo in two Wilms tumor cell lines. Furthermore, IFI16 augmented the transcriptional activity of WT1 on both synthetic and physiological promoters. Strikingly, short hairpin RNA (shRNA)-mediated knockdown of either IFI16 or WT1 led to decreased growth of Wilms tumor cells. These data suggest that IFI16 and WT1, in certain cellular context including sporadic Wilms tumors, may support cell survival.