Reza Kiarash

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.

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.

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.

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.

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.

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

Significance At present, microtubule-targeting agents are the most important antimitotic drugs used in the clinic. However, there is an urgent need for the discovery of new approaches to more effectively target tumor cells with less toxicity. Emerging strategies for anticancer therapy include exploiting cell-cycle checkpoint vulnerabilities and genomic instability in cancer cells. The spindle assembly checkpoint (SAC) is important for cell survival, and its inactivation generates lethal genomic instability in cancer cells. Inhibition of SAC signaling through targeting of monopolar spindle 1 (Mps1) has provided an indication of the feasibility of such an approach. We report here the cellular and antitumor effects of CFI-402257, a potent and specific small-molecule inhibitor of Mps1. CFI-402257 is currently in a phase I clinical trial (ClinicalTrials.gov ID: NCT02792465). 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.

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%).

Abstract LB-215: Inhibition of Polo-like kinase 4 as an anti-cancer strategy

Polo-like kinase 4 or PLK4 is a member of a conserved family of serine/threonine protein kinases that regulate multiple cellular processes, such as cell division and checkpoint regulation of mitosis. These kinases are often deregulated in cancer. PLK4 is the most structurally divergent PLK, localizes to centrosomes and is a critical regulator of centriole duplication. Over-expression of PLK4 leads to centrosome amplification and results in chromosome instability (CIN), a common characteristic observed in many types of cancers. We found that PLK4 is upregulated in breast cancer, specifically in the basal-like subtype. PLK4 expression is induced by hypoxia and suppressed by p53 in cancer cells. Consistent with this observation, PLK4 expression in cancer cells is upregulated when they are implanted and grown as xenografts in vivo. Furthermore, RNAi-mediated depletion of PLK4 inhibits the growth of cancer cells, but not normal cells (HMEC), in vitro, and tumor growth in vivo. Interestingly, siRNA knockdown of PLK4 sensitizes cancer cells to hypoxia. These findings suggest that targeting PLK4 may be a good therapeutic strategy in treating certain cancers. To this end, we initiated a discovery program that resulted in the identification of potent PLK4 inhibitors. These novel inhibitors are potent anti-prolifeartives, cause loss of mitotic checkpoint followed by apoptotic cell death, and suppress tumor growth in xenograft models. Mechanistically, inhibition of PLK4 suppresses phosphorylation of PLK4 and Histone H3, leads to failure of centrosome clustering and formation of multipolar spindles. Interestingly, breast cancer cell response to PLK4 inhibition appears to differ among subtypes of breast cancer cells and to be influenced by receptor and mutation status, such as ER and PTEN. Since multipolar division in cancer cells is not viable, due to massive missegregation of chromosomes, inhibition of PLK4 and formation of multipolar division followed by cell death may be a unique strategy for killing cancer cells. Implications of these findings in treating cancer will also be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2011-LB-215

Abstract 1642: Evaluation of ENMD-2076 in combination with anti-PD1 in syngeneic cancer models

ENMD-2076 is a clinical stage compound with potent activity towards Aurora A and angiogenic kinases. ENMD-2076 has shown promising activity in multiple Phase 1 clinical trials, as well as in a Phase 2 trial in advanced ovarian cancer. ENMD-2076 is currently the subject of several ongoing Phase 2 clinical trials including fibrolamellar carcinoma, triple-negative breast cancer (TNBC), advanced/metastatic soft tissue sarcoma (STS), and advanced ovarian clear cell carcinomas (OCCC). ENMD-2076 has been developed to date as a single agent, however ENMD-2076 inhibits a spectrum of targets including Aurora A, FAK, CSF1R, c-Kit, and KDR, that are potentially involved in immune evasion mechanisms. These kinases have been shown in published studies, when inhibited, to enhance or augment the activity of immune checkpoint inhibitors such as anti-PD1. A study was thereby conducted in syngeneic models to determine the utility of ENMD-2076 combined with immune checkpoint inhibition as a rational strategy for cancer therapy. The study evaluated the efficacy of ENMD-2076 administered daily by oral gavage in the MC38 and CT26 colon cancer models, alone and in combination with an anti-PD1 antibody. Xenografts were established in the appropriate mouse strain (C57BL/6 and BALBc, respectively) by the subcutaneous inoculation of MC38 or CT26 cells into the right flank of female mice. Treatment was initiated 7 days following inoculation when tumor volumes had reached a mean volume of approximately 85 mm3. All treatments were well tolerated, with no significant body weight loss seen during either study. While CT26 tumors were relatively refractory to single agent ENMD-2076, tumor regression was observed in several MC38-bearing animals suggesting an immune activating mechanism. In both models a trend was observed for an augmentation of anti-tumor response in combination relative to single agent ENMD-2076 and anti-PD1 alone. Further studies to evaluate mechanism and an assessment of re-challenge experiments in animals exhibiting complete regression of tumors will be discussed. These studies support the further evaluation of ENMD-2076 in combination with immune checkpoint inhibition as a strategy for cancer therapy. Citation Format: Graham C. Fletcher, Reza Kiarash, Mark R. Bray, Amanda S. Hu, Ken K. Ren. Evaluation of ENMD-2076 in combination with anti-PD1 in syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2017-1642

Abstract B267: Inhibition of PLK4 as a therapeutic strategy for genomically unstable cancers deficient for PTEN.

The unique Polo-like kinase family member, PLK4, was identified as a novel therapeutic target for breast cancer through a systematic approach that integrated functional RNAi viability profiles with molecular profiling data. PLK4 is a conserved upstream regulator of centriole duplication that is aberrantly expressed in several tumor types. Dysregulation of PLK4 activity causes loss of centrosome numeral integrity, thereby promoting genomic instability, but could also enable cancer cells to tolerate its effects. A drug discovery program was initiated that resulted in the identification of CFI-400945, a first-in-class, potent and selective PLK4 small molecule inhibitor (IC50 = 2.8 nM, Ki = 0.26 nM). CFI-400945 is highly selective towards other PLK family members (PLK1, PLK2 and PLK3 IC50s >50 µM) and numerous other protein and lipid kinase classes. Consistent with PLK4 loss-of-function studies, cancer cells treated with CFI-400945 exhibit dysregulated centriole duplication, mitotic defects and cell death. Oral administration of CFI-400945 as a single agent once daily to mice bearing human cancer-derived cell line xenografts and patient-derived xenografts (PDX) results in significant inhibition of tumor growth at doses that are well-tolerated. Superior antitumor activity in vivo is observed towards PTEN-deficient tumor models compared to PTEN wild-type tumor models. Analysis of tumor xenografts from CFI-400945-treated mice demonstrated an increase in tumor cells with aberrant mitoses compared to vehicle-treated mice, and is consistent with observations from cell culture experiments. Together, these pharmacologic results support observations from functional genetic screening that inhibition of PLK4 kinase activity is lethal for PTEN-deficient cancer cells, and suggest a new mechanism-based approach for the treatment of patients whose tumors have defective PTEN. PLK4 dysregulation may represent an adaptation cancer cells have made to tolerate the effects of genomic instability induced by loss-of-function of PTEN or other mediators, such as TP53, ATM, BRCA1 and BRCA2, and thereby is a vulnerability that may be exploited for the design of cancer cell-selective therapies. The CTA to Health Canada for CFI-400945 received approval in July 2013, and an IND has been filed at the FDA, clearing the way for clinical evaluation of the molecule in the near future. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B267. Citation Format: Jacqueline M. Mason, Dan C.-c. Lin, Xin Wei, Yi Che, Yi Yao, Reza Kiarash, Graham C. Fletcher, Mark R. Bray, Guohua Pan, Tak W. Mak. Inhibition of PLK4 as a therapeutic strategy for genomically unstable cancers deficient for PTEN. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B267.