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Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials., Finding therapeutic strategies for aggressive triple negative breast cancer (TNBC) is an important challenge in clinical practice. Here, the authors identify a multi-kinases inhibitor with antitumor activity and able overcome chemotherapy resistance of TNBC in vivo.

Aberrant signaling triggered by oncogenic or hyperactive RAS proteins contributes to the malignant phenotypes in a significant percentage of myeloid malignancies. Of these, juvenile myelomonocytic leukemia (JMML), an aggressive childhood cancer, is largely driven by mutations in RAS genes and those that encode regulators of these proteins. The Mx1-cre kras+/G12D mouse model mirrors several key features of this disease and has been used extensively to determine the utility and mechanism of small molecule therapeutics in the context of RAS-driven myeloproliferative disorders. Treatment of disease-bearing KRASG12D mice with rigosertib (RGS), a small molecule RAS mimetic that is in phase II and III clinical trials for MDS and AML, decreased the severity of leukocytosis and splenomegaly and extended their survival. RGS also increased the frequency of HSCs and rebalanced the ratios of myeloid progenitors. Further analysis of KRASG12D HSPCs in vitro revealed that RGS suppressed hyperproliferation in response to GM-CSF and inhibited the phosphorylation of key RAS effectors. Together, these data suggest that RGS might be of clinical benefit in RAS-driven myeloid disorders.

Overexpression and constitutive activation of CYCLIN D1 and Casein Kinase 2 are common features of many hematologic malignancies, including mantle cell lymphoma (MCL) and leukemias such as T-cell acute lymphoblastic leukemia (T-ALL). Although both CK2 and CDK4 inhibitors have shown promising results against these tumor types, none of these agents have achieved objective responses in the clinic as monotherapies. Because both proteins play key roles in these and other hematological malignancies, we have analyzed the therapeutic potential of ON108110, a novel dual specificity ATP-competitive inhibitor of protein kinase CK2 as well as CDK4/6 in MCL and T-ALL. We show that in cell growth inhibition assays, MCL and T-ALL cell lines exhibited increased sensitivity to ON108110 when compared to other tumor types. Treatment with ON108110 reduced the level of phosphorylated RB-family proteins. In addition, ON108110 treatment resulted in concentration dependent inhibition of PTEN phosphorylation and a concomitant decrease in PI3K-AKT signaling mediated by CK2. Accordingly, cells treated with ON108110 rapidly accumulated in the G0/G1 stage of the cell cycle as a function of increasing concentration followed by rapid onset of apoptosis. Together, these results indicate that dual inhibition of CK2 and CDK4/6 may be an efficient treatment of MCL and T-ALLs displaying upregulation of CK2/PI3K and CDK4 signaling pathways.

Four new organometallic triruthenium carbonyl incorporating 4-pyridones: [Ru 3 (CO) 8 (2L-2H)] ( 1 , L = N-benzyl-3-hydroxy-2-methyl-4-pyridone; 2 , L = 3-hydroxy-2-methyl-N-phenyl-4-pyridone; 3 , L = 3-hydroxy-N,2-dimethyl-4-pyridone; 4 , L = 2-methyl-4-pyridone) were synthesized and characterized. Proton NMR spectrum of 1 shows doublets for the two-nonequivalent benzylic hydrogens due to restricted rotation around C-N bond upon bonding to Ru(2)---Ru(3) as opposed to a singlet in the unbound N-benzyl-3-hydroxy-2-methyl-4-pyridone. Compounds 1 and 3 were characterized by X-ray diffraction analyses. Compounds 1 and 3 consists of two ruthenium-ruthenium bonds Ru(1)-Ru(2), Ru(1)-Ru(3) and a non-bonded separation of two ruthenium atoms defined by Ru(2) … Ru(3), which is bridged by two 3 electron donar oxygen atoms through two-pyridinonato ligands above and below triruthenium plane, and 8 terminal CO groups. Atoms Ru(2) and Ru(3) ligated by two CO ligands each and Ru(1) with four. Antitumor activity of the compounds 1–4 has been evaluated against five types of human cancer cell lines. Compounds 1 and 2 show excellent antitumor activity due to lipophilic nature of N-substituted arene pyridinonato ligands than compounds 3 and 4 . Structure-activity studies show that there is a correlation between the N-substituted arene pyridinonato ligands and an increase in activity following the order 1 > 2>3 > 4. The anticancer activity of compounds 1 and 2 is between 1–2 orders of magnitude more active than cisplatin. This is possibly due to substitution of CO ligands at Ru(1) by transferrin delivery vehicle transporting to cancer cells and lipophilic N-substituted arenes diffuse through lipophic membrane leading to apoptosis and other carbonyl groups under physiological conditions likely function as carbonyl releasing molecules (CORMs).

Four new triruthenium carbonyl containing 4-pyrones: Ru 3 (CO) 8 (2 L-2H) 1 (L = ethylmaltol), Ru 3 (CO) 7 (PTA) (2 L-2H) 2 (L = ethylmaltol, PTA = 1,3,5 triaza-7-phosphaadamantane), Ru 3 (CO) 8 (2 L-2H) 3 (L = allomaltol), Ru 3 (CO) 7 (PTA) (2 L-2H) 4 (L = allomaltol, PTA = 1,3,5 triaza-7-phosphaadamantane) have been synthesized and characterized. Anticancer activity of compounds 1–4 has been evaluated in vitro against five types of human cancer cell lines and compared with clinically used drug cisplatin. Anticancer activity of compound 1 is an order of magnitude more potent than cisplatin against five types of human cancer cell lines. Compounds 1 and 2 with ethyl group at 2-position is more active than the compounds 3 and 4 with methyl group at 6-position. Substitution of a CO ligand with PTA decreases the activity following the order 1 > 2 and 3 4 . The single crystal X-ray structure of compound 1 shows two Ru-CH 2 -H 2 C-H---O-Ru interactions: ethyl group C (217)-C (218) of first ethylmaltolato ligand bonded to Ru (2) folded towards ruthenium Ru (3) to form Ru (2)-CH 2 -H 2 C-H--O-Ru (3) interaction and the second ethylmaltolato ligand C (317)-C (318) bonded to Ru (3) folded towards Ru (2) to form Ru (3)-CH 2 -H 2 C-H---O-Ru (2). Proton NMR shows a well-resolved multiplet instead of a simple quartet for methylene protons indicating asymmetric environment.

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cellsin vitroandin vivoand is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity.In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.

Background/Rationale: Sensitivity to chemotherapy is a strong, consistent predictor of survival for patients diagnosed with triple negative breast cancer (TNBC) and chemotherapy resistance remains a critical problem for a subset of patients. Cancer cells with a stem cell-like profile are thought to represent a relatively chemotherapy resistant subpopulation. A screen for novel inhibitors of these cancer stem cell-like cells identified the multi-kinase inhibitor ON108600 that inhibits Casein kinase 2 (CK2), TNIK and Dyrk kinases. We sought to determine whether treatment with ON108600 enhances chemotherapy sensitivity of aggressive, multi-drug resistant TNBC, thereby offering a novel therapeutic option for patients with high risk TNBC. Methods: Patient-derived xenograft (PDX) models derived from patients with chemotherapy-resistant primary or metastatic TNBC were screened for expression of ON108600 targets prior to selection for in vivo studies. Studies to confirm target engagement in vivo were performed to optimize dosing schedules. Selected PDX models were expanded and treated with vehicle control, ON108600, Paclitaxel, or ON108600/Paclitaxel combination. Tumor growth was assessed. Tumors were harvested at end point for histological analysis. Results: Chemotherapy- resistant TNBC PDX models were screened for protein expression of CK2, Dyrk and TNIK kinases. Two models with the highest level of these kinases were selected and propagated for in vivo studies; one model is derived from a primary TNBC with intrinsic resistance to chemotherapy while the second is derived from a heavily pre-treated metastatic TNBC. Target engagement was confirmed by assessing levels of CK2 substrates in tumors treated with different doses of ON108600. Expanded cohorts of these PDX models were treated with ON108600, Paclitaxel or combined ON108600/Paclitaxel. While Paclitaxel chemotherapy had minimal effect on tumor growth in these PDX models, combined ON108600/Paclitaxel treatment significantly suppressed growth and/or caused regression of these aggressive tumors. Importantly, combination treatment was associated with minimal toxicity. RNASeq profiling of PDX tumors treated with ON108600 or vehicle control was performed, revealing suppression of multiple oncogenic pathways by ON108600 which are currently being validated. Conclusions: ON108600, identified as an inhibitor of a cancer stem cell-like population, overcomes Paclitaxel resistance to suppress growth of aggressive TNBC for which treatment options remain limited. These results support further study and clinical translation of ON108600. Citation Format: Katsutoshi Sato, Amol Padgaonkar, Stephen Cosenza, Stacey Baker, Ramana Reddy, E Premkumar Reddy, Hanna Y. Irie. A novel inhibitor of cancer stem cells overcomes chemotherapy resistance of triple negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-04.

This study describes the characterization of a novel kinase inhibitor, ON123300, which inhibits CDK4/6 (cyclin-dependent kinases 4 and 6) and phosphatidylinositol 3 kinase-δ (PI3K-δ) and exhibits potent activity against mantle cell lymphomas (MCLs) both in vitro and in vivo. We examined the effects of PD0332991 and ON123300 on cell cycle progression, modulation of the retinoblastoma (Rb) and PI3K/AKT pathways, and the induction of apoptosis in MCL cell lines and patient-derived samples. When Granta 519 and Z138C cells were incubated with PD0332991 and ON123300, both compounds were equally efficient in their ability to inhibit the phosphorylation of Rb family proteins. However, only ON123300 inhibited the phosphorylation of proteins associated with the PI3K/AKT pathway. Cells treated with PD0332991 rapidly accumulated in the G0/G1 phase of cell cycle as a function of increasing concentration. Although ON123300-treated cells arrested similarly at lower concentrations, higher concentrations resulted in the induction of apoptosis, which was not observed in PD0332991-treated samples. Mouse xenograft assays also showed a strong inhibition of MCL tumor growth in ON123300-treated animals. Finally, treatment of ibrutinib-sensitive and -resistant patient-derived MCLs with ON123300 also triggered apoptosis and inhibition of the Rb and PI3K/AKT pathways, suggesting that this compound might be an effective agent in MCL, including ibrutinib-resistant forms of the disease.

The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multikinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyano pyridopyrimidine compounds and identified structure–activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30–100 nM. In vitro kinase profiling revealed that 7x is a multikinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure–activity relationship, kinase inhibitory profile, in vitro cytotoxicity, and in vivo tumor regression studies by this lead compound.

Organometallic ruthenium complexes containing two dehydrogenated maltol ligands Ru3(CO)8(2L–2H) 1, Ru3(CO)7PPh3(2L–2H) 2, [Ru3(CO)7 (2L–2H)]2(dppm or dppe) 3,4 (L = Maltol) have been synthesized and characterized. The in vitro anticancer activity of compounds 1–4 against seven types of human cancer cell lines was assessed and compared to clinically used drug cisplatin. The anticancer activity of compound 1 ( Fig. 3 ) is many times more potent than cisplatin against seven types of human cancer cell lines. There is a correlation between substituting a CO ligand in 1 with different phosphines decreases the activity following the order 1 > 2 > 3 > 4. The X-ray crystal structures of complexes 1 and 2 are reported. The single crystal X-ray diffraction structure of 1 consists of a triangular ruthenium metal framework in which a Ru–Ru bond is bridged by two maltolate ligands with their two oxygen atoms in a μ−η2-bonding mode. The dihedral angle between Ru3 and maltol planes is 40.2°. The two ruthenium atoms bridged by maltol ligands each have two carbonyl ligands and the third ruthenium atom is bonded to four carbonyl ligands. The greatest structural difference between 1 and 2 is the angle between the best planes of the two coordinated C6H5O3−1 ligands; in 1 it is 27.1° while in 2 it is 42.8°. It is interesting to note that the phosphine substitution occurs at the ruthenium atom not bound by maltol ligands.

Cyclin D proteins are elevated in many cancer cells and targeted deletion of Cyclin D1 gene in the mammary tissues protects mice from breast cancer. Accordingly, there is an increasing awareness of this novel non-enzymatic target for cancer therapeutics. We have developed novel, non-alkylating styryl benzyl sulfones that induce cell death in wide variety of cancer cells without affecting the proliferation and survival of normal cells. The development of derivatized Styryl Benzyl Sulfones followed logically from a tumor cell cytotoxicity screen performed in our laboratory that did not have an a priori target profile. Modifications of some of the precursor molecules led to lead optimization with regard to tumor cell cytotoxicity. In this report we describe the synthesis and structure-activity relationships of novel, non-alkylating (E) styryl benzyl sulfones, and the development of the novel anti-cancer agent sodium (E)-2-{2-methoxy-5-[(2′,4′,6′-trimethoxystyrylsulfonyl)methyl]phenylamino}-acetate (ON 01910.Na), which is in Phase III trials for myelodysplastic syndromes (MDS) associated with aberrant expression of cyclin D proteins.

Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

Novel (E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.

ON 01910.Na is a novel targeted anti-cancer agent under clinical investigation in Phase I and II trials. The purpose of this research was to evaluate the pharmacokinetic profile of ON 01910.Na across several species, and to evaluate the effects of protein binding and duration of exposure on its in vitro cytotoxic activity. Data were collated from several preclinical investigations, where the plasma disposition and tissue distribution of ON 01910.Na were assessed after administration (10–150 mg/kg, IP or IV) to several species (mouse, rat, and dog). Plasma protein binding was assessed using ultrafiltration. Cytotoxic activity of ON 01910.Na was determined in DU145 cells, and activity was correlated to unbound drug concentration and the duration of exposure. ON 01910.Na exhibits extensive plasma protein binding and the compound displays rapid elimination from the circulation in all three animal species (t1/2 range 0.404–0.870 h). Tissue distribution studies in mice revealed highest drug accumulation in the liver, followed by the kidneys. ON 01910.Na is not extensively metabolized in vivo and urinary excretion is predominant at higher doses. ON 01910.Na cytotoxicity in DU145 cells was adversely affected by protein binding in the incubation medium. Drug cytotoxicity was greatly enhanced upon extending the duration of exposure at reduced drug concentrations. Due to the short half-life and rapid clearance of the drug, administration of ON 01910.Na by continuous IV infusion is a likely treatment option for cancer patients.

Four new chiral ruthenium carbonyl cluster complexes Ru3(μ-H)2(CO)9(L-2H) (1), Ru3(μ-H)2(CO)7(L-2H)(dppm) (2), Ru3(μ-H)2(CO)7(L-2H)(PPh3)2 (3), Ru3(μ-H)2(CO)7(L-2H)(dppe) (4) containing a dehydrogenated form (L-2H) of 3,4,6-tri-O-benzyl-d-galactal (L) as a chiral ligand have been prepared and characterized. The anticancer activity of five compounds 1–4 and Ru3(μ-H)2(CO)9(L-2H) 5 (L = tribenzyl glucal) against six types of human cancer cell lines was studied and compared to cisplatin. Compound 1 was chosen to produce more detailed growth curves based on overall highest activity profile. The structure of compound 2 was established by a single-crystal X-ray diffraction analysis. The structure based on triangular metal framework contains a bridging dehydrogenated tribenzyl galactal ligand bonded in a parallel μ3-η2-bonding mode and a bridging dppm ligand. Variable-temperature NMR studies show that the two hydride ligands in compounds 1 and 2 are dynamically active on the NMR time scale at room temperature.

Imatinib, which is an inhibitor of the BCR-ABL tyrosine kinase, has been a remarkable success for the treatment of Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemias (CMLs). However, a significant proportion of patients chronically treated with imatinib develop resistance because of the acquisition of mutations in the kinase domain of BCR-ABL. Mutations occur at residues directly implicated in imatinib binding or, more commonly, at residues important for the ability of the kinase to adopt the specific closed (inactive) conformation to which imatinib binds. In our quest to develop new BCR-ABL inhibitors, we chose to target regions outside the ATP-binding site of this enzyme because these compounds offer the potential to be unaffected by mutations that make CML cells resistant to imatinib. Here we describe the activity of one compound, ON012380, that can specifically inhibit BCR-ABL and induce cell death of Ph + CML cells at a concentration of in vitro and cause regression of leukemias induced by i.v. injection of 32Dcl3 cells expressing the imatinib-resistant BCR-ABL isoform T315I. Daily i.v. dosing for up to 3 weeks with a >100 mg/kg concentration of this agent is well tolerated in rodents, without any hematotoxicity.

We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

Orderly cell cycle progression of mammalian cells determines their ability to undergo regulated proliferation, differentiation, senescence, and apoptosis (43, 44). Perturbations in the expression and activities of proteins that regulate these cellular phenomena determine the emergence of a cancerous state. Two important tumor suppressor pathways, which depend on the expression and activity of two key cell cycle regulatory proteins, pRb and p53, serve as critical branch points that govern controlled cell growth (14, 17, 33, 43, 44, 49). In concordance with this, it has been observed that a majority of human tumors harbor mutations that debilitate the surveillance function of the pRb and/or p53 pathways (43, 49). Moreover, patients with inactivating mutations in both pathways have a much worse prognosis compared to patients with inactivation in just one of the two pathways. While the consequences of inactivation of p53 on cancer development has been exhaustively studied using mice that are nullizygous at the p53 loci, it has been difficult to examine the effects of simultaneous inactivation of the retinoblastoma (Rb) family proteins on cancer progression, as homozygous inactivation of both Rb alleles leads to early embryonic lethality (5, 9, 19, 27). This requirement of the Rb family proteins during embryogenesis and early development has precluded the examination of their role during adult development and on cancer predisposition by using conventional gene disruption techniques (5, 6, 19, 25–28). Since homozygous inactivation of Rb leads to early embryonic lethality, studies focused on examination of the role of Rb family proteins in development and cancer have been restricted to characterization of mouse models that either (i) harbor heterozygous inactivation of Rb family genes, (ii) harbor a heterozygous inactivation of Rb family genes in addition to disruption of genes that code for other key cell cycle regulators, such as members of the cyclin kinase inhibitor (CKI) family, or (iii) have inactivation of the Rb family proteins via expression of viral proteins, such as papillomavirus protein E7 or the simian virus 40 large T-antigen protein (14, 17). However, the unequivocal importance of the retinoblastoma proteins in regulating cell cycle progression and cell proliferation and its underlying effects on immortalization and tumorigenesis has been elucidated by studies that describe the disruption of the retinoblastoma family gene loci in fibroblasts (7, 39). Loss of expression of pRb, p107, and p130 proteins in fibroblasts resulted in increased cell proliferation, shortening of the cell cycle, and immortalization. However, these studies do not allow an extrapolation of the effects of simultaneous inactivation of the retinoblastoma proteins on adult development and cancer predisposition. Cdk4, along with Cdk6, is the chief catalytic subunit of the regulatory cyclin D family of proteins that govern G1-to-S phase progression of mammalian cells via phosphorylation and inactivation of retinoblastoma family proteins (43, 44). Members of the INK4 family of proteins, chiefly p16Ink4a, are specific inhibitors of the cyclin D/Cdk4 complexes (43, 44). Mutations in CDK4 and its key kinase inhibitor p16INK4A have been implicated in the genesis and progression of familial human melanoma (20, 31). The importance of the CDK4 locus in human cancer was further emphasized upon identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4A, predisposing humans to hereditary melanoma (51, 53). This observation suggests that a mutant CDK4 gene can function as a dominant oncogene that is resistant to normal physiological inhibition by p16INK4A. To determine the consequence of the R24C germ line mutation on mouse development and cancer susceptibility, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated “knock-in” technology (37). The presence of the Cdk4R24C mutation induced hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130, and mouse embryo fibroblasts (MEFs) derived from Cdk4R24C/R24C mice escape from replicative senescence and become insensitive to contact-induced growth arrest. Moreover, the presence of the Cdk4R24C mutation resulted in transformation in vitro and spontaneous and chemical carcinogen-induced tumorigenesis in vivo, suggesting that the Cdk4R24C mutation can serve as the primary event in the progression towards a fully transformed phenotype.

Approximately one third of patients suffering from Acute Myeloid Leukemia harbor a FLT3 Internal tandem duplication mutation (FLT3-ITD). When mutated, this receptor tyrosine kinase increases the activity of pathways for proliferation and blocks apoptosis. Quizartinib is a 2nd generation FLT3 inhibitor that inhibits FLT3-ITD in AML, but has a median duration response of 12.1 weeks. Studies revealed a secondary mutation in FLT3 at the aspartate of codon 835 (D835X) is responsible for relapse. The D835 substitution renders FLT3 constitutively active. Type 2 inhibitors like Quizartinib bind to FLT3 in its inactive state and fail to inhibit FLT3-ITD harboring a D835 mutation. Here, we tested the utility of ON150030, developed by our group, as a novel therapeutic agent to treat AML. Structural studies suggest ON150030 binds to the active form of FLT3 (Type 1 inhibitor) so mutations such as D835X do not affect the inhibitory activity of the compound. In vitro kinase assays demonstrate that ON150030 potently inhibits Wildtype and FLT3-D835Y forms, while Quizartinib fails to inhibit FLT3-D835Y. Additionally, ON150030 demonstrated time and temperature dependent inhibition of FLT3, suggesting that the compound is an irreversible inhibitor of FLT3. Biological studies reveal that ON150030 specifically inhibits the growth of MV4-11 cells harboring the FLT3-ITD mutation (GI50: 10nM). Western blot analysis demonstrates that MAPK and PI3K/AKT pathways in these cells are inhibited with increasing dose of ON150030. The JAK independent phosphorylation of STAT5 seen in the context of FLT3-ITD is also reduced in response to ON150030. Future goals are to introduce FLT3 and its various mutant isoforms into the mouse myeloid cells (32Dcl3) and examine how it affects proliferation and differentiation, and then compare the effects of ON150030 and Quizartinib. In addition to strongly inhibiting FLT3, ON150030 inhibits SRC, which was shown to induce resistance to targeted therapies in several leukemias including AML. We will introduce SRC into the 32D-FLT3-ITD cell lines sensitive to ON150030 and test whether these cells retain their sensitivity to the drug. Next, we will perform cytotoxicity and biochemical assays on patient-derived primary AML cells using ON150030. Mouse xenograft models will be used to determine if ON150030 synergizes with standard chemotherapy agents and inhibits cancer progression in vivo. At the conclusion of this project, we hope to demonstrate that ON150030 can be used in combination therapies in all AML patients harboring a FLT3 mutation, and result in sustained remission of disease. Citation Format: Helya Ghaffari, M.V. Ramana Reddy, Stephen C. Cosenza, Rodrigo Vasques del Carpio, E. Premkumar Reddy. Dual inhibition of FLT3 and Src pathways by ON150030, a type 1 inhibitor, as a novel strategy for relapsed and refractory AML therapy [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 2094. doi:10.1158/1538-7445.AM2017-2094

Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z -factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC 50 values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI 50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9–Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.

The v-fgr oncogene encodes a chimeric oncoprotein composed of feline sarcoma virus (FeSV)-derived gag and cellular-derived actin and c-Fgr sequences. v-Fgr is myristoylated and membrane bound, two criteria which must be met for src kinases to induce cellular transformation. Although inhibition of myristoylation resulted in a decreased ability of v-Fgr to sediment with membranes from an NIH-3T3 P100 fraction, deletion of the gag domain caused nearly all of the protein to remain unbound and cytosolic. Systematic deletions within gag indicate that while amino acids 3 through 9 are critical determinants of myristoylation and/or define a domain which directs membrane localization, these residues cooperate with additional gag sequences when anchoring the protein to the plasma membrane. Furthermore, nonmyristoylated and/or cytoplasmic variants of v-Fgr failed to induce anchorage-independent growth of NIH-3T3 cells, indicating that proper subcellular localization of v-Fgr is a key factor in its ability to induce transformation.

A series of novel (E)-N-aryl-2-arylethenesulfonamides (6) were synthesized and evaluated for their anticancer activity. Some of the compounds in this series showed potent cytotoxicity against a wide spectrum of cancer cell-lines (IC50 values ranging from 5 to 10 nM) including all drug resistant cell-lines. Nude mice xenograft assays with compound (E)-N-(3-amino-4-methoxyphenyl)-2-(2',4',6'-trimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction in tumor size, indicating their in vivo potential as anticancer agents. A preliminary drug development study with compound 6t is predicted to have increased blood-brain barrier permeability relative to many clinically used antimitotic agents. Mechanistic studies indicate that 6t and some other analogues disrupted microtubule formation, formation of mitotic spindles, and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in vitro and in vivo and circumvented drug resistance mediated by P-glycoprotein. Compound 6t specifically competed with colchicine binding to tubulin and with similar avidity as podophylltoxin, indicating its binding site on tubulin.

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against colorectal cancers both in vitro and in vivo. While overexpression of Cyclin D1 is closely correlated with the proliferative rate of these tumor cells, metastatic colorectal cancers over-express ARK5, a member of the AMPK family and mediator of AKT activation. In this study, we show that ON 123300, which inhibits both CDK4/6 and ARK5, is a potent inducer of apoptosis of colorectal cancer cells when compared to palbociclib, a highly selective inhibitor of CDK4/6 kinases that does not target ARK5. Results & Conclusions: We examined the effects of palbociclib and ON 123300 on cell cycle progression, modulation of Rb and PI3K/AKT pathways, and induction of apoptosis in multiple colorectal cancer cell lines. Comparative kinase inhibition assays showed that while palbociclib and ON 123300 exhibited equivalent inhibition against CDK4/CDK6, ARK5 activity was inhibited only by ON 123300. When DLD1 and SW480 cells were incubated with increasing concentrations of palbociclib or ON 123300, both compounds were equally efficient in their ability to inhibit phosphorylation of all three members of the Rb family of proteins. However, when the phosphorylation status of proteins associated with the PI3K/AKT pathway was measured by western blot, ON 123300 showed concentration-dependent inhibition of mTOR, AKT, 4EBP1 and S6RB phosphorylation while palbociclib had little or no effect on the phosphorylation of these proteins. Cells treated with palbociclib rapidly accumulated in the G0/G1 stage of the cell cycle with increasing drug concentrations. Although cells treated with ON 123300 also arrested in the G0/G1 phase at lower concentrations (01-0.5 uM), with increasing concentrations of drug there was an accumulation of cells with sub-G1 DNA content, suggesting induction of apoptosis. ON 123300-treated cells showed cleavage of PARP and Caspases (3, 7 and 9) as well as inhibition of FOXO1 phosphorylation, which was not observed in cells treated with palbociclib. Since ARK5 belongs to the AMPK family of kinases, we next examined the effects of ON 123300-mediated ARK5 inhibition on metabolic changes of tumor cells that over-express this gene. Treatment of SW-480 colorectal cancer cells with ON 123300 resulted in an increase in glucose uptake, profound inhibition of glutamine uptake and reduced ATP production. A detailed metabolomic study revealed significant alterations in the levels of metabolites associated with glutamine metabolism. Nude mouse xenograft assays using Colo-205 cells revealed strong inhibition of tumor growth following 100 mg/kg of ON 123300 given QD or QOD, with little evidence of toxicity as measured by change in body weight. Thus, dual inhibition of ARK5 and CDK4 pathways could be an effective therapeutic strategy for the treatment of colorectal cancers. Citation Format: Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Vinee Purohit, Venugopal Gunda, Pankaj K. Singh, E. Premkumar Reddy. Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3029.

ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6 Da) was found in the drug-resistant cells upon a 1 h incubation with ON 013100 (20 μg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84 μg/ml at 0 h through 0.21 μg/ml at 6 h to below detection limit of 8.0 ng/ml at 9 h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41 μg/ml (0 h) to 0.06 μg/ml (9 h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88 μg/ml to 0.94 μg/ml.

Tubulin, the major structural component of microtubules, is a target for the development of anticancer agents. A series of (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) were synthesized and evaluated for anti-proliferative activity in cell based assay. The most active compound (Z)-1-(2- bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)-prop-2-en-1-one (10ae) was tested in 20 tumor cell lines including multidrug resistant phenotype and was found to induce apoptosis in all these cell lines with similar GI50 values. Flow cytometry studies showed that 10ae arrested the cells in G2/M phase of cell cycle. In addition to G2/M block, these compounds caused microtubule stabilization like paclitaxel and induced apoptosis via activation of the caspase family. The observations made in this investigation demonstrate that (Z)-1-Aryl-3- arylamino-2-propen-1-one (10) represents a new class of microtubule – stabilizing agents.

To evaluate a mitotic inhibitor, ON01910.Na, as a potential chemotherapeutic agent for brain tumors using a series of PK/PD studies, which led to the evaluation of its structural analog, ON013105, a prodrug of the more lipophilic product, ON013100.Systemic PK characterization of ON01910 and ON013105 was completed in healthy mice. Using an orthotopic U87 glioma mouse model, brain and brain tumor distribution under steady-state conditions were evaluated for ON01910.Na and ON013105/ON013100; anticancer potential following a multiple-dose schedule of 250 mg/kg/day IP for 7 days was evaluated for ON01910.Na.ON01910 exhibited low brain and brain tumor distribution with quasi-steady-state brain/plasma (Css(brain)/Css(plasma)) and brain tumor/plasma (Css(brain tumor)/Css(plasma)) concentration ratios of 0.03 ± 0.02 and 0.14 ± 0.08, respectively. Significant antiangiogenic potential and antiproliferative capacity of ON01910 in the intracerebral model was absent. ON013100 showed high brain and brain tumor penetration with Css(brain)/Css(plasma) and Css(brain tumor)/Css(plasma) ratios of 0.92 ± 0.26 and 1.35 ± 0.40, respectively; its prodrug ON013105 showed negligible brain and brain tumor penetration.ON013105, not ON01910.Na, was identified as a potential anticancer drug candidate for further investigation in brain tumor chemotherapy based on the properties of ON013100.

Analysis of gene expression following stimulation of growth-arrested cells has been the main approach for identification of growth-associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and the state of quiescence of the cell, the activation and role of the same genes may be entirely different in non-growth arrested, actively proliferating cells. We have addressed the question of growth-associated gene expression during active growth by analyzing gene expression during G-1 of cells which have just exited mitosis without first leaving the cell cycle. We were able to isolate, by a non-inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitosis (greater than 90%) in numbers sufficient to determine the pattern of expression of a large number of representative growth-associated genes. Our results show that after replating the mitotic cells into conditioned medium: (1) growth-associated gene expression is not constant during G-1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c-myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur-77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G-1 progression during active growth.

The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.

Cell cycle-regulated gene expression is essential for normal cell growth and development and loss of stringent growth control is associated with the acquisition of the transformed phenotype. The selective synthesis of histone proteins during the S phase of the cell cycle is required to render cells competent for the ordered packaging of replicating DNA into chromatin. Regulation of H4 histone gene transcription requires the proliferation-specific promoter binding factor HiNF-D. In normal diploid cells, HiNF-D binding activity is regulated during the cell cycle; nuclear protein extracts prepared from normal cells in S phase contain distinct and measurable HiNF-D binding activity, while this activity is barely detectable in G1 phase cells. In contrast, in tumor-derived or transformed cell lines, HiNF-D binding activity is constitutively elevated throughout the cell cycle and declines only with the onset of differentiation. The change from cell cycle-mediated to constitutive interaction of HiNF-D with the promoter of a cell growth-controlled gene is consistent with, and may be functionally related to, the loss of stringent cell growth regulation associated with neoplastic transformation.

Cell cycle progression is regulated by cyclins and cyclin-dependent kinases, which are formed at specific stages of the cell cycle and regulate the G1/S and G2/M phase transitions, employing a series of "checkpoints" governed by phosphorylation of their substrates. Tumor development is associated with the loss of these checkpoint controls, and this provides an approach for the development of therapeutic agents that can specifically target tumor cells. Here, we describe the synthesis and SAR of a novel group of cytotoxic molecules that selectively induce growth arrest of normal cells in the G1 phase while inducing a mitotic arrest of tumor cells resulting in selective killing of tumor cell populations with little or no effect on normal cell viability. The broad spectrum of antitumor activity in vitro and xenograft models, lack of in vivo toxicity, and drug resistance suggest potential for use of these agents in cancer therapy.

Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as αvβ3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing αvβ3 and to cultured tumor cells which express αvβ3. Methods Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with 99m Tc and tested for binding to cells in vitro. Results 99m Tc-Kistrin, flavoridin and VLO4 had the highest binding, 99m Tc-echistatin had moderate binding, and 99m Tc-bitistatin and 99m Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to αvβ3 to various extents: echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, 99m Tc-kistrin was not displaced by competing peptide or plasma proteins. Conclusions These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting αvβ3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting αvβ3 on vasculature because they bind strongly to surface receptors and are not readily displaced.

A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by performing in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore, the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal human fibroblasts suggesting that these compounds are tumor-specific.

Synthesis of coumarin 3-(N-aryl) sulfonamides was accomplished either by Knoevenagel condensation of anilinosulfonylacetic acids with suitable salicylaldehydes or by the reaction of methyl anilinosulfonylacetates with substituted salicylaldehydes in presence of a catalytic amount of a base. All the compounds tested for antiproliferative activity in different cancer cell lines have shown GI(50) values less than 100 microM.

Introduction: The goal of this study was to develop a targeted therapy for triple negative breast cancer (TNBC) since these tumors do not respond to hormonal-related therapies or medications that target HER2. TNBC is typically treated with a combination of surgery, radiation, and chemotherapy albeit with worse outcome than patients with breast cancers of other subtypes. We have previously shown that GBO-006-1 (ON 1231320) is a highly selective, ATP competitive PLK2 inhibitor, which induces irreversible mitotic arrest of triple negative breast cancer (TNBC) cell lines and results in their apoptotic death. The goal of our current study was to characterize the drug-like and ADME properties of this molecule, and to optimize the in vivo exposure for preclinical evaluation. Experimental procedures: Drug-like properties were characterized by determining the pH solubility profile, log D, oral absorption potential using PAMPA, metabolic stability using rat and liver microsomes, plasma protein binding, and the inhibitory effect on various CYP isozymes. GBO-006-1 has weakly ionizable groups and did not show any improvement of solubility at various pH values. To overcome this limitation, we developed a co-solvent-based formulation that increased the solubility of the compound. A preclinical formulation was subsequently developed that allowed us to perform PK studies in mice, rats (IV, PO & IP) and dogs (IV) and to conduct efficacy studies with GBO-006-1 as a single agent (10, 30 and 75 mg/kg) and in combination using nude mouse xenograft models. In vitro safety pharmacology was assessed by performing hERG (patch clamp), and genotoxic potential was evaluated using the Ames test. Summary: The solubility of GBO-006-1 was enhanced using a co-solvent approach, which permitted the determination of ADME properties. The solution formulation provided good exposure and bioavailability upon IP administration (41% bioavailability at 3mg/Kg) and dose dependency up to 100 mg/kg. The compound showed moderate to high in vivo clearance in rat and mouse, respectively, and has high plasma protein binding and volume of distribution, with no CYP or hERG inhibition. Exploratory non-GLP toxicity studies are currently ongoing in three species, along with other safety pharmacological studies and ex vivo surrogate biomarker analysis. Conclusion: We were successful in developing a formulation forGBO-006-1, which allowed for parenteral administration. GBO-006-1 showed linear dose escalation in mice that translated into marked efficacy in a triple negative breast cancer xenograft model. Initial safety profiling suggested no hERG inhibition. IND directed studies are currently being conducted to facilitate Phase 1 clinical trials in the near future. Citation Format: ARNAB ROYCHOWDHURY, ATHISAYAMANI JEYARAJ DURAISWAMY, SRINIVASARAO MADDI, CHANDRA DEB, SAYAN MITRA, RAMANA REDDY, MANOJ MANIAR, SHASHIDHAR JATIANI, STEPHEN C. COSENZA, AMOL PADGAONKAR, PREMKUMAR REDDY. Pharmacokinetics of PLK2 inhibitor GBO-006-1, developed as a novel first-in-class molecule to treat triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4537. doi:10.1158/1538-7445.AM2014-4537

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against Mantle Cell Lymphomas (MCLs) both in vitro and in vivo. Mantle cell lymphoma is genetically characterized by the t(11;14)(q13;q32) chromosomal translocation which results in constitutive overexpression of cyclin D1. In addition, MCLs also activate other pathways, including aberrant B-Cell Receptor and PI3K/AKT/mTOR signaling. As a result, MCL has a poor clinical outcome with a median survival of 4-5 years. In this study, we show that ON123300, which inhibits both CDK4/6 and PI3K-α (the predominant PI3K catalytic subunit expressed in MCL cells), is a superior inducer of apoptosis of MCL cells when compared to PD0332991, a selective inhibitor of CDK4/6 kinases. Experimental Procedures: We examined the effects of PD 0332991 and ON123300 on cell cycle progression, modulation of the Rb and PI3K/AKT pathways, and the induction of apoptosis in the Granta 519 and Z138C mantle cell lymphoma cell lines. When Granta 519 and Z138C cells were incubated with increasing concentrations of PD 0332991 and ON 123300, both compounds efficienty inhibited the phosphorylation of the Rb family of proteins. However, ON123300 showed concentration-dependent inhibition of MTOR, AKT, 4EBP1 and S6RB phosphorylation while PD 0332991 had no effect on the phosphorylation status of these proteins. While cells treated with PD 0332991 rapidly accumulated in the G0/G1 stage of cell cycle, cells treated with ON123300 showed an accumulation of cells with a sub-G1 DNA content. These ON123300 treated cells showed cleavage of PARP as well as Caspases 3, 7 and 9 and inhibition of FOXO1 phosphorylation, which was not observed in cells treated with PD 0332991. We tested the effects of ON 123300 in nude mouse xenograft assays using Z138 MCL cells. These studies revealed a strong inhibition of tumor growth when tumor-bearing mice were treated daily with 100 mg/kg of ON123300. In addition, there was little evidence of toxicity as measured by change in the body weight in ON123300-treated mice. Conclusions: ON123300 targets the CyclinD/CDK/Rb pathway as well as the PI3K/AKT/MTOR pathway to induce apoptosis of MCL cells via intrinsic apoptotic pathways. Mouse xenograft assays show that ON 123300 is a strong inhibitor of MCL tumor growth in vivo. This dual activity against Rb and AKT pathways appears to be an effective therapeutic strategy for the treatment of MCL. Citation Format: E. Premkumar Reddy, Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Balaiah Akula, Samir Parekh. Targeting of cyclin D/Rb/E2F and PI3K/AKT/MTOR pathways with ON 123300 as a therapeutic strategy for mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2014-4519

e13562 Background: The signal transducer and activator of transcription (STAT) family of proteins are important intracellular mediators. Two of these family members, STAT3 and STAT5, have been foun...

Protein kinase CK2 (Casein kinase 2) is a highly conserved, constitutively active Ser/Thr protein kinase implicated in cellular transformation and tumorigenesis. CK2 regulates multiple oncogenic pathways involved in cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA repair. Unlike other signaling molecules such as PI3K, RAF, RAS, where genetic alterations lead to deregulated signaling pathways, in the case of CK2, only its high expression levels have been associated with a disease state and no mutations have been found to date. Here we describe the mechanism of action of ON108600 a potent small molecule inhibitor of both catalytic subunits of protein kinase CK2. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple cancer cell lines while having little or no effect on normal cells. CK2 regulates Akt signaling through phosphorylation and inactivation of PTEN and via direct and specific phosphorylation of Akt at S129. Treatment with ON108600 resulted in dramatic reductions of phosphorylation of PTEN and Akt S129 and downstream targets of Akt in multiple cancer cell lines. Further, phosphorylation of p21Cip1/Waf1 at Thr145 and its cytoplasmic localization has been shown to be a poor prognostic marker in breast cancer. ON108600 showed a dose and time dependant dephosphorylation of p21 at Thr145 and induced a potent mitotic cell cycle arrest in most cancer cell lines. CK2 has also been implicated in regulating Caspase signaling via phosphorylation of caspase cleavage sites and thus exerting its pro-survival effects. ON108600 treatment induced potent apoptosis by activating the Caspase 3/7 signaling cascade. To understand the structural basis of CK2 inhibition by ON108600 we performed x-ray crystallographic studies of ON108600-CK2. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of GTP in the CK2 active site. Structural studies also revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit which is known to interact with the β-regulatory subunit of CK2 and critical for substrate recognition and activation. CK2α has been reported to be a structural microtubule associated protein and the CK2 holoenzyme has been shown to regulate microtubule dynamics. Interestingly, ON108600 was found to inhibit CK2 holoenzyme mediated polymerization of β-tubulins in in-vitro assays. CK2 has emerged as novel druggable target and selective inhibitors of CK2 such as ON108600 may prove as a potential anti-cancer therapy. Our ongoing studies are focused towards identifying novel combinations of ON108600 with existing chemotherapeutic agents in multiple myeloma, prostate and breast cancer. Citation Format: Amol Padgaonkar, Olga Rechkoblit, Stephen Cosenza, Venkat R. Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, Aneel Aggarwal, E Premkumar Reddy. Discovery and biological characterization of ON108600, a small molecule inhibitor of protein kinase CK2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2013-3239 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Development of radio-protective agents that are non-toxic is critical in light of ever increasing threats associated with proliferation of nuclear materials, terrorism and occupational risks associated with medical and space exploration. In this communication, we describe the discovery, characterization and mechanism of action of ON01210.Na, which effectively protects mouse and human bone marrow cells from radiation-induced damage both in vitro and in vivo. Our results show that treatment of normal fibroblasts with ON01210.Na before and after exposure to ionizing radiation provides dose dependent protection against radiation-induced damage. Treatment of mice with ON01210.Na prior to radiation exposure was found to result in a more rapid recovery of their hematopoietic system. The mechanistic studies described here show that ON01210.Na manifests its protective effects through the up-regulation of PI3-Kinase/AKT pathways in cells exposed to radiation. These results suggest that ON 01210.Na is a safe and effective radioprotectant and could be a novel agent for use in radiobiological disasters.

A stereoselective and efficient method for free radical addition of benzyl thiol to aryl acetylene in the presence of Et3B-hexane has been developed for the synthesis of (Z) and (E)-styryl benzyl sulfides where base catalyzed hydrothiolations have failed. The scope of this reaction was successfully extended for the synthesis of (E)-ON 01910·Na, a phase III clinical stage anti-cancer agent and its inactive geometrical isomer (Z)-ON 01910·Na. It is interesting to note that all the E-isomers synthesized have shown better cytotoxicity profile on cancer cells compared to the Z-isomers.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor cell resistance towards chemotherapeutic agents is thought to arise through a number of different mechanisms, induced by prior exposure, activation of intrinsic resistance mechanisms or a combination of both and is a major problem in oncology. [ON013105][1] is a novel compound with a potent anti-tumor activity against both solid and leukemic tumor cell lines. This compound was found to induce mitotic arrest of tumor cells leading to activation of caspase activity and apoptotic death. This compound is currently in phase I clinical trials for mantle cell lymphoma therapy. While studying the kinetics of cell death induced by [ON013105][1] (phosphate prodrug) and [ON013100][2] (free acid), we identified a colorectal cancer cell line that was 1000 fold resistant to both forms of the compound. Studies were undertaken to identify the mechanism of resistance. We first sought to determine the frequency and degree of resistance by testing a large panel of cell lines from a variety of tumor types. We found that 4 out of 9 colon tumor lines were resistant while all other tumor cell types were sensitive. This led us to explore the possibility of metabolism and/or transport mechanisms that could result in drug resistance. The compounds are not substrates for the MDR-1 pumps, but transcriptome and protein expression studies identified a variety of UDP-glucuronosyltransferases (UGTs) expressed in the resistant cell types. Cross resistance studies with compounds that are known UGT substrates showed that only combretastatin A4 cell killing activity correlated with sensitivity towards our compounds. Uptake studies showed that sensitive cells retained [ON013100][2] while [ON013100][2] was rapidly removed in resistant cells. [ON013100][2]-glucuronide was found to be a metabolite (571 Da) in both the cell pellet and the medium and its formation correlated with cell sensitivity. In cell pellet, (a) from 0 to 2 h, metabolite quantities (in terms of peak intensities) were 50 to 250 times higher in HT-29 (resistant) than in HCT-15 (sensitive); (b) from 2 to 24 h, rapidly decreased to baseline; (c) metabolite quantities in HCT-15 were essentially zero with very small amount (peak intensities) detected from 0 to 4 h. We could also show specific competition by known acyl glycones whose treatment resulted in reversal of resistance. The recent identification of glucuronidation of combretastatin A4 by UGT1A9 also points to UGT activity as the mechanism of resistance. We find it interesting that both of these compounds are agents that interact with tubulins. Identification of the UGT isoforms responsible for [ON013100][2] glucuronidation demonstrate the possibility of intrinsic resistance due to UGT activity of colon tumors and should be considered as a clinically important factor when determining possible therapies that include tubulin poisons which are also UGT substrates. 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 695. doi:10.1158/1538-7445.AM2011-695 [1]: /lookup/external-ref?link_type=GEN&access_num=ON013105&atom=%2Fcanres%2F71%2F8_Supplement%2F695.atom [2]: /lookup/external-ref?link_type=GEN&access_num=ON013100&atom=%2Fcanres%2F71%2F8_Supplement%2F695.atom

Oncology drug discovery is constantly evolving by integrating new basic findings with established developmental practice. Two drug discovery platforms currently exist. One is an extension of basic research, rational drug design. Its alternative, one based on cytotoxicity, which qualifies leads by their ability to selectively kill cancer cells. Using this cytotoxicity based approach we recently discovered a novel synthetic small molecule we have called ON015640. This compound is highly effective at killing 15 different cancer cell lines with IC50 ranges from 20-90 nanomolar. The predominant phenotype associated with this compound is that of cellular rounding and detachment from substratum, a similar phenomenon to that observed during treatment of cultured cells with mitotic inhibitors like Taxol and Nocodazole. Using FACS, molecular mitotic indexing, and fluorescent microscopy we have subsequently demonstrated that ON015640 induces mitotic arrest and aberrant spindle formation. This mitotic arrest and spindle disruption can be attributed to this molecule's ability to depolymerize tubulin both in vitro and in vivo in a similar manner to Nocodazole. At effective concentrations this compound commences a program of mitotic catastrophe subsequently initiating effector caspases 3/7 and cleavage of Poly ADP Ribose Polymerase, primary steps in the activation of the intrinsic apoptotic pathway. Unlike classical tubulin depolymerizing agents, ON015640 is not a substrate for the ATP binding cassette family of efflux pumps and is capable of killing Multi Drug Resistant cell lines. This characteristic and the compound's nanomolar activity make it an attractive candidate for further development and characterization. To date, the exact site where ON015640 binds to tubulin remains unknown. Additionally, the pharmacokinetics and efficacy of this molecule have yet to be fully characterized. Thus, current and future efforts will be focused on establishing the dynamics by which ON015640 interacts with tubulin, while also developing this molecule into a clinically relevant drug. 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 4524. doi:10.1158/1538-7445.AM2011-4524

The Polo like kinases play key roles in mitosis. While the upregulation of PLK1 in cancer is well documented and PLK3 has been demonstrated to be a tumor suppressor, little is known about the oncogenic significance of PLK2. PLK2 kinase activity is essential for centriolar duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. Protein tyrosine hyper-phosphorylation and the upregulation of serine/threonine kinases associated with cell cycle progression have been linked to the etiology of cancer. In our attempt to develop ATP-mimetic compounds that are cytotoxic against a panel of cancer cell lines, we identified several sulfonyl pyridopyrimidines that possess nanomolar activity. The most potent of these compounds, ON1231320, was found to be a specific PLK2 inhibitor when profiled against a panel of 288 wild-type, 55 mutant and 12 special kinases. Further in vitro testing revealed that ON1231320 is a selective inhibitor of PLK2 with no inhibitory activity against PLK1, PLK3 and PLK4. Confirmation of this interaction and its selectivity was obtained from pull down studies using lysates of tumor cells and a biotinylated analog of ON1231320. The cytotoxic effect of the drug is mediated by apoptosis as evidenced by the induction of Caspase 3/7 activity and by the cleavage of PARP in a dose dependent manner. ON1231320 affects cell cycle progression by blocking tumor cells in the G2/M phase however it does not affect normal human fibroblasts. ON123120 exhibits an excellent safety profile with no overt signs of toxicity, no loss of body weight and 100% survival in mice given a single peritoneal dose of 200 mg/kg. Our ongoing efforts include efficacy studies in nude mouse models, identification of the structural determinants of the interaction between ON1231320 and PLK2 by computational and crystallographic methods and the identification of novel PLK2 substrates to elucidate its role in cancer biology. 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 643. doi:10.1158/1538-7445.AM2011-643

Abstract 3827 Poster Board III-763 Background ON 01910.Na, a small molecule multikinase inhibitor, promotes G2/M arrest and apoptosis. Key targets for this inhibitor include Plk1 (polo-like kinase, a cell cycle regulator), Cdk1, (cyclin dependent kinase, a mitotic regulator) and the PI-3 kinase pathway (Ramana Reddy et al. J. Med. Chem. 2008, Park et al, Oncogene, 2007, Gumireddy et al., Cancer Cell, 2005). The drug has been shown to have anti-tumor activity in in vitro and in vivo models. Phase I studies in >100 advanced cancer patients revealed that the drug is well tolerated. Further, in several ongoing Phase 1 clinical trials in patients with myelodysplastic syndromes (MDS), positive effects on hematological indicators have been noted (Sloand et al, ASH 2008). Based on these data, a Phase 2 single-arm study is in progress to assess the efficacy and safety of the drug in IPSS Intermediate-2 and High risk MDS patients. Single Cell Network Profiling (SCNP) using flow cytometry is a platform that measures multiple fluorescent parameters (up to 10) in each cell, including both surface markers and intracellular signaling proteins in response to extracellular network inputs. By simultaneously measuring the effects of drug exposure on several pathways within each cell type in a heterogeneous patient tissue sample, valuable data can be gained about drug interactions with specific cellular pathways and cell type selectivity. This information has potential implications for dose/schedule optimization and development of patient stratification biomarkers. Objectives Studies were designed to evaluate the in vitro effects of ON 01910.Na, at clinically relevant concentrations, on intracellular pathways in the human GM-CSF-dependent erythroblastic TF-1 cell line using SCNP in order to monitor transitional changes in the cell cycle, with a focus on the G2-M phase and to perform dose-dependent titrations of drug using these cell cycle readouts. Methods The reagents chosen to measure cell cycle readouts were fluorochrome-conjugated antibodies that recognize cyclin B1, p-histone H3(S28) and p-Cdk1(Y15) and 4'6'-diamino-2-phenylindole (DAPI), a fluorescent dye that binds strongly to DNA. The phosphorylation status of p-histone H3(S28) and p-Cdk1(Y15), and the level of cyclin B1 expression are all determinants of the G2-M and/or M phase of the cell cycle. Dose dependent titrations of ON 01910.Na and its inactive analog ON 01911 were performed over a dose range starting at 10-5 M and decreasing to 10-10 M (dose range which includes pharmacologically achievable concentrations in humans) with 3-fold serial dilutions for eleven points after an exposure to the drug for either 24 or 48 hrs. Cells were processed for multiparameter flow cytometry by fixation, permeabilization and incubation with fluorchrome-conjugated antibodies. Results The data showed that at 24 hours after ON 01910.Na exposure there was a simultaneous increase in phosphorylation of histone H3(S28), a decrease in phosphorylation of Cdk-1(Y15), and accumulation of cyclin B1. These data suggest that ON 01910 exposure disrupted the G2/M cell cycle transition leading to mitotic arrest with subsequent apoptosis. TF-1 cell DNA content measured by DAPI verified this to be the case as increases in G2/M and sub-G1 (a measure of apoptotic cell death) were simultaneously observed. No significant effects on G2/M targets were observed when TF-1 cells were exposed to ON 01911, indicating the effects of ON 01910.Na on the cell cycle were specific to the drug. Maximal effects of ON-01910.Na on cell cycle signaling molecules were observed at a drug concentration of 0.37 mM and no further changes were seen at higher concentrations. These effects were also observed at 48 hours, although with more cell death. Conclusions These data indicate that intracellular phosphorylation changes of histone H3(S28) and Cdk-1(Y15), in addition to accumulation of cyclin B1 with subsequent apoptosis, reflect possible mechanisms of action of ON 01910.Na. The assay will be used in ongoing clinical trials to measure the pharmacodynamic activity of the drug in MDS patient samples pre- and post-treatment. Disclosures: Soper: Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Wilhelm:Onconova Therapeutics Inc: Employment. Cosenza:Onconova Therapeutics Inc.: Consultancy. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Cesano:Nodality Inc.: Employment, Equity Ownership. Greenberg:Nodality Inc.: Research Funding; Onconova Therapeutics Inc.: Research Funding. Fantl:Nodality, Inc.: Employment, Equity Ownership.

2515 Background: ON 01910.Na induces G2/M cell cycle arrest, apoptosis, and cell death in a broad spectrum of cancer cells, but not in non-neoplastic cells. In vitro, cell killing is dependent on d...

3569 Background: Plk1 is a key mitotic regulator of the transition through the G2/M checkpoint in the cell cycle. This work aimed to evaluate the activity of ON 01910.Na, a Plk1 pathway modulator, in in vitro and in vivo models of pancreatic cancer (PaCa) and to discover biomarkers predictive of efficacy. Methods: ON 01910.Na was tested in 12 PaCa cell lines. Studies assessing Plk1 related markers were conducted to identify biomarkers. For validation a live collection of PaCa xenografts from fresh tumor samples obtained at the time of surgical resection was used (PancXenoBank). The ex vivo assay was based on fine-needle aspirate (FNA) biopsies. Results: ON 01910.Na showed equal activity to gemcitabine against PaCa cell lines. The activity of ON 01910.Na correlated with suppression of two downstream mediators of PLK1, CDC25C and cyclin B1 (by mRNA and protein). ON 01910.Na was tested in xenografts from representative pancreatic cell lines. The selected markers were evaluated in an ex vivo assay, using intra-tumor pharmacokinetics to select the dose of the assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Knockdown of cyclin B1 by siRNA had no effect per se or in the response of the resistant MiaPaca2 to either of the drugs. We next used the ex vivo assay to profile ten patient-derived cases from the PancXenoBank. Two cases were catalogued as potential responders. From each of these ten cases, a group of mice bearing at least 20 tumors received vehicle or ON 01910.Na for 28 days. There was a correlation between the ex vivo cyclin B1 assay and the sensitivity to the tested agent, as the 2 cases prospectively identified as sensitive met pre-specified criteria for response. Of the 8 tumors predicted to be resistant, only one was sensitive. In IHC testing cases showing ex vivo cyclin B1 down-regulation had also decreases in cyclin B1 protein, and there was a correlation between activity and IHC changes in cyclin B1. Conclusions: ON 01910.Na demonstrated significant activity in a preclinical model of PaCa. A rationally designed ex vivo cyclin B1-based assay not only identified cases sensitive to ON 01910.Na, but also replicated the pharmacodynamic events occurring after in vivo exposure. No significant financial relationships to disclose.