A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

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.

A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent.

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that 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. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.

B-myb is an essential regulator of hematopoietic stem cell and myeloid progenitor cell development.

The B-myb (MYBL2) gene is a member of the MYB family of transcription factors and is involved in cell cycle regulation, DNA replication, and maintenance of genomic integrity. However, its function during adult development and hematopoiesis is unknown. We show here that conditional inactivation of B-myb in vivo results in depletion of the hematopoietic stem cell (HSC) pool, leading to profound reductions in mature lymphoid, erythroid, and myeloid cells. This defect is autonomous to the bone marrow and is first evident in stem cells, which accumulate in the S and G2/M phases. B-myb inactivation also causes defects in the myeloid progenitor compartment, consisting of depletion of common myeloid progenitors but relative sparing of granulocyte-macrophage progenitors. Microarray studies indicate that B-myb-null LSK(+) cells differentially express genes that direct myeloid lineage development and commitment, suggesting that B-myb is a key player in controlling cell fate. Collectively, these studies demonstrate that B-myb is essential for HSC and progenitor maintenance and survival during hematopoiesis.

ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent.

Elevated expression of polo-like kinase1 (Plk1) has been reported in many human tumors, and inhibition of Plk1 activity results in their mitotic arrest and apoptosis. Here we describe the profile of ON01910, a small molecule inhibitor of Plk1 activity, which induces mitotic arrest of tumor cells characterized by spindle abnormalities leading to their apoptosis. This compound was not ATP-competitive, but competed for the substrate binding site of the enzyme. In vivo, this compound did not exhibit hematotoxicity, liver damage, or neurotoxicity, and was a potent inhibitor of tumor growth in a variety of xenograft nude mouse models. ON01910 showed strong synergy with several chemotherapeutic agents, often inducing complete regression of tumors.

CDK4: a master regulator of the cell cycle and its role in cancer.

The cell cycle is regulated in part by cyclins and their associated serine/threonine cyclin-dependent kinases, or CDKs. CDK4, in conjunction with the D-type cyclins, mediates progression through the G1 phase when the cell prepares to initiate DNA synthesis. Although Cdk4-null mutant mice are viable and cell proliferation is not significantly affected in vitro due to compensatory roles played by other CDKs, this gene plays a key role in mammalian development and cancer. This review discusses the role that CDK4 plays in cell cycle control, normal development and tumorigenesis as well as the current status and utility of approved small molecule CDK4/6 inhibitors that are currently being used as cancer therapeutics.

TGFß reprograms TNF stimulation of macrophages towards a non-canonical pathway driving inflammatory osteoclastogenesis.

It is well-established that receptor activator of NF-κB ligand (RANKL) is the inducer of physiological osteoclast differentiation. However, the specific drivers and mechanisms driving inflammatory osteoclast differentiation under pathological conditions remain obscure. This is especially true given that inflammatory cytokines such as tumor necrosis factor (TNF) demonstrate little to no ability to directly drive osteoclast differentiation. Here, we found that transforming growth factor ß (TGFß) priming enables TNF to effectively induce osteoclastogenesis, independently of the canonical RANKL pathway. Lack of TGFß signaling in macrophages suppresses inflammatory, but not basal, osteoclastogenesis and bone resorption in vivo. Mechanistically, TGFß priming reprograms the macrophage response to TNF by remodeling chromatin accessibility and histone modifications, and enables TNF to induce a previously unrecognized non-canonical osteoclastogenic program, which includes suppression of the TNF-induced IRF1-IFNß-IFN-stimulated-gene axis, IRF8 degradation and B-Myb induction. These mechanisms are active in rheumatoid arthritis, in which TGFß level is elevated and correlates with osteoclast activity. Our findings identify a TGFß/TNF-driven inflammatory osteoclastogenic program, and may lead to development of selective treatments for inflammatory osteolysis.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease.

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.

p89c-Myb is not required for fetal or adult hematopoiesis.

The c-myb gene encodes two proteins, termed p75 and p89. Of these, the larger isoform is transcribed from an alternatively spliced message that contains an additional exon, exon 9A. Disruption of the c-myb locus in mice results in embryonic lethality due to defective hematopoiesis and in the adult, tissue-specific inactivation of c-myb in hematopoietic tissues blocks differentiation along several lineages. The c-myb knock-out mouse models described thus far result in the disruption of both the p75 and p89 isoforms, making it impossible to assign a definitive role to p89(c-Myb) in development and hematopoiesis. We have therefore generated a null-mutant mouse where exon 9A has been systemically deleted that results in the absence of only the p89-myb transcript and protein. Unlike disruption of both forms of the c-myb gene, loss of only the p89-encoding isoform does not have any deleterious effects on mammalian hematopoiesis and development.

Rigosertib ameliorates the effects of oncogenic KRAS signaling in a murine model of myeloproliferative neoplasia.

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.

Targeting protein kinase CK2 and CDK4/6 pathways with a multi-kinase inhibitor ON108110 suppresses pro-survival signaling and growth in mantle cell lymphoma and T-acute lymphoblastic leukemia.

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.

Molecular mechanisms associated with the regulation of apoptosis by the two alternatively spliced products of c-Myb.

The c-myb proto-oncogene encodes two alternatively spliced mRNAs, which in turn code for proteins of 75 kDa and 89 kDa. It is at present unclear whether the two isoforms of c-Myb perform identical functions or whether they mediate different biological effects. To assess their role in apoptotic death of hematopoietic cells, we expressed the two isoforms of c-Myb in the murine myeloid cell lines 32Dcl3 and FDCP1. Our results show that while ectopic overexpression of p75 c-Myb results in the acceleration of cell death, similar overexpression of p89 c-Myb results in the protection of cells from apoptotic death. An analysis of gene expression changes with mouse cDNA expression arrays revealed that while p75 c-Myb blocked the expression of glutathione S-transferase micro mRNA, p89 c-Myb greatly enhanced the expression of this gene. These results were further confirmed by Northern blot analysis. Ectopic overexpression of the glutathione S-transferase micro gene in 32Dcl3 cells resulted in protection of cells from interleukin-3 withdrawal-induced cell death similar to that seen with the ectopic overexpression of p89 c-Myb. These results suggest that the two isoforms of c-Myb differentially regulate apoptotic death of myeloid cells through differential regulation of glutathione S-transferase micro gene expression.

Small unstable apoptotic protein, an apoptosis-associated protein, suppresses proliferation of myeloid cells.

Apoptosis, or programmed cell death, is a process where developmental or environmental stimuli activate a genetic program to implement a series of events that culminate in cell death. To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, representational difference analysis was performed using RNAs derived from 32Dcl3 myeloblastic cells that were proliferating in the presence of IL-3 and cells that were actively undergoing apoptosis as a result of interleukin 3 deprivation for 24 h. This report describes a novel gene [small unstable apoptotic protein (SUAP)] that is up-regulated in these cells after the removal of interleukin 3 and exposure to granulocyte colony stimulating factor. The protein encoded by this gene is a target of the proteasome and does not share homology with other previously characterized proteins. To further define SUAP's role in growth arrest and apoptosis, 32Dcl3 cells that ectopically express SUAP under the control of an inducible promoter were generated and tested for their ability to proliferate under conditions where SUAP expression is induced. These studies show that although the SUAP expressing cells exhibited suppressed proliferation rates, this was not attributable to alterations in cell cycle progression. Rather, SUAP appears to induce the appearance of Annexin V-positive cells, supporting a role for this protein in programmed cell death.

Discovery of 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x) as a potent inhibitor of cyclin-dependent kinase 4 (CDK4) and AMPK-related kinase 5 (ARK5).

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.

CDK4: A Key Player in the Cell Cycle, Development, and Cancer.

The cell cycle is regulated in part by cyclins and their associated serine/threonine cyclin-dependent kinases, or CDKs. CDK4, in conjunction with the D-type cyclins, mediates progression through the G1 phase when the cell prepares to initiate DNA synthesis. Although CDK4-null mutant mice are viable and cell proliferation is not significantly affected in vitro due to compensatory roles played by other CDKs, this gene plays a key role in mammalian development and cancer. This review discusses the role that CDK4 plays in cell cycle control, normal development, and tumorigenesis as well as how small molecule inhibitors of CDK4 can be used to treat disease.

Targeted inhibition of kinases in cancer therapy.

With an understanding of the molecular changes that accompany cell transformation, cancer drug discovery has undergone a dramatic change in the past few years. Whereas most of the emphasis in the past has been placed on developing drugs that induce cell death based on mechanisms that do not discriminate between normal and tumor cells, recent strategies have emphasized targeting specific mechanisms that have gone awry in tumor cells. However, the identification of cancer-associated mutations in oncogenes and their amplification in tumors has suggested that inhibitors against such proteins might represent attractive substrates for targeted therapy. In the clinic, the success of imatinib (Gleevec®, STI571) and trastuzumab (Herceptin®), both firsts of their kind, spurred further development of new, second-generation drugs that target kinases in cancer. This review highlights a few important examples each of these types of therapies, along with some newer agents that are in various stages of development. Second-generation kinase inhibitors aimed at overriding emerging resistance to these therapies are also discussed.

Jak/STAT pathways in cytokine signaling and myeloproliferative disorders: approaches for targeted therapies.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Studies conducted over the past 10 to 15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors, termed STATs (signal transducers and activators of transcription). Aberrations in these pathways, such as those caused by the recently identified JAK2(V617F) mutation and translocations of the JAK2 gene, are underlying causes of leukemias and other myeloproliferative disorders. This review discusses the role of JAK/STAT signaling in normal hematopoiesis as well as genetic abnormalities associated with myeloproliferative and myelodisplastic syndromes. This review also summarizes the status of several small molecule JAK2 inhibitors that are currently at various stages of clinical development. Several of these compounds appear to improve the quality of life of patients with myeloproliferative disorders by palliation of disease-related symptoms. However, to date, these agents do not seem to significantly affect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements, or significantly reduce allele burden. These results suggest the possibility that additional mutational events might be associated with the development of these neoplasms, and indicate the need for combination therapies as the nature and significance of these additional molecular events is better understood.

A Non-ATP-Competitive Dual Inhibitor of JAK2 and BCR-ABL Kinases: Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition.

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.

A non-ATP-competitive inhibitor of BCR-ABL overrides imatinib resistance.

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 <10 nM. Kinetic studies demonstrate that this compound is not ATP-competitive but is substrate-competitive and works synergistically with imatinib in wild-type BCR-ABL inhibition. More importantly, ON012380 was found to induce apoptosis of all of the known imatinib-resistant mutants at concentrations of <10 nM concentration 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.