Suman Kambhampati

Jak family of kinases in cancer.

The family of Jak kinases is composed from at least four different tyrosine kinases (Tyk2, Jak1, Jak2, Jak3) that share significant structural homology with each other. The members of this family of kinases associate constitutively with a variety of cytokine and hormone receptors. Upon binding of the specific ligands to their receptors, Jak kinases are rapidly activated and their kinase activities induced, to regulate tyrosine phosphorylation of various effectors and initiate activation of downstream signaling pathways. The discovery of this family of tyrosine kinases dates back in the early 1990s with the cloning of the Tyk-2 tyrosine kinase as a critical element of the Type I interferon signaling pathway. Extensive work over the last few years has provided evidence that Jak kinases play important roles in the generation of responses for interferons, which are cytokines that exhibit important antitumor activities. There is also accumulating evidence that constitutive activation of different Jaks and Stats mediates neoplastic transformation and promotes abnormal cell proliferation in various malignancies. This review summarizes the role of various Jak-kinase dependent pathways in malignancies and discusses the therapeutic implications of the recent advances in the field.

Activation of Protein Kinase Cδ by IFN-γ

Engagement of the type II IFN (IFN-γ) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-γ-activated site (GAS) elements in the promoters of IFN-γ-inducible genes. An important event in IFN-γ-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3′-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3′-kinase and is engaged in IFN-γ signaling. Our data demonstrate that PKCδ is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKCδ associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKCδ activity diminishes phosphorylation of Stat1 on Ser727 and IFN-γ-dependent transcriptional regulation via IFN-γ-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKCδ is activated during engagement of the IFN-γ receptor and plays an important role in IFN-γ signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-γ-stimulated genes.

Activation of Protein Kinase Cδ by All-trans-retinoic Acid

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.

Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells, providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-beta-mediated gene activation in BM stromal cells, and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

Cutting Edge: Activation of the p38 Mitogen-Activated Protein Kinase Signaling Pathway Mediates Cytokine-Induced Hemopoietic Suppression in Aplastic Anemia

Myelosuppressive cytokines, in particular IFN-γ and TNF-α, play an important role in the pathogenesis of idiopathic aplastic anemia in humans. It is unknown whether these negative regulators of hemopoiesis suppress stem cells by activating a common signaling cascade or via distinct nonoverlapping pathways. In this study, we provide evidence that a common element in signaling for IFN-γ and TNF-α in human hemopoietic progenitors is the p38/MapKapK-2 signaling cascade. Our studies indicate that pharmacological inhibition of p38 reverses the suppressive effects of IFN-γ and TNF-α on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 strongly enhances hemopoietic progenitor colony formation from aplastic anemia bone marrows in vitro. Thus, p38 appears to play a critical role in the pathogenesis of aplastic anemia, suggesting that selective pharmacological inhibitors of this kinase may prove useful in the treatment of aplastic anemia and other cytokine-mediated bone marrow failure syndromes.

Phase II study of the histone deacetylase inhibitor MGCD0103 in patients with previously treated chronic lymphocytic leukaemia

MGCD0103, an orally available class I histone deacetylase (HDAC) inhibitor, was examined for pre‐clinical activity in chronic lymphocytic leukaemia (CLL). A phase II clinical trial was performed, starting at a dose of 85 mg/d, three times per week. Dose escalation to 110 mg or the addition of rituximab was permitted in patients without a response after two or more cycles. MGCD0103 demonstrated pre‐clinical activity against CLL cells with a LC50 (concentration lethal to 50%) of 0·23 μmol/l and increased acetylation of the HDAC class I specific target histone H3. Twenty‐one patients received a median of two cycles of MGCD0103 (range, 0–12). All patients had previously received fludarabine, 33% were fludarabine refractory, and 71% had del(11q22·3) or del(17p13·1). No responses according to the National Cancer Institutes 1996 criteria were observed. Three patients received 110 mg and four patients received concomitant rituximab, with no improvement in response. Grade 3–4 toxicity consisted of infections, thrombocytopenia, anaemia, diarrhoea, and fatigue. HDAC inhibition was observed in six out of nine patients on day 8. Limited activity was observed with single agent MGCD0103 in high risk patients with CLL. Future investigations in CLL should focus on broad HDAC inhibition, combination strategies, and approaches to diminish constitutional symptoms associated with this class of drugs.

Activation of protein kinase C-delta (PKC-delta) by All-trans-retinoic acid

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.

Retinoic acid signaling pathways in development and diseases.

Retinoids comprise a group of compounds each composed of three basic parts: a trimethylated cyclohexene ring that is a bulky hydrophobic group, a conjugated tetraene side chain that functions as a linker unit, and a polar carbon-oxygen functional group. Biochemical conversion of carotenoid or other retinoids to retinoic acid (RA) is essential for normal regulation of a wide range of biological processes including development, differentiation, proliferation, and apoptosis. Retinoids regulate various physiological outputs by binding to nuclear receptors called retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which themselves are DNA-binding transcriptional regulators. The functional response of RA and their receptors are modulated by a host of coactivators and corepressors. Retinoids are essential in the development and function of several organ systems; however, deregulated retinoid signaling can contribute to serious diseases. Several natural and synthetic retinoids are in clinical use or undergoing trials for treating specific diseases including cancer. In this review, we provide a broad overview on the importance of retinoids in development and various diseases, highlighting various retinoids in the drug discovery process, ranging all the way from retinoid chemistry to clinical uses and imaging.

Boron chemicals in diagnosis and therapeutics

Advances in the field of boron chemistry have expanded the application of boron from material use to medicine. Boron-based drugs represent a new class of molecules that possess several biomedical applications including use as imaging agents for both optical and nuclear imaging as well as therapeutic agents with anticancer, antiviral, antibacterial, antifungal and other disease-specific activities. For example, bortezomib (Velcade(®)), the only drug in clinical use with boron as an active element, was approved in 2003 as a proteasome inhibitor for the treatment of multiple myeloma and non-Hodgkins lymphoma. Several other boron-based compounds are in various phases of clinical trials, which illustrates the promise of this approach for medicinal chemists working in the area of boron chemistry. It is expected that in the near future, several boron-containing drugs should become available in the market with better efficacy and potency than existing drugs. This article discusses the current status of the development of boron-based compounds as diagnostic and therapeutic agents in humans.

CCN5/WISP-2 expression in breast adenocarcinoma is associated with less frequent progression of the disease and suppresses the invasive phenotypes of tumor cells.

Although previous in vitro studies predicted that CCN5/WISP-2 may act as an anti-invasive gene in breast cancer, the distribution pattern of CCN5 in breast cancer samples is conflicting. Thus, we systematically investigated the CCN5 expression profile in noninvasive and invasive breast tumor samples and its functional relevance in breast cancer progression. The studies showed that CCN5 expression is biphasic, such that in normal samples CCN5 expression is undetectable, whereas its expression is markedly increased in noninvasive breast lesions, including atypical ductal hyperplasia and ductal carcinoma in situ. Further, CCN5 mRNA and protein levels are significantly reduced as the cancer progresses from a noninvasive to invasive type. Additionally, we showed that CCN5 mRNA and protein level was almost undetectable in poorly differentiated cancers compared with the moderately or well-differentiated samples and its expression inversely correlated with lymph node positivity. The result was further supported by evaluating the RNA expression profile in microdissected sections using real-time PCR analysis. Therefore, our data suggest a protective function of CCN5 in noninvasive breast tumor cells. This hypothesis was further supported by our in vitro studies illuminating that CCN5 is a negative regulator of migration and invasion of breast cancer cells, and these events could be regulated by CCN5 through the modulation of the expression of genes essential for an invasive front. These include Snail-E-cadherin signaling and matrix metalloproteinase (MMP)-9 and MMP-2. Collectively, these studies suggest that the protective effect of CCN5 in breast cancer progression may have important therapeutic implications.