Erik S. Knudsen

Nucleophosmin/B23 Is a Target of CDK2/Cyclin E in Centrosome Duplication

In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of centrosome duplication.

The history and future of targeting cyclin-dependent kinases in cancer therapy

Cancer represents a pathological manifestation of uncontrolled cell division; therefore, it has long been anticipated that our understanding of the basic principles of cell cycle control would result in effective cancer therapies. In particular, cyclin-dependent kinases (CDKs) that promote transition through the cell cycle were expected to be key therapeutic targets because many tumorigenic events ultimately drive proliferation by impinging on CDK4 or CDK6 complexes in the G1 phase of the cell cycle. Moreover, perturbations in chromosomal stability and aspects of S phase and G2/M control mediated by CDK2 and CDK1 are pivotal tumorigenic events. Translating this knowledge into successful clinical development of CDK inhibitors has historically been challenging, and numerous CDK inhibitors have demonstrated disappointing results in clinical trials. Here, we review the biology of CDKs, the rationale for therapeutically targeting discrete kinase complexes and historical clinical results of CDK inhibitors. We also discuss how CDK inhibitors with high selectivity (particularly for both CDK4 and CDK6), in combination with patient stratification, have resulted in more substantial clinical activity.

Aromatic Hydrocarbon Receptor Interaction with the Retinoblastoma Protein Potentiates Repression of E2F-dependent Transcription and Cell Cycle Arrest

Polyhalogenated aromatic hydrocarbons, of which 2,3,7,8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR·RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.

Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation.

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (i) the A/B pocket, which binds proteins with the LXCXE motif; (ii) the C pocket, which binds the c-Abl tyrosine kinase; and (iii) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. We have previously shown that LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). In this report, we show that the E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB.

Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

Tailoring to RB: tumour suppressor status and therapeutic response

The retinoblastoma tumour suppressor (RB) is a crucial regulator of cell-cycle progression that is invoked in response to a myriad of anti-mitogenic signals. It has been hypothesized that perturbations of the RB pathway confer a synonymous proliferative advantage to tumour cells; however, recent findings demonstrate context-specific outcomes associated with such lesions. Particularly, loss of RB function is associated with differential response to wide-ranging therapeutic agents. Thus, the status of this tumour suppressor may be particularly informative in directing treatment regimens.

RB-Dependent S-Phase Response to DNA Damage

ABSTRACT The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G1/S transition. RB plays an essential role in the G1 arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb+/+ but not in Rb−/− mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21Cip1-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G1 and in S phase.

The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.

Ketones and lactate increase cancer cell “stemness”, driving recurrence, metastasis and poor clinical outcome in breast cancer: Achieving personalized medicine via metabolo-genomics

Previously, we showed that high-energy metabolites (lactate and ketones) “fuel” tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with “stemness,” including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced “gene signatures” were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the “cancer stem cell” phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific “classical” gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and “mitochondrial poison” metformin. Finally, we propose that this new approach to personalized cancer medicine be termed “Metabolo-Genomics,” which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.

Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure.

A hallmark of cancer is the deregulation of cell-cycle machinery, ultimately facilitating aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 has a crucial role in the development of disease. Recently, a highly specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast cancer. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated on a panel of breast cancer cell lines. These analyses showed that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, ultimately, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies showed a highly specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F, and the ability to attenuate CDK2 activity. Acquired resistance to PD-03322991 was specifically associated with attenuation of CDK2 inhibitors, indicating that redundancy in CDK functions represents a determinant of therapeutic failure. Despite these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell-cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic.