Cyclin D1 sensitizes myeloma cells to endoplasmic reticulum stress-mediated apoptosis by activating the unfolded protein response pathway.

BACKGROUND: Cyclin D1 and its kinase partners control cell cycle progression. Cyclin D1 is frequently deregulated in various cancers, including malignant hemopathies, and tumor cells display uncontrolled cell proliferation. Cyclin D1 is not expressed in the B-cell lineage but is found in multiple myeloma (MM) cells in almost 50% of patients with this condition. Paradoxically, cyclin D1 expression is associated with a good prognosis and longer overall survival in MM patients. METHODS: We used two independent MM cell lines (RPMI 8226 and LP1) to generate several clones stably expressing either the green fluorescent protein (GFP) or a GFP-cyclin D1 fusion protein, and we analyzed the properties acquired following cyclin D1 expression. RESULTS: Whole-genome expression analysis in the cell clones indicated that cyclin D1 profoundly modified several cellular functions, including the regulation of apoptotic cell death. We studied the apoptotic response of GFP- and GFP-cyclin D1-expressing clones to bortezomib-treatment. We found that the apoptotic response occurred faster and was of a greater amplitude in cyclin D1-expressing cells. Cyclin D1 expression enhanced the caspase-dependent apoptosis mediated by the intrinsic mitochondrial pathway. More importantly, cyclin D1 also activated the unfolded protein response (UPR) and induced endoplasmic reticulum (ER) stress-mediated apoptosis. CONCLUSION: The ER is well known to be a crucial regulator of plasma cell death and it plays the same role in their malignant counterparts, myeloma cells. This role involves activation of the UPR controlled at least in part by cyclin D1.

Phenotypic variation among seven members of one family with deficiency of hypoxanthine-guanine phosphoribosyltransferase.

We describe a family of seven boys affected by Lesch-Nyhan disease with various phenotypes. Further investigations revealed a mutation c.203T>C in the gene encoding HGprt of all members, with substitution of leucine to proline at residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared by site-directed mutagenesis and the kinetics of the enzyme revealed that the catalytic activity of the mutant was reduced, in association with marked reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with guanine as substrate with associated reduced catalytic potential. Molecular modeling confirmed that the most prominent defect was the dramatically reduced affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a strong impact on PRPP binding and on stability of the active conformation. This suggests that factors other than HGprt activity per se may influence the phenotype of Lesch-Nyhan patients.

HIF-1α and rapamycin act as gerosuppressant in multiple myeloma cells upon genotoxic stress.

Multiple myeloma (MM) is still an incurable hematological malignancy. Despite recent progress due to new anti-myeloma agents, the pathology is characterized by a high frequency of de novo or acquired resistance. Delineating the mechanisms of MM resistance is essential for therapeutic advances. We previously showed that long-term genotoxic stress induces the establishment of a senescence-associated secretory phenotype, a pro-inflammatory response that favors the emergence of cells with cancer stem-like properties. Here, we studied the short-term response of MM cells following treatment with various DNA damaging agents such as the energetic C-ion irradiation. MM cells are highly resistant to all treatments and do not enter apoptosis after they arrest cycling at the G2 phase. Although the DNA damage response pathway was activated, DNA breaks remained chronically in damaged MM cells. We found, using a transcriptomic approach that RAD50, a major DNA repair gene was downregulated early after genotoxic stress. In two gerosuppression situations: induction of hypoxia and inhibition of the mammalian target of rapamycin (mTOR) pathway, we observed, after the treatment with a DNA damaging agent, a normalization of RAD50 expression concomitant with the absence of cell cycle arrest. We propose that combining inhibitors of mTOR with genotoxic agents could avoid MM cells to senesce and secrete pro-inflammatory factors responsible for cancer stem-like cell emergence and, in turn, relapse of MM patients.

Heat shock factor 1 is a potent therapeutic target for enhancing the efficacy of treatments for multiple myeloma with adverse prognosis.

Deregulated expression of heat shock proteins (HSPs) encoding genes is frequent in multiple myeloma. HSPs, which are molecular chaperones involved in protein homeostasis pathways, have emerged recently as promising therapeutic targets. Using human myeloma cell lines and primary myeloma cells belonging to various molecular groups, we tested the efficacy of HSP90, HSP70, and heat shock factor 1 (HSF1) inhibitors alone or associated with current antimyeloma drugs. We report here that KNK-437 (an inhibitor of HSF1) and bortezomib have additive effects on apoptosis induction in cells belonging to groups with bad prognosis.

New biomarkers for early diagnosis of Lesch-Nyhan disease revealed by metabolic analysis on a large cohort of patients.

BACKGROUND: Lesch-Nyhan disease is a rare X-linked neurodevelopemental metabolic disorder caused by a wide variety of mutations in the HPRT1 gene leading to a deficiency of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). The residual HGprt activity correlates with the various phenotypes of Lesch-Nyhan (LN) patients and in particular with the different degree of neurobehavioral disturbances. The prevalence of this disease is considered to be underestimated due to large heterogeneity of its clinical symptoms and the difficulty of diagnosing of the less severe forms of the disease. We therefore searched for metabolic changes that would facilitate an early diagnosis and give potential clues on the disease pathogenesis and potential therapeutic approaches. METHODS: Lesch-Nyhan patients were diagnosed using HGprt enzymatic assay in red blood cells and identification of the causal HPRT1 gene mutations. These patients were subsequently classified into the three main phenotypic subgroups ranging from patients with only hyperuricemia to individuals presenting motor dysfunction, cognitive disability and self-injurious behavior. Metabolites from the three classes of patients were analyzed and quantified by High Performance Ionic Chromatography and biomarkers of HGprt deficiency were then validated by statistical analyses. RESULTS: A cohort of 139 patients, from 112 families, diagnosed using HGprt enzymatic assay in red blood cells, was studied. 98 displayed LN full phenotype (86 families) and 41 (26 families) had attenuated clinical phenotypes. Genotype/phenotype correlations show that LN full phenotype was correlated to genetic alterations resulting in null enzyme function, while variant phenotypes are often associated with missense mutations allowing some residual HGprt activity. Analysis of metabolites extracted from red blood cells from 56 LN patients revealed strong variations specific to HGprt deficiency for six metabolites (AICAR mono- and tri-phosphate, nicotinamide, nicotinic acid, ATP and Succinyl-AMP) as compared to controls including hyperuricemic patients without HGprt deficiency. CONCLUSIONS: A highly significant correlation between six metabolites and the HGprt deficiency was established, each of them providing an easily measurable marker of the disease. Their combination strongly increases the probability of an early and reliable diagnosis for HGprt deficiency.

Deazaneplanocin a is a promising drug to kill multiple myeloma cells in their niche.

Tumoral plasma cells has retained stemness features and in particular, a polycomb-silenced gene expression signature. Therefore, epigenetic therapy could be a mean to fight for multiple myeloma (MM), still an incurable pathology. Deazaneplanocin A (DZNep), a S-adenosyl-L-homocysteine hydrolase inhibitor, targets enhancer of zest homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2) and is capable to induce the death of cancer cells. We show here that, in some MM cell lines, DZNep induced both caspase-dependent and -independent apoptosis. However, the induction of cell death was not mediated through its effect on EZH2 and the trimethylation on lysine 27 of histone H3 (H3K27me3). DZNep likely acted through non-epigenetic mechanisms in myeloma cells. In vivo, in xenograft models, and in vitro DZNep showed potent antimyeloma activity alone or in combination with bortezomib. These preclinical data let us to envisage new therapeutic strategies for myeloma.

Antitumoral activity of lenalidomide in in vitro and in vivo models of mantle cell lymphoma involves the destabilization of cyclin D1/p27KIP1 complexes.

PURPOSE: Clinical responses to the immmunomodulatory drug lenalidomide have been observed in patients with relapsed/refractory mantle cell lymphoma (MCL), although its mechanism of action remains partially unknown. We investigated whether the expression and subcellular localization of cyclin D1, a major cell-cycle regulator overexpressed in MCL, and the cyclin-dependent kinase inhibitor p27(KIP1), could identify MCL cases sensitive to lenalidomide, and whether the compound could modulate cyclin D1/p27(KIP1) complexes in MCL cells. EXPERIMENTAL DESIGN: MCL primary samples and cell lines were analyzed for subcellular levels of cyclin D1/p27(KIP1) complexes by Western blot, immunohistochemistry, immunoprecipitation, and flow cytometry. Activity of lenalidomide in vitro and its effect on cyclin D1/p27(KIP1) complexes were evaluated by real-time PCR, immunoprecipitation, immunofluorescence, and Western blot. In vivo validation was carried out in a mouse xenograft model of human MCL. RESULTS: We found cyclin D1 and p27(KIP1) to be coordinately expressed in all the MCL samples tested. Immunoprecipitation analyses and siRNA assays suggested a direct role of cyclin D1 in the regulation of p27(KIP1) levels. The nuclear accumulation of both proteins correlated with MCL cell tumorigenicity in vivo, and sensitivity to lenalidomide activity in vitro and in vivo. Lenalidomide mechanism of action relied on cyclin D1 downregulation and disruption of cyclin D1/p27(KIP1) complexes, followed by cytosolic accumulation of p27(KIP1), cell proliferation arrest, apoptosis, and angiogenesis inhibition. CONCLUSIONS: These results highlight a mechanism of action of lenalidomide in MCL cases with increased tumorigenicity in vivo, which is mediated by the dissociation of cyclin D1/p27(KIP1) complexes, and subsequent proliferation blockade and apoptosis induction.

Transcriptomic approach to Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is an X-linked metabolic disease caused by various mutations in the gene HPRT1 encoding an enzyme of purine metabolism, hypoxanthine guanine phosphoribosyltransferase (HPRT). In its most severe form, LND patients suffer from overproduction of uric acid along with neurological or behavioural difficulties including self-injurious behaviours. To gain more insight into pathogenesis, we compared the transcriptome from human LND fibroblasts to normal human fibroblasts using a microarray with 60,000 probes corresponding to the entire human genome. Using stringent criteria, we identified 25 transcripts whose expression was significantly different between LND and control cells. These genes were confirmed by quantitative RT-PCR to be dysregulated in LND cells. Moreover, bioinformatic analysis of microarray data using gene ontology (GO) highlighted clusters of genes displaying biological processes most significantly affected in LND cells. These affected genes belonged to specific processes such as cell cycle and cell-division processes, metabolic and nucleic acid processes, demonstrating the specific nature of the changes and providing new insights into LND pathogenesis.

A sensitive method to quantify senescent cancer cells.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce. Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required. To this end, the standard ß-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization in Xenopus egg extract.

The organization of the microtubule cytoskeleton depends crucially on crosslinking motors that arrange microtubules in space. Kinesin-5 is such an essential motile crosslinker. It is unknown whether its organizing capacity during bipolar spindle formation depends on its characteristic kinetic properties, or whether simply crosslinking combined with any plus-end-directed motility is sufficient for its function in a physiological context. To address this question, we replaced the motor domain of Xenopus Kinesin-5 by motor domains of kinesins belonging to other kinesin subfamilies, without changing the overall architecture of the molecule. This generated novel microtubule crosslinkers with altered kinetic properties. The chimeric crosslinkers mislocalized in spindles and consequently caused spindle collapse into tightly bundled microtubule arrays. This demonstrates that plus-end directionality and microtubule crosslinking are not the only characteristics required for proper functioning of Kinesin-5 during spindle assembly in Xenopus egg extract. Instead, its motor domain properties appear to be fine-tuned for the specific function of this kinesin.

Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles.

BACKGROUND: Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.