Dasatinib induces endothelial dysfunction leading to impaired recovery from ischaemia.

Chronic myeloid leukaemia (CML) management is complicated by treatment-emergent vascular adverse events seen with tyrosine kinase inhibitors (TKIs) such as nilotinib, dasatinib and ponatinib. Pleural effusion and pulmonary arterial hypertension (PAH) have been associated with dasatinib treatment. Endothelial dysfunction and impaired angiogenesis are hallmarks of PAH. In this study, we explored, at cellular and whole animal levels, the connection between dasatinib exposure and disruption of endothelial barrier integrity and function, leading to impaired angiogenesis. Understanding the mechanisms whereby dasatinib initiates PAH will provide opportunities for intervention and prevention of such adverse effects, and for future development of safer TKIs, thereby improving CML management.

Using natural killer cell-derived exosomes as a cell-free therapy for leukemia.

Natural killer (NK) cells are components of the innate immune system which play a pivotal role in cancer cell surveillance. Despite promising results in clinical trials, the use of NK-based therapies is limited due to unsatisfactory efficiencies and safety issues. In recent years, exosomes have emerged as a powerful, natural therapeutic tool. Since exosomes are known to carry cargos that reflect the cellular makeup of their cell of origin, we were prompted to test whether NK-derived exosomes (NKexo) maintain the anti-leukemia capacity of NK-cells. We found NK92MI-cells to secrete large amounts of 100-200 nm cap-shaped particles expressing exosomal and NK biomarkers (CD63, CD81, CD56). We demonstrated that NKexo exert a potent, selective, anti-leukemia effect on all leukemia cell-lines tested. Furthermore, NKexo eliminated leukemia cells isolated from patients with acute and chronic leukemia and inhibited hematopoietic colony growth. While leukemia cells were targeted and severely affected by NKexo, healthy B-cells remained unaffected, indicating a selective effect. This selectivity was further confirmed by demonstrating that NKexo were specifically taken up by leukemic cells but not by healthy B-cells. Our in vivo data support our in vitro and ex vivo findings and demonstrate improved human-CD45+ leukemia blast counts and overall survival in NKexo treated humanized acute myeloid leukemia (HL-60) xenograft mice thus supporting the assumption that NKexo possess an anti-leukemia effect. Pending further analyses, our findings provide the pre-clinical evidence needed to test the NKexo approach in future pre-clinical and clinical studies to ultimately develop an acellular "off-the-shelf" product to treat leukemia.

Extracorporeal photopheresis induces NETosis in neutrophils derived from patients with chronic graft-vs-host disease.

INTRODUCTION: Extracorporeal photopheresis (ECP) is considered an effective treatment for patients with chronic graft vs host disease (cGVHD) and demonstrates efficacy in ameliorating GVHD. The mechanism by which ECP acts against cGVHD is not fully understood. Preliminary observations have hinted at the potential involvement of neutrophil extracellular traps (NETs) formation in the pathogenesis of cGVHD. We aimed to assess the influence of ECP on the formation of NETs in patients with cGVHD as a potential mechanism in this setting. METHODS: Patients treated with ECP for cGVHD at the Rabin Medical Center were included in this study. Blood samples were obtained at three different time points: before starting an ECP cycle, at the end of the first day of treatment, and 24 h following the initiation of the ECP treatment cycle. Neutrophils were harvested from all blood samples. NET formation was assessed by measurement of NET-bound specific neutrophil elastase activity and by immunofluorescence staining. RESULTS: Six patients (two females and four males) with cGVHD were included in the study. We observed a significant increase in NET formation among all six patients following ECP. Net-bound specific neutrophil elastase activity was elevated from a median value of 2.23 mU/mL (interquartile range [IQR] 2.06-2.47 mU/mL) at baseline to a median value of 13.06 mU/mL (IQR 10.27-15.97 mU/mL) immediately after the treatment and to a peak median value of 14.73 mU/mL (IQR 9.6-22.38 mU/mL) 24 h following the initiation of the ECP cycle. A qualitative assessment of NET formation using immunofluorescence staining has demonstrated markedly increased expression of citrullinated histone H3, a marker of NET formation, following ECP treatment. CONCLUSIONS: Our preliminary data indicate that ECP induces NET formation among patients with cGVHD. The contribution of increased NET formation to the therapeutic effect of cGVHD should be further investigated.

Deferasirox induces cyclin D1 degradation and apoptosis in mantle cell lymphoma in a reactive oxygen species- and GSK3ß-dependent mechanism.

Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by cyclin D1 (CD1) overexpression. Targeting CD1 in MCL has been shown to be of therapeutic significance. However, treatment of MCL remains challenging since patients are still subject to early and frequent relapse of the disease. To ensure their high proliferation rate, tumour cells have increased iron needs, making them more susceptible to iron deprivation. Indeed, several iron chelators proved to be effective anti-cancer agents. In this study, we demonstrate that the clinically approved iron chelator deferasirox (DFX) exerts an anti-tumoural effect in MCL cell lines and patient cells. The exposure of MCL cells to clinically feasible concentrations of DFX resulted in growth inhibition, cell cycle arrest and induction of apoptosis. We show that DFX unfolds its cytotoxic effect by a rapid induction of reactive oxygen species (ROS) that leads to oxidative stress and severe DNA damage and by triggering CD1 proteolysis in a mechanism that requires its phosphorylation on T286 by glycogen synthase kinase-3ß (GSK3ß). Moreover, we demonstrate that DFX mediates CD1 proteolysis by repressing the phosphatidylinositol 3-kinase (PI3K)/AKT/GSK3ß pathway via ROS generation. Our data suggest DFX as a potential therapeutic option for MCL and paves the way for more treatment options for these patients.

Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors.

Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Profiling and bioinformatics analyses reveal chronic lymphocytic leukemia cells share a unique circular RNA expression pattern.

Approximately 10% of the human transcriptome is composed of circular RNAs (circRNAs). These are non-coding RNA molecules in which a covalent bond between the 3' and 5' forms a stable circular loop. Herein, we profiled the expression of 13,368 cricRNAS in 21 patients with chronic lymphocytic leukemia (CLL). Regardless of clinical, genetic, or prognostic characteristics, CLL cells share a unique expression profile distinguishable from that of normal B cells. Specifically, 859 circRNAs from 592 genes were differentially expressed (fold change ≥2 and false discovery rate ≤0.05). Whether dysregulation of circRNAs contributes to the pathogenesis of CLL remains to be determined.

Deferasirox selectively induces cell death in the clinically relevant population of leukemic CD34+CD38- cells through iron chelation, induction of ROS, and inhibition of HIF1α expression.

Despite a high remission rate after therapy, only 40-50% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. The main cause of treatment failure is thought to be insufficient eradication of CD34+CD38- AML cells. In order to induce preferential cell death in CD34+CD38- AML cells, two separate events may be necessary: (1) inhibition of survival signals such as nuclear factor kappa-beta (NF-κB) and (2) induction of stress responses such as the oxidative stress response. Therefore, regimens that mediate both effects may be favorable. Deferasirox is a rationally designed oral iron chelator mainly used to reduce chronic iron overload in patients who receive long-term blood transfusions. Our study revealed that clinically relevant concentrations of deferasirox are cytotoxic in vitro to AML progenitor cells, but even more potent against the more primitive CD34+CD38- cell population. In addition, we found that deferasirox exerts its effect, at least in part, by inhibiting the NF-κB/hypoxia-induced factor 1-alpha (HIF1α) pathway and by elevating reactive oxygen species levels. We believe that, pending further characterization, deferasirox can be considered as a potential therapeutic agent for eradicating CD34+CD38- AML cells.

Bosutinib, dasatinib, imatinib, nilotinib, and ponatinib differentially affect the vascular molecular pathways and functionality of human endothelial cells.

The tyrosine kinase inhibitors (TKIs), nilotinib, ponatinib, and dasatinib (but not bosutinib or imatinib), are associated with vascular adverse events (VAEs) in chronic myeloid leukemia (CML). Though the mechanism is inadequately understood, an effect on vascular cells has been suggested. We investigated the effect of imatinib, nilotinib, dasatinib, bosutinib, and ponatinib on tube formation, cell viability, and gene expression of human vascular endothelial cells (HUVECs). We found a distinct genetic profile in HUVECs treated with dasatinib, ponatinib, and nilotinib compared to bosutinib and imatinib, who resembled untreated samples. However, unique gene expression and molecular pathway alterations were detected between dasatinib, ponatinib, and nilotinib. Angiogenesis/blood vessel-related pathways and HUVEC function (tube formation/viability) were adversely affected by dasatinib, ponatinib, and nilotinib but not by imatinib or bosutinib. These results correspond to the differences in VAE profiles of these TKIs, support a direct effect on vascular cells, and provide direction for future research.

Ponatinib reduces viability, migration, and functionality of human endothelial cells.

Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.

MAP kinase activation by mu opioid receptor in cord blood CD34(+)CD38(-) cells.

OBJECTIVE: Opioid receptor expression and function traditionally have been studied in neuronal cells and recently in mature lymphoid cells; however, little is known about their possible functions in hematopoietic stem cells (CD34(+) cells). We studied the expression of the mu receptor on CD34(+) cells and assessed the signal transduction cascade it induces. MATERIALS AND METHODS: Mu-receptor expression on cord blood (CB) and peripheral blood (PB) CD34(+) cells was studied by microarrays, immunostaining, and fluorescence-activated cell sorting analysis. Signal transduction by the mu receptor was studied through Western blots and kinase assay of enkephalin-activated CB CD34(+) cells. Apoptotic, differentiation, and proliferation responses following mu-receptor activatioSn were studied by annexin V assay and inverted microscopy. RESULTS: A prominent difference in gene expression, in favor of CB compared to PB CD34(+) cells, was observed in the mu-receptor gene. This receptor was mainly expressed on the CB CD34(+)CD38(-) subpopulation. A MAP kinase signal transduction cascade was shown to be induced through activation of this receptor by enkephalin or morphine. CONCLUSIONS: We showed for the first time that the mu receptor is expressed on immature CB stem cells and that its activation by enkephalin or morphine induces a MAP kinase signal transduction cascade. Because the MAP kinase cascade is known to elicit proliferation and differentiation responses, these findings suggest a possible role of endogenous enkephalins in hematopoietic stem cell proliferation and differentiation and may lead to therapeutic applications of opiates in CB stem cell expansion and neuronal differentiation.

MiR-30e induces apoptosis and sensitizes K562 cells to imatinib treatment via regulation of the BCR-ABL protein.

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cell carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) of the BCR-ABL kinase are the treatment of choice for CML patients. Imatinib was the first TKI used in clinical practice with excellent results. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. Aberrant miRNA expression profiles have been shown to be characteristic of many cancers. Here, we demonstrate that miR-30e is expressed at low levels in CML cell lines and patient samples. Bioinformatics analysis reveals a putative target site for miR-30e in the 3'-untranslated region (UTR) of the ABL gene. In agreement, luciferase assay verified that miR-30e directly targets ABL. Enforced expression of miR-30e in K562 cells suppressed proliferation and induced apoptosis of these cells and sensitized them to imatinib treatment. These findings strongly suggest that miR-30e acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-30e in CML cells may therefore have a therapeutic efficacy against this disease.

Restoration of miR-424 suppresses BCR-ABL activity and sensitizes CML cells to imatinib treatment.

MicroRNAs (miRNAs) are small noncoding RNAs that participate in many biological processes by posttranscriptionally regulating gene expression. Dysregulation of miRNA expression has been shown to be typical of many neoplasms. Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL tyrosine kinase fusion gene. While the development of tyrosine kinase inhibitors (TKIs) like imatinib has revolutionized treatment of CML, it has become increasingly clear in recent years that TKI treatment alone will not be curative in many cases. Thus, further dissection of the regulatory networks that drive BCR-ABL-induced malignant transformation may help to identify other novel therapeutic approaches that complement TKI treatment. In this study we demonstrate that the expression of miR-424 is markedly low in CML cell lines and patient samples at time of diagnosis. With the aid of bioinformatics analysis we revealed a conserved target site for miR-424 in the 3'-untranslated region (UTR) of the ABL gene. Via luciferase assays, we showed that miR-424 directly targets BCR-ABL. Overexpression of miR-424 was shown to suppress proliferation and induce apoptosis of K562 cells as well as sensitize these cells to imatinib treatment. These findings strongly suggest that miR-424 acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-424 in CML cells may therefore have a therapeutic effect against this disease.

Is cure of chronic myeloid leukemia in the third millennium a down to earth target (ed) or a castle in the air?

The therapeutic approach in chronic myeloid leukemia (CML) has undergone a revolutionary change during the last 2 decades with the introduction of the tyrosine kinase inhibitors. The use of these agents has changed overall survival as well as the quality of life of CML patients. Since the introduction of imatinib, newer agents have been developed and their role as first line treatment was examined. The use of genomics and proteomics as a means to tailor treatment of CML patients is underway. Meanwhile, clinical parameters as disease phase and response to treatment are used to guide a personalized approach. In this review, we will discuss the various aspects of personalized medicine in CML, including the use of the different scoring systems to guide treatment, disease phase as a means to choose the proper approach and the value of early response evaluation in decision-making. The approach towards patients resistant to tyrosine kinase inhibitors (TKIs), individual strategies for discontinuation of TKIs and "operational" cure in CML will also be discussed and finally the personalized treatment for CML patients in the near future based on present outcomes.

Retinoic acid induces adhesion and migration in NB4 cells through Pyk2 signaling.

Since the introduction of all-trans-retinoic acid (ATRA) treatment for acute promyelocytic leukemia (APL) there has been increasing concern about extramedullary disease (EMD) progression despite favorable response in the bone marrow. We postulated that ATRA treatment enhances migration and adhesion abilities possibly enabling APL cells to inhabit extramedullary sites. We revealed an increase in adhesion, migration and invasion capabilities of NB4 cells following ATRA treatment. ATRA induced upregulation of Pyk2 mRNA, protein and phosphorylation levels and enhanced Pyk2 interaction with paxillin and vinculin. Pyk2 inhibition resulted in a reduction of NB4 cell adhesion and migration following ATRA treatment. These results indicate that in vitro Pyk2 might function to regulate cell adhesion and motility following ATRA treatment and its upregulated expression may contribute to EMD development in APL patients.

Enhanced adhesion/migration and induction of Pyk2 expression in K562 cells following imatinib exposure.

Concern about extramedullary relapse (EMR) despite favorable response in the bone marrow has been raised with the use of imatinib for treatment of chronic myeloid leukemia (CML). In the present study we show an increase in adhesion, migration and invasion capabilities of the CML cell line K562 following imatinib administration. Imatinib induced upregulation of Pyk2 mRNA and protein levels. Pyk2 inhibition resulted in a reduction of K562 cells' adhesion and migration subsequent to imatinib treatment. This effect was similar to that shown by us previously with all trans retinoic acid (ATRA) in the acute promyelocytic leukemia (APL) cell line NB4. Our data pinpoint Pyk2 as a shared key mediator of targeted-therapy induced adhesion and migration and may implicate that targeting Pyk2 may serve as an effective therapeutic strategy to reduce EMR in APL and CML.

Mechanism of the antitumoral activity of deferasirox, an iron chelation agent, on mantle cell lymphoma.

Mantle cell lymphoma (MCL) characterized by the t(11;14)(q13;q32) translocation, resulting in cyclin D1 overexpression, is one of the most challenging lymphomas to treat. Iron chelators, such as deferasirox, have previously been shown to exhibit anti-proliferative properties; however, their effect on MCL cells has never been investigated. We showed that deferasirox exhibited antitumoral activity against the MCL cell lines HBL-2, Granta-519 and Jeko-1, with 50% inhibitory concentration (IC(50)) values of 7.99 ± 2.46 µM, 8.93 ± 2.25 µM and 31.86 ± 7.26 µM, respectively. Deferasirox induced apoptosis mediated through caspase-3 activation and decreased cyclin D1 protein levels resulting from increased proteasomal degradation. We also demonstrated down-regulation of phosphor-RB (Ser780) expression, which resulted in increasing levels of the E2F/RB complex and G(1)/S arrest. Finally, we showed that deferasirox activity was dependent on its iron chelating ability. The present data indicate that deferasirox, by down-regulating cyclin D1 and inhibiting its related signals, may constitute a promising adjuvant therapeutic molecule in the strategy for MCL treatment.

Second-generation tyrosine kinase inhibitors reduce telomerase activity in K562 cells.

In this study we present the effects of nilotinib and dasatinib on telomerase activity and regulation. Nilotinib and dasatinib strongly reduced telomerase activity in BCR-ABL-positive (K562) and BCR-ABL-negative (HL60) cells, demonstrating that their effect on telomerase activity is uncoupled from their effect on BCR-ABL. Nilotinib and dasatinib caused a substantial decrease in hTERT mRNA expression. Phospho-Sp1 regulates hTERT transcription. We detected a considerable decrease in Sp1 nuclear expression and binding to the hTERT promoter following exposure to the drugs. We also detected a reduction in Map kinase, known to phosphorylate Sp1. Telomerase is also activated and translocated to the nucleus when phosphorylated by AKT. We detected a decrease in phospho-AKT and a reduction in the nuclear expression of hTERT following exposure to nilotinib and dasatinib. In conclusion, we provide evidence for transcriptional and post-translational inhibition of telomerase by nilotinib and dasatinib which is not necessarily mediated via known targets of these tyrosine kinase inhibitors.

Downregulation of miR-31, miR-155, and miR-564 in chronic myeloid leukemia cells.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. RESULTS: Using miRNA microarrays and miRNA real-time PCR we characterized the miRNAs expression profile of CML cell lines and patients in reference to non-CML cell lines and healthy blood. Of all miRNAs tested, miR-31, miR-155, and miR-564 were down-regulated in CML cells. Down-regulation of these miRNAs was dependent on BCR-ABL activity. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. As expected, in K562 cells, the expression of several of these targets was inverted to that of the miRNA putatively regulating them. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, ErbB, mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the signaling pathways-related miRNAs. CONCLUSIONS: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment.

NAD(P)H quinone oxidoreductase protects TAp63gamma from proteasomal degradation and regulates TAp63gamma-dependent growth arrest.

BACKGROUND: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. PRINCIPAL FINDINGS: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin. CONCLUSIONS/SIGNIFICANCE: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy.