The biguanide polyamine analog verlindamycin promotes differentiation in neuroblastoma via induction of antizyme.

Deregulated polyamine biosynthesis is emerging as a common feature of neuroblastoma and drugs targeting this metabolic pathway such as DFMO are in clinical and preclinical development. The polyamine analog verlindamycin inhibits the polyamine biosynthesis pathway enzymes SMOX and PAOX, as well as the histone demethylase LSD1. Based on our previous research in acute myeloid leukemia (AML), we reasoned verlindamycin may also unblock neuroblastoma differentiation when combined with all-trans-retinoic acid (ATRA). Indeed, co-treatment with verlindamycin and ATRA strongly induced differentiation regardless of MYCN status, but in MYCN-expressing cells, protein levels were strongly diminished. This process was not transcriptionally regulated but was due to increased degradation of MYCN protein, at least in part via ubiquitin-independent, proteasome-dependent destruction. Here we report that verlindamycin effectively induces the expression of functional tumor suppressor-antizyme via ribosomal frameshifting. Consistent with previous results describing the function of antizyme, we found that verlindamycin treatment led to the selective targeting of ornithine decarboxylase (the rate-limiting enzyme for polyamine biosynthesis) as well as key oncoproteins, such as cyclin D and Aurora A kinase. Retinoid-based multimodal differentiation therapy is one of the few interventions that extends relapse-free survival in MYCN-associated high-risk neuroblastoma and these results point toward the potential use of verlindamycin in this regimen.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

In Vivo Modeling of Chemoresistant Neuroblastoma Provides New Insights into Chemorefractory Disease and Metastasis.

Neuroblastoma is a pediatric cancer that is frequently metastatic and resistant to conventional treatment. In part, a lack of natively metastatic, chemoresistant in vivo models has limited our insight into the development of aggressive disease. The Th-MYCN genetically engineered mouse model develops rapidly progressive chemosensitive neuroblastoma and lacks clinically relevant metastases. To study tumor progression in a context more reflective of clinical therapy, we delivered multicycle treatment with cyclophosphamide to Th-MYCN mice, individualizing therapy using MRI, to generate the Th-MYCN CPM32 model. These mice developed chemoresistance and spontaneous bone marrow metastases. Tumors exhibited an altered immune microenvironment with increased stroma and tumor-associated fibroblasts. Analysis of copy number aberrations revealed genomic changes characteristic of human MYCN-amplified neuroblastoma, specifically copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. RNA sequencing revealed enriched expression of genes associated with 17q gain and upregulation of genes associated with high-risk neuroblastoma, such as the cell-cycle regulator cyclin B1-interacting protein 1 (Ccnb1ip1) and thymidine kinase (TK1). The antiapoptotic, prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, and treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival. Our results highlight that under treatment conditions that mimic chemotherapy in human patients, Th-MYCN mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease. The Th-MYCN CPM32 model therefore is a useful tool to dissect in detail mechanisms that drive metastasis and chemoresistance, and highlights dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease. SIGNIFICANCE: An in vivo mouse model of high-risk treatment-resistant neuroblastoma exhibits changes in the tumor microenvironment, widespread metastases, and sensitivity to JAK1/2 inhibition.

Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle.

MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease.

We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Retrospective analysis of earlobe keloids treated with the carbon dioxide laser ablation or cold steel debulking surgery.

BACKGROUND: Treatment of keloids is a therapeutic challenge. OBJECTIVES: To determine the outcome and the risk of recurrence after debulking cold steel surgery or carbon dioxide (CO2) laser ablation of earlobe keloids. MATERIAL AND METHODS: The case records of 16 patients with earlobe keloids managed at Changi General Hospital, Singapore, from 2003 to 2009 were reviewed retrospectively. RESULTS: Fourteen patients were females, and the mean age at presentation was 20 years. Eight patients underwent CO2 laser ablation, six patients underwent cold steel surgery, one patient underwent both surgery and CO2 laser ablation, and one patient received only 40 mg/ml of intralesional triamcinolone acetonide. Fourteen patients were followed up for 1-24 months post procedure, and two patients defaulted. Both modalities were equally effective in debulking the earlobe keloids. All 13 patients who had either CO2 laser ablation or cold steel surgery had recurrence of keloid growth at 2-18 weeks post procedure. The patient who received intralesional triamcinolone acetonide therapy alone had only partial response to the therapy. CONCLUSIONS: Both the CO2 laser ablation and cold steel surgery were equally useful in reducing the size of the earlobe keloids, but were not effective in preventing regrowth of the keloids, even with adjunctive intralesional steroids. Patients should be clearly counselled regarding this.

Fractional ablative carbon dioxide laser in the treatment of atrophic acne scarring in Asian patients: a pilot study.

INTRODUCTION: Ablative carbon dioxide resurfacing is the gold standard for treating atrophic acne scarring but is associated with prolonged recovery and many side effects. To address these limitations, newer modalities employing the principle of fractional photothermolysis have emerged. METHODS: We undertook a prospective study whereby five Asian patients of skin phototype IV with moderate to severe atrophic acne scarring received two sessions of a fractional carbon dioxide laser 6-8 weeks apart. Treatment parameters were: fluence, 28 J/cm(2); pulse width, 2.5 ms; spot size, 300 microm; penetration depth, up to 500 microm; degree of skin coverage, 20%; single pass. Photographic evaluation was done on the level of improvement according to a quartile grading scale: 75% (excellent). RESULTS: At 2 months post-treatment, all five subjects showed some clinical improvement (four: mild improvement; one: moderate improvement). The treatment was well tolerated. All patients had erythema, which lasted for a mean of 6 days. No other complications were observed. CONCLUSION: Our study has shown that in Asians the fractional ablative carbon dioxide laser produces mild to moderate improvement in acne scarring with the advantage of a quick recovery period with minimal adverse effects.

A novel radiofrequency device for the treatment of rhytides and lax skin: a pilot study.

BACKGROUND: Radiofrequency devices generate electromagnetic energy to induce dermal heating to a critical temperature of about 65 degrees C, causing collagen to shrink and contract, and this provides a non-ablative means to improve rhytides and lax skin. OBJECTIVE: To evaluate the efficacy and safety of a novel radiofrequency device (Photo Bio Care, Thailand) operating at a frequency of 1.75 MHz for the treatment of rhytides and lax skin in Asian patients. METHODS: Six female patients (age range, 30-60 years; mean age 48 years) with class I and II facial rhytides and Fitzpatrick skin types III and IV were treated six times at intervals of 2-3 weeks. The forehead, periorbital area, cheeks and nasolabial folds were treated using the following parameters: monopolar mode, pulse duration of 5 s, frequency of 18 Hz, and power of 100 W. No preoperative anesthesia or postoperative treatment were used. The handpiece was applied to the skin in a continuous sweeping motion until the baseline skin temperature was elevated to 40 degrees C, as monitored with a laser thermometer, and this was maintained for 3 minutes. Clinical photographs were taken before, after the sixth treatment and 2 months after the last treatment. Analysis was undertaken through photographic evaluation by non-treating physicians. RESULTS: At 2 months after the last treatment, mild to moderate (25-50%) clinical improvement was noted in all patients compared to baseline, and all patients were somewhat satisfied with the treatment. The procedure was well tolerated with no unexpected side effects. Discomfort was rated as mild and post-treatment erythema resolved within 1-2 hours. CONCLUSION: Treatment with the radiofrequency device can result in mild to moderate clinical improvement of age-related rhytides and lax skin. The system is safe and inexpensive with no consumables required.

Transforming activity of AML1-ETO is independent of CBFbeta and ETO interaction but requires formation of homo-oligomeric complexes.

Although both heterodimeric subunits of core binding factors (AML1/RUNX1 and CBFbeta) essential for normal hematopoiesis are frequently mutated to form different chimeric fusion proteins in acute leukemia, the underlying molecular mechanisms and structural domains required for cellular transformation remain largely unknown. Despite the critical role of CBFbeta for wild-type AML1 function and its direct involvement in chromosomal translocation, we demonstrate that both the expression and interaction with CBFbeta are superfluous for AML1-ETO (AE)-mediated transformation of primary hematopoietic cells. Similarly, the hetero-oligomeric interaction with transcriptional repressor ETO family proteins and the highly conserved NHR1 domain in AE fusion are also dispensable for transforming activity. In contrast, AE-mediated transformation is critically dependent on the DNA binding and homo-oligomeric properties of the fusion. Abolishment of homo-oligomerization by a small-molecule inhibitor could specifically suppress AML1 fusion-mediated transformation of primary hematopoietic cells. Together, these results not only identify the essential molecular components but also potential avenues for therapeutic targeting of AE-mediated leukemogenesis.

Minimally ablative erbium:YAG laser resurfacing of facial atrophic acne scars in Asian skin: a pilot study.

BACKGROUND: Atrophic scars are dermal depressions caused by collagen damage most commonly occurring after inflammatory acne vulgaris. There are little published data regarding the effectiveness and safety of minimally invasive lasers in the treatment of atrophic acne scars in darker skin types. OBJECTIVE: The purpose was to evaluate the efficacy and safety of a low-fluence 2,940-nm erbium:YAG laser in the treatment of atrophic acne scars in Asian patients. MATERIALS AND METHODS: Nine patients aged 19 to 45 years with mild to moderate atrophic facial scars and Skin Types IV and V were treated with topical anesthesia and one to two passes with an erbium:YAG laser two times at 1-month intervals. Treatment parameters were 6-mm spot size, fluence of 400 mJ, pulse duration of 300 micros, and repetition rate of 2 Hz. RESULTS: At 2 months after the last treatment, mild to moderate clinical improvement was noted in all patients compared to baseline. Treatment was well tolerated. Side effects consisted of posttreatment erythema, peeling, and crusting, which resolved within 1 to 2 weeks. There was no postinflammatory hyper- or hypopigmentation, blistering, or hypertrophic scarring. CONCLUSION: Low-fluence erbium:YAG facial resurfacing was effective and safe in patients with mild to moderately severe atrophic acne scarring.

Photodynamic therapy with 20% aminolevulinic acid for the treatment of recalcitrant viral warts in an Asian population.

BACKGROUND: Topical photodynamic therapy (PDT) is based on the principle of targeted tissue destruction using selective photosensitization via a topical porphyrin precursor, followed by light exposure. It is well established for the treatment of actinic keratoses and superficial nonmelanoma skin cancers. Some studies have reported good efficacy when using PDT to treat viral warts in the Western population. METHODS: We carried out a prospective, single-arm, phase II study of 5-aminolevulinic acid (5-ALA)-PDT in the treatment of recalcitrant viral warts in an Asian population. Recalcitrant viral warts were surgically pared, and then treated with 20% 5-ALA cream (Medac, Hamburg, Germany) under occlusion for 4 hours before irradiation with a red light source (Waldmann PDT1200; wavelength, 590-700 nm) at an irradiance of 50 mW/cm(2) and a total dose of 50 J/cm(2). PDT was repeated fortnightly for a maximum of four times. RESULTS: Twelve adult Asian patients were enrolled into the study (10 males, two females). The mean age of the patients was 32.8 years (range, 18-70 years). They had skin phototypes III-IV. Nine patients had plantar warts and three patients had hand warts (two had warts on the fingers, one had a wart on the palm). Five patients (42%) showed complete disappearance of their warts, one patient (8%) showed partial clearance (greater than 50% decrease in the wart area), five patients (42%) had stable disease (less than 50% decrease in the wart area), and one (8%) showed progressive disease (increase in the wart area). Adverse effects included mild to moderate pain and erythema, which lasted no longer than 48 hours and was well tolerated by all patients. None of the patients withdrew from the study because of side-effects. CONCLUSION: 5-ALA-PDT, given its noninvasiveness, minimal adverse effects, and good cosmetic results, is a promising alternative treatment for recalcitrant viral warts. Further studies with a larger cohort of patients would be of value.

Recruitment of RXR by homotetrameric RARalpha fusion proteins is essential for transformation.

While formation of higher-order oncogenic transcriptional complexes is critical for RARalpha fusion proteins in acute promyelocytic leukemia, the essential components and their roles in mediating transformation are still largely unknown. To this end, the present study demonstrates that homodimerization is not sufficient for RARalpha fusion-mediated transformation, which requires higher-order homotetramerization. Surprisingly, intrinsic homo-oligomeric DNA binding by the fusion proteins is also dispensable. Importantly, higher-order RXR/RARalpha fusion hetero-oligomeric complexes that aberrantly recruit transcriptional corepressors to downstream targets are essential for transformation. Intervention of RXR-dependent pathways by panRXR-agonists or RXRalpha shRNAs suppresses RARalpha fusion-mediated transformation. Taken together, these results define the oncogenic threshold for self-association and reveal the pathological significance of higher-order RARalpha fusion/RXR hetero-oligomeric complexes and their potential value as a therapeutic target.

Forced homo-oligomerization of RARalpha leads to transformation of primary hematopoietic cells.

Almost 100% of APL patients carry chimeric transcripts encoding truncated RARalpha fused to homo-oligomerization domains from partner proteins. To gain further insights into the cellular transformation mechanisms mediated by RARalpha fusion proteins, thorough structure/function analyses have been performed and identified the POZ homo-oligomerization domain as the minimal transformation domain that is necessary and sufficient for PLZF-RARalpha-mediated in vitro transformation of primary hematopoietic cells. A transformation-incompetent PLZF-RARalpha mutant defective in homo-oligomerization but not corepressor interaction could be rescued by synthetic FKBP-oligomerization domains. Furthermore, an artificial FKBP-RARalpha construct not only mimicked various biochemical properties of bona fide RARalpha fusion proteins but also mediated an ATRA-dependent transformation. Taken together, these findings endorse an oligomerization-dependent mechanism for RARalpha-mediated transformation and suggest a potential avenue for molecular therapy.

Non-ablative 1,450-nm diode laser treatment of striae distensae.

BACKGROUND AND OBJECTIVES: Striae distensae are dermal scars with flattening and atrophy of the epidermis. Successful treatment of these stretch marks has been disappointing. The non-ablative 1,450-nm diode laser has been shown to improve atrophic scars and may be expected to improve striae. As yet, no study has been published to document the effects of this laser on striae. Our aim is to evaluate the efficacy of the 1,450-nm diode laser in the treatment of striae rubra and striae alba in Asian patients with skin types 4-6. STUDY DESIGN/MATERIALS AND METHODS: Striae on one half of the body in 11 patients were treated with the 1,450-nm diode laser with cryogen cooling spray with the other half serving as a control. The following parameters were used: 6 mm spot size and dynamic cooling device (DCD) for 40 milliseconds to protect the epidermis. Patients were randomly assigned to receive either 4, 8, or 12 J/cm2. A total of three treatments were given at 6-week intervals. The following sites were treated: abdomen, arms, back, buttocks, and thighs. Two patients had striae rubra and nine striae alba. Clinical photographs were taken before and after each treatment and analysis was undertaken through photographic evaluation by non-treating physicians. RESULTS: At 2 months after the last treatment, no patients showed any noticeable improvement in the striae on the treated side compared to baseline and to the control areas. Side effects were limited to transient erythema and postinflammatory hyperpigmentation (PIH), which occurred in seven (64%) patients. CONCLUSIONS: The non-ablative 1,450-nm diode laser is not useful in the treatment of striae in patients with skin types 4, 5, and 6.

Long pulsed dye laser treatment of facial wrinkles.

BACKGROUND: The flashlamp pulsed dye laser has been used in the treatment of rhytids. OBJECTIVE: To evaluate the efficacy of the long pulsed dye laser in the treatment of mild to moderate wrinkles in Asian patients. METHODS: Wrinkles on one half of the face in 10 subjects were treated with the long pulsed dye laser (595 nm, 10 mm spot size, 10 ms, 7 J/cm2, 40 ms spray, 40 ms delay, single-pass, 30% overlap) with the other side serving as a control. A total of three treatments were given at 2 monthly intervals. The following sites were treated: periorbital area, six patients; forehead, two patients; cheek, two patients. No preoperative anesthesia or postoperative treatment were used. Clinical photographs were taken before and after each treatment, and analysis was undertaken through photographic evaluation by non-treating physicians. RESULTS: At 2 months after the last treatment, the clinical improvement of rhytids was noted in all patients compared with baseline. Four subjects had mild improvement (< or = 25%), five had moderate improvement (26-50%) and one had marked improvement (51-75%). The periorbital area was more responsive to treatment compared with the other sites. No clinical changes were noted in the control areas. No adverse effects were reported except for transient mild erythema in two patients which lasted for up to an hour. Nine patients were somewhat satisfied with the treatment and one was highly satisfied. All wanted the other half of the face to be treated. CONCLUSION: Treatment with a non-ablative 595 nm flashlamp pulsed dye laser can lead to mild to moderate clinical improvement in class I-II rhytids with minimal to no side effects in patients with darker skin types.