The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway. mammary stem cells | p53 family | breast cancer

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

Obesity is associated with a higher risk of developing many cancer types including acute promyelocytic leukaemia (APL), a subset of acute myeloid leukemias (AML) characterized by expression of the PML-RARα oncogene. The molecular mechanisms linking obesity and APL development are not known. To model clinical observations, we established a mouse model of diet-induced obesity using transgenic mice constitutively expressing PML-RARΑ α in the hematopoietic system (PML-RARα KI mice) fed either standard (SD) or high-fat (HFD) diets. HFD-fed PML-RARα KI mice developed leukaemia with reduced latency and increased penetrance, as compared to SD-fed mice. HFD leads to accumulation of DNA damage in hematopoietic stem cells (HSCs), but, surprisingly, this was not associated with mutational load gain, as shown by whole genome/exome sequencing of pre-leukemic and leukemic cells. Importantly, very few of the observed mutations were predicted to act as cancer drivers, suggesting the relevance of nongenetic mechanisms. HFD led to an expansion of hematopoietic progenitor cells with a concomitant reduction in long-term hematopoietic stem cells, and in the presence of PML-RARα this was also accompanied by an enhancement of in vitro and in vivo self-renewal. Interestingly, Linoleic Acid (LA), abundant in HFD, recapitulates the effect of HFD on the self-renewal of PML-RARα HPCs by activating the peroxisome proliferator-activated receptor delta (PPARδ), a central regulator of fatty acid metabolism involved in the promotion of cancer progression. Our findings have implications for dietary or pharmacological interventions aimed at counteracting the cancer-promoting effect of obesity.

Introduction. There is increasing interest in therapeutic modulation of metabolic pathways in cancer. Tumor cells preferentially use aerobic glycolysis to meet their energetic demands. However, glycolysis inhibition alone is unable to bring durable responses because of limited therapeutic index and because of previously underappreciated metabolic adaptability in tumor cells, which can switch to alternative substrate usage when specific nutrients are limiting. The molecular basis of metabolic adaptation is poorly understood. Recently, the histone demethylase LSD1 (Lysine-Specific Demethylase 1) has been implicated in the control of oxidative phosphorylation (OXPHOS) in adipocytes through its interaction with NRF1 (Nuclear Respiratory Factor 1), a master regulator of metabolic gene transcription (1). We hypothesized that LSD1 could regulate metabolic adaptability and be a therapeutic target upon metabolic modulation through Caloric Restriction (CR) in Acute Myeloid Leukaemia (AML) and specifically in APL (Acute Promyelocytic Leukaemia), which we showed to be sensitive to body fatness in the clinic (2). Methods. APLs were generated in mice expressing the PML-RARa fusion under the control of the Cathepsin G promoter (3). Primary leukemias were transplanted into recipients subjected to 30% CR or Standard Diet (SD). We scored the effect of CR alone or in combination with the LSD1 inhibitor IEO368 (4) on mouse survival, Leukemia Initiating Cell (LIC) frequency and epigenomic, transcriptomic and metabolic parameters. Results. Compared to SD controls, CR-fed recipients experienced an initial dramatic decrease in the total leukemic burden accompanied by cell cycle slowdown (“adaptation phase”); this was followed by a delayed disease progression that brought animals to death (“terminal phase”) (median survival 91 vs 51 days, p=0.038). Limiting-dilution transplantation of CR-conditioned leukemias revealed increased frequency of LICs (estimated frequency 1/3064 cells in SD vs 1/947 in CR, p=0.003) and increased aggressiveness (median survival reduced to 49 vs 70.5 days with 5000 cells injected, p<0.0001). Thus, CR limits the expansion of leukemic cells but enriches for cells with increased ability to regrow. RNAseq of leukemic cells purified during the terminal phase (but not earlier) showed that a dramatic transcriptional reprogramming in CR, characterized by upregulation of genes controlling OXPHOS, Krebs cycle and nucleotide and protein biosynthesis, and downregulation of insulin signaling and glucose transporters. Flow cytometry with Mitotracker Red confirmed increased mitochondrial activity. Thus, leukemic cells exposed to CR put in place adaptive transcriptional changes to allow survival in a nutrient/growth factor deprived environment. To investigate the basis of these transcriptional changes, we revised ChIPseq analysis of LSD1 binding in human APL cell lines and found a significant enrichment for i) NRF1 consensus binding motif and ii) promoters of genes encoding for OXPHOS and Krebs cycle enzymes. NRF1 binding to OXPHOS/Krebs enzymes was confirmed on mouse leukemias by ChIPseq. These data suggested that the CR-induced adaptive changes could be mediated by LSD1/NRF1. Strikingly, co-treatment of leukemic mice with CR and our LSD1 inhibitor IEO368 (4) resulted in macroscopic and microscopic eradication of disease (see figure, p=0.0018 compared to SD). In these conditions, leukemic cells completely disappeared in 4/6 mice after 4 weeks. LSD1 inhibition alone was also effective but did not produce bona fide disease eradication. Importantly, some of the features of the CR-LSD1 interaction could be modeled by combining LSD1 and an IGF1/Insulin inhibitor. In vivo, this combination was synergistic and led to durable responses (median survival 121 vs 50 days in untreated controls, p=0.0143, vs 65.5 and 78.5 days with Insulin/IGF1 Inhibitor and IEO368 respectively). Conclusion: the combination of LSD1 inhibition and insulin/IGF1 signaling reduction by pharmacological or dietary intervention appears as a highly effective therapeutic strategy and deserves further investigation. Ongoing preclinical studies will verify its applicability to other models of AML. References: 1. Duteil et al, Nat Commun. 2014 Jun 10;5:4093 2. Breccia et al, Blood. 2012 Jan 5;119(1):49-54 3. Westervelt et al, Blood. 2003 Sep 1;102(5):1857-65 4. Varasi et a,l Eur J Cancer Vol 50 supp 6: 185 Figure 1. Figure 1. Disclosures Pelicci: Rasna therapeutics: Membership on an entity's Board of Directors or advisory committees.

Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiovascular disease and the leading cause of sudden death in young adults. HCM is characterized by regional ventricular hypertrophy in the absence of cavitary dilation. Myofibrillar disarray associated with mutations of sarcomeric proteins and collagen accumulation are considered major determinants of the disease phenotype. Septal hypertrophy has been viewed as the result of an increase in size of preexisting myocytes, but whether myocyte formation contributes to septal thickening has never been determined. Thus, 29 patients with HCM undergoing septal myectomy were studied, and 10 sex-, age-matched septa, collected from patients who died from causes other than cardiovascular diseases, were used as controls. Mutations of genes coding for contractile proteins were present in all cases of HCM; 79% of patients were in NYHA class III, 17% in class II, and 4% in class IV. Arrhythmic events consisting of atrial fibrillation and ventricular tachycardia were found in 11 patients. In 8 cases, HCM was diagnosed in family members as well. Septal thickness is the product of myocyte diameter and the number of myocyte across the septum. The thickness of the septum increased 2.3-fold in HCM (Controls: 11±1 mm; HCM: 25±8 mm, p<0.001). Septal thickening in HCM was mediated by a 69% increase in myocyte number (Controls: 722±120; HCM: 1,217±375, p<0.001) and a 31% increase in cell diameter (Controls: 16±3 λm; HCM: 21±4 λm, p<0.001). Myocyte regeneration was confirmed by the expression of the cell cycle protein, Ki67. The number of Ki67-positive myocyte nuclei increased 15-fold in HCM (Controls: 400 myocytes/10 6 cells; HCM: 6,000 myocytes/10 6 cells, p<0.001). In humans, myocyte formation is regulated by commitment of resident c-kit-positive cardiac stem cells (CSCs). In comparison with control hearts, the number of CSCs increased 2.7-fold in patients with HCM (Controls: 11±5 cells/mm 3 ; HCM: 30±17 cells/mm 3 , p<0.01). Additionally, the expression of contractile proteins was detected in differentiating CSCs. In conclusion, myocyte regeneration and hypertrophy contribute to septal thickening in HCM and newly formed myocytes originate from growth activation and lineage specification of resident CSCs.

Aging is associated with alterations in the electrical properties of the heart resulting in an increased incidence of arrhythmic events and perturbation of cardiac performance. The aim of the present study was to determine whether the late sodium current (I NaL ), which presents slow inactivation kinetics, is upregulated in myocytes from old hearts contributing to the electrical remodeling of the senescent myocardium. For this purpose, the electrical profile of young (3-5 months) and old (24-30 months) C57BL6 mice was established at the organ and cellular levels. In comparison to young mice, old animals presented enlarged left ventricles and declined systolic and diastolic functions. The QRS complex and QT interval of the ECG were significantly prolonged in old animals, indicating that aging delays the electrical activation and recovery of the myocardium. The latter property was confirmed by slower repolarization phases of epicardial monophasic action potentials (AP) in old hearts, using perfused Langendorff preparations. Moreover, old hearts were more prone to develop arrhythmia by programmed electrical stimulation. By patch-clamp technique, myocytes from old mice showed a 2- and 1.7-fold increase in the time to 50% and 90% repolarization of the AP, respectively, than cells from young animals. In voltage-clamp mode, I NaL , which is operative during the repolarization phase of the AP, was 1.7-fold larger in cells obtained from old hearts with respect to young. Activation, steady state inactivation and time constants for I NaL were comparable in young and old myocytes, indicating that the enhanced late current was mediated by an increased maximal conductance. In old myocytes, blockade of I NaL with low doses of tetrodotoxin or mexiletine reduced by 24-48% the intermediate and late repolarization phases of the AP, whereas in young cells this intervention affected to a lesser extent only the late repolarization phase. Finally, administration of mexiletine in old mice shortened the QT interval by 25%, restoring ventricular electrical recovery. In conclusion, the late sodium current I NaL is upregulated with aging and contributes to the slower repolarization of the senescent myocardium.

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