Key message The circadian clock controls many molecular activities, impacting experimental interpretation. We quantify the genome-wide effects of time-of-day on the heat-shock response and the effects of “diurnal bias” in stress experiments. Abstract Heat stress has significant adverse effects on plant productivity worldwide. Most experiments examining heat stress are performed during daytime hours, generating a ‘diurnal bias’ in the pathways and regulatory mechanisms identified. Such bias may confound downstream interpretations and limit our understanding of the full response to heat stress. Here we show that the transcriptional and physiological responses to a sudden heat shock in Arabidopsis are profoundly sensitive to the time of day. We observe that plant tolerance and acclimation to heat shock vary throughout the day and are maximal at dusk. Consistently, over 75% of heat-responsive transcripts show a time of day-dependent response, including many previously characterized heat-response genes. This temporal sensitivity implies a complex interaction between time and temperature where daily variations in basal transcription influence thermotolerance. When we examined these transcriptional responses, we uncovered novel night-response genes and cis -regulatory elements, underpinning new aspects of heat stress responses not previously appreciated. Exploiting this temporal variation can be applied to most environmental responses to understand the underlying network wiring. Therefore, we propose that using time as a perturbagen is an approach that will enhance our understanding of plant regulatory networks and responses to environmental stresses. Electronic supplementary material The online version of this article (10.1007/s11103-019-00873-3) contains supplementary material, which is available to authorized users.

CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential. In this study, we hypothesized that combining 5F9 with azacytidine (AZA) would enhance therapeutic efficacy against AML. AZA (Vidaza®) is a hypomethylating and chemotherapeutic agent indicated for AML. AZA's anti-cancer mechanism of action is believed to be twofold, the first being induction of DNA demethylation and the second being its anti-metabolite activity. Interestingly, it has also been found that AZA can increase the expression of the anti-phagocytic signal, CD47, and the pro-phagocytic signal, calreticulin, in myeloid malignancies. Based on these previous findings, we hypothesized that AML cells may be more efficaciously eliminated using a combination of AZA and 5F9 through enhancement of AML cell phagocytosis. We first tested this hypothesis using an in vitro phagocytosis assay. AML cells (i.e. GFP-expressing HL60 cells) were incubated for 24 hours with 3µM AZA and afterwards, the HL60-GFP cells were co-cultured for 2 hours with either human macrophages plus IgG4 control or 5F9 (10µg/ml). Phagocytosis of HL60 AML cells was calculated as a percentage of GFP-positive macrophages (i.e. the amount of macrophages that engulfed GFP-positive HL60 cells), compared to total number of macrophages. Results were normalized to a condition that produced the maximum amount of phagocytosis (100%). We found that the combination of AZA with 5F9 enhanced the human macrophage-mediated phagocytic elimination of HL60-GFP cells compared to either agent alone (Fig. 1). Next, we asked whether we could confirm our in vitro findings in vivo utilizing an aggressive AML xenograft mouse model. HL60-GFP cells (500,000 cells/per mouse) expressing luciferase were engrafted by intravenous injection into 6 - 8 week old immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Three days post engraftment (PE), bioluminescence imaging was performed to assess AML engraftment based on total flux (photons/sec). Animals were randomized based on these values into 6 treatment cohorts with 8 animals per group. Treatment was performed as follows: (1) control (PBS) was initiated on day 4 PE and continued for 14 consecutive daily doses; (2) AZA (7.5 mg/kg) was initiated on day 4 PE and continued for 5 consecutive daily doses; (3) two cohorts of 5F9 (10mg/kg) were initiated at day 4 or day 7 PE and continued for 14 consecutive daily doses; and (4) two combination cohorts of AZA with 5F9 were initiated according to the 5F9 monotherapy dosing regimens. Routine bioluminescence imaging was performed during treatment and for several months after to assess AML burden and reoccurrence. Both combination cohorts inhibited AML growth as early as day 10 PE, and maintained elimination of growth and overall survival up to 255 days PE. In contrast, the AZA and 5F9 monotherapies initiated at day 7 PE (D7), decreased AML growth at day 10 PE, but failed to produce a durable response. Notably, as the AML expanded, all animals from the AZA cohort died by 46 days PE, and all animals from the 5F9 cohort died by 61 days PE. Of the 8 animals from the 5F9 cohort that received treatment on day 4 PE, only two animals demonstrated progressive disease and did not survive. The remaining animals from this cohort had no detectable AML cancer cells (Fig 2). In summary, the combination of 5F9 with AZA significantly enhanced the phagocytic elimination of AML cells by human macrophages in vitro, enhanced clearance of AML in vivo, and prolonged survival compared to single agent treatment with AZA or 5F9. These results support the rationale for investigating a combinatorial treatment of 5F9 and AZA in patients with AML. A clinical trial with this combination in patients with AML is currently ongoing (NCT03248479). Disclosures Feng: Forty Seven Inc: Employment, Equity Ownership. Gip:Forty Seven Inc: Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Zhao:Forty Seven Inc: Consultancy. Mata:Forty Seven Inc: Employment, Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc: Consultancy, Equity Ownership. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

We recently identified neuregulin-1 (NRG1) as a novel target of Notch1 required in Notch-dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1-dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1-ERBB3-ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.

Objectives: The present study aims to identify the genotype-phenotype correlation in children with Peutz-Jeghers Syndrome (PJS) through the analysis of STK11 gene mutations in the context of clinical and pathological characteristics. Method: In this observational cohort study, the clinical characteristics of 18 families diagnosed with pediatric PJS were collected. Genomic DNA from the peripheral blood of affected children and their family members was collected. The coding region of STK11 was amplified by PCR and screened for mutation by Sanger sequencing. The families that were negative for STK11 mutation were further assessed by multiplex ligation-dependent probe amplification (MLPA). Result: Initial presentation in affected children was at 1.6 to 14.2 years and included anemia in 8 patients whereas 6 presented for screening by virtue of family history. All patients underwent endoscopy, colonoscopy, and polypectomy. Polyps were distributed throughout the gastrointestinal (GI) tract, including the small intestine, stomach, colon, and rectum. In the 18 pediatric PJS families, STK11 mutations were detected in 8 families by Sanger sequencing, and large deletions were detected in 3 by MLPA, respectively. Nine of the 11 STK11 mutations were de novo, 3 were novel (c.419T>C:p.L140P, c.314T>G:p.L105X), and (c.488_489insACGG p.L164fs). Conclusions: Although the main clinical features of pediatric PJS were similar to those of PJS cases in adults, a high frequency of STK11 de novo mutations were encountered in our population of patients with PJS.