Emilie Bousquet Mur

Sara Bernardo

Laura Papon

Maicol Mancini

Eric Fabbrizio

Marion Goussard

Irene Ferrer

Anais Giry

Xavier Quantin

Jean-Louis Pujol

Olivier Calvayrac

Herwig P Moll

Yaël Glasson

Nelly Pirot

Andrei Turtoi

Marta Cañamero

Kwok-Kin Wong

Yosef Yarden

Emilio Casanova

Jean-Charles Soria

Jacques Colinge

Christian W Siebel

Julien Mazieres

Gilles Favre

Luis Paz-Ares

PhD Antonio Maraver
email: antonio.maraver@inserm.fr

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma

EGFR mutated lung adenocarcinoma patients treated with gefitinib and osimertinib showed a therapeutic benefit limited by the appearance of secondary mutations, such as EGFR T790M and EGFR C797S . It is generally thought that these secondary mutations render EGFR completely unresponsive to such inhibitors. Here, we found that gefitinib and osimertinib increased STAT3 phosphorylation (pSTAT3) in lung adenocarcinoma cells harboring the EGFR T790M and the EGFR C797S mutations. Moreover, concomitant treatment with a Notch inhibitor and gefitinib or osimertinib strongly reduced the expression of the transcriptional repressor HES1, in a pSTAT3-dependent manner.

Importantly, we show that tyrosine kinase inhibitor resistant tumors, with EGFR T790M and EGFR C797S mutations, are highly responsive to treatment with the combination of Notch inhibitors and gefitinib or osimertinib, respectively. Finally, in patients with lung adenocarcinoma harboring EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse, and high HES1 expression level correlated with shorter progression-free survival. Our results offer a proof of concept for a novel alternative to treat patients with lung adenocarcinoma after disease progression following osimertinib treatment.

INTRODUCTION

Lung cancer kills about a million people every year worldwide being the leading cause of death by cancer in the world. There are two main types of lung cancer: small cell lung carcinoma (about 20% of all lung cancers) and non-small-cell lung carcinoma that includes lung adenocarcinoma (40%), lung squamous carcinoma (30%), and large cell carcinoma (about 10% of all lung cancers) [START_REF] Herbst | Lung cancer[END_REF]. EGFR gene alterations are detected in about 20% of patients with lung adenocarcinoma in Western countries, and in up to 50% in some Asian countries, such as Korea. The most common EGFR alterations in lung adenocarcinoma are exon 19 deletion and the activating EGFR L858R mutation [START_REF] Rosell | Genetics and biomarkers in personalisation of lung cancer treatment[END_REF].

The life expectancy of patients has improved dramatically thanks to the development of EGFR tyrosine kinase inhibitors (TKI) [START_REF] Reck | Management of nonsmall-cell lung cancer: recent developments[END_REF]. Most of the patients treated with firstgeneration TKIs (i.e., gefitinib and erlotinib) initially respond well; however, their tumors rapidly develop resistance. This is explained, in about 60% of cases, by acquisition of the so-called 'gatekeeper' mutation EGFR T790M [START_REF] Tan | Treatment approaches for EGFR-inhibitorresistant patients with non-small-cell lung cancer[END_REF]. More recently, thirdgeneration TKIs that target EGFR T790M , such as osimertinib, have shown a very good therapeutic effect in patients with this mutation [START_REF] Janne | AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer[END_REF]. Unfortunately, tumors from patients treated with osimertinib also become resistant to this drug. In about 30% of cases, this is due to appearance of new gatekeeper mutations, such as EGFR C797S [START_REF] Thress | Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M[END_REF][START_REF] Le | Landscape of EGFR -dependent and -independent resistance mechanisms to osimertinib and continuation therapy post-progression in EGFR-mutant NSCLC[END_REF]. Thus, treatment of EGFR-driven lung adenocarcinoma with a single TKI might have limited value, and strategies based on combinational drug therapies could be more effective for controlling the effects of gatekeeper mutations.

The EGFR T790M gatekeeper mutation confers resistance to TKIs through different mechanisms, such as weaker drug binding due to steric hindrance and increased ATP affinity of mutated EGFR [START_REF] Yun | The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP[END_REF]. However, gefitinib binding to EGFR T790 protein is reduced, but not totally inhibited [START_REF] Yun | The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP[END_REF]. Moreover, X-ray structure analysis indicates that Bousquet Mur et al., submitted to Journal of Clinical Investigation 5 gefitinib binds in a similar manner to wild type EGFR and to EGFR T790M proteins [START_REF] Yoshikawa | Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor[END_REF].

Therefore, we hypothesized that in tumors with gatekeeper EGFR mutations, gefitinib could still affect EGFR downstream signaling pathways, although not reaching therapeutic effect, and that this could be exploited by concomitantly inhibiting other signaling pathways.!

The Notch signaling pathway is highly conserved among metazoans, and it is important during embryonic development as well as adult tissue homeostasis. In mammals, there are four NOTCH receptors (NOTCH1 to 4) that are activated upon interaction with their transmembrane ligands (DELTA and JAGGED). For this activation to occur, an intramembrane protease called γ-secretase, releases the Notch intracytoplasmic domain (NICD). Upon nuclear translocation and binding to its DNA binding partner RBPJ, NICD modulates the expression of target genes of the canonical Notch pathway, such as HES1 [START_REF] Bray | Notch signalling in context[END_REF]. Therefore, the Notch pathway can be inhibited by γsecretase inhibitors (GSIs), or by antibodies against the Notch ligands and receptors [START_REF] Takebe | Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update[END_REF].

By making use of genetically engineered mouse models, we and others demonstrated that KRAS-driven lung adenocarcinoma is dependent on Notch activity [START_REF] Licciulli | Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53[END_REF][START_REF] Baumgart | Opposing role of Notch1 and Notch2 in a Kras-driven murine non-small cell lung cancer model[END_REF][START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF]. Moreover, seminal in vitro and in vivo (mouse xenografts) studies using lung cancer cell lines showed that the combination of Notch inhibitors and EGFR TKIs gives a better anti-tumor response than single treatments in sensitive cells [START_REF] Konishi | Notch3 cooperates with the EGFR pathway to modulate apoptosis through the induction of bim[END_REF][START_REF] Arasada | EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling[END_REF][START_REF] Hu | Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumorinitiating cell frequency[END_REF]. However, the underlying mechanism is not fully understood, and the role of the Notch pathway in lung adenocarcinoma after relapse due to acquisition of gatekeeper mutations in EGFR remains largely unknown.

Here, to find some answers to these questions, we first performed a transcriptomic analysis in human lung adenocarcinoma PC9GR cells harboring the EGFR T790M gatekeeper mutation, and found that after gefitinib treatment, several pathways, including the KRAS signaling pathway, were downregulated. Therefore, based on our previous results concerning the importance of Notch in KRAS-driven lung adenocarcinoma [START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF], we tested the combined effect of TKI and Notch inhibition in different preclinical mouse models of lung adenocarcinoma with the EGFR T790M/L858R mutations. We found that in vivo Notch inhibition overcame resistance to gefitinib in human and murine lung adenocarcinoma cells via pSTAT3 binding to the HES1 promoter and HES1 decreased expression. Similarly, Notch inhibition re-sensitized also in vivo human lung adenocarcinoma cells harboring the EGFR C797S mutation, that cause resistance to osimertinib, which might become soon the first-line treatment for patients with EGFR-driven lung adenocarcinoma. Altogether, our data show that concomitant Notch inhibition with TKI, could be a potent strategy to treat EGFR-driven lung adenocarcinoma patients after TKI relapse.

RESULTS

Gefitinib treatment in human lung adenocarcinoma cells with the gatekeeper mutation EGFR T790M induces changes in several cancer-associated genetic signatures

To identify molecular changes upon gefitinib treatment in lung cancer cells harboring the EGFR T790M mutation that confers resistance to first-generation TKIs, we used the already described human EGFR-driven lung adenocarcinoma PC9GR cell line (EGFR T790M ) resistant to gefitinib [START_REF] Mancini | An oligoclonal antibody durably overcomes resistance of lung cancer to thirdgeneration EGFR inhibitors[END_REF]. Gene set enrichment analysis using the 1). Accordingly, among the eight downregulated gene sets in gefitinib-treated cells, we found "HALLMARK_KRAS_SIGNALING_UP" (Figure 1A and Supplemental Table 1).

This suggests that in PC9GR cells, gefitinib decreases the activity of the KRAS signaling pathway, a well-known EGFR downstream pathway [START_REF] Arteaga | ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics[END_REF].

We previously reported that the Notch pathway plays a major role in KRASdriven lung adenocarcinoma, and that its inhibition fully stops tumor growth in this setting [START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF]. Therefore, we hypothesized that gefitinib effects in PC9GR cells harboring the EGFR T790M gatekeeper mutation could be enhanced by Notch inhibition.

Inhibition of Notch signaling hampers tumor growth in EGFR T790M/L858R mice

Before directly testing this hypothesis, we studied the Notch pathway activation in EGFR-driven lung tumors in vivo, by crossing EGFR T790M/L858R [START_REF] Li | Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy[END_REF] and lung-specific CCSP-rtTA transgenic mice [START_REF] Tichelaar | Conditional expression of fibroblast growth factor-7 in the developing and mature lung[END_REF] to obtain mice in which EGFR T790M/L858R expression in lungs can be induced by treatment with doxycycline (EGFR T790M/L858R mice, hereafter).

After 8 weeks of doxycycline treatment, mice developed bronchial and peripheral EGFR T790M/L858R -driven tumors that are resistant to first-generation EGFR TKIs, such as gefitinib [START_REF] Li | Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy[END_REF]. Western blot analysis showed that N1ICD, the processed and active form of NOTCH1, and HES1, a Notch target gene, were strongly expressed in EGFR T790M/L858R -driven tumors compared with normal lung tissue from control mice (either littermates with the same genotype but not treated with doxycycline, or CCSP-rtTA transgenic mice treated with doxycycline) (Figure 1B). This finding is similar to what observed in the Kras G12V mouse model ( 14), and suggests that the Notch pathway may play a similar role in both tumor types.

As the NOTCH1 and NOTCH3 receptors promote Kras G12V -driven lung adenocarcinoma, whereas NOTCH2 has a tumor suppressive role [START_REF] Licciulli | Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53[END_REF][START_REF] Baumgart | Opposing role of Notch1 and Notch2 in a Kras-driven murine non-small cell lung cancer model[END_REF][START_REF] Zheng | A rare population of CD24(+)ITGB4(+)Notch(hi) cells drives tumor propagation in NSCLC and requires Notch3 for self-renewal[END_REF], we analyzed their expression in EGFR T790M/L858R -driven lung adenocarcinoma. The transmembrane forms of NOTCH1 and NOTCH3 (i.e., before γ-secretase cleavage)

were strongly expressed in tumor samples compared with controls (Figure 1B), whereas NOTCH2 expression was comparable in both groups (Figure 1B). Although the level of the transmembrane forms of NOTCH receptors does not reflect Notch activity, and NOTCH3 can be a direct target of NOTCH1 in some circumstances, this finding suggests that both NOTCH1 and NOTCH3 are mediators of the Notch pathway in EGFR-driven tumors in vivo.

To test whether Notch pathway activity is necessary for the growth of EGFRdriven tumors, we treated EGFR T790M/L858R mice with doxycycline for 8 weeks to induce tumor formation, and then randomly assigned them to three groups: i) control group, treated with vehicle and IgG antibody control; ii) GSI group, treated with dibenzazepine (DBZ), a potent and selective GSI; and iii) anti-NRR1/NRR3 group, treated with blocking antibodies against NOTCH1 and NOTCH3, according to previously described treatment regimens [START_REF] Choy | Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers[END_REF][START_REF] Rivera-Torres | Targeting gamma-secretases protect against angiotensin II-induced cardiac hypertrophy[END_REF][START_REF] Wu | Therapeutic antibody targeting of individual Notch receptors[END_REF]. After five weeks of treatment, tumors represented more than 40% of the lung area in the control group, but only 20% and 10% in the DBZ and anti-NRR1/NRR3 groups, respectively (Figure 1C). This indicates that the Notch pathway is required for EGFR T790M/L858R -driven tumor growth. Body weight was comparable in the three groups (Supplemental Figure 1A), suggesting the absence of the intestinal toxicity reported by other studies using regimens that led to stronger Notch inhibition [START_REF] Van Es | Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells[END_REF].

As expected, analysis of protein expression by immunohistochemistry (IHC) of tumors from anti-NRR1/NRR3-or DBZ-treated mice showed fewer HES1positive cells than in the vehicle-treated control group, implying that these treatments effectively inhibited the Notch pathway (Figure 1D). Moreover, the percentage of Ki67positive cells was lower in tumors from the anti-NRR1/NRR3 and DBZ groups than the control group, indicating that Notch activity promotes cell proliferation in EGFR T790M/L858R -driven tumors (Figure 1D). As the MAPK and AKT pathways are crucial downstream players of the EGFR signaling pathway [START_REF] Arteaga | ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics[END_REF], we also analyzed the expression of pERK and pAKT in the same samples. The percentage of pERK-positive cells was similarly reduced by treatment with the anti-NRR1 and -NRR3 antibodies and with DBZ compared control (Figure 1D), consistent with previous observations [START_REF] Baumgart | Opposing role of Notch1 and Notch2 in a Kras-driven murine non-small cell lung cancer model[END_REF][START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF]. Conversely, the percentage of pAKT-positive cells was comparable in all groups (Supplemental Figure 1B).

Notch inhibition overcomes resistance to gefitinib in EGFR T790M/L858R -driven lung adenocarcinoma

To study whether pharmacological inhibition of the Notch pathway in vivo had any impact on the resistance to gefitinib conferred by the gatekeeper mutation EGFR T790M , we randomized EGFR T790M/L858R mice (after 8 weeks of doxycycline treatment) in four treatment groups: i) vehicle (control), ii) gefitinib, iii) DBZ, and iv) gefitinib + DBZ.

For simplicity we decided to inhibit the Notch pathway hereafter only with a GSI.

As before, body weight was comparable in the different groups after the five weeks of treatment, suggesting that these drugs were well tolerated alone or in combination (Supplemental Figure 2A). In agreement with the previous findings (Figure 1C), tumor tissue occupied 42% of the lung in the control group, whereas it was decreased to 23% in the DBZ group (Figure 2A). As expected, gefitinib alone did not have any anti-tumor effect in EGFR T790M/L858R mice (52% of lung was tumor tissue).

Conversely, the DBZ and gefitinib combination led to a very significant reduction of the tumor area compared with DBZ alone (tumor tissue covered only 10% of the total lung area) (Figure 2A).

Histopathological analysis of lung adenocarcinoma samples (i.e., non-benign tumors, Supplemental Figure 2B) showed that the single treatments had no effect on the lung adenocarcinoma number compared with control (i.e., vehicle-treated mice) (Figure 2B). Importantly, animals treated with the combination of gefitinib and Notch inhibition had significantly fewer lung adenocarcinomas than vehicle-treated ones (a mean of 10 lung adenocarcinoma per mouse vs 31 in the control, Figure 2B).

IHC analysis showed that the percentage of HES1-, Ki67-, pERK-and pAKTpositive cells was comparable in tumors from the gefitinib group and from controls (Figure 2C). By contrast, the percentage of HES1-, Ki67-and pERK-positive cells was reduced in tumors from DBZ-treated mice (Figure 2C), as before (Figure 1D), although in this case the difference was not significant for pERK. The percentage of HES1-, Ki67-and pERK-positive cells tended to be lower in mice treated with the gefitinib and DBZ combination compared with DBZ-treated mice, particularly for pERK. Finally, the percentage of pAKT-positive cells was comparable in the DBZ, gefitinib and control groups, but interestingly, it was significantly reduced in the gefitinib + DBZ group compared with control mice (Figure 2C).

Altogether, these data demonstrate that inhibition of Notch signaling by DBZ restores sensitivity to treatment with gefitinib in EGFR T790M/L858R -driven lung adenocarcinoma in vivo.

Notch inhibition overcomes resistance to gefitinib in lung adenocarcinoma patientderived xenografts with EGFR T790M/L858R mutations

These results were very encouraging; however, it is considered that the best strategy for testing new cancer treatments is the combination of genetic mouse models and patientderived xenograft (PDX) preclinical models [START_REF] Day | Preclinical mouse cancer models: a maze of opportunities and challenges[END_REF]. Therefore, we developed a lung adenocarcinoma PDX that harbors the EGFR T790M/L858R mutations, like our transgenic mouse model. One week after subcutaneous grafting of the PDX, nude mice were randomized in four groups as before: i) vehicle alone (control), ii) gefitinib, iii) DBZ, and iv) gefitinib + DBZ. Tumor growth was monitored for 30 days (i.e., the treatment duration). As expected, the EGFR T790M mutation conferred resistance to gefitinib. On the other hand, DBZ inhibited tumor growth, and strikingly, the DBZ and gefitinib combination almost totally blocked tumor growth (Figure 3A).

As before, IHC analysis of tumors showed that DBZ (alone or in combination with gefitinib) efficiently decreased the percentage of HES1-positive cells compared with control (Figure 3B). Tumor cell proliferation (Ki67-positive cells) was reduced by DBZ alone, and this effect was increased by addition of gefitinib. Similarly, the

Combining EGFR TKIs and Notch inhibitors synergistically decreases HES1 expression

Our previous analysis showed that the DBZ and gefitinib combination is more efficient than each single treatment in reducing MAPK and AKT pathways. Previous reports, including work from our laboratory, identified HES1 as an important positive MAPK regulator in KRAS-driven lung adenocarcinoma [START_REF] Baumgart | Opposing role of Notch1 and Notch2 in a Kras-driven murine non-small cell lung cancer model[END_REF][START_REF] Maraver | Notching up a new therapeutic strategy for Non-Small Cell Lung Carcinoma (NSCLC)[END_REF]. Even more, HES1 has a similar effect on AKT signaling in T-cell acute lymphoblastic leukemia (T-ALL) [START_REF] Palomero | Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia[END_REF].

Therefore, we hypothesized that HES1 could be an important mediator of pERK and pAKT upon treatment with the DBZ and gefitinib combination. As the percentage of HES1-positive cells was similar in tumors from mice treated with DBZ alone and the DBZ and gefitinib combination in both preclinical models (Figure 2C and Figure 3B), we analyzed HES1 signal intensity in the same samples. Importantly, HES1 signal intensity was significantly lower in tumors from mice treated with the DBZ and gefitinib combination than from mice treated with DBZ alone in the PDX model, and followed a similar trend in EGFR T790M/L858R mice (Figure 4, A and B).

To further validate our data, we analyzed HES1 expression by western blotting in PC9GR cells (previously used for the RNA-seq analysis, Figure 1A and Supplemental Table 1) after incubation with the different drugs alone or in combination.

In accordance to our in vivo observation, HES1 expression was strongly reduced in cells exposed to the DBZ and gefitinib combination (Figure 4C).

Then, to explore HES1 role in PC9GR cells, we depleted HES1 using a pool of siRNAs targeting HES1 mRNA (siHES1) (Supplemental Figure !3). Of note, proliferation of siHES1-treated cells was impaired compared with control cells transfected with the non-targeted siRNA (siNT), and this effect was potentiated in the presence of gefitinib (Figure 4D).

To test whether gefitinib effect was EGFR-mediated, we used the Chinese hamster ovary (CHO) cell line that is a natural null for EGFR, and was previously used for EGFR gain of function analyses [START_REF] Lo | Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway[END_REF]. Interestingly, HES1 expression was not affected by co-treatment with DBZ and gefitinib in CHO cells transfected with empty vector, but was reduced in CHO cells that express EGFR T790M/L858R protein (Figure 4E).

We concluded that EGFR is needed for HES1 expression reduction by the DBZ and gefitinib combination.

Taken together, our data indicate that the DBZ and gefitinib combination synergistically reduces the expression of HES1, a major driver in lung adenocarcinoma.

pSTAT3 directly binds to the HES1 promoter and inhibits its expression

Previous studies have shown a benefit of combining EGFR TKIs and Notch inhibitors in TKI-sensitive cells, but the underlying mechanism was not fully described [START_REF] Konishi | Notch3 cooperates with the EGFR pathway to modulate apoptosis through the induction of bim[END_REF][START_REF] Arasada | EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling[END_REF][START_REF] Hu | Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumorinitiating cell frequency[END_REF].

On the basis of the EGFR-dependent HES1 decrease in EGFR T790M/L858R -expressing CHO cells upon incubation with the DBZ and gefitinib combination, we hypothesized that a common mechanism could be involved in the response to TKI treatment in TKIsensitive and -resistant lung adenocarcinoma cells. An increase in the phosphorylation of STAT3 protein (pSTAT3), dependent on both JAK and FGFR activities, is reported in sensitive lung adenocarcinoma cells upon treatment with first-generation (erlotinib)

and second-generation (afatinib) TKIs [START_REF] Kim | Activation of IL-6R/JAK1/STAT3 signaling induces de novo resistance to irreversible EGFR inhibitors in non-small cell lung cancer with T790M resistance mutation[END_REF][START_REF] Lee | Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells[END_REF][START_REF] Codony-Servat | Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC)[END_REF], hence, we investigated whether this occurred also in TKI-resistant cells.

Indeed, analysis of STAT3 phosphorylation status in PC9GR cells showed an increase in pSTAT3 levels upon gefitinib treatment (Figure 5A). This effect was partially inhibited by co-treatment with PD173074 or ruxolitinib, pan-inhibitors of FGFR and JAK pathways respectively. Even more, the combination of both inhibitors reduced pSTAT3 to levels lower than in control non-treated cells (Supplemental Figure 4). Moreover, we found that in the human HES1 and mouse Hes1 gene promoters, consensus binding sites for pSTAT3 (i.e., TTNNNNNAA) [START_REF] Darnell | STATs and gene regulation[END_REF] are close to RBPJ sites (i.e., where the Notch transcription complex binds) (Supplemental Figure 5, A and B).

To test whether pSTAT3 binds directly to the human HES1 promoter in PC9GR cells, we performed chromatin immunoprecipitation (ChIP) experiments using antibodies against pSTAT3 and against NOTCH1, which is known to bind to the HES1 promoter (positive control). NOTCH1 bound to the HES1 promoter, and this interaction was reduced by incubation with DBZ (Figure 5B). Importantly, pSTAT3 bound to the HES1 promoter only when cells were co-incubated with gefitinib and DBZ (Figure 5B). To determine whether pSTAT3 binding was critical for HES1 downregulation (Figure 4C), we incubated PC9GR cells with the various drug combinations after siSTAT3 treatment that efficiently reduced both pSTAT3 and STAT3 expression (Figure 5C). Co-incubation with gefitinib and DBZ strongly reduced HES1 protein level in control siNTtreated cells (Figure 5C), but strikingly, the same co-treatment kept HES1 levels in siSTAT3-treated cells (Figure 5C).

Altogether, these findings support that pSTAT3 decreases HES1 protein level by acting as a transcriptional repressor at the HES1 promoter.

Notch inhibition overcomes resistance to osimertinib in human lung adenocarcinoma cells harboring the EGFR C797S mutation

As various TKIs increase pSTAT3 levels in lung adenocarcinoma cells [START_REF] Kim | Activation of IL-6R/JAK1/STAT3 signaling induces de novo resistance to irreversible EGFR inhibitors in non-small cell lung cancer with T790M resistance mutation[END_REF][START_REF] Lee | Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells[END_REF][START_REF] Codony-Servat | Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC)[END_REF], we asked whether the pSTAT3-dependent mechanism observed for gefitinib applied also to osimertinib. To this aim, we used the PC9GROR cell line (previously generated from PC9GR cells) that is resistant to osimertinib and harbor the gatekeeper mutation EGFR C797S [START_REF] Mancini | An oligoclonal antibody durably overcomes resistance of lung cancer to thirdgeneration EGFR inhibitors[END_REF].

First, western blot analysis of PC9GROR cells incubated with DBZ and/or osimertinib showed that pSTAT3 levels increased upon osimertinib treatment.

Accordingly, the combination of DBZ and osimertinib reduced HES1 protein levels (Figure 6A).

To test whether DBZ re-sensitized EGFR C797S mutant human lung adenocarcinoma cells to osimertinib in vivo, we grafted PC9GROR cells subcutaneously in mice, and two weeks later, we treated them with DBZ and/or osimertinib for 3 weeks.

Body weight remained comparable in the different treatment groups (Supplemental Figure 6A). Osimertinib alone had no significant effect on growth of PC9GROR cell xenografts (Figure 6B), while it strongly inhibited the growth of PC9GR xenografts (Supplemental Figure 6B). Similarly, DBZ showed no effect on growth of PC9GROR cell xenografts, but importantly, tumor growth was strongly inhibited in mice treated with the osimertinib and DBZ combination (Figure 6B).

This finding demonstrates that treatment with DBZ restores sensitivity to osimertinib in human lung adenocarcinoma cells harboring the EGFR C797S mutation, confirming and extending our previous observations that DBZ sensitizes TKI-resistant tumors to TKIs.

Nirogacestat overcomes resistance to gefitinib in human lung adenocarcinoma cells harboring the EGFR T790M mutation

To strengthen the translational impact of our work, we wanted to confirm the Notch inhibitor sensitizing effect using a GSI under clinical trials. We chose nirogacestat because a recently finished phase 2 trial, showed that it has promising effects in patients with desmoid tumors, is well tolerated, and can be used for long-term treatments [START_REF] Kummar | Clinical Activity of the gamma-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis)[END_REF].

We randomized mice with subcutaneous PC9GR cell xenografts in six treatment groups: i) vehicle, ii) DBZ, iii) nirogacestat, iv) gefitinib, v) DBZ + gefitinib, vi) and nirogacestat + gefitinib. As gefitinib has some effect in PC9GR cells in vitro (Figure 4D), we used 10 mg/kg instead of the previously used dose of 20mg/kg. This lower concentration had a mild, non-significant effect on tumor growth compared with vehicle. Like in PC9GROR cells, the GSIs alone (DBZ and nirogacestat) did not have any effect. Conversely, gefitinib in combination with DBZ or nirogacestat strongly inhibited tumor growth (Figure 7A), as observed in mice harboring PDX and EGFR T790M/L858R -driven tumors treated with the gefitinib and DBZ combination.

Moreover, Kaplan-Meier survival analysis of mice treated or not with nirogacestat and/or gefitinib showed that survival rate was comparable in mice treated with vehicle, nirogacestat or gefitinib alone, although it tended to be higher in the gefitinib group (Figure 7B). By contrast, the nirogacestat with gefitinib combination increased survival compared with all other groups (median survival after treatment started: 24, 26.5, 32, and 39 days for vehicle, nirogacestat, gefitinib, and nirogacestat + gefitinib, respectively). For this analysis, we used only nirogacestat because at the used dose we could administer DBZ only for 5 weeks (26), while nirogacestat is well tolerated in patients for more than 2 years [START_REF] Kummar | Clinical Activity of the gamma-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis)[END_REF]. As before, body weight was not significantly different in all groups during the experiment (Supplemental Figure 7).

These results show that the combination of gefitinib and nirogacestat increases the survival of mice xenografted with human lung adenocarcinoma cells that carry the EGFR T790M mutation conferring resistance to EGFR TKIs.

High HES1 protein levels correlate with poor progression-free survival and relapse in patients with EGFR mutated lung adenocarcinoma treated with TKIs

Our findings showed that HES1 has a key role in the resistance of EGFR-driven lung adenocarcinoma to TKI therapy. To strengthen this observation, we analyzed the correlation between progression-free survival (PFS) and nuclear HES1 protein levels in 75 patients with lung adenocarcinoma harboring EGFR mutations and treated with TKIs. We found that patients with low nuclear HES1 expression had a median PFS of 14 months, whereas patients with high nuclear HES1 expression had a median PFS of 7 months (hazard ratio 2.77, 95% CI [1.4-5.5], p = 0.006) (Figure 7C). Moreover, analysis of HES1 protein in tumor biopsy samples from patients with lung adenocarcinoma harboring EGFR activating mutations and treated with TKIs taken at diagnosis and after disease progression showed that HES1 nuclear levels were increased in samples obtained at relapse in six of the seven patients (p = 0.034) (Figure 7D and Supplemental Figure 8).

These findings extend our previous study [START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF], and suggest a crucial role for HES1 in the relapse of patients with EGFR-driven lung adenocarcinoma under treatment with TKIs.

DISCUSSION

In this study, we confirmed and extended the observation that HES1 is a crucial mediator of the Notch pathway oncogenic activity in lung adenocarcinoma and uncovered its crucial role in resistance to EGFR TKIs.

We first observed that in mouse models of EGFR-driven lung adenocarcinoma, treatment with GSIs leads to decreased HES1 expression and pERK levels. This is consistent with studies in KRAS-driven lung adenocarcinoma mouse models showing that HES1-induced repression of DUSP1 increases pERK levels [START_REF] Baumgart | Opposing role of Notch1 and Notch2 in a Kras-driven murine non-small cell lung cancer model[END_REF][START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF]. Therefore, we hypothesize that this is the same mechanism for the antitumor effect of Notch inhibitors when used as single treatment in EGFR-driven tumors.

Next, as a proof of concept of sensitizing cells with EGFR gatekeeper mutations to TKIs upon Notch inhibition, we found that murine and human EGFR-driven lung tumors harboring the EGFR T790M gatekeeper mutation sensitized to gefitinib when it is administered in combination with the GSI DBZ. Concomitantly, pAKT and pERK were further decreased upon combined treatment with gefitinib and DBZ, compared with the other treatments. It is known that HES1 represses PTEN and increases AKT activity in T-ALL [START_REF] Palomero | Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia[END_REF], and represses DUSP1 and increases pERK levels in lung adenocarcinoma [START_REF] Maraver | Notching up a new therapeutic strategy for Non-Small Cell Lung Carcinoma (NSCLC)[END_REF]. Interestingly, we found in preclinical mouse models a higher HES1 decreased expression after treatment with the DΒΖ and gefitinib combination compared with DBZ alone. Moreover, HES1 loss of function sensitized human lung adenocarcinoma cells harboring the EGFR T790M mutation to gefitinib through a still unknown mechanism. In T-ALL, HES1 directly represses the BBC3 gene that encodes PUMA, an inducer of apoptosis [START_REF] Schnell | Therapeutic targeting of HES1 transcriptional programs in T-ALL[END_REF]. Therefore, it is tempting to speculate that in lung adenocarcinoma cells, HES1 could repress BBC3 or other genes encoding apoptosis-related factors, such as BCL2L11 (i.e., BIM), crucial for gefitinib-induced cell death (39-41). The EGFR T790M mutation does not totally inhibit gefitinib binding to EGFR [START_REF] Yun | The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP[END_REF][START_REF] Yoshikawa | Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor[END_REF], and mutated EGFR is needed in CHO cells to reduce HES1 expression upon coincubation with gefitinib and DBZ compared with DBZ alone. As the level of active, phosphorylated STAT3 is increased upon treatment with first-and second-generation TKIs [START_REF] Kim | Activation of IL-6R/JAK1/STAT3 signaling induces de novo resistance to irreversible EGFR inhibitors in non-small cell lung cancer with T790M resistance mutation[END_REF][START_REF] Lee | Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells[END_REF][START_REF] Codony-Servat | Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC)[END_REF], we hypothesized that this feature could explain the decreased levels of HES1 in our experimental setting. Indeed, incubation of EGFR T790M lung adenocarcinoma cells with gefitinib increased pSTAT3 levels in an FGFR-and JAKdependent manner. Moreover, pSTAT3 directly interacted with the HES1 promoter by ChIP analysis only when cells were gefitinib and DBZ co-treated. Finally, in PC9GR cells transfected with siRNAs against STAT3, HES1 levels were comparable in cells incubated with the combination of gefitinib and DBZ and with DBZ alone. These findings indicate that pSTAT3 represses HES1 expression effectively only when NOTCH processing is concomitantly inhibited, probably because the NOTCH transcriptional complex binds more efficiently to the HES1 promoter than pSTAT3. A previous study showed that in sensitive EGFR-driven lung adenocarcinoma cells, erlotinib promotes Notch pathway induction after several days [START_REF] Arasada | EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling[END_REF]. We did not observe such induction, and this discrepancy could be due to the different treatment kinetics and/or the resistant background of PC9GR cells. Our data are in accordance with previous studies showing that pSTAT3 acts as a transcriptional repressor [START_REF] Zhang | STAT3 restrains RANK-and TLR4-mediated signalling by suppressing expression of the E2 ubiquitin-conjugating enzyme Ubc13[END_REF], and has a tumor suppressive role in prostate cancer [START_REF] Pencik | STAT3 regulated ARF expression suppresses prostate cancer metastasis[END_REF], glioblastoma [START_REF] Peixoto | HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies[END_REF], and importantly, KRAS-driven lung adenocarcinoma [START_REF] Grabner | Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis[END_REF]. On the basis of these findings, the STAT3 inhibitors currently in clinical trials [START_REF] Johnson | Targeting the IL-6/JAK/STAT3 signalling axis in cancer[END_REF] should be used with caution, at least in tumors where the Notch pathway and consequently HES1 have a pro-tumorigenic role, as for instance in lung adenocarcinoma.

Our findings provided the proof of concept that lung adenocarcinoma cells with between HES1 mRNA levels and PFS in a cohort of 64 patients with EGFR mutated non-small cell lung carcinoma treated with TKIs (49).

Our findings might be very relevant for patients with EGFR-driven lung adenocarcinoma that becomes resistant to osimertinib through acquisition of gatekeeper mutations, such as EGFR C797S , and for whom treatment possibilities are limited to conventional therapies, because checkpoint inhibitors are mostly ineffective in this context. A phase 1 clinical trial should now be put in place to confirm the efficacy of the GSI-TKI combination in patients. Interestingly, a phase I/II trial in 16 patients with lung adenocarcinoma showed that the TKI erlotinib and GSI RO4929097 (Roche) combination is safe and feasible [START_REF] Gold | A phase I/II trial combining erlotinib with gamma secretase inhibitor RO4929097 in advanced non-small cell lung cancer (NSCLC)[END_REF]. As the side effects associated with erlotinib are higher than those with osimertinib (47), the osimertinib and GSI combination also might be safe in patients, particularly when using nirogacestat. Indeed, this GSI displays longterm efficacy and is well tolerated by patients [START_REF] Kummar | Clinical Activity of the gamma-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis)[END_REF]. Moreover, the present study showed that similarly to DBZ, nirogacestat also overcomes resistance to TKIs in human lung adenocarcinoma cells harboring gatekeeper mutations.

MATERIAL AND METHODS

Mice

Tet-on-EGFR T790M/L858R and CCSP-rtTA mice were described previously [START_REF] Li | Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy[END_REF][START_REF] Tichelaar | Conditional expression of fibroblast growth factor-7 in the developing and mature lung[END_REF]. For in vivo PC9GROR lung adenocarcinoma cell xenograft growth assays, 3.5 x 10 6 PC9GR or PC9GROR cells were injected subcutaneously in the flank of 6-week-old, female athymic Nude-Foxn1 mice (Envigo). Drug treatments were started when tumors reached the volume of 200 mm 3 . For Kaplan-Meier analyses, mice were killed when tumors reached the volume of 1200 mm 3 .

Animal procedures were performed according to protocols approved by the French national committee of animal care.

Western blotting

Western blotting was performed as previously described [START_REF] Maraver | Therapeutic Effect of gamma-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK[END_REF]. The following antibodies were used for the analysis: anti-N1ICD, -HES1, -NOTCH1, -NOTCH2, -NOTCH3 and -pSTAT3 (Cell Signaling Technology), -STAT3 (BD), and -tubulin (Sigma). Secondary antibodies were horseradish peroxidase-linked anti-rabbit or anti-mouse (Cell Signaling Technology). Tubulin was used as loading control for all blots.

Treatments in mice

Dibenzazepine (DBZ) (Syncom) was administered 4 days per week (3.3 mg/kg/day) by intraperitoneal (IP) injection. Gefitinib and osimertinib (Cliniscience) were administered by gavage 4 days per week (20 mg/kg/day) and 5 days per week (5 mg/kg/day), respectively. Antibodies against NOTCH1 (NRR1) and NOTCH3 (NRR3)

were administered by IP injection: NRR1 at 5 mg/kg/day every 5 days, and NRR3 at 15 mg/kg/day every Monday and Thursday (Genentech).

Histopathology and immunohistochemistry

Lung lobes were fixed, embedded in paraffin and stained with hematoxylin and eosin (HE) or used for immunohistochemistry. Tumor area and total lung area were measured using the Image J software. For pathological analysis of HE-stained tissue sections, classical cytological and architectural features (tumor cell invasion and high mitotic rate) were evaluated by our expert pathologist (M.C.). For immunohistochemistry, the following antibodies were used: anti-HES1 (Cell Signaling Technology), -Ki67

(Agilent), -pERK (Cell Signaling Technology), and -pAKT (Novus Biologicals). For each tumor, five 10X magnification fields were scored using the Image J software.

Signal intensity in murine and human tissue samples was scored as follows: 0 = lowest intensity, and 5 = highest intensity.

Cell culture and transfection reagents

PC9GR (resistant to gefitinib), and PC9GROR (resistant to gefitinib and osimertinib) cells were obtained from Yarden's laboratory [START_REF] Mancini | An oligoclonal antibody durably overcomes resistance of lung cancer to thirdgeneration EGFR inhibitors[END_REF]. The control siRNA (non-targeting, siNT), and the siRNAs against HES1 (siHES1) and STAT3 (siSTAT3) (Dharmacon) were transfected at 20 nM with Dharmafect1 following the manufacturer's instructions.

For western blotting, RNA-seq or ChIP, cells were incubated with DBZ (250 nM), gefitinib (1 µM), osimertinib (250 nM), PD173074 (2µM), ruxolitinib (0.25 µM),

or DMSO (vehicle).

For the proliferation assays, siRNA-transfected cells were incubated with gefitinib (15 nM) (or DMSO). Then, cells were fixed at various time points and stained with sulforhodamine B (SRB). Absorbance was measured at 560 nm in a microplate reader (Glomax, Promega).

RNA sequencing

RNA was sequenced by Fasteris (Switzerland) using Next-Generation DNA sequencing (NGS) based on the Illumina technology. The RNA-seq data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (accession number GSE117846).

Reads were aligned against the Ensembl Homo sapiens genome assembly (GRCh38). Read counts were extracted from the STAR output file with HTSeq and only the protein-coding genome features were taken into account in the final count matrix. Sample counts were normalized by summing read counts for each sample

( 𝑠 ! , 𝑖 = 1, … ,12
), and computing a first factor for each sample

𝑓 ! = 𝑠 ! /median !!!,…,!" (𝑠 !
). These factors were normalized such that the product of all the normalized factors 𝑔 ! was equal to 1:

𝑔 ! = ! ! ! ! !!!,..,!" !/!"
. Finally, each column (each sample) of the read count matrix was divided by the corresponding 𝑔 ! . Gene set enrichment analysis [START_REF] Subramanian | Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles[END_REF] was performed using «The Molecular Signatures Database Hallmark Gene Set Collection» [START_REF] Liberzon | The Molecular Signatures Database (MSigDB) hallmark gene set collection[END_REF].

Chromatin immunoprecipitation (ChIP)

Chromatin was prepared as described previously [START_REF] Fabbrizio | Negative regulation of transcription by the type II arginine methyltransferase PRMT5[END_REF]. Statistical significance was determined with the paired two-tailed Student's t test.

  Mur et al., submitted to Journal of Clinical Investigation 12 percentage of pERK-positive cells was decreased by treatment with DBZ alone and even more by the DBZ and gefitinib combination compared with control. This indicated that the DBZ and gefitinib combination was more effective in reducing MAPK signaling than Notch inhibition alone. Finally, the percentage of pAKT-positive cells also was efficiently and similarly reduced by DBZ and by the DBZ and gefitinib combination. Altogether, our results provide strong preclinical evidence for the likely therapeutic benefit of Notch inhibition and gefitinib combination in patients with TKIresistant EGFR-driven lung adenocarcinoma harboring the gatekeeper mutation EGFR T790M .

  gatekeeper mutations conferring resistance to TKIs can be sensitized to such compounds by inhibiting the γ-secretase activity. To extend and validate our findings, we performed additional experiments using the lung adenocarcinoma PC9GROR cell line with the gatekeeper mutation EGFR C797S that confers resistance to the thirdgeneration TKI osimertinib. The relevance of this experiment relies in results from a recent phase 3 clinical trial showing that the PFS of patients with EGFR mutated lung adenocarcinoma was significantly longer in the group with osimertinib as a first-line treatment than in the group with gefitinib or erlotinib[START_REF] Soria | Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer[END_REF]. This might soon lead to the use of osimertinib as first-line treatment in patients with EGFR-mutated lung adenocarcinoma. Our data shows that osimertinib treatment in lung adenocarcinoma cells harboring the EGFR C797S mutation induced also pSTAT3 and when combined with DBZ further inhibited HES1 expression. Moreover, the DBZ and osimertinib combination strongly inhibited the growth of PC9GROR cell xenografts in vivo. This suggests that although osimertinib binding is highly reduced by the EGFR C797S!! gatekeeper mutation, it can still promote similar changes as observed in EGFR T790M cells treated with gefitinib. Moreover, these effects are exacerbated by Notch inhibition. Our results question the effect of the Notch pathway in the drugtolerant state[START_REF] Sharma | A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations[END_REF] in lung adenocarcinoma cells treated with osimertinib and this is currently an important area of study in the laboratory.Overall, our new mechanistic findings highlight the role of HES1 in tumor relapse after EGFR TKI therapy. This was confirmed by the negative correlation between HES1 expression and PFS, as well as HES1 upregulation upon disease progression in patients with EGFR-mutated lung adenocarcinoma treated with TKIs. Our results are in agreement with a recent publication showing the negative correlation Bousquet Mur et al., submitted to Journal of Clinical Investigation 22

FIGURESFigure. 1 .Figure. 2 .

 12 FIGURES

Figure. 3 .

 3 Figure. 3. Notch inhibition overcomes resistance to gefitinib in human EGFR T790M/L858R -driven lung adenocarcinoma. (A) Growth of EGFR T790M/L858R lung adenocarcinoma PDX implanted in the right flank of nude mice. When tumors were approximately 100 mm 3 in volume, mice were treated

Figure. 4 .

 4 Figure. 4. Combining EGFR TKIs and Notch inhibitors synergistically decreases

Figure. 5 .

 5 Figure. 5. pSTAT3 directly binds to the HES1 promoter and inhibits its expression.

Figure. 6 .

 6 Figure. 6. Notch inhibition overcomes resistance to osimertinib in EGFR C797S

Figure. 7 .

 7 Figure. 7. High HES1 protein levels correlate with poor progression-free survival

Bousquet Mur et al., submitted to Journal of Clinical Investigation

ACKNOWLEDGMENTS

We thank Daniel Herranz, Laurent Le Cam, Daniel Fisher, Hélène Tourriere, and Manuel Serrano for helpful discussion and critical reading of the manuscript. Elisabetta Andermarcher professionally edited the manuscript. We thank Dom Helmlinger for technical help with the RNA-seq. We thank the IRCM animal facility members for their outstanding work. We thank the Cell Signaling Technology immunohistochemistry technical service for their help. E.B. was supported by a contract from Fondation de

Conflict of interest statement:

The authors have declared that no conflict of interest exists.


Viktoriia Ohorodniichuk

Soufiane El-Oualid

Anne Dauscher

Christophe Candolfi

Philippe Masschelein

Sylvie Migot

Pascal Dalicieux

Philippe Baranek

Bertrand Lenoir
email: bertrand.lenoir@univ-lorraine.fr

Viktoriia Ohorodniichuk1

Soufiane El-Oualid1

Anne Dauscher1

Christophe Candolfi1

Philippe Masschelein1

Sylvie Migot1

Pascal Dalicieux2

Philippe Baranek3

Bertrand Lenoir1

Structural and transport properties of quenched and melt-spun

Keywords: thermoelectric, melt-spinning, bismuth telluride

Introduction

Due to its unique advantages such as high reliability, no emissions of toxic gases, long lifetime and absence of moving parts, solid-state thermoelectric energy conversion has gained a renewed interest over the last decades [START_REF]Thermoelectric Handbook: Macro to nano[END_REF][START_REF]Thermoelectrics for power generation[END_REF][START_REF]Bringing thermoelectricity into reality[END_REF]. This versatile technology offers the possibility to cool or heat systems or to produce electricity directly from any heat source. The potential of a given material to convert a thermal gradient into electrical energy (or vice-versa) at a given absolute temperature T is determined by the so-called dimensionless thermoelectric figure of merit ZT defined as 𝑍𝑇 = 𝛼 2 𝜌𝜅 𝑇 = 𝑃 𝜅 𝑇 where  is the thermopower (or Seebeck coefficient),  is the electrical resistivity, 𝜅 is the total thermal conductivity, and 𝑃 = 𝛼 2 𝜌 is the power factor [START_REF] Goldsmid | [END_REF][START_REF]Thermoelectrics and Its Energy Harvesting[END_REF].

Following the early developments of semiconductors in the 1950s, Bi2Te3-based alloys were identified to be suitable materials for thermoelectric applications near room temperature [START_REF] Eibl | Thermoelectric Bi2Te3 nanomaterials[END_REF][START_REF] Goldsmid | Bismuth telluride and its alloys as materials for thermoelectric generation[END_REF]. Even after decades of intensive research activities, these materials still remain nowadays the reference thermoelectric materials in commercial devices. Bi2Te3, along with its solid solutions with Sb2Te3 and Bi2Se3, belong to the class of narrow-band-gap semiconductors that crystallize in the 𝑅3𝑚 space group. The best p-type thermoelectric properties are achieved in the Sb2Te3-rich solid solutions BixSb2-xTe3 for x ~ 0.4 -0.5 with a peak ZT of around 0.9 -1.0 near 300 K in commercial materials [START_REF] Goldsmid | Bismuth telluride and its alloys as materials for thermoelectric generation[END_REF].

Over the past few years, several studies have reported ZT values substantially higher than unity in these p-type compounds. Most of these advances have been attributed to the beneficial influence of nanostructuration, achieved by various synthesis methods [START_REF] Eibl | Thermoelectric Bi2Te3 nanomaterials[END_REF]. Along this line, the most impressive improvements in thermoelectric properties were obtained by applying the melt-spinning (MS) technique [START_REF] Xie | Unique nanostructures and enhanced thermoelectric performance of melt-spun BiSbTe alloys[END_REF][START_REF] Fan | P-type Bi0.4Sb1.6Te3 nanocomposites with enhanced figure of merit[END_REF][START_REF] Xie | Identifying the specific nanostructures responsible for the high thermoelectric performance of (Bi,Sb)2Te3 nanocomposites[END_REF][START_REF] Xie | High performance Bi2Te3 nanocomposites prepared by single-element-melt-spinning spark-plasma sintering[END_REF]. ZT values as high as 1.56 at 300 K were reported in a series of studies, making MS an innovative route for developing high performance Bi2Te3-based alloys. These studies followed pioneering works performed in the 1980s and 1990s on the binaries Bi2Te3, Sb2Te3 and Bi2Se3 prepared by MS [START_REF] Glazov | Structural features of alloys of the Bi-Te system, produced by ultrafast quenching of the liquid-state[END_REF][START_REF] Glazov | Features of the breakdown of semiconducting metastable solid-solutions of the systems BiSe(Te) and SbTe prepared by quenching from the liquid[END_REF][START_REF] Koukharenko | Thermoelectric Properties of Bi2Te3 Material Obtained by the Ultrarapid Quenching Process Route[END_REF][START_REF] Koukharenko | Electrical properties of Bi2-xSbxTe3 materials obtained by ultrarapid quenching[END_REF][START_REF] Horio | Microstructure and crystal orientation of rapidly solidified (Bi,Sb)(2)(Te,Se)(3) alloys by the liquid quenching technique[END_REF]. Even if these remarkable values have been tempered recently with ZT values close to 1.1 -1.2 [START_REF] Zheng | Mechanically robust BiSbTe alloys with superior thermoelectric performance: A case study of stable hierarchical nanostructured thermoelectric materials[END_REF][START_REF] Ohorodniichuk | Investigation of the nozzle diameter as a control parameter of the properties of melt-spun Sb2-xBixTe3[END_REF][START_REF] Ohorodniichuk | Influence of preparation processing on the transport properties of melt-spun Sb2-xBixTe3+y[END_REF][20][START_REF] Zheng | Thermal stability of p-type BiSbTe alloys prepared by melt spinning and rapid sintering[END_REF][START_REF] Deng | Modulation of carrier concentration and microstructure for high performance BixSb2-xTe3 thermoelectrics prepared by rapid solidification[END_REF][START_REF] Deng | Thermal conductivity in Bi0.5Sb1.5Te3+x and the role of dense dislocation arrays at grain boundaries[END_REF][START_REF] Yoon | High thermoelectric performance of melt-spun BixSb2-xTe3 by synergistic effect of carrier tuning and phonon engineering[END_REF], all the studies showed that MS is a worthwhile mean for further optimizing the thermoelectric performances of Sb2Te3 and Bi2Te3-based materials near room temperature.

As the thermoelectric properties are strongly sensitive to the Bi content in these alloys, several attempts at investigating the influence on the thermoelectric properties of the Bi-to-Sb ratio of the initial ingot for MS have been performed [START_REF] Xie | High performance Bi2Te3 nanocomposites prepared by single-element-melt-spinning spark-plasma sintering[END_REF][START_REF] Deng | Modulation of carrier concentration and microstructure for high performance BixSb2-xTe3 thermoelectrics prepared by rapid solidification[END_REF][START_REF] Murmu | Multifold improvement of thermoelectric power factor by tuning bismuth and antimony in nanostructured n-type bismuth antimony telluride thin films[END_REF]. These studies have evidenced that i) the best composition falls within the range 0.4 ≤ x ≤ 0.5, and ii) the thermoelectric performances achieved are significantly improved in MS samples compared to standard zoneleveling ingots. In a prior study on the melt-spun Bi0.48Sb1.52Te3 ribbons themselves, we showed that they are highly-textured and exhibit more pronounced metallic transport properties with respect to samples of similar chemical compositions prepared by conventional powder metallurgy, that is, quenched from the melt followed by fast sintering by spark plasma sintering [20]. A direct comparison between samples prepared by quenching in room-temperature water and by MS could provide additional information on the exact impact of the MS process on the transport properties, which are not directly accessible by a comparison to zone-levelingprepared polycrystals.

Here, we address this as-yet unexplored issue via a detailed investigation of the structural, electrical, thermal and galvanomagnetic properties (5 -473 K) of BixSb2-xTe3 with x = 0.40 and 0.48 prepared by water-quenching and MS. The similar chemical compositions of both series of samples allowed for a systematic comparison of the transport properties, thereby clearly highlighting the influence of the MS process. In addition, the anisotropy on the electrical and thermal transport and its evolution with temperature has been assessed by measurements on samples cut parallel and perpendicular to the pressing direction. This point is pivotal to determining the most favorable direction and evaluating correctly the ZT values. Measurements performed on different batches of MS samples demonstrate that our preparation process leads to highly-reproducible results with a maximum ZT value of 1.1 achieved at 340 K in the MS x = 0.48 sample.

Experimental details

Synthesis

The synthesis of the initial ingots was performed from high-purity (5N +, 99.999%) Bi, Te and Sb elements in the form of granules. Stoichiometric amounts of these elements for the targeted BixSb2-xTe3 (x = 0.40 and 0.48) compositions were placed in cleaned and degassed silica tubes of 15 mm inner diameter. The tubes were sealed under an argon-hydrogen atmosphere (95/5%), placed in a vertical rocking furnace, heated to 983 K for 5 h, finally followed by quenching in room-temperature water. The resulting ingots were cut in pieces that were subsequently used either to prepare reference samples or as the starting material for the melt-spinning process. The reference samples were obtained by hand-grinding the ingot into fine powders that were consolidated by spark plasma sintering (SPS) under a pressure of 30 MPa at 773 K for 5 min. The consolidated cylinders were 7 mm in height and 10 mm in diameter with a relative density, determined by weight and geometrical dimensions, higher than 97 %.

The melt-spinning process was applied to the ingot using an Edmond Bühler apparatus under the following conditions: heating at 883 K, copper wheel of 19.8 cm in diameter rotating at 36 m.s-1 linear speed and an argon overpressure of 0.8 bar. The nozzle diameter of the quartz tube was set to 1 mm as it showed to be the best experimental diameter to obtain enhanced thermoelectric properties [START_REF] Ohorodniichuk | Investigation of the nozzle diameter as a control parameter of the properties of melt-spun Sb2-xBixTe3[END_REF]. The obtained melt-spun ribbons were hand-crushed and further densified by SPS under the same conditions used for the reference samples. More details on the preparation method can be found in [START_REF] Ohorodniichuk | Investigation of the nozzle diameter as a control parameter of the properties of melt-spun Sb2-xBixTe3[END_REF][START_REF] Ohorodniichuk | Influence of preparation processing on the transport properties of melt-spun Sb2-xBixTe3+y[END_REF][20]. Hereafter, the four synthesized samples will be labelled as Ref0.40, Ref0.48, MS0.40 and MS0.48 for the reference and MS samples, respectively, with 0.40 and 0.48 referring to the Bi content x in BixSb2-xTe3.

Structural and chemical characterizations

Powder X-ray diffraction (PXRD) was used to assess the phase purity of the four samples (Bruker-D8 Advance diffractometer, CuKα1 radiation, λ = 1.54056 Å, 300 K). The chemical homogeneity and morphology of the samples was probed by scanning electron microscopy (SEM) ((FEI-Quanta FEG 650) on polished (back-scattered mode: BSE) and fractured (secondary electrons mode: SE) samples, respectively. The chemical compositions were determined by wavelength dispersive X-ray spectroscopy (WDXS) (JEOL-J7600F instrument) using Bi, Sb and PbTe as standards for determining the concentrations of Bi, Sb and Te, respectively. Around 100 points were measured on each samples. The micro-nanostructure of the bulk MS samples has been observed by transmission electron microscopy (TEM) (JEOL-CM200) on thin slices obtained by micro-cleavage and deposited on a carbon-covered copper grid.

Transport properties measurements

The thermoelectric and galvanomagnetic properties were measured along (that is, parallel) or perpendicular to the pressing direction to probe the anisotropy. Several samples were cut with a diamond wire saw from the dense SPS samples into bar, disk and/or squareshaped samples (typical dimensions of 2.5×2×8 mm3, Ø = 10 mm and 6×6×1 mm3, respectively) as illustrated in Figure 1. Measurements in both directions are critical in mixed Sb2Te3-Bi2Te3 polycrystals since an erroneous combination of transport properties measured along different directions can lead to an overestimation of ZT of up to 100% due to the significant anisotropy in the transport properties.

Low-temperature thermoelectric properties (2 -300 K) were measured using the thermal transport option (TTO) of the physical properties measurement system (PPMS, Quantum Design) on the bar-shaped samples. The contacts were realized by soldering four copper leads onto the sample using a low-melting-point braze. Hall coefficient measurements were conducted between 5 and 300 K on the same sample using the AC transport option of the PPMS.

A five-probe method was utilized by soldering copper wires to the sample with the same lowmelting-point braze. Transverse electrical resistivity 𝜌 𝑥𝑦 data were collected while sweeping the magnetic field 𝜇 0 𝐻 from -1 up to +1T. The data were corrected to dismiss possible magnetoresistive contribution due to contacts misalignment following the formula 𝜌 𝐻 = [𝜌 𝑥𝑦 (+𝜇 0 𝐻) -𝜌 𝑥𝑦 (-𝜇 0 𝐻)]/2 where 𝜌 𝐻 is the Hall voltage. Specific heat was measured on a small square-shaped sample (mass of  15 mg) from 2 to 300 K using the dedicated option of the PPMS.

High-temperature thermoelectric measurements were carried out in the 300 -500 K temperature range. Thermopower and electrical resistivity were measured simultaneously with a ZEM3 system (ULVAC-RIKO) under helium atmosphere on the bar-shaped samples.

Thermal diffusivity a was determined in the same temperature range using a Netzsch laser flash instrument (LFA 427). The samples (cylinder or square-shaped samples) were coated on both sides with a thin layer of graphite to optimize absorption of the laser shot and emission. The data were analyzed by the Cowan model including pulse correction. The specific heat 𝐶 𝑝 was measured by differential scanning calorimetry (DSC Netzsch Pegasus) on small cylindershaped samples. Assuming a constant density 𝜌 𝑉 , the thermal conductivity 𝜅 was calculated from 𝜅 = 𝑎𝜌 𝑉 𝐶 𝑝 .

An overall good match between the low-and high-temperature data sets was observed, the deviation being at most 10% at 300 K. The combined uncertainty in the determination of ZT is estimated to be  17% [START_REF] Alleno | A round robin test of the uncertainty on the measurement of the thermoelectric dimensionless figure of merit of Co0.97Ni0.03Sb3[END_REF].

Results and discussion

Microstructure and chemical homogeneity

As the Sb2Te3-Bi2Te3 pseudo-binary phase diagram shows a solid solution with liquidus and solidus lines separated by a temperature of around 5 K for x = 0.4 and 0.48, both Bi0.4Sb1.6Te3 and Bi0.48Sb1.52Te3 compounds do not melt congruently [START_REF] Caillat | Study of the ternary Bi-Sb-Te phase diagram[END_REF]. Thus, chemical segregations are possible in the initial ingots obtained from a rapid solidification of the melt during water quenching. This possibility was checked on the quenched ingots by EDXS. These analyses revealed the presence of micro-segregations of Bi and Sb elements in the ingot suggesting that the cooling rate achieved by water quenching, estimated to be between 600 -1200 K s-1, is too low to yield a homogenous bulk material. However, as shown below, these inhomogeneities do not have any significant effect on the MS samples. (indexed according to Pearson's crystal data, powder pattern n°310785). A magnification of the main peak (015) highlights the displacement of the peak position towards lower angles for the Ref0.48 and MS0.48 samples with respect to the 0.40 samples (Figure 2b). This trend is consistent with the larger atomic radius of Bi (RBi = 1.63 Å) compared to that of Sb (RSb = 1.53 Å). MS samples exhibit broader peaks than the Ref samples due to the reduced grain size, as expected from the MS process.

Figure 3 shows SEM images taken on polished and fractured SPS samples. In good agreement with the peak broadening observed by PXRD, the MS samples show smaller grain sizes compared to the corresponding reference samples (10-15 m versus < 2 m, see fracture images on Fig. 3). The grain size in the MS samples is smaller than that reported by Zheng et al. (about 8.8 m) despite a similar speed of the copper roller [START_REF] Zheng | Mechanically robust BiSbTe alloys with superior thermoelectric performance: A case study of stable hierarchical nanostructured thermoelectric materials[END_REF]. Our values are, however, consistent with those determined by Deng et al. [START_REF] Deng | Modulation of carrier concentration and microstructure for high performance BixSb2-xTe3 thermoelectrics prepared by rapid solidification[END_REF][START_REF] Deng | Thermal conductivity in Bi0.5Sb1.5Te3+x and the role of dense dislocation arrays at grain boundaries[END_REF]. Similar to what has been observed by all these authors, no preferred orientation could be detected upon closer inspection of the grains that show a lamellar structure. Both reference samples exhibit some phase contrast in backscattered electron images, with features appearing as compositional stripes oriented perpendicular to the pressing direction. For example, compositions varying from Bi0.36Sb1.60Te3.04 to Bi0.50Sb1.47Te3.03 (observed in the whiter places) have been determined by WDXS analyses performed on the Ref0.40 sample. It is interesting to note that the SPS process improves the chemical homogeneity of the initial water-quenched ingots, despite the presence of these compositional stripes. The origin of such original features is however not fully understood. One possibility is that they originate from the micro-segregations observed in the water-quenched ingot which are not fully suppressed by the SPS process. In contrast, the chemical homogeneity of the MS samples after SPS is excellent with no obvious Te-rich phases detectable. This result can be attributed to the very high cooling rate undergone by the melt during the MS process that leads to homogeneous ribbons, as shown in our previous study [20].

The average actual compositions, determined by WDXS analyses and normalized to 5 atoms per formula unit, were measured by averaging about 90 measurement points (Table 1).

A good correlation between the nominal and actual compositions is observed for the Ref and MS samples. Further insights into the variations in composition all over the surface can be obtained by sorting out the data according to an increase in the Bi content, as exemplified in Fig. 4 for the x = 0.40 sample. Although the Bi content corresponds to the expected concentration for nearly all the points, a small excess is nevertheless observed for about 15 points, which is to the detriment of the Sb content. Moreover, the actual Sb content is systematically lower than the nominal value leading to an increased Te content (Table 1, Fig. 4).

Figure 5 shows TEM images collected on two different micro-cleaved fragments that are typical of what could be observed on several fragments of the MS0.40 sample. The micronsized particles seen by SEM in the MS samples could not be observed by TEM since thin enough large fragments were not achieved with the micro-cleavage technique. On one hand, grains of about several hundreds of nm are visible (Fig. 5b). On the other hand, some fragments clearly display features indicative of closely packed, seemingly aggregated nano-grains with no amorphous structure (Fig. 5a). The electron diffraction pattern taken on these fragments exhibits nearly bright continuous diffraction rings that could all be indexed within the 𝑅3 ̅ 𝑚 space group of Bi0.4Sb1.6Te3. By selecting spots on two different rings, the corresponding dark field images highlight the presence of the diffracting nanoparticles. Their size is less than 20 nm, confirming that the MS process is an efficient way to preserve nano-particles and that the SPS process does not significantly affect the size of these nanoparticles. The densely-packed nano-grains form a large number of grain boundaries that can impede the movement of dislocations [START_REF] Li | Mechanical properties of BixSb2-xTe3 nanostructured thermoelectric materials[END_REF] and significantly affect phonon scattering, ultimately enhancing the thermoelectric properties.

Transport properties

The temperature dependence of the electrical resistivity  of the reference and MS BixSb2-xTe3 samples for x = 0.40 and 0.48 are shown in Figures 6a and6b, respectively.

Measurements were performed parallel and perpendicular to the pressing direction. For the reference samples, the  values are constant below T < 20 K and increase with increasing temperature above this temperature. Taking into account the magnitude of  (from 2 to 24 .m), this behavior is typical of heavily-doped semiconductors. We note that  is anisotropic for both x = 0.40 and 0.48 with values significantly higher in the pressing direction than in the perpendicular direction. Furthermore, the anisotropy ratio (par/perp) increases with temperature to reach 1.4 and 1.7 at 300 K for x = 0.40 and 0.48, respectively. This feature, in line with the results obtained on single crystals [START_REF] Caillat | Transport properties analysis of single crystals grown by the traveling heater method[END_REF], indicates that the SPS process induces a preferred orientation along the direction of the applied SPS pressure. Increasing the Bi content from x = 0.40 to x = 0.48 results in a slight increase in  in both directions.

Although the MS samples show similar general trends compared to the reference samples (Figs 6a and6b), a noticeable difference can be observed: the anisotropy ratio is significantly lower in the x = 0.40 and 0.48 samples, with values close to 1 between 300 and 460 K. This result is in good agreement with the aforementioned structural analyses.

The temperature dependence of the Hall coefficient RH, measured between 5 and 300 K, is shown in Fig. 7. All the samples exhibit a smooth temperature dependence, with a slight increase upon warming. This behavior is in good agreement with those obtained on single crystals [START_REF] Caillat | Transport properties analysis of single crystals grown by the traveling heater method[END_REF][START_REF] Champness | Thermoelectric properties of Bi2Te3-Sb2Te3 alloys[END_REF][START_REF] Testardi | Density anomalies in the Bi2Te3-Sb2Te3 system[END_REF][START_REF] Volotskii | Investigation of complex structure of band edges and of mechanism of carrier scattering in Bi-Sb-Te single crystals[END_REF], for which, an increase in the Hall coefficient was explained using a two valence-band model. For all samples, RH is positive indicating that the electrical transport is dominated by holes. The RH values of the x = 0.40 and x = 0.48 Ref and MS samples are very close in both directions suggesting that RH is nearly isotropic. However, we should stress here that some anisotropy may nevertheless exist as reported by Gerovac et al. [START_REF] Gerovac | Thermoelectric properties of n-type polycrystalline BixSb2-xTe3 alloys[END_REF] on polycrystalline samples prepared by mechanical alloying. The reason comes from the fact that two Hall coefficients are present in BixSb2-xTe3 single crystals due to their layered crystal structure: R123 and R231 (in this notation the first index refers to the direction of the electrical field, the second to the direction of the current and the third one to the direction of the magnetic field; the numbers 1, 2 and 3 referring to the binary, bisectrix and trigonal axis, respectively). Stördeur et al. [START_REF] Stordeur | Valence band-structure of (Bi1-xSbx)2Te3 single crystals[END_REF] showed that these two coefficients are not equal in single crystals in the low field limit (𝜇𝐵 << 1 where 𝜇 is the carrier mobility and 𝐵 is the magnetic field) due to the peculiarities of the valence band structure. In addition, the difference between these two coefficients depends on the composition, that is, on the x value. For x = 0.40, the difference at 300 K was found to be only about 9% but can be as high as 34% for x = 1 [START_REF] Stordeur | Valence band-structure of (Bi1-xSbx)2Te3 single crystals[END_REF]. Based on these considerations, it cannot be strictly ruled out that a difference exists between the Hall coefficients measured on samples cut perpendicular (where roughly RH-perp ~ R123) or parallel (where roughly RH-par ~ R231) to the pressing direction.

Following several prior investigations, the hole concentration can be determined from RH assuming a single-parabolic-band model. In this approximation, the (apparent) hole concentration 𝑝 is simply defined by 𝑝 = 1 𝑅 𝐻 𝑒 where e is the elementary charge. In single crystals, the Hall coefficient RH = R123, corresponding to a magnetic field along the trigonal axis and a current and Hall field along the basal planes, is usually used. In polycrystalline samples, the orientation closest to this configuration is obtained in samples cut perpendicular to the pressing direction. For this reason, we used RH-perp for the calculation of p.

Within this approximation, the temperature dependence of 𝑝 was inferred and is shown in Figure 8. 𝑝 is constant below 20 K and decreases slowly between 20 and 200 K. In Ref0.40 and Ref0.48 samples, 𝑝 is equal to 3.91019 cm-3 and 3.61019 cm-3 at 300 K, respectively. The MS process does not seem to strongly impact the hole concentration even though no clear trend emerges from these data. While 𝑝 increases in the x = 0.40 sample (4.81019 cm-3 at 300 K), it decreases for x = 0.48 (2.41019 cm-3 at 300 K). In our previous study, we showed that the MS ribbons for x = 0.48 exhibit a more pronounced metallic character (𝑝 ~ 91019 cm-3) compared to the reference sample, presumably due to extra holes associated with Te vacancies [20]. The results obtained herein show that the consolidation of the ground ribbons by SPS reduces the initial charge carrier concentration. A possible scenario could be that the Te precipitates observed in the ribbons dissolve into the matrix during the SPS treatment, thereby reducing 𝑝.

Regardless of the synthetic process used, the x = 0.40 sample exhibits higher 𝑝 than x = 0.48. This behavior can be explained qualitatively if we assume, as in single crystals, that holes are mainly created in bulk polycrystals by antisite defects which originate from the substitution of Bi or Sb for Te atoms with the driving force to create such defects being the small difference of electronegativity between these atoms. Hence, increasing the Bi2Te3 content should lead to a lower amount of antisite defects due to the larger difference in electronegativity between Bi and Te with respect to Sb and Te.

From the Hall and electrical resistivity measurements, the Hall mobility along the perpendicular direction 𝜇 𝐻 = 𝑅 𝐻 𝜌 ⁄ can be calculated (Fig. 9). At 300 K, the 𝜇 𝐻 values reach nearly 200 cm2 V-1 s-1 in the reference samples, which are comparable to those observed in single crystals of the same composition [START_REF] Caillat | Study of the ternary Bi-Sb-Te phase diagram[END_REF]. Near 300 K, 𝜇 𝐻 follows a T-3/2 dependence and saturates to a constant value below about 15 K. This behavior is suggestive of a combination of scattering by neutral impurities below 15 K while acoustic phonon scattering dominates at higher temperatures. These trends are similar in the MS samples, even though the values, while still large, are somehow degraded by the MS process due to differences in the microstructure.

The thermopower  is shown as a function of temperature in Fig. 10. All the samples show positive  values, which vary linearly from 5 to 40 K. Above this temperature,  increases logarithmically with T up to 300 K. Above 300 K, the slope of (T) decreases, vanishes and eventually becomes negative for some samples. This behavior is consistent with the thermal excitation of minority carriers across the band gap. As a consequence, the higher the hole concentration, the higher the temperature at which the samples enter the intrinsic regime. From this simple picture, we can safely conclude that the MS0.48 sample has the lowest hole concentration, which is consistent with Hall effect data. From the position of the maximum  value (max), the band gap energy Eg (in eV) can be estimated from the formula derived by Goldsmid and Sharp [START_REF] Goldsmid | Estimation of the thermal band gap of a semiconductor from Seebeck coefficient[END_REF], Eg = 2emaxTmax where Tmax is the absolute temperature at which max occurs. Taking max = 220 V.K-1 at Tmax = 420 K for the MS0.48 sample yields Eg = 185 meV.

This value is in excellent agreement with those determined using more sophisticated techniques [START_REF] Stölzer | IR transmission investigations of (Bi1-xSbx)2Te3 single crystals[END_REF][START_REF] Süssmann | Seebeck coefficient and electrical conductivity in p-(Bi1-xSbx)2Te3 at room temperature[END_REF]. The lower  values measured in the MS0.40 sample compared to the MS0.48 sample are consistent with a higher hole concentration in the former.

The  data also evidence the absence of anisotropy below 300 K in the reference samples.

At higher temperatures, however, the values measured in the parallel direction exceed those measured perpendicular to the pressing direction (the maximum anisotropy ratio is around 10%). In Sb2Te3-rich BixSb2-xTe3 single crystals,  can be anisotropic depending on the carrier concentration with values along the trigonal axis being higher than those perpendicular to it, possibly due to an anisotropy in the effective masses [START_REF] Caillat | Transport properties analysis of single crystals grown by the traveling heater method[END_REF]. Considering that our reference samples show some degree of texture as evidenced by the electrical resistivity, an anisotropy in  is thus possible. Interestingly, this anisotropy is suppressed over the entire temperature range in the MS samples.

The temperature dependence of the total thermal conductivity 𝜅 for the reference and MS samples is shown in Fig. 11. Upon warming, 𝜅(𝑇) increases with increasing temperature following approximately a T dependence, shows a maximum near 20 K and then decreases smoothly up to 350 -400 K depending on the samples before eventually increasing again. At 300 K, the 𝜅 value of the samples measured in the perpendicular direction ranges between 1.2 and 1.6 W m-1 K-1. Another clear evidence of the anisotropy in the Ref samples is provided by a comparison of the thermal conductivity measured in both directions. The anisotropy ratio 𝜅 𝑝𝑒𝑟𝑝 𝜅 𝑝𝑎𝑟 ⁄ is around 1.3 -1.5 at 300 K and is higher for x = 0.48 than for x = 0.40. This ratio evolves with temperature and peaks near the highest 𝜅 value for which 𝜅 𝑝𝑒𝑟𝑝 𝜅 𝑝𝑎𝑟 ⁄ = 1.6 -1.7.

Of note, the anisotropy ratio is opposite to that of the electrical resistivity, similar to what has been widely observed in single crystals. No evidence of anisotropy in the MS samples seems to emerge from these data (Fig. 11b). This result is consistent with the conclusions drawn from the  data and in agreement with the conclusions of Xie et al. [START_REF] Xie | Investigation of the sintering pressure and thermal conductivity anisotropy of melt-spun spark-plasmasintered (Bi,Sb)2Te3 thermoelectric materials[END_REF], who reported for the composition x = 0.48, an anisotropy of 2 -3% for samples sintered by SPS at a pressure of 30

MPa. 𝜅 of the MS samples does not show any clear trend compared to the reference samples.

The enhancement of 𝜅 above 350 -400 K, which is sample dependent, is due to an additional contribution of electron-hole pairs to the thermal conduction as a result of the excitation of minority carriers across the band gap. The temperature at which this bipolar contribution occurs matches well the temperature at which  starts decreasing.

Based on these results, the temperature dependence of ZT is shown for all samples in Fig.

12. The bell-shaped peak observed near room temperature in the ZT(T) curves is characteristic of these p-type ternary compounds. For the reference samples, maximum ZT values of 1.1 (x = 0.4) and 1.05 (x = 0.48) are achieved near T = 400 K in samples cut parallel to the pressing direction. The MS samples show similar thermoelectric performances in both directions. The peak ZT of 1.1 is shifted to 340 K in the MS0.48 sample while the MS0.40 sample shows a slightly lower peak ZT value of 0.9 due to the increased hole concentration in this sample. These results highlight the strong sensitivity of ZT to small variations in the chemical composition and, for a given composition, to small variations in the carrier concentration and microstructure.

Of note, although we do observe interesting thermoelectric performances for one MS composition (x = 0.48), our findings contrast substantially with the results of Xie et al. [START_REF] Xie | Unique nanostructures and enhanced thermoelectric performance of melt-spun BiSbTe alloys[END_REF][START_REF] Xie | Identifying the specific nanostructures responsible for the high thermoelectric performance of (Bi,Sb)2Te3 nanocomposites[END_REF][START_REF] Xie | High performance Bi2Te3 nanocomposites prepared by single-element-melt-spinning spark-plasma sintering[END_REF][START_REF] Xie | Investigation of the sintering pressure and thermal conductivity anisotropy of melt-spun spark-plasmasintered (Bi,Sb)2Te3 thermoelectric materials[END_REF], who reported ZT values higher than 1.2 at 300 K for 0.4 ≤ x ≤ 0.52 with a maximum of 1.5 for x = 0.48. The reasons behind such discrepancies are yet to be understood.

Nevertheless, several studies have recently reported experimental results for this family of materials prepared by MS that are in better agreement with our results [START_REF] Zheng | Mechanically robust BiSbTe alloys with superior thermoelectric performance: A case study of stable hierarchical nanostructured thermoelectric materials[END_REF][START_REF] Zheng | Thermal stability of p-type BiSbTe alloys prepared by melt spinning and rapid sintering[END_REF][START_REF] Deng | Modulation of carrier concentration and microstructure for high performance BixSb2-xTe3 thermoelectrics prepared by rapid solidification[END_REF][START_REF] Deng | Thermal conductivity in Bi0.5Sb1.5Te3+x and the role of dense dislocation arrays at grain boundaries[END_REF][START_REF] Yoon | High thermoelectric performance of melt-spun BixSb2-xTe3 by synergistic effect of carrier tuning and phonon engineering[END_REF][START_REF] Ebling | Structure and thermoelectric properties of nanocomposite bismuth telluride prepared by melt spinning or by partially alloying with IV-VI compounds[END_REF][START_REF] Böttner | Melt spinning preparation of bismuth telluride and partially alloying with IV-VI compounds for thermoelectric application[END_REF][START_REF] Ivanova | Thermoelectric and Mechanical Properties of the Bi0.5Sb1.5Te3 Solid Solution Prepared by Melt Spinning[END_REF].

Because various methods used to prepare p-BixSb2-xTe3 ternary compounds often led to thermoelectric properties that were not reproducible, it is essential to determine the robustness of the experimental protocol described herein to produce MS samples. In this context, we prepared two MS samples (x = 0.48) coming from two distinct batches and characterized their electronic and thermal properties along and perpendicular to the pressing direction. The transport and galvanomagnetic data obtained on these samples, gathered in Fig. 13, demonstrate the good reproducibility of the techniques used to fabricate these materials. In each case, the deviations observed in the transport properties are below few percent. These results further

show that MS provides a fruitful approach to optimize and control the thermoelectric properties of these technologically-important chalcogenides.

Tables Table 1. Nominal and actual compositions in the four samples studied determined by WDXS.

The actual chemical compositions have been obtained by averaging around 90 different measurement points and were calculated by normalizing the chemical formulae to 5 atoms per formula unit.

Sample Nominal Actual

Figure captions

Fig. 1. Sketch of the adopted scheme for cutting the samples parallel (red color) and perpendicular (blue color) to the pressing direction. Low temperature measurements: barshaped samples for both electrical and thermal properties cut in "perp" and "par" directions (PPMS, TTO), bar-shaped samples cut in "perp" direction for galvanomagnetic measurements (PPMS, ACT). High temperature measurements: bar-shaped samples for electrical properties (ZEM3); cylindrical ("par") or square-("perp") shaped samples for thermal diffusivity measurements (LFA).

  The PXRD patterns of the four different samples (Ref and MS samples with x = 0.40 and 0.48) are shown in Figure 2a. Both Ref and MS samples are well crystallized, with all the observed peaks corresponding to the rhombohedral 𝑅3 ̅ 𝑚 crystal structure of Bi0.5Sb1.5Te3

Fig. 2 .

 2 Fig. 2. PXRD patterns of the BixSb2-xTe3 samples: a) reference and melt-spun samples for x =

Fig. 3 .

 3 Fig. 3. a, b, c, d: backscattered electron images collected on polished samples; e, f: secondary

Fig. 4 .

 4 Fig. 4. Compositions determined by WDXS on roughly 100 points over the surface on reference

Fig. 5 .

 5 Fig. 5. TEM images collected on the MS0.40 sample. a) Bright field (BF) image showing an

Fig. 6 .

 6 Fig. 6. Temperature dependence of the electrical resistivity  for the (a) reference and (b) MS

Fig. 7 .

 7 Fig. 7. Temperature dependence of the Hall coefficient RH for the (a) reference and (b) MS

Fig. 8 .

 8 Fig. 8. Temperature dependence of the hole concentration p for the (a) reference and (b) MS

Fig. 9 .

 9 Fig. 9. Temperature dependence of the Hall mobility 𝜇 𝐻 for the (a) reference and (b) MS BixSb2-

Fig. 10 .

 10 Fig. 10. Temperature dependence of the thermopower S for the (a) reference and (b) MS BixSb2-

Fig. 11 .

 11 Fig. 11. Temperature dependence of the total thermal conductivity  for the (a) reference and

Fig. 12 .

 12 Fig. 12. Temperature dependence of ZT for the (a) reference and (b) MS BixSb2-xTe3 samples

Fig. 13 .

 13 Fig. 13. Temperature dependence of the (a) electrical resistivity, (b) thermopower, (c) hole

Figure 2 28 Figure 3 29 Figure 4 30

 283294 Figure 2

Figure 5 Figure 6

 56 Figure 5

Figure 7 32

 7 Figure 7

Figure 8

 8 Figure 8

Figure 9 33 Figure 10

 3310 Figure 9

Figure 11

 11 Figure 11

Figure 13

 13 Figure 13

Acknowledgement V.O. would like to acknowledge the support of EDF through the CIFRE convention N°2011/1329. The authors acknowledge support through the "Contrat-Plan-Etat-Region" 2014-2020 ENARBATIM and MAT-DS.

Compliance with Ethical Standards:

Summary and conclusions

We have synthesized polycrystalline and melt-spun BixSb2-xTe3 samples for x = 0.40 and x = 0.48 consolidated by spark plasma sintering. The systematic comparison of the microstructural and transport properties of the Ref and MS samples evidences differences that can be ascribed to the peculiar microstructure achieved by MS. Although both series of samples are well crystallized, the chemical homogeneity is not equivalent. The SPS-processed ribbons yield bulk dense samples highly homogeneous while the reference samples exhibit some segregations at the microscale that might be due to the specific characteristics of the pseudobinary Bi2Te3 -Sb2Te3 phase diagram. Variations in the chemical composition of the initial ingot has a significant influence on the transport properties of the final samples as revealed by our detailed transport property measurements. Our results are in line with recent investigations on melt-spun materials. A peak ZT value of 1.1 at 340 K was achieved in the MS x = 0.48 sample. Our results demonstrate that the MS technique is a powerful tool to produce polycrystalline samples with high thermoelectric performances and with a very good reproducibility from sample to sample.

Conflict of Interest:

The authors declare that they have no conflict of interest.


Damien Pellier
email: damien.pellier@imag.fr

Humbert Fiorino
email: humbert.fiorino@imag.fr

Totally and Partially Ordered Hierarchical Planners in PDDL4J Library

In this paper, we outline the implementation of the TFD (Totally Ordered Fast Downward) and the PFD (Partially ordered Fast Downward) hierarchical planners that participated in the first HTN IPC competition in 2020. These two planners are based on forwardchaining task decomposition coupled with a compact grounding of actions, methods, tasks and HTN problems.

Introduction

The TFD (Totally Ordered Fast Downward) and PFD (Partial ordered Fast Downward) hierarchical planners are based on forward-chaining task decomposition used by the SHOP2 planner [START_REF] Nau | SHOP2: an HTN planning system[END_REF]) coupled with a compact grounding of actions, methods, tasks and HTN problems. Both planners accept as input HDDL (Hierarchical Domain Description Language) proposed by [START_REF] Höller | HDDL: an extension to PDDL for expressing hierarchical planning problems[END_REF] and are implemented on top of the PDDL4J library [START_REF] Pellier | PDDL4J: a planning domain description library for java[END_REF]. In this short paper we present first the compact representation used by TFD and PFD as well as the grounding procedure implemented. Finally, we conclude with a brief presentation of the search strategy implemented in both planners.

Grounding technique

Most modern planners work with grounded representations of the planning problem. However, planning domains are commonly defined with a lifted description language such as PDDL [START_REF] Ghallab | PDDL: The Planning Domain Definition Language[END_REF] or HDDL [START_REF] Höller | HDDL: an extension to PDDL for expressing hierarchical planning problems[END_REF]). Thus, planning systems have to generate a grounded representation of the lifted domain in a preprocessing step, the objective of which is to generate the most compact grounded representation possible without removing any action, method or fluent needed for a solution plan. The more compact the grounded representation is, the more efficient is the search for a solution plan as reducing the size of the search space speeds up search and heuristic value computation. In practice, computing a grounded representation from a lifted representation is quite straightforward. All possible Copyright c 2020, Association for the Advancement of Artificial Intelligence (www.aaai.org). All rights reserved. instantiations of ground predicates, primitive actions, abstract tasks and methods must be computed, and appropriately replaced by their ground versions in the lifted representation.

In the context of classical (non hierarchical) planning, the planners FF [START_REF] Hoffmann | The FF planning system: Fast plan generation through heuristic search[END_REF] and FastDownward (Helmert 2006) have implemented techniques for transforming lifted to ground planning representation that are still used in many planners today. Regarding hierarchical planning, [START_REF] Ramoul | Grounding of HTN planning domain[END_REF]) inspired from [START_REF] Koehler | Handling of inertia in a planning system[END_REF] have been the first to propose an efficient grounding preprocessing, and it was successfully applied to the planners proposed by [START_REF] Schreiber | Tree-rex: SAT-based tree exploration for efficient and high-quality HTN planning[END_REF]. Recently, [START_REF] Behnke | On succinct groundings of HTN planning problems[END_REF]) has proposed novel techniques.

In the TFD and PFD planners, the grounding combines the approches proposed by [START_REF] Ramoul | Grounding of HTN planning domain[END_REF]) and [START_REF] Behnke | On succinct groundings of HTN planning problems[END_REF]

Search procedures

The non-deterministic TFD procedure for solving a HTN planning problem is given in Algorithm 1. This procedure is based directly on the recursive definition of a solution plan for HTN planning problems.

The TFD procedure takes as input a problem P = (s 0 , T, A, M ) where s 0 is the initial state, T = t 1 , t 2 , ..., t k is a list of tasks, A the set of actions, and M the set of methods, all in their ground representation. First, the procedure tests if the list of tasks T is empty (line 2). In this case, no task has to be executed, thus the empty plan is returned. Then the procedure gets the first task t 1 of the list T . Two cases must be considered depending on the type of t 1 : Case 1. If t 1 is primitive (line 3) then the procedure computes the set of all the ground actions that accomplishes t 1 and that are applicable in s 0 (line 4). If there is no action (line 5), the procedure fails because no action accomplishes the goal task t 1 . Then the procedure nondeterministically chooses an action that accomplishes the task (line 6), and calls itself recursively on the planning problem P = (γ(s 0 , a 1 ), T -{t 1 }, A, M ) (line 7). Finally, if the recursive call to the procedure fails to find a plan π, it returns failure (line 8); otherwise it returns the plan that is the concatenation of a and π (line 9). Case 2. If t 1 is non-primitive (line 10) then the procedure computes the set of ground decompositions that accomplish t 1 and that are applicable in s 0 (line 11). If there is no decomposition to accomplish t 1 (line 12) then the procedure returns failure. Otherwise the procedure nondeterministically chooses a decomposition d that accomplishes the task t 1 (line 13), and recursively returns the solution plan for the problem P = (s 0 , subtasks(d) ⊕ t 2 , . . . , t k , A, M ) (line 14). Practically non-deterministic choices are made by systematically choosing the task networks with the least amount of non-decomposed tasks. In the case where several networks have the same number of tasks remaining to be decomposed, the task network containing the least number of actions is chosen.

The search procedure implemented in PFD is almost similar. The main difference is no longer to choose the first t 1 task in the task network but to choose the first task that does not have any predecessor task in the task network. In addition, each time case 2 applies, it is necessary to check the consistency of the ordering constraints of the task network in order to generate a-cyclic task networks. This check is performed before line 14 by calculating the transitive closure of the ordering constraints. The computation of the transitive closure is based on Warshall algorithm. The complexity is O(n) where n is the number of tasks of the task network.

A ← the set of relevant actions for t 1 and applicable in s 0

  choose an action a ∈ RA 7 π ← T F D(γ(s 0 , a), t 2 , . . . , t k , A, M ) 8 if π = failure then return failure 9 else return a ⊕ π 10 else if t 1 is a non-primitive task then 11 M ← the set of relvant methods for t 1 and applicable in s 0 12 if M = ∅ then return failure 13 non-deterministically choose a decomposition m ∈ M