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Next-generation therapies for adrenocortical carcinoma

Barbara Altieri, MD, PHD, Post-doc in Endocrinology, Cristina L. Ronchi, MD, PHD, Consultant in Endocrinology, Matthias Kroiss, MD, PHD, Consultant in Endocrinology, Martin Fassnacht, MD, Professor of Medicine, Chief Division of Endocrinology and Diabetes

EARCH

BEST PRACTICE & RESEARCH

Clinical Endocrinology & Metabolism

PRA

Editor-in-Chief Christoph A. Meler

PII:	S1521-690X(20)30061-0
DOI:	https://doi.org/10.1016/j.beem.2020.101434
Reference:	YBEEM 101434
To appear in:	Best Practice & Research Clinical Endocrinology & Metabolism

Please cite this article as: Altieri B, Ronchi CL, Kroiss M, Fassnacht M, Next-generation therapies for adrenocortical carcinoma, Best Practice & Research Clinical Endocrinology & Metabolism, https:// doi.org/10.1016/j.beem.2020.101434.

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@ 2020 Elsevier Ltd. All rights reserved.

Next-generation therapies for adrenocortical carcinoma

Barbara Altieri MD, PhD (Post-doc in Endocrinology)ª, Cristina L. Ronchi MD, PhD (Consultant in Endocrinology)a,b, Matthias Kroiss MD, PhD (Consultant in Endocrinology) a,c,d, Martin Fassnacht MD (Professor of Medicine, Chief Division of Endocrinology and Diabetes) a,c,d

ª Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Würzburg, Würzburg, Germany

b Institute of Metabolism and System Research, University of Birmingham, Birmingham, UK.

· Comprehensive Cancer Mainfranken, University of Würzburg, Würzburg, Germany

d Central Laboratory, University Hospital Würzburg, Würzburg, Germany

Word Count: 6032

ournal Pre- center, es Corta,

Corresponding author:

Altieri Barbara, MD, PhD Division of Endocrinology and Diabetes, Department of Internal Medicine I University Hospital, University of Würzburg Oberduerrbacher-Str 6 97080 Würzburg, Germany Tel number: +49-931-201-39704 e-mail: Altieri_B@ukw.de

Abstract

Almost one decade ago, etoposide, doxorubicin, cisplatin and mitotane (EDP-M) has been established as first-line systemic therapy of metastatic adrenocortical carcinoma (ACC). Although heterogeneous, the prognosis of advanced stage ACC is still poor and novel treatments are urgently needed. This article provides a short summary of current systemic ACC treatment and provides a comprehensive overview of new therapeutic approaches that have been investigated in the past years, including drugs targeting the IGF pathway, tyrosine kinase inhibitors, radionuclide treatment, and immunotherapy. The results of most of these trials were disappointing and we will discuss possible reasons why these drugs failed (e.g. drug interactions with mitotane, disease heterogeneity with exceptional responses in very few patients, and resistance mechanisms to immunotherapy). We then will present potential new drug targets that have emerged from

Key words: adrenal cancer, treatment, mitotane, targeted therapy, immunotherapy, tyrosine kinase inhibitors next-generation therapies of ACC.

Introduction

Adrenocortical carcinoma (ACC) is a rare tumour with an annual incidence of 0.7-2.0 cases per million per year [1, 2] that can occur at any age with women being more often affected than men (55-60%) [3, 4]. Some 55% of ACC patients present with symptoms of steroid hormone excess (most frequently with hypercortisolism and/or hyperandrogenism), whereas 30-40% of cases experience symptoms associated to tumour mass, and the remaining 10% are incidentally discovered [5].

The prognosis of ACC is heterogeneous, with a 5-year survival ranging from 80% in patients with localized disease to 15% in those patients with advanced European Network for the Study of Adrenal Tumors (ENSAT) tumour stage [6]. In addition to ENSAT tumour stage, age [7], hormone- or tumour mass-related symptoms [7, 8], tumour resection status [9], and Ki-67 index [10], as well as molecular markers [11, 12] have been shown to be of prognostic relevance in ACC patients. The integration of both clinical and molecular prognostic markers appears to improve prognostication compared to these two components evaluated independently [13].

Due the rarity of the disease, most recommendations for ACC treatment are derived from retrospective studies, and only a limited number of therapeutic options is available. Complete en bloc tumour resection, including the resection of peritumoural fat and loco-regional lymphadenectomy, when feasible, is currently the only curative option for ACC patients [5]. The reported frequency of recurrence after complete resection is quite variable (30-85%) [14-16]. Referral bias may account for most of the variability and the actual recurrence rate will likely be somewhat in between. The rarity of the disease is one reason why optimal patient care is required to involve centres with special expertise in ACC and to discuss every case in a multidisciplinary team meeting according to current guidelines.

We performed a systematic review of the literature evaluating the medical therapies currently available for the treatment of ACC, with a particular focus on new strategies of treatment and predictive markers of response, trying to give suggestions that could be used in the clinical practice.

1. Current treatment of ACC and predictive biomarkers of response

1.1. Mitotane

Mitotane is the only approved drug for treatment of advanced ACC and is advocated by many centres for patients with high risk of recurrence (including those with ENSAT stage III, incomplete resection, or Ki67 >10) [5].

The question whether mitotane is of benefit for patients with low risk of recurrence, is currently addressed in a multicentre randomized phase III trial (ADIUVO, NCT00777244). According to multiple studies, the rate of objective tumour response in advanced ACC ranges between 13% and 31%, but these are often short-lived [4, 17]. Several studies have shown the importance of attaining plasma mitotane levels above 14 mg/L [18- 20].

While at the molecular level mitotane has been shown to be an inhibitor of sterol-O-acyl transferase (SOAT1) [21], other targets may also be important [22]. SOAT1 tissue protein expression was found not to correlate with mitotane response [23, 24] (Weigand et al., under review).

Few other molecular markers have been proposed to predictive mitotane levels and the response to treatment. A low expression of ribonucleotide reductase large subunit (RRM1) at mRNA levels correlated with a better disease-free survival (DFS) in ACC patients treated adjuvantly with mitotane [25]. Some small studies have analysed the impact of different CYP enzymes as predictors of mitotane response [23, 26, 27]. A multicentre ENSAT study evaluated the role of CYP2W1 and CYP2B6 single nucleotide polymorphisms (SNPs) in a large cohort treated with mitotane monotherapy both after radical resection (n=103) or in advanced disease (n=79) [28]. In advanced ACC, patients with CYP2W1*6 SNP showed a significantly reduced probability to reach mitotane therapeutic range and a significantly lower response rate compared to patients wild type; on the contrary, CYP2B6*6 significantly correlated with higher mitotane levels [28]. Therefore, the evaluation of these two SNPs could be a first step towards personalized medicine in mitotane treatment.

1.2. Cytotoxic chemotherapies

Platinum-based chemotherapy, mostly in combination with etoposide and doxorubicin plus mitotane (EDP- M scheme) is the first line of treatment in advanced ACC [5]. This recommendation derives from the FIRM- ACT trial that demonstrated a greater efficacy of EDP-M compared to the combination of streptozotocin plus mitotane arm, with a progression-free survival (PFS) of 5.5 vs 2.1 months [29]. A later phase II study investigating the combination of cisplatin and docetaxel as first line of chemotherapy reported a PFS of 3 months and partial response in 21% of cases [30]. However, since none of those patients reported a complete response, this regimen is not used as standard treatment. Cytotoxic chemotherapy is sometimes used in the clinical practice also as adjuvant therapy in patients with very high risk of recurrence. An on ongoing clinical trials (ADIUVO II, NCT03583710) is comparing the efficacy of EP-M scheme vs mitotane alone in this population.

Predictive biomarkers to conventional cytotoxic chemotherapy have been difficult to establish due to frequently discrepant and non-replicable findings. Both topoisomerase II alpha (TOP2A) [31] and excision repair cross complementing group 1 (ERCC1) [32-34] have been suggested to predict response, but could not be confirmed.

The current ACC guidelines suggest the use of additional therapies in patients with tumour progression after EDP-M [5]. Two different well-studied cytotoxic regimens are suggested as second-line chemotherapy: streptozotocin plus mitotane [29] and gemcitabine plus capecitabine with or without mitotane [35, 36]. For gemcitabine-based chemotherapy our group previously excluded the value of the human equilibrative nucleoside transporter type 1 (hENT1) and RRM1 expression as predictive biomarkers of treatment response [36]. It is important to underline that, even though few patients experienced long-term disease control, both cytotoxic regimens are modestly active in the majority of cases, showing an objective response rate below 10% and a median PFS generally <4 months. The combination of systemic chemotherapies and local treatment (e.g. surgery, radiation, chemoembolization, radiofrequency thermal ablation) is therefore often advisable. This evidence was very recently confirmed by a monocentric series including advanced/metastatic ACC patients that reported a better PFS and overall survival in patients who underwent surgery of the residual disease after responding to EDP-M compared to those receiving chemotherapy only [37]. Due to the

unsatisfactory results with first- and second-line chemotherapies, salvage therapies, including trofosfamide or thalidomide, are often applied albeit with limited efficacy [38-40].

2. Experience and current trials with targeted therapies in ACC

Treatment of patients with advanced ACC refractory to standard therapies remains challenging. ACC guidelines suggest to consider the enrolment of patients in clinical trials whenever there is the possibility and offer to patients therapeutic options based on published trials or evidence from preclinical studies [5].

2.1. Insulin-like growth factor (IGF) pathway

The insulin-like growth factor (IGF) system plays a major role in the regulation of different biological functions of the normal adrenal gland and is one of the most frequently altered pathways in ACC [41, 42]. IGF2, IGF1 receptor (IGF1R) as well as the insulin receptor (IR) and its isoform A are frequently overexpressed in ACC [43], which leads to an auto-paracrine loop where IGF2, through the stimulation of IGF1R and IR that activates IGF-downstream pathways, which promote cell proliferation, motility, survival, and prevent cell apoptosis [41, 42].

The presumed key role of the IGF system in ACC encouraged the assessment of many IGF-target molecules in this tumour entity. NVP-AEW541, a selective IGF1R inhibitor, and cixutumumab, an IGF1R monoclonal antibody, reduced NCI-H295R cell proliferation both in vitro and in vivo in mouse xenograft model [44, 45]. These encouraging preclinical data and promising results in some, but not all early phase trials [46-50] (Table 1), prompted to start a phase III trial with linsitinib, a specific inhibitor of both, IGF1R and IR. However, this multicentre, randomized placebo-controlled phase III trial failed to demonstrate any effect of linsitinib in OS and PFS [51]. Of note, among the intervention arm (n=90), 4 patients remained on linsitinib treatment with a disease control from 23 to more than 45 months (Table 1). One patient finally even had complete response, stopped treatment after 75 months and is still free of disease 111 months after starting the trial (personal observation). Therefore, it can be speculated that a small subset of patients can be effectively

treated with IGF1R inhibitors. It is now the task to identify the molecular basis of this subgroup and initiate another molecular-based trial. However, this trial clearly demonstrated the heterogeneity of ACC.

Another drug that - among other targets - interferes with the IGF1R/IR pathway is metformin, commonly used in the treatment of type 2 diabetes mellitus, which present also direct antitumour effect. A recent in vitro and in vivo study showed an anti-proliferative effect of metformin in ACC NCI-H295R cells and mouse xenograft model [52]. However, clinical data evaluating the efficacy of metformin in ACC have been not yet reported.

2.2. Mammalian target of rapamycin (mTOR) pathway

A dysregulation of the mTOR pathway has been described in a subset of ACC and the administration of mTOR inhibitors, including sirolimus, temsirolimus and everolimus, alone or in combination with mitotane or other drugs, had an anti-proliferative effect in in vitro and in vivo [53].

Only few clinical studies with mTOR inhibitor have been reported in ACC [47, 54, 55] (Table 1). Although the total number of ACC patients is very limited, it seems unlikely that monotherapy with mTOR targeting drugs will have clinically relevant efficacy.

2.3. Tyrosine kinase inhibitors (TKI)

Receptor tyrosine kinases (RTK) are composed of an extracellular ligand binding domain, a signal transducing transmembrane region, and an intracellular kinase domain that selectively phosphorylates tyrosine residues of the receptor itself and substrate targets which engage a cascade of signalling events resulting in the activation of transcription factors which regulate cell growth and differentiation. Both selective and multi-tyrosine kinase inhibitors (TKI) have been developed as anti-tumour treatment.

2.3.1. Multi-tyrosine kinase inhibitors

Tumoural neoangiogenesis is an established drug target in several tumour entities. Most approved drugs target vascular endothelial growth factor (VEGF) and its receptor VEGFR2, but usually inhibit other kinases

such as platelet derived growth factor receptors (PDGFRs), fibroblast growth factor receptors (FGFRs) and epidermal growth factor receptors (EGFRs) to a varying degree. Using data from The Cancer Genome Atlas (TGCA), we have summarized in Figure 1 overexpressed RTK in ACC that could be targeted by TKI. Earlier study have already demonstrated that VEGR and its receptor VEGFR2 are high expressed at protein levels in many ACC [56, 57], suggesting that antagonizing angiogenesis could be successful for ACC treatment.

Among the inhibitors with broad kinase specificity, sorafenib and sunitinib have been evaluated for the treatment of ACC (Table 2). For both drugs, preclinical data suggested some efficacy [56, 57]. However, a phase II study in 9 patients with advanced ACC treated with sorafenib plus metronomic paclitaxel was precociously terminated due to early disease progression in all patients [58]. In the SIRAC phase II trial, only 5 out 35 patients with refractory ACC treated with sunitinib experienced stable disease after the first evaluation at 12 weeks, but in the overall trial population median PFS was only 2.8 months [59]. It was the merit of this trial to confirm a previous incidental observation in 2 patients [60] that mitotane is associated with low plasma concentrations of sunitinib ant its active metabolite N-desethylsunitinib. This association was shown to be caused by mitotane induced CYP3A4 activity [61, 62], which led to pharmacokinetic drug interaction with rapid metabolism of sunitinib.

Furthermore, axitinib [63] and dovitinib [64], two other multi-TKI failed to demonstrate convincing efficacy, similarly to the monoclonal anti-VEGF antibody bevacizumab [65] (Table 2).

Cabozantinib is an inhibitor of c-MET, VEGFR2, AXL, and RET and approved for several solid tumours. C- Met is the receptor for hepatocyte growth factor (HGF) and research from the Habra group has demonstrated increased phosphorylation of c-Met in ACC by immunohistochemistry of tissue samples compared to adrenocortical adenomas (ACA) [66]. By knocking down c-Met expression in NCI-H295 cells the authors demonstrated decreased cell proliferation and treatment of a NCI-H295 mouse xenograft model with cabozantinib reduced growth of implanted tumours significantly [66]. We recently published the overall encouraging international experience with cabozantinib in 16 ACC patients [67] (Table 2). Of note, cabozantinib was offered only patients in whom mitotane had been discontinued for a prolonged period of time or low mitotane plasma concentration had been demonstrated. Cabozantinib resulted in objective

tumour response in three patients, and PFS of more than four months in eight patients suggested some clinical efficacy.

Two parallel phase II trials with cabozantinib in ACC patients without mitotane exposure are currently recruiting in Europe (NCT03612232) and the U.S. (NCT03370718).

2.3.2. Selective tyrosine kinase inhibitors

EGFR inhibitors

A strong EGFR membrane staining was reported in 36% of ACC samples [68] and inhibition of EGFR signalling in vitro reduced cell viability in tumour primary ACC and SW13 cells [69]. However, none of the two EGFR TKI, erlotinib and gefitinib, has been reported to have clinical benefit in advanced ACC [70, 71] (Table 2). The inefficacy of EGFR inhibitors in ACC patients could be due to the rarity of activating EGFR mutations [12, 68], which has been shown to determine response to these compounds in cancer types like non-small cell lung cancer [72]. Thus, overexpression of EGFR alone may be not sufficient to determine response to EGFR inhibitors since compensatory mechanisms may lead to treatment resistance.

FGFR inhibitors

Dysregulation of the FGF system has been associated with tumour development and progression. An amplification of FGFR1 was described in 3 out 28 (10.7%) patients with ENSAT tumour stage III-IV by comparative genomic hybridization [73]. Moreover, FGFR1 and 4 and mRNA levels were overexpressed in ACC compared to ACA and normal adrenal glands [74], and were found in up to 12% in TCGA data sets (Figure 1) [12].

Up to now, only derazantinib (ARQ 087), a pan-FGFR inhibitor has been tested in a phase I/II study that included 4 ACC patients (Table 2) [75]. Among those patients, one with FGFR1 amplification and one without apparent FGFR genetic alterations, had stable diseases longer than 12 months. As for EGFR inhibitors, the efficacy of FGFR inhibitors could not only dependent on FGFR pathway overexpression, but also on genetic alteration (including amplification, fusion, and mutation). In ACC, genetic alteration of the FGFR pathway has been described in a range of 0% (for FGFR1) to 6% (for FGFR4) of cases [12].

3. SOAT inhibitor

Nevanimibe (ATR-101), developed as a SOAT1 inhibitor, was shown to potently inhibit adrenal steroidogenesis and induced apoptosis in adrenal cells [76], but also in the adrenal cortex of dogs [77]. Similarly to mitotane, it seems to have mitochondrial targets as well [76]. In a recent phase I study, it was not possible to reach in ACC patients enough drug exposure and no clear signal of efficacy was observed [78].

4. Immunotherapy

4.1. Preclinical rationale for immunotherapy of ACC

Preclinical evidence to apply immunotherapy in ACC is scarce. In a pan-cancer analysis in TCGA, ACC was among the tumour types with low degree of T cell infiltration [79] when using PD1 mRNA expression as a marker. In the same data set and by using six immune-related signatures, ACC has been shown to be leukocyte depleted in general with a bipartite phenotype of immune cell exclusion vs. inflammation [80]. The molecular underpinnings of these two phenotypes are not yet understood, but intratumoural glucocorticoid exposure and genetic alterations might play a role here.

As mentioned above, steroid hormone secretion is present in ~60% of ACC cases and dominated by glucocorticoids and androgens. Obviously, intra-tumoural steroid hormone concentrations can be supposed to be markedly higher than measured in the circulation. Based on this reasoning, we used immunohistochemistry of immune cell marker proteins to demonstrate that on average the degree of immune-cell infiltration of ACC is low. We identified an inverse relationship of steroid hormone secretion and immune infiltration [81] and hypothesized that co-treatment with anti-glucocorticoid drugs might be pivotal to reactivate the immune system in immunologically “cold” ACC by immune checkpoint inhibition. Yet, there are no in vitro or in vivo data that would validate this strategy.

Again, by using TCGA data sets it has been shown that ß-catenin signalling is associated with impaired immune cell infiltration. By considering both somatic mutations or somatic copy number alterations (SCNA)

in ß-catenin signalling elements and pathway prediction from RNA sequencing data, ACC was the tumour entity in which the strongest inverse relationship between ß-catenin signalling and immune infiltration [82]. In other tumours such as melanoma it appears that activation of the ß-catenin pathway can lead to dendritic cell inactivation through chemokine signalling [82, 83].

4.2. Markers of response to immunotherapy in ACC

Three main markers of treatment response are currently used in other types of cancers and investigated in ACC: expression of PD1 and PD-L1, microsatellite instability (MSI) and - closely linked with MSI - tumour mutational burden.

To evaluate the potential of PD1/PD-L1 directed therapy, an early small cohort of ACC samples that was not part of TCGA was assessed for expression of programmed death receptor 1 ligand PD-L1 and found to generally have low expression with 3/28 samples considered positive [84]. No larger independent series has yet been published.

Germ line mutations in the mismatch repair (MMR) genes MLH1, PMS2, MSH2 and MSH 6 predispose to Lynch syndrome (LS) and LS-like tumour syndromes. MMR deficiency leads to a high number of tumoural mutations that can be detected through increased variability in the length of repetitive DNA sequences (microsatellites) leading to the term microsatellite instability (MSI). MSI can also be caused by somatic inactivation of MMR protein expression. The PD1 antagonist pembrolizumab has been approved by the U.S. Food and Drug Administration (FDA) for treatment of tumours with MSI independent of the histology. This approval was based on a series of clinical trials in which MSI was evaluated by immunohistochemistry or PCR and an objective response rate of~40% was demonstrated in this population [85]. ACC has been shown to be a LS-related cancer although a positive family history is present in exceptional cases only [86]. Several reports in the literature [87, 88] have reported long-lasting responses to immune checkpoint inhibition in ACC patients with germ line MMR mutations. From one of these patients even a patient derived xenograft moue model could be established [88].

Tumour mutational burden (TMB, usually expressed as mutations per megabase with >10 being considered high) is a surrogate marker for the potential of a tumour to express and present mutant peptides in the context of human leukocyte antigen (HLA) type I molecules. Presentation of mutant peptides is a prerequisite for tumour reactive T cells and hence response to immune therapy. While generally the number of mutations in ACC is small, it has been suggested in a small series that a higher number of mutations may be present in advanced ACC [89]. It has not been tested whether high TMB can predict response to immune therapy of ACC.

4.3. Clinical trials of immunotherapy in ACC

To date, four clinical trials have been published that investigated three different inhibitors of PD1/PD-L1 [90-93]. The characteristics of these reports are detailed in Table 3. It is noteworthy that none of the clinical trials required the presence of markers of response to immune therapy established in other tumour entities. All trials have explored those markers but none of the markers was predictive of treatment response in ACC. A selection of commonly investigated markers is given in Table 3.

Remarkably, in the study by Raj et al, 2 out of six patients with MSI tumours, both of them with germ line mutation in an MMR gene, responded to treatment while 5 of the seven responders did not show MSI. Both trials of pembrolizumab excluded concomitant mitotane treatment. While this may have favourably impacted on tolerability of the treatment it cannot be determined whether this impacts on the unprecedented response rate in one of the studies [93]. Taken together response to current immunotherapy in ACC is not understood. Some explanation for the variable outcome might lie in the genetic background of the given tumour or in the role of glucocorticoids as both discussed previously.

Many centres use immunotherapy as salvage treatment. However, it seems to be reasonable to test for tumoural microsatellite instability and germ line mutations in MMR genes to identify patients with higher likelihood of response. In the U.S., pembrolizumab is approved for MSI tumours regardless of histology and therefore is available for MSI ACC.

5. Radiopharmaceuticals

5.1. Iodometomidate

[123I] iodometomidate (IMTO), through its binding the adrenocortical enzymes CYP11B1+B2 can be used for imaging of benign and malignant adrenocortical lesions and in 36.2% of patients with advanced ACC relevant radiotracer uptake in all metastatic lesions ≥2 cm were detectable [94, 95]. By replacing [123I] by [13]]] IMTO can be used for targeted radionuclide therapy. In a small group of 11 patients with advanced ACC receiving up to 20GBq [13]]]IMTO, stable disease or partial response was achieved in 6 (54.5%) patients [96].

5.2. 90Y/177Lu-DOTATOC

Very recently, a study investigated the potential use of yttrium-90/lutetium-177 (90Y/177Lu-DOTATOC) peptide receptor radionuclide therapy (PRRT) in a group of 19 patients with advanced ACC. Before performing the PRRT, only patients who showed a strong uptake distribution of the Gallium 68 (68Ga)- DOTATOC, indicating high expression of somatostatin receptors, underwent the treatment. With this selection criterion, 2 (11%) patients were treated with PRRT, obtaining an overall disease control lasting 4 and 12 months, respectively [97]. SSTRs-based PRRT may represent a potential treatment opportunity for a subgroup of ACC patients with high expression of somatostatin receptors.

We then will present potential new drug targets that have emerged from many molecular studies (e.g. wnt/ß- catenin, cyclin-dependent kinases, PARP1) that may be the foundation of next-generation therapies of ACC.

6. Future molecular targets in ACC

Thanks to both pan-genomic and targeted studies, a number of specific genetic alterations have been recognized in subgroups of ACC that may represent potentially anti-cancer drug targetable events [11-13,

73]. These have been nicely summarized in a recent review by Crona and Beuschlein [98]. Here we focus on genes that we judge particularly promising and review available in vitro, pre-clinical and clinical data with presumably implication for future ACC treatment (Figure 2).

6.1. Wnt signaling inhibitors

The Wnt/B-catenin signaling pathway is developmentally important in many organs, including the adrenal gland. Somatic activating mutations in the gene CTNNB1 encoding ß-catenin (i.e. p.S45P) are recognized in about 40% of ACC [11-13]. In addition, there are several other alterations leading to an activated Wnt/B- catenin signalling of which the most frequent (about 20%) are loss-of-function mutations or copy number losses in ZNRF3, a Wnt/ß-catenin-related proposed tumour suppressor gene.

Blocking of the Wnt/ ß-catenin signalling has shown to increase apoptosis and impair adrenal steroidogenesis in NCI-H295R ACC cells that also harbour the p.S45P mutation [99]. More recently, two studies aiming to investigate drug repurposing (an emerging approach for identifying new indications for existing drugs) identified two compounds that play an anti-cancer effect via suppression of the Wnt/B-catenin pathway. Rottlerin, a natural plant polyphenol, inhibits cell proliferation and induces cell apoptosis both in ACC cell lines and xenograft models [100], niclosamide reduced ß-catenin expression and inhibited level of mediators of epithelial-to-mesenchymal transition [101].

Altogether these studies suggest that inhibition of ß-catenin-dependent transcription or of autocrine/paracrine Wnt signaling may be therapeutically efficacious in ACC. However, until now, direct targeting of Wnt/ ß-catenin remains challenging and no specific CTNNB1 inhibitor demonstrated to be promising enough to be tested in clinical trials. New approaches to target the Wnt/B-catenin pathway are under development including inhibitors of porcupine (an acyltransferase enzyme essential for the secretion of all Wnt ligands) and Frizzled proteins, which may be more effective in malignant tumours such as ACC with somatic alterations in ZNRF3 [102-104]. However, even these approaches fail to totally are tissue unselective [105] and hence render it unlikely that these approaches would yield sufficient therapeutic benefit as monotherapy. Therefore, it may be better to with limit toxicity by in combining with other treatments [102].

6.2. Cyclin dependent kinase inhibitors

Cell cycle is controlled by several key proteins, including CDKs (cyclin-dependent kinases), which are the target of recently discovered cell-cycle checkpoint inhibitors.

CDK4/6:Copy number gains or amplifications in the CDK4 oncogene locus have been reported in several studies as recurrent in ACC with prevalence between 7% and 40% [11, 12, 73, 98, 106] [13]. Deletions at CDKN2A or CDKN2B, that inhibit CDK4, have been found in>10% of ACC [11, 73]. We recently demonstrated that CDK4 is highly expressed in ACC also at the protein level, especially in a subgroup of ACC tumours [107]. Finally, Hadjadj et al did an in silico study TGCA and found that high mRNA levels of CDK6 gave the most significant association with shorter time to relapse and poorer survival of patients [108].

These observations suggested a potential role for CDK4 and CDK6 inhibitors (CDK4/6i), such as palbociclib, ribociclib and abemaciclib, as candidate drugs for the treatment of ACC [98]. This might be of particular interest since different in vitro studies showed that palpociclib reduced cell viability in ACC standard cell lines (NCI-H295R, SW13, and MUC1) and primary cell cultures [107-109]. Moreover, it is approved by FDA for specific types of locally advanced or metastatic breast cancer [110] and has an acceptable toxicity, being neutropenia the most relevant adverse effect. [107-109].

Thus, current preclinical evidence demonstrates that CDK4/6 targeting agents could be effective in the ACC treatment, and Palbociclib deserves to be further explored as a new therapeutic option in ACC.

CDK1: Besides CDK4 and CDK6, also CDK1 has been proposed as a candidate drug target in ACC. A recent study investigated the effects of flavopiridol (as CDK inhibitor) and carfilzomib, previously identified as candidate agent by quantitative HTS, in three ACC cell lines [111]. The authors demonstrated high CDK1 and CDK2 expression in human ACC samples and a dose-dependent, anti-proliferative effect, as well as a synergistic activity of the combination in monolayer cell culture as well as in three-dimensional tumour spheroids. Flavopiridol and carfilzomib showed anticancer activity also in mice with human ACC xenografts [111], thus supporting future evaluation of this combination therapy in patients with advanced ACC.

6.3. Notch inhibitors

Previous SNP array analyses have revealed genomic alteration of the Notch pathway (i.e. copy number gains in JAG1 and NOTCH1 locus) as being frequent in ACC [13, 112]. In addition, protein expression of several members of the Notch pathway (e.g. JAG1, activated NOTCH1 (aNOTCH1), and HEY2) has been reported to be higher in ACC than in normal adrenal glands and adenomas [113], suggesting a role for the pathway in malignant transformation. At the DNA level, we found recurrent somatic mutations in NOTCH1 gene, even if the biological role of these alterations remains partially unclear [13]. Another study demonstrated that JAG1 enhances ACC cell proliferation and tumour aggressiveness in a non-cell-autonomous manner through activation of Notch signalling in adjacent cells [114]. The authors showed that inhibition of Notch signalling by DNMaml resulted in inhibition of cell proliferation, suggesting a potential role for Notch inhibitors in treatment of ACC.

In clinical trials, several gamma-secretase inhibitors (GSI), that inhibit Notch signalling by preventing cleavage of transmembrane domain of Notch protein, have been tested in patients with different solid tumours [115, 116] but not specifically in ACC. Thus, further investigations are needed to explore the role of Notch inhibitors/GSI in patients with ACC.

6.4. MAPK/ERK pathway

The Ras/Raf/MEK/ERK pathway is a chain of proteins in the cell that communicates a signal from the surface of the cell to its nucleus.

NF1: Neurofibromin 1 (NF1) is involved in this pathway by inhibiting the RAS proto-oncogene. Thus, an underlying defect of the NF1 gene results in an activation of the key tumourigenetic Ras/Raf/MEK/ERK pathway. Neurofibromatosis type 1 (NF1) is a frequent neurocutaneous syndrome that predisposes for various benign and malignant tumours. ACC has been rarely reported in patients with NF1. Considering sporadic ACC and somatic alterations, loss-of-function NF1 mutations have been observed in approximately 10% of cases (Figure 2) [11-13]. This is of particular interest as NF1 represents a target for MAPK/ERK kinase (MEK1) inhibitors (MEKi). Moreover, it has been reported that MEK1 inhibition with PD184352

significantly decreases cell proliferation as well as steroidogenesis and also increased the redox state of NCI- H295R cells [117]. Further preliminary studies on the potential role of MEKi in ACC management are needed in order to propose clinical trials in patients with ACC.

BRAF: The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK. Activating mutations in the proto-oncogene BRAF are well known driver alterations in several cancer types. It is known from direct DNA sequencing data that activating BRAF mutations may occur in a subset of ACC, ranging from 2 to 6% of investigated cases (Figure 2) [12, 13, 118, 119]. This observation is of particular interest since the BRAF V600E mutation a well-recognized drug target for BRAF inhibitors. In fact, vemurafenib, encorafenib and dabrafenib are all FDA-approved drugs for the treatment of metastatic melanoma specifically expressing BRAF V600E. Therefore, specific inhibitors of the Ras/Raf/MEK/ERK may represent candidate targeted therapies for future clinical trials in carefully selected patients harbouring specific activating mutations.

6.5. MDM2 inhibitors

The murine double minute (MDM) family member MDM2 acts as regulators of p53 pathway and is frequently found amplified/overexpressed in cancer. The pharmacological targeting of MDM2 in order to restore or increase P53 expression and activity therefore holds promise for cancer therapy [120].

A recent in vitro study demonstrated that MDM2 inhibitor (MDM2i) compound nutlin-3a inhibited cellular proliferation in the ACC cell line NCI-H295R. Nutlin-3a treatment also inhibited steroid secretion in vitro and ACC tumour growth with no observed toxicity in mice in vivo [121]. Another study demonstrated that inhibition of polo-like kinase 1 (that negatively modulates p53 functioning) by BI-2536 sensitized the ACC cell lines NCI-H295R and SW13 cells to MDM2 inhibition. This dual inhibition resulted in an additive apoptotic response in NCI-H295R cells with wild-type p53 [122].

Several phase I/II clinical trials with MDM2i, such as the “nutlin” compounds RG7112 and RG7388 (idasanutlin) as well as novel small molecules, have been completed for different malignancies with low levels of TP53 mutations [123, 124]. These studies highlighted key aspects i.e. drug-related toxicity and

development of drug resistance, that should be taken into account before proposing MDM2i for treatment of advanced ACC.

6.6. PARP inhibitors

DNA damage repair (DDR) genes play key roles in maintaining human genomic stability. Loss of DDR function, conversely, is an important determinant of cancer risk, progression, and therapeutic response. DDR genes can be grouped into functional pathways, among which the homology-dependent recombination repair (HRR) comprises a series of genes involved in the repair of DNA double-stranded breaks and interstrand crosslinks. Important HRR-related genes include RAD51, BRCA1, BRCA2 and PALB2.

Drugs that inhibit protein Poly ADP-ribose polymerase (PARP1) cause multiple double strand breaks and in tumours with BRCA1, BRCA2 or PALB2 mutations, these double strand breaks cannot be efficiently repaired, leading to the death of the cells. Treatment with PARP1 inhibitors (PARPi) are FDA-approved for germline BRCA1/2 mutated solid tumours (olaparib and rucaparib for ovarian carcinoma, talazoparib for breast cancer). In addition, deficiency in ATM, a key activator of the DNA-damage response to double-strand breaks, has been associated with increased sensitivity of olaparib treatment in different cancer types [125, 126].

In ACC, it has been reported that loss-of-function somatic mutations in genes such as BRCA1, BRCA2 and ATM may be observed in circa 4% of cases [98]. However, no specific pre-clinical studies have been performed with PARPi in ACC, yet.

6.7. Hedgehog pathway inhibitors

Sonic hedgehog (SHH) signalling is involved in adrenal development [127]. In addition, it is aberrantly activated in many cancers and has been shown to be involved in chemotherapy resistance. The hedgehog receptor Ptch1, which is over-expressed in many recurrent and metastatic cancers, including ACC, is a multidrug transporter and it contributes to the efflux of chemotherapeutic agents such as doxorubicin, leading to chemotherapy resistance [128]. This makes Ptch1 a particularly attractive therapeutic target for cancers

expressing Ptch1. Hasanovic et al. showed that the use of methiothepin, an inhibitor of Ptch1, inhibited the doxorubicin efflux, enhancing the cytotoxic and antiproliferative effects of doxorubicin on NCI-H295R cells [129]. Moreover, in vivo the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumours more efficiently than doxorubicin alone. Of note, loss-of-function PTCH1 mutations have been reported in 2-3% of ACC [98, 106]. This might be of interest, as SHH inhibitors, such as smoothened (SMO) inhibitors vismodegib and erismodegib, are FDA-approved for basal cell carcinoma [130]. In addition, multiple other hedgehog pathway inhibitors are in different phases of clinical trials.

7. Summary

Despite efforts of many researcher, standard medical treatment for advanced ACC is still mitotane with or without EDP chemotherapy (etoposide, doxorubicin, and cisplatin). Several attempts to improve therapeutic options by investigating more targeted therapies failed so far. Among several potential reasons, we would like to emphasize at least two: (1) many “new” cancer drugs (including all TKI) are metabolized by CYP3A4 and even small concentrations of mitotane (with its long half-life) in the body might be enough to diminish the efficacy of these drugs. (2) ACC is a very heterogeneous disease and several trials demonstrated that a “one fits all” approach failed. Therefore, it is crucial for the next decade to learn more about the molecular basis of the heterogeneity in ACC. Furthermore, we are convinced that drug combinations are required to simultaneously attack this aggressive tumour from different angles. Finally, it will be important to establish reliable predictive biomarkers to allow true personalized medicine against this rare disease. All these studies can only succeed if they are performed as large multicentre efforts (e.g. within the ENSAT and the American-Australian-Asian Adrenal Alliance). Taking into account these considerations, future clinical trials should be based on molecular phenotyping and pay attention to pharmacokinetic interactions. Although these trials will take longer, we are optimistic that finally we will make progress.

Practice Points

· ACC is a rare disease, and for optimal patient care it is recommended to involve a centre with special expertise in ACC.

· Mitotane is still the backbone of medical therapy and is indicated in the majority patients both in adjuvant setting and in advanced disease.

· In advanced disease, mitotane is usually combined with etoposide, doxorubicin, cisplatin.

· After failure of platinum-based chemotherapy, clinical trials should be considered.

· For current patient management, but also for the design of new trials, it is important to acknowledge that mitotane is very strong inducer of CYP3A4, which lead to increased metabolism of many drugs (including all anti-cancer TKI).

· Immunotherapy of ACC is unlikely to be efficacious in unselected patients, but testing for Lynch Syndrome, related germline mutations and microsatellite instability in tissue is likely to increase the proportion of responders.

· ACC is a heterogeneous disease and an improved understanding of inter-, but probably also intra- individual heterogeneity will be crucial to develop improved treatment strategies.

· Resistance to single agent therapies through pathway redundancy likely requires combination approaches for which clinical trials are difficult to organize.

· To facilitate progress in ACC it is important to include as many patients as possible in clinical trials.

Research Agenda

Significant progress has been made in the molecular analysis of ACC, but this did not lead to improved clinical outcome yet. Here will list some suggestions to make progress:

· Although mitotane is an old drug, a better understanding of the pharmacology and mode of action is probably essential to improve clinical management of this drug, but also to develop a second- generation mitotane.

· For improved understanding of the heterogeneity of ACC, single-cell analyses as well as large-scale molecular comparisons of primary tumours and metastases are probably important approaches.

· Liquid biopsies (steroid metabolomics, cell-free DNA, microRNA, etc.) are promising tools and should be further investigated.

· The establishment of biomarkers that allow the prediction of response to certain treatment (including the “standard therapies”) would be a major step towards an individualized medicine.

· To make immunotherapy more efficient against ACC, we have to move beyond single agent checkpoint inhibitors and attack for instance in parallel the intratumoural glucocorticoid excess in many tumours.

· It should be a high priority to move now from identified drug targets to proper designed clinical trials.

· New concepts for clinical trials (e.g. so-called registry-based randomized trials) are probably needed to facilitate the above-mentioned studies in a time- and economically reasonable manner.

Conflict of interest:

Matthias Kroiss received institutional research support for a clinical trial with cabozantinib in ACC from Ipsen Pharma. Martin Fassnacht is an investigator in this trial. The other authors have no conflict of interest.

Funding:

This work has been supported by the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio (project number: 314061271 - TRR 205 to C.L.R., M.K. and M.F.), the project FA-466/8-1 and RO-5435/3- 1 (M.F. and C.L.R., respectively), FA-466-4-2 to M.F. and KR 4371/1-2 to M.K., and the Deutsche Krebshilfe (70113526 to C.L.R. and M.F.).

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Legend to Figures

Figure 1. Alteration of mRNA levels of receptors tyrosine kinases in ACC in the TGCA series. Overview of mRNA levels potential druggable receptors tyrosine kinases (RTK) in adrenocortical carcinoma from The Cancer Genome Atlas (TCGA) [12] considering patients with available data of RNA expression (n=78) in correlation with clinical parameters of sex, age, disease-specific survival and progression-free survival, by using the cBioPortal (cbioportal.org). We evaluated the mRNA expression z-scores relative to diploid samples (RNA Seq V2 RSEM) and a threshold ±2.0 was considered to determine high/low mRNA levels.

Figure 2. Mutations and copy number variations of potential druggable target genes in ACC. Overview of mutations and copy number variations (CNV) of potential targeted genes in adrenocortical carcinomas (ACC) from different genetic series [11-13] including a total number of 241 ACC patients (n=45 from Assié et al., Nat Genet 2014 [11], n=89 from Zheng et al., Cancer Cell 2016 [12] and n=107 from Lippert et al., J Clin Endocrinol Metab 2018 [13]). To note, CDK6 and JAG1 were not evaluated in the targeted panel used by Lippert et al., thus the reported results derived from the first two studies including 134 patients.

Table 1. Clinical trials and case series investigating drug targeting IGF/mTOR signaling pathways in patients with advanced adrenocortical carcinoma.

Intervention	Target	Phase	Population	Primary outcome	PR	SD	PD	Comments	Reference
Figitumumab	IGF1R	I	Advanced ACC (n=14)	Safety, tolerability, and efficacy	0	8/14	6/14	SD only for short time	Haluska et al., 2010
temsirolimus + cixutumumab	IGF1R + mTOR	I	Advanced solid tumors, including ACC (n=10)	Safety and tolerability. Tumor response as secondary outcome	0	4/10	6/10	Long SD >8 months.	Naing et al., 2011
Temsirolimus + lenalidomide	Temsirolimus: mTORC1. Lenalidomide: IL-6, VEGFR, TNFa, IGF1	I	Advanced cancers, including ACC (n=3)	Safety, pharmacokinetic.	0	1/3	2/3	SD >6 months	Ganesan et al., 2013
Everolimus + pazopanib	Everolimus: nTOR. Pazopanib: VEGFR, PDGFR, c-Kit	I	Advanced cancers, including ACC (n=1)	Safety, pharmacokinetic.	0	1/1	0	SD >13 months	Wagle et al., 2014
Cixutumumab	IGF1R	II	Solid tumors and recurrent ACC (n=10, including 8 pediatric ACC).	Response rate and toxicities	0	0	10/10	All patients showed progression.	Weigel et al., 2014
Mitotane + IMC-A12	IGF1R	II	Advanced ACC (n=20)	PFS	1/20	7/20	12/20	Median PFS was 6 weeks. The study was terminated before randomization because of limited efficacy.	Lerario et al., 2014

OSI-906 (linsintinib) in 3 different schedules (S)	IGF1R + IR	I	Advanced solid tumors, including ACC (n=15; 9 in S1, 1 in S2 and 5 in S3).	Safety, pharmacokinetic.	0	2/15	13/15	SD was observed within S1.	Jones et al., 2015
OSI-906 (linsitinib) vs placebo.	IGF1R + IR	III	ACC (90 treated with linsitinib vs 49 with placebo).	OS	3/90	6/90	81/90	No differences in OS and PFS between the two groups.	Fassnacht et al., 2015

Abbreviation: ACC, adrenocortical carcinoma; c-Kit, stem cell ligand receptor; IGF1, insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; IL-6, Interleukin 6; IR, insulin receptor; mTORC1, mammalian target of rapamycin complex 1; n, number of patients; OS, overall survival; PDGFR, platelet derived growth factor receptors; PFS, progression-free survival; PD, progressive disease (including also patients who interrupted treatment due to adverse events); PR, partial response; SD, stable disease; TNFa, Tumor necrosis factor-alpha; VEGFR, vascular endothelial growth factor receptor.

Table 2. Clinical trials and case series investigating drug targeting VEGFR-, PDFGR-, c-MET-, EGFR-, FGFR-signaling pathways in patients with advanced adrenocortical carcinoma.

Intervention	Target	Phase	Population	Primary outcome	PR	SD	PD	Comments	Reference
Gefitinib (Iressa)	EGFR	II	Advanced ACC previously treated with standard regimes (n=19)	Objective response rate	0	0	19/19	No efficacy in all patients.	Samnotra et al., 2007
Erlotinib + gemcitabine ± mitotane	Erlotinib: EGFR. Gemcitabine: inhibition of DNA synthesis	Case series	Advanced ACC previously treated with standard regimes (n=10)	PFS	0	1/10	9/10	9/10 died after a median of 5.5 months.	Quinkler et al., 2008
Bevacizumab + metronomic capecitabine	Bevacizumab: monoclonal antibody anti-VEGFR. Capecitabine: thymidylate synthase inhibitor	Case series	Advanced ACC previously treated with standard regimes (n=10)	PFS	0	0	10/10	Median survival=124 days.	Wortmann et al., 2010
Sorafenib + metronomic paclitaxel	Sorafenib: multi-TKI (VEGFR2-3, PDGFR, RAF-1). Paclitaxel: microtubules	II	Advanced ACC previously treated with standard regimes (n=10)	PFS	0	0	10/10	Study was terminated earlier because of limited efficacy	Berruti et al., 2011
Sunitinib ± mitotane	multi-TKI: VEGFR1-2, PDGFR, c-Kit	II	Advanced ACC previously treated with standard regimes (n=35)	PFS	0	5/35	24/35	6 died from ACC before the first evaluation. Median PFS=2.8 months.	Kroiss et al., 2012

Axitinib	VEGFR1, -2, and -3	II	Advanced ACC previously treated with standard regimes (n=30)	PFS	0	8/30	22/30	SD>3 months. Median PFS=5.48 months	O'Sullivan et al., 2014
Dovitinib	multi-TKI: VEGFR, FGFR and PDGFR	II	Advanced ACC previously treated only with mitotane (n=17)	PFS	1/17	4/17	12/17	SD>6 months. Median PFS=1.8 months	García-Donas et al., 2014
Derazantinil (ARQ 087)	Pan-FGFR inhibitor	I/II	Advanced cancers, including ACC (n=4)	Safety and tolerability	0	2/4	2/4	SD>12 months.	Papadopoulos et al., 2017
Cabozantinib	c-MET, VEGFR2, AXL, and RET	Case series	Advanced ACC with mitotane discontinuation for long time or low mitotane plasma concentration (n=16)	PFS	3/16	5/16	8/16	Median PFS=16.2 weeks	Kroiss et al., 2020

Abbreviation: ACC, adrenocortical carcinoma; c-Kit, stem cell ligand receptor; c-MET, receptor for hepatocyte growth factor; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; n, number of patients; receptor; PFS, progression-free survival; PD, progressive disease (including also patients who interrupted treatment due to adverse events); PDGFR, platelet derived growth factor receptor; PR, partial response; SD, stable disease; TKI, tyrosine kinase inhibitors; VEGFR, vascular endothelial growth factor receptor.

Table 3. Clinical trials of immunotherapy in adrenocortical carcinoma.

ClinicalTrials.gov Identifier	NCT01772004	NCT02721732	NCT02720484	NCT02673333
Compound	avelumab	pembrolizumab	nivolumab	pembrolizumab
Phase	1b extended	2	2	2
Part of a basket trial	yes	yes	no	no
n	50	16	10	39
Marker driven?	no	no	no	no
Marker analysis?	yes	yes	yes	yes
PD-L1 expression	yes	yes	yes	yes
Tumor infiltrating lymphocytes	no	yes	yes	yes
MMR-D/MSI	yes	yes	no	yes
TMB	no	no	no	yes
median PFS	2.6 months	n.r.	1.8 months	2.1 months
median OS	10.6 months	n.r.	n.r.	24.9 months
ORR	6%	14%	(10%)	23%
Concomitant mitotane	yes (25/50)	no	not reported	no
Reference	LeTourneau et al., JIC 2018	Habra et al., JIC 2019	Carneiro et al., JCEM 2019	Raj et al., JCO 2019

Abbreviation: MMR-D mismatch repair deficiency; MSI microsatellite instability; n, number of patients, n.r., not reported; OS, overall survival; ORR, objective response rate; PFS, progression-free survival; TMB tumor mutational burden.

Diagnosis Age

Sex

Overall Survival Status

Progression Free Status

VEGFA

5%

VEGFB

6%

KDR

5%

PDGFA

4%

PDGFB

4%

PDGFRA

1.3%

PDGFRB KIT

4%

6%

MET

9%

AXL

2.6%

EGFR

6%

FGF1

8%

FGFR1

12%

FGFR2

5%

FGFR3

2.6%

FGFR4

10%

Genetic Alteration

mRNA High

mRNA Low No alterations

Diagnosis Age

14

77

Sex

Female

Male

Overall Survival Status

DECEASED

LIVING

Progression Free Status

CENSORED PROGRESSION

Journal Pre-proof

PTCH1-

☐ CNV

ATM-

BRCA2-

☒ Mutations

BRCA1.

MDM2-

BRAF.

NF1.

JAG1-0

NOTCH1.

CDK6-

CDK4-

ZNRF3-

CTNNB1.

TP53-

0

5

10

15

20

25

% of patients

Journal Pre-proof