ADRENOCORTICAL CARCINOMA: AN ORPHAN MALIGNANCY: FROM THE PATIENT TO THE BENCH AND BACK
MARGARET E. WIERMAN, MD, and (by invitation) KATJA KISELJAK VASSILIADES, DO
AURORA, COLORADO
ABSTRACT
Adrenocortical carcinoma (ACC) is an orphan cancer with 35% five-year survival that has been unchanged for last five decades. Patients often pres- ent with severe hypercortisolism or with mass effects. The only Food and Drug Administration (FDA)-approved drug for ACC is mitotane, an insecti- cide derivative, which provides only limited additional months of survival, but with toxicities. Little progress in the field has occurred due to a lack of preclinical models. We recently developed new human ACC in vitro and in vivo research models. We produced the first two new ACC cell lines for the field, CU-ACC1 and CU-ACC2, which we have distributed for global collaborations. In addition, we developed 10 ACC patient-derived xenograft (PDX) and two humanized ACC-PDX models to test new therapeutics and examine the mechanism of mitotane action in combination with immuno- therapy. These new preclinical models allow us to identify novel targets and test new therapeutics for our patients with adrenal cancer.
INTRODUCTION: LIFE AS A PHYSICIAN-SCIENTIST
Throughout my career as a physician-scientist, my research efforts have started first with a patient. During my fellowship, I was fortunate to care for patients with precocious puberty and idiopathic hypogonado- tropic hypogonadism as two ends of the spectrum of reproductive axis disorders with onset too early or too late (1-3). We were the first to use pulsatile GnRH to restore reproductive axis function in humans and to employ the first GnRH agonists to turn off the early activation of the
Correspondence and reprint requests: Margaret E. Wierman, MD, Professor in Medicine and Integrative Physiology, Division of Endocrinology, Metabolism and Diabetes, Director of Pituitary and Adrenal Tumor Program, Staff Physician and Merit Investigator, Rocky Moun- tain Regional VAMC, University of Colorado School of Medicine, Endocrinology MS8106, 12801 East 17th Ave, RC1 South, Aurora, CO 80045, Tel: 303-399-2769, E-mail: margaret.wierman@ cuanschutz.edu.
Potential Conflicts of Interest: None Disclosed.
reproductive axis in children in precocious puberty (1-3). These experi- ences in clinical research led me to expand my foundation in molecular biology, participate in the cloning of the gonadotropin subunit FSHB, and study the effects of sex steroid regulation on the pituitary axis in rodents (4). Armed with new molecular approaches, in my own lab, we cloned the rat GnRH gene and examined its promoter regulation (5,6). To better understand the physiology of reproductive axis development, my lab focused on the factors that impact GnRH neuronal migration and survival (7). Using cell and animal models, we contributed to the many factors that ensure targeting of the neurons to the hypothala- mus to connect to the pituitary to trigger sexual development. Stud- ies of the (AXL) family in this process were complemented by human studies showing that mutations occur in patients who fail to undergo normal pubertal development (8). Next, I returned to the pituitary to focus on endocrine neoplasia as we built a large multidisciplinary clinic for patients with pituitary tumors. In addition to our clinical and educational center, we have created one of the largest pituitary tumor banks in the world, with over 750 tumor specimens and 100 normal pituitaries to identify molecular signatures that may give insight in the pathophysiology of pituitary tumorigenesis and/or potential treatment targets. Gonadotrope tumors are most common in men, are large, and have no medical therapies. Our lab identified a novel kinase MST4 as a potential therapeutic target for these tumors, and studies are underway to show the effect of a MST4 inhibitor to inhibit tumor growth in mouse models (9). Each of these research areas started with carefully pheno- typing the patient and then approaching the disorder at the bench to understand the disease and open new areas for therapeutic research.
Understanding Adrenocortical Carcinoma: Clinical Presentation and Current Treatment Options
Most recently, my lab’s focus has been on ACC. After a clinical obser- vation that patients with ACC presented to our institution with greater frequency than 1 case per million per year, as reported in literature, and faced with limited therapeutic options, we set up a multidiscipli- nary clinic with endocrinologists, endocrine surgeons, oncologists, adre- nal pathologists, and radiation oncologists to improve the care of these patients and investigate pathophysiology of ACC (10,11). ACC is an orphan cancer with a reported incidence of about 1/1million; however, a much higher prevalence has occurred in recent years. It is an aggres- sive cancer of the adrenal cortex that occurs in children, adults in their 20s and 30s, and those over 55. It occurs more commonly in women
than men at a ratio of 2-3:1 for unclear reasons. ACC is a deadly cancer with a five-year survival of less than 35%. No progress in the field of ACC had been made in the last 50 years, in part because of a lack of preclinical models.
Clinically, patients with ACC present with evidence of hormone overproduction in 40-60% of cases, usually cortisol causing Cushing’s syndrome, hyperandrogenism in 40%, hyperaldosteronism <5%, or estrogen production in 1-3% (12). Other patients experience abdomi- nal or flank pain, fullness, early satiety, or sometimes an incidental finding on imaging. Surgical resection is the first line of treatment for patients with ACC. However, 45% of the surgeries are performed in community hospitals, 30% in academic institutions, and only 15% in National Cancer Institute (NCI)-designated Cancer Centers. The expertise of the surgeon significantly impacts on recurrence and over- all survival for this rare cancer (11). Since 40% of patients have hyper- cortisolism, the effects of high cortisol impact morbidity and mortality if not aggressively treated. To date, the only FDA-approved drug is mitotane, an adrenolytic with many endocrine side effects, with 30% of patients showing stable disease or partial response. The one large therapeutic trial used mitotane with chemotherapy including etopo- side, doxorubicin, and cisplatin and demonstrated a 20% response with 50% stable disease short term (13). There was a dismal progression free survival of only five months. Many single agent drugs have been tried but without a clear hypothesis, no preclinical data to support the effort and demonstrable effectiveness.
Molecular Signatures in ACC
Molecular profiling of 90 ACC tumors by the Tumor Cell Genomic Atlas (TCGA) from the NCI confirmed previous studies that about 60% of ACCs have mutations in the WNT/B catenin pathway or TP53 (14,15). Unfortunately, these pathways are not currently targetable. Forty percent of the tumors in TCGA analysis had no known drivers. We recently collaborated with Foundation Medicine (Cambridge, Mas- sachusetts) and, using its platform FoundationOne Cdx, examined a cohort of 364 ACC tumors for molecular sequencing (16). We were able to show that, in addition to the previously identified common drivers, a large proportion of ACCs have alterations in the mismatch repair (MMR) pathway associated with greater mutational burden compared to wild type ACC tumors. However, in contrast to other solid tumors, the MMR dysregulated ACC tumors did not demonstrate concomitant microsatellite instability (Figure 1).
60
Gene % in pathway
40
20
0
Immune evasion
Hedgehog signaling pathway
DNA base excision repair
DNA methylation
Notch signaling pathway
MAPK signaling pathway
DNA mismatch repair
Telomere lengthening
SWI/SNF
PI3K/AKT signaling pathway
Double strand break repair
Cell cycle
Histone modification
WNT signaling pathway Tumor suppressor genes
Enriched Pathways
Development of Preclinical ACC Models
We hypothesized that development of preclinical models would advance the field of ACC to identify potential new therapeutics to then translate into clinical trials for our patients. We designed a pro- tocol in which human tumor samples were taken at the time of sur- gery and placed in in vitro cell culture with various matrices and then a portion of tissue was injected into the flanks of nude mice to cre- ate patient-derived xenografts. The tumors were passed in mice, and some were then grown in culture. With these methods, we developed the first two new ACC cell lines for the field in 50 years: CU-ACC1 and CU-ACC2 (Figure 2) (17). These cell lines have now been distrib- uted to investigators globally to advance our understanding of adre- nal cancer and to investigate new treatment options. We now have also generated 10 distinct PDX in nude mice available to confirm or refute studies performed in the ACC cell lines (unpublished data).
In vitro culture
In vivo model
Human ACC cell lines
ACC Patient derived xenograft PDX in nude mice
Feeder cells +
Collagen
ROCK inhibitor
Collagen
Feeder cells +
ROCK inhibitor
Each tumor is molecularly profiled to help predict response to various potential targeted agents.
Targeting Mitotic Kinases Dysregulated in ACC
Using publicly available databases, we identified that there are sev- eral mitotic kinases that are highly dysregulated in ACC compared to normal adrenal. In ACC cells and in vivo PDX models, we have shown that pharmacologic targeting of the oncogenic mitotic kinases, PBK and MELK, showed an ability to increase apoptosis and inhibit tumor growth in vitro and in vivo, in multiple models that we have developed (18,19). The putative MELK inhibitor, OTSSP167, appears to work through additional pathways independent of suppression of the MELK kinase activity to promote anti-tumor effects (unpublished observa- tions), which may be useful in clinical medicine. The inhibitor is now in human trials in solid tumors. We hope that targeting these kinases alone or in combination with mitotane or chemotherapy may prove to be a rational therapeutic option for some patients with ACC in the future.
Investigating the Effects of Mitotane and Checkpoint Inhibitors in ACC
When checkpoint inhibitors became available to test in solid tumors, our preliminary clinical experience demonstrated that a subset of ACC patients with progressive disease had a partial response or sta- ble disease when pembrolizumab was added to ongoing mitotane ther- apy with therapeutic levels (Figure 3) (20). The graph demonstrates responses of an early cohort of individual patients treated with pem- brolizumab added to ongoing mitotane therapy. The arrow identifies a patient who never achieved a therapeutic level of mitotane; when she stopped the drug, the metastatic lesions progressed (see imaging). Of interest, the responders were not predicted by PD1 expression in the tumor or if they had a mutation in the mismatch repair pathway. The mechanism of the response to combination therapy was more complex.
A.
6
Partial respose
5
Stable disease
Patient
Progressive disease
4
3
2
1
0
10
20
30
40
B.
Months
Patient 4
Pembrolizumab started
Mitotane stopped
Mitotane treatment
Mitotane + Pembrolizumab
Pembrolizumab alone
Feb 2017
May 2017
Nov 2017
We hypothesized that mitotane may alter the tumor microenvironment to augment the response to checkpoint inhibitors.
To understand the mechanisms by which mitotane could alter the tumor microenvironment to enhance checkpoint inhibitor immuno- therapy efficacy, a humanized mouse model of ACC was developed (21). The humanized mouse model (hu-CB-BRGS) was generated using sub- lethally irradiated BRGS strain mice transplanted with human um- bilical cord blood from healthy donors at the University of Colorado. At 19 weeks post-transplantation, hu-CB-BRGS mice were examined for their level of human chimera and randomized into two groups based on their chimerism. The mice were then injected with CU-ACC2-PDX human tumors tissue previously established in nude mice. Once the PDX tumors reached 150-300 mm (3), the mice were treated with either control or pembrolizumab. The checkpoint inhibitor, pembroli- zumab, showed significant inhibition of in vivo tumor growth with con- comitant increase in immune infiltrate in the tumor samples. However, the pembrolizumab treatment alone had short-lasting effects with tumor growth escape observed at five weeks. Around the same time, the matching ACC2 patient had disease progression. The patient was already on mitotane with therapeutic level (>14 ug/mL) and pembroli- zumab was added. The patient responded to a combination of mitotane and pembrolizumab with regression of metastatic disease; compared to the matching humanized mouse treated with pembrolizumab alone, the patient has had long-lasting remission. Ongoing studies are inves- tigating what aspects of the tumor microenvironment are altered after mitotane exposure in human tissue and mouse models and whether we can predict which tumors may respond to this combination approach. In a unique case where we had tumor samples before and after mito- tane exposure, mitotane increased the immune cell infiltrates in the human ACC tumor microenvironment with increase predominantly in CD8+ T cells (CD3+ CD8+) (unpublished observations). The human- ized mouse model allows us to further investigate combinations of im- mune checkpoint inhibitors, chemotherapy, mitotane, and other agents for future therapeutic options.
DISCUSSION
Our investigation into adrenocortical cancer started with our patients. We reviewed the current state of the field and current thera- peutic options. We hypothesized that, at least in part, the slow pro- gress in the ACC field was due to lack of preclinical models compared to most other cancers. We have initially focused our efforts to develop
additional preclinical cell models and PDX models that can be used by us and others to expand our understanding of ACC and test new therapeutic single or combination agents. In addition, we have focused our studies on identifying dysregulated mitotic kinases in ACC and evaluating pharmacologic targeting in the cell and animal models to support future studies in humans. More recent efforts have been active in elucidating the mechanism of mitotane alone or in combina- tion with checkpoint inhibitors on ACC tumor progression. Develop- ment of humanized ACC models has allowed us to begin to understand how mitotane alters the tumor microenvironment and will allow future testing of different combinations of therapeutics with immune modula- tion with or without mitotane. Distributing our models globally should hopefully advance the field by allowing others to test their hypotheses for future agents in preclinical models before testing in humans. Fund- ing of this orphan cancer research is needed to move the field forward to help our patients with ACC.
ACKNOWLEDGMENTS AND FINANCIAL SUPPORT
We thank the Wierman lab personnel and collaborators including Adwita Kar, Tapahsama Banerjee, Tessa Holmstoen, Lauren Fishbein, and Nikita Pozdeyev and importantly our patients.
Funding has been provided by VA Merit to MEW, K08 to KKV, and the Adrenal Tumor Program fund at the University of Colorado Endocrine Division, Anschutz Medical Campus.
REFERENCES
1. Boepple PA, Mansfield MJ, Wierman ME, et al. Use of a potent, long acting agonist of gonadotropin-releasing hormone in the treatment of precocious puberty. Endocr Rev 1986;7:24-33.
2. Santoro N, Wierman ME, Filicori M, Waldstreicher J, Crowley WF, Jr. Intravenous administration of pulsatile gonadotropin-releasing hormone in hypothalamic amen- orrhea: effects of dosage. J Clin Endocrinol Metab 1986;62:109-16.
3. Wierman ME, Beardsworth DE, Crawford JD, et al. Adrenarche and skeletal maturation during luteinizing hormone releasing hormone analogue suppression of gonadarche. J Clin Invest 1986;77:121-6.
4. Gharib SD, Wierman ME, Badger TM, Chin WW. Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat. J Clin Invest 1987;80:294-9.
5. Bruder JM, Krebs WD, Nett TM, Wierman ME. Phorbol ester activation of the protein kinase C pathway inhibits gonadotropin-releasing hormone gene expression. Endocrinol 1992;131:2552-8.
6. Kepa JK, Wang C, Neeley CI, et al. Structure of the rat gonadotropin releasing hormone (rGnRH) gene promoter and functional analysis in hypothalamic cells. Nucleic Acids Res 1992;20:1393-9.
7. Wierman ME, Pawlowski JE, Allen MP, Xu M, Linseman DA, Nielsen-Preiss S. Molecular mechanisms of gonadotropin-releasing hormone neuronal migration. Trends Endocrinol Metab 2004;15:96-102.
8. Salian-Mehta S, Xu M, Knox AJ, et al. Functional consequences of AXL sequence vari- ants in hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2014;99:1452-60.
9. Xiong W, Knox AJ, Xu M, et al. Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation and survival under hypoxia. Mol Endocrinol 2015;29:460-72.
10. Kiseljak-Vassiliades K, Bancos I, Hamrahian A, et al. American Association of Clinical Endocrinology Disease State Clinical Review on the Evaluation and Management of Adrenocortical Carcinoma in an Adult: a Practical Approach. Endocr Pract 2020;26: 1366-83.
11. Fassnacht M, Dekkers OM, Else T, et al. European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2018;179:G1-G46.
12. Else T, Kim AC, Sabolch A, et al. Adrenocortical carcinoma. Endocr Rev 2014;35: 282-326.
13. Fassnacht M, Terzolo M, Allolio B, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012;366:2189-97.
14. Zheng S, Cherniack AD, Dewal N, et al. Comprehensive pan-genomic characteriza- tion of adrenocortical carcinoma. Cancer Cell 2016;29:723-36.
15. Assie G, Letouze E, Fassnacht M, et al. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet 2014;46:607-12.
16. Pozdeyev N, Fishbein L, Gay LM, et al. Targeted genomic analysis of 364 adrenocorti- cal carcinomas. Endocr Relat Cancer 2021;28:671-81.
17. Kiseljak-Vassiliades K, Zhang Y, Bagby SM, et al. Development of new preclinical models to advance adrenocortical carcinoma research. Endocr Relat Cancer 2018;25: 437-51.
18. Kiseljak-Vassiliades K, Zhang Y, Kar A, et al. Elucidating the role of the maternal embryonic leucine zipper kinase (MELK) in adrenocortical carcinoma. Endocrinol 2018;159:2532-44.
19. Kar A, Zhang Y, Yacob BW, et al. Targeting PDZ-binding kinase is anti-tumorigenic in novel preclinical models of ACC. Endocr Relat Cancer 2019;26:765-78.
20. Head L, Kiseljak-Vassiliades K, Clark TJ, et al. Response to immunotherapy in com- bination with mitotane in patients with metastatic adrenocortical cancer. J Endocr Soc 2019;3:2295-304.
21. Lang J, Capasso A, Jordan KR, et al. Development of an adrenocortical cancer human- ized mouse model to characterize anti-PD1 effects on tumor microenvironment. J Clin Endocrinol Metab 2020;105:26-42.
DISCUSSION
Gravallese, Boston: I’m very interested in the mitotane story. Can you tell us a little bit more about the mechanism by which mitotane might be leading to this increased immune response?
Wierman, Aurora: Despite mitotane being used for 50 years, no one knows at all how it works. We know it inhibits steroidogenesis, and some data suggest that it may work through certain mitochondrial pathways in the cell, but we still don’t understand its exact mechanism of action.
Lippman, Washington, DC: Thank you for your very interesting presentation. One aspect of trying to examine the effects of immune components as part of the response to anticancer therapy is the immune status of the host mouse. Recent work by Profes- sor Elizabeth Repasky at Roswell Park (which we have replicated) reveals that mice housed at normal animal house conditions are under extreme thermal stress which has an incapacitation effect on their immune responses. Animals housed at their “preferred” temperature, which is 82-85° C, are able to elicit far more robust responses to agents such as checkpoint inhibitors. I strongly suggest that you consider this step in pursuing your exciting experiments with mitotane.
Wierman, Aurora: Thank you for your comments about temperature-modulating response to immunotherapy. Radiation can be effective for local disease, but, as far as I know, no information is available on whether radiation would enhance immunotherapy or vice versa.