Phase I Study of Domatinostat (4SC-202), a class I Histone Deacetylase Inhibitor in Patients with Advanced Hematological Malignancies

Bastian von Tresckow1*, Cyrus Sayehli2, Walter E. Aulitzky3, Maria-Elisabeth Goebeler2, Matthias

4,5,6*
Schwab
, Eunice Braz7, Babett Krauss7, Rolf Krauss7, Frank Hermann7, René Bartz7, Andreas

Engert1

1: University Hospital Cologne, Department of Internal Medicine I, Kerpener Str. 62, 50937 Köln; Germany
2: Comprehensive Cancer Center Mainfranken, Early Clinical Trial Unit, Josef-Schneider Str. 6, 97080 Würzburg; Germany
3: Robert-Bosch-Hospital, Auerbachstraße 110, 70376 Stuttgart; Germany

4: Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology Stuttgart, Germany and University of Tübingen, Germany
5: Department of Clinical Pharmacology, University Hospital Tübingen; Germany 6: Department of Pharmacy and Biochemistry, University of Tübingen; Germany 7: 4SC AG, Fraunhoferstrasse 22, 82152 Planegg-Martinsried; Germany *corresponding author: [email protected]

*M.S. was supported by the Robert Bosch Foundation.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejh.13188
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Running Title: Phase I study domatinostat

2.Abstract & Key Words

Objectives – Domatinostat (4SC-202) is a selective class I histone deacetylase inhibitor (HDACi). This phase I study investigated safety, tolerability, pharmacokinetics (PK), pharmacodynamics and anti- tumor activity in patients with advanced hematological malignancies.

Methods – Domatinostat was administered orally once (QD) or twice daily (BID) on days 1-14 with 7 days off or continuously days 1-21 in a 3+3 design at 7 dose levels from 25 to 400 mg total daily dose (TDD). 24 patients were treated with domatinostat.

Results – No formal maximum tolerated dose (MTD) was determined. 1 dose-limiting toxicity (DLT, grade 4 hypercalcemia) occurred during 200 mg BID continuous treatment. 6 patients were reported with > grade 3 treatment-related adverse events (TRAE; grade 3 hematological in three patients, grade 3 and 4 liver enzyme increase in 2 patients, grade 4 pulmonary embolism and grade 4 hypercalcemia in 1patient each). Higher grade hepatic TRAE occurred in the 200 mg BID continuous treatment cohort. Out of 24 patients 1 achieved a complete response, 1 a partial response and 18 had stable disease as best response.

Conclusion – Administration of domatinostat was safe, well tolerated with signs of anti-tumor activity. 400 mg TDD in a 200 mg BID schedule (14+7) is the recommended phase II dose for monotherapy.

ClinicalTrials.gov Identifier: NCT01344707

Keywords: domatinostat, 4SC-202, epigenetics, HDAC inhibition, phase I study, hematological malignancies

3.Introduction

The accumulation of genetic alterations drives the transformation of normal cells through different stages of cancer to a metastatic disease. Carcinogenesis however, cannot only be explained by genetic alterations, but also involves different epigenetic processes, like DNA methylation, histone modification and non-coding RNA deregulation. (1),(2) Post-translational modification of histone proteins (e.g. by acetylation or methylation) has a direct impact on gene expression and therefore is believed to be crucial for the regulation of transcription (reviewed in (3)). Addition of acetyl-groups from an Acetyl-CoA donor to specific lysine residues on histones is carried out by histone acetyltransferases (HATs) while histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in histone proteins. Since transcriptional regulation by epigenetic modifiers directly impacts important cellular processes in development, differentiation and homeostasis, it is not surprising that these functions are compromised during various diseases. Especially in cancer, cellular processes are often aberrant due to underlying genetic changes and malfunctions in epigenetic modulation as reviewed in (2). Overexpression of HDACs is well described as a negative prognostic factor for overall survival (OS) in multiple cancers including breast, lung, liver, colorectal, pancreatic, gastric cancer and melanoma (2,4,5). Furthermore, significantly elevated expression levels of HDACs have been observed in a broad range of hematological malignancies, for example elevated expression levels of HDAC 1, 2 and 6 in patients with cutaneous T-cell lymphoma (CTCL) (6). Consequently, a variety of HDACi have been approved for hematological malignancies: As monotherapy, vorinostat by FDA for the treatment of cutaneous T cell lymphoma (CTCL) (7), romidepsin by the FDA for the treatment of CTCL and peripheral T-cell lymphoma (PTCL) (8), chidamide in China by the CFDA for treatment of PTCL (9) and belinostat by the FDA for the use against PTCL (10) and for combination treatment, panobinostat by the FDA and EMA for the treatment of multiple myeloma (11). Those and many others, broad-spectrum as well as selective HDACi are in clinical trials either as monotherapy or in combination in hematological and solid tumor indications (12–14).

Domatinostat (4SC-202, (E)-N-(2-aminophenyl)-3-(1-((4-(1-methyl-1H-pyrazol-4-yl)phenyl)sulfonyl)-

1H-pyrrol-3-yl)acrylamide tosylate; CAS number 1186222-89-8) is an orally (PO) available small molecule selectively targeting HDAC class I proteins. It was tested in an in vitro panel of human recombinant HDACs 1-11 and showed high specificity against HDAC class I (HDAC 1, 2 and 3) with inhibitory constant (Ki) values ranging from 7.9 nM (HDAC 3), 14.8 nM (HDAC 1) to 38.8 nM for HDAC 2. Activity against other HDACs was either low (Ki > 1.8 µM) or not detected (data not shown). Additionally, some inhibitory activity against Lysine-specific histone demethylase 1A (LSD1) in the low µM range was observed. The compound was further tested in cell-based viability assays using different hematological-derived cell lines. Domatinostat showed a high potency against all tested cell lines (Table 1A) with IC50 values ranging from 0.11 µM (HL60) to 0.52 µM in the CCRF-CEM cell line. Additionally, several different xenograft models were tested. Animals were subcutaneously implanted with different human cancer cells and treated with different doses of domatinostat. Robust anti-tumor activity was observed at doses of 40 mg/kg or higher (Table 1B). The GRANTA-519 B-cell lymphoma model displayed higher sensitivity against treatment with domatinostat however an increased body weight loss was also observed in this model (see supplemental methods for additional details).

Here we report results from a first-in-man phase I study (‘TOPAS’ ClinicalTrials.gov Identifier: NCT01344707). The primary objective was to investigate the safety, tolerability and pharmacokinetics (PK) of repeated daily oral doses of domatinostat using a 3+3 dose-escalation design. Secondary objectives included assessment of pharmacodynamic (PD) profiles and anti-cancer activity in advanced hematological malignancies. PD biomarker assays investigating lysine acetylation, HDAC inhibition (HDACi), gene regulation and cytokine modulation were included to provide evidence for on-target activity of domatinostat to adequately guide the choice of the recommended phase II dose (RP2D).

4.Patients and Methods

Study design and patient selection

This phase I study was conducted in adult patients with advanced hematological malignancies – treatment-refractory, relapsed and/or with no established treatment option in a 3+3 dose escalation design starting at 25 mg as the lowest dose. The main study phase comprised the initial two cycles until first tumor assessment after six weeks. All patients had an Eastern Cooperative Group (ECOG) performance status (PS) of 0-2, a life expectancy of at least 12 weeks, adequate liver, renal and bone marrow function including normal serum electrolytes.

The protocol was approved by the Institutional Review (IRB) board of each participating institution and was conducted in accordance with Good Clinical Practice (GCP) guidelines and the Declaration of Helsinki. All patients provided written informed consent before any trial procedure was performed.
Treatment

The results from 2 x 5-days pivotal repeated-dose studies in mice and rats as well as from a 28-days pivotal repeated-dose study in monkeys (not shown, IND) indicated a clinical starting daily dose of 25 mg per patient for a repeated consecutive administration over 14 days. Consequently, treatment schedules for 14 consecutive dosing days followed by a 7-day off-treatment period in a three-week treatment cycle were chosen for the initial dose cohorts and a 21 consecutive dosing days schedule for the later dose escalation as alternative escalation schedule.

Domatinostat was administered as tablets and dose cohorts of 3 to 6 patients were intended for treatment with escalating doses of domatinostat until maximum tolerated dose (MTD), whereas the MTD was defined as the dose preceding those at which at least 2 of 3 or at least 2 of 6 patients experience DLT or further escalation is not seen as indicated due to pharmacodynamic, pharmacokinetic and/or safety reasons resulting in the RP2D. Dose modifications were allowed and dose interruption criteria were predefined.

Assessment

Safety. Safety evaluations included medical history, physical examination, vital signs, weight, temperature, review of concomitant medications, assessment of ECOG PS, laboratory studies (incl. troponin) and repetitive 12-lead electrocardiographs (ECGs). Dose-limiting toxicities (DLTs) were defined as any of the following conditions occurring during the first 21 days of treatment (Cycle 1) including ≥ grade 3 non-hematological toxicity; ≥ grade 3 nausea, vomiting or diarrhea uncontrollable by intensive treatment; grade 4 granulocytopenia lasting ≥ 7 days or febrile neutropenia of any duration in case of OD or BID treatment schedule. Furthermore, grade 4 thrombocytopenia, grade 3 anemia lasting ≥ 7 days despite of intervention, inability to begin the next dosing of treatment within two weeks of the scheduled dose due to unresolved toxicity (max. 7+14 = 21 days resting period); ≥ Grade 2 non-hematological toxicity that persisted beyond the first cycle (21 days) and grade 2 toxicities which in the judgement of the investigator/sponsor were dose limiting.

Pharmacokinetics. The PK analysis set was defined as all patients who received more than 80% of the full domatinostat medication at consistent doses during treatment cycles 1 and 2 and from whom more than 80% of all planned PK samples were taken. Blood samples for PK analysis of 4SC- 202 were taken at cycle 1 day 1 (C1D1), C1D14 and at C2D1 for at different time points for up to 24 h. Blood samples were stored on ice and plasma was then obtained by centrifugation at 4°C within max. 15 min after the collection. Plasma was then immediately stored at -20°C or below and 4SC-202 plasma concentration was quantified using a validated HPLC-MS/MS method.

Pharmacodynamics. Blood samples for PD assessment were drawn pre-dose and after 2h, 5h, 24 h at C1D1, C1D14 and C2D1. A single pre-dose value was taken at C1D5. Patient samples were assayed for HDAC inhibition, lysine acetylation, gene/microRNA (miRNA) expression and cytokine modulation using validated enzyme-linked immunosorbent assays (ELISA), quantitative polymerase-chain reaction (qPCR) methodologies and immunoblotting techniques.

Antitumor activity. Tumor response evaluation was conducted according to clinical practice for the underlying haematological malignancy. Patients were examined for treatment response according to the judgement of the investigator preferably every six weeks for six months, thereafter tumor assessments were performed in a repeated manner. In case of clinical note of progression, a tumor assessment should have been performed immediately.

5.Results

Patient characteristics

Twenty-four patients with advanced hematological malignancies were enrolled in three German Cancer Centers and treated with domatinostat at doses between 25 mg QD in a 14 days on- and 7 days off schedule and 400 mg TDD continuous treatment. Details on patient demographics, baseline criteria, disease characteristics and prior treatments are summarized in Table 2.
Treatment

A total of 209 treatment cycles were administered to twenty-four patients at seven different dose levels (14 days on-treatment with 7 days off in a 21-day cycle: 25 mg QD [n=3]; 50 mg QD [n=3]; 100 mg QD [n=3]; 200 mg QD [n=3]; 400 mg QD [n=3]; 200 mg BID [n=3] and 21-day continuous treatment: 200 mg BID [n=6]). A median duration of treatment of five cycles was recorded. No clear

correlation of duration of treatment to dose levels was seen in the 14 days on- and 7-days off- treatment schedule while the lowest median duration of treatment was seen in the 200 mg BID continuous treatment cohort (Table 2 and Figure 1)
Safety and tolerability

Administration of domatinostat in this study was safe and well tolerated. Most adverse events were mild to moderate. Six patients were reported with > grade 3 treatment-related adverse events (TRAE; grade 4 pulmonary embolism in one patient, grade 4 hypercalcemia [dose-limiting toxicity (DLT)] in one patient, grade 3 hematological laboratory decreases in three patients and grade 3 and 4 liver enzyme increases in two patients). All > grade 3 hepatic TRAE and one grade 4 hypercalcemia were seen in the 200 mg BID continuous dosing cohort. No treatment-related deaths occurred. Two patients (exacerbation of skin and mucosa graft-versus-host disease (GvHD) to grade 2 and grade 1 paresthesia) discontinued from the trial due to TRAEs. All TRAEs in ≥ 10% of patients are summarized in Table 3 (Supplement Table S1: All Treatment-related adverse events (TRAE) per SOC PT).

One DLT occurred (grade 4 hypercalcemia) and no formal MTD was reached. In the 200 mg BID continuous treatment cohort two patients with high-grade hepatic enzyme elevation adverse events (AEs) were reported which would have qualified as DLTs but occurred outside the predefined DLT period.

The most frequently reported AEs (≥ 20%) were fatigue (45.8%), diarrhea (37.5%), nausea (29.2%) and decreased appetite, peripheral edema, urinary tract infection or nasopharyngitis (each 20.8%). 87 (26.4%) of all reported AEs were considered to have a causal relationship to domatinostat treatment. Seven patients experienced 17 serious adverse events (SAEs), one was considered possibly related to treatment (grade 4 pulmonary embolism in a patient in the 50 mg QD cohort). Two patients were withdrawn during the study treatment due to mild paresthesia and moderate GvHD, which were both considered possibly related.

Most of the grade ≥ 3 AEs reported in 13 patients were considered unrelated to treatment with domatinostat. 12 grade 3/4 AEs in six patients were considered likely, probably or possibly related to treatment. 3 TRAEs concerned hematological toxicities (grade 3 thrombocytopenia, neutrophil count decreased and neutropenia, in 3 patients) and seven events concerned grade 3 and 4 increases in liver enzymes and values (ALT, AST, GGT, alkaline phosphatase, bilirubin and LDH) in 2 patients. Additionally, one event of grade 4 pulmonary embolism was reported in one patient with underlying diffuse large B-cell lymphoma (DLBCL) and one grade 4 hypercalcemia in one patient. An accumulation of higher grade hepatic enzyme elevations in two patients in the 200 mg BID continuous dosing cohort was identified, suggesting a possible treatment effect whereas for both patients unrelated differential diagnoses were discussed and considered by the investigator due to personal medical history and subsequent further diagnostics. One patient had previously undergone allogeneic stem cell transplantation and had suffered from acute and chronic intestinal, skin and mucosal GvHD. The time of increase of liver enzymes, the symptoms and signs of skin and mucosal GvHD worsened and so a differential diagnosis of hepatic GvHD involvement was discussed and considered another possible etiology. Another patient had shown an immediate combined rapid increase of liver enzymes and cholestasis parameters bilirubin and GGT. Study drug was temporarily interrupted, and the patient was discontinued from study due to progression prior to recovery of the TRAEs. Over time a liver biopsy was conducted and had shown histologic signs of chronic cholestasis and an intrahepatic-vascular intima proliferation compatible to a previous veno-occlusive disease (VOD), possibly caused by prior systemic cancer agents – so the investigator subsequently considered this another possible etiology.

There was no correlation of incidence and severity of treatment-emergent adverse events (TEAEs) to either an age group, prior cancer treatment, general medical history or an underlying hematological malignancy and no other clinically significant findings in clinical laboratory data that indicated any relationship to the study drug or dose (Supplement Table S2: Related TEAEs in patients per dose level by SOC (Safety Analysis Set).

Neither newly diagnosed relevant morphological changes nor newly diagnosed relevant arrhythmias were observed during the study. Overall, no consistent relation between electrocardiographic changes (morphology and/or QT interval prolongation) or arrhythmias and administration of domatinostat could be noted. Abnormal ECG signs were most likely related to the patients´ disease status, but not to the study treatment.

PK studies

Twenty-one patients treated were included in the PK analysis set (80% compliance) and data were evaluated from all available concentration data. The PK was linear up to 200 mg but displayed a capacity limitation at 400 mg. Additionally, a considerable inter-individual variability at both 200 mg BID 14+7 and once daily was noticed. A dose dependent half-life was noted with half-lives up to 146 h in case of 200 mg BID dosing. Indeed, patients enrolled into the 200 mg BID 14+7 cohort exhibited measurable drug concentrations on C2D1 pre-dose after a seven day wash out period. On the contrary, the exposure at C1D14 might not reflect steady state conditions for dose level 200 mg BID (Figure 2 A).

PD studies

Several markers were analyzed to determine their utility as pharmacodynamic biomarkers, namely lysine acetylation, HDAC inhibition (HDACi), gene regulation and cytokine modulation.

Total lysine acetylation

Total lysine acetylation was measured in peripheral blood monocytic cells (PBMCs). A clear

exposure-response relationship could not be derived. However, in the case of AUC-PD at C1D1 evaluable data could be fitted with a simple Emax model. Overall, a 2 to 3-fold increase (relative to

pre-dose C1D1) in acetylation at C1D14 was observed at dose levels greater than 25 mg. In addition, increased AUC-PD values at C1D14 and C2D1 relative to pre-dose C1D1 were observed, independent of dose level (Figure 2 B).

HDAC inhibition

HDAC inhibition could be demonstrated in nine out of 17 patients at day 1, and in 13 out of 17 patients at day 14. Therefore, the number of patients which demonstrated inhibition of HDAC activity increased from day 1 to day 14. Additionally, maximum inhibition increased from day 1 to day 14. HDAC inhibition by domatinostat can be demonstrated in the patient samples already in the lowest dose cohort, but no correlation with dose, exposure or clinical outcome can be stated (Figure 2C).

Recommended phase II dose

Taking all safety parameters and PK conclusions into account, 400 mg TDD in a 200 mg BID schedule for 14 days on-treatment with consecutive 7 days off-treatment is the recommended administration dose and schedule of for further monotherapy clinical studies with domatinostat. Despite this, for potential combination treatment studies lower doses might be taken into consideration depending on the safety profile of the combination compounds.

Antitumor activity

Twenty patients (83%) had at least stable disease (SD) after two treatment cycles at the first tumor response evaluation after six weeks and 19 of them continued treatment. Median duration of treatment was 15 weeks, exceeding one year in three patients with a maximum of 122 weeks. Most of the patients (75%) showed SD as their best response, lasting for more than six months in five patients with a maximum of more than two years (24,6 months). Four patients did not respond to

treatment and had a progressive disease (PD) after two treatment cycles. Progression-free survival (PFS) of all patients ranged from 43 to 859 days.
Objective responses (OR), one complete response (CR) and one partial response (PR), were achieved in two patients. The onset of the OR was detected after six weeks of treatment for both patients. The duration of response was more than two years (745 days) for the complete responder and almost six months (172 days) for the partial responder (Figure 3).
 69-year-old patient with stage IV early relapsed angioimmunoblastic T-cell lymphoma having received three prior systemic therapies, including high dose chemotherapy with autologous stem cell transplantation, showed rapid remission of all lymphoma manifestations under treatment with domatinostat at a dose of 200 mg twice daily for 14 days. He achieved a good PR after two cycles and even a CR. Clinically the patient´s condition rapidly improved from initial ECOG PS2 to ECOG PS0 (Figure 3 A).

 29-year-old patient with stage IV nodular-sclerosis HL having received 8 prior systemic therapies, three cycles of radiation, one autologous and one allogenic stem cell transplantation showed a partial response with decrease in size of lymph node packages after two weeks of treatment with domatinostat at a dose of 200 mg BID for consecutive 21 days (Figure 3 B).

Detailed data for patients with Non-Hodgkin lymphoma, Hodgkin´s lymphoma and multiple myeloma are listed in Table 2. In addition, both patients enrolled with untreated MDS stopped treatment presenting with SD for six months, the one patient enrolled with AML had PD at the end of the main study phase and discontinued the study.

6.Discussion

HDACi are able to influence diverse biological processes and therefore may have pleiotropic anti- tumor effects (15). Induction of cancer cell cycle arrest, differentiation and cell death as well as reduction of angiogenesis and modulation of immune responses are well described and render HDACi promising therapeutic agents in oncology (16). Hypothesis of “epigenetic vulnerability of cancer cells”, which has been proposed by Dawson and Kouzarides (17), is a cause of relative specificity of HDACi. This hypothesis supposed that normal cells have, in contrast to some cancer cells, multiplied epigenetic regulatory mechanisms. Therefore, HDACs may be essential for the maintenance of a set of key genes required for survival and growth of cancer but not normal cells. (17)
Several classes of HDACi have been identified and different HDACi appear to inhibit different HDAC subgroups (18). Chemically, four classes of HDACi have been developed so far. While early developed compounds, belonging to the groups of short-chain fatty acids like valproic acid or hydroxamate acids like vorinostat, resminostat or panobinostat share a broad-spectrum HDAC inhibition pattern – more recent developments belonging to the depsipeptide group like romidepsin or belonging to the benzamide group like entinostat, mocetinostat and domatinostat are characterized by a more selective HDAC inhibition profile, mainly targeting class I HDACs (19). Not only by chemical structure but also by binding kinetics, e.g. hydroxamate acid broad-spectrum HDACi and selective benzamide HDACi show differences while compounds from the benzamide class of inhibitors display slow-on/slow-off binding properties (20). In this regard selective HDAC class I inhibitor are often described as promising selective agents to treat cancer (21), respectively act as an ideal combination partner with established treatment approaches to synergize anti-tumor activity or overcome resistance (22). HDAC family members 1, 2 and 3 are located in the nucleus, show ubiquitous tissue expression and build subunits of multi-protein nuclear complexes that are crucial for transcriptional repression and epigenetic landscaping (23).

Here we characterize and clinically validate domatinostat (4SC-202) as an orally available small molecule, selective class I HDACi that efficiently and selectively blocks HDAC 1,2 and 3. Although some activity against LSD1 was observed using purified enzyme, the dual inhibitory activity against HDAC and LSD1 of domatinostat remains ambiguous and may require additional experiments (24). In cell-based assays, the compound showed potent inhibition of viability in several human hematological tumor cell lines. These effects could also be translated to in vivo models and domatinostat showed robust anti-tumor activity in several xenograft mouse models.
Based on the results of several pivotal repeated-dose studies in mice, rats and monkeys which indicated a clinical starting daily dose of 25 mg per patient for a repeated consecutive administration over 14 days, the here reported data from the first-in-man TOPAS study have shown that domatinostat can be given safely and is well tolerated up to 400 mg TDD in a 14-day daily QD or BID treatment and a consecutive 7-day pause schedule in mostly heavily pretreated hematological cancer patients.
The PK analysis after oral administration of the drug were linear up to 200 mg but displayed a capacity limitation at 400 mg. A considerable inter-individual variability at both 200 mg BID 14+7 and once daily was noticed. A dose dependent half-life was noted with half-lives up to 146 h in the case of 200 mg BID dosing. In conclusion, 400 mg TDD in a 200 mg BID schedule for 14 days on-treatment with consecutive 7 days off-treatment was suggested as the recommended administration dose and schedule of domatinostat for further monotherapy clinical studies. Despite this, for potential combination treatment studies lower doses could be considered depending on the safety profile of the combination compounds.
Most frequent toxicities were gastrointestinal disorders like diarrhea and nausea, as well as fatigue while hematological toxicities up to grade 3 could be observed in these patients with mostly pretreated, underlying hematological malignancies. High grade (> grade 3) hepatic TRAEs were limited to two patients in the 200 mg BID continuous treatment cohort which would have principally

qualified for DLTs but occurred after the defined DLT observation period. Therefore, and in conjunction with the pharmacokinetic saturation observed beginning at 200 mg QD, no further escalation was performed. In addition, one patient with DLBCL as underlying hematological disease experienced a grade 4 pulmonary embolism and one patient experienced grade 4 hypercalcemia which was the only DLT seen in the study, so no formal MTD was reached. As cardiotoxicity, especially QT-prolongation, is seen as a potential class-specific adverse event for HDACi, it is of note that sinus tachycardia in one patient was the only observed cardiac adverse event in the TOPAS study. Domatinostat has shown the expected organ distribution pattern of treatment-related adverse events known and described for HDACi in general and selective class I HDACi in particular worth mentioning that the safety profile of HDACi seems independent from selectivity or chemical class (19). However and taken with caution due to the limited number of patients treated, domatinostat has shown a comparably low rate of grade 3/4 treatment-related adverse events up to the RP2D and no accumulation of cardiotoxic adverse events (25),(26). Additionally, the ability of daily and even twice daily administration is unique for this class I selective HDACi and offers a variety of treatment schedules from safety perspective for potential combinations approaches. Continuous treatment with lower daily doses was not investigated in the TOPAS study but might be favorable for maintaining the constant inhibition of HDAC activity and should be considered being tested in upcoming clinical studies. However, there´s only limited knowledge to date about favorability of constant or pulsatile HDACi and more insights need to be gained to optimize clinical use of HDACi in monotherapy and combination approaches.
Although considering the small number of patients in a phase I study, anti-tumor activity could be demonstrated in this heavily pretreated patient population with at least stabilization in 83% of the patients with one patient showing PR and one patient even a CR with a duration of almost six months and over two years, respectively.

The results of this phase I study of the selective HDAC class I inhibitor domatinostat clearly merit further clinical development of this compound for treating cancer patients. Besides the potential in advanced hematological malignancies, particularly in T-cell lymphoma and HL as monotherapeutic agent, evidence is emerging that due to its pleiotropic effects epigenetic modulation, and in particular HDAC inhibition, might be a promising combination partner in hematology but also in solid tumor indications. Particularly, some potential to overcome resistance and favorable immune modulatory effects have been described for HDAC class I inhibitor warranting combination approaches, e.g. with immunotherapy. (27),(28),(29).

7.Acknowledgements

We are grateful to our patients, their families, investigators and co-investigators for their participation and contributions to this research. We thank Jonas Wohlfarth for his support in writing of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8.Conflict of Interest and Sources of Funding

E.B., B.K., R.K., F.H. and R.B. are all full-time employees of 4SC AG and may own shares of the company. B.V.T., C.S., W.E.A., ME.G., M.S. and A.E. declare no conflict of interest with relevance to this work. M.S. was supported by the Robert Bosch Foundation.

9.References

1.Allis CD, Jenuwein T. The molecular hallmarks of epigenetic control. Nat Rev Genet.
2016;17(8):487–500.

2.West AC, Johnstone RW. New and emerging HDAC inhibitors for cancer treatment. J Clin Invest. 2014;124(1):30–9.

3.Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer [Internet]. 2004;4(2):143–53. Available from: http://dx.doi.org/10.1038/nrc1279

4.Sarkar D, Leung EY, Baguley BC, Finlay GJ, Askarian-Amiri ME. Epigenetic regulation in human melanoma: Past and future. Epigenetics. 2015;2294(May 2015):37–41.

5.Weichert W, Röske A, Gekeler V, Beckers T, Ebert MP, Pross M, et al. Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis. Lancet Oncol. 2008;9(2):139–48.

6.Marquard L, Gjerdrum LM, Christensen IJ, Jensen PB, Sehested M, Ralfkiaer E. Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma. Histopathology. 2008;53(3):267–77.

7.Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R. FDA Approval Summary: Vorinostat for Treatment of Advanced Primary Cutaneous T-Cell Lymphoma. Oncologist [Internet]. 2007;12(10):1247–52. Available from: http://theoncologist.alphamedpress.org/cgi/doi/10.1634/theoncologist.12-10-1247

8.Iyer SP, Foss FF. Romidepsin for the Treatment of Peripheral T-Cell Lymphoma. Oncologist [Internet]. 2015;20(9):1084–91. Available from: http://theoncologist.alphamedpress.org/cgi/doi/10.1634/theoncologist.2015-0043

9.Lu X, Ning Z, Li Z, Cao H, Wang X. Development of chidamide for peripheral T-cell lymphoma, the first orphan drug approved in China. Intractable Rare Dis Res. 2016;5(3):185–91.

10.Lee H-Z, Kwitkowski VE, Del Valle PL, Ricci MS, Saber H, Habtemariam BA, et al. FDA Approval: Belinostat for the Treatment of Patients with Relapsed or Refractory Peripheral T-cell Lymphoma. Clin Cancer Res [Internet]. 2015;21(12):2666–70. Available from: http://clincancerres.aacrjournals.org/cgi/doi/10.1158/1078-0432.CCR-14-3119

11.Tzogani K, van Hennik P, Walsh I, De Graeff P, Folin A, Sjöberg J, et al. The European Medicines Agency Review of Panobinostat (Farydak) for the Treatment of Adult Patients with Relapsed and/or Refractory Multiple Myeloma. Oncologist [Internet]. 2018;23:631–6. Available from: http://theoncologist.alphamedpress.org/lookup/doi/10.1634/theoncologist.2017-0301

12.Xiao Pfister S, Ashworth A. Marked for death: targeting epigenetic changes in cancer. Nat Publ Gr [Internet]. 2017;16(4):241–63. Available from: http://www.nature.com/nrd/journal/vaop/ncurrent/pdf/nrd.2016.256.pdf

13.Thurn K., Thomas S, Moore A, Munster PN. Rational therapeutic combinations with histone deactylase inhibitors for the treatment of cancer. Futur Oncol. 2011;7(2):263–83.

14.Mazzone R, Zwergel C, Mai A, Valente S. Epi-drugs in combination with immunotherapy: a new avenue to improve anticancer efficacy. Clin Epigenetics [Internet]. 2017;9(1):59.

Available from: http://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-017-0358-y

15.Li Z, Zhu W-G. Targeting Histone Deacetylases for Cancer Therapy: From Molecular Mechanisms to Clinical Implications. Int J Biol Sci [Internet]. 2014;10(7):757–70. Available from: http://www.ijbs.com/v10p0757.htm

16.Newbold A, Falkenberg KJ, Prince HM, Johnstone RW. How do tumor cells respond to HDAC inhibition? FEBS Journal. 2016.

17.Dawson MA, Kouzarides T. Cancer epigenetics: From mechanism to therapy. Cell [Internet]. 2012;150(1):12–27. Available from: http://dx.doi.org/10.1016/j.cell.2012.06.013

18.Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer,
neurological diseases and immune disorders. Nat Rev Drug Discov [Internet]. 2014;13(9):673– 91. Available from: http://dx.doi.org/10.1038/nrd4360

19.Subramanian S, Bates SE, Wright JJ, Espinoza-Delgado I, Piekarz RL. Clinical toxicities of histone deacetylase inhibitors. Pharmaceuticals. 2010;3(9):2751–67.

20.Lauffer BEL, Mintzer R, Fong R, Mukund S, Tam C, Zilberleyb I, et al. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J Biol Chem. 2013;288(37):26926–43.

21.Karagiannis TC, El-Osta A. Will broad-spectrum histone deacetylase inhibitors be superseded by more specific compounds? Leukemia [Internet]. 2007;21(1):61–5. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17109024

22.Kroesen M, Gielen PR, Brok IC, Armandari I, Hoogerbrugge PM, Adema GJ. HDAC inhibitors and immunotherapy; a double edged sword? Oncotarget. 2014;5(16):6558–72.

23.Witt O, Deubzer HE, Milde T, Oehme I. HDAC family: What are the cancer relevant targets? Cancer Lett [Internet]. 2009;277(1):8–21. Available from: http://dx.doi.org/10.1016/j.canlet.2008.08.016

24.Kalin JH, Wu M, Gomez A V., Song Y, Das J, Hayward D, et al. Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors. Nat Commun. 2018;9(1).

25.Ryan QC, Headlee D, Acharya M, Sparreboom A, Trepel JB, Ye J, et al. Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma. J Clin Oncol. 2005;23(17):3912–22.

26.Lillian L. Siu, Roberto Pili, Ignacio Duran, Wells A. Messersmith, Eric X. Chen R, Sullivan, Martha MacLean, Serina King, Shirley Brown, Gregory K. Reid, Zuomei Li AM, Kalita, Eric J. Laille, Jeffrey M. Besterman, Robert E. Martell and MAC. Phase I Study of MGCD0103 Given As a Three-Times-Per-Week Oral Dose in Patients With Advanced Solid Tumors. J Clin Oncol. 2008;26(12):1940–7.

27.Gallagher SJ, Tiffen JC, Hersey P. Histone modifications, modifiers and readers in melanoma resistance to targeted and immune therapy. Cancers (Basel). 2015;7(4):1959–82.

28.Epner EM, Saroya BS, Hasanali ZS, Loughran TP. Combination epigenetic and immunotherapy overcomes resistance to monoclonal antibodies in hematologic malignancies: A new therapeutic approach. Exp Hematol [Internet]. 2016;44(3):157–60. Available from: http://dx.doi.org/10.1016/j.exphem.2015.12.011

29.Zheng H, Zhao W, Yan C, Watson CC, Massengill M, Xie M, et al. HDAC inhibitors enhance T- cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma. Clin Cancer Res. 2016;22(16):4119–32.

10.Legends

Table 1: in-vitro and in-vivo characterization of domatinostat (4SC-202)
(A)Human cell lines from the indicated hematological malignancies were seeded into 96-well plates and incubated with concentration gradient of domatinostat as described in the Methods section of the manuscript. After 72 h viability was determined via Alamar blue assay and corresponding IC50 values were calculated.
(B)Domatinostat was administered at the indicated doses as aqueous suspension with 20% PEG and 3.2% Methocel at a dose volume of 10 ml/kg and the indicated schedule. Tumor volume and body weight was determined 2-3 times per week. Tumor growth inhibition T/C was calculated as described in the Methods section.

Table 2: Diagnosis, demographics, baseline characteristics, duration of treatment and anti-tumor activity * Patients with MDS (2) and AML (1) not separately listed
** Three Screening failure: High-risk MDS (1); ECOG too high (1); QT prolongation (1)
*** Five B-cell NHL: Three chronic lymphatic leukemia (CLL), one DLBCL, one mantle-cell lymphoma (MCL) and four T-cell lymphoma: two AITL, one T-cell lymphoma no other specified (NOS), 1 Sezary Syndrome)

Table 3: Treatment-related adverse events (TRAE) in > 10% of patients

Figure 1: Duration of treatment per dose cohort.
Duration of treatment in weeks is plotted against the respective treatment regimen

Figure 2: Evaluation of PK and PD parameters
(A) Clinical PK was determined and plotted as mean log (AUClast) against the administered dose. (B) Box plots of AUC-PD of all evaluable patients sorted by occasion of measurement, i.e. sorted by C1D1, C1D14 and C2D1. Dashed horizontal lines denote median, solid horizontal lines denote mean values. (C) HDAC activity was determined at day 14 of C1D1 and set relative to values determined at pre-dose (100%). HDAC activity was plotted to exposure (AUClast) of each individual patient.

Figure 3a: CT scan at entry and after two cycles of treatment; a 69-year-old male patient with stage IV angioimmunoblastic T-cell lymphoma. CT scan at entry (left) shows bilateral cervical lymph node packages (arrows); corresponding scan after two cycles of treatment [200 mg BID; 14+7; right] shows regression of lymph node packages (arrows).
Figure 3b: CT scan at entry and after two cycles of treatment; a 29-year-old male patient with stage IV nodular- sclerosis HL. CT scan at entry (left) shows contrast-medium enhancing lymph node packages (arrows); corresponding scan after two cycles of treatment [200 mg BID; 21 days; right] shows regression of lymph node packages (arrows).

Supplement Table S1: All Treatment-related adverse events (TRAE) * DLT in one patient in 200 mg BID continuous cohort
** all events in two patients in 200 mg BID continuous treatment cohort

Supplement Table S2: Related TEAEs in patients per dose level by SOC (Safety Analysis Set) * DLT in one patient in 200 mg BID continuous cohort
** all events in two patients in 200 mg BID continuous treatment cohort

Table 1: in vitro and in vivo characterization of domatinostat (4SC-202)

(A)Domatinostat inhibits viability of cancer cells

Cancer cell line
Cell line (Disease)
Domatinostat IC50 [µM]
CCRF-CEM acute lymphoblastic leukemia 0.52
EOL-1 acute myeloid leukemia 0.11

K-562
chronic myelogenous leukemia
0.31

MV-4-11 peripheral blood / biphenotypic B myelomonocytic
0.33

MOLM-13 Peripheral blood /
acute myeloid
0.26

RS-4-11 bone marrow /
acute lymphoblastic
0.11

HL60 peripheral blood /
acute promyelocytic
0.27

(B)Anti-tumor effects of domatinostat in preclinical in vivo models

Human Cancer Cells
Species/
Number/ Gender
Oral Dose (mg/kg)/ Schedule [d]

T/C Maximal Weight
1)
Loss
% [d]

GRANTA-519

SCID mice 12 F/group

120+80 SID / 11+8 d 80 BID / 4 d
vehicle

0.01
0.13
1
17% (d11) 16% (d4) 6% (d5)

RPMI8226

SCID mice 12 F/group

60 BID / 2×4 d 80 SID / 19 d 40 SID / 19 d

0.24
0.37
0.47 -
-
-

RPMI8226

SCID mice
13-14 F/group

60 BID / 2×4 d 80 SID / 19 d

0.3
0.02

5% (d4)
-

Table 2: Diagnosis, demographics, baseline characteristics, duration of treatment and anti-tumor activity

Overall
(screened/treated)
Hodgkin´s Lymphoma
Multiple Myeloma
Non-Hodgkin Lymphoma

Number of Patients* 27/24** 8 4 9*** Age

Median in years [Min-Max]
> 65 years
68.5 [29 – 96]
13[54%]
42 [29 – 68]
1[13%]
66 [62-79]
2[50%]
73 [59 – 96]
8 [89%]

Gender (Male/Female) ECOG Performance Status
14/10 3/5 3/1 8/1

PS 0 PS 1 PS 2
8 [33%]
12 [50%]
4 [17%]
2 [25%]
6 [75%]
0
1[25%]
1[25%]
2[50%]
3[33%]
4[44%]
2[22%]

Prior Treatment

Number of prior systemic therapies, Median [Min-Max]
Prior radiotherapy Prior autologous SCT
Prior additional allogeneic SCT
3
[0-11]
14[58%]
11 [46%]
1 [4%]
7.5
[2-11]
8 [100%]
6[75%]
1[13%]
1.5
[0-2]
2[50%]
3 [75%]
0
2
[1-8]
4 [44%]
1 [11%]
0

Tumor Response
Best Overall Response Complete Response Partial Response
Stable Disease (> 6 months) Progressive Disease

Domatinostat
1
1
18 (5) 4

0
1
6 (2) 1

0
0
3(2) 1

1
0
7(0) 1

Progression-free Survival (median PFS; days median [CI])
94.5 [43-859]
135.5 [43-737]
182 [54-283]
86 [50-859]

Duration of Treatment

mean in weeks (standard deviation) median in weeks [Min-Max]
26 [30.1]
15[3.7 – 122]
31.1 [35.1]
18.7 [3.7–104.7]
24.7 [16.22]
25.5 [5.9 – 41.9]
24.4 [36.8]
12.1 [7.3-122]

Table 3: Treatment-related adverse events (TRAE) in > 10% of patients
Adverse Events No. of patients (events) % Grade 3 (Pt./E.) Grade 4 (Pt./E)

Blood and lymphatic system disorders
 Thrombocytopenia/ Platelet countdecreased
o Thrombocytopenia
o Platelet count decreased
 Neutropenia/ Neutrophil count decreased
o Neutropenia
o Neutrophil count decreased

4 (5)

1 (1) 3 (4)

3 (3) 1 (1) 2 (2)

20.8

4.2
12.5

12.5
4.2
8.3

1 (1)

1 (1)

2 (2) 1 (1) 1 (1)

-
Gastrointestinal disorders  Diarrhea
 Nausea
5 (5) 3 (3)
20.8
12.5
-
-
-
-
General disorders and administration site conditions  Fatigue

5 (7)

20.8

-

-
Investigations
 Hepatic enzyme elevation
o Alanine aminotransferase increased
o Aspartate aminotransferase increased
o Gamma-glutamyltransferase increased
3 (11)
3 (3) 3 (3) 3 (5)
29.2
8.3

12.5

12.5
2 (5)

1 (1)
1 (2)
1 (1) 1(1)
Nervous system disorders  Paresthesia
3 (4)
12.5
-
-
Skin and subcutaneous tissue disorders  Dry skin
3 (5)
12.5
-
-