Clinical trials assessing hypomethylating agents combined with other therapies: causes for failure and potential solutions

Abstract

Purpose:

Azacitidine and decitabine are hypomethylating agents (HMAs), that is, inhibit and deplete DNA methyltransferase 1 (DNMT1). HMAs are standard single-agent therapies for myelodysplastic syndromes and acute myeloid leukemias. Several attempts to improve outcomes by combining HMAs with investigational agents, excepting with the BCL2-inhibitor venetoclax, have failed in randomized clinical trial (RCT) evaluations. We extract lessons from decades of clinical trials to thereby inform future work.

Experimental Design:

Serial single-agent clinical trials were analyzed for mechanism and pathway properties of HMAs underpinning their success, and for rules for dose and schedule selection. RCTs were studied for principles, dos and don’ts for productive combination therapy.

Results:

Single-agent HMA trial results encourage dose and schedule selection to increase S-phase dependent DNMT1-targeting, and discourage doses that cause indiscriminate anti-metabolite effects/cytotoxicity, since these attrit myelopoiesis reserves needed for clinical response. Treatment-related myelosuppression should prompt dose/frequency-reductions of less-active investigational agents rather than more active HMA. Administering cytostatic agents concurrently with HMA can antagonize S-phase dependent DNMT1-targeting. Supportive care that enables on-time administration of S-phase (exposure-time) dependent HMA could be useful. Agents that manipulate pyrimidine metabolism to increase HMA pro-drug processing into DNMT1-depleting nucleotide, and/or inhibit other epigenetic enzymes implicated in oncogenic silencing of lineage-differentiation, could be productive, but doses and schedules should adhere to therapeutic-index/molecular-targeted principles already learned.

Conclusion:

>40 years of clinical trials history indicate mechanism, pathway and therapeutic-index properties of HMAs that underpin their almost exclusive success and teaches lessons for selection and design of combinations aiming to build on this treatment foundation.

Introduction

The small molecule drugs azacitidine (5-azacytidine) and decitabine (5-aza-2’-deoxycytidine) are DNA hypomethylating agents (HMAs). That is, both agents inhibit and deplete the key epigenetic regulator DNA methyltransferase enzyme 1 (DNMT1). DNMT1 is the ‘maintenance methyltransferase’ that copies methylation marks of the parental DNA strand onto the newly synthesized strand during cell cycle S-phase, and is also a corepressor (a protein mediating gene repression) recruited to gene loci by lineage master transcription factors. The HMAs are the most effective single-agents for treating myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), with response rates of ~35–60%. Numerous attempts to further improve outcomes by combining HMAs with investigational drugs, however, have failed in randomized controlled clinical trials (RCTs), except for combinations with the BCL2-inhibitor venetoclax. We extract from this hard-fought history 1. Properties of HMAs that can explain their success versus numerous other single agents, so that future trials can seek to preserve or enhance these properties; 2. Principles for HMA dose and schedule selection, taught by serial HMA single-agent studies; 3. Classes of agents to consider for combination therapy, suggested by successful RCTs; 4. Lessons learned from unsuccessful RCTs, to avoid repeating previous miscalculations. In this way, we can hopefully increase likelihoods of future success.

1. Mechanism and pathway properties of HMAs to preserve or enhance

Azacitidine and decitabine are the only drugs approved, and routinely used as single-agents, to treat all sub-types of MDS, succeeding where cytarabine, hydroxyurea, topotecan, fosteabine, gemcitabine, irinotecan, daunorubicin, etoposide, and many other drugs failed over several decades of clinical trials. These other drugs are cytotoxic – that is, their intended mechanism is DNA damage that upregulates the transcription factor p53 (TP53) and its downstream apoptosis transcriptional programs (reviewed in(1)). Attenuation of p53 or its major cofactors by mutations/deletion of TP53, amplifications of MDM2/4, and/or by other means (1–3), is, however, characteristic of malignant transformation of most tissue lineages including myeloid. Thus, there can be upfront resistance to cytotoxic treatments (primary refractory), or ready selection by first-line cytotoxic treatments for the most apoptosis-attenuated malignant sub-clones that then resist next-line cytotoxic treatments also (relapsed-refractory) (1). TP53 mutations are hence negative-prediction biomarkers for cytarabine-based cytotoxic therapy, the historical standard for treating AML (4). Meanwhile, normal dividing hematopoietic stem and progenitor cells (HSPC) do undergo apoptosis, since these have intact p53, thereby exacerbating or causing low blood counts (3,5). This is problematic because morbidity and death in MDS/AML is usually from low normal blood counts to begin with, and clinical response/benefit requires not just cytoreduction of malignant clones, but blood count recoveries mounted by functional HSPC. Amplifying this problem, reserves of functional HSPC diminish markedly with age (reviewed in (6)) and the majority of MDS/AML patients are >60 years of age.

By contrast, HMA efficacy does not depend on p53-dependent apoptosis: DNMT1-targeting prompts MDS/AML cells to terminal-differentiation, exits from cell cycle that do not require p53 (1–3) (Figure 1, 2). In normal HSPCs, DNMT1-targeting is not inherently cytotoxic but preserves lineage-maturation in the case of committed progenitors, or self-renewal in the case of stem cells, thus sparing these vital cells (5,7–10) (Figure 1). That is, DNMT1-targeting can terminate malignant but not normal hematopoietic stem cell self-replications (good therapeutic index). Accordingly, TP53 mutation and/or deletion is not a negative predictive biomarker for HMA clinical activity: in a randomized comparison of azacitidine 75 mg/m²/day for 7 days every 28 days vs conventional care regimens including standard cytotoxic induction therapy with cytarabine and daunorubicin, in patients with newly diagnosed AML, sub-set analysis of patients with chromosome 17p (TP53) abnormalities (n=46) found median overall survival of 5 vs 2.8 months (Log-rank p-value =0.07)(11). Although this analysis was limited by the small patient number, single-arm studies from more than one other institution have also documented meaningful responses to HMA in patients with TP53-mutated disease, and more generally, in the very elderly with high risk disease and diminished myelopoietic reserves (2,3,12,13). Having said this, TP53 abnormalities are still very much an adverse prognostic biomarker in HMA-treated patients – although clinically meaningful hematologic responses occur, these responses are short-lived (11). Even though HMAs have been shown scientifically to not require p53 to antagonize MYC and terminate malignant replication, p53-inactivation upregulates the endogenous de novo pyrimidine synthesis pathway that directly antagonizes the DNMT1-depleting nucleotide (Figure 2E, F) (14)(reviewed in (15)). In sum, there remains a need to enhance therapy: mechanism and clinical trials data thus far suggest that such efforts should seek to preserve or enhance a normal HSPC-sparing, p53-independent mechanism-of-action of HMAs, properties that distinguish HMAs from numerous less or non-beneficial evaluated drugs (Figure 1, 2, Table 1).

Figure 1. Decitabine (Dec) and azacitidine (5Aza) are processed by pyrimidine metabolism into various metabolites, causing various molecular pharmacodynamic and pathway effects.

A) Dec and 5Aza have the identical pyrimidine ring modification, but Dec has a deoxyribose and 5Aza a ribose sugar moiety - this channels their metabolism differently. B) One metabolic product is Aza-dCTP, that after incorporation into DNA during cell cycle S-phase, depletes DNMT1. Relative to decitabine, the fraction of an administered dose of 5-azacytidine that is processed into Aza-dCTP is ~1/10th. That is, for DNMT1-depleting goals, decitabine:5-azacytidine dose-equivalence is ~1:10, e.g., decitabine 7.5 mg/m² ≈ 5-azacytidine 75 mg/m². C) Other metabolites impact DNA and RNA metabolism in different ways – most of 5Aza is incorporated into RNA. D) The DNMT1-depleting action uniquely can terminate malignant self-replication and proliferation, via a p53-independent pathway, while sparing the self-replication of normal hematopoietic stem cells (good therapeutic index). This DNMT1-depleting action/pathway is saturated at relatively low Dec or 5Aza concentrations, and is S-phase dependent. In addition to inducing terminal-differentiation of malignant cells, DNMT1-depletion in cancer cell lines has been shown to induce expression of self, double-stranded RNA from hypomethylated repetitive elements, that in turn can trigger type 1 interferon production and Rnase L mediated cell death.

Figure 2. Known mechanisms of resistance to cytarabine (Ara-C), decitabine (Aza-dC), and 5-azacytidine (Aza-C).

A) In addition to inactivating mutations in the p53-system, e.g., TP53 mutation, resistance to Ara-C is mediated by changes in pyrimidine metabolism, e.g., upregulation of the catabolic enzyme cytidine deaminase (CDA) or of de novo pyrimidine synthesis that generates natural dCTP that competes with Ara-CTP for incorporation into DNA. dCTP also allosterically regulates DCK activity (although Ara-C appears to be a ribonucleotide by chemical formula, by structure it is processed as a deoxyribonucleotide). B) Pyrimidine metabolism shifts mediate resistance to Aza-dC. C) Pyrimidine metabolism shifts mediate resistance to Aza-C. D) These pyrimidine metabolism shifts can occur automatically (auto-Resistance): TYMS-depletion by Dec (or Ara-C), and RRM1-depletion by 5Aza, increase and decrease dCTP levels respectively, prompting automatic compensatory metabolic shifts. Aza-dC and Aza-C drive DCK and UCK2 in opposite directions, but both agents upregulate the catabolic enzyme CDA and the initial enzyme in de novo pyrimidine synthesis - carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD). E) Even though Aza-dC and Aza-C do not require p53 to antagonize MYC, p53-inactivation upregulates CAD to potentially antagonize Aza-dCTP with endogenous dCTP. Mean±SD. TCGA RNA-Seq, n=167, p-value 2-sided t-test. F) This observation is recapitulated in hematopoietic malignancy cell lines. CCLE RNA-Seq, n= 175.

Table 1.

Molecular-targets and downstream pathways of investigational agents evaluated in combination with HMAs

#	Drug	Molecular-target/molecular-pharmacodynamic effect	Pathway activated in malignant myeloid cells	Pathway activated in normal hematopoietic cells	References
1	Decitabine (HMA)	DNMT1 depletion (at <0.5–1 μM)	S-phase dependent; terminal-differentiation (p53-independent cytoreduction)	Self-replication in normal HSC, terminal-differentiation in normal committed progenitors	(3,5,10)
		TYMS depletion; DNA-protein adducts exceeding cellular capacity for repair (at > 0.5–1 μM)	Cell-cycle dependent; DNA damage and apoptosis (p53-dependent cytoreduction)	DNA-damage and apoptosis (cytotoxicity)
2	Azacitidine (HMA)	DNMT1 depletion (at <1–5 μM)	S-phase dependent; terminal-differentiation (p53-independent)	Self-replication in normal hematopoietic stem cells, terminal-differentiation in normal committed progenitors
		RRM1 depletion; DNA-protein adducts exceeding cellular capacity for repair (at > 0.5–1 μM)	Cell-cycle dependent; DNA damage and apoptosis (p53-dependent)	DNA-damage and apoptosis (cytotoxicity)
3	Histone deacetylase inhibitors (entinostat, pracinostat, vorinostat etc.)	Histone deacetylase enzyme (HDAC1, HDAC2, HDAC3, HDAC6, HDAC7) inhibition	Terminal-differentiation; DNA damage; other cellular damage (because of diverse cell physiology roles of HDACs not confined to epigenetic regulation)(p53-independent and p53-dependent cytoreduction)	Self-replication in normal HSC, terminal-differentiation in normal committed progenitors; also DNA and other cellular damage causing apoptosis (cytotoxicity) and cytostasis (mixed effects that cannot be separated using different concentrations or dosages)	(47–50)
4	Lenalidomide	Cerebelon ubiquitin ligase (CRBN) gain-of-function	Altered substrate specificity of cerebelon, increases proteosomal degradation of substrates that vary with cellular context, triggering apoptosis (p53-dependent); may alter progenitor lineage-commitment decisions	Apoptosis (cytotoxicity) and cytostasis (but not profound)	(51)
5	Gilteritinib	Fms related receptor tyrosine kinase 3 (FLT3) inhibition	Augments activity of the myeloid master transcription factor CEBPA to promote terminal-differentiation; may inhibit de novo pyrimidine synthesis, and in this way augment activity of pyrimidine nucleoside analogs	Decrease normal hematopoietic precursor replication	(41)
6	Eprenetapopt	Stabilizer of p53 protein	Apoptosis in stressed cells	Not described	(42)
7	Eltrombopag	Thrombopoietin receptor agonist; TET dioxygenase enzyme inhibition	Inhibition of TET enzymes might be synthetic lethal in TET-deficient malignant myeloid cells, cytostatic effects by unknown mechanism	Increase normal HSC self-replication, increase multi-potent progenitor commitment into megakaryocyte lineage, increase megakaryocyte differentiation
8	Venetoclax	BCL2-antagonist	Mitochondrial depolarization that depowers several metabolic functions of mitochondria, and that at its most extreme activates apoptosis (Figure 3)		(25–27)

Abbreviations: HSC = hematopoietic stem cells; TYMS = thymidylate synthase; RRM1 = ribonucleotide reductase catalytic subunit M1

2. Principles of HMA dose and schedule selection

Dose selection.

The earliest trials of azacitidine were based on conventional oncotherapy objectives of apoptosis/cytotoxicity, since high concentrations of azacitidine or decitabine do cause nucleotide imbalances, damage DNA and can thereby activate p53/apoptosis (Figure 1, Table 1). Doses were therefore based on maximum tolerated levels (16)(reviewed in(17)). These trials were unsuccessful. DNMT1-targeting, however, is achieved and saturated at relatively low azacitidine concentrations (Figure 1), and 10-fold reductions from initially evaluated doses produced clinical success: standard azacitidine regimens today administer 50–75 mg/m²/day (17). Decitabine clinical development was similar: initial, unsuccessful trials administered daily doses as high as 600 mg/m² (17); the first approval used a >10-fold lower dose of 45 mg/m², and a 2^nd-approved regimen uses an even lower dose of 20 mg/m²/day (18,19). These dose-reductions were arrived at empirically, therefore, we conducted studies to formally identify minimum decitabine doses needed to deplete DNMT1 without cytotoxicity to normal bone marrow/HSPC in non-human primates and humans: 0.1–0.2 mg/kg/day (~5 mg/m²/day) (2,20,21).

Schedule selection.

In the earliest HMA trials, cytotoxic daily doses could only be tolerably administered for a few days, e.g., 3 days, followed by intervals of ~6 weeks needed to recover from the cytotoxic side-effects (schedules were necessarily pulse-cycled). One benefit of dose-reductions that decreased cytotoxicity was that intervals between treatment pulses could be shortened: standard azacitidine regimens administer 50–75 mg/m2/day for 7–10 days every 4 weeks; the most commonly used standard regimen of decitabine administers 20 mg/m²/day for 5 days every 4 weeks. This more frequent administration is beneficial because DNMT1-targeting by azacitidine or decitabine requires overlap between treatment exposure windows and malignant cell S-phase entries. That is, DNMT1-targeting is S-phase or exposure-time dependent, and lowering the decitabine dose from 45 mg/m2/day to 20 mg/m²/day, and increasing the frequency of administration from 3 days every 6 weeks to 5 days every 4 weeks produced 2–3-fold improvements in remission and hematologic improvement rates (18,19). Building on this logical progression, we scheduled non-cytotoxic, DNMT1-depleting doses of decitabine 0.1–0.2 mg/kg/day (~5 mg/m²/day) for frequent and distributed administration 1–2X/week. The overall response rate per International Working Group criteria was 44%, including complete cytogenetic remissions of TP53-mutated disease containing complex cytogenetic abnormalities (2,12,22). This non-cytotoxic regimen has been effectively and safely administered for >5 years in MDS patients >80 years old (12).

Thus, pulse-cycled schedules of HMA administration reflect conventions inherited from cytotoxic treatments. Lower doses that target DNMT1 without cytotoxicity enable alternative schedules of administration that increase drug exposure durations and distributions to increase S-phase dependent DNMT1-targeting.

3. Successful HMA combinations: lessons learned

HMAs can produce non-toxic clinical responses but relapse/resistance is routine. Resistance, in vitro, in mice, and in patients, is characterized by failure to deplete DNMT1 (23,24). To deplete DNMT1, azacitidine, a cytidine analog, and decitabine, a deoxycytidine analog, must be processed by pyrimidine metabolism into a nucleotide analog that incorporates into the newly synthesized DNA strand during S-phase (Figure 1). Pyrimidine metabolism is a network evolved for nucleotide homeostasis. The interactions between decitabine and azacitidine and this network perturb nucleotide balances, and thus trigger automatic, balancing responses that dampen decitabine or azacitidine pro-drug processing into DNMT1-depleting nucleotide (Figure 2)(23). One key pyrimidine metabolism adaptation underlying this mode of resistance is upregulated de novo pyrimidine synthesis, that competes directly with the DNMT1-depleting nucleotide Aza-dCTP by building natural cytidines and deoxycytidines from amino acid building blocks (Figure 2). Inhibiting de novo pyrimidine synthesis can thus restore AML cell sensitivity to HMAs (14)(15). Venetoclax is a BCL2-inhibitor that dislodges from BCL2 sequestration BH3-only proteins; BH3-only proteins then bind to and enable the effector-proteins BAX and BAK1 to permeabilize mitochondrial membranes (Figure 3). This depowers mitochondrial processes including de novo pyrimidine synthesis (25) (Figure 3). Also decreased are mitochondrial outputs of nicotinamide adenine dinucleotide (NAD+/NADH) and flavin adenine dinucleotide (FAD+/FADH), mandatory cofactors for the transcription repressing enzymes C-terminal binding protein 1 (CTBP1) and lysine demethylase 1A (KDM1A), that like DNMT1, are implicated in oncogenic, aberrant epigenetic repression of hundreds of lineage-differentiation genes in oncogenesis (Figure 3) (15,25–27). Thus, successful mitochondrial membrane depolarization by venetoclax, short-of triggering full-blown apoptosis, can augment DNMT1-targeting/terminal-differentiation in several ways (Figure 3).

Figure 3. Mechanisms by which mitochondrial-targeting by venetoclax may cooperate with DNMT1-targeting by Aza-dCTP.

A) De novo pyrimidine synthesis manufactures dCTP that competes with Aza-dCTP, and requires the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), powered by the membrane electron gradient. Also powered this way are mitochondrial outputs of epigenetic enzyme cofactors, that similar to DNMT1, inhibit terminal-differentiation of malignant cells (Ac-CoA=acetyl-CoA; HAT = histone acetyltransferase; AKG=alpha-ketoglutarate; TET=ten-eleven translocation methylcytosine dioxygenases; NAD=nicotinamide adenine dinucleotide; CTBP1=C-terminal binding protein 1 corepressor; FAD=flavin adenine dinucleotide; KDM1=lysine demethylase 1; CTP=cytidine triphosphate; dCTP=deoxycytidine triphosphate). B) The anti-apoptotic BCL2 protein family (BCL2, BCL2L2, BCL2L1, MCL1) sequester BH3-only proteins (BID, BAD etc.) that otherwise activate the effector-proteins BAX and BAK1 that permeabilize mitochondrial membranes. C) BAX, a key effector-protein in the pathway downstream of venetoclax, is significantly decreased in primary AML cells containing mutated or deleted TP53. Mean±SD. TCGA RNA-Seq, n=167, p-value 2-sided t-test. F) This observation is recapitulated in hematopoietic malignancy cell lines. CCLE RNA-Seq, n= 175.

Other cofactors produced by mitochondria, e.g., alpha-ketoglutarate (AKG), serve epigenetic enzymes that promote terminal-differentiation, e.g., ten-eleven translocation 2 (TET2) that demethylates DNA (Figure 3). AKG is antagonized by an oncometabolite 2-hydroxyglutarate (2HG) that is produced by mutant IDH1 or IDH2 (IDH1 and IDH2 mutations are recurrent in myeloid malignancies). Accordingly, inhibitors of mutant IDH1/2 align with therapeutic-index/pathway goals of HMA therapy, and early clinical trials data for such combinations are encouraging (Figure 3).

The pyrimidine metabolism enzyme, cytidine deaminase (CDA), rapidly catabolizes azacitidine and decitabine into uridine counterparts that do not target DNMT1, and also contributes to HMA resistance (23) (Figure 2). CDA in the liver and gastro-intestinal tract moreover severely limits HMA plasma half-life and oral bioavailability (28,29). A fixed-dose co-formulation of the CDA-inhibitor cedazuridine with oral decitabine was recently approved for MDS treatment in the United States based on pharmacokinetic equivalence to parenteral decitabine (30). Another CDA-inhibitor, tetrahydrouridine, has also been co-formulated with oral decitabine and 5-azacytidine, with weight-based dosing and schedules of administration designed for non-cytotoxic DNMT1-targeting (28,31,32).

Clinical trials experience thus suggests utility in combining HMAs with agents aiming to manipulate pyrimidine and mitochondrial metabolism, to thereby enhance pro-drug processing and non-cytotoxic corepressor targeting, with open opportunities to refine and optimize such combinations.

4. Unsuccessful RCTs of combination therapy: lessons learned

We analyzed nine completed, unsuccessful, combination RCTs (Table 2) and identified departures from, and workable design modifications to better align with the mechanism and pathway principles learned thus far, to in this way inform future efforts.

Table 2.

Randomized clinical trials of HMA combination therapy to treat MDS/AML – results

#	Arms, ClinicalTrials#	Patients	Schedule (28 day cycles)	Phase II/III results
1	Aza + Entinostat vs Aza alone NCT00313586	97 MDS 52 AML	Concurrent: Aza D1–10 Entinostat D3 & 10	1o End-point: HN (CR + PR + HI) Aza: HN 32% (95% CI: 22%−44%), median OS 18m Combination: HN 27% (95%CI 17% – 39%), median OS 13m
2	Aza + Pracinostat vs Aza + Placebo NCT01873703	102 MDS IPSS Int. or high risk	Concurrent: Aza D1–7 or D1–5 Pracinostat QOD D1–21	1o End-point: CR by cycle 6 Aza: CR 18%, median OS 16m Combination: CR 33% (p=0.07), median OS 19m (HR 1.21; 95% CI 0.66–2.23)
3	Aza + Pracinostat vs Aza + Placebo NCT03151408	500 AML ineligible for standard induction	Concurrent: Aza D1–7 Pracinostat QOD or 3X/wk D1–21	1o End-point: OS Terminated prematurely because unlikely to meet 1o End-point8
4	Aza + Vorinostat vs Aza alone NCT01617226	217 AML 42 MDS	Concurrent: Aza D1–7 Vorinostat D3–9	1o End-point: ORR and OS Aza: ORR 41%, CR/CRi/mCR 22%, median OS 9.6m Combination: ORR 42%, CR/CRi/mCR 26%, median OS 11.0m (HR 1.15, 95%CI 0.87–1.51)(p=0.32)
5	Dec + Valproate vs Dec alone NCT0l305499	87 MDS R-IPSS Int.or high risk 62 AML ≥60 y old	Concurrent: Dec D1–5 Valproate D1–7	1o End-point: ORR Dec: CR 31%, ORR 51%, median OS 11.9m Combination: CR 37% (p=0.49), ORR 58% (p=0.41), median OS 11.2m (p=0.92)
6	Aza + Lenalidomide vs Aza + Vorinostat vs Aza alone NCT01522976	224 MDS IPSS high risk 53 CMML	Concurrent: Aza D1–7 Lenalidomide D1–21 Vorinostat D3–9	1o End-point: ORR Aza: 38%, median OS 15m Aza + Lenalidomide: 49% (p=0.14), median OS 19m (p=0.68) Aza + Vorinostat: 27% (p=0.16), median OS 17m (p=0.22)
7	Aza + Gilteritinib vs Aza alone NCT02752035	114 AML ineligible for standard induction	Concurrent: Aza D1–7 Gilteritinib D1–28	1o End-point: OS Terminated prematurely because unlikely to meet primary endpoint
8	Aza + Eprenetapopt vs Aza alone NCT03745716	154 MDS, TP53 mutant	Sequential: Aza D4–10 or D4–5 + D8–12 Eprenetapopt D1–4	1o End-point: CR Aza: CR 22.4% Combination: CR 33.3% (p = 0.13) Terminated prematurely because unlikely to meet primary endpoint.
9	Aza + Eltrombopag vs Aza + Placebo NCT02158936	356 MDS with thrombocytopenia	Concurrent: Aza D1–7 Eltrombopag daily D1–28	1o End-point: Platelet-transfusion freedom in cycles 1–4 Aza: ORR 35%, median OS 78 weeks Combination: ORR 20% (p=0.005), median OS 60 weeks

Abbreviations: Aza, azacitidine; Dec, decitabine; D, Day; MDS, myelodysplastic syndromes; AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; CR, complete remission; PR, partial remission; CRi, CR with incomplete blood count recovery; mCR, marrow CR; OS, overall survival; HR, hazard ratio; PFS, progression-free survival; ORR, overall response rate; HN, hematologic normalization; HI, hematologic improvement; Int, intermediate; R-IPSS, Revised International Prognostic Scoring System;

HMA combinations with histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACi), by promoting acetylation of histones and other proteins, can also target the epigenome to reverse oncogenic gene silencing. HMA combination with HDACis was found synergistic in pre-clinical studies, and produced high response rates in small, single arm studies (33). Entinostat is a potent oral inhibitor of HDAC class I and was combined with azacitidine in the E1905 RCT in patients with MDS and AML with myelodysplasia related changes (34). Azacitidine alone was favored with hematologic normalization (HN) in 32% versus 27% for the combination (Table 2). Pracinostat is a class I/II/IV HDACi and was combined with azacitidine in a RCT in patients with International Prognostic Scoring System (IPSS) intermediate (INT)-2 or high-risk MDS (35). The combination did not provide a significant overall response rate (ORR) or overall survival (OS) benefit versus azacitidine plus placebo (Table 2). Recently, a RCT of pracinostat plus azacitidine versus placebo plus azacitidine in AML patients was terminated prematurely, due to failure to meet its primary endpoint of complete remission (CR) rate superiority (36). Vorinostat is a HDAC class I/II inhibitor; a multicenter open-label RCT compared vorinostat plus azacitidine versus azacitidine monotherapy in patients with newly diagnosed or relapsed/refractory AML or IPSS INT-2 or high-risk MDS (37). Unfortunately, this trial also failed to meet its ORR and OS primary endpoints. Valproic acid is a pan HDAC inhibitor (38); a RCT evaluation of valproic acid plus decitabine versus decitabine alone in INT or high-risk MDS and AML patients older than 60 failed to demonstrate significant ORR or OS benefit for the combination (39) (Table 2). Lastly, SWOG-S1117 randomized patients with higher risk MDS (81%) or chronic myelomonocytic leukemia (CMML) (19%) into 3 arms: azacitidine plus lenalidomide versus azacitidine plus vorinostat versus azacitidine monotherapy (40). No significant improvement in the ORR primary endpoint was observed between either combination arm versus azacitidine monotherapy (Table 2). An ORR difference was observed in sub-set analyses of patients with CMML, favoring azacitidine plus lenalidomide (ORR 68%, n=19) over azacitidine alone (ORR 28%, n=18) (p=0.02), but without a difference in OS (p=0.87). Based on the overall non-superior ORR and OS outcomes, the phase 3 part of the study was not conducted.

HMA combinations with other targeted agents.

HMAs have also been combined with drugs targeting mutant-FMS-like tyrosine kinase 3 (FLT3) or mutant-p53, and with thrombopoietin (TPO)-receptor agonists. Again, single-arm trial results were encouraging, but not RCT readouts. FLT3 is amongst the most recurrently mutated genes in AML and encodes for a transmembrane ligand-activated receptor. Gilteritinib is a potent 2^nd-generation FLT3-inhibitor active against both FLT3 internal tandem duplication and FLT3 tyrosine kinase domain mutations. Several RCTs have failed to demonstrate superiority of FLT3-inhibitors combined with HMAs versus HMAs alone: most recently, the phase 3, multicenter, open-label LACEWING RCT compared gilteritinib plus azacitidine to azacitidine alone in patients with newly diagnosed FLT3-mutated AML ineligible for induction chemotherapy. Although the phase 1–2 results looked promising, the phase 3 failed to meet its primary endpoint – there was no OS benefit (41)(Table 2).

TP53 mutations in MDS or AML predict poor prognoses, and a small molecule, APR-246 (eprenetapopt), has been postulated to convert the mutant-p53 protein to its original wild type form. In RCT evaluation, patients with TP53-mutated MDS received APR-246 plus azacitidine versus azacitidine alone. Again, phase 1B/2 results were encouraging but phase 3 failed to show significantly higher CR rate for the combination versus azacitidine alone (42) (Table 2). Insofar as Aza/Dec success to treat MDS/AML is because of a p53-independent mechanism-of-action, it may be more rational to combine this class of agent (p53-stabilizer) with agents intending p53-dependent cytotoxicity, e.g., cytarabine (Figure 2).

Thrombocytopenia in MDS/AML patients is a major source of morbidity, and single-agent TPO-receptor agonists have shown efficacy in raising platelet counts. A double-blind RCT assigned IPSS INT-1, INT-2 and high-risk MDS patients with baseline platelet count less than 75 × 10⁹/L to eltrombopag plus azacitidine versus placebo plus azacitidine (43). The study ended prematurely because it did not meet its primary endpoint of platelet-transfusion independency, and there was no significant improvement in ORR or OS with the combination (Table 2).

Why did these RCTs fail?

These combinations thus did not add benefit and sometimes demonstrated worse responses than with HMAs alone (34,40). Here are reasons:

(i) Direct antagonism of S-phase dependent HMA by concurrent administration of cytostatic/cytotoxic investigational agent. A common attribute of these RCT is that the investigational agents can have cytostatic effects - even eltrombopag has cytostatic effects on leukemia cells (44,45) - and were given concurrently/overlapping with the HMA (Table 2). Such scheduling has the potential to antagonize the mainstay HMAs, e.g., correlative analysis of DNA methylation showed that combination entinostat plus azacitidine produced less demethylation than the azacitidine alone arm (34). It would be reasonable therefore to avoid concurrent (same day) scheduling of less active agents that have cytostatic effects, including HDACi, IMiD, or kinase inhibitors, with the HMA backbone, since this would antagonize S-phase dependent DNMT1-depletion (Figure 1).

(ii) Investigational agent suppression of functional myelopoiesis - known side-effects of HDACi as single-agents, even in patients without myeloid malignancies, are thrombocytopenia, neutropenia and anemia (40). Such myelosuppression may delay or decrease feasible exposure-times to the more active HMA. How often an HMA is administered dictates the fraction of the malignant cell population that has the possibility of being treated because DNMT1-targeting is S-phase dependent. In successful RCT evaluations of azacitidine plus venetoclax, low blood counts attributed to therapy were managed by reducing venetoclax frequency and/or dose (46). By contrast, in other RCTs, myelosuppression was managed by reducing azacitidine frequency and dose, even discontinuing azacitidine altogether, favoring continuation of investigational agents shown to be less active as single-agents (40). Thus, myelo-suppression attributed to treatment should be managed as much as possible with dose and/or frequency reductions of the less-active investigational agent, not the HMA.

(iii) Destruction of normal HSPC (Figure 1, Table 1) – Separation of epigenetic pharmacodynamic effects that terminate malignant self-replications, from cytotoxic effects that destroy normal HSPC, has been shown for HMA but not for HDACi (47–50). This could be because HDACs have pleotropic cell physiology functions, not restricted to epigenetic repression of genes (HDAC deacetylate substrates other than histones) (47–50). HDACi thus have significant side-effects but modest activity for treating myeloid malignancies (reviewed in(33)). If biochemically possible, it would be useful to design, identify and use doses of the HDACi that inhibit HDAC, or the IMiD that alters cereblon substrate-specificity, or the kinase inhibitor to inhibit FLT3, without cytotoxicity to normal HSPC (dosage selection based on pharmaco-dynamics instead of maximum-tolerated levels). Investigational agent dose, as per the HMA, should be selected per molecular-targeted pharmacodynamic (‘optimal-biological-dose’) not maximum-tolerated-dose (MTD) principles, to limit or avoid indiscriminate anti-metabolite effects and cytotoxicity to normal HSPC needed for response and durable benefit.

(iv) Fidelity to schedules of administration. Non-superiority with investigational agent addition to HMAs in RCTs implies the HMA backbone also largely accounts for preceding Phase 2 results that were found exciting. One reason for apparently better results than historic for the same HMA regimen could be fidelity to schedules-of-administration, important for S-phase dependent DNMT1-targeting: in one Phase 2 evaluation of decitabine 20 mg/m²/day for 5 days every 28 days, growth factor support was used to alleviate neutropenia that might otherwise threaten on-time administration of cycles every 28 days (18,19), while in another Phase 2 evaluation of the same regimen neutropenia was managed by cycle-delays (19) – the trial pursuing on-time HMA administration reported overall response rates of 63% versus 35% for the trial using cycle delays (18,19) (Figure 1, Table 1). Hence, it could be useful to consider growth factor support, or other measures, to enable on-time administration of the HMA - avoid cycle delays (17).

Another observation is lower response rates for the same combination treatment in Phase 3 versus prior Phase 2, e.g., eprepranocept + azacitidine produced a CR rate of 33.3% in the Phase 3 RCT (n=77) but 50% in the prior single-arm Phase 2 (n=40) (42). A straightforward explanation is less selected and larger (more representative) sample sizes in Phase 3.

Conclusion

In sum, >40 years of clinical trial evidence, complemented by mechanism-studies, suggests selecting HMA dose and schedule to increase S-phase dependent DNMT1-targeting, manipulating pyrimidine metabolism to increase HMA pro-drug processing into DNMT1-depleting nucleotide, and targeting of other epigenetic enzymes implicated in oncogenic repression of lineage-maturation programs, all the while adhering to basic therapeutic index principles that are especially critical when treating myeloid malignancies in the elderly.

Statement of Translational Relevance.

Hypomethylating agents (HMAs), the small molecules azacitidine and decitabine that target DNA methyltransferase 1 (DNMT1), are mainstays for treating myeloid malignancies, e.g., myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). Several attempts to improve outcomes by combining HMAs with investigational agents, excepting with the BCL2-inhibitor venetoclax, have failed in expensive, randomized clinical trial (RCT) evaluations. We extract lessons from successes and failures over >40 years of clinical trials and mechanism-studies to inform and guide future combination therapy trials.