BMI1-Inhibitor PTC596 in Combination with MCL1 Inhibitor S63845 or MEK Inhibitor Trametinib in the Treatment of Acute Leukemia

Simple Summary

Prognosis for acute myeloid leukemia (AML) patients is poor, particularly in TP53 mutated AML, secondary, relapsed, and refractory AML, and in patients unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. Targeting the stem cell oncoprotein BMI1 in leukemic cells may represent a promising novel treatment option for poor risk AML patients, especially in combination with other targeted therapies. Here we tested the BMI1 inhibitor PTC596 in combination with a variety of targeted therapies in AML cell lines and patient samples in vitro. In addition, we defined the biomarkers of response to the combination treatments in the leukemic cells. The combination treatment with the BMI1 inhibitor PTC596 and the MCL1 inhibitor S63845 may be an effective treatment in CD34+ adverse risk AML with elevated MN1 gene expression and MCL1 protein levels, while combination treatment with BMI1 inhibitor PTC596 and the MEK inhibitor trametinib may be more effective in CD34+ adverse risk AML with elevated BMI1 gene expression and MEK protein levels. The determination of gene and protein expression levels in leukemic cells as biomarkers of response to targeted combination therapies may be helpful to optimize treatment efficacy.

Abstract

Purpose: Prognosis for acute myeloid leukemia (AML) patients is poor, particularly in TP53 mutated AML, secondary, relapsed, and refractory AML, and in patients unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. The combined use of compounds targeting the stem cell oncoprotein BMI1 and activating the tumor suppressor protein p53 may represent a promising novel treatment option for poor risk AML patients. Experimental Design: The BMI1 inhibitor PTC596, MCL1 inhibitor S63845, and MEK inhibitor trametinib, as well as the p53 activator APR-246 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 mutant and wild type AML cell lines and a variety of patient derived AML cells. Results: AML cell lines were variably susceptible to PTC596 and to combination treatments with PTC596 and MCL1 inhibitor S63845, MEK inhibitor trametinib, or TP53 activator APR-246, independent of TP53 mutational status. Susceptibility of patient samples for PTC596 in combination with S63845 or trametinib was significant for the majority of adverse risk primary and secondary AML with minimal efficacy in favorable risk AML, and correlated significantly with CD34 positivity of the samples. BMI1 and MN1 gene expression, and MCL1 and MEK1 protein levels were identified as biomarkers for response to PTC596 combination treatments. Conclusions: The combination of PTC596 and S63845 may be an effective treatment in CD34+ adverse risk AML with elevated MN1 gene expression and MCL1 protein levels, while PTC596 and trametinib may be more effective in CD34+ adverse risk AML with elevated BMI1 gene expression and MEK protein levels.

1. Introduction

Acute myeloid leukemia (AML) is a clonal blood malignancy characterized by arrested maturation and abnormal proliferation of hematopoietic precursor cells. At the cellular level, specific genetic and epigenetic alterations lead to changes in cellular signaling pathways, including the common inactivation of the TP53 tumor suppressor axis, thereby contributing to the blockade of differentiation and accumulation of leukemic blasts in the blood and bone marrow [1].

The meningioma 1 (MN1) gene is expressed in CD34 positive hematopoietic stem cells and down-regulated during myeloid differentiation [2]. Increased MN1 gene expression in myeloid stem cells leads to enhanced proliferation and loss of myeloid differentiation [3]. MN1 overexpression has been linked to shorter overall (OS) and disease-free survival (DFS) of patients diagnosed with acute myeloid leukemia with normal cytogenetics [4,5,6,7,8]. Correlation analysis of MN1 with myeloid gene expression levels revealed the positive association of MN1 and BMI1, CD34, FOXP1, and MDM2 expression indicating the stem cell gene BMI1 as putative MN1 target gene [9]. While there are currently no compounds targeting MN1, inhibitors targeting BMI1 are being tested in clinical studies for various solid tumors.

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1), the first functional gene in the PcG family, was identified in a mouse lymphoma in 1993. BMI1 induces self-renewal of hematopoietic (HSC) and leukemic stem (LSC) cells [10]. BMI1 gene expression levels correlate well with progression and prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [11]. BMI1 plays an important role in the development of malignant tumors including solid tumors of the brain, breast, colon, head and neck, liver, lung, and prostate, as well as hematologic malignancies including lymphoma and leukemia [12,13]. Increased BMI1 expression in tumor cells is associated with poor prognosis. BMI1 is a novel target for cancer therapy [14] and may be a valid target in AML therapy. BMI1 inhibition eliminates cancer stem cells [15]. PTC596 is a novel BMI1 inhibitor currently in clinical trials for ovarian, fallopian, and peritoneal cancer, glioma, and advanced solid tumors (NCT02404480, NCT03605550, NCT03761095). PTC596 is tolerable with manageable gastrointestinal side effects [16].

Targeting mutated TP53 is a novel approach to restore the crucial p53 tumor suppressor function. APR-246 triggers an upregulation of genes involved in cell cycle control and apoptosis in both TP53 mutant and wild type cancer cells [17]. APR-246 has various other effects, including induction of oxidative and ER stress [18]. The first-in-human study of APR-246 in hematologic malignancies (NCT00900614) demonstrated good tolerance and a favorable pharmacokinetic profile, with upregulation of p53 target genes and induced apoptosis [19]. Three clinical trials have been recruiting to test the safety and efficacy of APR-246 treatment in advanced esophageal carcinoma (NCT02999893), high grade ovarian cancer (NCT02098343), and mutant TP53 hematologic myeloid malignant disease (NCT 03072043). Finally, a phase I/II study to investigate the safety and clinical activity of APR-246 in combination with a BRAF inhibitor in patients with mutant-BRAF unresectable metastatic melanoma resistant to anti-BRAF/anti-MEK inhibitors has started (NCT03391050).

In previous studies, we observed a considerable anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of FLT3 and TP53 [20].

Here we investigated the combined treatment of PTC-596 with the MCL1-inhibitor S63845 and the MEK-inhibitor trametinib, as well as the p53 activator APR-246 on AML cells in order to identify a potentially effective treatment specifically for aggressive AML, in particular in patients with refractory disease and in patients unfit for intensive chemotherapy. The study might provide the rationale for initiating a clinical study in aggressive AML evaluating PTC596 combination therapies.

2. Materials and Methods

2.1. Patient Samples

Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2005 and 2018 were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland, decision number #221/15. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state of the art techniques [21]. Mutational screening for FLT3, NPM1, TP53, and conventional karyotype analysis of at least 20 metaphases were performed in all samples. The recent samples were analyzed by NGS sequencing of the myeloid panel genes.

2.2. AML Cell Lines

OCI-AML2 (AML-M4, FLT3wt, DNMT3A R882C, NPM1wt, TP53wt); OCI-AML3 (AML-M4, FLT3wt, DNMT3A R882C, NPM1mut, TP53wt), MOLM-13 (AML-M5, t(9;11), FLT3-ITD, TP53wt), MOLM-16 (AML-M0, FLT3wt, TP53mut), ML-2 (AML-M4, t(6;11), FLT3wt, TP53mut), PL-21 (AML-M3, FLT3-ITD), and HL-60 (AML-M2, FLT3wt, TP53 null) cells were supplied by the Leibniz Institute DSMZ, German Collection of Micro-organisms and Cell Cultures. AML cells were grown in RPMI 1640 media (SIGMA-ALDRICH, St. Louis, MO USA) supplemented with 20% fetal bovine serum (FBS, Biochrom GmbH, Germany) in a standard cell culture incubator at 37 °C with 5% CO₂.

2.3. Cytotoxicity Assays

For assays with AML cell lines, cells were plated at a density of 5 × 10⁵/mL and treated with targeted compounds or conventional induction therapy. For assays with patient derived mononuclear cells, the cells were cultured for 2 h prior to treatment. The BMI1 inhibitor PTC596, the FLT3 inhibitor midostaurin (PKC412), the MCL1 inhibitor S63845, the MEK inhibitor trametinib, and the p53 activator APR-246 were purchased at MedChemExpress (Monmouth Junction, NJ, USA). Conventional induction therapy consisted of equimolar solution of cytarabine and idarubicin purchased at Sigma-Aldrich (St.Louis, MO, USA) and SelleckChem (Houston, TX, USA). Cell viability was determined after 20 h of treatment using the MTT-based cell proliferation kit I (Roche). This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays (biological replicates) with four measurements (technical replicates) per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. For the calculation of combination indexes three dosages of PTC596 and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on Compusyn software. Data are depicted as XY graphs, column plots, or scatter plots with mean values and SD. Statistical analysis was done on GraphPad Prism (GraphPad Software, San Diego, CA, USA) in grouped analysis and significance calculated by t-test for column graphs or Mann–Whitney test for scatter plots.

2.4. Measurement of mRNA Expression by qPCR

RNA was extracted from AML cells and quantified using qPCR. The RNA extraction kit was supplied by Macherey-Nagel, Düren, Germany. Reverse transcription was done with MMLV-RT (Promega, Madison, WI, USA). Real-time PCR was performed on the ABI7500 Real-Time PCR Instrument using ABI universal master mix (Applied Biosystems, Austin, TX, USA) and gene specific probes Hs01104728_m1 (ABL1), Hs00180411_m1 (BMI1), Hs00923894_m1 (CDKN2A), Hs00159202_m1 (MN1), and Hs02758991_g1 (GAPDH).

Measurements for BMI1 and MN1 were normalized with ABL1 values, measurements for CDKN2A were normalized with GAPDH values (ddCt relative quantitation). Assays were performed in three or more independent experiments. Statistical analysis was done on GraphPad Prism software using two-tailed t-tests. Data are depicted in column bar graphs plotting mean with SD values.

2.5. Measurement of Protein Levels by Enzyme-Linked Immunosorbent Assay (ELISA)

Protein extraction was done according to standard protocol. In short, cell pellets were lysed in RIPA buffer. GAPDH, MCL1, and MEK1 protein levels were determined with double-antibody Sandwich ELISA (SEB932Hu, SEC615Hu, SED559Hu, Cloud-Clone Corp., Houston, TX, USA). MCL1 and MEK1 values were normalized with GAPDH values. Two independent assays with three technical replicates were performed per sample. Statistical analysis was done using averaged normalized values and linear regression on GraphPad Prism software. Data are depicted as XY graphs with linear regression.

2.6. Antibodies and Cytometry

Staining for apoptosis was done using AnnexinV-CF488A (Biotium, Germany) in AnnexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min. at 37 °C, followed by several washes. Propidium iodide was added shortly before imaging on the Nucleocounter NC-3000 (ChemoMetec, Denmark). For cell cycle analysis, cells were incubated in lysis buffer with DAPI (10 μg/mL) for 5 min. at 37 °C and analyzed on NC-3000 imager.

3. Results

3.1. Susceptibility of AML Cell Lines to PTC596 and APR-246

To determine the sensitivity of AML cells to the BMI-1 inhibitor PTC596 and the p53 activator APR-246 AML cells were subjected to in vitro cytotoxicity assays. AML cells were treated with the compounds for 20 h in dose escalation experiments before cell viability assessment. The AML cell lines covered the major morphologic and molecular subtypes including, particularly, FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 wild type, mutant, hemizygous, and null cells (

Table 1. Genetic variants in acute myeloid leukemia (AML) cell lines.

ID	AML (FAB) Classification	Origin	FLT3 Gene	TP53 Gene	Other Gene Mutations	Karyotype
HL-60	M2	de novo	wt	null	NRAS Q61L CDKN2A R80X	hypotetraploid
ML-2	M4	T-ALL	wt	wt	MLL-AF6/(t6;11) KRAS A146T	t6;11
MOLM-13	M5a, relapse	MDS	ITD	wt	MLL-AF9/(t9;11)	t9;11
MOLM-16	M0, relapse	de novo	wt	V173M C238S	MLL V1368L MTOR T571K	hypotetraploid
OCI-AML2	M4	de novo	wt A680V	wt	DNMT3A R635W MLL K1751 *	+6, +8, 3q26
OCI-AML3	M4	de novo	wt	wt	DNMT3A R882C NRAS Q61L NPM1 L287fs	+1. +5, +8
PL-21	M3	de novo	ITD P336L	wt P36fs	KRAS A146V	hypertetraploid
SKM-1	M5, refractory	MDS	wt	R248Q	ASXL1, TET2	del9

Abbreviations: Wild type (wt); internal tandem duplication (ITD). * is the official denotation for a stop codon.

).

The susceptibility to PTC596 was elevated in TP53 null HL-60 cells with IC50 of 220 nM, intermediate in TP53 wild type MOLM-13 and OCI-AML3 cell lines with IC50 values of 300–500 nM, and rather low in TP53 mutant cells lines PL-21, MOLM-16, and SKM-1 with IC50 values of 800–1200 nM. ML-2 cells with MLL-AF6 transfusion oncogene and adverse risk were least susceptible to PTC596 with IC50 at 1500 nM (Figure 1A,B). With respect to APR-246, the susceptibility was elevated in TP53 mutant cell lines with IC50 values of 20–30 μM, intermediate in TP53 wild type and hemizygous cell lines with IC50 values of 50–70 μM, and reduced in TP53 null HL-60 cells with IC50 of 80 μM. The TP53 wild type ML-2 cells were most susceptible to APR-246 with IC50 of 22 μM (Figure 1C,D).

In order to define the most effective treatment combinations, we focused on inhibitors expected to elicit synergistic effects in combination with PTC596 based on previous studies with FLT3-, MDM2-, MCL1-, and MEK-inhibitors [20,22,23] as presented in Figure 2.

3.2. PTC596 Combination Treatment in AML Cell Lines

Cell viability was determined in AML cell lines treated with increasing dosages of single compounds and in combination treatments using the BMI-1 inhibitor PTC596 and a variety of targeted therapies including the TP53 activator APR-246 (Figure 3A), the MCL1 inhibitor S63845 (Figure 3B), and the MEK inhibitor trametinib (Figure 3C). Combination indexes were calculated according to Chou Talalay (

Table 2. Combination index values AML cell lines (Chou and Talalay).

Cell Line	PTC596 + APR246	PTC596 + S63845	PTC596 + Trametinib	PTC596 + PKC412	PTC596 + HDM201	PTC596 + CI *
OCI-AML2	0.9–1.1	0.9–1.1	0.9–1.1	nd	0.9–1.1	nd
OCI-AML3	0.9–1.1	0.3–0.5	0.1–0.3	0.9–1.1	0.9–1.1	1.1–1.5
MOLM-13	0.9–1.1	0.4–0.6	0.3–0.5	0.3–0.5	0.7–0.9	1.1–1.5
HL-60	0.7–0.9	0.8–1.0	0.8–1.0	nd	nd	1.3–2.1
PL-21	0.4–0.6	0.7–0.9	0.4–0.6	nd	nd	>3
MOLM-16	0.7–0.9	0.5–0.7	0.3–0.5	nd	nd	nd
SKM-1	0.7–0.9	0.3–0.5	0.1–0.3	nd	nd	nd
ML-2	0.7–0.9	0.1–0.3	0.4–0.6	nd	nd	nd

Combination indexes were calculated according to Chou Talalay [24]. Interpretation of combinatorial effects: Strong synergy CI = 0.1–0.3, moderate synergy CI = 0.3–0.7, mild synergy CI = 0.7–0.9, additive effects CI = 0.9–1.1, antagonism CI > 1.1. * Conventional induction therapy (idarubicin, cytarabine).

, Figure S1). Some cell lines were additionally treated with PTC596 in combination with the FLT3-ITD inhibitor PKC-412 (midostaurin), the MDM2 inhibitor HDM201, and conventional induction therapy (CI). For the TP53 wild type cell lines, overall solid response to combination treatments was detected in the MDS-AML cell line MOLM-13, which presented moderate synergy to PTC596 in combination with S63845, trametinib, or PKC-412, as well as mild synergy to PTC596 in combination with HDM201 and APR-246. Maximal synergistic effects were present in OCI-AML3 cells treated with PTC596 and trametinib and in ML-2 cells treated with PTC596 and S63845. OCI-AML3 cells also presented moderately synergistic effects to PTC596 in combination with S63845. ML-2 response was moderately synergistic to PTC596 in combination with trametinib and mildly synergistic in combination with APR-246. Cytotoxic effects in the TP53 wild type OCI-AML2 and the TP53 null HL-60 cell responses were additive in the combination of PTC596 with S63845 or trametinib (Table 2).

For the TP53 mutant cell lines, maximal synergistic effects to combination treatments were detected in SKM-1 cells with PTC596 and trametinib. Moderate synergies were present in the combinations of PTC596 and trametinib in PL-21 and MOLM-16 cells, as well as in the combination of PTC596 and S63845 in MOLM-16 and SKM-1 cells, with mildly synergistic effects in the TP53 hemizygous PL-21 cells. PTC596 and APR-246 elicited moderate synergistic effects in PL-21 cells and mildly synergistic effects in MOLM-16 and SKM-1 cells (Table 2).

The combination of PTC596 and conventional induction therapy consisting of equimolar cytarabine and idarubicin (20–100 nM) yielded overall antagonistic effects in the tested AML cell lines (Table 2).

3.3. Dose-Dependent Induction of Apoptosis and Cell Death in AML Cell Lines

The effects of PTC596 and trametinib or S63845 alone and in combination on induction of apoptosis, cell cycle arrest, and cell death were determined in cytometric assays. In the TP53 wild type cell lines strong response to combination treatments were detected in MOLM-13 cells with induction of G2 arrest by PTC596, induction of apoptosis and cell death when treated with PTC596 in combination with S63845, HDM-201, PKC-412, and trametinib (Figure 4A,B, Figures S2 and S3). OCI-AML3 cells also responded with enhanced apoptosis and cell death when treated with PTC596 in combination with S63845 or trametinib (Figure 4C,D). OCI-AML3 cells showed minor induction of G2 arrest in the cytometric assays, however, the BMI1 target gene CDKN2A expression was strongly upregulated in the combination treatments (Figure 4E,F) indicating induction of cell cycle arrest.

In the TP53 mutant cell lines, strong responses to combination treatments were detected in SKM-1 cells with induction of G2 arrest by PTC596, apoptosis, and cell death when treated with PTC596 in combination with S63845 and trametinib (Figure 4G,H).

Induction of apoptosis and cell death was also present in TP53 mutant (MOLM-16 and SKM-1) and TP53 wild type cells (ML-2 and OCI-AML3) when treated with PTC596 and APR-246 (Figure 5).

3.4. PTC596 Combination Treatments in Leukemic Cells In Vitro

Strong synergistic effects were elicited in the combination of PTC596 with trametinib or S63845 in the majority of the tested AML cell lines, these treatment combinations were applied to patient derived hematological cells and normal bone marrow. A total of ten primary AML, seven secondary AML post MDS (MDS-AML), one MDS, two B-ALL, and five normal bone marrow samples were subjected to single compound and combination treatments. In the combination of PTC596 and S63845 strong cytotoxic effects were detected in two AML and one MDS-AML: AML1, classified as FLT3 mutant adverse de novo AML with 90% blast count; AML6, a relapsed t-AML; MDS-AML5, a DNMT3A, TET2, NPM1 mutant secondary AML. Moderate cytotoxic effects were present in AML2 to 5, all classified as FLT3 and NPM1 mutant de novo AML with intermediate risk, and in the two B-ALL samples. Mild cytotoxic effects were detected in the rest of the AML and MDS-AML samples, AML7 to 9, classified as AML with NPM1 mutant or inv(16)/CBFB-MYH11 and favorable risk, and AML 10, a TP53 mutant AML (Figure 6A,C).

In the combination treatments with PTC596 and trametinib strong cytotoxic effects were present in four MDS-AML and one B-ALL: MDS-AML 1, 3, 6 with adverse risk, the TP53 mutant MDS-AML2, and the B-ALL2. The FLT3 and NPM1 mutant AML samples (AML2 to 5) and the TP53 mutant AML10, as well as MDS-AML4, 7, and 8 showed moderate response to PTC596 and trametinib. MDS-AML5, the DNMT3A, TET2, NPM1 mutant secondary AML, was not susceptible to trametinib. AML7 to 9, classified as AML with mutated NPM1 or inv(16) and favorable risk showed mild cytotoxic effects (Figure 6B,D). The cytotoxic effects elicited by the combination treatments were analyzed in the hematological cell samples correlated to CD34 positivity (Figure 6E,F) and grouped into leukemic cells with CD34 > 30% and CD34 < 30%, compared to normal bone marrow. The group with elevated CD34 positivity consisted of seven MDS-AML, one MDS, three AML, and two B-ALL samples, the group with reduced CD34 positivity consisted of eight AML samples. There was a significant correlation of CD34 positivity and response to PTC596 combination treatments (Figure 6G,H).

3.5. Biomarkers of Response to PTC Combination Treatments in Leukemic Cells

In order to define biomarkers of response the gene expression of BMI1 and MN1 as well as the protein levels of MCL1 and MEK1 were determined in the mononuclear cells isolated from AML patients at diagnosis (

Table 3. Clinical characteristics, gene expression, and protein levels in the hematological samples.

ID	Class	Stage	Mutation Profile	Karyo-Type	Risk	Sorce	Blast Count	CD34+	BMI1	MN1	MCL1	MEK1
A1	AML M1	de novo	FLT3	inv(4)	adv	PB	90%	55%	1.2	3.1	2.0	0.1
A2	AML M5	de novo	FLT3 NPM1	norm	inter	PB	40%	9%	2.1	0.1	1.0	0.1
A3	AML M1	de novo	FLT3 NPM1	norm	inter	PB	91%	1%	0.39	0.01	0.9	0.01
A4	AML M1	de novo	FLT3 NPM1	norm	inter	PB	90%	1%	1.43	0.01	0.8	0.01
A5	AML M5	de novo	FLT3 NPM1	norm	inter	PB	90%	1%	1.26	0.02	1.1	0.01
A6	tAML	relapse	NPM1	norm	adv	PB	56%	68%	0.82	7.9	2.2	0.01
A7	tAML	sec	norm	inv(16)	fav	BM	30%	3%	0.32	0.01	0.5	0.01
A8	AML M5	de novo	NPM1	norm	fav	PB	38%	1%	0.61	0.01	0.4	0.01
A9	AML M4	de novo	NPM1	norm	fav	PB	26%	7%	0.25	0.01	0.6	0.01
A10	AML M4	sec	FLT3 TP53-G245S	norm	adv	PB	46%	1%	0.51	0.01	0.7	0.14
MA1	AML M0	post MDS	norm	norm	adv	PB	90%	98%	4.2	3.9	0.5	0.2
MA2	AML M4	post MDS	TP53-N259V	T(2;5)	adv	PB	25%	71%	1.9	0.01	0.6	0.18
MA3	AML M2	post MDS	norm	del5, del7	adv	PB	60%	80%	4.2	5.1	0.5	0.19
MA4	AML	post MDS	CEBPA ASXL1 EZH2 RUNX1	norm	adv	BM	13%	26%	1.3	1.1	1.5	0.15
MA5	AML M1	post MDS	DNMT3A TET2 NPM1	norm	fav	PB	81%	80%	2.0	14	2.5	0.01
MA6	MDS	de novo	KRAS	del(7q)	adv	PB	19%	60%	0.63	2.9	1.1	0.16
MA7	AML	post MDS	JAK2	norm	inter	PB	8%	17%	1.43	0.5	0.9	0.19
MA8	AML M4	post MDS	norm	norm	inter	PB	20%	53%	1.38	1.9	0.8	0.1
TCL	TCL	CR	norm	norm	na	BM	0%	0%	0.41	0.01	0.5	0.01
MCL	MCL	de novo	norm	norm	na	BM	15%	5%	0.32	0.01	1.2	0.02
BA1	B-ALL	de novo	BCR-ABL1	t(9;22)	na	PB	82%	63%	0.83	3.9	1.5	0.02
BA2	B-ALL	de novo	MLL-AF4	t(4;11)	na	BM	51%	85%	5.52	5.8	1.4	0.2
M 1–4	MM	CR	norm	norm	na	BM	0%	1%	na	na	na	na

Abbreviations: Classification (class), acute myeloid leukemia (AML), MDS-AML (M-AML), myelodysplastic syndrome (MDS), T-cell lymphoma (TCL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), multiple myeloma (MM), secondary (sec), adverse (adv), intermediate (inter), favorable (fav), peripheral blood (PB), bone marrow (MB), complete remission (CR). Gene expression levels for BMI1 and MN1 genes, protein levels for MCL1 and MEK1.

), and correlated to the response to PTC596 combination treatments (Figure 7A–H). BMI1 gene expression and MEK1 protein levels were positively associated to the response to PTC596 and trametinib (Figure 7B,H), while MN1 gene expression and MCL1 protein levels were positively associated to the response to PTC596 and S63845 (Figure 7C,E). MN1 and BMI1 gene expression were positively correlated (Figure 7I). There was a robust correlation of MN1 gene expression with C34 positivity (Figure 7J). There was also a positive association of BMI1 gene expression and MEK1 protein levels with CD34 positivity (Figure 7K,L).

4. Discussion

In order to determine the susceptibility of AML cell lines to the BMI1 inhibitor PTC596 and the TP53 activator APR-246, a dose escalation screening was performed. Susceptibility to PTC596 varied in the tested AML cell lines with elevated susceptibility of the TP53 null cell line HL-60, the TP53 wild type AML cell line OCI-AML3, as well as the MDS-AML cell line MOLM-13, and reduced susceptibility of the TP53 mutant cell lines MOLM-16, SKM-1, and PL-21 as well as the TP53 wild type cell line ML-2. The susceptibility of TP53 null cells to PTC596 indicated a p53 independent mechanism of action. Susceptibility of MOLM-13 and OCI-AML3 cells had been previously described for the BMI1 inhibitors PTC-209 [25] and PTC596 [26].

Susceptibility to the p53 activator APR-246 was comparable in all AML cell lines with elevated susceptibility in the TP53 wild type ML-2 and the TP53 mutant MOLM-16 cells indicating a p53 independent mechanism of action. APR-246 has been described as a small molecule that restores and enhances the function of mutated or wild type p53 [17,27]. APR-246 triggers an upregulation of genes involved in cell cycle control and apoptosis in TP53 wild type and TP53 mutant cancer cells. Moreover, APR-246 elicits p53-independent effects including production of ROS, thereby inducing oxidative stress, as well as upregulation of heat shock proteins and UPR response genes, thereby inducing ER stress [17].

In order to define the most effective treatment combinations, we focused on inhibitors expected to elicit synergistic cytotoxic effects in combination with PTC596 based on previous studies with FLT3-, MDM2-, MCL1-, and MEK- inhibitors [20,22,23]. In the current study, we found synergistic cytotoxic effects for combinations of PTC596 with APR-246, S63845, trametinib, in many AML cell lines, including the TP53 double mutant MOLM-16, which turned out to be resistant to various other targeted therapies in the past. Here for the first time, MOLM-16 cells were susceptible to a targeted treatment with PTC596, and the effects were enhanced in combination with S63845 or trametinib.

Synergistic effects of PTC596 with S63845 and trametinib were to be expected, as PTC596 reduces MCL-1 expression in AML cells and may influence expression of MCL1 inducers including MEK, ERK, AKT, STAT3, and STAT5 [26], while S63845 inhibits MCL1 directly, and trametinib inhibits MEK. In a previous study, we found that susceptibility to the MCL-1 inhibitor S63845 and the MEK inhibitor trametinib correlated to the cellular MCL1 and MEK1 protein levels [20]. AML cell lines MOLM-13 and OCI-AML3, which were susceptible to S63845, had elevated MCL1 levels, while OCI-AML2 and MOLM-16, which were resistant to S63845, had reduced MCL1 levels. AML cell lines MOLM-13 and OCI-AML3, which were susceptible to trametinib, had elevated MEK levels, while OCI-AML2 and MOLM-16, both unsusceptible to trametinib, had very low MEK levels.

In the current study, we found antagonistic effects in the combination of PTC596 and conventional induction therapy in various AML cell lines. Similar antagonistic effects of PTC596 and doxorubicin or cytarabine had been previously described in mantle cell lymphoma [28]. The molecular mechanism underlying this antagonism is unclear. However, it seems that BMI1 inhibitors should not be applied in combination with doxorubicin or cytarabine, and by inference, the combination of PTC596 with other anthracyclines or nucleoside derivatives may also be unfavorable.

The effects of PTC596 and trametinib or S63845 alone and in combination on induction of apoptosis, cell cycle arrest, and cell death were determined in cytometric assays. PTC596 induced G2 arrest and apoptosis, as published previously [26]. Effects on cell cycle arrest, apoptosis, and cell death induced by PTC596 were enhanced by APR-246, S63845, trametinib, PKC412, or HDM201, as expected from the synergistic effects in the cytotoxicity assays.

Susceptibility of patient samples for PTC596 in combination with S63845 or trametinib was significant for the majority of adverse risk AML and MDS-AML samples, with least efficacy in favorable risk AML. In previous studies, BMI-1 protein levels were described to be significantly higher in patients with unfavorable cytogenetics compared with those with intermediate or favorable cytogenetics [25], and CD34 positive AML cells were more susceptible to PTC596 than mature AML cells [26]. The novel BMI-1 inhibitor PTC596 downregulated MCL-1 and induced mitochondrial apoptosis in a p53-independent manner. PTC596 effectively killed CD34 positive AML stem/progenitor cells while sparing normal hematopoietic stem/progenitor cells. In a recent phase I study, the most frequently reported PTC596-related treatment-emergent adverse events were mild to moderate gastrointestinal symptoms, including diarrhea, nausea, vomiting, and fatigue. Only one patient treated with 10.4 mg/kg experienced dose-limiting toxicity of neutropenia and thrombocytopenia, both of which were reversible [16]. In our study, the susceptibility of leukemic cells to PTC596 in combination with S63845 or trametinib also correlated significantly to CD34 positivity of the hematological samples. As secondary AML post MDS (MDS-AML) and B-ALL are characterized by a high degree of CD34 positivity these patient subsets may profit most from a PTC596 combination treatment. There is an apparent expansion of CD34 cells during the evolution from MDS to secondary AML [29], and CD34 is a prognostic biomarker for ALL [30], hence MDS-AML and B-ALL may both be valid targets for combination treatments with PTC596 and S63845 or trametinib. In addition to CD34 positivity, MN1 gene expression and MCL1 protein levels were biomarkers for response to PTC596 combination treatments with S63845, while BMI1 gene expression and MEK1 protein levels were biomarkers of response to PTC596 combination treatments with trametinib. The determination of gene and protein expression levels in leukemic cells as biomarkers of response to targeted combination therapies may be helpful to optimize treatment efficacy. The combination of PTC596 and S63845 may be an effective treatment in CD34 positive AML with elevated MCL1 protein levels, while PTC596 and trametinib may be more effective in CD34 positive AML with elevated MEK1 protein levels, including TP53 mutant AML.

5. Conclusions

In this study, we tested the BMI1 inhibitor PTC-596 alone and in combination with a variety of novel targeted agents in AML cell lines and primary AML blast cells in cell viability assays. The AML cell lines included a variety of cyto-morphologic FAB subtypes as well as molecular subtypes, including FLT3-ITD and FLT3 wild type, NPM1 mutant and NPM1 wild type, TP53 mutant and TP53 wild type cell lines. The results suggest that BMI1-inhibition by PTC-596 can induce cell cycle arrest and apoptosis in AML cells independent of TP53 status. In fact, best response to PTC-596 was detected in TP53 null cells, in minimally differentiated AML and in secondary AML post MDS. Induction of apoptosis and cell cycle arrest were analyzed. Effective combination treatments were validated in cytotoxicity assays with normal bone marrow and AML patient samples. Strong synergistic effects were detected in both TP53 wild type and TP53 mutant AML cell lines treated with PTC596 in combination with the MCL1 inhibitor S63845 or the MEK-inhibitor trametinib. In addition, mild synergistic effects were detected in both TP53 wild type and TP53 mutant AML cell lines treated with PTC596 and the TP53 activator APR-246. Strong cytotoxic effects were also detected in some AML patient cells treated with PTC596 in combination with S63845 or trametinib, in particular in adverse risk AML including a TP53 mutant MDS-AML and a relapsed t-AML. The susceptibility of leukemic cells to PTC596 in combination with S63845 or trametinib correlated significantly to CD34 positivity of the hematological samples. These results indicate that the combination of PTC596 and S63845 or trametinib may be both effective and specific treatments to target adverse risk AML, especially with elevated CD34 positivity, thus providing the rationale for initiating clinical studies evaluating these treatment combinations.