Repression of Mcl-1 expression by the CDC7/CDK9 inhibitor PHA-767491 overcomes bone marrow stroma-mediated drug resistance in AML

Acute myeloid leukaemia (AML) is an aggressive cancer with 50–75% of patients relapsing even after successful chemotherapy. The role of the bone marrow microenvironment (BMM) in protecting AML cells from chemotherapeutics and causing consequent relapse is increasingly recognised. However the role that the anti-apoptotic Bcl-2 proteins play as effectors of BMM-mediated drug resistance are less understood. Here we show that bone marrow mesenchymal stromal cells (BMSC) provide resistance to AML cells against BH3-mimetics, cytarabine and daunorubicin, but this is not mediated by Bcl-2 and/or Bcl-XL as previously thought. Instead, BMSCs induced Mcl-1 expression over Bcl-2 and/or Bcl-XL in AML cells and inhibition of Mcl-1 with a small-molecule inhibitor, A1210477, or repressing its expression with the CDC7/CDK9 dual-inhibitor, PHA-767491 restored sensitivity to BH3-mimetics. Furthermore, combined inhibition of Bcl-2/Bcl-XL and Mcl-1 could revert BMSC-mediated resistance against cytarabine + daunorubicin. Importantly, the CD34+/CD38− leukemic stem cell-encompassing population was equally sensitive to the combination of PHA-767491 and ABT-737. These results indicate that Bcl-2/Bcl-XL and Mcl-1 act in a redundant fashion as effectors of BMM-mediated AML drug resistance and highlight the potential of Mcl-1-repression to revert BMM-mediated drug resistance in the leukemic stem cell population, thus, prevent disease relapse and ultimately improve patient survival.

behind. Underlining its importance, bone marrow-mediated protection was found to be the major cause of low FLT3-inhibitor efficacy 5,6 . The most studied mechanism by which bone marrow stromal cells (BMSCs) induce drug resistance is the activation of pro-survival signal transduction, typically culminating in the upregulation of Bcl-2 (BCL2) and/or Bcl-X L (BCL2L1) 7,8 . Induction of anti-apoptotic Bcl-2 proteins is an inherent feature of normal differentiation of leukocytes as Bcl-2 proteins provide survival advantage to the properly formed mature cells. For example, Mcl-1 (MCL1) is required for the survival of hematopoietic stem cells (HSC) 9 , common myeloid progenitors (CMP) and common lymphoid progenitors (CLP), Bcl-2 is induced during the selection of T and B lymphocytes while Bcl-X L (BCL2L1) is critical for erythrocyte- 10,11 , megakaryocyte-12 and platelet survival 13 , and A1 (BCL2A1) supports neutrophil survival 14 .
Increased Bcl-2 expression is also a characteristic of several haematological malignancies, including chronic lymphocytic leukemia (CLL) and AML. The notion that leukemic cells become dependent on anti-apoptotic Bcl-2 protein expression for survival is proven by the potent effect of the Bcl-2/Bcl-X L /Bcl-W inhibitor, ABT-737 and its Bcl-2-selective variant, ABT-199 15 . The ability of anti-apoptotic Bcl-2 proteins to drive drug resistance is also well established. Accordingly, ABT-737 and/or  have been shown to sensitise isolated AML cells to 5-azacytidine 16 , FLT3 inhibitors 17 as well as docetaxel 18 .
Here we determined the role of anti-apoptotic Bcl-2 proteins as effectors of bone marrow stroma-mediated drug resistance in AML blasts and the CD34 + /CD38 − cells representing a population enriched for leukemic stem cells (LSC) 19 . We show that bone marrow stromal cells (BMSCs) provide resistance against BH 3 -mimetics, cytarabine (AraC) and daunorubicin (DnR) and that this protection is also pronounced in the CD34 + /CD38 − cell population. We show that inhibition of Bcl-2 and Bcl-X L with ABT-737 is not sufficient to revert BMSC-mediated drug resistance against AraC + DnR. On the other hand, BMSC-mediated drug resistance was associated with increased Mcl-1 expression. Furthermore, Mcl-1 inhibition with A1210477 or repression with PHA-767491 could revert drug resistance mediated by BMSCs. Importantly, repression of Mcl-1 expression with the dual CDC7/ CDK9 inhibitor PHA-767491 equally sensitised the CD34 + /CD38 − cell population offering a strategy to eradicate the main cell population responsible for disease relapse.

Bone marrow mesenchymal stromal cells protect AML cells from therapeutic drugs. In order
to determine the effect of anti-apoptotic Bcl-2 proteins in drug resistance mediated by the BMM, a layered stroma-AML co-culture system has been set up. AML cell lines or primary AML blasts were cultured on a monolayer of BMSCs in direct contact. As a model of BMSCs, HS-5 cells, an immortalised healthy donor-derived BMSC cell line, were used. HS-5 cells were chosen over primary BMSCs of AML patients, as the latter were found to be prone to senescence under ex vivo culture 20 . As HS-5 cells are only a clone of immortalised BMSCs, they may not represent the full spectrum of function that primary BMSCs have. Thus we tested how faithfully they could replicate the effect of patients' own BMSCs. To this end, primary AML cells were cultured alone, on the patients' own BMSCs or on two different immortalised BMSC lines; the commercially available HS-5 cells and a non-commercially available hTERT immortalised BMSC cell line that we named iMSC. After 24 h, the cultures were treated with ABT-737 or ABT-199 and induction of cell death was quantified. Both HS-5 cells and iMSCs could both replicate the effect of the patients' own BMSCs in providing a comparable level of resistance to AML cells against both drugs ( Fig. 1A-D, Suppl. Fig. 1).
The broader effect of BMSCs on AML drug sensitivity was next tested using AraC, DnR and ABT-737 in both AML cell lines and primary AML cells. OCI-AML2, ML-1 and Molm-13 cells were co-cultured with HS-5 cells for 24 h followed by treatment with a dosage of AraC or ABT-737 for 24 h and cell viability was determined with Annexin V staining. With the exception of ABT-737 treatment of OCI-AML2 cells, all co-cultures showed a significantly reduced sensitivity ( Fig. 1E-J).
Similar effects were found using AML blasts. The mononuclear cell fraction isolated from bone marrow aspirates of AML patients (AML blasts) were cultured on HS-5 cells for 24 h followed by exposure to a 3:1 molar ratio of AraC and DnR (corresponding to the molar ratio of the two drugs used in the clinic (7 + 3 therapy 22 ) or a dosage of ABT-737 for 24 h. Induction of cell death in the AML blasts was determined using the viability dye, ToPro-3. Similar to the cell lines, AML blasts gained resistance against both AraC + DnR and ABT-737 when cultured with BMSCs ( Fig. 2A-F). Of note, the resistance against DnR + AraC provided by HS-5 BMSCs was also pronounced in the CD34 + /CD38 − population shown by their enrichment in the surviving cell fraction (Fig. 2G,H).

AML cells residing in the bone marrow microenvironment have increased Mcl-1 expression.
To determine the contribution of anti-apoptotic Bcl-2 proteins to BMM-driven drug resistance, we determined the effect of HS-5 BMSCs on the expression of anti-apoptotic Bcl-2 proteins. A panel of AML cell lines (OCI-AML2, OCI-AML3, HL60, ML-1, Molm-13) were cultured with HS-5 cells for 24 h and changes in the expression of Bcl-2, Bcl-X L and Mcl-1 were determined with Western blotting. Interestingly, while HS-5 BMSCs did not induce significant change in the expression of Bcl-2 and Bcl-X L , in 4 out of the 5 cell lines tested, Mcl-1 induction was detected (Fig. 3A).
To gain insight whether the BMM exerts the same effect in vivo, we compared the mRNA expression of Bcl-2, Bcl-X L and Mcl-1 between bone marrow (BM)-residing and peripheral blood (PB)-circulating AML blasts. To   To assess the contribution of anti-apoptotic Bcl-2 proteins to BMM-mediated AML drug resistance, we tested if inhibition of Bcl-2/Bcl-X L can revert BMSC-mediated resistance to chemotherapeutics. To this end, AML cell lines were cultured with HS-5 BMSCs for 24 h before exposing the cells to AraC for an additional 24 h in the presence or absence of ABT-737. The doses of ABT-737 for each cell line has been selected based on their sensitivity profile (Suppl. Fig. 3). All cell lines, except OCI-AML3 (Fig. 4B), showed sensitivity to ABT-737 administered as a single agent, indicating that AML cells depend on Bcl-2/Bcl-X L expression for survival even in the BM environment. At the same time, ABT-737 could not revert BMSC-mediated resistance to AraC and DnR, indicating that Bcl-2 and Bcl-X L are not the sole effectors of BMSC-mediated drug resistance (Fig. 4).   (Fig. 5A) and induction of cell death was quantified with Annexin V staining. Treatment with 2 µM of PHA-767491 alone triggered a modest induction of apoptosis, but, it potently sensitised the cells to ABT-737 (Fig. 5B,C). Importantly, the drug combination retained full efficacy in the presence of HS-5 BMSCs (Fig. 5C). At this concentration, PHA-767491 downregulated Mcl-1 expression after 3 h of exposure onward (Fig. 5A). Using the Chou-Talalay median effect equation, the combination index (CI) was calculated (table under the graphs in Fig. 5B,C). At each drug concentration, the CI index was well below 1.0, indicating a potent synergy between ABT-737 and PHA-767491.
To confirm that the mechanism of PHA-767491-mediated sensitization involved Mcl-1, a small molecule Mcl-1 inhibitor, A1210477, was employed. A1210477 is a selective, small-molecule inhibitor of Mcl-1 developed by the Soeurs lab 27 shown to have a high selectivity to Mcl-1 over other Bcl-2 family members, multiple kinases and G-protein coupled receptors. Similarly to treatment with PHA-767491, A1201477 was able to sensitise OCI-AML3 cells to ABT-737 (Fig. 5D).
In order to corroborate the above result, the role of Mcl-1 in BMSC-mediated drug resistance was tested in a second AML cell line, with lower Mcl-1 expression. As opposed to OCI-AML3 cells, Molm-13 cells have a low baseline Mcl-1 expression but, they show Mcl-1 induction upon contact with BMSCs (Fig. 3A) and increased resistance to ABT-737 and AraC (Fig. 1G,J). Molm-13 cells were cultured alone or in contact with HS-5 BMSCs as before and exposed to ABT-737, with or without a 4 h pre-treatment with A1210477, for 20 h and induction of cell death was determined with Annexin V staining (Fig. 6A). Co-culture with BMSCs induced a moderate, but consistent protection against both ABT-737 and A1210477. At the same time, A1210477 pre-treatment sensitised Molm-13 cells to ABT-737 both in single culture and in co-culture with HS-5 cells (Fig. 6A, CI values are under the graph). Notably, the effect of the Mcl-1 inhibitor was more pronounced under the co-culture conditions, reflected also by lower CI indices, indicating that Mcl-1 is an effector of BMSC-mediated ABT-737 resistance (Fig. 6A).  The finding that AML cells have enhanced sensitivity to the combined inhibition of Bcl-2/Bcl-X L and Mcl-1 when they are in contact with BMSCs was further explored using AML blasts (Fig. 7). Samples from 10 patients were cultured on a HS-5 BMSC monolayer for 24 h, followed by treatment with ABT-737 for 24 h (30 nM and 300 nM) with or without a 4 h pre-treatment with PHA-767491 (1-4 µM). Except for one patient (Patient 2), whose cells displayed a high sensitivity for ABT-737, the combination of ABT-737 and PHA-767491 showed a synergistic effect (Fig. 7A-D, Suppl. Fig. 4, CI indices in Suppl. Table 2). We also carried out these experiments using the patients' own BMSCs and found a similar synergistic effect (Suppl. Fig. 5A, C, E and G), corroborating the findings. Because the LSC population often has higher drug resistance driving relapse, the sensitivity of the  CD34 + /CD38 − LSC-encompassing population was also determined (Suppl. Fig. 4). Looking at the effect of the two drugs individually, the CD34 + /CD38 − population showed higher resistance to ABT-737 in 3 out of the 10 samples (Patient 24, 25 and 6) and in 2 samples, they were more resistant to PHA-767491 (Patient 24 and 5). Importantly, the combination treatments showed that PHA-767491 could sensitise the ABT-737 resistant cells and the drug combination had a potent cytotoxic effect. The clinical data (Suppl. Table 3) of the tested samples shows that patients with refractory AML are also sensitive to the combination of ABT-737 and PHA-767491, highlighting a potential patient cohort that could benefit from this drug combination. Because PHA-767491 is likely to repress the expression of a number of genes, not only Mcl-1, Mcl-1 was also inhibited using A1210577 to prove that sensitisation to ABT-737 required Mcl-1 repression. 4 patient samples were treated as before, only replacing PHA-767491 with A1210577 (5 μM, Fig. 7E-H). As a single agent, A1210477 had a much lower cytotoxic effect than PHA-767491 in line with its expected, lower non-specific (Mcl-1 independent) effects. A1210477 sensitised 2 out of the 4 samples to ABT-737 (patient 20 and 26), confirming that repression of Mcl-1 is a key mechanism through which PHA-767491 sensitises AML cells under BMSC support to ABT-737. The combination of ABT-737 with A1210477 was also tested using matched BMSCs. Inhibition of Mcl-1 was equally efficient in sensitizing the AML blast cultured with their matched BMSCs, giving an indication for similar potency in vivo (Suppl. Fig. 5B, D, F and H). To confirm that repression or inhibition of Mcl-1 sensitizes AML blasts under stromal support, a knockdown of Mcl-1 was carried out on primary AML blasts. AML blasts from 2 patients, whose blasts showed resistance to ABT-737 were transfected with siRNA against Mcl-1 using nucleofection (Suppl. Fig. 6A) and co-cultured with iMSCs for 24 h. Cells were then treated with ABT-737 (30 and 300 nM) for 24 h. While we noticed that the transfection itself stressed the cells and reduced Mcl-1 expression, knockdown of Mcl-1 enhanced sensitivity to ABT-737 (Suppl. Fig. 6B).
As ABT-737 is only a proof-of-concept drug, we also tested the clinically relevant variant, ABT-199 (Bcl-2 selective inhibitor). In the 4 patient samples tested, pre-treatment with both PHA-767491 or A1210477 enhanced sensitivity to ABT-199. Of note, the sensitisation appears to be lower than to ABT-737, indication that Bcl-X L may also contribute to drug resistance (Suppl. Fig. 7).
Since Bcl-2 proteins, including Mcl-1, play fundamental roles in hematopoietic lineage cells, the potential toxic effect of the combined treatment on normal HSCs was measured. For this purpose, we obtained CD34 + HSCs from patients with a disease not associated with aberrant HSCs (the disease profile of these samples is listed in Suppl. Table 3, patient [27][28][29][30]. The samples were analysed in the same co-culture settings as before. Non-malignant HSC-1 and -3 showed a partial sensitivity to high dose PHA767491 (Fig. 8A,C) and non-malignant HSC-2 and -4 showed a partial sensitivity to ABT-737 (Fig. 8B,D). However, the drug combination failed to induce a synergistic effect, except in non-malignant HSC-1. Of note, both HSC-1 and HSC-3 samples were from multiple myeloma patients, and the disease background might have had an impact on the behaviour of the HSCs. Overall, these results indicate a possible therapeutic window for the targeting of LSCs with the ABT-737 and PHA-767491 drug combination.

Discussion
Hematopoietic lineage cells are dependent on the expression anti-apoptotic Bcl-2 proteins for survival. Individual Bcl-2 family members are upregulated at different stages of hematopoietic differentiation as well as by cytokine signals, which allows the survival of the properly formed or antigen-reactive cells.
Malignantly transformed leukemic cells appear to have a tighter dependency on anti-apoptotic Bcl-2 proteins than normal leukocytes. The reason for this is that oncogenic stress and other cellular stresses associated with malignant transformation lead to the induction of pro-apoptotic BH 3 -only proteins, such as Bim in lymphoma development, and unless these are neutralised by anti-apoptotic family members the transformed leukemic cells may not survive 28 . In line with this notion, BH 3 -mimetic drugs, such as the mainstream Bcl-2/Bcl-X L /Bcl-W inhibitors ABT-737 and ABT-263 and their Bcl-2-selective derivative, ABT-199 showed remarkable efficacy in clinical trials 29 .
High anti-apoptotic Bcl-2 protein expression does not only promote survival of leukemic cells, but it is also associated with treatment-resistance. High percentage of Bcl-2 positive AML cells correlate with low complete remission rates after intensive chemotherapy 30 and high Bcl-2 expression levels in AML cells were found to drive resistance against cytarabine 31 . Bcl-X L expression has also been associated with chemoresistance, for example against 5-azacytidine in AML or the topoisomerase inhibitor, etoposide, in B cell leukemia 16,32 .
The effect of the bone marrow microenvironment on drug resistance is well established. However, its effect on the expression of anti-apoptotic Bcl-2 proteins and the impact of BMM-mediated anti-apoptotic Bcl-2 protein expression on resistance against classical chemotherapeutics or BH 3 -mimetics is much less understood. Equally, the effect of the BMM on anti-apoptotic Bcl-2 protein expression in the most resistant LSC population and drug resistance conveyed by them is also poorly investigated.
Here we show that BMSCs drive resistance against the mainstream chemotherapeutics AraC + DnR with the CD34 + /CD38 − cells, representative of an LSC-enriched population, displaying higher resistance than the AML blasts. We found that inhibition of Bcl-2/Bcl-X L /Bcl-W with ABT-737 could not reverse this resistance. We also found that the BMM induced Mcl-1 expression both in vitro in AML cell lines and in vivo in a subset of primary samples, while BMM-mediated Bcl-2 and Bcl-X L induction was less consistent. In line with these findings, Garrido and colleagues have also reported inconsistent or low level induction of Bcl-2 in primary patient AML cells in ex vivo BMSC co-cultures 33  Mcl-1 has a number of distinguishing features that separates it from Bcl-2 and Bcl-X L . It lacks a conserved BH4 domain and it has a very short half-life time due to a P-E-S-T motif in its N-terminal portion (resulting a half-life time of only 2-4 h in most cells) 40,41 . This, combined with the multiple pathways that control its (1) transcription (such as the transcription factors ATF5, E2F1, STAT3, PU.1 and NF-kB), (2) regulate its translation (3) as well as protein stability, create a very dynamic control of Mcl-1 protein levels in response to a wide variety of cellular stresses and signals 41,42 .
To date, there was little focus on the role of Mcl-1 in BMM-driven AML drug resistance. Here we found that Bcl-2 and Mcl-1 are both effectors of bone marrow stroma-mediated drug resistance and they act in a redundant manner, i.e. the presence of either Bcl-2 or Mcl-1 was sufficient to provide resistance since simultaneous inhibition of both proteins was required to restore drug-sensitivity.
The CDC7/CDK9 inhibitor, PHA-767491 represses Mcl-1 expression 43 . CDK9 forms the catalytic core of the positive transcription elongation factor b (P-TEFb). P-TEFb phosphorylates RNA polymerase II and thus initiates the elongation phase of transcription 44 . Inhibition of CDK9 thus leads to the depletion of proteins with short half-life times, such as Mcl-1 25 . Previous studies have shown that combining CDK9 inhibitors with Bcl-2 inhibitors, such as ABT-737 and ABT-199, can overcome drug resistance in a synergistic manner [45][46][47] . We found that PHA-767491-mediated downregulation of Mcl-1 was very potent in sensitising both AML cell lines and primary AML blasts to ABT-737 and to the recently FDA-approved ABT-199. It has to be noted, that ABT-199 appeared to be inferior in inducing AML cell death compared to ABT-737 in our studies, indicating a possible role of Bcl-X L as well. As shown by Jilg and colleagues, the CD34 + population showed sensitivity to ABT-737 48 . Our studies confirm these findings, but also show that in some patients the CD34 + /CD38 − population is resistant to ABT-737. Importantly, PHA-767491 restored the sensitivity of resistant CD34 + /CD38 − cells to ABT-737. These results highlight the role of Mcl-1 in addition to Bcl-2 as a key effector of BMM-mediated pro-survival signalling and consequent drug resistance. At the same time, non-malignant CD34 + HSCs were not sensitised to ABT-737 by PHA-767491, indicating that Mcl-1 has a differential role in CD34 + cells of AML versus non-malignant HSCs thus offering a therapeutic window to target LSCs. Chemoresistant AML may be a potential patient cohort who could benefit from treatment with ABT-737 and PHA-767491 or other CDK9 inhibitors, as samples from refractory AML patients showed equally high sensitivity to the ABT-737 + PHA-767491 treatment. In this regard, a recent report highlighted that feedback activation of STAT3, a key transcriptional regulator of Mcl-1 was induced by the BMM and drove chemotherapy-resistance 49 .
Mcl-1 has been shown to play a central role in emergency hematopoiesis, i.e. the regeneration of hematopoietic lineage cells after stress. It is likely that chemotherapy triggers the same effect 50 . This emphasizes the need to consider pharmaceutical inhibition of Mcl-1 to target LSCs and reduce the occurrence of relapse. However, the potential toxic effect of Mcl-1 inhibitors cannot be neglected.
Therapies that target pathways that upregulate Mcl-1 expression in neoplastic cells as opposed to direct inhibition of Mcl-1 may be less toxic. In this regard, CDK9 has been shown to be aberrantly activated by oncogenic fusion proteins (such as MLL-fusion proteins) driving lymphoid and myeloid leukemia [51][52][53] . NF-κB (p65) also requires P-TEFb for transcription elongation 54 . As NF-κB signalling is often elevated in AML and it can drive Mcl-1 expression, this represents another mechanism how inhibition of CDK9 can repress abnormal Mcl-1 expression 54 . Finally the Gandhi laboratory reported, although in CLL not AML, that stroma-driven resistance to apoptosis of CLL cells was associated with a cascade of transcriptional events that included increased phosphorylation of RNA Pol II on serine residues at positions 2 and 5 (the phosphorylation site of CDK9), leading to increased rate of global RNA synthesis, and amplification of Mcl-1 transcript levels 55 . These reports provide a strong rationale for targeting Mcl-1 gene expression as opposed to direct inhibition of the Mcl-1 protein (like a BH 3 -mimetic) In conclusion, the results presented here indicate that Bcl-2/Bcl-X L and Mcl-1 act in a redundant fashion as effectors of BMM-mediated AML drug resistance and indicate that for BH3-mimetic-based treatment of AML the focus must be broadened from sole targeting of Bcl-2 to the additional inhibition or repression of Mcl-1.

Materials and Methods
Reagents. All reagents were purchased from Sigma, unless stated otherwise. ABT-737 and A1210477 from Selleck Chemicals, ABT-199 from Active Biochem and PHA-767491 (provided by Corrado Santocanale, NUI Galway) were dissolved in dimethyl sulfoxide (DMSO). AraC and DnR (Sigma) were dissolved in water. Carboxyfluorescein succinimidyl ester (CFSE) (Biolegend) was dissolved in DMSO. Annexin V was purchased from Immunotools. ToPro-3 was purchased from Molecular Probes.
Primary AML samples were generated from bone marrow aspirates by isolating the mononuclear cell (MNC) fraction with Ficoll gradient-centrifugation as described before 56 . MNCs were stored in liquid nitrogen until use. Upon revival of the cells, viability was determined with trypan blue staining. Only samples with viability above 60% were used. Primary AML blasts were grown in αMEM (Sigma) containing 10% HI-FBS, penicillin (100 U/ml), streptomycin (100 μg /ml), L-glutamine (2 mg/ml) and sodium pyruvate (1 mM).

Ethics Statement. Ethical approval was obtained from the Research Ethics Committee of University College
Hospital, Galway and NUI Galway. The ethical approval confirmed that all methods were carried out according to the regulation of NUI Galway and all applied protocols have been approved by the research ethics committees. Informed consent was obtained from all participants.
Immunophenotyping and viability assay. MNCs were incubated with fluorochrome-conjugated anti-CD34 ((phycoerythrin, PE) and anti-CD38 (allophycocyanine (APC)-H7) antibodies (BD Bioscience, San Diego, USA) in PBS/1% BSA (Sigma) for 30 minutes on ice in the dark. After washing off unbound antibodies, cells were stained with ToPro-3 (Molecular Probes) according to the manufacturers' protocol. The samples were analysed using BD FACS Canto II flow cytometer (BD Bioscience, San Diego, USA) by collecting 100,000 events AML blast gate. HS-5 cells were excluded by gating out GFP + /FSС high events. The detailed gating strategy is shown in Suppl. Fig. 8.
In the AML cell lines cell death was quantified with Annexin V-FITC or propidium iodide (PI) staining. Cells were collected and stained with Annexin V-FITC in Annexin V buffer (10 mM HEPES/NaOH, pH 7.5, 140 mM NaCl, 2.5 mM CaCl 2 ) or propidium iodide (1 μg/ml) for 15 min on ice in the dark. Samples were analyzed on a