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Targeting PKC and BET induces differentiation of AML
3D, E). As BET inhibitors, such as CPI-203, are known to downregulate MYC expression,32 we next investigated the role of MYC in H4-dependent differentiation of primary AML-3 cells. While MYC expression increased upon the addition of H4, treatment with CPI-203 alone or in combi- nation efficiently downregulated MYC (Figure 5F), indicat- ing that upregulation of MYC is dispensable in H4-induced differentiation of AML-3 cells. Notably, the M5 FLT3 wild- type leukemic cell line THP1 responded with MYC down- regulation upon H4 treatment, while the combination with CPI-203 further suppressed MYC expression (Figure 5F). These results suggest that the effect of H4 can be enhanced with the BET inhibitor CPI-203 in a MYC-independent process. We also provide supporting evidence that the monoblastic nature of AML is a potential prognostic mark- er of drug sensitivity to H4 and CPI-203. Overall, our study highlights the power and robustness of multiplexed screening on individual primary samples and demonstrates that mutation agnostic combined pathway-targeting can lead to efficient leukemic differentiation.
Discussion
The 10-year survival of patients with AML between 55 and 64 years is as low as 20%.2 These patients often develop co-morbidities, and their treatment must be cho- sen with care and often terminated in advance. Therefore, new and more AML-specific treatments with fewer side effects are greatly needed. However, due to the underly- ing diversity in the regulation of AML, the “one size fits all” strategy will be challenging to pursue, and the key to suc- cess is to identify subpopulations of patients with similar responses to selective treatments. Thus, better ways to develop patient-specific treatments with good translation from research to the clinic are needed. In this study, we performed a screen of the effects of natural products on primary AML cells using a co-culture system that simu- lates the microenvironment in the bone marrow. The screen identified a novel natural compound, called H4, which induced differentiation of FLT3 wild-type AML. Furthermore, using a combinatorial screen with H4 and molecules with defined targets, we identified the mecha- nism of H4 and determined that PKC activation, together with BET inhibition, further promoted the induction of leukemia differentiation. This demonstrates that our strategy based on a multiplex screening platform can be used for developing combined targeted therapy as well as for gaining mechanistic insights into leukemic regulation.
The novel macrocyclic diterpene identified in this study belongs to the jatrophane class of compounds, pre- viously unknown for its effect on AML. Jatrophanes structurally resemble phorbol molecules known as PKC activators.33 In line with this, H4 activated PKC followed by the downstream MAPK-ERK/JNK pathways that have previously been shown to be crucial to the induction of monocytic differentiation by additional agents such as vitamin D3, iron-chelating drugs, reactive oxygen species-inducing agents, as well as other PKC agonists.34- 38 Furthermore, the clinical potential of activating this pathway was demonstrated in a retrospective case-con- trol study of AML patients treated with vitamin D3 and an iron-chelating agent which resulted in induced blast cell differentiation and increased overall survival.38,39 Hence, further studies are warranted to evaluate H4 as a
clinical candidate, including defining the therapeutic window in vivo.
H4-induced differentiation was found to be dependent on FLT3 status, in that FLT-ITD or FLT3 mutations inhib- ited the differentiation-inducing capacity of H4, with the strongest responders being monocytic (M5) FLT3 wild- type leukemias. This indicates that the predicted response is dependent on both the genetic aberrations and in what stage in the hierarchy the differentiation is arrested. The mechanism by which FLT3-ITD or FLT3 mutant signaling inhibits the effect of H4 is not clear, but since these mutations are often seen as secondary muta- tions, they may be central in the leukemic transformation and thus directly control the differentiation block.40 As such, several essential myeloid transcription factors have been shown to be directly regulated by FLT-ITD such as RUNX1, PU.1 and CEBP/α, which may confer resistance to PKC signling.41,42 Since FLT-ITD and FLT3 mutations are among the most common aberrations in AML (being present in around 30% of all AML patients)7 this could limit the use of H4 clinically. However, in FLT3-ITD or FLT3-mutated AML, it would be interesting to evaluate if H4 combined with FLT3 inhibitors or molecules specifi- cally targeting the downstream effector genes to FLT3 such as CKI,43 could unlock the differentiation block, allowing differentiation also in these leukemias.
Gene expression analysis revealed that inflammatory signaling was the primary cellular response to H4, which, together with the upregulation of CD64, suggests mono- cytic differentiation towards non-classical inflammatory monocytes by M1 polarization.44,45 We also observed a strong activation of MYC and MYC target genes. Consistent with M1 polarization, MYC is involved in metabolic reprogramming, leading to increased use of glutamine, whose process, when inhibited, also reduced the effect of H4.46,47 However, we and others found that MYC is downregulated in leukemic cell lines upon PKC activation,35 suggesting that the differentiation response in cell lines and primary cells can be mechanistically dif- ferent. This points to the importance of using individual AML samples for mechanistic studies.
Combinatorial treatment is a strategy to overcome therapy resistance to directed therapy.13,15 Nevertheless, identifying combinations based on mutational profile alone is difficult, especially in the context of complex karyotypes. In contrast, using our platform, we could rap- idly evaluate hundreds of combinations on primary patients’ cells, revealing the combination of CPI-203 (a BET4 inhibitor) and H4. Intriguingly, the combination of PKC agonists with BET inhibitors is an effective strategy to activate latent human immunodeficiency virus (HIV) in CD4 T cells and monocytes.48 Studies on HIV also showed that the combination of BET and PKC enabled optimal efficacy at a lower concentration than using the individual molecules, which is important for reducing potential side effects. In line with this, the optimal con- centration for CPI-203 in our study was 50 nM, which is non-toxic for healthy bone marrow cells, and below the concentrations used in other studies in which CPI-203 was given alone.49,50
Our combination not only improves the effect of H4 but also potentially improves its clinical potential by removing unwanted side effects such as MYC expression. Furthermore, it would be interesting to determine whether cancer patients with specific MYC amplifica-
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