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S. Hultmark et al.
euthanized because of the development of AML, while only two out of five mice transplanted with H4-treated cells had leukemic cells, at barely detectable levels of engraft- ment (>0.02% engraftment in spleen and bone marrow) (Figure 2F and Online Supplementary Figure S2C). These results demonstrate that H4 can induce differentiation of primary leukemia-propagating cells.
To further investigate the leukemia-differentiating poten- tial of H4, we next assessed the response of an additional 17 (20 in total) primary samples and seven AML cell lines. Four of the 20 primary samples and two of the cell lines respond- ed with increased CD11b expression (> 20% increase) (Figure 2G, Table 1, Online Supplementary Table S1, Online Supplementary Figure S3A, B), still without any major toxici- ty recorded for the tested samples (Table 1, Online Supplementary Figures S2D and S3B). For healthy control cells (CD34+ bone marrow cells, CD34+ peripheral mononu- clear cells and peripheral mononuclear cells), only a small increase in CD11b was detected, without altered cell mor- phology, size, or granularity (Figure 2G, Table 1, Online Supplementary Figure S2E-F). Next, to establish a link between drug response and genetic aberrations, the sam- ples were grouped based on their FLT3 status, which revealed a significant correlation to drug response in pri- mary samples lacking FLT3 mutations (Figure 2G), with a similar trend in AML cell lines (Online Supplementary Figure S3C). These data suggest that the FLT3-ITD/FLT3 muta- tions confer resistance to the effect of H4 and that the ther- apeutic potential is effective in patients with normal FLT3 alleles. In addition, two of the four responding primary samples (with >20% increase in CD11b) and the three strongest responding AML cell lines (Figure 2G, Table 1 and Online Supplementary Figure S3A, B), were diagnosed as acute monocytic leukemia (AML subtype M5 according to the French-American-British [FAB] classification) suggesting a favorable drug response for AML blocked at the monoblastic stage. Taken together, we identified H4 as a differentiating agent of FLT3 wild-type AML.
H4 treatment activates the gene expression program of monocytic differentiation
To further characterize H4-induced leukemic differentia- tion, we performed global gene expression profiling on pri- mary AML-3 cells 16 h after treatment (Figure 3A). The genes most significantly modulated (42 unique ID, adjusted P-value -log10 >1) were mainly upregulated, indicating that H4 treatment leads to transcriptional activation rather than repression (Figure 3B). Next, by comparison of the genes with the greatest fold change in expression to those in a dataset of normal human hematopoiesis24 (using CellRadar, developed by G. Karlsson’s laboratory at Lund University; unpublished data from Dhapola et al.) we found that the upregulated genes were associated with monocyte matura- tion and downregulated genes with myeloid progenitor cell identity (Figure 3B, C), supporting that H4 induces leukemic differentiation towards monocytes. In line with this, gene set enrichment analysis (GSEA) also showed strong enrich- ment for processes involved in monocytic differentiation and M1 polarization such as interferon-α/γ, tumor necrosis factor-α signaling via NF-κB, and reactive oxygen species (Figure 3D, E).14,25 In addition, among the upregulated genes, we identified FcγRI (CD64), which is known to be specifi- cally expressed by monocytes and macrophages.26 The upregulation was confirmed at the protein level, with there being a 4-fold change in CD64 protein levels 4 days after
treatment in comparison to the level in control experiments with DMSO (Figure 3F). Furthermore, gene ontology analy- sis of the upregulated genes demonstrated significant enrichment of genes coding for ribosomal biogenesis and translational elongation (Online Supplementary Table S2). In line with this, AML-3 cells treated for 19 h with H4 showed increased incorporation of OP-Puro, indicating an increased rate of protein translation (Figure 3G). Collectively, these findings demonstrate that H4 can alter the leukemic molec- ular program of primary AML to induce leukemic differen- tiation towards the monocytic lineage.
H4 induces leukemic differentiation through activation of protein kinase C
To identify candidate signaling pathways enabling H4- driven leukemic differentiation, we screened cells from patient AML-3 with H4 (at 10 mM) in combination with 176 compounds (Selleck Chem L3000) with defined tar- gets (tested at 0.05 mM, 0.5 mM, and 10.0 mM) (Figure 4A). Several molecules had an inhibitory effect on H4-depen- dent CD11b upregulation, with an enrichment of com- pounds targeting the RAF/MEK/ERK signaling pathway together with JNK and glutaminase 1 (GLS1), all of which are downstream of PKC (Figure 4B and Online Supplementary Figure S4A, B).27,28 In line with this, the addi- tion of the specific PKC inhibitor GF109203X29 prevented CD11b upregulation and the morphological changes of H4-treated AML-3 cells (Figure 4C and Online Supplementary Figure S4C), as well as in the AML cell lines THP-1 and MM6 (Online Supplementary Figure S5A). H4 treatment of 293T cells transfected with PKC-EGFP fusion protein led to a rapid translocation (within 3 min) from the cytoplasm to the plasma membrane indicative of direct activation of both conventional PKCα and b, as well as novel e and d isoforms30 (Figure 4D, E and Online Supplementary Video S1-4). Additionally, H4 treatment of FLT3-wild type primary AML-3 cells and the FLT3-ITD AML cell line MOLM-13 led to increased endogenous phospho-PKC activation of both novel and conventional isoforms 30 min after H4 treatment (Figure 4F). Notably, the PKC inhibitor efficiently prevented CD11b upregula- tion by H4 in the FLT3-mutated AML cell line MM6, while the inhibitors of ERK and MEK only had limited effects on H4-induced differentiation. This is in contrast to AML-3 and THP1 where the inhibitors of ERK and MEK efficiently prevented H4-induced differentiation (Online Supplementary Figure S5). Furthermore, the FLT3 inhibitor quizartinib reduced the H4-induced differentiation in both FLT3 wild-type AML-3 cells and THP1 cells while quizar- tinib was toxic to the FLT3-mutated MM6 cell line as expected (Figure 4B and Online Supplementary Figure S5). These data support the concept that, mechanistically, H4 relies on activating PKC to promote leukemic differentia- tion while downstream signaling may be affected differ- ently depending on inter-sample differences. Furthermore, these data underline the robustness of our model as a high throughput platform for mechanistic studies on primary cells.
Combinatorial screening identified a BET inhibitor as an enhancer of H4-induced differentiation
To identify molecules and pathways that can synergize with H4, we analyzed the above combinatorial screen for compounds that enhanced H4-driven CD11b upregulation of primary AML-3 cells (Figure 5A). Among the top candi-
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haematologica | 2021; 106(10)