Therapeutic target identification and drug-target validation for acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy characterized by accumulation of immature myeloid cells with aberrant proliferation, differentiation and survival capacity in the patient’s bone marrow (BM) and peripheral blood. Most AML patients are treated with standard combination chemotherapy consisting of cytarabine and an anthracycline, aiming at achieving complete remission (CR, <5% leukemic blasts). Although most AML patients achieve CR after initial treatment, the overall survival is less than 40%. The major cause of treatment failure in AML is intrinsic heterogeneity in treatment response. After therapy, a small population of leukemic cells survives (minimal or measurable residual disease, MRD) and has the capacity to develop into a relapse. Therapy-resistant AML cells might exist prior to drug treatment or might become resistant upon treatment exposure. Relapse-initiating AML cells residing within MRD have stem cell features and are therefore called leukemic stem cells (LSCs). For development of successful treatment strategies that potentially delay or prevent relapse initiation, unraveling the mechanisms that drive (chemo)therapy resistance and leukemia-initiating potential during the disease course and unraveling the mechanisms that drive persistence of AML MRD and LSCs after therapy are crucial. The overall aim of the studies described in this thesis is to identify novel therapeutic targets and to validate these targets as potential effective and safe treatment options for AML patients. In chapter 2 we reviewed the different faces of (chemo)therapy-resistant AML (stem) cell populations, how these mechanisms affect treatment outcome of AML. and we discussed potential strategies to target dynamic treatment resistance and LSCs. In chapter 3 we identified insulin-like growth factor binding protein 7 (IGFBP7) as an efficient therapeutic approach to specifically eliminate AML (stem) cells. Low IGFBP7 is a feature of LSCs and is correlated with reduced chemotherapy sensitivity. In chapter 4 we identified a subpopulation of reversible anthracycline-tolerant leukemia cells (ATCs) and elucidated their transcriptional and epigenetic features. The ATCs lacked upregulation of H3K27 methylation after doxorubicin, and could be eliminated by targeting the H3K27 demethylase KDM6B using the inhibitor GSK-J4. GSK-J4 could also reduce MRD load and eradicated LSCs at diagnosis and after chemotherapy, suggesting that there is a potential for epigenetic-based therapy to eliminate chemotherapy-resistant AML cells. Acute promyelocytic leukemia (APL) is the only curable AML subtype, due to effective application of all-trans retinoic acid (ATRA). In chapter 5 we discussed the current knowledge on molecular, transcription and epigenetic mechanisms and potential biomarkers associated with ATRA susceptibility in APL and non-APL AML. In chapter 6 we identified IGFBP7 as part of ATRA-induced responses in APL cells, and we showed that IGFBP7 has the potential to induce susceptibility for ATRA-driven responses in non-APL AML patients by lowering the transcription factor GFI1. We found that high RARA expression is a biomarker predicting sensitivity for rhIGFBP7-induced activation of ATRA-driven responses in non-APL AML patients. In chapter 7 we studied whether AML (stem) cells could be efficiently targeted using epigenetic drugs directed to proteins involved in the regulation of transcription by chromatin modulation. In AML cells, we characterized the biological activity of two inhibitors targeting both bromodomain and extraterminal domain (BET) proteins and cyclic AMP response binding protein-binding protein (CBP) and its paralogue E1A interacting protein of 300 kDa (p300), NEO1132 and NEO2734. Additionally, we examined whether NEO2734 could serve as novel therapeutic strategy to increase effectiveness of combination chemotherapy in AML. The studies performed in this thesis contribute to improved knowledge on the biological properties of therapy-resistant AML cells. In chapter 8 we further evaluated the potential of the identified novel therapeutic strategies to target AML (stem) cells and to enhance (chemo)therapy efficacy in relation to the improvement of AML prognosis

Escape From Treatment; the Different Faces of Leukemic Stem Cells and Therapy Resistance in Acute Myeloid Leukemia

Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells (minimal residual disease, MRD). Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia "persisters" are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment "persisters" and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs

Reprogramming acute myeloid leukemia into sensitivity for retinoic-acid-driven differentiation

The success of all-trans retinoic acid (ATRA) therapy for acute promyelocytic leukemia (APL) provides a rationale for using retinoic acid (RA)-based therapy for other subtypes of acute myeloid leukemia (AML). Recently, several studies showed that ATRA may drive leukemic cells efficiently into differentiation and/or apoptosis in a subset of AML patients with an NPM1 mutation, a FLT3-ITD, an IDH1 mutation, and patients overexpressing EVI-1. Because not all patients within these molecular subgroups respond to ATRA and clinical trials that tested ATRA response in non-APL AML patients have had disappointing results, the identification of additional biomarkers may help to identify patients who strongly respond to ATRA-based therapy. Searching for response biomarkers might also reveal novel RA-based combination therapies with an efficient differentiation/apoptosis-inducing effect in non-APL AML patients. Preliminary studies suggest that the epigenetic or transcriptional state of leukemia cells determines their susceptibility to ATRA. We hypothesize that reprogramming by inhibitors of epigenetic-modifying enzymes or by modulation of microRNA expression might sensitize non-APL AML cells for RA-based therapy. AML relapse is caused by a subpopulation of leukemia cells, named leukemic stem cells (LSCs), which are in a different epigenetic state than the total bulk of the AML. The survival of LSCs after therapy is the main cause of the poor prognosis of AML patients, and novel differentiation therapies should drive these LSCs into maturity. In this review, we summarize the current knowledge on the epigenetic aspects of susceptibility to RA-induced differentiation in APL and non-APL AML

Cellular Assay to Study β-Arrestin Recruitment by the Cannabinoid Receptors 1 and 2

Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here

The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

Acute myeloid leukemia (AML) is a disease characterized by transcriptional dysregulation that results in a block in differentiation and aberrant self-renewal. Inhibitors directed to epigenetic modifiers, aiming at transcriptional reprogramming of AML cells, are currently in clinical trials for AML patients. Several of these inhibitors target bromodomain and extraterminal domain (BET) proteins, cyclic AMP response binding protein-binding protein (CBP), and the E1A-interacting protein of 300 kDa (p300), affecting histone acetylation. Unfortunately, single epigenetic inhibitors showed limited efficacy due to appearance of resistance and lack of effective eradication of leukemic stem cells. Here, we describe the efficacy of 2 novel, orally available inhibitors targeting both the BET and CBP/p300 proteins, NEO1132 and NEO2734, in primary AML. NEO2734 and NEO1132 efficiently reduced the viability of AML cell lines and primary AML cells by inducing apoptosis. Importantly, both NEO drugs eliminated leukemic stem/progenitor cells from AML patient samples, and NEO2734 increased the effectiveness of combination chemotherapy treatment in an in vivo AML patient-derived mouse model. Thus, dual inhibition of BET and CBP/p300 using NEO2734 is a promising therapeutic strategy for AML patients, making it a focus for clinical translation

Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38− cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9–218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36–99%) and 71% (95% CI: 58–81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker

Structural basis of ligand interaction with atypical chemokine receptor 3

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor

Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38− cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9–218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36–99%) and 71% (95% CI: 58–81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker

Structural basis of ligand interaction with atypical chemokine receptor 3.

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor

Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells.Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016).To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination.In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome.Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson &amp; Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.↵* Asterisk with author names denotes non-ASH members