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

Activated intrinsic apoptosis pathway is a key related prognostic parameter in acute myeloid leukemia

Purpose: By parallel assessment of multiple apoptosis-related transcripts, we aimed to refine the current concept of apoptosis resistance in acute myeloid leukemia (AML) and identify the combination of genes best predicting overall survival (OS). Patients and Methods: The reverse transcriptase multiplex ligation-dependent probe amplification technique was used for simultaneous quantification of 31 apoptosis-related transcripts in viable (7AAD -/AnnexinV-) blasts (CD45dim) from bone marrow aspirates of 120 newly diagnosed AML patients. By forward selection, a prognosis-predicting gene expression profile was constructed. The predictive validity of this profile was assessed by cross validation. Results: High transcript levels were associated with poor OS for seven of 31 genes, three of which were proapoptotic. The average expression of all 12 antiapoptotic genes was associated with poor OS (P = .029). A similar association with poor OS was found for the average expression of all 19 proapoptotic genes (P = .009). Forward selection and cross validation revealed the antiapoptotic gene BIRC3 and the proapoptotic genes BAX-(I) and BMF to optimally predict OS. Three equally sized patient groups, constructed by ranking the cross-validated prognoses of the patients, were clearly distinct (median OS times were 8.2, 16.7, and 85.6 months). Conclusion: High expression of both pro- and antiapoptotic genes predicted poor OS, which postulates a mechanism of activation of the apoptosis pathway as a whole. This mechanism, which culminates in a three-gene expression signature, allows accurate clinical outcome prediction in AML and puts efforts to target single antiapoptosis genes in a new perspective

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

Quantitative molecular detection of minimal residual head and neck cancer in lymph node aspirates

Purpose: Staging of the clinically N0 neck in patients with head and neck squamous cell carcinoma (HNSCC) using ultrasound-guided, fine needle aspiration cytology (USgFNAC) has a false-negative rate of ∼ 20% that might be caused by inaccurate cytology. Molecular analysis of aspirate residues might reduce the false-negative rate, and we therefore set up a quantitative reverse transcription-PCR (Q-RT-PCR) assay based on TaqMan technology using the squamous cell-specific antigen E48 (Ly-6D) as molecular marker. Experimental design: The detection limit of the assay was determined in reconstruction experiments. The sensitivity of the assay was tested on cytological tumor-positive aspirate residues and the specificity on lymph node aspirate residues of noncancer controls. Subsequently, 235 lymph node aspirate residues of 64 HNSCC patients staged with USgFNAC were examined for the presence of E48 mRNA. E48 Q-RT-PCR results of the aspirated lymph nodes were compared with cytology and clinical outcome. Results: The detection limit of E48 Q-RT-PCR was a single tumor cell in a background of 106 peripheral blood mononuclear cells. From the 41 aspirates that were not evaluable at cytology, 24 (59%) could be diagnosed with E48 Q-RT-PCR. In the 191 aspirates that were tumor negative or not evaluable at cytology, 8 samples from 6 patients were E48 positive. These results were confirmed by histology or clinical outcome in 3 of 6 patients. E48 Q-RT-PCR showed an increase in sensitivity from 56 to 67% and an increase in frequency of reached diagnosis from 97 to 100% compared with cytology. The specificity decreased from 100 to 92%. Conclusions: Real-time E48 Q-RT-PCR is an accurate technique for squamous cell detection in lymph node aspirates of HNSCC patients. The assay shows an increase in sensitivity and frequency of reached diagnosis compared with cytology. The test could be implemented routinely in USgFNAC to diagnose cases for which cytological examination is not conclusive

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 & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.↵* Asterisk with author names denotes non-ASH members