Abstract
In pediatric acute myeloid leukemia (AML), fusions involving lysine methyltransferase 2A (KMT2A) are considered hallmarks of aggressive AML, for whom the development of targeted specific therapeutic agents to ameliorate classic chemotherapy and obtain a complete eradication of disease is urgent. In this study, we investigated the antiapoptotic proteins in a cohort of 66 pediatric AML patients, finding that 75% of the KMT2A-r are distributed in Q3 + Q4 quartiles of BCL-2 expression, and KMT2A-r have statistically significant high levels of BCL-2, phospho-BCL-2 S70, and MCL-1, indicating a high anti-apoptotic pathway activation. In an attempt to target it, we tested novel drug combinations of venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, in KMT2A-MLLT3, for being the most recurrent, and KMT2A-AFDN, for mediating the worst prognosis, rearranged AML cell lines. Our screening revealed that both the bromodomain and extra-terminal domain (BET) inhibitor, I-BET151, and kinase inhibitor, sunitinib, decreased the BCL-2 family protein expression and significantly synergized with venetoclax, enhancing KMT2A-r AML cell line death. Blasts t (6; 11) KMT2A-AFDN rearranged, both from cell lines and primary samples, were shown to be significantly highly responsive to the combination of venetoclax and thioridazine, with the synergy being induced by a dramatic increase of mitochondrial depolarization that triggered blast apoptosis. Finally, the efficacy of novel combined drug treatments was confirmed in KMT2A-r AML cell lines or ex vivo primary KMT2A-r AML samples cultured in a three-dimensional system which mimics the bone marrow niche. Overall, this study identified that, by high-throughput screening, the most KMT2A-selective drugs converged in different but all mitochondrial apoptotic network activation, supporting the use of venetoclax in this AML setting. The novel drug combinations here unveiled provide a rationale for evaluating these combinations in preclinical studies to accelerate the introduction of targeted therapies for the life-threatening KMT2A-AML subgroup of pediatric AML.
Highlights
Leukemia is the most common type of cancer in childhood, accounting for 25–30% of cancers in children and adolescents aged 0–18 years (Kuhlen et al, 2019).Despite the refinement of risk stratification on the basis of clinical characteristics, molecular profiling, and detection of minimal residual disease after induction therapy, to date the overall survival for children with acute myeloid leukemia (AML) has not exceeded 70% (Pui et al, 2011; Pession et al, 2013; Elgarten and Aplenc, 2020)
The 11q23-rearranged AML subgroup represents more than 20% of pediatric cases, with >80 recognized fusion partners, the majority of leukemias result from KMT2A fusions with one of about six common partner genes, with KMT2A-MLLT3 being the most recurrent, and the prognostic significance is strictly dependent on the fusion partner, with the KMT2A-AFDN fusion associated with a poor prognosis (Balgobind et al, 2009; Coenen et al, 2011; Pigazzi et al, 2011; Manara et al, 2014a; Meyer et al, 2018)
We found that KMT2A-rearranged AML patients were prevalent in the higher quartiles Q3 + Q4 with respect to the lower Q1 and Q2 (KMT2A-r cases: Q3 + Q4, n = 12, 75% vs. Q1 + Q2, n = 4, 25%; Figure 1A, Supplementary Table S1)
Summary
Leukemia is the most common type of cancer in childhood, accounting for 25–30% of cancers in children and adolescents aged 0–18 years (Kuhlen et al, 2019).Despite the refinement of risk stratification on the basis of clinical characteristics, molecular profiling, and detection of minimal residual disease after induction therapy, to date the overall survival for children with acute myeloid leukemia (AML) has not exceeded 70% (Pui et al, 2011; Pession et al, 2013; Elgarten and Aplenc, 2020). The 11q23-rearranged AML subgroup represents more than 20% of pediatric cases, with >80 recognized fusion partners, the majority of leukemias result from KMT2A fusions with one of about six common partner genes, with KMT2A-MLLT3 being the most recurrent, and the prognostic significance is strictly dependent on the fusion partner, with the KMT2A-AFDN fusion associated with a poor prognosis (Balgobind et al, 2009; Coenen et al, 2011; Pigazzi et al, 2011; Manara et al, 2014a; Meyer et al, 2018) This ever-expanding knowledge on leukemia biology is crucial to identify those molecular targets useful for the development of therapeutic agents to ameliorate classic chemotherapy and obtain complete eradication of the disease while reducing toxicity when possible (Mercher and Schwaller, 2019). While lymphoid malignancies nearly universally overexpress BCL-2 (Stilgenbauer et al, 2016; Ashkenazi et al, 2017), in myeloid leukemias, BCL-2 expression is heterogeneous and not always upregulated, with relapses showing higher percentages of positive expression than those seen at leukemia onset, suggesting that BCL-2-expressing blasts might be those escaping apoptosis in first-line treatments (Bensi et al, 1995; Testa and Riccioni, 2007; Kuusanmäki et al, 2020)
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