P435: novel compounds to target kmt2a-rearranged pediatric acute myeloid leukemia

Background: In pediatric acute myeloid leukemia (AML), fusions involving lysine methyltransferase 2A (KMT2A) are considered hallmarks of aggressive AML, representing a challenge for the development of targeted specific therapeutic agents to ameliorate classic chemotherapy and obtain a complete eradication of disease. Among them, t(9;11)KMT2A::MLLT3 represents the most recurrent rearrangement, whereas t(6;11)KMT2A::AFDN-r is associated with the a dismal prognosis below 25%. We previously described KMT2A-AFDN mediating a peculiar gene expression, the hyper activation of MAPK signaling and marked adhesion properties. Through a high-throughput drug screening (HTS) we identified thioridazine (TDZ) as an efficacious compound, but its use is limited by the huge blood brain barriers crossing and cardiotoxic effects. Aims: We generated a novel compound to treat KMT2A-AFDN-r AML to find promising therapeutic strategies for KMT2A-r AML. Methods: We investigated the levels of BCL-2, phospho-BCL-2 S70 and MCL-1 in a cohort of 66 pediatric AML patients previously analyzed by Reverse Phase Protein Array. We selected KMT2A-specific drugs resulted from a previously performed HTS. To overcome cytotoxic effects of TDZ, we synthesized an analogous namely TDZ6 by chemically modifying the molecule structure to increase branching and polarity. We validated novel drug combinations in AML cell lines and ex vivo primary cells in a three-dimensional (3D) scaffold seeded with mesenchymal stromal cells mimicking the bone marrow niche and in vivo. Results: We found that 75% of the KMT2A-r are distributed in Q3+Q4 quartiles of BCL-2 expression, with high levels of phospho-BCL-2 S70 and MCL-1 as well, indicating a high anti-apoptotic pathway activation. In attempt to target it, we tested novel drug combinations of venetoclax (VEN), a BCL- 2 inhibitor, with KMT2A-specific drugs, revealing that the Bromodomain and Extra-Terminal Domain (BET) inhibitor I-BET151, and the kinase inhibitor sunitinib, decreased BCL-2 family protein expression and synergized with VEN enhancing KMT2A-r AML cell death, also in the 3D model. Regarding KMT2A-AFDN, we validated that TDZ6 maintained the specific cytotoxic activity on AML cell lines and on ex-vivo PDX cells, with IC50 <10µM. The combination of TDZ6 and VEN, both acting on mitochondria, resulted synergistic through a strong increase of mitochondrial depolarization that triggered blasts apoptosis (ZIP-synergy score 22.2; Combination Index 0.83), confirmed in the 3D model. In vivo, TDZ6 resulted efficacious on NSG mice with KMT2A-AFDN-AML cell line flank injection (8mg/kg i.p.), whereas it was not confirmed in a pilot study on one AML-PDX. Thus, we increased TDZ dose, showing that AML decreased at 70 mg/kg i.p. (46% of leukemia reduction) as maximum tolerated dose. We documented that this latter dose was devoid of cardiotoxic effects, as revealed by QTc measurement during electrocardiogram, whereas TDZ resulted cardiotoxic at 12 mg/kg (p<0.00006, QTc ctr: 77.9msec, TDZ6: 75.7msec, TDZ: 93.7msec). Summary/Conclusion: Overall, this study identified novel drug combinations of KMT2A-selective drugs together with VEN converging in different but all mitochondrial apoptotic networks activation, supporting the use of VEN in this AML setting. We demonstrate that TDZ6 reduces the AML growth in vitro and an enlarged in vivo study will support its efficacy and safety. This study highlights the importance of having a bona-fide model to test drugs and produce reliable results, to accelerate the introduction of targeted therapies for the life-threatening KMT2A-AML subgroup of pediatric AML. Keywords: Venetoclax, KMT2A, Acute myeloid leukemia, BCL2