Interrogating bromodomain inhibitor resistance in KMT2A-rearranged leukemia through combinatorial CRISPR screens.

Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.

The Combination of Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Delays KMT2A-Rearranged Leukemia Progression.

Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.

Only Hematopoietic Stem and Progenitor Cells from Cord Blood Are Susceptible to Malignant Transformation by MLL-AF4 Translocations.

Mixed lineage leukemia (MLL) (KMT2A) rearrangements (KMT2Ar) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear a MLL translocation and half of them are t(4;11), resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are poorly understood. Recently, we established an efficient CRISPR/Cas9-based KMT2Ar model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) and faithfully mimicked the underlying biology of the disease. Here, we applied this model to HSPCs from adult bone marrow (huBM) to investigate the impact of the cell of origin and fusion partner on disease development. Both genome-edited infant and adult KMT2Ar cells showed monoclonal outgrowth with an immature morphology, myelomonocytic phenotype and elevated KMT2Ar target gene expression comparable to patient cells. Strikingly, all KMT2Ar cells presented with indefinite growth potential except for MLL-AF4 huBM cells ceasing proliferation after 80 days. We uncovered FFAR2, an epigenetic tumor suppressor, as potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions.