Michael L. Cleary

A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR⁺/E2A-PBX1⁺ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR⁺/E2A-PBX1⁺ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR⁺/E2A-PBX1⁺ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

Acute myeloid leukemia (AML) with mixed-lineage leukemia (MLL) gene rearrangement (MLLr) comprises a cellular hierarchy in which a subpopulation of cells serves as functional leukemia stem cells (LSCs). They are maintained by a unique gene expression program and chromatin states, which are thought to reflect the actions of enhancers. Here, we delineate the active enhancer landscape and observe pervasive enhancer malfunction in LSCs. Reconstruction of regulatory networks revealed a master set of hematopoietic transcription factors. We show that EP300 is an essential transcriptional coregulator for maintaining LSC oncogenic potential because it controls essential gene expression through modulation of H3K27 acetylation and assessments of transcription factor dependencies. Moreover, the EP300 inhibitor A-485 affects LSC growth by targeting enhancer activity via histone acetyltransferase domain inhibition. Together, these data implicate a perturbed MLLr-specific enhancer accessibility landscape, suggesting the possibility for disruption of the LSC enhancer regulatory axis as a promising therapeutic strategy in AML.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. While frontline chemotherapy regimens are generally very effective, the prognosis for patients whose leukemia returns remains poor. The presence of measurable residual disease (MRD) in bone marrow at the completion of induction therapy is the strongest predictor of relapse, suggesting that strategies to eliminate the residual leukemic blasts from this niche could reduce the incidence of recurrence. We have previously reported that toll-like receptor (TLR) agonists achieve durable T cell-mediated protection in transplantable cell line-based models of B cell precursor leukemia (B-ALL). However, the successful application of TLR agonist therapy in an MRD setting would require the induction of anti-leukemic immune activity specifically in the bone marrow, a site of the chemotherapy-resistant leukemic blasts. In this study, we compare the organ-specific depletion of human and mouse primary B-ALL cells after systemic administration of endosomal TLR agonists. Despite comparable splenic responses, only the TLR9 agonist induced strong innate immune responses in the bone marrow and achieved a near-complete elimination of B-ALL cells. This pattern of response was associated with the most significantly prolonged disease-free survival. Overall, our findings identify innate immune activity in the bone marrow that is associated with durable TLR-induced protection against B-ALL outgrowth.

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.

The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL. Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia. Graphical http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg. Cancer Res; 78(22); 6497–508. ©2018 AACR.

Key Points: Genome editing induces t(9;11) chromosomal translocations and transforms primary CD34+ human cord blood cells leading to acute leukemia. CD9 is upregulated in primary t(9;11) cells and is a useful marker for enrichment of genome-edited MLL-rearranged cells in vitro.

There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%–7% of pediatric and 50% of pre-B-cell receptor (preBCR⁺) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR⁺/E2A-PBX1⁺ leukemias in vitro and in vivo. Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937–49. ©2016 AACR.