Acute myeloid leukemia (AML) is a fatal blood cancer with cytotoxic chemotherapy offering at best 25% 5-year survival. While targeted BCL2 and FLT3 inhibitors venetoclax and gilteritinib are used upfront in the treatment of a subset of adult patients with AML and help to extend the survival of some patients, a curative treatment combination with minimal side effects has yet to be discovered. We find that use of the dual histone acetyltransferase p300/CBP bromodomain inhibitor CCS1477 (inobrodib), together with venetoclax and gilteritinib, virtually eliminates leukemia stem cells in an aggressive preclinical model of DNMT3A/FLT3-mutant AML by impairing pro-oncogenic survival and proliferation factors to effectively block leukemogenesis. This work identifies potential clinical utility of a targeted, triplet combination therapy for treatment of AML.

Abstract: The consequences of activated innate immune signaling in acute myeloid leukemia (AML) is not well understood. Using ligands directed at the toll-like family receptors (TLR) in models of high-risk AML, we uncover that TLR2 ligands exert unique antileukemic effects that are distinct from other TLRs. Although TLR2 signaling broadly induces inflammatory gene expression in AML cells, at the single-cell level, cell-type–dependent, divergent transcriptional responses coordinate cellular outputs of proliferation, differentiation, cell death, and activation of immune cell function. TLR2 ligands were the only TLR agonists capable of extending survival of AML-bearing mice through leukemia stem cell (LSC) reprogramming that elevated major histocompatibility complex (MHC) class II surface expression and ultimately impaired self-renewal. We find that the coexpression of TLR2 and MHCII genes is associated with better overall survival in patients with AML, which is consistent with our observations of activated TLR2 signaling in mice. These data reveal functional TLR2 signaling critically antagonizes leukemogenesis and emphasizes a role for TLR2 agonism in AML.

Cancer evolution is a multifaceted process leading to dysregulation of cellular expansion and differentiation through somatic mutations and epigenetic dysfunction. Clonal expansion and evolution is driven by cell-intrinsic and -extrinsic selective pressures, which can be captured with increasing resolution by single-cell and bulk DNA sequencing. Despite the extensive genomic alterations revealed in profiling studies, there remain limited experimental systems to model and perturb evolutionary processes. Here, we integrate multi-recombinase tools for reversible, sequential mutagenesis from premalignancy to leukemia. We demonstrate that inducible Flt3 mutations differentially cooperate with Dnmt3a, Idh2, and Npm1 mutant alleles, and that changing the order of mutations influences cellular and transcriptional landscapes. We next use a generalizable, reversible approach to demonstrate that mutation reversion results in rapid leukemic regression with distinct differentiation patterns depending upon co-occurring mutations. These studies provide a path to experimentally model sequential mutagenesis, investigate mechanisms of transformation and probe oncogenic dependency in disease evolution.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 ( FLT3-ITD ) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3 - ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Aberrant inflammatory signaling is a hallmark of myeloid malignancies and is generally thought to promote disease. Few studies have systematically dissected the molecular and cellular mechanisms of pathogen-induced inflammation in myeloid disease settings. Using our previously established immune-competent mouse model of DNTM3A/FLT3-mutant acute myeloid leukemia (FD AML), we screened a panel of synthetic Toll-like receptor (TLR) agonists to mimic pathogen-specific responses. Activation of TLR2 heterodimers, but not other TLRs, significantly extended overall survival of leukemia bearing mice. Moreover, TLR2 is the highest expressed TLR in AML patients regardless of mutation profile. These data led us to explore the impact of TLR2 mediated inflammatory signaling on AML disease processes. To understand AML-cell intrinsic versus extrinsic effects, we conducted a series of reciprocal syngeneic transplants using FD AML and Tlr2-/-FD AML into wild-type (WT) or Tlr2-/- recipients. Note, Tlr2 deletion does not significantly alter the development or progression of FD AML. After AML establishment, mice were treated with a single dose of Tlr1/2 specific agonist, Pam3CSK4. This revealed Tlr2 expression on AML cells is essential for significantly prolonged survival with Pam3CSK4 treatment. Given that TLRs have direct and indirect signaling with functional phenotypic consequences, we employed single-cell RNA sequencing coupled with cell surface proteins (CITE-seq) to unbiasedly investigate the pleiotropic effects of Tlr2 activation. We identified 21 clusters that recapitulate the hierarchical nature of FD AML. Pam3CSK4 significantly decreased the fraction of AML cells in stem/progenitor clusters, expanded AML-derived monocyte precursor clusters, and gave rise to a new AML-derived macrophage cluster. There were also decreased AML-derived mature neutrophil clusters despite expansion of neutrophil precursor clusters. We validated these AML cell states using the reciprocal Tlr2 proficient/deficient transplantation models. Our data indicate that Tlr2 activation drove effector macrophages (CD11b+F4/80+ MHCII+ CD80+/86+) with elevated phagocytic activity from AML. In contrast, Pam3CSK4 blocked maturation of AML-derived neutrophils in an AML-non-autonomous manner. Cytokine analyses showed G-CSF production downstream of Tlr2 activation in the microenvironment prevented the cell intrinsic Tlr2-mediated neutrophil maturation of AML cells. By CITE-seq, Cepbewas significantly reduced in AML-derived neutrophil precursors, indicating the block by G-CSF may be happening at a precursor stage. Indeed, FD AML co-treated with anti-G-CSF neutralizing antibodies and Pam3CSK4 restored AML-derived neutrophil differentiation, but at the expense of monocyte differentiation, through a bipotential intermediate. Lastly, to understand whether Tlr2 activation on AML cells causes a transient reduction in stem/progenitor numbers or a sustained functional impairment of AML stem cell activity, we transplanted limiting numbers of FD AML stem/progenitor cells from Pam3CSK4 and vehicle treated mice into WT recipients. We observed a 5-fold decrease in leukemia initiating cell activity with Pam3CSK4 treated donors. In summary, we discovered Tlr2 activation induces pleiotropic effects on a variety of cell types in AML, ultimately impacting overall survival. Our results shed light on the functional consequences of pathogen recognition by AML cells that may be leveraged for novel treatments of this deadly disease.

Clinical mutation data on primary human AML samples.

DMR in DNMT3A rescued c-Kit+ Flt3ITD/ITD;Dnmt3afl/fl MxCre cells versus c-Kit+ Flt3ITD/ITD;Dnmt3afl/fl MxCre empty vector control cells.

ICGS HOPACH gene expression clustering results performed on RNA-Seq data from 96 single Flt3ITD/ITD;Dnmt3afl/fl MxCre AML cells.

Supplementary Table from miR-196b–TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML

Frequencies of monocyte and myelomonocytic populations in the bone marrow and spleen of Flt3ITD/ITD;Dnmt3afl/fl MxCre mice and age-matched Flt3ITD/ITD;Dnmt3afl/fl and wild-type control mice by flow cytometric analyses.

Supplementary Figure S1. Dnmt3a haploinsufficiency transforms Flt3ITD MPN into an AML with clonogenic c-Kit+ leukemic stem and progenitors in the spleen. Supplementary Figure S2. Comparative patterns of differential global DNA methylation in DNMT3A-mutant human and murine AML. Supplementary Figure S3. Hematopoietic stem cells/progenitor and c-Myc signatures are upregulated in murine AML. Supplementary Figure S4. DNMT3A rescued murine Flt3ITD/ITD;Dnmt3afl/fl MxCre AML hypermethylated regions correspond to HSPC-like populations.

In this issue of Blood, Li et al 1 report age and co-mutation contexts wherein FLT3 internal tandem duplication (ITD) orchestrates unique transcriptional and epigenetic programs to deliver distinct functional outputs from myeloid progenitor cells.

Supplementary Data from KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs

Background: Myeloproliferative neoplasms (MPN) are hematologic malignancies that present with excessive production of mature myeloid blood cells. MPN are characterized by somatic mutations in JAK2, CALR or MPL, which result in constitutive activation of JAK2 signaling including STAT3/5, MAPK and PI3K/Akt pathways. JAK2 inhibitors like ruxolitinib effectively inhibit the JAK-STAT axis, but signaling via the MAPK pathway remains activated in vivo, thus limiting therapeutic effects. SHP2, Gab1 and Grb2 have been implicated in the signal transduction from JAK2 to the MAPK pathway, but the molecular connection is not fully clarified. Due to its important function in hematopoiesis, SHP2 is of special interest in an MPN setting. Aims: We study the role of the protein tyrosine phosphatase SHP2 in activating the MAPK pathway in MPN and evaluate its translational potential as a therapeutic target. Methods: SHP2 was depleted by shRNA in Ba/F3 cells stably expressing Jak2V617F or wildtype Jak2 along with EPOR. Pharmacologic targeting by SHP2 inhibitors TNO155 or IACS-13909 as well as dual SHP2/JAK2 targeting was evaluated in MPN cell lines and primary MPN patient cells. Corrective effects were characterized in vivo by targeting SHP2 in a Jak2V617F mutant transgenic MPN mouse model presenting with a polycythemia vera phenotype and in a MPLW515L mutant retroviral transplant mouse model with a myelofibrosis phenotype. Results: SHP2 was expressed at substantial mRNA and protein levels in MPN cell lines along with MAPK pathway activation. shRNA-induced SHP2 depletion reduced activation of MAPK pathway kinases including ERK1/2 and RSK as well as expression of MAPK downstream effectors such as DUSP6. Pharmacologic SHP2 inhibition with TNO155 or IACS-13909 was similarly able to interfere with MAPK pathway activation as shown by reduced pERK1/2 and pRSK. Of note, signaling effects were most pronounced after combined JAK2/SHP2 inhibitor exposure (A). Both SHP2 knock-down and pharmacologic targeting sensitized MPN cells to JAK2 inhibition with ruxolitinib, as reflected by inhibited proliferation at significantly lower IC50 compared to ruxolitinib as single agent (B). More moderate inhibition of proliferation was observed in Ba/F3 cells expressing wild-type JAK2 (C). In a Jak2V617F mutant MPN mouse model, SHP2 inhibition by TNO155 promptly mediated corrective effects on the MPN phenotype including splenomegaly, erythrocytosis and leukocytosis. Of note, TNO155 as single agent showed similar effects as ruxolitinib, while combined JAK2/SHP2 inhibition enhanced efficacy and was tolerable (D). In a MPLW515L mutant MPN mouse model with extensive splenomegaly and leukocytosis, combined JAK2/SHP2 inhibition promptly normalized leukocyte counts, which is not seen to this extent with ruxolitinib (E). Similarly, MPN infiltration of the spleen and extramedullary hematopoiesis in the liver was markedly reduced after one week of combination treatment. Importantly, myelo-erythroid colony formation from primary MPN patient cells was more effectively suppressed by combined JAK2/SHP2 inhibition as compared to JAK2 inhibitor single agent exposure. Summary/Conclusion: Our findings suggest a relevant role of SHP2 in MPN given enhanced MAPK pathway suppression and corrective effects when SHP2 is targeted in MPN cells, mouse models and primary patient isolates. Further studies will delineate the involvement of SHP2 phosphatase vs. non-phosphatase functions and detail the potential of JAK2/SHP2 inhibition as therapeutic approach in MPN.Keywords: SHP-2, Ruxolitinib, Myeloid malignancies

Peripheral blood findings in moribund Flt3ITD/ITD;Dnmt3afl/fl MxCre mice and age-matched Flt3ITD/ITD;Dnmt3afl/fl and wild-type control mice.

Abstract Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. Significance: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587