FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

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.

Location, location, location: A mini-review of CEBPA variants in patients with acute myeloid leukemia.

CEBPA variants are frequently recurring in acute myeloid leukemia (AML). The prognostic significance of CEBPA mutations has recently undergone a major shift in the 5th edition of WHO classification of hematological neoplasms and ELN 2022 classification. Whereas prior iterations did not specify the type of CEBPA mutation, the updated schema specify that only mutations localized to the C-terminal basic zipper (bZIP) domain are considered prognostically favorable. This change is based primarily on three recently published large datasets evaluating the prognostic significance of mutation location in CEBPA mutant AML. Here, we review the evolution of the prognostic classification of CEBPA variants.

LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway.

Despite recent therapeutic advances, the 5-year survival rate for adults with acute myeloid leukemia (AML) is poor and standard-of-care chemotherapy is associated with significant toxicity, highlighting the need for new therapeutic approaches. Recent work from our group and others established that the G protein-coupled estrogen receptor (GPER) is tumor suppressive in melanoma and other solid tumors. We performed a preliminary screen of human cancer cell lines from multiple malignancies and found that LNS8801, a synthetic pharmacologic agonist of GPER currently in early phase clinical trials, promoted apoptosis in human AML cells. Using human AML cell lines and primary cells, we show that LNS8801 inhibits human AML in preclinical in vitro models, while not affecting normal mononuclear cells. Although GPER is broadly expressed in normal and malignant myeloid cells, this cancer-specific LNS8801-induced inhibition appeared to be independent of GPER signaling. LNS8801 induced AML cell death primarily through a caspase-dependent apoptosis pathway. This was independent of secreted classical death receptor ligands, and instead required induction of reactive oxygen species (ROS) and activation of endoplasmic reticulum (ER) stress response pathways including IRE1α. These studies demonstrate a novel activity of LNS8801 in AML cells and show that targeting ER stress with LNS8801 may be a useful therapeutic approach for AML. Significance: Previous work demonstrated that LNS8801 inhibits cancer via GPER activation, especially in solid tumors. Here we show that LNS8801 inhibits AML via GPER-independent mechanisms that include ROS induction and ER activation.

Evaluation of hypereosinophilia in a case of FLT3-mutant acute myeloid leukemia treated with gilteritinib.

Acute myeloid leukemias (AMLs) frequently harbor activating mutations in Fms-like tyrosine kinase 3 (FLT3). The use of FLT3 inhibitors (FLT3i) is the standard of care for treatment of newly diagnosed and relapsed patients with AML. Differentiation responses including clinical differentiation syndrome have been previously reported with FLT3i when used as single agents in relapsed disease. We present a case of hypereosinophilia in a patient on FLT3i therapy with persistent FLT3 polymerase chain reaction (PCR) positivity in peripheral blood. We sorted mature leukocytes by lineage to determine if the eosinophils were leukemia-derived. FLT3 PCR and next-generation sequencing analysis demonstrated monocytic differentiation of the FLT3-ITD leukemic clone with reactive hypereosinophilia that was derived from a preleukemic SF3B1, FLT3 wild-type clone. Our case is the first to definitively demonstrate the emergence of clonal FLT3-ITD monocytes with FLT3i and the first to demonstrate a differentiation response following decitabine, venetoclax, and gilteritinib triplet therapy.

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

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.

Hematopoietic cytokines mediate resistance to targeted therapy in FLT3-ITD acute myeloid leukemia.

Activating mutations in Fms-like tyrosine kinase 3 (FLT3) occur in ∼30% of adult cases of acute myeloid leukemia (AML). Selective second- and third-generation FLT3 inhibitors have shown significant clinical activity in patients with relapsed FLT3-mutant AML. However, clearance of FLT3-mutant clones does not consistently occur, and disease will progress in most patients after an initial response. This scenario challenges the model of FLT3-mutant AML being oncogene addicted, and it suggests that redundant signaling pathways regulate AML cell survival after FLT3 inhibition. We show that primary FLT3-mutant AML cells escape apoptosis induced by FLT3 inhibition in vitro in the presence of cytokines produced normally in the bone marrow, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). Despite reactivating canonical FLT3-signaling pathways, GM-CSF and IL-3 maintain cell survival without rescuing proliferation. Cytokine-mediated resistance through GM-CSF and IL-3 is dependent on JAK kinase, STAT5, and proviral integration site of Moloney murine leukemia virus (PIM) but not MAPK or mammalian target of rapamycin signaling. Cotreatment with FLT3 inhibitors and inhibitors of JAK or PIM kinases blocks GM-CSF and IL-3 rescue of cell survival in vitro and in vivo. Altogether, these data provide a strong rationale for combination therapy with FLT3 inhibitors to potentially improve clinical responses in AML.

Minimal Residual Disease in Acute Myeloid Leukemia.

OPINION STATEMENT: New technology and improved understanding of the pathogenesis of acute leukemias have allowed for sensitive detection of minimal residual disease (MRD). Despite many years of research demonstrating the prognostic value of MRD, there is no standard of care for measurement of MRD in acute myeloid leukemia. The techniques for assessment are continuing to improve at a rapid pace; however, the benefit of risk-adapted approaches for MRD positive disease remains a major question. This review focuses on recent methodological advances for MRD detection, the role of MRD in prognostication, and current application of the available evidence in guiding therapy decisions.

Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho-K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL-dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP3 regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.

Cytosolic Ras supports eye development in Drosophila.

Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development.

Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis.

K-Ras is a member of a family of proteins that associate with the plasma membrane by virtue of a lipid modification that inserts into the membrane and a polybasic region that associates with the anionic head groups of inner leaflet phospholipids. In the case of K-Ras, the lipid is a C-terminal farnesyl isoprenoid adjacent to a polylysine sequence. The affinity of K-Ras for the plasma membrane can be modulated by diminishing the net charge of the polybasic region. Among the ways this can be accomplished is phosphorylation by protein kinase C (PKC) of serine 181 within the polybasic region. Phosphorylation at this site regulates a farnesyl-electrostatic switch that controls association of K-Ras with the plasma membrane. Surprisingly, engagement of the farnesyl-electrostatic switch promotes apoptosis. This chapter describes methods for directly analyzing the phosphorylation status of K-Ras using metabolic labeling with (32)P, for indirectly assessing the farnesyl-electrostatic switch by following GFP-tagged K-Ras in live cells, for artificially activating the farnesyl-electrostatic switch by directing the kinase domain of a PKC to activated K-Ras using a Ras-binding domain, and for assessing apoptosis of individual cells using a YFP-tagged caspase 3 biosensor.