Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF–induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

Mixed lineage leukemia (MLL) gene translocations are found in ~75 % infant and 10 % adult acute leukemia, showing a poor prognosis. Lysine-specific demethylase 1 (LSD1) has recently been implicated to be a drug target for this subtype of leukemia. More studies using potent LSD1 inhibitors against MLL-rearranged leukemia are needed. LSD1 inhibitors were examined for their biochemical and biological activities against LSD1 and MLL-rearranged leukemia as well as other cancer cells. Potent LSD1 inhibitors with biochemical IC50 values of 9.8–77 nM were found to strongly inhibit proliferation of MLL-rearranged leukemia cells with EC50 of 10–320 nM, while these compounds are generally non-cytotoxic to several other tumor cells. LSD1 inhibition increased histone H3 lysine 4 (H3K4) methylation, downregulated expression of several leukemia-relevant genes, induced apoptosis and differentiation, and inhibited self-renewal of stem-like leukemia cells. Moreover, LSD1 inhibitors worked synergistically with inhibition of DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, against MLL-rearranged leukemia. The most potent LSD1 inhibitor showed significant in vivo activity in a systemic mouse model of MLL-rearranged leukemia without overt toxicities. Mechanistically, LSD1 inhibitors caused significant upregulation of several pathways that promote hematopoietic differentiation and apoptosis. LSD1 is a drug target for MLL-rearranged leukemia, and LSD1 inhibitors are potential therapeutics for the malignancy.

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

Key Points: IL-6 levels in bone marrow predict event-free survival in pediatric AML. Exogenous IL-6 protects AML blasts from chemotherapy-induced apoptosis.

Key Points: Atovaquone induces AML blast apoptosis and prolongs survival in AML xenografts. Atovaquone induces proapoptotic signaling and inhibits the mTOR pathway through upregulation of ATF4 and also suppresses OXPHOS.

Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, α (p92) and β (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3α is deleted in Stat3β and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3α and Stat3β into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3α or GFP-Stat3β revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3α or GFP-Stat3β. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3α, it was >180 min for GFP-Stat3β. Furthermore, although the intranuclear mobility of GFP-Stat3α was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3β in unstimulated cells was slower than that of GFP-Stat3α in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3α and Stat3β have distinct intracellular dynamics, with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3β.