RUNX2 regulates leukemic cell metabolism and chemotaxis in high-risk T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

Drug repurposing for targeting cyclic nucleotide transporters in acute leukemias - A missed opportunity.

While current treatment regimens for acute leukemia can dramatically improve patient survival, there remains room for improvement. Due to its roles in cell differentiation, cell survival, and apoptotic signaling, modulation of the cyclic AMP (cAMP) pathway has provided a meaningful target in hematological malignancies. Several studies have demonstrated that gene expression profiles associated with increased pro-survival cAMP activity or downregulation of various pro-apoptotic factors associated with the cAMP pathway are apparent in acute leukemia patients. Previous work to increase leukemia cell intracellular cAMP focused on the use of cAMP analogs, stimulating cAMP production via transmembrane-associated adenylyl cyclases, or decreasing cAMP degradation by inhibiting phosphodiesterase activity. However, targeting cyclic nucleotide efflux by ATP-binding cassette (ABC) transporters represents an unexplored approach for modulation of intracellular cyclic nucleotide levels. Preliminary studies have shown that inhibition of cAMP efflux can stimulate leukemia cell differentiation, cell growth arrest, and apoptosis, indicating that targeting cAMP efflux may show promise for future therapeutic development. Furthermore, inhibition of cyclic nucleotide transporter activity may also contribute multiple anticancer benefits by reducing extracellular pro-survival signaling in malignant cells. Hence, several opportunities for drug repurposing may exist for targeting cyclic nucleotide transporters.

Clioquinol: To harm or heal.

Clioquinol, one of the first mass-produced drugs, was considered safe and efficacious for many years. It was used as an antifungal and an antiprotozoal drug until it was linked to an outbreak of subacute myelo-optic neuropathy (SMON), a debilitating disease almost exclusively confined to Japan. Today, new information regarding clioquinol targets and its mechanism of action, as well as genetic variation (SNPs) in efflux transporters in the Japanese population, provide a unique interpretation of the existing phenomena. Further understanding of clioquinol's role in the inhibition of cAMP efflux and promoting apoptosis might offer promise for the treatment of cancer and/or neurodegenerative diseases. Here, we highlight recent developments in the field and discuss possible connections, hypotheses and perspectives in clioquinol-related research.

High-Throughput Flow Cytometry Drug Combination Discovery with Novel Synergy Analysis Software, SynScreen.

Classical therapeutic regimens are subject to toxicity, low efficacy, and/or the development of drug resistance. Thus, the discovery of synergistic drug combinations would permit treatment with lower, tolerable dosages of each agent and restored sensitivity. We describe the development and use of the SynScreen software application, which allows for visual and mathematical determinations of compound concentrations that produce super-additive effects. This software uses nonlinear regression fits of dose responses to determine synergism by the Bliss independence and Loewe additivity analysis models. We demonstrate the utility of SynScreen with data analysis from in vitro high-throughput flow cytometry (HTFC) combination screens with repurposed drugs and multiplexed synergy analysis of multiple biologic parameters in parallel. The applicability of SynScreen was confirmed by testing open-source data sets used in published drug combination literature. A key benefit of SynScreen for high-throughput drug combination screening is that observed measurements are graphically depicted in comparison with a three-dimensional surface that represents the theoretical responses at which Bliss additivity would occur. These images and summary tables for the calculated drug interactions are automatically exported. This allows for substantial data sets to be visually assessed, expediting the quick identification of efficacious drug combinations and thereby facilitating the design of confirmatory studies and clinical trials.

High-Throughput Flow Cytometry Identifies Small-Molecule Inhibitors for Drug Repurposing in T-ALL.

Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.

Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia.

Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.