Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and novel therapeutics are much needed. Profiling patient leukemia' drug sensitivities ex vivo, we discovered that 44.4% of childhood and 16.7% of adult T-ALL cases exquisitely respond to dasatinib. Applying network-based systems pharmacology analyses to examine signal circuitry, we identified preTCR-LCK activation as the driver of dasatinib sensitivity, and T-ALL-specific LCK dependency was confirmed in genome-wide CRISPR-Cas9 screens. Dasatinib-sensitive T-ALLs exhibited high BCL-XL and low BCL2 activity and venetoclax resistance. Discordant sensitivity of T-ALL to dasatinib and venetoclax is strongly correlated with T-cell differentiation, particularly with the dynamic shift in LCK vs. BCL2 activation. Finally, single-cell analysis identified leukemia heterogeneity in LCK and BCL2 signaling and T-cell maturation stage, consistent with dasatinib response. In conclusion, our results indicate that developmental arrest in T-ALL drives differential activation of preTCR-LCK and BCL2 signaling in this leukemia, providing unique opportunities for targeted therapy.

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia.

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

LFA-1 co-stimulation inhibits T(h)2 differentiation by down-modulating IL-4 responsiveness.

Initial T cell activation in the context of different co-stimulatory receptors can influence subsequent lineage commitment into T(h) effector cell subtypes. Specifically, CD28 co-stimulation promotes T(h)2 differentiation, whereas leukocyte function-associated antigen-1 (LFA-1) co-stimulation promotes T(h)1 differentiation and inhibits T(h)2 differentiation. In this report, we have addressed the mechanism of LFA-1-mediated inhibition of T(h)2 responses. We show that co-stimulation through LFA-1 does not decrease early IL-4 secretion, but rather induces a loss in IL-4 responsiveness. T cells primed in the context of LFA-1 co-stimulation require a 5-fold increase in the concentration of IL-4 required to drive T(h)2 differentiation, which is not mediated by a loss in IL-4R expression. To determine whether LFA-1 co-stimulation impacts on proximal signaling from the IL-4R, we first identified a kinetic window where we could separate IL-4-driven T(h)2 differentiation from initial T cell priming. T cells were primed for 2 days under different co-stimulation conditions and re-cultured in the presence of IL-4. Subsequent T(h)2 differentiation was absolutely dependent on addition of IL-4. Proximal IL-4R signaling, as evidenced by tyrosine phosphorylation of signal transducer and activator of transcription-6 (STAT6), was not inhibited by initial co-stimulation through LFA-1, yet these T cells still required higher amounts of IL-4 and corresponding higher levels of STAT6 activation to up-regulate GATA-3 and induce T(h)2 differentiation. Thus, LFA-1 co-stimulation appears to interfere with GATA-3 expression downstream of STAT6. These results suggest that LFA-1 co-stimulation functions as a threshold modulator of T(h)2 differentiation, increasing the effective concentration of IL-4 required to drive T(h)2 responses.

The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana.

Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.